Cytoskeleton modulating compounds for neuroprotection and AXON regeneration

Abstract

Compositions and methods for treating a mammalian subject for axonopathies by administering an effective dose of an agent or combination of agents that inhibit actin polymerization or enhance F-actin depolymerization. In some embodiments an axonopathy affects the optic nerve, e.g. degeneration of axons and / or soma of retinal ganglion cells (RGCs). The subject may be treated after a determination of IOP elevation, where the agent is delivered intravitreally, by injection-mediated retroorbital RGC targeting, and the like. The agent may comprise a small molecule agent, an AAV vector with a promoter operably linked to a gene, e.g. gelsolin, or a combination thereof.

Description

CYTOSKELETON MODULATING COMPOUNDS FOR NEUROPROTECTION AND AXON REGENERATIONGOVERNMENT SUPPORT RESEARCH

[0001] This invention was made with Government support under contract EY024932 awarded by the National Institutes of Health. The Government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the filing of United States Provisional Application Serial No. 63 / 748,364, filed January 22, 2025, the disclosure of which application is herein incorporated by reference.INTRODUCTION

[0003] Axonopathy is a common early feature of central nervous system (CNS) neurodegenerative diseases, characterized by CNS axon degeneration followed by progressive neuronal cell body death. The axons of adult CNS neurons do not regenerate spontaneously after degeneration, which causes irreversible neuronal function deficits. The axonopathy may be the result of disease or trauma, such as amyotrophic lateral sclerosis (ALS); optic nerve diseases including optic nerve traumatic injury, glaucoma, NAION (Non-arteritic Anterior Ischemic Optic Neuropathy), Arteritic anterior ischemic optic neuropathy (AAION), and optic neuritis; multiple sclerosis; hereditary spastic paraplegia (HSP); traumatic brain injury; spinal cord injury; or neuronal injury induced by a toxic agent such as a chemotherapeutic agent. Neural repair therapies that promote neuroprotection and axon regeneration are desperately needed.

[0004] Mammalian central nervous system (CNS) axons, which play a pivotal role in forming and maintaining neural circuits and functions, do not regenerate after injury or in disease- induced degenerations. Deletion of Pten (phosphatase and tensin homolog) was initially found to promote mouse optic nerve (ON) regeneration after ON crush (ONC) injury, and it remains by far the most potent single gene manipulation strategy to promote ON regeneration. Similar axon regeneration phenotypes induced by Pten inhibition have also been reported for rodent and C. elegans motor neuronsand drosophila sensory neurons.

[0005] A number of molecules have been determined to provide pro-axon regeneration, however, it remains to be determined how these molecules achieve significant axon regeneration and neuroprotection. Elucidation of the common molecular mechanisms responsible for the pro-regeneration / pro-neuroprotection phenotypes of these downstream molecules of Pten deletion may shed light on novel neural repair strategies and avoid side effects coming from upstream signaling master regulators.

[0006] Provided herein are methods and neuroprotective agents for neuroprotection.SUMMARY

[0007] Compositions and methods for treating a mammalian subject for axonopathies by administering an effective dose of an agent or combination of agents that inhibit actin polymerization, or that enhance F-actin depolymerization. It is shown herein that actin depolymerization is a driving force of axons (nerves), for example optic nerve, regeneration. Pro-regeneration / pro-actin depolymerization therapies promotes substantial axon regeneration, at least in part by significantly enhancing axonal mitochondria transport.

[0008] Conditions of interest for treatment include axonopathy that is a result of disease or trauma, such as amyotrophic lateral sclerosis (ALS); optic nerve diseases including optic nerve traumatic injury, glaucoma, NAION (Non-arteritic Anterior Ischemic Optic Neuropathy),Arteritic anterior ischemic optic neuropathy (AAION), and optic neuritis; multiple sclerosis; hereditary spastic paraplegia (HSP); traumatic brain injury; spinal cord injury; or neuronal injury induced by a toxic agent. In some embodiments an axonopathy affects the optic nerve, e.g. degeneration of axons (optic nerves) and / or soma of retinal ganglion cells (RGCs). In some embodiments an axonopathy affects the motor neurons, e.g. degeneration of axons (such as sciatic nerves) and / or soma of spinal cord or motor cortex motor neurons. In some embodiments ON neuropathies, including without limitation retinal ganglion cell degeneration, glaucoma, optic neuritis, ON traumatic injury and other ON-related diseases, are treated by practicing the methods disclosed herein. In some embodiments ALS or HSP, including without limitation motor neuron degeneration, motor axon degeneration, sciatic nerve degeneration, and Neuromuscular junction (NMJ) degeneration in ALS or HSP, are treated by practicing the methods disclosed herein. It is further shown herein that there is a correlation between extracellular levels of F-actin and the severity of glaucomatous neurodegeneration, which may be treated by the methods of the disclosure. The subject being treated may be diagnosed with an axonopathy prior to treatment.

[0009] Treatment comprises administration of an effective dose of an agent targeting the cytoskeleton. In some embodiments an agent targets F-actin depolymerization. In some such embodiments an agent enhances actin depolymerization, or inhibits actin polymerization.

[0010] In some embodiments an agent that targets active depolymerization is a small molecule that enhances actin depolymerization or inhibits actin polymerization. Agents of interest include, without limitation, Latrunculin B, Latrunculin A, Cytochalasin D, Cytochalasin B, Blebbistatin, CK-666, BMS-754807, etc. Aspects of the disclosure include formulations of an agent that targets actin depolymerization, and a pharmaceutically acceptable excipient that is suitable for delivery to the nerves, and which may provide for sustained release delivery to the nerve. In some embodiments the formulation is suitable for ocular delivery. In some embodiments, anagent is combined with administration of a vector, e.g. an AAV vector, that expresses a gene that targets actin depolymerization.

[0011] Genes of interest include one or a combination of: Gsn, Anxa2, Dstn, Cfl1 , CapG, Advillin / Avil, and Adseverin / Scin. In some such embodiments the gene is one or a combination of Gsn, Dstn, Cfl1 and CapG. Alternative vectors of interest disrupt expression of genes that enhance polymerization, e.g. profilin 1 (Pfn1), Arp2 / 3 or tropomyosin (Tpm1,3). In some embodiments the vector is delivered by intravitreal injection. In some embodiments the vector is delivered by injection-mediated retrograde RGC targeting.

[0012] In some embodiments a gene of interest for treatment of an axonopathy is gelsolin (Gsn), which may be operably linked to a selective promoter in the context of an AAV vector. In some embodiments treatment with Gsn is combined with treatment with a small molecule, including for example Latrunculin B. In certain embodiments, treatment is initiated for an individual after diagnosis of elevated IOP.

[0013] Aspects of the disclosure include formulations of a vector that targets actin depolymerization, and a pharmaceutically acceptable excipient that is suitable for delivery to the nerves, and which may provide for sustained release delivery to the nerve. In some such embodiments the agent enhances actin depolymerization, or inhibits actin polymerization. In some embodiments the vector expresses a gene that targets actin depolymerization, including one or a combination of: Gsn, Anxa2, Dstn, Cfl1 , CapG, Advillin / Avil, and Adseverin / Scin. In some such embodiments the gene is one or a combination of Gsn, Dstn, Cfl1 and CapG. In some embodiments Gsn is administered in combination with a gene encoding Anxa2.

[0014] The neuroprotective effects can include a range of outcomes. For instance, neuroprotective and regenerative effects may include, without limitation, a reduction in neuronal cell body death, a reduction in neuronal axon death, a promotion of axon regeneration, an improvement in visual acuity when compared to the absence of treatment, an increase in axonal mitochondria transport relative to the absence of treatment, and the like. In an embodiment, a method is provided for treating a mammalian subject for optic nerve (ON) neuropathies, and / or reducing or ameliorating degeneration of axons and / or soma of retinal ganglion cells (RGCs) and promoting optic nerve regeneration by administering an effective dose of an agent that targets actin polymerization as described herein. In some embodiments administration is performed after a determination that IOP is elevated.

[0015] In some embodiments a formulation is provided, comprising an agent that targets actin polymerization as described above. The formulation may be provided in a unit dose for delivery to the eye. The formulation may be provided topically for intra-vitreal injection or for retroorbital injection, or systemic delivery. The formulation may be provided for sustained release to the eye. In some aspects, provided herein is a method of inducing neuroprotection / increasing survival I promoting functional recovery of RGC somata and axons, comprising intravitreallyadministering the composition into a mammalian subject experiencing or at risk of an ON axonopathy. In some embodiments of the method, the ON neuropathy is retinal ganglion cell degeneration, including glaucoma, optic neuritis, ON traumatic injury and other ON-related diseases.

[0016] In an embodiment, a method is provided for treating a mammalian subject for optic nerve (ON) neuropathies, and / or reducing or ameliorating degeneration of axons and / or soma of retinal ganglion cells (RGCs) and promoting optic nerve regeneration by administering an effective dose of a therapeutic gene therapy viral vector, comprising a murine y-synuclein promoter, or functional fragment thereof, that promotes expression of a transgene specifically in RGCs, said promoter in operable linkage with an expression cassette encoding the transgene where the transgene inhibits active polymerization. The vector may be administered in combination with a small molecule agent, as disclosed herein. In some embodiments, the therapeutic gene therapy vector is an AAV virus comprising a therapeutic sequence. In some embodiments, the therapeutic vector comprises a CRISPR / Cas9 system and at least one guide RNA (gRNA) directed to a gene that enhances actin polymerization, e.g. profilin 1 (Pfn 1 ) , Arp2 / 3 or tropomyosin (Tpm1 ,3).

[0017] In some embodiments a formulation is provided, comprising an effective dose of a small molecule, and / or therapeutic gene therapy viral vector, comprising a murine y-synuclein promoter, or functional fragment thereof, that promotes expression of a transgene specifically in RGCs, said promoter in operable linkage with an expression cassette encoding the transgene, wherein the expressed transgene inhibits active polymerization. The formulation may be provided in a unit dose for delivery to the eye. The formulation may be provided for intra-vitreal injection. The formulation may be provided for sustained release to the eye. In some aspects, provided herein is a method of inducing neuroprotection / increasing survival I promoting functional recovery of RGC somata and axons, and promoting optic nerve regeneration, comprising intravitreally administering the composition into a mammalian subject experiencing or at risk of an ON axonopathy. In some embodiments of the method, the ON neuropathy is retinal ganglion cell degeneration, including glaucoma, optic neuritis, ON traumatic injury and other ON-related diseases

[0018] Aspects of the disclosure include administering a formulation disclosed herein to treat the subject for the ON neuropathy either intravitreally or systemically. When the formulation is administered, it may be administered at a time that is dependent on the type of ON neuropathy being treated. For instance, if the ON neuropathy is the result of traumatic injury, the composition may be administered within hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, one week, two weeks or more than two weeks after traumatic injury.

[0019] In some embodiments, the composition of the present disclosure may be administered with a secondary treatment modality that is used to further treat the optic neuropathy. If the optic neuropathy is glaucoma, a number of different secondary treatment modalities may be administered. For instance, secondary treatment modalities for glaucoma may include, without limitation, prostaglandins (e.g. latanoprost, travoprost, tafluprost, or bimatoprost), rho kinase inhibitors (e.g. netarsudil), nitric oxides (e.g. latanoprostene bunod), miotic or cholinergic agents (e.g. pilocarpine), alpha-adrenergic agonists (e.g. apraclonidine or brimonidine), beta blockers (e.g. betaxolol or timolol), carbonic anhydrase inhibitors (e.g. dorzolamide or brinzolamide), etc.BRIEF DESCRIPTION OF THE FIGURES

[0020] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0021] FIGS. 1A-1 F. Actin depolymerization promotes significant ON regeneration in ONG mouse model. (A) Scatter plot of differentially expressed genes (DEGs, adjusted p value < 0.05, avg_log2FC > 0.25) of regRGCs compared to non-regenerating surRGCs. The x-axis represents the change in the percentage of cells expressing each gene, and the y-axis shows the average log fold change in gene expression. Orange, gray, and green dots represent up- regulated, non-significantly changed, and down-regulated genes, respectively. Red dots highlight up-regulated genes in regRGCs involved with actin dynamics. The red-coded genes, including Spp1, Anxa2, Ecm1 , Gsn, and Mpp1 , have been experimentally confirmed in axon regeneration. (B) Protein protein interaction (PPI) network diagram of regRGC-enriched genes’ that was analyzed using the STRING database and visualized with Cytoscape. Nodes represent genes and edges indicate interactions, with thicker lines indicating stronger or more significant interactions. Genes are clustered based on their roles in actin-based processes. Additional proregeneration genes Ilk and Plat (tPA) are also incorporated into the analysis. Experimentally proved pro-axon regeneration genes are highlighted by red. (C) Representative confocal images of ON wholemounts after optical clearance showing maximum intensity projection of regenerating fibers labeled with CTB-Alexa 555 at 14dpc. AAV intravitreal injection to deliver Gsn, Dstn, Cfl1 or Ctrl was performed two weeks before ONC. Scale bar, 100 pm. *: crush site. (D) Quantification of regenerating fibers at different distances distal to the lesion site, compared to AAV-ctrl.. n=8 in all groups. (E) Representative confocal images of ON wholemounts as in (D) harvested from animals after vehicle or compounds (LatB or CytoD) treatment or Pten deletion. Compound intravitreal injection was performed twice weekly starting two days beforeONC. (F) Quantification of regenerating fibers from wholemounts as in (F). Vehicle, n=5; CytoD, n=8; LatB, n=5; Pten KO, n=10. Data in (D, F) are presented as means ± s.e.m p<0.01 , ***: p<0.001 , “**: p<0.0001 , two-way ANOVA with Sidak’s multiple comparisons test.

[0022] FIGS. 2A-2F. Inhibition of actin polymerization promotes significant ON regeneration.(A) Representative confocal images of ON wholemounts after optical clearance showing maximum intensity projection of regenerating fibers labeled with CTB-Alexa 555 at 14dpc. AAV intravitreal injection to deliver Pfn1 , Pfn2, Arp2 / 3, or Ctrl was performed two weeks before ONC; compound Jasp intravitreal injection was performed twice weekly starting two days before ONC. Scale bar, 100 pm. *: crush site. (B) Quantification of regenerating fibers from wholemounts as in (A). (C) Representative confocal images of ON wholemounts after optical clearance showing maximum intensity projection of regenerating fibers labeled with CTB-Alexa 555 at 14dpc. AAV intravitreal injection to deliver Cas9 and gRNAs was performed two weeks before ONC. Scale bar, 100 pm. *: crush site. (D) Quantification of regenerating fibers from wholemounts as in (C). (E) Representative confocal images of ON wholemounts after optical clearance showing maximum intensity projection of regenerating fibers labeled with CTB-Alexa 555 at 14dpc. Compound CK666 intravitreal injection was performed twice weekly starting two days before ONC. Scale bar, 100 pm. * crush site. (F) Quantification of regenerating fibers from wholemounts as in (E). Data in (B, D, F) are presented as means ± s.e.m, n =5-9, **: p<0.01 , p<0.0001 , two-way ANOVA with Sidak’s multiple comparisons test.

[0023] FIGS. 3A-3M. Actin depolymerization disrupts the axonal actin ring and increases axonal mitochondria motility and axon growth in cultured hippocampal neurons. (A) Superresolution images of F-actin in the axons of cultured hippocampal neurons treated with vehicle, Gsn, or LatB. The z-positions in the STORM images are color-coded according to the color scale, with violet and red indicating positions closest to and farthest from the substratum, respectively. The enlarged images show periodic actin ring-like MPS, submembrane longitudinal long F-actin, and filopodia F-actin in a growth cone outlined by dashed lines and indicated by a red arrow. (B) Average one-dimensional (1 D) autocorrelation functions of the imaged periodic actin rings, calculated from randomly selected axonal regions under the three indicated conditions (left), with corresponding autocorrelation amplitudes reflecting their structural integrity (right). Data are presented as means ± s.e.m. Vehicle group: n =24 axons; Gsn-treated group: n=23 axons; LatB- treated group: n=24 axons; all from 4 experimental replicates. (C) Quantification of the length of axonal longitudinal submembrane F-actin. Vehicle group: n =17 axons; Gsn-treated group: n=15 axons. LatB-treated group: n= 15 axons; all from 4 experimental replicates. (D) Quantification of the area of filopodia F-actin in growth cones. Vehicle group: n =8 growth cones; Gsn-treated group: n=7 growth cones; LatB-treated group: n=5 growth cones; all from 3 experimental replicates. Data in B-D are presented as means ± s.e.m. *: p<0.05, **: p<0.01 , p<0.001 , *”*: p<0.0001, one-way ANOVA with Tukey multiple comparisons test.(E) In vitro reconstitution assay of cargo transport along microtubules: Cartoon illustration of cargo-bound KIF5B-GFP on microtubules and timelapse snapshots of the KIF5B-GFP coated beads (2.8pm diameter) walking along in vitro polymerized microtubules immobilized on a coverslip, with or without F-actin / Gsn (100nM); KIF5B-GFP coated beads (cyan); F-actins (yellow); and microtubules (magenta); scale bar is 10pm. (F) Quantification of percentage of moving beads. Each dot in the graph represents one experiment. The quantities of analyzed beads in each group: 201 beads in three experiments without F-actin; 160 beads in seven experiments with F-actin; and 149 beads in eight experiments with F-actin+Gsn. (G) Quantification of moving beads’ velocity. Each dot in the graph represents one bead. The quantities of data points in each group: 63 beads in three experiments without F-actin, 69 beads in three experiments with F-actin; and 48 beads in three experiments with F-actin+Gsn. Data in F,G are presented as means ± s.e.m. **** p<0.0001 , one-way ANOVA with Tukey multiple comparisons test. (H) Axonal mitochondrial kymograph (anterograde: blue, retrograde: orange) in hippocampal neuron transfected with GFP or Gsn-GFPplus MitoDsRed for mitochondria labeling. The first frame (time = Os) of a live imaging series is shown with the kymograph. Horizontal scale bar is 10 pm; vertical sale bar is 60s. (I) Quantifications of average speed, move length, and percentage of mobile mitochondria. GFP control group: n=171 mitochondria from 17 axons of 4 experimental repeats; Gsn-treated group: n=419 mitochondria from 34 axons of 12 experimental repeats. *: p<0.05, **: p<0.01, *** p<0.001, p<0.0001 , two-way ANOVA with Sidak’s multiple comparisons test. (J) Quantification of total axonal mitochondria numbers per 100pm of axons. GFP control group: n =58 axons of 4 experimental repeats; Gsn-treated group: n=50 axons of 12 experimental repeats. (K) Timelapses images of growth cone extension. (L) Temporal color projection and kymograph of growth cones extension. (M) Quantification of growth cone extension speed and neurite length. Growth Speed, GFP: n =45 growth cones of 6 experimental repeats; Gsn: n=58 growth cones of 8 experimental repeats; Neurite Length, GFP: n =695 axons of 15 experimental repeats; Gsn: n=400 axons of 34 experimental repeats. (J and M) Data are presented as mean s.e.m, ****: p<0.0001 , non- parametric Mann-Whitney test.

[0024] FIGS. 4A-4F. Axonal mitochondria transport is essential for actin depolymerization- mediated axon regeneration. (A) Representative confocal images of ON wholemounts after optical clearance showing maximum intensity projection of regenerating fibers labeled with CTB- Alexa 555 at 14dpc. AAV intravitreal injection to deliver Gsn, Cre, or Ctrl was performed two weeks before ONC. Scale bar, 100 pm. *: crush site. (B) Quantification of regenerating fibers from wholemounts as in (A). (C) Representative confocal images of ON wholemounts after optical clearance showing maximum intensity projection of regenerating fibers labeled with CTB- Alexa 555 at 14dpc. AAV intravitreal injection to deliver Cre was performed two weeks before ONC; compound LatB or vehicle intravitreal injection was performed twice weekly starting two days before ONC. Scale bar, 100 pm. *: crush site. (D) Quantification of regenerating fibers fromwholemounts as in (C). (E) Representative confocal images of ON wholemounts after optical clearance showing maximum intensity projection of regenerating fibers labeled with CTB-Alexa 555 at 14dpc. Compound BMS754807 or vehicle intravitreal injection was performed twice weekly starting two days before ONC. Scale bar, 100 gm. *: crush site. (F) Quantification of regenerating fibers from wholemounts as in (E). Data in (B, D, F) are presented as means ± s.e.m, n =6-7. *: p<0.05, p<0.01, ****: p<0.0001 , two-way ANOVA with Sidak’s multiple comparisons test.

[0025] FIGS. 5A-5G. Combinatory genetic modulations act synergistically to functionally reinnervate SCN. (A) Light-sheet fluorescent image of iDISCO cleared whole brain with attached ON showing regenerating axons in ON, optic chiasm, SCN, and optic tract in Pten / Socs3 / Gsk3|3 triple floxed mice injected with AAVs to overexpress Gsn, Anxa2, Ecm1 , and Cre together with Plat treatment at 8 weeks post crush (8wpc). The red dashed circles outline suprachiasmatic nucleus (SCN). *:crush site. Scale bar, 500pm. Pre-OX: pre-optic chiasm; Post-OX: post optic chiasm; Ipsi-SCN: ipsilateral SCN; Cont-SCN: contralateral SCN; Cont-ON: contralateral optic nerve. (B) Z-stack of regenerating axons at different locations along the projection pathway in the brain. (C) Quantification of regenerating axons from the Z-stacks in B. (D) Schematic illustrating the experimental procedures for detecting light-evoked c-fos expression in SCN. (E) Brain sections from naive mice, P / S / G floxed mice with AAV-Ctrl, or with AAV- Cre+Gsn+Anxa2+Ecm1 -i-Plat showing c-fos+ cells in SCN in response to light stimulation. Scale bar, 200 pm. SCN is outlined by a white dash circle; c-fos+ cells are marked by red arrows. Data are presented as mean s.e.m. AAV-Ctrl: n=8 SCNs; Combined treatment: n=10 SCNs. Each dot represents an individual SCN. **: p<0.01 , non-parametric Mann-Whitney test. (F) Schematic of animal preparation for fiber cannula implantation and fiber photometry of SCN in response to light stimulation. The brain slice image showing SCN was labeled by GCaMP7s and fiber cannula insertion to approach SCN. (G) Left: SCN Ca2+ waveforms in response to light stimulation at 3-second On and 5-second Off intervals recorded by fiber photometry Right: Quantification of AF / F0. Data are presented as mean s.e.m. n=7 SCNs, *: p<0.05, **: p<0.01 , paired student’s t-test.

[0026] FIGS. 6A-6H. RGC-specific Gsn overexpression ameliorates ON mitochondria transport deficits, visual functions and survival of RGCs and ONs in the mouse SOHU glaucoma model. (A) Left panel of each group: 5 minutes color-coded temporal projection of MitoTracker Orange- labeled mitochondria in ex vivo ON wholemounts from naive mice, SOHU glaucoma mice with AAV-Ctrl, or SOHU glaucoma mice with AAV-mSncg-Gsn at 1wpi. Scale bars, 50pm; color scale, 1-300s. Right panel of each group: 2D mitochondrial movement tracing in ONs, each trace represents an individual mitochondrion’s moving trajectory. Time scale bar, 100s. (B) Quantifications of average speed, move length, and percentage of mobile mitochondria in groups as in (A). In average speed quantification, naive: n = 1795 mitochondria from 8 ONs of4 mice; SOHU: n = 217 mitochondria from 6 ONs of 6 mice; Gsn-treated SOHU: n = 1192 mitochondria from 6 ONs of 6 mice; In moving length quantification, naive: n = 1114 mitochondria from 8 ONs of 4 mice; SOHU: n = 172 mitochondria from 6 ONs of 6 mice; Gsn- treated SOHU: n = 473 mitochondria from 6 ONs of 6 mice. For percentage of mobile mitochondria quantification, naive: n = 8 ONs; SOHU: n = 6 ONs; Gsn-treated SOHU: n = 6 ONs. Data are presented as means ± s.e.m, *: p<0.05, ** p<0.01 , “*: p<0.001 , p<0.0001 , with one-way ANOVA with Turky multiple comparisons test. (C) Quantification of axonal mitochondria total numbers per 10000 pm2 ON. Naive: n = 8 ONs; SOHU: n = 6 ONs; Gsn- treated SOHU: n = 6 ONs. Data are presented as means ± s.e.m, *: p<0.05, p<0.001 , with one-way ANOVA with Tukey multiple comparisons test. (D) Representative in vivo OCT images of mouse retinas from contralateral (CL) naive, SOHU glaucomatous, and Gsn-treated SOHU glaucomatous eyes at three weeks post SO injection (3wpi). GCC: ganglion cell complex, including RNFL, GCL and I PL layers; indicated as double end arrows. Quantification of GCC thickness measured by OCT at 3wpi, represented as percentage of GCC thickness in the SOHU eyes compared to the sham CL eyes. (E) Upper: cartoon illustration of visual acuity measurement by OKR equipment. Lower: visual acuity measured by OKR at 3wpi, represented as percentage of glaucomatous eyes compared to the sham CL eyes. (F) Upper: representative wave forms of PERG at baseline and 3wpi; blue traces represent glaucomatous eyes; black traces represent contralateral control (CL) eyes. Lower: quantification of P1 -N2 amplitude of PERG at 3wpi, represented as a percentage of glaucomatous eyes compared to the sham CL eyes. All quantification data are presented as means ± s.e.m, n=12-15 in each group. (G) Representative confocal images of the whole flat-mounted retinas (scale bar, 500pm) and central, middle and peripheral flat-mounted retinas (scale bar, 50pm) showing surviving RBPMS+ RGCs at 3wpi. Quantification of surviving RGC somata in peripheral, middle, and central retinas at 3wpi, represented as percentage of glaucomatous eyes compared to the sham CL eyes. (H) Light microscope images of semi-thin transverse sections of ON and enlarged regions with PPD staining at 3wpi. Scale bar, 10pm. Quantification of surviving axons in ONs at 3wpi, represented as percentage of glaucomatous eyes compared to the sham CL eyes. n=12- 15 in each group. All quantification data in D-H are presented as means ± s.e.m, *: p<0.05, ** p<0.01 , p<0.001 , p<0.0001 , with non-parametric Mann-Whitney test.

[0027] FIGS. 7A-7H. Delayed actin depolymerization treatment by LatB ameliorates ON mitochondria transport deficits, visual functions and survival of RGCs and ONs in the mouse SOHU glaucoma model. (A) Upper: 5-minute color-coded temporal projection of MitoTracker Orange- labeled mitochondria in ex vivo ON wholemounts of SOHU mice at 1wpi. Scale bars, 50pm; color scale, 1-300s. Lower: 2D mitochondrial movement tracing in ONs, each trace represents an individual mitochondrion’s moving trajectory. Time scale bar, 100s. (B) Quantifications of average speed, move length, and percentage of mobile mitochondria in eachON. In average speed quantification, vehicle-treated SOHU group: n = 377 mitochondria from 6 ONs of 6 mice; LatB- treated SOHU group: n = 1742 mitochondria from 10 ONs of 10 mice. In moving length quantification, vehicle-treated SOHU group: n = 577 mitochondria from 6 ONs of 6 mice; LatB- treated SOHU group: n = 6049 mitochondria from 10 ONs of 10 mice. For percentage of mobile mitochondria quantification, vehicle-treated SOHU group: n = 6 ONs; LatB- treated SOHU group: n = 10 ONs. (C) Quantification of axonal mitochondria total numbers per 10000 pm2 ON. Vehicle- treated SOHU group: n = 6 ONs of 6 mice; LatB-treated SOHU group: n = 10 ONs of 10 mice. (D) Representative in vivo OCT images of mouse retinas from CL naive, vehicle-treated or delayed LatB-treated SOHU glaucomatous eyes at 3wpi. GCC: indicated as double end arrows. Quantification of GCC thickness measured by OCT at 3wpi, represented as percentage of GCC thickness in the SOHU eyes compared to the sham CL eyes. (E) Quantification of P1-N2 amplitude of PERG at 3wpi, represented as a percentage of glaucomatous eyes compared to the sham CL eyes. (F) Visual acuity measured by OKR at 3wpi, represented as percentage of glaucomatous eyes compared to the sham CL eyes. (G) Representative confocal images of the whole flat-mounted retinas (scale bar, 500pm) and central, middle and peripheral flat-mounted retinas (scale bar, 50pm) showing surviving RBPMS+ RGCs at 3wpi. Quantification of surviving RGC somata in peripheral, middle, and central retinas at 3wpi, represented as percentage of glaucomatous eyes compared to the sham CL eyes. (H) Light microscope images of semi-thin transverse sections of ON and enlarged regions with PPD staining at 3wpi. Scale bar, 10pm. Quantification of surviving axons in ONs at 3wpi, represented as percentage of glaucomatous eyes compared to the sham CL eyes. All the quantification data are presented as means ± s.e.m, n=12-15 in each group, ns: p>0.05, *: p<0.05, **: p<0.01 , ***: p<0.001 , “**: p<0.0001 , with non-parametric Mann-Whitney test.

[0028] FIGS. 8A-8G. F-actin depolymerization increases axon growth and axonal mitochondria transport in human RGCs. (A) Fluorescence images of human retinal organoids containing tdTomato+ RGC axons and quantification of the numbers of RGC axons at different lengths. Scale bar, 200 pm. Data are presented as means ± s.e.m. DMSO: n =7, LatB: n=8; all from 2 experimental repeats. *: p<0.05, p<0.01, p<0.001 , **“: p<0.0001, two-way ANOVA with Sidak’s multiple comparisons test. (B) Neurite outgrowth of human RGCs isolated from retinal organoids (left) and quantification of the neurite length (left). Scale bar is 50 pm. Data are presented as means ± s.e.m. DMSO: n =107, LatB: n=142; all from 3 experimental repeats. *“*: p<0.0001 , non-parametric Mann-Whitney test. (C) Mitochondrial movement (anterograde: blue, retrograde: orange) in human RGC axons treated with vehicle (DMSO) or LatB plus MitoTracker for mitochondria labeling. The first frame (time = 0s) of a live imaging series is shown with the kymograph. Horizontal scale bar is 20 pm; vertical scale bar is 1 minute. (D) Quantification of average speed, move length, and percentage of mobile mitochondria: vehicle group: n=79 mitochondria from 22 axons of 4 experimental repeats; LatB-treated group: n=173 mitochondriafrom 50 axons of 6 experimental repeats. Data are presented as means ± s.e.m., ns: p>0.05, *: p<0.05, **: p<0.01 , p<0.001 , “**: p<0.0001 , with two-way ANOVA with Sidak’s multiple comparisons test. (E) Quantification of axonal mitochondria total numbers per 100pm of axons. Vehicle group: n =29 axons; LatB-treated group: n=58 axons. Data are presented as means ± s.e.m. DMSO: n =107, LatB: n=142. ****: p<0.0001 , non-parametric Mann-Whitney test. (F) F- actin ELISA assay of human aqueous humor. Left, the standard curve of F-actin used to measure the F- actin concentrations in human aqueous humor. Right, the F-actin concentration in aqueous humor of control (cataract patients), mild, and severe glaucoma patients. Cataract control, n=10; mild glaucoma, n=12; Severe glaucoma, n=7. Data are presented as means ± s.e.m, ns: no significance, *: p<0.05, ** p<0.01 , with one-way ANOVA with Tukey multiple comparisons test. (G) Two factors distribution plotting of F-actin concentration and visual field mean deviation of human patients.

[0029] FIGS. 9A-9H. AAV-mediated RGC specific expression of actin depolymerization molecules in ON regeneration after ONC. (A) Violin plot of Gsn mRNA levels of regRGCs and surRGCs. (B) Retinal wholemounts of Pten KO mice at 14 days post crush (14dpc) showing Gsn labeling (green) in regRGCs (retrogradely traced with Dextran-red), blue arrows show colabeling of Gsn with dextran+ / RBPMS+ regRGCs. Scale bar, 50pm. (C) Cartoon illustration of the genes and small molecules that act on F-actin severing and depolymerization. (D) AAV- mediated transgene expression in RGCs labeled by HA antibodies two weeks after intravitreal injection of HA-tagged genes. Scale bar, 50 pm. (E) Representative confocal images of ON wholemounts after optical clearance showing maximum intensity projection of regenerating fibers labeled with CTB-Alexa 555 at 14dpc. AAV intravitreal injection to deliver the individual genes was performed two weeks before ONC. Scale bar, 100 pm. *: crush site. (F) Quantification of regenerating fibers at different distances distal to the lesion site from wholemounts as in (E). Data are presented as means ± s.e.m., WT: n = 8; Scin: n = 8; CapG: n =16; Avil: n = 10, ***; p<0.001 , two-way ANOVA with Sidak’s multiple comparisons test. (G) F-actin levels in mouse hippocampal neurons detected by SiR-actin with or without AAV-mediated expression of Avil or Scin. Scale bar, 100pm. Zoomed view of the white dashed boxes shown in left upper corner. (H) Quantification of F-actin fluorescence intensity of individual neurons’ somata and neurites. Data are presented as means ± s.e.m., WT: n = 241 neurons; Scin: n = 80 neurons; Avil: n = 238 neurons; 4 experimental repeats, ns: no significance, one-way ANOVA with Tukey multiple comparisons test.

[0030] FIGS. 10A-10D. AAV-mediated RGC-specific expression of actin polymerization / stabilization molecules in ON regeneration after ONC. (A) Cartoon illustration of the genes and small molecules acting on F-actin polymerization, branching, and stabilization. (B) AAV-mediated transgene expression in RGCs labeled by HA antibodies two weeks after AAV intravitreal injection of HA-tagged genes. Scale bar, 50pm. (C) Representative confocalimages of ON wholemounts after optical clearance showing maximum intensity projection of regenerating fibers labeled with CTB-Alexa 555 at 14dpc. AAV intravitreal injection to deliver the individual genes was performed two weeks before ONC. Scale bar, 100 pm. *: crush site. (D) Quantification of regenerating fibers at different distances distal to the lesion site from wholemounts as in (C). Data are presented as means ± s.e.m., n = 7-10; two-way ANOVA with Sidak’s multiple comparisons test.

[0031] FIGS. 11 A-11 E. F-actin levels in growth cones and axon shafts of cultured embryonic hippocampal neurons after treatment with actin depolymerization and polymerization molecules. (A) Endogenous Anxa2 and Gsn distribution in growth cones of E16 hippocampal neurons. Scale bar, 20pm. (B) F-actin levels in growth cones and axon shafts of hippocampal neurons transfected with HA-tagged Gsn, Anxa2, and Pfn1, detected by SiR-actin with or without treatments. Scale bar, 20pm. Dashed yellow boxes represent axon shafts shown at higher magnification in the lower panel. Dashed white boxes represent growth cones shown at higher magnification in the lower panel. (C) Quantification of F-actin levels in growth cones and axon shafts of hippocampal neurons from images as in (B). Growth cone (upper): Ctrl, n= 42 neurons; Gsn, n= 62 neurons; Anxa2, n= 28 neurons; Pfn1 , n= 45 neurons; all from 4-6 experimental repeats. Axon Shaft (lower): Ctrl, n= 42 neurons; Gsn, n= 58 neurons; Anxa2, n= 45 neurons; Pfn1 , n= 54 neurons; all from 3-4 experimental repeats. (D) F-actin levels in growth cones and axon shafts of hippocampal neurons treated with small molecules, detected by SiR-actin with or without treatments. Scale bar, 20pm. Dashed yellow boxes represent axon shafts shown at higher magnification in the lower panel. Dashed white boxes represent growth cones shown at higher magnification in the lower panel. (E) Quantification of F-actin levels in growth cones and axon shafts of hippocampal neurons from images as in (D). Growth cone (upper): DMSO, n= 47 neurons; LatB, n= 95 neurons; CytoD, n= 95 neurons; CK666, n= 22 neurons; Jasp, n= 32 neurons ; all from 2-4 experimental repeats. Axon Shaft (lower): DMSO, n= 35 neurons; LatB, n= 22 neurons; CytoD, n= 54 neurons; CK666, n= 36 neurons; Jasp, n= 39 neurons; all from 2-4 experimental repeats. All quantification data are presented as means ± s.e.m, *: p<0.05; **“: p<0.0001 , with one-way ANOVA, Tukey’s multiple comparisons test.

[0032] FIGS. 12A-12L. F-actin does not significantly affect microtubule-based movement of KIF5B-GFP without cargo or axonal recycling endosomes but significantly inhibits axonal lysosome transport. (A,B) In vitro reconstitution assay of transport along immobilized microtubules: Cartoon illustration of individual KIF5B-GFP molecules moving on microtubules in the presence or absence of F-actin; timelapse snapshots of KIF5B-GFP walking along the microtubules and kymographs of KIF5B, with or without F-actin. Cyan dots are KIF5B-GFP; yellow filaments are F-actins; and magenta lines are microtubules. Both scale bars represent 2 urn, timescale is 30 sec. (C) Quantification of percentage of moving KIF5B molecules per microtubule in the absence and in the presence of F-actin, burgundy, n=27; yellow, n=19. Eachdot in the graph represents one microtubule, Wilcoxon test, p = 0.14. (D) Quantification of moving KIF5B velocity, burgundy, n=226; yellow, n=234. Each dot in the graph represents one KIF5B molecule, Wilcoxon test, p = 0.0002. (E) Lysosome movement (anterograde: blue, retrograde: orange) in hippocampal neuron axons transfected with GFP or Gsn-GFP, plus SiR- Lysotracker labeled lysosomes (red). The first frame (time = 0s) of the live imaging series is shown with the kymograph. Vertical scale bar is 60s. Quantification of average speed, move length, and percentage of the mobile lysosomes. GFP group: n=126 lysosomes from 19 axons; Gsn-GFP group: n=149 lysosomes from 21 axons. 3 experimental repeats. (F) Recycling endosome (RE) movement (anterograde: blue, retrograde: orange) in hippocampal neuron axons transfected with GFP or Gsn-GFP, plus Rabi 1a-mCherry labeled REs. The first frame (time = 0s) of the live imaging series is shown with the kymograph. Vertical scale bar is 60s. Quantification of average speed, move length and percentage of the mobile Res: GFP group: n=98 REs from 16 axons; Gsn-GFP group: n=163 REs from 27 axons; 3-4 experimental repeats. In E,F, quantification data are presented as means ± s.e.m, ns: no significance, *: p<0.05, ** p<0.01 , p<0.001 , **** p<0.0001 , with two-way ANOVA with Sidak’s multiple comparisons test. (G) Axonal mitochondrial kymograph (anterograde: blue, retrograde: orange) in hippocampal neuron transfected with GFP or Anxa2-GFP plus MitoDsRed for mitochondria labeling. The first frame (time = 0s) of a live imaging series is shown with the kymograph. Horizontal scale bar is 10 pm; vertical sale bar is 60s. (H) Quantifications of average speed, move length, and percentage of mobile mitochondria. GFP control group: n=81 mitochondria from 17 axons; Anxa2 -treated group: n=503 mitochondria from 31 axons. 3-6 experimental repeats. *: p<0.05, **: p<0.01 , ***: p<0.001 , two-way ANOVA with Sidak’s multiple comparisons test. (I) Quantification of total axonal mitochondria numbers per 100pm of axons. GFP control group: n =75 axons; Anxa2-treated group: n=77 axons. (J) Timelapses images of growth cone extension. (K) Temporal color projection and kymograph of growth cones extension. (L) Quantification of growth cone extension speed and neurite length. Growth Speed, GFP: n =78 growth cones; Anxa2 n=44 growth cones; Neurite Length, GFP: n =17 axons; Anxa2 n=19 axons. 3 experimental repeats. (I and L) Data are presented as meant s.e.m, **: p<0.01 , p<0.0001 , non-parametric Mann-Whitney test.

[0033] FIGS. 13A-13I. Confirmation that LatB and BMS754807 increase axonal mitochondria motility. (A) Mitochondrial movement (anterograde: blue, retrograde: orange) in hippocampal neuron axons treated with vehicle or LatB plus MitoTracker for mitochondria labeling. The first frame (time = 0s) of a live imaging series is shown with the kymograph. Horizontal scale bar is 10 pm; vertical sale bar is 60s. (B) Quantifications of average speed, move length, and percentage of mobile mitochondria: vehicle control group: n=162 mitochondria from 37 axons; LatB-treated group: n=262 mitochondria from 24 axons. 4-5 experimental repeats. Data are presented as means ± s.e.m, *: p<0.05, ** p<0.01 , ***: p<0.001 , p<0.0001 , two-wayANOVA, Sidak’s multiple comparisons test. (C) Quantification of axonal mitochondria total numbers per 100pm of axons. Control group: n =43 axons; LatB-treated group: n=67 axons. 4- 5 experimental repeats. Data are presented as means ± s.e.m, p<0.0001 , with Student’s t- test. (D) Timelapse images, temporal color projection, and kymograph of growth cone extension with or without LatB treatment. (E) Quantification of growth cone extension speed. DMSO: n =114 growth cones; LatB: n=318 growth cones. 4-5 experimental repeats. Data are presented as meant s.e.m, p<0.0001 , non-parametric Mann-Whitney test. (F) Mitochondrial movement (anterograde: blue, retrograde: orange) in hippocampal neuron axons transfected with GFP, Pfn1-GFP, plus MitoDsRed labeled mitochondria. The first frame (time = Os) of live imaging series is shown with the kymograph. Vertical scale bar is 60s. (G) Quantification of average speed, move length, and percentage of the mobile mitochondria. For average speed and move length quantification, control group: n=81 mitochondria from 17 axons; Pfn1-GFP group: n=241 mitochondria from 53 axons. 3-6 experimental repeats. Quantification data are presented as means ± s.e.m, ns: no significance, *: p<0.05, with two-way ANOVA with Sidak’s multiple comparisons test. (H) Mitochondrial movement (anterograde: blue, retrograde: orange) in hippocampal neuron axons treated with DMSO vehicle or BMS754807, plus MitoTracker Orange to label mitochondria. The first frame (time = Os) of the live imaging series is shown with the kymograph. Vertical scale bar is 60s. (I) Quantification of average speed, move length, and percentage of the mobile mitochondria: DMSO group: n=20 mitochondria from 7 axons; BMS754807 group: n=43 mitochondria from 10 axons. Quantification data are presented as means ± s.e.m, ns: no significance, *: p<0.05, non-parametric Mann-Whitney test.

[0034] FIGS. 14A-14F The effects of combinatory genetic modulations on ON regeneration.(A) Confocal images of ON wholemounts after optical clearance showing maximum intensity projection of regenerating fibers labeled with CTB-Alexa 555 at 14dpc. AAV-Anxa2 and / or Gsn intravitreal injection was performed two weeks before ONC. Scale bar, 100 pm. *: crush site. (B) Quantification of regenerating fibers at different distances distal to the lesion site. Data are presented as means ± s.e.m., n = 8 of each group; two-way ANOVA with Sidak’s multiple comparisons test. In B, green * is comparison of Gsn+Anxa2 vs. AAV-Ctrl; blue * is comparison of Anxa2 vs. Ctrl; red * is comparison of Gsn vs. Ctrl; green # is comparison of Gsn+Anxa2 vs. either one. (C) AAV-mediated HA-Ecm1 expression in RGCs labeled by HA antibodies two weeks after AAV intravitreal injection. Scale bar, 50pm. (D) Confocal images of ON wholemounts after optical clearance showing maximum intensity projection of regenerating fibers labeled with CTB-Alexa 555 at 14dpc. AAV intravitreal injection was performed two weeks before ONC. Scale bar, 100 pm. *: crush site. Quantification of regenerating fibers at different distances distal to the lesion site. All the quantification data are presented as means ± s.e.m., n = 8 of each group; *: p<0.05; “: p<0.01 ; ***: p<0.001 ; p<0.0001 ; two-way ANOVA with Sidak’s multiple comparisons test. (E) Intravitreal injection of CTB647 to label the normal ONprojection pathway showing optic chiasm, suprachiasmatic nucleus (SON) and optic tract. Scale bar, 500 pm. (F) Light-sheet fluorescent images of iDISCO-cleared whole brain with attached ON showing lengthy regenerating axons (green) from the crushed eye and intact axons from the contralateral eye (red) in ON, optic chiasm, and SON in Pten / Socs3 / Gsk3|3 triple floxed mice injected with AAVs to overexpress Ore alone or together with Ecm1 + tPA or Gsn + Anxa2, at 8 weeks post crush (8wpc). The white dashed circles outline suprachiasmatic nucleus (SON). Scale bar, 500pm.

[0035] FIGS. 15A-15D. Identification of RGCs sending regenerating axons into SON by retrograde tracing. (A) Scheme of SON retrograde tracing with Dextran in Pten / Socs3 / Gsk3p triple KO mice treated with Gsn+Anxa2+Ecm1 -i-tPA at 8wpc. (B) SON injected with Dextran. Left panels, red arrows indicate injection sites on a ventral view and an enlarged ventral view; middle panel, the injection sites on a coronal view; right panel, brain coronal view from Allen Brain Atlas showing SON location. (C) SLO fundus images of retinas retrogradely labeled by SON Dextran injection. (D) Confocal images of Dextran retro-labeled RGCs co-labeled by RGC markers. Upper panel, white arrows point to a Dextran+ / RBPMS+ / Spp1 + RGC; lower panel, white arrows point to a Dextran+ / RBPMS+ RGC negative for Spp1 or Opn4. Scale bar, 10 pm.

[0036] FIGS. 16A-16J. AAVretro-mediated delayed Gsn overexpression also achieves significant neuroprotection in mouse SOHU glaucoma model. (A) Scheme of AAV2-Gsn intravitreal injection two weeks before SO intracameral injection and evaluation of SOHU glaucomatous eyes at three weeks post SO injection (3wpi). (B) IOP of naive and glaucomatous eyes with or without AAV2-Gsn treatment at 3wpi. (C) Live SLO images of retina two weeks after stereotaxic superior colliculus (SC) injection with AAVretro-hSyn-H2B-Clover3. (D) Left panel: representative confocal images of flat-mounted retina 2 weeks after SC injection with AAVretro-HA-Gsn. Right panel: quantification of Gsn+ RGCs in RBPMS+ RGCs. Data presented as means ± s.e.m, n=4. (E) Scheme of delayed treatment using AAVretro-Gsn retrogradely deliveried by stereotaxic superior colliculus (SC) injection three days after SO intracameral injection. (F) IOP of naive and glaucomatous eyes with or without AAVretro-Gsn treatment at 3wpi. All the quantification data are presented as means ± s.e.m, n=8-15 in each group. **“: p<0.0001 , with one-way ANOVA Tukey’s comparisons test. (G) Representative in vivo OCT images of mouse retinas from CL naive, SOHU glaucomatous, and delayed Gsn- treated SOHU glaucomatous eyes at 3wpi. GCC: indicated as double end arrows. Quantification of GCC thickness measured by OCT at 3wpi, represented as percentage of GCC thickness in the SOHU eyes compared to the sham CL eyes. (H) Visual acuity measured by OKR at 3wpi, represented as percentage of glaucomatous eyes compared to the sham CL eyes. (I) Left: representative wave forms of PERG at baseline and 3wpi; blue traces represent glaucomatous eyes; black traces represent CL eyes. Right: quantification of P1 -N2 amplitude of PERG at 3wpi, represented as a percentage of glaucomatous eyes compared to the sham CL eyes. (J)Representative confocal images of the whole flat-mounted retinas (scale bar, 500pm) and central, middle and peripheral flat-mounted retinas (scale bar, 50pm) showing surviving RBPMS+ RGCs at 3wpi. Quantification of surviving RGC somata in peripheral, middle, and central retinas at 3wpi, represented as percentage of glaucomatous eyes compared to the sham CL eyes. Light microscope images of semi-thin transverse sections of ON and enlarged regions with PPD staining at 3wpi. Scale bar, 10pm. Quantification of surviving axons in ONs at 3wpi, represented as percentage of glaucomatous eyes compared to the sham CL eyes. All the quantification data are presented as means ± s.e.m, n=8-15 in each group, ns: p>0.05, *: p<0.05, *“*: p<0.0001 , with non-parametric Mann-Whitney test.

[0037] FIGS. 17A-17I. Long term effects of Gsn and Anxa2 overexpression on mouse retina.(A,E) OCT of retinas and quantification of the thickness of GCC, IS / OS (inner segment / outer segment of photoreceptors), and ONL (outer nuclear layer). The AAVs were intravitreally injected 16 weeks before euthanization. (B,F) Quantification of P1-N2 amplitude of PERG at 16wpi. (C,G) Quantification of visual acuity by OKR at 16wpi. (D,H) Representative mouse ERG traces and quantification of dark-adapted A-wave and B-wave amplitude at 16wpi. The dark- adapted ERG were detected at 0.01 cd*s / m2, 0.1 cd*s / m2, or 1 cd*s / m2. In A-H, data are presented as means ± s.e.m, n=4-5 in each group, ns: no significance, with Student’s t-test. (I) Representative confocal images of whole flat-mounted retinas (scale bar, 100pm) and peripheral retinas (scale bar, 50 pm), and quantification of peripheral RGC density. The retinas are co-stained with HA antibody for Gsn and Anxa2 expression and RBPMS as RGC marker. Data are presented as means ± s.e.m, n=4-8 in each group, ns: p>0.05, one-way ANOVA, Tukey’s multiple comparisons test.

[0038] FIGS. 18A-18E. Delayed topical delivery of LatB after SO injection. (A) Cartoon illustration of retro-orbital injection of LatB. (B) Scheme of timeline of delayed daily treatment with LatB starting at one day after SO intracameral injection and continuing for seven days. Ex vivo ON imaging of mitochondria movement. (C) Scheme of timeline of delayed daily LatB treatment starting at three days after SO intracameral injection and continuing for three weeks. (D) IOP of naive and glaucomatous eyes with or without LatB treatment at 3wpi. Data are presented as means ± s.e.m, *“*: p<0.0001 , with one-way ANOVA, Tukey’s multiple comparisons test. (E) The working model of the common molecular mechanisms of axon regeneration and neuroprotection generated from this study. The pro-regeneration / pro- neuroprotection molecules act on ECM-membrane-cytoskeleton dynamics to converge on actin depolymerization, which is the direct driving force of axon regrowth and protection through enhancing axonal mitochondria motility. The cartoons of growth cone and axonal mitochondria transport are modified from BioRender.DET ILED DESCRIPTION OF THE EMBODIMENTS

[0039] Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0040] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0041] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0043] All publications and patents cited in this specification are herein incorporated by reference in their entirety, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0044] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement isintended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0045] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0046] While the compositions and methods have been or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §112, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.

[0047] As used herein, compounds which are "commercially available" may be obtained from commercial sources including but not limited to Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee Wl, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester PA), Crescent Chemical Co. (Hauppauge NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester NY), Fisher Scientific Co. (Pittsburgh PA), Fisons Chemicals (Leicestershire UK), Frontier Scientific (Logan UT), ICN Biomedicals, Inc. (Costa Mesa CA), Key Organics (Cornwall U.K.), Lancaster Synthesis (Windham NH), Maybridge Chemical Co. Ltd. (Cornwall U.K.), Parish Chemical Co. (Orem UT), Pfaltz & Bauer, Inc. (Waterbury CN), Polyorganix (Houston TX), Pierce Chemical Co. (Rockford IL), Riedel de Haen AG (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland OR), Trans World Chemicals, Inc. (Rockville MD), Wako Chemicals USA, Inc. (Richmond VA), Novabiochem and Argonaut Technology.

[0048] Compounds can also be made by methods known to one of ordinary skill in the art. As used herein, "methods known to one of ordinary skill in the art" may be identified though various reference books and databases. Suitable reference books and treatises that detail the synthesis of reactants useful in the preparation of compounds of the present invention, or provide references to articles that describe the preparation, include for example, "Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York; S. R. Sandler et al., "Organic Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; H. O. House, "Modern Synthetic Reactions", 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L. Gilchrist, “HeterocyclicChemistry”, 2nd Ed., John Wiley & Sons, New York, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanisms and Structure”, 4th Ed., Wiley-lnterscience, New York, 1992. Specific and analogous reactants may also be identified through the indices of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through on-line databases (the American Chemical Society, Washington, D.C. may be contacted for more details). Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis houses, where many of the standard chemical supply houses (e.g., those listed above) provide custom synthesis services.Genes of interest

[0049] Gelsolin is a multifunctional actin-binding protein that plays a central role in the regulation of actin filament dynamics in both the cytoplasm and extracellular space. Encoded by the GSN gene, gelsolin belongs to a family of proteins characterized by multiple homologous domains that interact with actin in a calcium-dependent manner. In response to elevated intracellular calcium, gelsolin binds to filamentous actin (F-actin), severs actin filaments, and caps the newly generated barbed ends, thereby promoting rapid cytoskeletal remodeling. This activity is essential for processes such as cell motility, shape changes, phagocytosis, and apoptosis. The Genbank refseq for the human mRNA is NM 000177.5, and for the protein is NP 000168.1.

[0050] Beyond its intracellular functions, gelsolin also exists as a secreted plasma protein, where it participates in the extracellular actin-scavenging system. Following tissue injury or cell death, actin released into the circulation can form aggregates that contribute to inflammation and vascular dysfunction. Plasma gelsolin binds and clears this extracellular actin in cooperation with vitamin D-binding protein, helping to maintain blood fluidity and modulate inflammatory responses. Gelsolin has additionally been shown to interact with bioactive lipids, such as phosphatidylinositol 4,5-bisphosphate, further influencing signaling pathways linked to cytoskeletal regulation.

[0051] Cofilin (Cfl1) is a highly conserved actin-binding protein that plays a critical role in the dynamic regulation of the actin cytoskeleton by promoting actin filament turnover. It binds preferentially to ADP-bound actin within filamentous actin (F-actin), inducing a conformational change that weakens inter-subunit interactions and leads to filament severing and depolymerization. Through this activity, cofilin generates new barbed ends for actin polymerization while simultaneously enhancing actin disassembly, enabling rapid remodeling of the cytoskeleton required for processes such as cell migration, endocytosis, cytokinesis, andneuronal growth cone dynamics. The Genbank refseq for mRNA is NM 005507.3, and for the protein is NP 005498.1.

[0052] The activity of cofilin is tightly regulated by multiple signaling pathways to ensure precise spatial and temporal control of actin dynamics. A key regulatory mechanism is reversible phosphorylation at serine 3: phosphorylation by LIM kinases inactivates cofilin, whereas dephosphorylation by phosphatases such as slingshot and chronophin restores its actin-binding activity. Cofilin function is also modulated by interactions with phosphoinositides, changes in intracellular pH, and redox state, allowing cells to rapidly adjust actin behavior in response to extracellular cues and mechanical forces.

[0053] Destrin (Dstn), also known as actin depolymerizing factor (ADF) is a member of the ADF / cofilin family of actin-binding proteins that regulate actin filament dynamics. Like cofilin, destrin binds preferentially to ADP-bound actin within filamentous actin (F-actin), promoting filament severing and depolymerization. Through this activity, destrin facilitates rapid actin turnover, which is essential for maintaining cytoskeletal plasticity during processes such as cell shape changes, migration, and intracellular trafficking. Destrin is widely expressed across tissues and contributes to the fine-tuning of actin dynamics in both proliferative and differentiated cells. The Genbank refseq for human destrin is NM 006870.

[0054] The activity of destrin is tightly controlled by post-translational and biochemical mechanisms similar to those regulating cofilin. Phosphorylation at a conserved serine residue inhibits its actin-binding and depolymerizing activity, while dephosphorylation restores function, allowing cells to dynamically regulate actin remodeling in response to signaling cues. Destrin activity is also influenced by intracellular pH and interactions with phosphoinositides, providing additional layers of regulation that coordinate actin dynamics with cellular metabolism and membrane signaling.

[0055] Macrophage-capping protein (CapG) is an actin-binding protein of the gelsolin superfamily that regulates cytoskeletal dynamics by capping the barbed ends of actin filaments without severing them. CapG is predominantly expressed in cells of the myeloid lineage, including macrophages, neutrophils, and dendritic cells, where rapid actin remodeling is essential for motility, phagocytosis, and immune responses. By binding to the fast-growing ends of actin filaments in a calcium-dependent manner, CapG limits filament elongation and helps coordinate cycles of actin assembly and disassembly at the cell cortex. Genbank refseq for the human mRNA is NM_001256139; NM_001256140; NM_001747; NM_001320732; NM 001320733; and the protein is NP_001243068, NP_001243069, NP_001307661 , NP 001307662, NP_001307663.

[0056] Unlike gelsolin, CapG lacks actin-severing activity and does not form stable caps, allowing for more dynamic and reversible control of filament growth. Its activity is regulated by intracellular calcium levels and by interactions with phosphoinositides, particularly phosphatidylinositol 4,5-bisphosphate, which can inhibit its capping function and promote actin polymerization. CapG is found in both the cytoplasm and the nucleus, and nuclear CapG has been implicated in transcriptional regulation and chromatin-associated processes, although these functions are less well understood than its cytoskeletal roles.

[0057] Advillin, also known as AVIL, is a neuron-specific actin-binding protein belonging to the gelsolin superfamily and is primarily expressed in sensory neurons of the peripheral nervous system. Advillin shares structural and functional similarities with other gelsolin family members, including the ability to bind actin in a calcium-dependent manner and regulate actin filament dynamics. Through its interactions with both globular and filamentous actin, advillin contributes to cytoskeletal remodeling processes that are essential for neurite outgrowth, growth cone motility, and maintenance of sensory neuron morphology.

[0058] Functionally, advillin is considered a highly selective molecular marker for dorsal root ganglion neurons and other peripheral sensory neuron populations, including nociceptors and mechanoreceptors. Its expression begins early during sensory neuron development and persists into adulthood, making it particularly useful for lineage tracing and targeted genetic manipulation in neurobiology research. By modulating actin organization within axons and growth cones, advillin supports processes such as axonal guidance, synaptic connectivity, and responses to peripheral nerve injury.

[0059] Adseverin (Soin), also known as scinderin, is a calcium-dependent actin-binding protein belonging to the gelsolin superfamily that plays a key role in regulating actin filament dynamics during regulated exocytosis. Like other members of this family, adseverin binds to filamentous actin (F-actin) and severs actin filaments while capping their barbed ends, leading to localized actin disassembly. This activity is particularly important in secretory cells, where dense cortical actin networks act as a physical barrier to vesicle fusion with the plasma membrane. The Genbank refseq for the human mRNA is NM 001112706, NM 033128 and the protein is NP 001106177, NP 149119.

[0060] Functionally, adseverin is best known for its role in neuroendocrine and secretory systems, including chromaffin cells, endocrine cells, and certain immune cells. Upon stimulation and the resulting rise in intracellular calcium, adseverin rapidly reorganizes cortical actin, creating permissive zones that allow secretory vesicles to access docking and fusion sites at the cell surface. This actin remodeling is a critical step in stimulus-dependent hormone,neurotransmiter, or granule release, distinguishing adseverin’s role from more general actin regulators involved in cell motility or shape.Definitions

[0061] The terms "treatment," "treating," "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and / or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect atributable to the disease. The term “treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and / or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom(s) but has not yet been diagnosed as having it; (b) inhibiting the disease and / or symptom(s), i.e., arresting development of a disease and / or the associated symptoms; or (c) relieving the disease and the associated symptom(s), i.e., causing regression of the disease and / or symptom(s). Those in need of treatment can include those already inflicted (e.g., those with optic neuropathies) as well as those in which prevention is desired (e.g., those with increased susceptibility to optic neuropathies; those with optic neuropathies; those suspected of having optic neuropathies; etc.).

[0062] The terms “recipient,” “individual,” “subject,” “host,” and “patient” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In some embodiments, the mammal is human.

[0063] The terms “specific binding,” “specifically binds,” and the like, refer to non-covalent or covalent preferential binding to a molecule relative to other molecules or moieties in a solution or reaction mixture (e.g., a neuroprotective agent specifically binds to a HRH1 relative to other available polypeptides or small molecules). In some embodiments, the affinity of one molecule for another molecule to which it specifically binds is characterized by a KD(dissociation constant) of 10'5M or less (e.g., 10-6M or less, 10-7M or less, 10’8M or less, 10’9M or less, 10'1° M or less, 10’11M or less, 10-12M or less, 10'13M or less, 10'14M or less, 10'15M or less, or 10’16M or less). "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower KD.

[0064] The terms "co-administration" and "in combination with" include the administration of two or more therapeutic agents either simultaneously, concurrently or sequentially within no specific time limits. In one embodiment, the agents are present in the cell or in the subject's body at the same time or exert their biological or therapeutic effect at the same time. In oneembodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, a first agent can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to {e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) after the administration of a second therapeutic agent.

[0065] The term “sample” as used herein relates to a material or mixture of materials, typically, although not necessarily, in fluid, i.e., aqueous, form, containing one or more components of interest. Samples may be derived from a variety of sources such as from food stuffs, environmental materials, a biological sample or solid, such as tissue or fluid isolated from an individual, including but not limited to, for example, plasma, serum, spinal fluid, semen, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, and also samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, putatively virally infected cells, recombinant cells, and cell components). In certain embodiments of the method, the sample includes a cell. In some instances of the method, the cell is in vitro. In some instances of the method, the cell is in vivo.

[0066] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms “polynucleotide” and “nucleic acid” should be understood to include, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and doublestranded polynucleotides.

[0067] The terms "polypeptide," "peptide," and "protein", are used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like. The term “polypeptide” includes lipoproteins, glycoproteins, and the like.

[0068] A “host cell,” as used herein, denotes an in vivo or in vitro eukaryotic cell, or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, which eukaryotic cells can be, or have been, used as recipients for a nucleic acid (e.g., an expression vector), and include the progeny of the original cell which has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. A “recombinant host cell” (also referred to as a “genetically modified host cell”) is a host cell into which has been introduced a heterologous nucleic acid, e.g., an expression vector, a guide RNA, a donor DNA template, and the like. For example, a subject eukaryotic host cell is a genetically modified eukaryotic host cell, by virtue of introduction into a suitable eukaryotic host cell of a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to the eukaryotic host cell, or a recombinant nucleic acid that is not normally found in the eukaryotic host cell.

[0069] As used herein, “CRISPER / Cas9 system” typically includes a polynucleotide sequence (which may, together, be referred to as an expression cassette, or one or more gRNAs may be separately encoded and referred to as a cassette), wherein the polynucleotide sequence encodes the Cas9 nuclease alone, or also encodes one or more guide RNAs (gRNAs) as well as the Cas9 nuclease. In some instances, the expression cassette encoding the CRISPER / Cas9 system is referred to as a “transgene.”

[0070] The term 'neuroprotective' as used herein refers to the ability to protect neurons or their axons or synapses in the central or peripheral nervous system from damage or death. Many different types of insult can lead to neuronal damage or death, for example: metabolic stress caused by hypoxia, hypoglycemia, diabetes, loss of ionic homeostasis or other deleterious process, physical injury of neurons, exposure to toxic agents and numerous diseases affecting the nervous system including inherited disorders. The presence of an agent that is neuroprotective enables a neuron to remain viable upon exposure to insults that would otherwise cause a loss of functional integrity in an unprotected neuron.

[0071] The term 'injury' as used herein refers to damage inflicted on the neuron, whether in the cell body or in axonal or dendritic processes. This can be a physical injury in the conventional sense i.e. traumatic injury to the brain, spinal cord or peripheral nerves caused by an external force applied to a subject. Other damaging external factors are for example environmental toxins such as mercury and other heavy metals, pesticides and solvents. Alternatively, injury can result from an insult to the neuron originating from within the subject, for example: reduced oxygen and energy supply as in ischemic stroke and diabetic neuropathy, autoimmune attack as in multiple sclerosis or oxidative stress and free-radical generation as is believed to be important in amyotrophic lateral sclerosis. Injury is also used here to refer to any defect in the mechanism of axonal transport.

[0072] Axonopathy, broadly defined as functional or structural defects in the axon or its terminal, has been established as a major early contributor to the genesis, progression, and symptomology of neurodegenerative disorders. Axon degeneration is an active process, as demonstrated in Wallerian degeneration, which involves the fragmentation and disintegration of an axon distal to the site of an injury. Axonopathy is often considered in the context of peripheral motor and sensory neurons, given their length, the presence of diseases that specifically affect these systems, and their sensitivity to challenges such as chemotherapy drugs or metabolic disorders such as diabetes. However, these characteristics are not limited to the peripheral nervous system. Glaucoma, a neuropathy affecting axons of the optic nerve, one of the few central nervous system components outside of the brain and spinal cord. Glaucoma shares commonalities with other central neurodegenerations such as amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia (HSP), Alzheimer's, Parkinson's, and Huntington's diseases, often exhibiting comorbidity with those conditions, as well as exhibiting similar mechanisms with these and other axonopathies.Compositions

[0073] Compounds that target F-actin depolymerization. Molecules that target actin depolymerization play crucial roles in modulating the dynamics of the actin cytoskeleton, which is essential for various cellular processes such as migration, division, and intracellular trafficking. These compounds generally destabilize actin filaments by either promoting their depolymerization or preventing polymerization. Latrunculin A and B, derived from marine sponges, bind to actin monomers (G-actin) and sequester them, preventing their incorporation into filaments (F-actin) and accelerating depolymerization. Cytochalasin D, a fungal metabolite, caps the barbed (growing) end of actin filaments, blocking further polymerization and destabilizing the filament structure. Swinholide A, another marine-derived compound, severs actin filaments and promotes depolymerization by creating new filament ends. Bistramide A disrupts actin dynamics by depolymerizing filaments and binding monomeric actin. Blebbistatin is a chemical compound that selectively inhibits non-muscle myosin II (NMI I) ATPase activity, therefore preventing NMII from interacting with the F-actin cytoskeleton and loosening actin network.

[0074] In some embodiments an agent that targets active depolymerization for use in the methods of the disclosure is a small molecule that enhances actin depolymerization or inhibits actin polymerization. Agents of interest include, without limitation, Latrunculin B, Latrunculin A, Cytochalasin D, Cytochalasin B, Blebbistatin, CK-666, BMS-754807, Swinholide A, Bistramide A, etc. The agents may be formulated for ocular administration.

[0075] AAV gene therapy. Utilizing a viral vehicle to deliver genetic material into cells allows direct targeting of pathogenic molecules and restoration of function. The retina is an advantageous target for gene therapy due to its easy access, confined non-systemic localization, partial immune privilege, and well-established definitive functional readouts. The success of adeno-associated virus (AAV)-mediated gene replacement in treating inherited retinal disease makes RGC-specific therapy with AAV a promising gene therapy strategy for optic neuropathies. Because AAV is non-pathogenic and cannot reproduce itself without helper viruses, it has served as a primary vehicle for gene therapy. It is a single-stranded DNA virus that stably and efficiently infects a wide variety of cells in multiple tissues. AAV2, the best- characterized AAV serotype, efficiently infects RGCs in retina after intravitreal injection.

[0076] In some embodiments, the vector is a recombinant adeno-associated virus (AAV) vector.AAV vectors are DNA viruses of relatively small size that can integrate, in a stable and site specific manner, into the genome of the cells that they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies. The AAV genome has been cloned, sequenced and characterized. It encompasses approximately 4700 bases and contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as an origin of replication for the virus. The remainder of the genome is divided into two essential regions that carry the encapsidation functions: the left-hand part of the genome, that contains the rep gene involved in viral replication and expression of the viral genes; and the right-hand part of the genome, that contains the cap gene encoding the capsid proteins of the virus.

[0077] The application of AAV as a vector for gene therapy has been rapidly developed in recent years. Wild-type AAV can infect, with a comparatively high titer, dividing or non-dividing cells, or tissues of mammal, including human, and also can integrate into in human cells at specific site (on the long arm of chromosome 19) (Kotin et al, Proc. Natl. Acad. Sci. U.S.A., 1990. 87: 2211- 2215; Samulski et al, EMBO J., 1991. 10: 3941-3950 the disclosures of which are hereby incorporated by reference herein in their entireties). AAV vector without the rep and cap genes loses specificity of site-specific integration, but may still mediate long-term stable expression of exogenous genes. AAV vector exists in cells in two forms, wherein one is episomic outside of the chromosome; another is integrated into the chromosome, with the former as the major form. Moreover, AAV has not been found to be associated with any human disease, nor any change of biological characteristics arising from the integration has been observed. There are sixteen serotypes of AAV reported in literature, respectively named AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, AAV14, AAV15, and AAV16, wherein AAV5 is originally isolated from humans (Bantel-Schaal, and H. zur Hausen. Virology, 1984. 134: 52-63), while AAV1-4 and AAV6 are all found in the study of adenovirus (Ursula Bantel-Schaal, Hajo Delius and Harald zur Hausen. J. Viral., 1999. 73: 939-947).

[0078] AAV vectors may be prepared using any convenient methods. Adeno-associated viruses of any serotype are suitable (See, e.g., Blacklow, pp. 165-174 of "Parvoviruses and Human Disease" J. R. Pattison, ed. (1988); Rose, Comprehensive Virology 3:1, 1974; P. Tattersall "The Evolution of Parvovirus Taxonomy" In Parvoviruses (J R Kerr, S F Cotmore. ME Bloom, RMLinden, C RParrish, Eds.) p 5-14, Rudder Arnold, London, UK (2006); and D E Bowles, J E Rabinowitz, R J Samulski "The Genus Dependovirus" (J R Kerr, SF Cotmore. ME Bloom, R M Linden, C R Parrish, Eds.) p 15-23, Rudder Arnold, London, UK (2006), the disclosures of which are hereby incorporated by reference herein in their entireties). Methods for purifying for vectors may be found in, for example, U.S. Pat. Nos. 6,566, 118, 6,989,264, and 6,995,006 and W0 / 1999 / 011764 titled "Methods for Generating High Titer Helper-free Preparation of Recombinant AAV Vectors", the disclosures of which are herein incorporated by reference in their entirety. Preparation of hybrid vectors is described in, for example, PCT Application No. PCTIUS2005 / 027091 , the disclosure of which is herein incorporated by reference in its entirety. The use of vectors derived from the AAVs for transferring genes in vitro and in vivo has been described (See e.g., International Patent Application Publication Nos: 91 / 18088 and WO 93 / 09239; U.S. Pat. Nos. 4,797,368, 6,596,535, and 5,139,941 ; and European Patent No: 0488528, all of which are herein incorporated by reference in their entirety). These publications describe various AAV-derived constructs in which the rep and / or cap genes are deleted and replaced by a gene of interest, and the use of these constructs for transferring the gene of interest in vitro (into cultured cells) or in vivo (directly into an organism). The replication defective recombinant AAVs according to the invention can be prepared by co-transfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes (rep and cap genes), into a cell line that is infected with a human helper virus (for example an adenovirus). The AAV recombinants that are produced are then purified by standard techniques.

[0079] In some embodiments, the vector(s) for use in the methods of the invention are encapsidated into a virus particle (e.g. AAV virus particle including, but not limited to, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVIO, AAVII, AAV12, AAV13, AAV14, AAV15, and AAV16). Accordingly, the invention includes a recombinant virus particle (recombinant because it contains a recombinant polynucleotide) comprising any of the vectors described herein. Methods of producing such particles are known in the art and are described in U.S. Pat. No. 6,596,535.

[0080] A neuron-specific promoter allows precise manipulation of gene expression without affecting other cell types. Aspects of the present invention encompass expression cassettes and / or vectors comprising polynucleotide sequences of interest for expression in targeted cells. The polynucleotides can comprise promoters operably linked to RAG coding sequence.Targeted expression is accomplished using a cell-selective or cell-specific promoter. Examples are promoters for somatostatin, parvalbumin, GABAa6, L7, and calbindin. Other cell specific promoters can be promoters for kinases such as PKC, PKA, and CaMKII; promoters for other ligand receptors such as NMDAR1 , NNIDAR2B, GluR2; promoters for ion channels including calcium channels, potassium channels, chloride channels, and sodium channels; and promoters for other markers that label classical mature and dividing cell types, such as calretinin, nestin, and beta3-tubulin.

[0081] Promoters of particular interest are RGC specific promoters, e.g. murine y-synuclein (mSncg) promoter, which drives specific, potent and sustained transgene expression in rodent RGCs, nonhuman primate RGCs, and human primary RGCs, as well as human induced Pluripotent Stem Cell (iPS) stem cell-derived RGCs.

[0082] In some embodiments a promoter is used for the selective expression of an operably linked gene in retinal ganglion cells (RGCs). In some embodiments the promoter comprises or consists of an mSncg promoter, see for example US Patent no. 12,496,356, herein specifically incorporated by reference. In some embodiments the promoter sequence is provided in the context of a vector for expression, including without limitation a viral vector, e.g. an AAV vector. Cells of interest for expression include, without limitation, cells in the eye and progenitors thereof, e.g. retinal cells, particularly retinal ganglion cells, and their progenitors.

[0083] As used herein, the term “wildtype” generally refers to a gene, or sub-portion thereof, in the subject that is not mutated, or not substantially mutated (e.g., at either allele) so as to affect the function of the gene. Accordingly, a wildtype locus may contain the common (i.e., most prevalent, normal, etc.) sequence of the gene, or essentially the common sequence of the gene, without mutation, or without substantial mutation, affecting the function of the gene. The “common sequence”, as used in this context, generally refers to the gene sequence as it most frequently occurs in a natural population. In some instances, common sequences may be represented by a reference sequence, e.g., a reference sequence as it appears in a sequence database, such as but not limited to e.g., GenBank database (NCBI), UniProt database (EBI / SI B / PI R) , or the like. In some instances, a wildtype locus may be identical or substantially identical to a reference sequence.

[0084] A promoter is operably linked to a gene of interest for targeting actin depolymerization.In some embodiments the gene is selected from Gsn, Dstn, Cfl1 , CapG, Advillin / Avil, and Adseverin / Scin or a combination thereof. In some such embodiments the gene is one or a combination of Gsn, Dstn, Cfl1 and CapG. In some embodiments the gene is a human gene. In some embodiments the gene is Gsn.

[0085] The formulation can be administered by any suitable means, including ocular, intra- vitreal, oral, parenteral, etc. Parenteral infusions include intramuscular, intravenous (bolus or slow drip), intraarterial, intraperitoneal, intrathecal or subcutaneous administration. An agent can be administered in any manner which is medically acceptable. Sustained release administration is also specifically included in the disclosure, by such means as depot injections or erodible implants.

[0086] As noted above, an agent can be formulated with a pharmaceutically acceptable carrier (one or more organic or inorganic ingredients, natural or synthetic, with which a subject agent is combined to facilitate its application). A suitable carrier includes sterile saline although other aqueous and non-aqueous isotonic sterile solutions and sterile suspensions known to be pharmaceutically acceptable are known to those of ordinary skill in the art. An "effective amount" refers to that amount which is capable of ameliorating or delaying progression of the diseased, degenerative or damaged condition. An effective amount can be determined on an individual basis and will be based, in part, on consideration of the symptoms to be treated and results sought. An effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.

[0087] In some embodiments an effective dose of an agent is from 0.1 pg / retina to about 1 mg / retina or more, e.g. from about 0.5 pg, about 1 pg, about 5 pg, about 10 pg, about 25 pg, about 50 pg, about 75 pg, about 100 pg, about 250 pg, about 500 pg, about 750 pg, about 1 mg. In some embodiments the agent is provided in a sustained release formulation that delivers the agent of a period of over about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or more, e.g. over 2 weeks, 3 weeks 4 weeks, or more.

[0088] The volume in intravitreal injection, per injection, may be not more than about 500 pl, not more than about 200 pl, not more than about 100 pl, and may be from about 1 pl to about 200 pl, from about 5 pl to about 100 pl, from about 25 pl to about 100 pl, and may be around 50 pl.

[0089] In one embodiment a therapeutic vector or agent is intended for use as a neuroprotective medicament in the treatment of a neurodegenerative disorder resulting from neuronal injury. The term 'injury' as used herein refers to damage inflicted on the neuron, whether in the cell body or in axonal or dendritic processes. This can be a physical injury in the conventional sense i.e. traumatic injury to the brain, spinal cord or peripheral nerves caused by an external force applied to a subject. Other damaging external factors are, for example, environmental toxins such as mercury and other heavy metals, pesticides and solvents. Alternatively, injury can result from an insult to the neuron originating from within the subject, for example: reduced oxygen andenergy supply as in ischemic stroke and diabetic neuropathy, autoimmune attack as in multiple sclerosis or oxidative stress and free-radical generation as is believed to be important in amyotrophic lateral sclerosis. Injury is also used here to refer to any defect in the mechanism of axonal transport.

[0090] In another embodiment, a therapeutic vector comprising an actin depolymerization targeting gene coding sequence is intended for use as a neuroprotective medicament wherein the neurodegenerative disorder is caused by a neuronal injury resulting from a disease. In some instances, the optic neuropathy and / or neurodegenerative disorder treated according to the methods described herein may be an optic neuropathy such as Leber’s hereditary optic neuropathy (LHON), Anterior ischemic optic neuropathy (AION), Non-arteritic anterior ischemic optic neuropathy (NAION), optic disc drusen (ODD), dominant optic atrophy (DOA), ON damage associated with glaucoma, or other CNS neurodegenerative disorder leading to ON degeneration. In some instances of the methods disclosed herein, the disease or disorder may involve inflammation leading to degeneration of the ON.

[0091] In one embodiment, the neurodegenerative disorder is an ophthalmic disorder such as glaucoma. Glaucomas are a group of eye disorders characterized by progressive optic nerve damage in which an important part is a relative increase in intraocular pressure (IOP) that can lead to irreversible loss of vision. Glaucomas are categorized as open-angle glaucoma or angleclosure glaucoma. The “angle” refers to the angle formed by the junction of the iris and cornea at the periphery of the anterior chamber. The angle is where > 98% of the aqueous humor exits the eye via either the trabecular meshwork and the Schlemm canal or the ciliary body face and choroidal vasculature. Glaucomas are further subdivided into primary (cause of outflow resistance or angle closure is unknown) and secondary (outflow resistance results from a known disorder), accounting for > 20 adult types. Another group of glaucoma patients does not have IOP elevation, which in general is called normal tension glaucoma (NTG). NTG is also associated with progressive optic nerve degeneration and RGC death. Thus they are also subject to this gene therapy treatment.

[0092] Axons of retinal ganglion cells travel through the optic nerve carrying visual information from the eye to the brain. Damage to these axons causes ganglion cell death with resultant optic nerve atrophy and patchy vision loss. Elevated intraocular pressure (IOP; in unaffected eyes, the average range is 11 to 21 mm Hg) plays a role in axonal damage, either by direct nerve compression or diminution of blood flow. However, the relationship between externally measured pressure and nerve damage is complicated. Of people with IOP > 21 mm Hg (ie, ocular hypertension), only about 1 to 2% / year (about 10% over 5 years) develop glaucoma. Additionally, about one third of patients with glaucoma do not have IOP > 21 mm Hg (known as low-tension glaucoma or normal-tension glaucoma).

[0093] IOP is determined by the balance of aqueous secretion and drainage. Elevated IOP is caused by inhibited or obstructed outflow, not oversecretion; a combination of factors in the trabecular meshwork (eg, dysregulation of extracellular matrix, cytoskeletal abnormalities) appear to be involved. In open-angle glaucoma, IOP is elevated because outflow is inadequate despite an angle that appears unobstructed. In angle-closure glaucoma, IOP is elevated when a physical distortion of the peripheral iris mechanically blocks outflow.

[0094] Symptoms and signs of glaucoma vary with the type of glaucoma, but the defining characteristic is optic nerve damage as evidenced by an abnormal optic disk and certain types of visual field deficits. Glaucoma is diagnosed when characteristic findings of optic nerve damage are present and other causes have been excluded. Elevated IOP makes the diagnosis more likely, but elevated IOP can occur in the absence of glaucoma and is not essential for making the diagnosis.

[0095] Formulations suitable for injection can be administered by an intravitreal, intraocular, or other route of administration, e.g., injection into the retina.

[0096] An agent can be administered as a pharmaceutical composition comprising a pharmaceutically acceptable excipient. The preferred form depends on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

[0097] In some embodiments, pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SepharoseTM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).

[0098] A carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group, and non-covalent associations. Suitable covalent-bond carriers include proteins such as albumins, peptides, and polysaccharides such as aminodextran, each of which have multiple sites for the attachment of moieties. The nature of the carrier can be either soluble or insoluble for purposes of the invention.

[0099] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organicacids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG). Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0100] The active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0101] Compositions can be prepared as injectables, either as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0102] Toxicity of the active agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in further optimizing and / or defining a therapeutic dosage range and / or a sub-therapeutic dosage range (e.g., for use in humans). The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.Conditions for Treatment

[0103] In another embodiment, the neuroprotective agent is intended for use as a neuroprotective medicament wherein the optic neuropathy is caused by a neuronal injury resulting from a disease. In some instances, the optic neuropathy and / or neurodegenerative disorder treated according to the methods described herein may be an optic neuropathy such as Leber’s hereditary optic neuropathy (LHON), Anterior ischemic optic neuropathy (AION), optic disc drusen (ODD), dominant optic atrophy (DOA), Non-arteritic anterior ischemic optic neuropathy (NAION), ON damage associated with glaucoma, or other CNS neurodegenerative disorder leading to ON degeneration. In some instances of the methods disclosed herein, the disease or disorder may involve inflammation leading to degeneration of the ON.

[0104] In one embodiment, the neurodegenerative disorder is an ophthalmic disorder such as glaucoma. Glaucomas are a group of eye disorders characterized by progressive optic nerve damage in which an important part is a relative increase in intraocular pressure (IOP) that can lead to irreversible loss of vision. Glaucomas are categorized as open-angle glaucoma or angleclosure glaucoma. The “angle” refers to the angle formed by the junction of the iris and cornea at the periphery of the anterior chamber. The angle is where > 98% of the aqueous humor exits the eye via either the trabecular meshwork and the Schlemm canal or the ciliary body face and choroidal vasculature. Glaucomas are further subdivided into primary (cause of outflow resistance or angle closure is unknown) and secondary (outflow resistance results from a known disorder), accounting for > 20 adult types. Another group of glaucoma patients does not have IOP elevation, which in general is called normal tension glaucoma (NTG). NTG is also associated with progressive optic nerve degeneration and RGC death. Thus they are also subject to this gene therapy treatment.

[0105] Axons of retinal ganglion cells travel through the optic nerve carrying visual information from the eye to the brain. Damage to these axons causes ganglion cell death with resultant optic nerve atrophy and patchy vision loss. Elevated intraocular pressure (IOP; in unaffected eyes, the average range is 11 to 21 mm Hg) plays a role in axonal damage, either by direct nerve compression or diminution of blood flow. However, the relationship between externally measured pressure and nerve damage is complicated. Of people with IOP > 21 mm Hg (ie, ocular hypertension), only about 1 to 2% / year (about 10% over 5 years) develop glaucoma. Additionally, about one third of patients with glaucoma do not have IOP > 21 mm Hg (known as low-tension glaucoma or normal-tension glaucoma).

[0106] IOP is determined by the balance of aqueous secretion and drainage. Elevated IOP is caused by inhibited or obstructed outflow, not oversecretion; a combination of factors in the trabecular meshwork (eg, dysregulation of extracellular matrix, cytoskeletal abnormalities) appear to be involved. In open-angle glaucoma, IOP is elevated because outflow is inadequatedespite an angle that appears unobstructed. In angle-closure glaucoma, IOP is elevated when a physical distortion of the peripheral iris mechanically blocks outflow.

[0107] Symptoms and signs of glaucoma vary with the type of glaucoma, but the defining characteristic is optic nerve damage as evidenced by an abnormal optic disk and certain types of visual field deficits. Glaucoma is diagnosed when characteristic findings of optic nerve damage are present and other causes have been excluded. Elevated IOP makes the diagnosis more likely, but elevated IOP can occur in the absence of glaucoma and is not essential for making the diagnosis.METHODS

[0108] As summarized above, aspects of the instant disclosure include methods of treating a subject for an axonopathy, e.g. an optic neuropathy. A variety of neurodegenerative disorders also may be treated by practice of the methods described herein, particularly glaucoma, e.g. open-angle glaucoma or angle-closure glaucoma. In some embodiments, provided herein is a method of treating an optic nerve (ON) neuropathy in a mammalian subject in need thereof, comprising intravitreally or systemically administering the composition into the subject, thereby treating the ON neuropathy. In some embodiments, provided herein is a method of reducing or ameliorating degeneration of axons and / or soma of RGCs, comprising intravitreally or systemically administering the composition into a mammalian subject experiencing or at imminent risk of an ON neuropathy.

[0109] In some aspects provided herein is a method of inducing neuroprotection and regeneration / increasing survival I promoting functional recovery of RGC somata and axons, comprising intravitreally or systemically administering the composition into a mammalian subject experiencing or at risk of an ON neuropathy. In some embodiments of the method, the ON neuropathy is retinal ganglion cell degeneration, including glaucoma, optic neuritis, ON traumatic injury and other ON-related diseases.

[0110] In some embodiments, the composition comprises a neuroprotective agent and a pharmaceutically acceptable excipient.[oom] Various subjects may be treated in the methods of the present disclosure. In some instances, treated subjects may be mammals, including but not limited to e.g., rodents (e.g., rats, mice, etc.), non-human primates (e.g., macaques, marmosets, tamarins, spider monkeys, owl monkeys, vervet monkeys, squirrel monkeys, baboons, chimpanzees, etc.), humans, and the like. In some instances, a treated subject may be an animal model (e.g., a rodent model, a non-human primate model, etc.) of an optic neuropathy and / or neurodegenerative disorder. In some instances, a treated subject may be a human subject, including but not limited to e.g., a human subject having an optic neuropathy and / or neurodegenerative disorder, a human subject at increased risk of developing an optic neuropathy and / or neurodegenerative disorder, a humansubject of advanced age (e.g., at least 60 years of age, at least 65 years of age, at least 70 years of age, at least 75 years of age, at least 80 years of age, at least 85 years of age, at least 90 years of age, etc.), or a combination thereof. Treated subjects may or may not be symptomatic, e.g., subject may or may not display or have previously displayed one or more symptoms of an optic neuropathy and / or neurodegenerative disorder, including but not limited to e.g., those optic neuropathies and / or neurodegenerative disorders described herein.

[0112] Methods of the present disclosure may include administering to a subject a neuroprotective agent that specifically targets actin depolymerization, as disclosed herein.

[0113] The compositions of this disclosure can be supplied in the form of a pharmaceutical composition. Any suitable pharmaceutical composition may be employed, described in more detail below. As such, in some instances, methods of the present disclosure may include administering one or more agents in a composition comprising an excipient (e.g., an isotonic excipient) prepared under sufficiently sterile conditions for administration to a mammal, e.g., a human.

[0114] Administration of an agent to a subject, as described herein, may be performed employing various routes of administration. The route of administration may be selected according to a variety of factors including, but not necessarily limited to, the condition to be treated, the formulation and / or device used, the patient to be treated, and the like. Routes of administration useful in the disclosed methods include but are not limited to oral and parenteral routes, such as intravenous (iv), intraperitoneal (ip), rectal, topical, ophthalmic, nasal, and transdermal. Formulations for these dosage forms are described herein.

[0115] Where the agent is a polypeptide, polynucleotide, analog or mimetic thereof, it may be introduced into tissues or host cells by any number of routes, including particularly viral infection, microinjection, or fusion of vesicles. Jet injection may also be used for intramuscular administration, as described by Furth et al., Anal Biochem. (1992) 205:365-368. The DNA may be coated onto gold microparticles, and delivered intradermally by a particle bombardment device, or "gene gun" as described in the literature (see, for example, Tang et al., Nature (1992) 356:152-154), where gold microprojectiles are coated with the DNA, then bombarded into skin cells. For nucleic acid therapeutic agents, a number of different delivery vehicles find use, including viral and non-viral vector systems, as are known in the art.

[0116] Those of skill in the art will readily appreciate that dose levels can vary as a function of the specific compound, the nature of the delivery vehicle, and the like. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

[0117] In those embodiments where an effective amount of an active agent is administered to the subject, the amount or dosage is effective when administered for a suitable period of time, such as one week or longer, including two weeks or longer, such as 3 weeks or longer, 4 weeks or longer, 8 weeks or longer, etc., so as to evidence a reduction in the disorder, e.g., a reductionin a symptom of the disorder or in a marker of disease pathology. For example, an effective dose is the dose that, when administered for a suitable period of time, such as at least about one week, and maybe about two weeks, or more, up to a period of about 3 weeks, 4 weeks, 8 weeks, or longer, will reduce a symptom of the disorder, for example, by about 10% or more, by about 20% or more, e.g., by 30% or more, by 40% or more, or by 50% or more, in some instances by 60% or more, by 70% or more, by 80% or more, or by 90% or more, for example, and will halt progression of the disorder in the subject. In some instances, an effective amount or dose of active agent will not only slow or halt the progression of the disease condition but will also induce the reversal of the condition, i.e., will cause an improvement in the neurological health of the subject. For example, in some instances, an effective amount is the amount that when administered for a suitable period of time, for example, at least about one week, and / or about two weeks, or more, up to a period of about 3 weeks, 4 weeks, 8 weeks, or longer will improve, stabilize, or at least reduce the progression of a disorder in subject, for example 1 .5- fold, 2-fold, 3-fold, 4-fold, 5-fold, in some instances 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more relative to the subject’s condition prior to administration.

[0118] In some instances, in those embodiments where an effective amount of an active agent is administered to the subject, the amount or dosage is effective when administered for a suitable period of time to result in increased RGC survival in the subject. Such an increase may manifest in various ways, including but not limited to e.g., or a reduction in the amount of degeneration of RGCs, or their axons or soma, or the like. In some instances, methods of the present disclosure may result in at least a 5%, e.g., at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 55%, at least a 60%, at least a 65%, at least a 70% at least a 75%, at least a 80%, e.g., increase in RGC survival. In some instances, methods of the present disclosure may result in at least a 5%, e.g., at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 55%, at least a 60%, at least a 65%, at least a 70% at least a 75%, at least a 80%, e.g., increase in RGC number, size or length of RGC axons or somata. Various methods of assessing the amount of RGC degeneration or increase in number, size or length of RGC axons or somata may be employed, including invasive and non-invasive techniques, such as electrophysiology measurement for RGC neuronal function, visual acuity, OCT imaging, fundus imaging, histology studies of RGC somata and axons morphology.

[0119] A “therapeutically effective amount”, a "therapeutically effective dose" or “therapeutic dose” is an amount sufficient to effect desired clinical results (i.e., achieve therapeutic efficacy, achieve a desired therapeutic response, etc.). A therapeutically effective dose can be administered in one or more administrations. For purposes of this disclosure, a therapeutically effective dose of an agent that inhibits expression of a target gene (e.g., Pfn1 ) or that increasesexpression of a protective gene, e.g. Gsn, is an amount that is sufficient, when administered to the individual, to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of the disease state (e.g., an optic axonopathy) by, for example, inhibiting gene expression product formation, increases actin depolymerization, or otherwise preventing the symptoms or clinical progression of a neurodegenerative disorder present in the subject.

[0120] An effective amount of a composition comprising a neuroprotective agent will depend, at least, on the particular method of use, the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic composition. A "therapeutically effective amount" of a composition is a quantity of a specified compound sufficient to achieve a desired effect in a subject (host) being treated.

[0121] Therapeutically effective doses of a composition comprising a neuroprotective agent or pharmaceutical composition can be determined by one of skill in the art, with a goal of achieving local (e.g., tissue) concentrations 7that are at least as high as the IC50 of an applicable compound disclosed herein.

[0122] The specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the composition comprising a neuroprotective agent, the metabolic stability and length of action of that composition, the age, body weight, general health, sex and diet of the subject, mode and time of administration, rate of excretion, drug combination, and severity of the condition of the host undergoing therapy.Pharmaceutical Compositions

[0123] A pharmaceutical composition comprising a neuroprotective agent (i.e., an agent or an AAV virus comprising a therapeutic vector) may be administered to a patient alone, or in combination with other supplementary active agents. The pharmaceutical compositions may be manufactured using any of a variety of processes, including, without limitation, conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, and lyophilizing. The pharmaceutical composition can take any of a variety of forms including, without limitation, a sterile solution, suspension, emulsion, lyophilisate, tablet, pill, pellet, capsule, powder, syrup, elixir or any other dosage form suitable for administration.

[0124] A composition comprising a neuroprotective agent may be administered to the host using any convenient means capable of resulting in the desired reduction in disease condition or symptom. Thus, a neuroprotective agent can be incorporated into a variety of formulations for therapeutic administration. More particularly, a neuroprotective agent can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid orgaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.

[0125] Formulations for pharmaceutical compositions are well known in the art. For example, Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995, describes exemplary formulations (and components thereof) suitable for pharmaceutical delivery of disclosed compositions. Pharmaceutical compositions comprising at least one of the neuroprotective agents can be formulated for use in human or veterinary medicine. Particular formulations of a disclosed pharmaceutical composition may depend, for example, on the mode of administration and / or on the location of the infection to be treated. In some embodiments, formulations include a pharmaceutically acceptable carrier in addition to at least one active ingredient, such as a composition comprising a neuroprotective agent. In other embodiments, other medicinal or pharmaceutical agents, for example, with similar, related or complementary effects on the affliction being treated can also be included as active ingredients in a pharmaceutical composition.

[0126] Pharmaceutically acceptable carriers useful for the disclosed methods and compositions are conventional in the art. The nature of a pharmaceutical carrier will depend on the particular mode of administration being employed. For example, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can optionally contain minor amounts of nontoxic auxiliary substances (e.g., excipients), such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like; for example, sodium acetate or sorbitan monolaurate. Other non-limiting excipients include, nonionic solubilizers, such as cremophor, or proteins, such as human serum albumin or plasma preparations.

[0127] Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters,polycarbonates and / or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

[0128] The disclosed pharmaceutical compositions may be formulated as a pharmaceutically acceptable salt of a disclosed neuroprotective agent. Pharmaceutically acceptable salts are nontoxic salts of a free base form of a compound that possesses the desired pharmacological activity of the free base. These salts may be derived from inorganic or organic acids. Non-limiting examples of suitable inorganic acids are hydrochloric acid, nitric acid, hydrobromic acid, sulfuric acid, hydroiodic acid, and phosphoric acid. Non-limiting examples of suitable organic acids are acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, methyl sulfonic acid, salicylic acid, formic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, asparagic acid, aspartic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, and the like. Lists of other suitable pharmaceutically acceptable salts are found in Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, Pa., 1985. A pharmaceutically acceptable salt may also serve to adjust the osmotic pressure of the composition.

[0129] A composition comprising a neuroprotective agent can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents. Such preparations can be used for oral administration.

[0130] A composition comprising a neuroprotective agent can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles. Formulations suitable for injection can be administered by an intravitreal, intraocular, intramuscular, subcutaneous, sublingual, or other route of administration, e.g., injection into the gum tissue or other oral tissue. Such formulations are also suitable for topical administration.

[0131] In some embodiments, a composition comprising a neuroprotective agent can be delivered by a continuous delivery system. The term "continuous delivery system" is usedinterchangeably herein with "controlled delivery system" and encompasses continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are known in the art.

[0132] Furthermore, a composition comprising a neuroprotective agent can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. A composition comprising a neuroprotective agent or therapeutic vector can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

[0133] The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a composition comprising a neuroprotective agent calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for a composition comprising a neuroprotective agent depend on the particular neuroprotective agent employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0134] The dosage form of a disclosed pharmaceutical composition will be determined by the mode of administration chosen. For example, in addition to injectable fluids, topical or oral dosage forms may be employed. Topical preparations may include eye drops, ointments, sprays and the like. In some instances, a topical preparation of a medicament useful in the methods described herein may include, e.g., an ointment preparation that includes one or more excipients including, e.g., mineral oil, paraffin, propylene carbonate, white petrolatum, white wax and the like, in addition to one or more additional active agents.

[0135] Oral formulations may be liquid (e.g., syrups, solutions or suspensions), or solid (e.g., powders, pills, tablets, or capsules). Methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.

[0136] Certain embodiments of the pharmaceutical composition comprising a neuroprotective agent may be formulated in unit dosage form suitable for individual administration of precise dosages. The amount of active ingredient administered will depend on the subject being treated, the severity of the affliction, and the manner of administration, and is known to those skilled in the art. Within these bounds, the formulation to be administered will contain a quantity of the extracts or compounds disclosed herein in an amount effective to achieve the desired effect in the subject being treated.

[0137] Each therapeutic composition can independently be in any dosage form, such as those described herein, and can also be administered in various ways, as described herein. For example, the compositions may be formulated together, in a single dosage unit (that is, combined together in one form such as capsule, tablet, powder, or liquid, etc.) as a combinationproduct. Alternatively, when not formulated together in a single dosage unit, an individual composition comprising a neuroprotective agent may be administered at the same time as another therapeutic composition or sequentially, in any order thereof.

[0138] In some instances, methods of treating a subject as described herein may include administering to the subject an effective amount of an agent that reduces RGC degeneration in the subject, as identified in a method of screening described herein.REAGENTS, DEVICES AND KITS

[0139] Also provided are reagents, devices and kits thereof for practicing one or more of the above-described methods. The subject reagents, devices and kits thereof may vary greatly. Reagents and devices of interest include those mentioned above with respect to the methods of treating a neurodegenerative condition in a subject, including by administering to the subject an effective amount of an agent that reduces the prevalence of RGC degeneration and / or increases RGC survival. The subject kits may include any combination of components (e.g., reagents, cell lines, etc.) for performing the subject methods, such as e.g., methods of treating a neurodegenerative condition and / or methods of identifying a target gene.

[0140] In addition to the above components, the subject kits will further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, portable flash drive, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.EXAMPLES

[0141] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0142] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBorLaboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.Example 1Actin depolymerization promotes axon regeneration and protection by restoring axonal mitochondria transport in mouse optic neuropathies.

[0143] We recently identified multiple pro-axon regeneration genes that promote optic nerve regeneration, protect retinal ganglion cell (RGC) somata and axons, and preserve visual function in mouse glaucoma models. However, the molecular mechanisms downstream of these pro-regeneration and pro-neuroprotection genes remain unclear, elucidating of which will shed light on novel therapeutic strategies for axon regeneration and neuroprotection. Here, by extensively characterizing another regeneration-associated gene, gelsolin, and other functionally relevant molecules using AAV-mediated specific expression in mouse RGCs, we show that actin depolymerization is a mechanistic nodal point on which all these proregeneration molecules converge to drive optic nerve regeneration and glaucoma neuroprotection by enhancing axonal mitochondria transport in optic nerve crush model and ocular hypertension glaucoma model. We also demonstrate use of a natural compound, latrunculin B, which precisely targets this unified mechanism in both mouse and human RGCs, to establish the neural repair potential of targeting actin depolymerization / axonal mitochondria transport and a drug candidate. Additionally, we detected upregulated F-actin in aqueous humors of patients with severe glaucoma that may be a valuable biomarker for monitoring glaucoma progression and the effects of neuroprotection treatments.

[0144] Using a high throughput process, we were recently able to profile the single cell transcriptomes of regenerating and non-regenerating RGCs from the same retina. We revealed multiple pro-axon regeneration genes downstream of Pten deletion, including Annexin A2 (Anxa2), its downstream effector integrin linked kinase (Ilk) and its upstream ligand plasminogen activator tissue (Plat), membrane palmitoylated protein 1 (Mpp1), secreted phosphoprotein 1 (Spp1), and galectin-1 (Lgalsl). Elucidation of common molecular mechanisms responsible for the pro-regenerative and neuroprotective properties of these molecules downstream of Pten deletion can shed light on novel neural repair strategies. Targeting these downstream molecules could avoiding side effects coming from upstream signaling master regulators.

[0145] One common feature of the pro-regenerative genes identified is that they are membrane or sub-membrane molecules involved in membrane-actin cytoskeleton-based bioprocesses. ANXA2 is a phospholipid-binding and F-actin-binding plasma membrane protein. It interacts with cell surface integrins to activate ILK, a vital sub-membrane structural / signaling molecule that mediates cell adhesion and migration through regulating actin-cytoskeleton dynamics. MPP1 is also a plasma membrane protein that affects cell polarity and migration through a direct interaction with actin. In addition, PTEN itself can be recruited to membrane, where it dephosphorylates membrane PIP3 (phosphatidylinositol trisphosphate) into PIP2 (phosphatidylinositol bisphosphate). It is well known that membrane PIP2 directly binds to and regulates many acting- binding proteins to control the actin network dynamics, including inhibition of actin depolymerization molecules gelsolin (GSN), cofilin (CFL), and ANXA2, and activation of actin polymerization molecules formin (FMN), and WASP / ARP2 / 3 (actin-related protein 2 / 3 complex). The deletion of Pten decreases PIP2 levels on cell membrane, which has the potential, by releasing and activating GSN, CFL, and ANXA2, to initiate actin depolymerization locally.

[0146] Previous studies suggest a complex and paradoxical role of actin dynamics in axon regeneration: F-actin depolymerization molecule CFL promotes CNS and PNS axon regeneration; actin polymerization molecule profilin (PFN) promotes sciatic nerve regeneration; but F-actin cross-linking molecules RhoA, non-muscle myosin II (NMI I), and FMN2 inhibit axon regeneration. We reason that cell membrane and sub-membrane molecules may coordinate local actin network to initiate membrane-cytoskeleton remodeling and therefore, axon regrowth.

[0147] Maintaining axon integrity and function, as well as axon regeneration, demands large amounts of energy generated by mitochondria. F-actin participates in mitochondria docking and immobilization and, therefore, restricts mitochondria movement in axons. Many CNS neurodegenerative diseases have deficits in axonal mitochondria delivery, and boosting mitochondria axonal targeting has been linked to CNS axon regeneration.

[0148] Our recent study demonstrated that decreased ON mitochondria transport is a common neurodegeneration mechanism for both optineurin (Optn) mutation-induced normal tension glaucoma and ocular hypertension glaucoma. We also found that boosting ON mitochondria transport achieves dramatic neuroprotection in glaucoma and significant ON regeneration. In this study we characterized the major actin depolymerization molecule Gsn, one of the top genes significantly upregulated in regenerating RGCs from our previous study, and other actin regulatory molecules in in vivo models of CNS traumatic axon injury and ocular hypertension- induced glaucomatous neurodegeneration. Together with ex vivo mechanistic studies, we elucidated a common molecular mechanism downstream of various pro-axon regeneration and pro-neuroprotection genes on axonal actin depolymerization and mitochondria transport. Byproviding additional evidence in clinically relevant mouse and human diseases’ conditions, we established a potent therapeutic strategy for CNS axonopathies.Results

[0149] Gsn and other actin depolymerization molecules promote ON regeneration in a mouse ON crush (ONC) model We previously developed a retrograde tracing-sequencing method (Retro-Seq) to differentially label and purify regenerating (regRGCs) and non-regenerating RGCs (surRGCs) from Pten KO mice with the same genetic background / modification / injuries. Profiling and comparing the transcriptomes of regRGCs and surRGCs at single cell level allowed us to acquire a list of genes uniquely associated with axon regeneration (Fig. 1A). Gene function annotation of the upregulated genes in regRGCs showed enrichment of genes regulating actin dynamics (Fig. 1B), including experimentally proved pro-axon regeneration genes Anxa2 and its upstream ligand Plat and downstream effector Ilk, Mpp1, Spp1, and Pten itself, suggesting a critical role of the actin cytoskeleton in CNS axon regeneration. We next focused on one of the top upregulated genes in regRGCs, Gsn (Fig. 1A, and FIG. 9A), which is a well-known actin depolymerization actin- binding protein that also has been detected in regenerating zebrafish RGCs.

[0150] We first confirmed that Gsn protein is highly expressed in retrograde tracing labeled regRGCs (FIG. 9B). Gsn belongs to the Gsn superfamily, which contains eight members that sever and cap the barbed end of F-actin. Another major actin depolymerization protein family contains Cfl and its paralog gene Dstn, which sever at the pointed end of F-actin (FIG. 9C). Additionally, natural small molecule compounds latrunculin B (LatB) and cytochalasin D (CytoD) have been widely used to induce actin depolymerization through sequestering G-actin monomers (LatB) and caps / blocks barbed ends of F-actin (CytoD) (FIG. 9C). We therefore tested these actin depolymerization molecules in mouse eyes in vivo.

[0151] We successfully generated AAVs with RGC-specific mSncg promoter to express Gsn, Dstn, Cfl1 , and three other Gsn superfamily genes (CapG, Advillin / Avil, and Adseverin / Scin) in mouse RGCs by intravitreal injection (FIG. 9D). Compared to control mice injected with AAV- Ctrl, both Gsn and Dstn induced a potent ON regeneration phenotype; Cfl1 generated modest axon regeneration after ONC injury (Fig. 1 C,D); CapG also had a modest axon regeneration effect, but the other two Gsn superfamily members, Avil and Scin, did not (FIG. 9E,F). Avil and Scin did also not alter actin depolymerization significantly (FIG. 9G,H). Intravitreal delivery of the actin depolymerization small molecules LatB and CytoD also produced potent ON regeneration that is similar to Pten deletion (Fig. 1 E,F).

[0152] In summary, we demonstrate that promoting actin depolymerization by overexpression of actin severing genes Gsn, Dstn, and Cfl1 or topical treatment with actin depolymerization small molecules LatB and CytoD achieves significant ON regeneration. Inhibition but notactivation of actin polymerization molecules promotes ON regeneration in a mouse ONG model Previous studies suggest that increased actin turnover / dynamics in the growth cone is critical for axon regeneration.

[0153] We next evaluated the effects of actin polymerization molecules (FIG. 10A) on ON regeneration in vivo. AAV-mediated overexpression of actin polymerization genes Pfn1 , Pfn2, and Arp2 / 3 in RGCs (FIG. 10B) or intravitreal delivery of F-actin stabilizing / polymerization small molecule jasplakinolide (Jasp) did not promote ON regeneration (Fig. 2A,B). We also tested several barbed end capping genes that stabilize F-actin, including members of the CapZ and adducin (Add) families (FIG. 10A,B). None of these F- actin capping / stabilizing genes induced significant ON regeneration (FIG. 10C, D), additional evidence that actin polymerization / stabilization does not promote axon regeneration.

[0154] We reasoned that inhibition of actin polymerization, similar to promoting actin depolymerization, might promote axon regeneration. We previously demonstrated effective gene knockdown (KD) in RGCs by AAV-Cas9+AAV-gRNAs. Here, we found that AAV-mediated CRISPR KD of Pfn1 induced modest ON regeneration, compared to control gRNA-treated mice (Fig. 2C, D).

[0155] The tropomyosin (Tpm) family binds to and stabilizes F-actin. Tpm1 ,3 double KD did not lead to ON regeneration (Fig. 2C,D). ARP2 and ARP3 form an actin nucleation / polymerization complex to establish F-actin branches and F-actin meshworks. Inhibition of ARP2 / 3 increases axon length in cultured neurons. Consistently, we demonstrated that CK666, an ARP2 / 3 small molecule inhibitor (fig. A), induced a potent ON regeneration, compared to vehicle treated mice (Fig. 2E,F), similar to actin depolymerization small molecules LatB and CytoD (Fig. 1 E,F). Taken all together, our results demonstrate that inhibition but not activation of actin polymerization / stabilization molecules promotes significant ON regeneration, further confirmation that actin depolymerization but not actin polymerization drives CNS axon regeneration.

[0156] Pro-regeneration molecules cause actin depolymerization in growth cones and axon shafts of cultured hippocampal neurons We then used cultured hippocampal neurons to evaluate the effect of pro-regeneration molecules on axonal F-actin levels. Endogenous ANXA2 and GSN were detected in shafts and growth cones of the axons of cultured hippocampal neurons (FIG. 11 A). Overexpression of Gsn and Anxa2 consistently and significantly decreased F-actin levels labeled by the F-actin-specific probe SiR- actin in the growth cones and axon shafts, whereas the actin polymerization gene Pfn1 increased the F-actin level in growth cones (FIG. 11 B,C). This finding validates a previous report that pro-axon regeneration molecule Anxa2 itself acts as an actin depolymerization molecule and supports the emerging theme from our studies that actin depolymerization is a common molecular mechanism for axon regeneration. Pro-regeneration small molecules LatB, CytoD, and CK666 also consistently andsignificantly decreased growth cone and axon shaft F- actin levels whereas the actin stabilizing small molecule Jasp had no effect on F-actin levels (FIG. 11D,E).

[0157] Actin depolymerization increases mitochondria motility in the axon shaft An axon shaft structure recently revealed by super-resolution microscope, the membrane- associated periodic skeleton (MPS), is an actin ring-like structure formed by F-actin and spectrin underneath the plasma membrane. The MPS spans the entire axonal shaft at a roughly 190-nm period to connect the adjacent actin rings. MPS / actin rings were previously reported to restrict axonal organelle transport; loosening them by inhibiting the F-actin cross-linking molecule, NMII, facilitates axonal transport of large intra-axonal organelles, such as mitochondria. We therefore used the STORM super-resolution microscope to explore the effect of Gsn or LatB on the structure of axonal MPS in cultured hippocampal neurons.

[0158] Consistent with the effect of NMII KD in widening the axon shaft to create space for mitochondria to pass through, Gsn or LatB treatment significantly disrupted MPS, disassembled longitudinal sub-membrane actin filaments, and reduced the size of growth cones (Fig. 3A-D). F-actin prevents axonal mitochondria movement in axon shafts by intracellular organelle docking and immobilization. We postulated that actin depolymerization in axon shafts may induce axon regeneration by promoting axonal mitochondria transport in addition to its local effects in growth cones. To test this possibility, we first performed an in vitro reconstitution motility assay with purified recombinant motor protein KIF5B and polymerized microtubules immobilized on coverslips. Using microbeads (~ 2.8pm diameter to mimic large cargos, such as mitochondria) conjugated with GFP- KIF5B, we observed baseline migration of KIF5B-cargos towards the microtubule plus-end, mimicking axonal anterograde trafficking (Fig. 3E-G). Adding F-actin almost entirely blocked the motility of KIF5B-cargos: percentage and speed of migration events on microtubules were markedly decreased. In contrast, Gsn treatment that severed F- actin led to a recovery of the motility of KIF5B-cargos (Fig. 3E-G). We attributed this effect on microtubule-based cargo transportation to the physical blockage of large cargos by F-actin because F-actin did not affect the percentage of individual KIF5B-GFP molecules moving along the microtubules without cargos, although it modestly decreased their velocity (fig. 12A-D).

[0159] To further confirm the effects of the pro-regeneration / pro-actin depolymerization genes Gsn and Anxa2 on axonal mitochondria transport, we performed mitochondria kymography assays in cultured hippocampal neurons. As in the in vitro motility assays, Gsn overexpression significantly increased the motility of axonal mitochondria, including the speed, percentage, and move length of the mobile mitochondria, and the total number of axonal mitochondria (Fig. 3H- J), concurrent with increasing the neurite growing speed and length (Fig. 3K-M). Gsn treatment also significantly increased axonal transport of lysosomes (fig. 12E), which are similar in size to mitochondria; but only slightly increased axonal transport of recycling endosomes, which are much smaller (fig. 12F), again indicating the importance of cargo size in F-actin-regulated axonaltransport. Anxa2 consistently showed similar effects: it significantly increased the speed, percentage, and move length of the mobile axonal mitochondria, and the total number of axonal mitochondria (fig. 12G-I), concurrent with increasing speed of growth and length of axons (fig.12J-L). LatB treatment also consistently increased axonal mitochondria transport (FIG. 13A-C) and the migration speed of growth cones (FIG. 13D,E). In contrast, actin polymerization / anti- axon regeneration gene Pfn1 did not affect the speed or move length of mobile mitochondria and decreased the percentage of total moving mitochondria in axons (FIG. 13F,G). All these results suggest that actin depolymerization is a driving force of ON regeneration through boosting axonal mitochondria transport.

[0160] Axonal mitochondria transport is necessary and sufficient for promoting axon regeneration To confirm that axonal mitochondria transport contributes to actin depolymerization-induced axon regeneration, we next tested the effect of blocking mitochondria transport on Gsn or LatB- mediated ON regeneration. The family of kinesin- 1 microtubule motor proteins (KIF5A-C) is responsible for anterograde axonal transport of mitochondria, and we previously found that both KIF5A and KIF5B are critical for ON mitochondria transport and integrity. KIF5A and KIF5B double KO (DKO) significantly decreased Gsn- or LatB-induced ON regeneration (Fig. 4A-D), indicating that KIF5-mediated axonal mitochondria transport is necessary for actin depolymerization-induced axon regeneration. We recently demonstrated that increasing axonal mitochondria transport by overexpression of OPTN / KIF5B / TRAK1 achieves significant ON regeneration.

[0161] To further confirm that directly enhancing axonal mitochondria transport is sufficient to promote axon regeneration, we also tested a small molecule enhancer of axonal mitochondria transport, BMS754807, which was identified through high-throughput screening (HTS). We first established that BMS754807 modestly increases axonal mitochondria transport in cultured hippocampal neurons (FIG. 13H,I) . Intravitreal delivery of BMS754807 promoted significant ON regeneration after ONC (Fig. 4E,F). Taken all together, these findings demonstrate that the pro- regeneration / pro-actin depolymerization genes promote substantial axon regeneration at least in part by enhancing axonal mitochondria transport.

[0162] Combinatory genetic modulation of extracellular matrix (ECM)-membrane-actin cytoskeleton dynamics achieves ON regeneration and functional re-innervation of the suprachiasmatic nucleus (SON) Since both Gsn and Anxa2 accelerate actin depolymerization and axonal mitochondria transport, we tested the combination of these two genes and found significantly more and longer axon regeneration than with either alone (FIG. 14A,B). Because of this additive effect, we explored combinations of multiple pro-regeneration genetic manipulations. We previously found that the ECM regulator PLAT alone can promote ON regeneration and can also produce longer ON regeneration together with its cell surface receptor Anxa2 and Pten deletion. Another ECM molecule, extracellular matrix protein 1 (Ecm1) is alsohighly upregulated in regRGCs (Fig. 1A,B). Consistently, we found that Ecm1 overexpression also promotes significant ON regeneration (FIG. 14C,D).

[0163] Synergistic axon regeneration has been found in Pten / Socs3 DKO mice, and we previously showed a similar synergistic effect of Pten / Gsk3p inhibition on ON regeneration. Therefore, we combined genetic modulations of ECM (Plat and Ecm1), membrane / sub- membrane (Anxa2), and actin cytoskeleton (Gsn) to synergistically boost axonal membrane- actin dynamics in the Pten / Socs3 / Gsk3p triple KO mice (P / S / G TKO). We observed robust and lengthy ON regeneration at 8 weeks post ON crush injury (8wpc) (Fig. 5A). Regenerating axons grew past the optic chiasm (OX) into the optic tract (OT); along the way some projected into the SCN and branched heavily there (Fig. 5A-C), resembling the normal ON projection to SCN (FIG.14E). In contrast, P / S / G TKO alone or combined with Ecm1+Plat treatment caused no regenerating axons to reach SCN (FIG. 14F). Combined Gsn+Anxa2 treatment induced some regenerating axons to grow into SCN, and 3-D reconstruction enabled us to quantify regenerating axons at different locations surrounding OX and SCN (Fig. 5B,C). Adult SCN was reported to express repulsive axon guidance cues, which were subsequently observed to prevent regenerating axons of Pten / Socs3 DKO mice treated with CNTF and c-Myc from growing into SCN.

[0164] To confirm the re-innervation of SCN by regenerating axons, we performed retrograde tracing with Dextran injection at SCN (FIG. 15A,B) and demonstrated that some regRGCs in the retina sent axons into SCN (FIG. 15C). We found retrogradely labeled SPP1 + / RBPMS+ a-RGCs and RBPMS+ but SPP1 / OPN4 negative RGCs, but not detect OPN4+ ipRGCs (FIG. 15D). This result shows that the long regenerating axons originate primarily in a- RGCs and ectopically innervate SCN, rather than in the ipRGCs that normally populate SCN. It also indicates the importance of characterizing the regenerating axons and understanding their physiological roles.

[0165] For additional evidence that the regenerating axons form functional synapses, we measured light-evoked expression of the immediate early gene c-fos in SCN: light stimulation significantly elevated c-fos in SCN of P / S / G TKO mice co-treated with Gsn / Anxa2 / Ecm1 / Plat at 8wpc, whereas there was scant response in control mice (Fig. 5D,E). We further recorded light- evoked Ca2+ responses of SCN neurons by fiber photometry in the P / S / G TKO mice co-treated with Gsn / Anxa2 / Ecm1 / Plat (Fig. 5F): we found fast and strong elevation of Ca2+ before ONC, no elevation at 1wpc (Fig. 5G), and delayed but partially recovered Ca2+ elevation in the same mice at 8wpc (Fig. 5G), indicating functional re-innervation. This result is consistent with a previous report showing that regenerating RGC axons can reach and form functional synapses in SCN. In summary, combinatory genetic modulation to boost actin cytoskeleton dynamics achieves lengthy ON regeneration; encouragingly, some of the regenerating axons re-innervate and form functional synapses within the SCN.

[0166] Gsn reverses ON mitochondria transport deficits and neurodegeneration in a glaucoma model Glaucoma is characterized by ON degeneration followed by progressive RGC death, and is the leading cause of irreversible blindness. Our previous studies showed that the proregeneration genes Anxa2, Mpp1 , and Spp1 protect RGC somata and axons and preserve visual function in a mouse glaucoma model, raising the possibility that axon regeneration contributes to neuroprotection or the molecular mechanism mediating the axon regeneration phenotype of these pro-regeneration genes may also be critical for neuroprotection in glaucoma. Both Gsn and Anxa2 promote axonal mitochondria transport through actin depolymerization, and, our recent study found a significant deficit of ON mitochondria transport in glaucomatous mice. We therefore first evaluated the effect of Gsn on glaucomatous ON mitochondria motility. We took advantage of our recently developed silicone oil-induced ocular hypertension (SOHU) mouse glaucoma models (FIG. 16A,B) to test whether overexpression of Gsn would benefit the ocular hypertension-induced ON mitochondria transport deficit. Using MitoTracker in freshly isolated ONs for an ex vivo mitochondria kymograph assay, we replicated ON mitochondria transport deficits that we found before (Fig. 6A-C): compared to naive ONs, speed, percentage, and move length of the mobile mitochondria, and the total number of ON mitochondria were significantly decreased in the glaucoma ONs treated with AAV-Ctrl at 1-week post SO injection / lOP elevation (1wpi). These decreases preceded significant axon degeneration.

[0167] We then intravitreally injected AAV-Gsn to infect RGCs 14 days before inducing SOHU glaucoma to allow transgene expression. Gsn treatment significantly reversed the deficits of glaucomatous ON mitochondria motility (Fig. 6A-C).

[0168] We then performed in vivo imaging and visual function assays to further evaluate Gsn’s effect in glaucomatous neurodegeneration. In vivo OCT imaging showed significant thinning of the retinal ganglion cell complex (GCC), including retinal nerve fiber layer (RNFL), ganglion cell layer (GCL) and inner plexiform layer (IPL), in SOHU glaucoma eyes injected with control AAVs compared to contralateral naive control eyes, at 3wpi (Fig. 6D), indicating significant glaucomatous RGC / ON degeneration. Gsn treatment significantly increased GCC thickness in SOHU glaucoma eyes (Fig. 6D). In vivo assessment of visual acuity by optokinetic tracking response (OKR) and of RGC electrophysiologic function by pattern electroretinogram (PERG) demonstrated that Gsn treatment significantly preserved visual function of the SOHU glaucoma eyes (Fig. 6E,F).

[0169] We next investigated RGC soma and axon morphology by histological analysis of postmortem retina wholemounts and ON semi-thin sections. Quantification of surviving RGC somata in retina and surviving RGC axons in ONs consistently showed that Gsn treatment increased RGC survival throughout the peripheral, middle, and central regions of the glaucomatous retinas and RGC axon survival throughout the glaucomatous ONs (Fig. 6G,H). Taken together, these results demonstrated that the axonal mitochondria deficit plays a key rolein glaucomatous neurodegeneration. Similar to Anxa2, Gsn treatment reversed this trend and achieved neuroprotection in the SOHU glaucoma model.

[0170] Delayed Gsn treatment also provides significant neuroprotection in glaucoma To simulate the clinical scenario, we tested whether delivering Gsn after IOP elevation also protects RGC somata, axons, and visual functions. Unfortunately, we cannot use routine intravitreal injection of AAV to express Gsn in glaucomatous RGCs after IOP elevation, because the intravitreal injection procedure itself decreases IOP, compounding the treatment effect of the transgene with the effect of reducing IOP. Taking advantage of AAVretro, an AAV2 capsid variant that permits robust retrograde access to projection neurons from their axon target regions, we first confirmed the efficiency of AAVretro superior colliculus (SC, a target of RGC projection axons) injection-mediated retrograde RGC targeting. We used in vivo SLO imaging (FIG. 16C) and histological quantification (FIG. 16D), which showed about 45% RBPMS+ RGC targeting. Our delayed treatment strategy consisted of an intracameral injection of SO to induce IOP elevation, followed by SC injection of AAVretro-Ctrl or AAVretro-Gsn 3 days later (FIG.16E). The delayed SC injection of AAV did not affect IOP (FIG. 16F). Because AAV-mediated gene expression in RGCs normally appears one week after SC injection, we were delaying the Gsn treatment for more than a week after SO injection and IOP elevation. Encouragingly, the delayed Gsn treatment also significantly increased GCC thickness in the glaucomatous eyes and preserved visual functions measured by OKR and PERG (FIG. 16G-I). Histological analysis confirmed significant neuroprotection of RGC somata and axons by the delayed Gsn treatment (FIG. 16J). In fact, long- term Gsn or Anxa2 overexpression in naive mice had no detectable detrimental effect on retina structure or function (FIG. 17A-I), validating the therapeutic potential of targeting actin depolymerization.

[0171] Delayed treatment of glaucoma with LatB increases ON mitochondria motility and promotes significant neuroprotection LatB is a classical and potent actin depolymerization small molecule that has been shown to lower human IOP through relaxing the trabecular meshwork (TM). We then tested topical delivery of LatB in the mouse SOHU glaucoma model. To avoid IOP reduction due to intravitreal injection, we delivered LatB into the glaucomatous eyes by daily retro-orbital injection, starting one day after SO injection (FIG. 18A,B). Using MitoTracker in freshly isolated ONs for the ex vivo mitochondria kymograph assay, we found that delayed topical LatB treatment replicated the effect of Gsn on rescuing glaucomatous ON mitochondria transport deficits (Fig. 7A-C).

[0172] We next tested delayed (by three days after induction) treatment of LatB in the SOHU glaucoma model (FIG. 18C). LatB retro-orbital injection did not affect IOP (FIG. 18D), which is consistent with the idea that IOP elevation in the SOHU glaucoma model is due to pupil blocking rather than to TM blocking. In vivo imaging and visual function assays demonstrated increased GCC thickness detected by in vivo OCT imaging (Fig. 7D); increased PERG amplitude (Fig. 7E);and increased visual acuity detected by OKR (Fig. 7F). Histological analysis of postmortem retina wholemounts and ON semi-thin sections consistently showed increased RGC somata survival throughout the peripheral, middle, and central regions of the whole retinas and dramatic axon protection in the glaucomatous ONs (Fig. 7G,H).

[0173] Taken all together, these results demonstrated that LatB is a potent enhancer of axonal mitochondria transport that can effectively reverse glaucomatous ON mitochondria motility deficits and significantly protect glaucomatous RGCs and ONs, and preserve visual functions.

[0174] LatB increases human RGC axon growth and axonal mitochondria transport To validate the effect of actin depolymerization in human neurons, we generated retinal organoids using human ESC). The attached retinal organoids were treated with DMSO or LatB (1 pM) for 3-hour and LatB treatment significantly increased axon outgrowth of human RGCs (hRGCs) within or isolated from retinal organoids (Fig. 8A,B), concurrent with increased total axonal mitochondria transport and total mitochondria numbers in isolated human RGCs neurites (Fig. 8C-E), although the retrograde transport is more significantly affected by LatB treatment than the anterograde transport

[0175] F-actin is significantly elevated in the aqueous humor of patients with severe glaucoma Dysregulation of F-actin has been associated with multiple neurodegenerative diseases and serum actin level has emerged as a potential biomarker for neurodegeneration. We therefore compared levels of F-actin in the aqueous humor of control patients who needed cataract surgery and patients with glaucoma who needed glaucoma surgery. We categorized glaucoma severity based on the mean deviation (MD) of Humphrey Visual Field (HVF) testing: mild / moderate glaucoma was between -6 dB to -12 dB and severe glaucoma was higher than - 12 dB. We found significantly higher levels of F-actin in patients with severe glaucoma than in control patients with cataracts or in patients with mild glaucoma (Fig. 8F,G), suggesting a potential correlation between extracellular levels of F-actin and the severity of glaucomatous neurodegeneration.

[0176] In this study we demonstrated that overexpression of Gsn and other actin depolymerization molecules, or inhibition of actin polymerization molecules, promotes significant ON regeneration. Consistently, the pro-regeneration molecules we identified before, Anxa2, Ilk, and Mpp1 , interact with actin and Gsn directly. Anxa2 acts as an actin depolymerization molecule, which is confirmed by our finding that it decreases F-actin levels in growth cones and axon shafts as Gsn. Pten itself is also involved in actin network control through regulating membrane PIP2 levels and its interaction with many acting-binding proteins. In addition to actin remodeling at growth cones, we found that these pro-regeneration molecules decrease F- actin in axon shaft and disrupt axonal actin rings, which leads to striking increases in axonal mitochondria motility. Based on these findings, we propose a general mechanism in whichdiverse anti- and pro-regeneration signaling pathways converge on cytoskeleton dynamics, immediately upstream of axon regeneration: extracellular molecules, including ECM components, growth factors, signaling molecules, and myelin molecules act on membrane receptors / regulators to directly or indirectly regulate cell / axonal membrane structural phospholipid (PIP2) and membrane dynamics, which modulate the sub-membrane actin cytoskeleton. When the convergence favors actin depolymerization, the local actin dynamics accelerate and axonal mitochondria transport increases, initiating and driving axon regrowth. Some of the modulators may act synergistically at the serial steps to potentiate their effect and produce the more potent and lengthy axon regeneration. The increased actin depolymerization- mediated axonal mitochondria transport also achieved dramatic neuroprotection and visual function preservation in an experimental model of glaucoma, regardless of whether the treatment was delivered before or after the onset of intraocular pressure (IOP) elevation, suggesting a potential actin-based neuroprotection strategy (FIG. 18E).

[0177] Although F-actin is enriched in growth cones and actin dynamics in growth cones have been credited with a key role in axon regeneration, we have demonstrated that the MPS, a unique axon shaft actin structure also known as the actin ring, is disturbed by Gsn or LatB treatment, concurrent with an increase in axonal mitochondria transport and axon growth. This is consistent with the effect of another pro-axon regeneration manipulation, NMII knockdown, which loosens the actin ring and widens the axon locally to create space for the passage of larger organelles like mitochondria. In the present report, we demonstrated that F-actin blocks microtubule-based large cargo transportation and that F-actin depolymerization by Gsn / Anxa2 / LatB increases axonal mitochondria motility in vitro and in vivo, which is critical for their function in both ON regeneration and neuroprotection. This notion is further supported by the ON regeneration and neuroprotection induced by LatB and BMS75480, mitochondria motility enhancers identified through unbiased HTS.

[0178] Our study suggests that decreased ON mitochondria transport is a common neurodegeneration mechanism shared by both OPTN mutation-induced normal tension glaucoma and ocular hypertension induced glaucoma. The striking regeneration and neuroprotection effects of Gsn / Anxa2 / LatB in the present study strongly support that targeting axonal mitochondrial transport represents a potentially powerful neural repair strategy for glaucoma, and possibly for other CNS neurodegenerative diseases as well.

[0179] We detected elevated F-actin in the aqueous humor of patients with severe glaucoma, consistent with a previous report that actin is upregulated in the aqueous humor in glaucoma. Given our demonstration that F-actin depolymerization has significant effects on axon regeneration and neuroprotection, the F-actin level is a promising biomarker for both disease severity and treatment efficacy. Dysregulation of F-actin has also been linked with multiple otherneurodegenerative illnesses, and GSN, that degrades F-actin, has been suggested as a prognostic disease marker, especially in CNS neurodegenerations.

[0180] Remarkably, actin polymerization is also required for mitochondria fission, whereas actin depolymerization promotes mitochondria fusion and mitochondrial metabolic function. Many CNS neurodegenerative diseases are associated with defective anterograde axonal mitochondria transport, and increased fission and decreased fusion, including glaucoma. It would be of great interest to characterize the morphological modifications of mitochondria in glaucomatous ONs and to investigate the roles of pro- regeneration / neuroprotection actin modulators in ON mitochondria fission, fusion, and metabolic changes.

[0181] In summary, we have used a potent Retro-Seq strategy and detailed characterization of the biological activities of multiple pro-regeneration molecules to identify key regeneration mediators downstream of Pten deletion, including Gsn and Anxa2. We have also demonstrated that enhanced mitochondria transport associated with actin depolymerization is a unified mechanism on which various ECM-plasma membrane-cytoskeleton modulators converge that is responsible for both axon regeneration and neuroprotection. Moreover, by targeting this mechanism, we also show that Gsn and LatB are very promising neuroprotectants due to their striking protection of glaucomatous RGCs / ONs and visual function. Although a Phase 1 human clinical trial has shown that LatB safely and effectively reduces IOP and intravitreal injection of LatB in monkey eyes has no detectable retina function deficit, its strong effect on actin depolymerization may cause damages to other retinal neurons or glial cells. It will, therefore, be critically important that treatment strategies optimize the delivery method and concentration to minimize potential toxicity while preserving the beneficial effects on RGC neuroprotection and regeneration. Because axonopathy is a common early feature of various CNS neurodegenerative diseases, the neural repair strategy we developed here, modulating actin dynamics and enhancing axonal mitochondria motility, is applicable to other neurodegenerative diseases and neural injuries.Materials and Methods

[0182] Study design This study prospectively tested whether reducing F-actin by modulating actin-regulatory genes promotes axon regeneration and protection through enhancing axonal mitochondrial transport. Both in vivo mouse models and ex vivo cell-based assays were used to demonstrate the neural repair effects and molecular mechanisms of these pro-regeneration and pro-neuroprotection genes and small molecules. For axon regeneration in traumatic optic nerve crush mouse model, sample size of 5 mice per group produces 10 retinas and 10 optic nerves, which was calculated by power analysis (a = 0.05, power = 0.8) and increased to account for attrition. For the SOHU glaucoma model, sample size of 15 mice per group was determined by our previous reports. Animals were randomly assigned to treatment groups, stratified by sex,and all procedures and analyses were blinded to the investigators in group allocation during outcome assessments, including RGC counting, regenerating axon counting and histological evaluation. Endpoint readouts included retinal morphology and visual function, mitochondrial transport, and safety metrics. Animal sample was excluded if surgical-related secondary eye complication happened, such as cornea ulcer or eye collapse, which is rare. There is no sample exclusion for cell-based assays. Detailed information regarding the number of replicates, the statistical test used, and the corresponding P values are provided in the figure legends. Statistics were conducted using analysis of variance (ANOVA) or non-parametric with appropriate corrections. All experimental procedures in mice were performed in compliance with animal protocols approved by the IACUC (#32093) at Stanford University School of Medicine with implementation of ARRIVE guidelines. Human retinal organoid / iRGC studies were approved by Stem Cell Research Oversight (SCRO #586) of Stanford University IRB. Human aqueous fluids were collected strictly following protocols approved by the University of California San Francisco Institution Review Board and adhered to the Declaration of Helsinki for research involving human subjects. All the patients agreed with written informed consent.

[0183] Statistical analyses. GraphPad Prism 7 was used to generate graphs and for statistical analyses. Data are presented as means ± s.e.m. For two groups comparison, normality and lognormality tests were performed first using Shapiro-Wilk normality test. Student’s Unpaired two-tailed Student’s t-test was used for two groups comparison if both groups passed the normality test, otherwise the non-parametric Mann-Whitney test was used. For comparisons among more than two groups, one- way ANOVA followed by Dunnett’s multiple comparisons test was used when comparing each treatment group with a single control. For all pairwise comparisons, like axon regeneration, mitochondria mobile speed and percentage in total / anterograde / retrograde, two-way ANOVA followed by T ukey or Sidak multiple comparisons test was performed. Significant P values were indicated in the graphs (*P < 0.05, **P < 0.01 , ***P < 0.001 , and *‘**P < 0.0001).

[0184] Mice. C57BL / 6J WT, B6.129S4-Pten,m1 Hwu / J mutation (Ptenflox / flox, 006440) and B6;129S4-Socs3tm1Ayos / J (SOCS3flox / flox, 010944), and B6.129S1-Kif5btm1 Njen / J (KIF5B floxed, 008637) male and female mice were purchased from Jackson Laboratories (Bar Harbor, Maine). Gsk3flox / ,loxmice with C57BL / 6 background were originally developed by Dr. Jim Woodgett and were described by us before. KIF5A floxed mice were described before. We crossed them to generate ptenflox / ,loxSocs3flox / ,loxGsk3|3,lox / floxtriple flox mice and KIF5A / B double flox mice. All mice were housed in standard cages on a 12-hour light-dark cycle. All experimental procedures were performed in compliance with animal protocols approved by the IACUC at Stanford University School of Medicine.

[0185] Constructs and AAV production. The coding regions of Gsn, Dstn, Cfl1, CapG, Avil, Scin, Pfn1 , Pfn2, Arp2 / Arp3, CapZal, CapZa2, CapZp, Add1 , Add3-isoform2 and Ecm1 geneswere amplified from different mouse tissue cDNA with Q5 high-fidelity DNA Polymerase (NEB, M0491L) or Phanta Super-fidelity DNA Polymerase (Vazyme, P501) and cloned into the pAM- AAV-mSncg-3HA-WPRE, pAM-AAV-mSncg-0.27K-3HA-WPRE, and pAM-AAV- mSncg- 0.27K-MCS1-3HA-MCS2-stp-WPRE backbone. The Gsn, Anxa2, and Pfn1 were also fused to GFP and with the same procedure cloned into AAV-mSncg-GFP-WPRE backbone. The Mito- DsRed plasmid (#44386) and the Rabi 1 -mCherry (#55124) were purchased from Addgene. The pAM-AAV-mSncg-Cre-WPRE plasmid was published previously.

[0186] The CRISPR-Cas9 system was used for gene knock-out in mouse retinas. As described previously, the Cas9 was cloned into pX551 driven by 0.27k mSncg promoter, and the top four guide RNAs of each gene were designed by CHOPCHOP online website and inserted into pX552 backbone. The four constructs of pX552-gRNAs were further combined by cutting the U6-guide RNA and inserting into another pX552 to generate two gRNAs in one pX552 plasmid. After maxi-prep of each pX552-gRNAs construct, the two pairs of gRNAs were pooled in a 1 :1 ratio for further AAV packing.

[0187] The maxi-precipitation of the constructs was performed by following the manual of Endo-Free Plasmid Maxi Kit (Omega Bio-tek, D6926-03 / 101319-342). The detailed procedure of the AAV production has been described previously. Briefly, AAV plasmids containing the target genes were co-transfected with pAAV2 (pACG2)-RC triple mutant (Y444, 500, 730F) or AAVretro, and the pHelper plasmid (StrateGene) into HEK293T cells by the PolyJet (SignaGen Laboratories, SL100688) transfection reagent. After transfection for 72 hours, the cells were lysed and purified by two rounds of cesium chloride density gradient centrifugation. The AAV titers of target genes were determined by real-time PCR and diluted to 1 .5 x 10'2vector genome (vg) / ml for intravitreal injection, 2 pl / eye.

[0188] Intravitreal injection. Mice were anesthetized by xylazine and ketamine based on their body weight (0.01 mg xylazine / g + 0.08mg ketamine / g). For AAV intravitreal injection, a pulled and polished microcapillary tube was inserted into the peripheral retina of about 4-week-old mice just behind the ora serrata. Approximately 2 pl of the vitreous was removed to allow injection of 2 pl AAV into the vitreous chamber. The mice were housed for an additional two weeks after AAV injection to achieve stable target genes expression. In mouse retina genes knock-out by CRISPR-Cas9, the AAV-Cas9 and AAV-gRNAs were mixed in a 2:1 ratio of viral genome copy (total 3x109 vg / eye) and intravitreally injected into each mouse eye for four weeks to knock-out the targets. For anterograde tracing of regenerating axons, 1 pl of 2pg / pl cholera toxin subunit B-Alexa 555 or 647 (CTB555 and CTB647, Invitrogen) was used for intravitreal injection.

[0189] For axon regeneration after ONC, 2pl solutions of latrunculin B (LatB: 6.95pM), cytochalasin D (CytoD: 4.92pM), CK666 (101.2pM), jasplakinolide (Jasp: 0.99pM), orBMS754807 (10pM), were delivered by intravitreal injection, two injections / week for two weeks. LatB, CK666, Jasp, BMS754807, and CytoD were dissolved in DMSO (or in vehicle with10% HS15, 5% DMSO, and 85% PBS). For the combinatory treatment of Gsn, Anxa2 and Ecm1 overexpression, tissue plasminogen activator (tPA, Sigma, 612200-m), an AAV mixture with equal amount of Anxa2 and Gsn as the first shot was intravitreally injected into the Pten / Scos3 / Gsk3p floxed mouse eyes, then an AAV mixture with equal amounts of Ecm1 and Cre was injected as the second shot on the third day after the first shot. ONG was performed at 14 days post the second injection to allow gene expression, and 2pl of 30 U / pil tPA was intravitreally injected twice / week after ONG.

[0190] To label ON mitochondria ex vivo, 2pl MitoTracker Orange (Thermo Fisher Scientific, 0.15mM) was injected intravitreally per eye at four hours before tissue collection. Retro-orbital (RO) injection. LatB was dissolved in a mixture solvent (5% DMSO / 10% Kolliphor HS15 / 85% PBS) and used to treat SOHU glaucoma by RO injection. After mice were anesthetized with a sustained flow of isoflurane (3% isoflurane at 2 L / min mixed with oxygen) delivered to the nose by a special rodent nose cone (Xenotec, Rolla, Missouri), the drug solutions (8.42 pM, 100 pl) were injected once daily in the SOHU eyes through the inferior palpebral subconjunctiva using a 30G disposable syringe. The contralateral control eyes received retro-orbital injection with 100 pl mixture solvent as vehicle control. Optic nerve crush (ONC). ONC was performed two weeks post-AAV injection when mice were between seven and eight weeks of age. The ON was accessed through an intraorbital approach at the 12 o’clock position, ensuring that the retro- orbital sinus remained intact. The nerve was subsequently crushed using jeweler's forceps (Dumont #5; Fine Science Tools, Foster City, California) for five seconds at approximately 0.5 mm posterior to the globe. Post-operatively, a neomycin-containing ophthalmic ointment (Akorn, Somerset, New Jersey) was applied to the cornea to prevent damage.

[0191] Retrograde labeling of regenerating RGCs by intraorbital optic nerve injection. The regenerating RGCs were labeled retrogradely as previously described. Pten knockout (KO) mice were anesthetized with xylazine and ketamine at doses adjusted for body weight (0.01 mg xylazine / g + 0.08 mg ketamine / g). The ON was exposed intraorbitally approximately 1.5-2 mm distal to the globe by lateral canthotomy through the conjunctiva and underlying soft tissues and muscles at the 9 o’clock position, without damaging the retro-orbital sinus. To stabilize the optic nerve, a piece of tissue paper was placed between the nerve trunk and surrounding soft tissue, and the dura was penetrated at the injection site, located ~1 mm distal to the ONC site, using a 33-gauge needle. A glass micropipette connected to a 50 pl microsyringe (80900, Hamilton), controlled by a Micro4 device, was employed to inject ~60 nL of dextran Texas red into the optic nerve through the pre-formed opening at a rate of 200 nL / min. Any dye leakage at the injection site was absorbed with fine tissue paper. Following the injection, a neomycin- containing ointment was applied to protect the cornea, and the mice were placed on a heating pad to recover. Mice were housed 24 hours before tissue collection.

[0192] Retrograde labeling or gene delivery specifically to RGCs by superior colliculus (SC) injection. The detailed procedure of the micro-injection into the SC has been described previously. In brief, the adult mice were anesthetized by xylazine and ketamine based on their body weight (0.01 mg xylazine / g+0.08mg ketamine / g) and fixed on a mouse adaptor attached to a digital stereotaxic instrument (68025, RWD Life Science). The bregma was set as the origin of anterior to posterior (AP), medial to lateral (ML) and dorsal to ventral (DV), and the same ML and DV of the lambda was aligned to the bregma. The horizontal plane of the mouse skull was calibrated by adjusting the left hemisphere point (AP:-2.00, ML:2.50) to the same DV as the contralateral point (AP:-2.00, ML:-2.50). The SC coordinates for four sites and three depths were located and drilled: AP:-3.55, ML:0.6, DV-1 .25 / -1.60 / -2.00, AP:-3.55, ML:-0.6, DV-1 .25 / - 1.60 / -2.00, AP:-3.92, ML:0.8, DV-1.25 / -1.50 / -1 .75 and AP:-3.92, ML:-0.8, DV-1.25 / -1.50 / - 1 .75. A pulled-glass micropipette fused to a 10 pl syringe (80314, Hamilton) filled with mineral oil was controlled by a micro syringe pump (Micro4™, World Precision Instruments, LLC) at the speed of 150nL / min for one minute per site. About 4pl AAVretro-hSyn-H2B-Clover3 and AAVretro-mSncg0.27K-3HA-Gsn were injected into both SCs.

[0193] Stereotaxic injection of AAV and Dextran; implantation of optic cannula implantation into SCN. The mouse was mounted on a stereotaxic instrument following the same procedure as described for the superior colliculus injection in the Methods section. The bregma was set as the origin for all stereotaxic coordinates. The injection site for the suprachiasmatic nucleus (SCN) was located 15° from the midline, and bilateral injections of AAV9-hSyn-jGCaMP7s virus and Dextran-Texas Red were performed at the following coordinates: AP, -0.20 mm; ML, ±1.70 mm; DV, -5.65 mm from bregma of the wildtype and P / S / G TKO+Gsn+Anxa2+Ecm1 +tPA 8wpc mice. 200 nL GCaMP7s and 50nL Dextran were delivered per side. After the injection, the glass pipette was left in place for 5 minutes to ensure adequate viral diffusion. For optic cannula implantation, optic cannula (NA 0.39, OD: 200 pm; RWD Life Science) were bilaterally implanted above the SCN region using the same 15° tilt angle and the same AP and ML coordinates as for the viral injection. The DV position was adjusted to be 0.30 mm above the viral injection site.

[0194] Brain tissue extraction, sectioning, and immunostaining for c-Fos. Mice with ON regenerating (P / S / G / TKO+Gsn+Anxa2+Ecm1+tPA, 8wpc) or wildtype control mice with ONC only were housed in full darkness from 6wpc till tissue harvest day (8wpc) to abolish the effect of light on SCN rhythmic activity. At 8wpc, the mice were exposed to light (20mW 395nm UV + 100 lux. white light) at 12:00 AM for 1 hour. After the light stimulation, the animals were anesthetized and perfused through the heart with PBS, 4% PFA as previously described. The mouse brains were dehydrated in 30% sucrose overnight, embedded with O.C.T. compound (Sakura, Cat:4583-118ML), and cryo-sectioned with Leica CM3050S. The brain slice samples were washed with PBS for 15min, blocked with blocking buffer (Triton x-100: 0.25%, goat serum: 5% in PBS), immune-stained with primary antibodies, c-fos antibody (Rabbit, Synaptic system,Cat# 226008, 1 :1000), or GFP antibody (Chicken, Aves Lab / Fisher, Cat# GFP-1010, 1 :500), and incubated with secondary antibodies, Alexa Fluor 594 goat anti-rabbit (Jax Immunoresearch, Cat# 111-585-003, 1 :200) or Alexa Fluor 488 goat anti-rabbit (Jax Immunoresearch, Cat# 111-545-003, 1 :200). The samples were sealed with FluoroMount-G before imaging.

[0195] Fiber photometry recording and data processing. Pten / Socs3 / Gsk3 flox / flox 8-12 weeks old mice with SCN injected with AAV9-jGCaMP7s and implanted with fiber cannula were further delivered AAV2-Cre and Ecm1 by intravitreal injection at Day 1 , and AAV2-Anxa2 and AAV2-Gsn at Day 3. A pre-ONC light-evoked Ca2+ responses of SCN were recorded by fiber photometry device (RWD, Multi-channel Fiber Photometry) to screen the best SCN responsive mice. Briefly, the mice were anesthetized with ketamine -i-xylazine as previous dosage used and fixed to the mice adaptor with eye faced 5cm distance to the 395nm UV light source (20 pW). The light stimulation pattern was adjusted as 3 seconds ON and 5 seconds OFF and repeated for 20 cycles. Foil cover of the 2 fiber cannulas, mouse head with dental cement, and part of the fiber are necessary to avoid the UV stimulation light caused artifact response. After intravitreal injection on Day 14, the optic nerve crush surgery was performed by double-blind seasoned hands as same as previously described. The tPA was intravitreally injected into mouse eyes twice / week till to 8 weeks post ONC. Follow-up recording on 1 week and 8 weeks post ONC were performed to evaluate the functional recovery. For preprocessing the raw fiber photometry data, we used the pMAT tool to correct the signals from the 470 nm channel by subtracting the isobestic control data obtained from the 410 nm channel. Fluorescence changes were quantified using the formula: A F / F0 = (F - F0) / F0 where the baseline (F0) was defined as the signal recorded 5 s before UV light stimulus presentation. The average Ca2+traces were generated using GraphPad Prism, with shaded areas representing the standard error of the mean (s.e.m.). The maximum amplitude within the light stimulus train was used for statistical comparison of baseline, post-1 -week, and post-8-week timepoints through one-way ANOVA.

[0196] Super-resolution Imaging Assay Primary culture of mouse hippocampal neurons All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the Pennsylvania State University. Primary cultures of hippocampal neurons were prepared as previously described. Briefly, timed- pregnant CD-1 IGS mice (Charles River Laboratories, Wilmington, MA; 022) were euthanized, and hippocampi were isolated from E18 mouse embryos. The dissected tissues were enzymatically dissociated with 0.25% trypsin-EDTA (1 x) (Sigma, T4549) at 37 °C for 15 minutes. Following digestion, the hippocampal tissues were washed three times with Hanks' Balanced Salt Solution (HBSS) (Thermo Fisher Scientific, 14175095), and then transferred to NbActivI culture medium (Transnetyx, NB1500 ml), a pre-mixed formulation comprising Neurobasal, B27,and Glutamax, with the addition of 100 pg / ml Primocin (InvivoGen, ant-pm-2). The tissues were gently triturated in the culture medium until a single-cell suspension was achieved, ensuring no tissue clumps remained. Dissociated cells were then counted and plated onto poly-D-lysine- coated 18-mm coverslips (Neuvitro, GG-18-1 ,5-PDL). Cultures were maintained in a humidified incubator at 37 °C with 5% CO2. Half of the medium volume was replaced every five days to maintain optimal culture conditions. Neurons were fixed at eight days in vitro for subsequent experiments described in this study.

[0197] Gsn overexpression and LatB treatment. To induce Gsn overexpression, 1 pL of AAV- mSncg-0.27K-3HA-Gsn (titer: 6.5E+12 v.g. / mL) was added to neuronal cultures 36 hours before fixation. For actin depolymerization, neurons were treated with 1 pM Latrunculin B (LatB) for 30 minutes before fixation. As a negative control, the same volume of DMSO was added for 30 minutes prior to fixation.

[0198] Neuron fixation and staining for fluorescence imaging. Cultured hippocampal neurons were fixed as previously described. Briefly, neurons were initially fixed and extracted for 1 min in a solution containing 0.3% (v / v) glutaraldehyde and 0.25% (v / v) Triton X-100 in cytoskeleton buffer (CB, 10 mM MES, pH 6.1 , 150 mM NaCI, 5 mM EGTA, 5 mM glucose and 5 mM MgCh), followed by post-fixation in 2% (v / v) glutaraldehyde in CB for 15 minutes. The samples were then treated with freshly prepared 0.1% (w / v) sodium borohydride for seven minutes to reduce background fluorescence. For neurons treated with LatB or DMSO, actin filaments were stained with Alexa Fluor 647-conjugated phalloidin (Invitrogen A22287) at ~0.5 pM in phosphate- buffered saline (PBS) for one hour at room temperature. After final washes in PBS, samples were processed for imaging. For neurons overexpressing AAV-mSncgO.27K-3HA-Gsn, samples were permeabilized in 0.2% (v / v) Triton X-100 in PBS for 10 minutes, then blocked in 3% (w / v) bovine serum albumin (BSA) in PBS for 1 hour at room temperature. Neurons were incubated overnight at 4°C with anti-HA primary antibody (Invitrogen, 26183-1 MG) diluted in blocking buffer, followed by three PBS washes and a 1 -hour incubation with Cy3-conjugated secondary antibody (Jackson ImmunoResearch, 715-165-151) at room temperature. After additional PBS washes, samples were stained with Alexa Fluor 647-phalloidin as described above.

[0199] STORM imaging The STORM setup was built on a Nikon Eclipse-Ti2 inverted microscope, equipped with a 100x (N.A. 1 .45) Plan Apo oil-immersion objective (Olympus) and an EM-CCD camera (Andor IXon Life 897). 405-nm (Coherent, 1265577, 140 mW), 488-nm (Coherent, 1416094 ,300 mW), 560-nm (MPB Communications Inc., 2RU-VFL-P-1500-B1 R, 1500 mW) and 640-nm (MPB Communications Inc., 2RU-VFL-P-2000-B1 R, 2000 mW) lasers were introduced through the back port of the microscope. The laser beams were shifted toward the edge of the objective to achieve near-total internal reflection (TIR) illumination, ensuring selective excitation of fluorophores within a few micrometers of the coverslip surface. For single-color 3D STORM imaging, Alexa Fluor 647 was imaged. A cylindrical lens was inserted into the detection path to introduce ellipticity into the point spread function, allowing determination of the z-positions of single molecules from the ellipticity of their images. Samples were imaged in an oxygen scavenging imaging buffer, containing 100 mM Tris-HCI (pH 7.5), 100 mM cysteamine (Sigma-Aldrich, M9768-5G), 5% (w / v) glucose (Sigma, G8270-100G), 0.8 mg / mL glucose oxidase (Sigma-Aldrich, G2133-1 OKU), and 40 pg / mL catalase (Sigma-Aldrich, C100-50MG). During imaging, continuous illumination of 640-nm (-2 kW / cm2) was used to excite AF647, switching into the dark state. Continuous illumination of the 405-nm laser (0-1 W / cm2) was used to reactivate the fluorophores to the emitting state, and the illumination power was controlled so that at any given time, only a small, optically resolvable fraction of the fluorophores in the field of view were in the emitting state. Atypical single-color 3D STORM image was reconstructed from -60,000 image frames acquired at a frame rate of 110 Hz. The recorded STORM movies were analyzed using established methods. Super-resolution images were reconstructed from the molecular coordinates by depicting each location as a 2D Gaussian peak.

[0200] Data quantification and analysis. 1 D autocorrelation analysis of the periodic actin lattice was performed using custom MATLAB scripts. Signals from STORM images were projected to the longitudinal axis of the axon segments, and autocorrelation functions were calculated from these 1 D projected signals. To obtain the average autocorrelation function, the autocorrelation curves from around 30 randomly selected axon segments (-3 pm in length) were averaged for each condition. The average 1 D autocorrelation amplitude was defined as the difference between the first peak (at -190 nm) and the average of the two first valleys (at -95 nm and -285 nm) in the average 1 D autocorrelation curve, which provides a quantitative measure of periodicity of the MPS lattice in neurites. The length of submembrane F-actin and the size of growth cones were measured manually in Imaged by outlining the morphology of F-actin and growth cone regions.

[0201] Human iRGC and retinoid preparation. The Brn3b-tdTomato H9 ESC line was a gift.Retinal organoids were differentiated as previously described. Briefly, human ESCs were maintained in StemFlex (Thermo Fisher Scientific, A3349401) at 37 °C in the presence of 5% CO2. Embryoid bodies were formed by cultures in low-attachment dishes in StemFlex with Blebbistatin (STEMCELL Technologies, 72402). Subsequently, embryoid bodies were attached to Matrigel-coated dishes and cultured in neural-inducing medium with N2 (Thermo Fisher Scientific, 17502-048) and retinal-differentiation medium with B27 (Thermo Fisher Scientific, 12587-010). On the 28th day of differentiation, retinal organoids gained their 3D structure and were lifted off manually to establish floating cultures in organoid culture medium (RC2 media). To induce axon extension, 40-45 days old retinal organoids were manually dissected into 3-4 pieces and attached to Matrigel-coated dishes for another 3 days culture in RC2 medium. The attached retinal organoids were treated with DMSO and Latrunculin B (1 pM) for a 3-hourincubation in 37 °C with 5% CO2, then fresh RC2 medium was added to replace the treatment medium. The retinal organoids were fixed with 4% PFA for 20 minutes at room temperature, and further stitch-imaged with Keyence fluorescent microscopy. Human iRGCs were dissected from retinal organoids and seeded on plates.

[0202] To promote RGC differentiation, 40-45 days old retinal organoids were manually dissected into 3-4 pieces and attached to Matrigel-coated dishes for another 7 days culture in RC2 medium. Subsequently, the pre-attached organoids were dissociated in Accutase (Innovative Cell Technologies, AT104) for 30 minutes at 37°C and resuspended in RC2 and quantified with an automatic cell counter. 5000 cells were then replated on PDL / laminin-coated plates and cultured in human iRGC culture medium for the following experiment. The human iRGCs were treated with LatB and DMSO as previously described. After the mitochondria were labeled with Mito-Tracker and time-lapse imaged with confocal microscopy, axonal mitochondria transportation was analyzed with a Kymolyzer and quantified as described above.

[0203] The human iRGCs were fixed with 4% PFA and further imaged by stitch with Keyence microscopy for neurite length. The length measurement of the retinoid neurites was performed with Simple Neurite Tracer (SNT) and Concentric circles plugins of Fiji-lmage J software. Briefly, whole retinal organoids neurites were outlined with SNT to generate a skeleton image, and concentric rings spaced at 100 pm. Neurites numbers of each ring were counted manually and quantified with GraphPad Prism. The neurite lengths of human iRGC were directly measured with the Fiji line outline and measure function. Human aqueous humor F-actin ELISA.

[0204] Human aqueous fluids were collected strictly following protocols (17-22840) approved by the University of California San Francisco Institution Review Board and adhered to the Declaration of Helsinki for research involving human subjects. All the patients agreed with written informed consent. All clinical data were anonymously extracted from the records. The detailed procedure of aqueous humor acquisition is well described in a previous publication. Briefly, aqueous fluid (50-200 pL) was collected by paracentesis of the anterior chamber using a 27- gauge needle prior to any intraocular injection. Care was taken to avoid blood or cellular debris contamination. Samples were labeled with ID numbers, transported on dry ice, and stored at -80°C until analysis. F-actin levels were measured in aqueous fluid from patients undergoing cataract surgery without glaucoma or other intraocular diseases (controls; n = 10) and from glaucoma patients undergoing surgery (Ahmed valve implantation, micro-invasive procedures, or trabeculectomy with orwithout cataract extraction and IOL placement) between October 2024 and June 2025.

[0205] Glaucoma patients were classified as mild (n = 12; MD -6 to -12) or severe (n = 7; MD < -12) primary open-angle glaucoma (POAG) based on Humphrey Visual Field mean deviation. F-actin was quantified using a human F-actin ELISA kit (MBS3803242, MyBioSource, San Diego) on 2.5-fold diluted aqueous samples, following the manufacturer’s protocol.Fluorescence at 450 nm was measured with a microplate reader (Tecan Spark, Multimode Microplate Reader), and F-actin concentrations (ng / mL) were calculated using a standard curve of recombinant F-actin. The patient MD and F-actin level were plotted with 2-factor distribution. Brain-optic nerve clearance and light sheet microscopy imaging. The attached ON and whole brain were carefully dissected with fine forceps and scissors, and embedded in 1.5% agarose gel block. The tissue embedded gel block was cleared with a modified iDISCO method(88 : PBS for four hours; a series of 20%, 40%, 60%, 80%, and 100% methanol in 1xPBS for one day at each concentration; dichloromethane (DCM) / methanol (2:1) for one day; 100% DCM for one day and dibenzyl ether (DBE) for one day. The ventral side of the tissue gel block was faced up and fixed on a spike holder, then placed into the imaging chamber immersed in the DBE buffer. The UltraMicroscope II generated six bi-directional 4pm thin light sheets to illuminate the tissue gel block from both sides while imaging the excited plane with a 2x objective microscope perpendicular to the sample using a 0.63x zoom for whole tissue and a 6.3x zoom for regenerating axons. Tissue was imaged with the diode 561 nm laser, emission filter 620 / 60nm or 633 nm laser, emission filter 670 / 20nm, and sheet numerical aperture (NA) 0.149 through a 4pm step-size of the Z-stack. The multiple optical sliced images of the whole tissue were collected and further maximum projections were processed by Fiji / I mage J.

[0206] SOHU glaucoma model and IOP measurement. The detailed procedure has been published before. In brief, 9-week-old mice were anesthetized by an intraperitoneal injection of Avertin (0.3mg / g) before a tunnel was made by a 32G needle through the layers of the cornea on the superotemporal side close to the limbus to reach the anterior chamber without injuring lens or iris. Then, ~ 2pl silicone oil (1 ,000 mPa.s, Silikon, Alcon Laboratories, Fort Worth, Texas) was injected slowly into the anterior chamber using a homemade sterile glass micropipette, until the oil droplet expanded to cover most areas of the iris (diameter ~ 1.8-2.2mm). After the injection, veterinary antibiotic ointment (BNP Ophthalmic Ointment, Vetropolycin, Dechra, Overland Park, Kansas) was applied to the surface of the injected eye. The contralateral control eyes received 2pl normal saline into the anterior chamber. Throughout the procedure, artificial tears (Systane Ultra Lubricant Eye Drops, Alcon Laboratories, Fort Worth, Texas) were applied to keep the cornea moist. The IOP of both eyes was measured before SO injection and at 3wpi by the TonoLab tonometer (Colonial Medical Supply, Espoo, Finland) according to product instructions.

[0207] Briefly, in the morning, mice were anesthetized with a sustained flow of isoflurane (3% isoflurane at 2 L / minute mixed with oxygen) delivered to the nose by a special rodent nose cone (Xenotec, Inc., Rolla, Missouri), which left the eyes exposed for IOP measurement. 1% T ropicamide sterile ophthalmic solution (Akorn, Somerset, New Jersey) was applied three times at 3-minute intervals to fully dilate the pupils (about 10 minutes) before taking measurements. The average of six measurements by the TonoLab was considered as one machine-generatedreading, and three machine-generated readings were obtained from each eye; the mean was calculated to determine the IOP. During this procedure, artificial tears were applied to keep the cornea moist. Spectral-domain optical coherence tomography (SD-OCT) imaging and scanning laser ophthalmoscopy (SLO) fundus imaging. Fundus OCT imaging was performed under OCT mode by switching to a 30° licensed lens (Heidelberg Engineering) as in the previously described procedure. Briefly, the mouse retina was scanned with the ring scan mode centered by the optic nerve head at 100 frames average under high-resolution mode (each B-scan consisted of 1536 A scans). The ganglion cell complex (GCC) includes retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), and inner plexiform layer (IPL). The average thickness of GCC around the optic nerve head was measured manually with the Heidelberg software. The mean of the GCC thickness in the injured retina was compared to that in the contralateral control retina to yield a percentage of GCC thickness value. The investigators who measured the thickness of GCC were blinded to the treatment of the samples.

[0208] For SLO retinal fundus imaging, the fundus labeled with green fluorescent dye was imaged under FA mode by switching to a 55° non-contact lens and a customized +10D contact lens (3.0 mm diameter, 1.6 mm BC, PMMA clear, Advanced Vision Technologies) (Heidelberg Engineering) as previously described. The mouse retina was imaged under the high-resolution mode (1536 x 1536 pixels) and 100 frames average with 488 nm excitation laser.

[0209] Pattern electroretinogram (PERG) recording. PERG recording of both eyes was performed at the same time with the Miami PERG system (Intelligent Hearing Systems, Miami, FL), as described in our previous publications. In brief, a feedback-controlled heating pad (TCAT- 2LV, Physitemp Instruments Inc., Clifton, New Jersey) maintained animal core temperature at 37°C. A small lubricant eye drop (Systane) was applied before recording to prevent corneal opacities. The reference electrode was placed subcutaneously on the back of the head between the two ears, the ground electrode was placed at the root of the tail and the active steel needle electrode was placed subcutaneously on the snout for the simultaneous acquisition of left and right eye responses. Two 14 cm x 14 cm LED-based stimulators were placed in front so that the center of each screen was 10 cm from each eye. The pattern remained at a contrast of 85% and a luminance of 800 cd / m2, and consisted of four cycles of black-gray elements, with a spatial frequency of 0.052 c / d.

[0210] Upon stimulation, the independent PERG signals were recorded from the snout and simultaneously by asynchronous binocular acquisition. With each trace recording up to 1020 ms, two consecutive recordings of 200 traces were averaged to achieve one readout. The first positive peak in the waveform was designated as P1 and the second negative peak as N2. P1 was typically around 100 ms. The mean amplitude of the P1-N2 amplitude in the injured eye was compared to that in the contralateral control eye to yield a percentage of amplitude change. The investigators who measured the amplitudes were blinded to the treatment of the samples.

[0211] Electroretinogram (ERG) recording. ERG recording of both eyes was performed at the same time with an ERG stimulator (Celeris, Diagnosys LLC) according to the manufacturer's instructions. Mice dark-adapted for 12 h before ERG recording were anesthetized by ketamine based on their body weight (0.08 mg ketamine / g + 0.01 mg xylazine), and their pupils were dilated by 1%tropicamide. Mice were stimulated with flashes of 0.01 , 0.1 , and 1 cd s1m2light intensity. The raw data and traces were exported as PDF format, and the response amplitude data were quantified with Graphpad Prism. OKR measurement.

[0212] The spatial vision of both eyes was measured using the opto-kinetic response (OKR) as described in our previous publications. In brief, mice were placed unrestrained on a platform in the center of four 17-inch LCD computer monitors (Dell, Phoenix, AZ); their movement was captured by a video camera above the platform. A rotating cylinder with vertical sine wave grating was computed and projected to the four monitors by OptoMotry software (CerebralMechanics Inc., Lethbridge, Alberta, Canada). The sine wave grating provides a virtual-reality environment to measure the spatial acuity of left eye when rotated clockwise and right eye when rotated counterclockwise. When the mouse calmed down and stopped moving, the gray of the monitor immediately switched to a low spatial frequency (0.1 cycle / degree) for five seconds, in which the mouse was assessed by judging whether the head turned to track the grating. The short time frame of assessment ensured that the mice did not adapt to the stimulus, which would lead to false readouts. The mice were judged to be capable of tracking the grating. The spatial frequency was increased repeatedly until a maximum frequency was identified and recorded. The % of vision acuity was yielded by comparing the maximum frequency of the experimental eye to that of the contralateral eye. The mice were tested in the morning and the investigator who judged the OKR was blinded to the treatment of the mice.

[0213] Embryonic hippocampal neuron culture and time-lapse imaging acquisition and quantification. Embryonic hippocampal neurons were dissociated from day 14-16 wildtype (WT) mouse embryos and cultured in 35 mm glass-bottom dishes (MatTek) pre-coated with poly-L- lysine (0.5 mg / ml in ultrapure water) using Neurobasal Plus medium supplemented with 2% IB- 27, 1x Glutamax (Gibco) for 5-7 days. Neurons were then transfected with Lipofectamine LTX (Invitrogen) using 0.5pg AAV-mSncg-GFP-Gsn, 0.5pg AAV-mSncg-GFP-Anxa2 or 0.5pg AAV- mSncg-GFP-Pfn1 and 0.5pg pLV-MitoDsRed. Two hours post transfection, the transfection complex was replaced with fresh Neurobasal Plus / B-27 / Glutamax medium. Twenty-four hours post-transfection, time-lapse confocal imaging was performed using a Zeiss LSM880 confocal microscope equipped with an incubation chamber set to 37°C and 5% CO2. Images were captured with a 40x oil immersion objective lens at 1 or 2 frames per second for 5 minutes.

[0214] Mitochondrial transport events were traced, and kymograph analyses were carried out using the Kymolyzer plugin in Fiji / lmageJ. Briefly, mitochondria with average instantaneousvelocity higher than 0.05pm / s were categorized as motile. Mitochondria with average instantaneous velocity lower than 0.05pm / s were considered as zero. The following parameters were determined using Kymolyzer plugin: 1) percentage of mobile time of each mitochondrion; 2) percentage of stationary time of each mitochondrion; 3) average speed of each mitochondrion in motion; 4) move length of each mitochondrion in motion; and 5) percentage of mitochondria in motion. Mitochondrial density was determined by manually counting the total number of mitochondria from each 100-pm-long distalmost axonal segment using Fiji / lmageJ software (NIH). The compounds, LatB, CytoD, CK666 and Jasp, were used to treat the embryonic neurons at 1 pM for 30 minutes before they were fixed by 2% PFA / 2% glutaraldehyde for 20 minutes at room temperature. The F-actin labeling dye, 1 pl SIR-Actin (1 pg / pl), was used in fixed neurons for 30 minutes. The neuronal growth cones were imaged with 40x objective lens as described before, and F-actin levels in axon shafts and growth cones were quantified by arbitrary fluorescence intensity of the mean region of interests (ROIs) by measurement function in Zen3.02. The neurites length of neurons were measured by Fiji / lmage J with freehand line function outlining and measuring. During live-cell imaging analysis, 5 minutes of 300 frame neuronal time-lapse data were recorded of E165DIV neurons by LSM880 confocal as described above. Growth cone (GO) extension distance was defined by GO moving distance measured with Zen3.02 (L: distance of GO center at 1 st frame to last frame; protrusion extension is defined as + growing, contraction is defined - growing). Growing speed of the GO was calculated as S=L / 5 (pm / min).

[0215] Ex vivo time-lapse imaging of mitochondrial transport in the optic nerve. The SOHU was induced at 2 weeks after AAV-mSncgO.27K-3HA-Gsn and Capsid injection into the eyes of WT mice. To visualize axonal mitochondrial transport in the optic nerve, 2 pl of MitoTracker Orange (0.15 mM) was injected into the vitreous chamber 3 hours prior to ex vivo time-lapse imaging when the SOHU is one week post injection (1wpi). Optic nerves of naive, SOHU and Gsn treatment groups were rapidly harvested, maintained in Hibernate E low-fluorescence medium (BrainBits) at 37°C on a heated stage, and subsequently transferred to 35 mm glassbottom dishes (MatTek) pre-coated with poly-L-lysine (0.5 mg / ml in ultrapure water) using a methyl cellulose-based coating medium (8 mg / ml in Hibernate E medium). Time-lapse imaging was conducted with a Zeiss LSM880 confocal microscope equipped with an incubation chamber, using a 40x oil immersion objective to capture frames at 1 frame per 2 seconds for 5 minutes. The x-y shifting time-lapse images were processed with MATLAB movie correction algorithm before further analysis.

[0216] In the studies of LatB treatment, LatB delivery to SOHU mice was delayed until 1dpi and continued by daily RO injection until 1 wpi, when tissue was collected for ex vivo imaging. The raw time-lapse sequencing data were converted into Imaris format by ImarisFileConverter x64 9.0.0. By using the spots tracking function of lmaris9.01 , the mitochondria motions of each opticnerve were traced and outlined automatically, then the quantification results of mean speed of moving dots and track displacement length were exported, and the snapshot of 2-D mitochondria motion plotted with time since track start as the X-axis and image height as the Y- axis was taken for the representative image of mito-motion. Mitochondria with average speed higher than 0.15pm / s were categorized as motile. Mitochondria moving length greater than a 5pm cutoff threshold was considered as true move length.

[0217] QUANTIFICATION AND STATISTICAL ANALYSIS Optic nerve clearance and axon regeneration quantification. The CTB anterograde labeled optic nerve was trimmed and cleared by a modified iDISCO method: PBS for 20mins; a series of 20%, 40%, 60%, 80%, and 100% methanol in 1xPBS for 20mins at each concentration; dichloromethane (DCM) / methanol (2:1) for 30mins; 100% DCM for 30mins and dibenzyl ether (DBF) for 30mins. The cleared optic nerve was mounted on slides between two 22x22mm cover slips supported with DBE, covered with a 22x22mm cover slip, and sealed with clear nail polish. The mounted whole nerve was imaged with a 25x oil immersion objective lens, using the airy scan mode (6pm per stack), Z stack and tile scan. The number of CTB labeled axons was quantified as described previously. Briefly, we counted the fibers that crossed perpendicular lines drawn on the optic nerve optical sections distal to the crush site at 250, 500, 1000, and 1500pm, and then every 250pm until no fibers were visible. Three Z-stacks at depths of 60, 120 and 180pm were sampled to acquire the mean axon density of the optic nerve, (axon number) / (R*t). The width of the stack (R) was measured at the point (d) at which the counts were taken and used together with the thickness of the optical section (t = 6 pm) to calculate the number of axons / pm2area of the stack. The mean axon density of the 3 stacks was used to calculate the total axon number, ad= nr2* mean axon density, r is the radius of the optic nerve. All CTB signals that were in the range of intensity that was set from lowest intensity to the maximum intensity after background subtraction were counted as individual fibers. The investigators who counted the cells or axons were blinded to the treatment of the samples.

[0218] Immunohistochemistry of retinal wholemounts and RGC counts. After transcardiac perfusion with 4% paraformaldehyde (PFA) in PBS, the eyes were dissected out, post-fixed with 4% PFA for 2 hours, at room temperature, and cryoprotected in 30% sucrose overnight. Retinas were then dissected out and washed extensively in PBS before blocking in staining buffer (10% normal donkey serum and 2% T riton X-100 in PBS) for 30 minutes. RBPMS guinea pig antibody (ProSci, California) and HA antibody were used at 1 :4000, and 1 :500to label RGCs and target gene expressing cells, respectively. Floating retinas were incubated with primary antibodies overnight at 4°C and washed 3 times for 30 minutes each with PBS. Secondary antibodies (Alexa Fluor 647-goat anti-guinea pig, Cy3-goat anti-rat and Cy2-goat anti-mouse) were then applied (1 :200; Jackson ImmunoResearch, West Grove, Pennsylvania) and incubated for 1 hour at room temperature. Primary antibody for Anxa2 (ab178677, 1 :200) and Gsn (11644-2-AP, 1 :200;PA5-18605, 1 :100) and secondary antibody (1 :200 of Alexa488 donkey anti-rabbit or Alexa647- donkey anti-goat) were used to label mouse regenerating RGCs in retinal wholemounts or cultured hippocampal neurons. Retinas were again washed 3 times for 30 minutes each with PBS before a cover slip was attached with Fluoromount-G (Southernbiotech, Birmingham, Alabama). Images of immunostained wholemounts were acquired with a Keyence epifluorescence microscope 20x lens and Zeiss confocal microscope (LSM 880) with 40x oil lens and serial filters (BP520-550 for Alexa Fluor 488, GFP, Clover3 or Cy2, BP565-650 for Cy3, Dylight594, Dextran Texas Red and BP650-750 for Alexa Fluor 647). For RGC counting, 12-16 fields of 332 pmx332 pm area from peripheral, 8-12 fields of area from middle and 4-6 fields of area from central of each whole retina were sampled for imaging and the number of surviving RGCs was counted by RGCode software. The whole flat mounted retinas were imaged and stitched by a 20x lens and a Keyence fluorescence microscope (Itasca, BZ-X800). The percentage of RGC survival was calculated as the ratio of surviving RGC numbers in injured eyes compared to contralateral uninjured eyes. The investigators who counted the cells were blinded to the treatment of the samples.

[0219] Optic nerve semi-thin sections and quantification of surviving axons. The detailed procedure has been described previously. Briefly, transverse semi-thin (1 pm) sections of optic nerve were cut on an ultramicrotome (EM UC7, Leica, Wetzlar, Germany) from tissue collected 2 mm distal to the eye (about 1.5 mm distal to the crush site) and stained with 1% paraphenylenediamine (PPD) in methanol: isopropanol (1 :1). Whole optic nerves were imaged and stitched through a 100x lens with 2.5x zoom in by Keyence fluorescence microscopy. Eight areas of 21.4 pm x 29.1 pm from the entire optic nerve were cropped on average and counted with Axonet, a Fiji / lmageJ plugin. After counting all the images taken from a single nerve, the mean of the surviving axon number was calculated for each optic nerve, and compared to that in the contralateral control optic nerve to yield a percentage of axon survival value.

[0220] Kinesin beads transportation assay. Protein constructs and purification Plasmid for bacterial expression of KIF5B-eGFP (truncated kinesin 1, aminoacids 1-430) with eGFP and 6xHis tag at its C-terminus was a kind gift from Stefan Diez’s group. After expression in a vector backbone pET-17b in E. coli (strain BL21 (DE3), #230280, Altium International), the bacterial pellet was resuspended in lysis buffer (50 mM Na-phosphate buffer, pH 7.5, 30 mM imidazole, 5% glycerol, 300 mM KOI, 1 mM MgCh, 0.1% Tween-20, 10 mM p-mercaptoethanol, 0.1 mM ATP, benzonase (0.63 U / mL, #70664, Novagen) supplemented with 1x Protease inhibitor cocktail (#34044100, Roche Diagnostics GmBH)) and lysed by sonication and Igepal treatment. After spinning at 40 OOOx g for 1 h at 4 °C, supernatant containing expressed KIF5B-eGFP was loaded on NiNTA agarose resin HisTrap (#XF340049, Thermo Scientific) and incubated with the resin for 2 h at 4 °C while being slowly rotated. Resin-bound KIF5B-eGFP was washed with 10 column-volumes of wash buffer (50 mM Na-phosphate buffer, pH 7.5, 30 mM imidazole, 5%glycerol, 300 mM KCI, 1 mM MgCh, 0.1% Tween-20, 10 mM p-mercaptoethanol, 0.1 mM ATP) and eluted by increasing imidazole concentration to 300 mM. Eluted fractions containing KIF5B- eGFP protein (SDS-PAGE based) were pooled, aliquoted, snap frozen in liquid nitrogen, and stored at -80 °C. Human recombinant gelsolin was purchased from Cytoskeleton, Inc. (#HPG6), resuspended, and stored according to the manufacturer’s instructions.

[0221] Microtubule Assembly Porcine brains were obtained from a local abattoir and used within approximate 4 hrs of death. Porcine brain unlabeled tubulin was isolated using the high- molarity PIPES procedure. HILyte647 labeled tubulin was purchased from Cytoskeleton, Inc. (#TL670M) and mixed with unlabeled tubulin in a ratio of 1 :10, respectively. Taxol microtubules (GTP polymerized, then taxol-stabilized; stored and imaged in the presence of taxol) were polymerized from 4 mg / ml tubulin for 30 min at 37C in BRB80 buffer supplemented with 4 mM MgCb, 5% DMSO, and 1mM GTP (#NU-101 , Jena Bioscience). The polymerized microtubules were diluted in BRB80T buffer (80mM PIPES pH 6.9, 1 mM EGTA, 2 mM MgCh, supplemented with 10 mM paclitaxel (#17191 , Sigma)) and centrifuged for 30 min at 18000 x g in a Microfuge 18 Centrifuge (Beckman Coulter). After centrifugation, the pellet with microtubules was resuspended, kept in BRB80T at room temperature and used within two weeks.

[0222] Assembly of stabilized actin filaments Lyophilized unlabeled rabbit muscle actin was purchased from Cytoskeleton (#AKL99), resuspended, and stored following the manufacturer’s instructions. Actin filaments were polymerized by mixing the aliquoted unlabeled actin (400 mg / ml final concentration) in polymerization buffer (5 mM Tris-HCI pH 7.8, 0.2 mM CaCh, 50 mM KCI, 2 mM MgCl2, 1 mM ATP) with phalloidin-rhodamine (10 mM final concentration) for filament stabilization and labeling. Phalloidin-stabilized actin filaments were polymerized overnight at 4C and used within one month. For use in the assays, actin filaments were further diluted as described below.

[0223] Preparation of labeled G-actin Lyophilized unlabeled rabbit muscle actin and rhodamine-labeled rabbit muscle actin were purchased from Cytoskeleton (#ALK99 and #AR05- B), resuspended, mixed (10:1 ratio of unlabeledJabeled) and stored following the manufacturer’s instructions. G-actin was obtained by diluting the stored actin mixture to 400 mg / mL in general actin buffer (GAB; 5 mM TRIS-HCI (pH 8.0), 0.2 mM CaCI2, 0.2 mM ATP and 5 mM DTT) in which actin filaments depolymerized for 2 days at 4C. Any residual filaments were pelleted by spinning the actin solution at 200 OOOx g for 1 h. Supernatant containing only G-actin was separated and used within 2 weeks for experiments with dynamically growing actin filaments.

[0224] Coating of beads with KIF5B-GFP Magnetic beads of 2.8pm diameter coated with sheep anti-rabbit IgG (#11203D, Thermo Fisher Scientific) were washed in BRB80 buffer by repeated pelleting using a magnet and then incubated with 1 .7 mg / mL anti-GFP antibodies produced in rabbit (#ab290, Abeam) for 10 min on ice, followed by repeated washes with BRB80buffer. Anti-GFP coated beads were then incubated with 20pM KIF5B-eGFP on ice for 20 min (beads were gently stirred every 10 min). Final wash was performed by repeated pelleting and resuspending beads in BRB80T buffer.

[0225] Imaging - TIRF microscopy setup Total internal reflection fluorescent (Tl RF) microscopy experiments were performed on an inverted microscope (Nikon-Ti E, Nikon-Ti2 E) equipped with 60x NA 1.49 oil immersion objectives (Apo TIRF, Nikon) and sCMOS Hamamatsu Orca Flash 4.0 LT, or PRIME BSI (Hamamatsu Photonics, Teledyne Photometries) cameras. Fluorescently labeled proteins were visualized by switching between microscope filter cubes for GFP, rhodamine, and HiLyte647 channels or by using a quad band set (405 / 488 / 561 / 640). The microscopes were controlled with Nikon NIS Elements software (v5.20). All experiments were performed at room temperature over the course of multiple months. No data was excluded from the study.

[0226] Imaging - TIRF experimental chamber preparation For TIRF experiments, chambers were assembled by melting thin strips of parafilm in between two glass coverslips (18 and 24 mm, Marienfield High Precision) to form ~2.5 mm wide parallel flow channels. Prior to the chamber assembly, the coverslips were extensively cleaned in “piranha” solution (consisting of one part 30% hydrogen peroxide and 2.5 parts 95-97% sulfuric acid) and silanized with 0.05% dichlorodimethylsilane (DDS, #440272, Sigma) mixed in 350 ml trichloroethylene. The chambers were then incubated based on experiments performed as described below. All experiments were quantified by pooling data from multiple independent chambers. Chambers were never re-used for additional experiments.

[0227] Imaging - TIRF in vitro reconstitution assays KIF5B-eGFP-beads measurements Experimental chambers were assembled as described above and incubated with 20ug / mL anti- ptubulin antibodies (in PBS, #B3640, Sigma) for 5 min, followed by incubation with 20ug / mL anti-biotin antibodies (in PBS, #B3640, Sigma) for 5 min, and then the glass surface was blocked by incubation with 1% Pluronic (F127 in PBS, #P2443, Sigma) for at least 30 min. Pluronic was washed out by TIRF assay buffer (TAB: BRB80 buffer, 10 pM paclitaxel, 10 mM dithiothreitol (DTT), 20 mM D-glucose, 0.1% Tween 20, 1 mM Mg- ATP, 0.5 mg / ml casein, 40 mM NaCI, 0.1 mM CaCh, supplemented with 0.22 mg / mL glucose oxidase and 0.02 mg / mL catalase). HiLyte647-labeled taxol-stabilized polymerized microtubules were introduced into the chamber and allowed to attach for 1 min; unattached microtubules were subsequently washed out by TAB. KIF5B-eGFP beads (in TAB) were flushed into the chamber and the “Without F-actin” condition was imaged. The “F-actin” condition was achieved in two different ways. Either 200 mg / mL phalloidin-stabilized rhodamine-labeled actin filaments (in TAB) were introduced into the chamber to the KIF5B-eGFP beads and the condition was imaged, or 200 mg / mL G-actin was added to the chamber and allowed polymerize into F-actin, whereas the “F-actin” condition was considered to form after 1 min of polymerization. Both approaches showed similar results, hencethe data were pooled. The “F-actin + Gsn” condition was also approached in two ways. Either 100 nM Gelsolin was flushed into the chamber with F-actin already present (in TAB) and the condition imaged immediately afterward, or a drop of 100 nM Gelsolin was placed at the end of the experimental chamber (with F-actin already present) and allowed to diffuse into the field of view. Both approaches showed similar results, hence the data were pooled.

[0228] KIF5B-eGFP single molecules measurements Experimental chambers were prepared as described above for KIF5B-eGFP-beads measurements (incubated with anti-ptubulin antibodies, anti-biotin antibodies, and 1% Pluronic). Hil_yte647-labeled taxol-stabilized polymerized microtubules were introduced into the chamber and allowed to attach for 1 min, then the unattached microtubules were washed out by TAB. 4nM KIF5B-eGFP in TAB was flushed in and imaged for 30 sec with no delay for “Without F-actin” condition. Subsequently, 200 mg / mL phalloidin-stabilized rhodamine-labeled actin filaments were introduced into the chamber, while keeping the stable concentration of KIF5B-eGFP, and the condition “with F- actin” was imaged for 30 sec with no delay.

[0229] Image Analysis - TIRF in vitro reconstitution assays Microscopy data were analyzed using ImageJ 2.9.0 / 1 ,53t(79), utilizing also Imaged plugin “TrackMate”. Graphs were generated in Matlab R2023b, and R4.4.1 was used for statistical analysis.

[0230] Count and velocities of KIF5B-eGFP-beads Number of the “walking” vs “non-walking" beads was counted manually, using “Maximum Intensity Projection” function in ImageJ. Only beads encountering microtubules throughout the video were taken into account. “Walking” beads were those that walked along a microtubule at least for a distance larger than the bead diameter. One dot in the percentage graph represents one independent experimental chamber. Velocities of the KIF5B-eGFP beads in each condition were measured by ImageJ plugin “TrackMate”, with “LogG detector” algorithm for particle detection and “Simple LAP detector” algorithm for trajectory tracking. The detected tracks were checked manually one by one and cleared from the ones not encountering a microtubule and from beads that remained stationary at the end of a microtubule or diffused away from the microtubules.

[0231] Count and velocities of KIF5B-eGFP single molecules Kymographs were generated by manually drawing a line along the microtubule lattice and using “KymographBuilder” plugin in ImageJ. Traces of kinesins visible in kymographs were detected and marked manually. To exclude background signal, the intensity threshold was set according to the kymograph generated from the background area. Fluorescent signals lasting for one pixel in time axis were not taken into consideration. The velocities of single molecules were calculated from run-length [pm] over run-time [s]; one data point in the velocity graph represents one kinesin molecule. As non-walking kinesins were considered the ones with run-length below 2 pixels, one data point in the percentage graph represents results from one microtubule. For each condition, two independent experimental chambers were analyzed.References

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[0378] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0379] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e. , any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0380] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

Claims

WHAT IS CLAIMED IS:1 . A method of treating an axonopathy in a mammalian subject in need thereof, the method comprising:administering an effective dose of an agent that enhances F-actin depolymerization or inhibits actin polymerization, thereby treating the axonopathy.

2. The method of claim 1 , wherein the agent is a small molecule that enhances actin depolymerization or inhibits actin polymerization.

3. The method of claim 2, wherein the agent is selected from Latrunculin B, Latrunculin A, Cytochalasin D, Cytochalasin B, Blebbistatin, CK-666, and BMS-754807.

4. The method of claim 2 or claim 3, wherein the small molecule is administered in combination with an AAV vector.

5. The method of claim 1 or claim 4, wherein the agent comprises an AAV vector that expresses a gene that targets actin depolymerization.

6. The method of claim 5, wherein the gene is operably linked to a neuron specific promoter.

7. The method of any of claims 4-6, wherein the gene is one or a combination of gelsolin (Gsn), Anxa2, destrin (Dstn), cofilin (Cfl1), Macrophage-capping protein (CapG), Advillin / Avil, and Adseverin / Scin.

8. The method of any of claims 4-6, wherein the gene is one or a combination of Gsn, Anxa2, Dstn, CfH and CapG.

9. The method of any of claims 4-6, wherein the gene is human Gsn.

10. The method of claim 1 or claim 4, wherein the agent is an AAV vector that disrupts expression of a gene that enhances actin polymerization.11 . The method of claim 10, wherein the gene is profilin 1 (Pfn1 ), Arp2 / 3 (Actin Related Protein 2 / 3), or tropomyosin (Tpm1 ,3).

12. The method of any of the previous claims, wherein the administration reduces or ameliorates degeneration of axons and / or soma of retinal ganglion cells (RGCs).

13. The method of any of the previous claims, wherein the administration promotes axon regeneration.

14. The method of any of the previous claims, wherein the axonopathy is associated with amyotrophic lateral sclerosis (ALS); glaucoma and other optic nerve diseases; hereditary spastic paraplegia (HSP); traumatic brain injury; spinal cord injury; or neuronal injury induced by a toxic agent.

15. The method of any of the previous claims, wherein the composition is administered intravitreally , topically, or systemically.

16. The method of any of the previous claims, wherein the agent is delivered by injection-mediated retrograde RGC targeting.

17. The method of any of the previous claims, wherein the composition administered systemically.

18. The method of any of the previous claims, wherein the optic neuropathy is retinal ganglion cell degeneration, including glaucoma, optic neuritis, optic nervetraumatic injury and other optic nerve-related diseases.

19. The method of any of the previous claims, wherein the optic neuropathy is glaucoma.

20. The method of any of the previous claims, wherein the subject is human.21 . The method of any of the previous claims, wherein administration is performed after a determination of elevated IOP.

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