Methods for reducing degeneration of retinal ganglion cells - Patents.com

Abstract

A method for reducing degeneration of retinal ganglion cells in a subject is provided, the method comprising administering to the subject a composition that increases activity of calcium-calmodulin-dependent kinase (CaMK) or cyclic AMP response element binding protein (CREB), the composition comprising CaMK or CREB, or a polynucleotide encoding CaMK or CREB. Also provided is a method for treating vision loss in a subject, the method comprising administering to the subject a composition that increases activity of CaMK or CREB, the composition comprising CaMK or CREB, or a polynucleotide encoding CaMK or CREB. Also provided is a pharmaceutical composition comprising a polynucleotide and a vector, the polynucleotide comprising a retinal ganglion cell promoter and encoding CaMK or CREB.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is an international application filed pursuant to the Patent Cooperation Treaty, and claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 154,432, filed February 26, 2021, and U.S. Provisional Patent Application No. 63 / 177,230, filed April 20, 2021, the entire contents of which are incorporated herein by reference.

[0002] Government Rights Statement This disclosure was made with Government support under Grant No. EY028921 awarded by the National Institutes of Health. The Government has certain rights in this disclosure.

[0003] Sequence Listing This application contains a sequence listing, created on February 3, 2022, an ASCII file named H2257236.txt, 119.4 KB in size. The file is incorporated by reference in its entirety into this application. The sequence listing submitted herein is identical to the sequence listing forming part of the international application. [Background technology]

[0004] Visual information is transmitted from the eye to higher processing centers in the brain via the optic nerve, a bundle of axons emerging from the output neurons of the retina: the retinal ganglion cells (RGCs). Loss of RGCs is the main cause of visual impairment and blindness in various pathological conditions. Some conditions damage the cell bodies of RGCs (including excitotoxicity and retinal ischemia), whereas others damage the axons of RGCs (including optic nerve section, optic nerve compression, papilledema, and glaucoma). Indeed, glaucoma is the leading cause of irreversible visual impairment worldwide.

[0005] A barrier to vision recovery after RGC injury is inducing axonal regeneration. There is an unmet clinical need to develop effective neuroprotective approaches to preserve RGCs and their function. Early neuroprotective treatment is needed to prevent acute and massive RGC loss in individuals at high risk of retinal ischemia and excitotoxicity. RGC neuroprotective interventions are also needed for the significant proportion of glaucoma patients who still progress to blindness despite treatment to reduce intraocular pressure. For patients with traumatic optic nerve injury, promoting RGC survival may aid efforts to regenerate retina-brain connections.

[0006] The present disclosure is directed to overcoming these and other deficiencies in the art. Summary of the Invention

[0007] Therefore, it is desirable to identify therapeutic approaches for vision disorders associated with RGC degeneration or loss, such as identifying targets that are effective in protecting RGC cell bodies and axons from various insults in a wide range of pathological spectrum.The present disclosure includes the identification of pharmaceutical compositions that increase the activity of Ca2+ / calmodulin-dependent protein kinase and their use in treating RGC degeneration or loss and in treating vision disorders, which is surprising in light of existing literature showing that CaMK activity promotes excitotoxic cell death, including RGCs.The present disclosure also includes the identification of pharmaceutical compositions that increase the activity of cyclic AMP response element binding protein (CREB), for use in treating RGC degeneration or loss and in treating vision disorders.

[0008] In one aspect, a method for reducing degeneration of retinal ganglion cells in a subject is provided herein, the method comprises administering to the subject a composition that increases the activity of CaMK, the composition comprises CaMK or a polynucleotide that encodes CaMK. In one example, the composition further comprises a vector, for example, a viral vector. The viral vector can comprise an adeno-associated viral vector (AAV).

[0009] In another example, the CaMK is selected from one or more of CaMKI, CaMKII, and CaMKIV. In a further example, the CaMK is selected from one or more of CaMKIIα, CaMKIIβ, CaMKIIγ, and CaMKIIδ. In yet another example, the CaMK is constitutively active. In yet another example, the CaMKII is selected from one or both of CaMKIIα containing a T286D substitution and CaMKIIβ containing a T287D substitution.

[0010] In another example, the composition comprises a polynucleotide encoding CaMK. In a further example, the polynucleotide further comprises a retinal ganglion cell promoter. In yet another example, the retinal ganglion cell promoter comprises a gamma-synuclein promoter, or a synapsin 1 promoter, or a neurofilament heavy chain promoter, or a Thy-1 cell surface antigen promoter. In yet another example, the retinal ganglion cell promoter comprises a gamma-synuclein promoter. In yet another example, the composition comprises CaMK.

[0011] In another example, the administration is selected from intraocular administration and systemic administration. In yet another example, the subject has or is at risk of having one or more of glaucoma, diabetic retinopathy, retinal ischemia, and optic nerve damage. In yet another example, preventing degeneration includes preventing the loss of one or both of retinal ganglion cell bodies and retinal ganglion cell axons.

[0012] In another aspect, a method for treating vision loss in a subject is provided, the method comprises administering to the subject a composition that increases the activity of CaMK, the composition comprises CaMK or a polynucleotide that encodes CaMK. In one example, the composition further comprises a vector, for example, a viral vector. The viral vector can comprise AAV.

[0013] In another example, the CaMK is selected from one or more of CaMKI, CaMKII, and CaMKIV. In a further example, the CaMK is selected from one or more of CaMKIIα, CaMKIIβ, CaMKIIγ, and CaMKIIδ. In yet another example, the CaMK is constitutively active. In yet another example, the CaMKII is selected from one or both of CaMKIIα containing a T286D substitution and CaMKIIβ containing a T287D substitution.

[0014] In another example, the composition comprises a polynucleotide encoding CaMK. In a further example, the polynucleotide further comprises a retinal ganglion cell promoter. In yet another example, the retinal ganglion cell promoter comprises a gamma-synuclein promoter, or a synapsin 1 promoter, or a neurofilament heavy chain promoter, or a Thy-1 cell surface antigen promoter. In yet another example, the retinal ganglion cell promoter comprises a gamma-synuclein promoter. In yet another example, the composition comprises CaMK.

[0015] In another example, the administration is selected from intraocular administration and systemic administration.In yet another example, the subject has or is at risk of having one or more of glaucoma, diabetic retinopathy, retinal ischemia, and optic nerve damage.In yet another example, preventing degeneration includes preventing the loss of one or both of retinal ganglion cell bodies and retinal ganglion cell axons.In yet another example, treating includes preventing vision loss.

[0016] In yet another aspect, a pharmaceutical composition is provided that includes a polynucleotide and a vector, the polynucleotide comprising a retinal ganglion cell promoter and encoding CaMK. In yet another example, the retinal ganglion cell promoter comprises a gamma-synuclein promoter, or a synapsin 1 promoter, or a neurofilament heavy chain promoter, or a Thy-1 cell surface antigen promoter. In one example, the retinal ganglion cell promoter comprises a gamma-synuclein promoter. In another example, the vector comprises a viral vector. In yet another example, the vector comprises an adeno-associated vector. In a further example, the CaMK is selected from one or more of CaMKI, CaMKII, and CaMKIV. In yet another example, the CaMK is selected from one or more of CaMKIIα, CaMKIIβ, CaMKIIγ, and CaMKIIδ.

[0017] In another example, the CaMK is constitutively active. In yet another example, the CaMKII is selected from one or both of CaMKIIα that includes a T286D substitution and CaMKIIβ that includes a T287D substitution.

[0018] In yet another aspect, a method for reducing degeneration of retinal ganglion cells in a subject is provided, the method comprising administering to the subject a composition that increases the activity of CREB, the composition comprising CREB or a polynucleotide encoding CREB. In one example, the composition further comprises a vector, e.g., a viral vector. The viral vector can comprise an adeno-associated viral vector (AAV).

[0019] In another example, the CREB is constitutively active. In yet another example, the CREB comprises VP-16 CREB. In yet another example, the composition comprises a polynucleotide encoding CREB. In a further example, the polynucleotide further comprises a retinal ganglion cell promoter. In yet another example, the retinal ganglion cell promoter comprises a gamma-synuclein promoter, or a synapsin 1 promoter, or a neurofilament heavy chain promoter, or a Thy-1 cell surface antigen promoter. In yet another example, the retinal ganglion cell promoter comprises a gamma-synuclein promoter. In yet another example, the composition comprises CREB.

[0020] In another example, the administration is selected from intraocular administration and systemic administration. In yet another example, the subject has or is at risk of having one or more of glaucoma, diabetic retinopathy, retinal ischemia, and optic nerve damage. In yet another example, preventing degeneration includes preventing the loss of one or both of retinal ganglion cell bodies and retinal ganglion cell axons.

[0021] In another aspect, a method of treating vision loss in a subject is provided, the method comprising administering to the subject a composition that increases the activity of CREB, the composition comprising CREB or a polynucleotide encoding CREB. In one example, the composition further comprises a vector, e.g., a viral vector. The viral vector can comprise AAV.

[0022] In another example, the CREB is constitutively active. In yet another example, the CREB comprises VP-16 CREB. In yet another example, the composition comprises a polynucleotide encoding CREB. In a further example, the polynucleotide further comprises a retinal ganglion cell promoter. In yet another example, the retinal ganglion cell promoter comprises a gamma-synuclein promoter, or a synapsin 1 promoter, or a neurofilament heavy chain promoter, or a Thy-1 cell surface antigen promoter. In yet another example, the retinal ganglion cell promoter comprises a gamma-synuclein promoter. In yet another example, the composition comprises CREB.

[0023] In another example, the composition comprises a polynucleotide encoding CREB. In a further example, the polynucleotide further comprises a retinal ganglion cell promoter. In yet another example, the retinal ganglion cell promoter comprises a gamma-synuclein promoter, or a synapsin 1 promoter, or a neurofilament heavy chain promoter, or a Thy-1 cell surface antigen promoter. In yet another example, the retinal ganglion cell promoter comprises a gamma-synuclein promoter. In yet another example, the composition comprises CREB.

[0024] In another example, the administration is selected from intraocular administration and systemic administration.In yet another example, the subject has or is at risk of having one or more of glaucoma, diabetic retinopathy, retinal ischemia, and optic nerve damage.In yet another example, preventing degeneration includes preventing the loss of one or both of retinal ganglion cell bodies and retinal ganglion cell axons.In yet another example, treating includes preventing vision loss.

[0025] In yet another aspect, a pharmaceutical composition is provided comprising a polynucleotide and a vector, wherein the polynucleotide comprises a retinal ganglion cell promoter and encodes CREB. In yet another example, the retinal ganglion cell promoter comprises a gamma-synuclein promoter, or a synapsin 1 promoter, or a neurofilament heavy chain promoter, or a Thy-1 cell surface antigen promoter. In one example, the retinal ganglion cell promoter comprises a gamma-synuclein promoter. In another example, the vector comprises a viral vector. In yet another example, the CREB is constitutively active. In yet another example, the CREB comprises VP-16 CREB. [Brief description of the drawings]

[0026] These and other features, aspects, and advantages of the present disclosure will be better understood when the following detailed description is read in conjunction with the accompanying drawings.

[0027] [Fig. 1A-1Y]According to an embodiment of the present disclosure, examples of excitotoxicity and optic nerve injury resulting in loss of CaMKII activity in RGCs are disclosed. (A-F) Confocal images of retinal whole mounts showing CaMKII phosphorylation (CaMKIIα at T286 + CaMKIIβ at T287) in Tuj1-labeled RGCs 2 hours after PBS (A-C) or NMDA (D-F) injection. Arrows, Tuj1+RGCs maintain (A-C) or lose (D-F) CaMKII activity. Scale bar, 20 μm. (GH) Quantification of CaMKII phosphorylation in RGCs after excitotoxic injury. (G) Number of total Tuj1+RGCs and pCaMKII+ / Tuj1+RGCs 2 hours after injection of PBS control or NMDA. Data are presented as mean ± standard deviation (n=5 retinas per group). (H) Percentage of pCaMKII+ / Tuj1+RGCs 2 hours after injection of PBS control or NMDA. Data are presented as mean ± standard deviation (n = 5 retinas per group). Unpaired t test, *P < 0.0001. (I-T) Confocal images of retinal whole mounts showing CaMKII phosphorylation (CaMKIIα at T286 + CaMKIIβ at T287) in Tuj1-labeled RGCs, (I-K) uninjured or 5 (L-N), 7 (O-Q), and 9 (R-T) days (dpc) after optic nerve crush. Arrows, Tuj1+ RGCs that have lost CaMKII activity (L-T). Scale bar, 20 μm. (U-V) Quantification of CaMKII phosphorylation in RGCs after optic nerve injury. (U) Number of total Tuj1+ RGCs and pCaMKII+ / Tuj1+ RGCs in uninjured retinas and 5, 7, and 9 days after crush. Data are presented as mean ± standard deviation (n = 6 retinas per group). (V) Percentage of pCaMKII+ / Tuj1+RGCs in intact retinas and retinas 5, 7, and 9 days after crush. Data are presented as mean ± standard deviation (n=6 retinas per group). One-way ANOVA with Tukey's multiple comparison test, F: 36.22, R2: 0.8445, *P<0.0001. (W-X) Confocal images of retinal whole mounts showing viable RGCs labeled by Tuj1 immunoreactivity 7 days after daily injections of PBS (W) or AIP (X). Scale bar, 40 μm. (Y) Quantification of RGC survival.The numbers of RGCs (left Y-axis) and the percentage of RGCs relative to those in intact retinas (right Y-axis) are presented. Data are presented as mean ± standard deviation (n = 5 retinas per group). Unpaired t-test, *P < 0.0001. [Figure 2A-2H] According to an embodiment of the present disclosure, examples of excitotoxicity and optic nerve injury leading to loss of CaMKII activity in RGCs are disclosed. (A-F) Confocal images of retinal whole mounts showing pCaMKII immunoreactivity in Tuj1-labeled RGCs without (B) or with (E) blocking of peptide phosphorylated at Thr286 for CaMKIIα (Thr287 for CaMKIIβ). Scale bar, 20 μm. (G) Western blot showing pCaMKII and GAPDH in purified RGCs from uninjured retina and injured retina 2 hours after NMDA injury. (H) Relative pCaMKII levels in purified RGCs from uninjured retina and injured retina 2 hours after NMDA injury. Data are presented as mean ± standard deviation (n=3 blots). Unpaired t-test, *P<0.0001. [Fig. 3A-3T]According to an embodiment of the present disclosure, an example of CaMKII reactivation protecting RGCs from excitotoxicity and optic nerve damage is disclosed. (A-D) Confocal images of retinal whole mounts showing surviving RGCs labeled by Tuj1 immunoreactivity 7 days after NMDA injection in control (AAV-EBFP) or AAV-CaMKIIα WT, AAV-CaMKIIα K42R, and AAV-CaMKIIα T286D treated eyes. Scale bar, 40 μm. (E) Quantification of RGC survival after treatment with CaMKIIα variants 7 days after NMDA injection. Expressed as number of RGCs (left Y-axis) and percentage of RGCs relative to those in uninjured retinas (right Y-axis). Data are presented as mean ± standard deviation (n=5 retinas per group). One-way ANOVA with Tukey's multiple comparison test, F: 515.5, R2: 0.9898, *P<0.0001. (F-I) Confocal images of retinal whole mounts showing surviving RGCs labeled by Tuj1 immunoreactivity 7 days after NMDA injection in control (AAV-EBFP) or AAV-CaMKIIβ WT, AAV-CaMKIIβ K43R, and AAV-CaMKIIβ T287D treated eyes. Scale bar, 40 μm. (J) Quantification of RGC survival after treatment with CaMKIIβ variants 7 days after NMDA injection. Expressed as number of RGCs (left Y-axis) and percentage of RGCs relative to those in uninjured retinas (right Y-axis). Data are presented as mean ± standard deviation (n = 5 retinas per group). One-way ANOVA with Tukey's multiple comparison test, F: 423.3, R2: 0.9876, *P < 0.0001. (K-N) Confocal images of retinal whole mounts showing surviving RGCs labeled by Tuj1 immunoreactivity 2 weeks after optic nerve crush in control (AAV-EBFP) or AAV-CaMKIIα WT, AAV-CaMKIIα K42R, and AAV-CaMKIIα T286D treated eyes. Scale bar, 40 μm. (O) Quantification of RGC survival after treatment with CaMKIIα variants 2 weeks after optic nerve crush. Expressed as number of RGCs (left Y-axis) and percentage of RGCs relative to those in uninjured retinas (right Y-axis). Data are presented as mean ± standard deviation (n = 5 retinas per group).One-way ANOVA with Tukey's multiple comparison test, F: 379.0, R2: 0.9861, *P<0.0001. (P-S) Confocal images of retinal whole mounts showing surviving RGCs labeled by Tuj1 immunoreactivity 2 weeks after optic nerve crush in control (AAV-EBFP) or AAV-CaMKIIβ WT, AAV-CaMKIIβ K43R, and AAV-CaMKIIβ T287D treated eyes. Scale bar, 40 μm. (T) Quantification of RGC survival after treatment with CaMKIIβ variants 2 weeks after optic nerve crush. Represented as number of RGCs (left Y-axis) and percentage of RGCs relative to those in uninjured retinas (right Y-axis). Data are presented as mean ± standard deviation (n = 5 retinas per group). One-way ANOVA with Tukey's multiple comparison test, F: 361.3, R2: 0.9855, *P<0.0001. [Figures 4A-4K] According to an embodiment of the present disclosure, an example of AAV-mediated gene transfer in RGCs is disclosed. (A-C) Confocal images of retinal whole mounts showing GFP expression in Tuj1+ RGCs 2 weeks after intravitreal injection of AAV-GFP. Scale bar, 40 μm. (D) Transduction efficiency is expressed as the percentage of GFP+ RGCs in total RGCs. Data are presented as mean ± standard deviation (n=5 retinas). (E-J) Confocal images of retinal whole mounts showing pan-CaMKII levels in RGCs 2 weeks after injection in control (AAV-EBFP) or AAV-CaMKIIα T286D treated eyes. Scale bar, 20 μm. (K) Quantification of pan-CaMKII intensity in RGCs. Data are presented as mean ± standard deviation (n=3 retinas per group). Unpaired t-test, *P=0.0033. [Figures 5A-5O]According to an embodiment of the present disclosure, we disclose examples of the performance of more CaMKII variants as well as the RGC-specific promoter mSncg in RGC protection. (A-E) Confocal images of retinal whole mounts showing surviving RGCs labeled by Tuj1 immunoreactivity 7 days after NMDA injection in control (AAV-EBFP) or AAV-CaMKIIα K42D, AAV-CaMKIIα T286A, AAV-CaMKIIα T286D / T305A / T306A, and CaMKIIα T286D / T305D / T306D treated eyes. Scale bar, 40 μm. (F) Quantification of RGC survival after treatment with CaMKIIα variants 7 days after NMDA injection. Expressed as number of RGCs (left Y-axis) and percentage of RGCs (right Y-axis) of those in uninjured retinas. Data are presented as mean ± standard deviation (n=5 retinas per group). One-way ANOVA with Tukey's multiple comparison test, F: 761.4, R2: 0.9935, *P=0.0001. (GH) Confocal images of retinal whole mounts showing surviving RGCs labeled by Tuj1 immunoreactivity 7 days after NMDA injection in control (AAV-mSncg-EBFP) or AAV-mSncg-CaMKIIα T286D treated eyes. Scale bar, 40 μm. (I) Quantification of RGC survival 7 days after NMDA injection. Expressed as number of RGCs (left Y-axis) and percentage of RGCs relative to those in uninjured retinas (right Y-axis). Data are presented as mean ± standard deviation (n=4 retinas per group). Unpaired t-test, *P<0.0001. (J-N) Confocal images of retinal whole mounts showing viable RGCs labeled by Tuj1 immunoreactivity 7 days after NMDA injection in control (AAV-EBFP) or AAV-CaMKIIβ K43D, AAV-CaMKIIβ T287A, CaMKIIβ T287D / T306A / T307A, and CaMKIIβ T287D / T306D / T307D treated eyes. Scale bar, 40 μm. (O) Quantification of RGC survival after treatment with CaMKIIβ variants 7 days after NMDA injection. Represented as number of RGCs (left Y-axis) and percentage of RGCs relative to those in uninjured retinas (right Y-axis). Data are presented as mean ± standard deviation (n=5 retinas per group).One-way ANOVA with Tukey's multiple comparison test, F: 579.0, R2: 0.9914, *P = 0.0002. [Figures 6A-6O] According to an embodiment of the present disclosure, an example of reactivation of CaMKII providing long-term RGC protection after injury and after excitotoxicity or axonal injury is disclosed. (A-B) Confocal images of retinal whole mounts showing surviving RGCs labeled by Tuj1 immunoreactivity 2 weeks after optic nerve crush after control (AAV-EBFP) or AAV-CaMKIIα T286D injury treatment. Scale bar, 40 μm. (C) Quantification of RGC survival 2 weeks after optic nerve crush. Expressed as number of RGCs (left Y-axis) and percentage of RGCs relative to those in uninjured retinas (right Y-axis). Data are presented as mean ± standard deviation (n=5 retinas per group). Unpaired t-test, *P<0.0001. (D-G) Confocal images of retinal whole mounts showing surviving RGCs labeled by Tuj1 immunoreactivity 2 and 12 months after NMDA injection in control (AAV-EBFP) and AAV-CaMKIIβ T287D treated eyes. Scale bar, 40 μm. (H) Quantification of RGC survival 2 and 12 months after NMDA injection. Expressed as number of RGCs (left Y-axis) and percentage of RGCs relative to those in uninjured retinas (right Y-axis). Data are presented as mean ± standard deviation (n = 4 retinas per group). One-way ANOVA with Tukey's multiple comparison test, F: 1370, R2: 0.9971, *P < 0.0001. (I-N) Confocal images of retinal whole mounts showing surviving RGCs labeled by Tuj1 immunoreactivity 1, 2, and 6 months after optic nerve crush in control (AAV-EBFP) or AAV-CaMKIIβ T287D-treated eyes. Scale bar, 40 μm. (O) Quantification of RGC survival 1, 2, and 6 months after optic nerve injury. The numbers of RGCs (left Y-axis) and the percentage of RGCs relative to those in intact retinas (right Y-axis) are presented. Data are presented as mean ± standard deviation (n = 4 retinas per group). One-way ANOVA with Tukey's multiple comparison test, F: 523.2, R2: 0.9932, *P < 0.0001. [Figures 7A-7Z]According to an embodiment of the present disclosure, an example is disclosed in which CREB acts downstream of CaMKII to protect RGCs from excitotoxicity and optic nerve injury. (A-C) Confocal images of retinal whole mounts showing CREB phosphorylation in RGCs from uninjured eyes (A) and 2 hours after NMDA injection in control (AAV-EBFP) (B) or AAV-CaMKIIα T286D (C) treated eyes. Arrows, Tuj1+RGCs that maintained (A) or lost (B) CREB activity. Lost CREB activity was restored after treatment with CaMKIIα T286D (C). Scale bar, 20 μm. (D-E) Quantification of CREB phosphorylation in RGCs 2 hours after NMDA-induced excitotoxic injury. (D) Number of total Tuj1+RGCs and pCREB+ / Tuj1+RGCs in uninjured or NMDA injured eyes. Data are presented as mean ± standard deviation (n=6 retinas per group). (E) Percentage of pCREB+ / Tuj1+ RGCs in uninjured or NMDA-injured eyes. Data are presented as mean ± standard deviation (n = 6 retinas per group). One-way ANOVA with Tukey's multiple comparison test, F: 6139, R2: 0.9988, *P < 0.0001. (F-G) Confocal images of retinal whole mounts showing surviving RGCs labeled by Tuj1 immunoreactivity 7 days after NMDA injection in AAV-CaMKIIα T286D + control (AAV-EBFP) or AAV-CaMKIIα T286D + AAV-A-CREB treated eyes. Scale bar, 40 μm. (H) Quantification of RGC survival. Expressed as number of RGCs (left Y-axis) and percentage of RGCs relative to those in uninjured retinas (right Y-axis). Data are presented as mean ± standard deviation (n = 5 retinas per group). Unpaired t-test, *P<0.0001. (I-J) Confocal images of retinal whole mounts showing viable RGCs labeled by Tuj1 immunoreactivity 7 days after NMDA injection in control (AAV-EBFP) or AAV-VP16-CREB treated eyes. Scale bar, 40 μm. (K) Quantification of RGC survival. Expressed as number of RGCs (left Y-axis) and percentage of RGCs relative to those in uninjured retinas (right Y-axis). Data are presented as mean ± standard deviation (n=5 retinas per group). Unpaired t-test, *P<0.0001.(L-R) Confocal images of retinal whole mounts showing CREB phosphorylation in RGCs from uninjured eyes (L) and in control (AAV-EBFP) (M-O) or AAV-CaMKIIα T286D (P-R) treated eyes 5, 7, and 9 days after optic nerve crush. Arrows, Tuj1+RGCs losing CREB activity (M-O). Scale bar, 20 μm. (S-T) Quantification of CREB phosphorylation in RGCs after optic nerve injury. (S) Number of total Tuj1+RGCs and pCREB+ / Tuj1+RGCs in uninjured and injured retinas 5, 7, and 9 days after crush. Data are presented as mean ± standard deviation (n=6 retinas per group). (T) Percentage of pCREB+ / Tuj1+RGCs in uninjured and injured retinas 5, 7, and 9 days after crush. Data are presented as mean ± standard deviation (n = 6 retinas per group). One-way ANOVA with Tukey's multiple comparison test, F: 89.58, R2: 0.9389, *P < 0.0001. (U-V) Confocal images of retinal whole mounts showing surviving RGCs labeled by Tuj1 immunoreactivity 2 weeks after optic nerve crush in AAV-CaMKIIα T286D + control (AAV-EBFP) or AAV-CaMKIIα T286D + AAV-A-CREB treated eyes. Scale bar, 40 μm. (W) Quantification of RGC survival. Expressed as number of RGCs (left Y-axis) and percentage of RGCs relative to those in uninjured retinas (right Y-axis). Data are presented as mean ± standard deviation (n = 5 retinas per group). Unpaired t-test, *P < 0.0001. (X-Y) Confocal images of retinal whole mounts showing surviving RGCs labeled by Tuj1 immunoreactivity 2 weeks after optic nerve crush in control (AAV-EBFP) or AAV-VP16-CREB treated eyes. Scale bar, 40 μm. (Z) Quantification of RGC survival. Expressed as number of RGCs (left Y-axis) and percentage of RGCs relative to those in uninjured retinas (right Y-axis). Data are presented as mean ± standard deviation (n=5 retinas per group). Unpaired t-test, *P<0.0001. [Figure 8A-8V]According to an embodiment of the present disclosure, an example of a signaling mechanism downstream of CaMKII in RGC protection is disclosed. (A-B) Confocal images of retinal whole mounts showing CREB phosphorylation in RGCs 2 hours after NMDA injection in AAV-CaMKIIα T286D+control (AAV-EBFP) or AAV-CaMKIIα T286D+AAV-A-CREB treated eyes. Arrows, Tuj1+RGCs that have lost CREB activity. Scale bar, 20 μm. (C-D) Quantification of CREB phosphorylation in RGCs after excitotoxic injury. (C) Number of total Tuj1+RGCs and pCREB+ / Tuj1+RGCs 2 hours after NMDA injection. Data are presented as mean ± standard deviation (n=4 retinas per group). (D) Percentage of pCREB+ / Tuj1+RGCs 2 hours after NMDA injection. Data are presented as mean ± standard deviation (n=4 retinas per group). Unpaired t-test, *P<0.0001. (E-F) Confocal images of retinal whole mounts showing CREB phosphorylation in RGCs 2 h after NMDA injection in control (AAV-EBFP) or AAV-VP16-CREB treated eyes. Arrows, Tuj1+RGCs that have lost CREB activity. Scale bar, 20 μm. (G-H) Quantification of CREB phosphorylation in RGCs after excitotoxic injury. (G) Number of total Tuj1+RGCs and pCREB+ / Tuj1+RGCs 2 h after NMDA injection. Data are presented as mean ± standard deviation (n=3 retinas per group). (H) Percentage of pCREB+ / Tuj1+RGCs 2 h after NMDA injection. Data are presented as mean ± standard deviation (n=3 retinas per group). Unpaired t-test, *P=0.0003. (I) Confocal images of retinal whole mounts showing TrkB phosphorylation in RGCs 2 hours after NMDA injection in control (AAV-EBFP) or AAV-CaMKIIα T286D treated eyes. Scale bar, 20 μm. (J) Quantification of pTrkB intensity in RGCs. Data are presented as mean ± standard deviation (n = 3 retinas per group). One-way ANOVA with Tukey's multiple comparison test, F: 19.26, R2: 0.8652, *P = 0.0124.(K-L) Confocal images of retinal whole mounts showing CREB phosphorylation in RGCs 5 days after optic nerve crush in AAV-CaMKIIα T286D+control (AAV-EBFP) or AAV-CaMKIIα T286D+AAV-A-CREB treated eyes. Arrows, Tuj1+RGCs that have lost CREB activity. Scale bar, 20 μm. (M-N) Quantification of CREB phosphorylation in RGCs 5 days after nerve injury. (M) Number of total Tuj1+RGCs and pCREB+ / Tuj1+RGCs 5 days after optic nerve crush. Data are presented as mean ± standard deviation (n=4 retinas per group). (N) Percentage of pCREB+ / Tuj1+RGCs 5 days after optic nerve crush. Data are presented as mean ± standard deviation (n=4 retinas per group). Unpaired t-test, *P<0.0001. (O-P) Confocal images of retinal whole mounts showing CREB phosphorylation in RGCs 5 days after optic nerve crush in control (AAV-EBFP) or AAV-VP16-CREB treated eyes. Arrows, Tuj1+RGCs that have lost CREB activity. Scale bar, 20 μm. (Q-R) Quantification of CREB phosphorylation in RGCs 5 days after nerve injury. (Q) Number of total Tuj1+RGCs and pCREB+ / Tuj1+RGCs 5 days after optic nerve crush. Data are presented as mean ± standard deviation (n=3 retinas per group). (R) Percentage of pCREB+ / Tuj1+RGCs 5 days after optic nerve crush. Data are presented as mean ± standard deviation (n=3 retinas per group). Unpaired t-test, *P=0.0002. (S) Confocal images of retinal whole mounts showing DLK staining in RGCs from uninjured eyes, and in RGCs 3 days after optic nerve crush in control (AAV-EBFP) or AAV-CaMKIIα T286D-treated eyes. Scale bar, 20 μm. (T) Quantification of DLK intensity in RGCs. Data are presented as mean ± standard deviation (n = 3 retinas per group). One-way ANOVA with Tukey's multiple comparison test, F: 192.3, R2: 0.9846, ns (not significant, P = 0.18).(U) Confocal images of retinal whole mounts showing c-Jun phosphorylation in RGCs from uninjured eyes and in RGCs 3 days after optic nerve crush in control (AAV-EBFP) or AAV-CaMKIIα T286D-treated eyes. Scale bar, 20 μm. (V) Quantification of pc-Jun intensity in RGCs. Data are presented as mean ± standard deviation (n = 3 retinas per group). One-way ANOVA with Tukey's multiple comparison test, F: 87.73, R2: 0.9669, ns (not significant, P = 0.09). [Figure 9A-9I] According to an embodiment of the present disclosure, examples of CaMKII-mediated protection of RGCs in induced and genetic models of glaucoma are disclosed. (A) Images of magnetic microbeads distributed uniformly around the anterior chamber using a magnet after injection. (B) Images of eye sections after H&E staining show accumulation of microbeads at the iridocorneal angle. Scale bar, 100 μm. (C) Quantification of intraocular pressure (IOP) after injection of PBS (sham) or microbeads. Data are presented as mean ± standard deviation (n=6 eyes per group). (D-E) Confocal images of retinal whole mounts showing surviving RGCs labeled by Tuj1 immunoreactivity 2 months after induction of elevated IOP in control (AAV-EBFP) or AAV-CaMKIIα T286D treated eyes. Scale bar, 40 μm. (F) Quantification of RGC survival. Expressed as number of RGCs (left Y-axis) and percentage of RGCs relative to those in uninjured retina (right Y-axis). Data are presented as mean ± standard deviation (n = 6 retinas per group). Unpaired t test, *P < 0.0001. (GH) Confocal images of retinal whole mounts from 2-month-old GLAST- / - mice showing surviving RGCs labeled by Tuj1 immunoreactivity in control (AAV-EBFP) or AAV-CaMKIIα T286D-treated eyes. Scale bar, 40 μm. (I) Quantification of RGC survival in GLAST- / - retinas. Expressed as number of RGCs (left Y-axis) and percentage of RGCs relative to those in uninjured wild-type retinas (right Y-axis). Data are presented as mean ± standard deviation (n = 5 retinas per group). Unpaired t test, *P < 0.0001. [Figure 10A-10P]According to an embodiment of the present disclosure, examples of CaMKII-mediated protection of RGC axons in induced and genetic models of glaucoma are disclosed. (A-D) Confocal images of retinal whole mounts showing pan-CaMKII levels in RGCs of uninjured retinas or AAV-CaMKIIα T286D-treated retinas 2 weeks after microbead injection. Scale bar, 20 μm. (E) Quantification of pan-CaMKII intensity in RGCs. Data are presented as mean ± standard deviation (n=3 retinas per group). Unpaired t-test, *P<0.0001. (F-H) Light microscopy images of semi-ultrathin sections of optic nerves stained with toluidine blue from uninjured eyes and 2 months after induction of IOP elevation in control (AAV-EBFP) or AAV-CaMKIIα T286D-treated eyes. Scale bar, 4 μm. (I) Quantification of axonal survival. Number of axons (left Y-axis) and percentage of axons relative to those in uninjured eyes (right Y-axis). Data are presented as mean ± standard deviation (n = 4 nerves per group). Unpaired t-test, *P = 0.0056. (J-M) Confocal images of retinal whole mounts showing pan-CaMKII levels in RGCs of uninjured retinas or AAV-CaMKIIα T286D-treated retinas of GLAST- / - mice 3 weeks after AAV injection. Scale bar, 20 μm. (N) Quantification of pan-CaMKII intensity in RGCs. Data are presented as mean ± standard deviation (n = 3 retinas per group). Unpaired t-test, *P = 0.0055. (O-P) Images of sections from 8-month-old GLAST- / - mice showing optic nerve head morphology (bright field) and the nuclear layer stained with DAPI (blue) in control (AAV-EBFP) or AAV-CaMKIIα T286D-treated eyes. Scale bar, 80 μm. [Fig. 11A-11M]According to an embodiment of the present disclosure, an example of CaMKII reactivation protecting RGC axons and their target projections to the brain is disclosed. (A) Schematic of anterograde cholera toxin subunit B (CTB) tracing of the optic nerve, lateral geniculate nucleus (LGN), and superior colliculus (SC). (B-D) Confocal images of anterograde CTB tracing of RGC axons in the optic nerve from uninjured eyes and 7 days after NMDA injection in control (PBS) or AAV-CaMKIIα T286D treated eyes. Scale bar, 300 μm. Inset: Whole mount retinal image showing CTB filling in the retina. (E) Quantification of CTB intensity in the optic nerve. Data are presented as mean ± standard deviation (n=4 nerves per group). One-way ANOVA with Tukey's multiple comparison test, F: 281.7, R2: 0.9843, *P<0.0001. (F-H) Confocal images of anterograde CTB tracing of RGC axons projecting to the contralateral LGN from uninjured eyes and 7 days after NMDA injection in control (PBS) or AAV-CaMKIIα T286D-treated eyes. Scale bar, 300 μm. (I) Quantification of CTB intensity in the contralateral LGN. Data are presented as mean ± SD (n = 4 brains per group). One-way ANOVA with Tukey's multiple comparison test, F: 155.8, R2: 0.9719, *P < 0.0001. (J-L) Confocal images of anterograde CTB tracing of RGC axons projecting to the contralateral SC from uninjured eyes and 7 days after NMDA injection in control (PBS) or AAV-CaMKIIα T286D-treated eyes. Scale bar, 300 μm. (M) Quantification of CTB intensity in the contralateral LGN. Data are presented as mean ± standard deviation (n = 4 brains per group). One-way ANOVA with Tukey's multiple comparison test, F: 226.9, R2: 0.9805, *P < 0.0001. [Fig. 12A-12H]According to an embodiment of the present disclosure, an example of CaMKII reactivation protecting RGC axon projections to the ipsilateral hemisphere is disclosed. (A-C) Confocal images of anterograde CTB tracing of RGC axons projecting to the ipsilateral LGN from uninjured eyes and 7 days after NMDA injection in control (PBS) or AAV-CaMKIIα T286D-treated eyes. Scale bar, 300 μm. (D) Quantification of CTB intensity in the ipsilateral LGN. Data are presented as mean ± standard deviation (n=4 brains per group). One-way ANOVA with Tukey's multiple comparison test, F: 145.0, R2: 0.9699, *P<0.0001. (E-G) Confocal images of anterograde CTB tracing of RGC axons projecting to the ipsilateral SC from uninjured eyes and 7 days after NMDA injection in control (PBS) or AAV-CaMKIIα T286D-treated eyes. Scale bar, 300 μm. (H) Quantification of CTB intensity in the ipsilateral SC. Data are presented as mean ± standard deviation (n = 4 brains per group). One-way ANOVA with Tukey's multiple comparison test, F: 162.2, R2: 0.9730, *P < 0.0001. [Fig. 13A-13Q]According to an embodiment of the present disclosure, an example of CaMKII reactivation that maintains functional vision is disclosed. (A-C) Representative responses of PERG recordings from uninjured eyes and 7 days after NMDA injection in control (PBS) or AAV-CaMKIIα T286D treated eyes. (D) Quantification of PERG amplitude. Data are presented as mean ± standard deviation (n=4 mice per group). One-way ANOVA with Tukey's multiple comparison test, F: 47.95, R2: 0.9142, *P<0.0001. (E-G) Representative responses of PVEP recordings from uninjured eyes and 10 days after NMDA injection in control (PBS) or AAV-CaMKIIα T286D treated eyes. (H) Quantification of PVEP amplitude. PVEP amplitude is shown for each animal (red) and averaged across groups (blue). Data are presented as mean ± standard deviation (n=4 mice per group). One-way ANOVA with Tukey's multiple comparison test, F: 40.67, R2: 0.9004, *P=0.0002. (I) Schematic of the visual water task. (J-L) Visual water task performance as a function of spatial frequency from uninjured mice and 4-14 days after NMDA injection in control (PBS) or AAV-CaMKIIα T286D-treated (binocular) mice. For each column, each row shows results from one mouse. For each animal, a best-fit trend line was generated and the point on the curve that intersected with 70% correct choices was taken as the visual acuity threshold. (M) Visual acuity (spatial frequency threshold) measured in the visual water task. Data are presented as mean ± standard deviation (n=4 mice per group). One-way ANOVA with Tukey's multiple comparison test, F: 529.8, R2: 0.9916, *P<0.0001. (N) Schematic of the visual cliff test. (O) Visual cliff performance from uninjured mice and 7 days after NMDA injection in control (PBS) or AAV-CaMKIIα T286D-treated mice. Data show number (left Y-axis) and percentage (right Y-axis) of shallow / deep choices. Fisher's exact test, *P=0.0373. (P) Schematic of the rooming response test.(Q) Performance in response to rooming stimulation 7 days after NMDA injection from uninjured mice and in control (PBS) or AAV-CaMKIIα T286D-treated mice. Data show the number (left Y-axis) and percentage (right Y-axis) of responders and non-responders. Fisher's exact test, *P=0.0028. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] References throughout this specification to "one example," "another example," "an example," etc. mean that particular elements (e.g., features, structures, and / or characteristics) described in connection with an example are included in at least one example described herein and may or may not be present in other examples. In addition, unless the context clearly dictates otherwise, it should be understood that the elements described with any example may be combined in any suitable manner in the various examples.

[0029] The present disclosure relates to a method for reducing degeneration of retinal ganglion cells in a subject, a method for treating vision loss in a subject, and a pharmaceutical composition. In one example, the pharmaceutical composition comprises one or more components applicable for use in the methods disclosed herein.

[0030] It is disclosed herein that CaMK and CREB signaling are significantly impaired after excitotoxic damage to RGC cell bodies or optic nerve damage to RGC axons, and it is disclosed herein that increasing the activity of these pathways robustly protects RGCs from damage. It is disclosed herein that CaMK protects RGCs in induced and genetic models of glaucoma, a major cause of blindness characterized by loss of RGC cell bodies and axons. It is also disclosed herein that increasing the activity of CaMK protects long-distance RGC axon projections and restores visual function throughout the visual pathway from the retina to the primary visual cortex of the brain. It is also disclosed herein that increasing the activity of CREB protects RGCs.

[0031] The protective effect of the present disclosure of increasing CaMK activity on RGCs and vision is particularly surprising given previous evidence that inhibiting CaMK blunts cytotoxicity. Calcium is a highly versatile intracellular signal involved in regulating a range of cellular processes (Berridge et al., 2000). Loss of Ca2+ homeostasis leads to cellular damage, often in the form of increased cytoplasmic Ca2+ (Dong et al., 2006). Abnormal Ca2+ activation is known to be involved in the death of RGCs after insults such as excitotoxicity and optic nerve injury (Hartwick et al., 2008, Prilloff et al., 2007). CaMKII is a central coordinator and executor of Ca2+ signaling (Hudmon and Schulman, 2002a). Thus, previous studies have shown that inhibiting CaMKII protects RGCs from excitotoxic cell death, suggesting that increasing CaMK activity promotes RGC degeneration. Conversely, surprisingly, promoting CaMK activity, as disclosed herein, protects RGCs and vision.

[0032] As disclosed herein, excitotoxic injury to RGC cell bodies or optic nerve damage to RGC axons resulted in the inactivation of CaMK and its downstream target CREB in RGCs. Increasing CaMK or CREB activity protected RGCs from both injuries. Furthermore, CaMK-mediated RGC protection slowed disease progression in induced and genetic animal models of glaucoma. Increasing CaMK activity not only rescues RGC cell bodies but also protects long-distance RGC axon projections from the retina to the visual relay center of the brain. Enhancing CaMK-mediated RGC protection also restores functional vision throughout the visual pathway, as evidenced by improved visual responses in the retina and primary visual cortex of the brain, as well as visually guided behavior. Also disclosed are targeted increases in CaMK or CREB activity as a method to reduce RGC degeneration and treat vision loss, as well as pharmaceutical compositions comprising compositions for increasing CaMK activity or CREB activity (including activity in RGCs).

[0033] CaMK isoforms include CamKI, CaMKII, and CaMKIV. As disclosed herein, increasing the activity of any of these CaMKs prevents RGC degeneration. For example, increasing the activity of CaMKI, CaMKII, or CaMKIV prevents RGC degeneration. CaMKII includes several isoforms, including CaMKIIα, CaMKIIβ, CaMKIIγ, and CaMKIIδ. Increasing the activity of CaMKIIα or CaMKIIβ prevents RGC degeneration. Increasing CaMKII activity also improves vision after injury known to damage RGCs by exposure to treatment that increases CaMK activity, compared to subjects exposed to such injury. Given the known shared cellular functions among CaMKs, including CaMKI, CaMKII (including but not limited to CaMKIIα, CaMKIIβ, CaMKIIγ, and CaMKIIδ), and CaMKIIIV, and the ability of various CaMKs to prevent degeneration of RGCs and treat vision loss, as disclosed herein, it will be understood that increasing the activity of any one or more of CaMKI, CaMKII (including but not limited to CaMKIIα, CaMKIIβ, CaMKIIγ, and CaMKIIδ), and CaMKIV will prevent degeneration of RGCs, prevent loss of RGC cell bodies, prevent loss of RGC axonal projections in the brain, prevent loss of RGC axons, prevent vision loss, treat vision loss, and any one or more of the foregoing.

[0034] In some examples, increasing the activity of CaMK can include increasing the activity of a variant of CaMK having an amino acid sequence different from the CaMK expressed by the subject or the CaMK expressed by one or more cells of the subject whose activity of CaMK is increased according to the methods disclosed herein. For example, increasing the activity of CaMK can include increasing the activity of a CaMK different from the CaMK disclosed in this disclosure, or increasing the activity of a CaMK different from the CaMK encoded by the genome of the subject, or increasing the activity of a CaMK different from the CaMK that would otherwise be expressed in one or more cells of the subject whose activity of CaMK is increased according to the methods disclosed herein by about 1% or more, or about 2% or more, or about 3% or more, or about 4% or more, It may include an increase of about 5% or more, or about 6% or more, or about 7% or more, or about 8% or more, or about 9% or more, or about 10% or more, or about 11% or more, or about 12% or more, or about 13% or more, or about 14% or more, or about 15% or more, or about 16% or more, or about 17% or more, or about 18% or more, or about 19% or more, or about 20% or more, or about 21% or more, or about 22% or more, or about 23% or more, or about 24% or more, or about 25% or more, or about 30% or more, or about 35% or more.

[0035] In other examples, increasing the activity of a CaMK can include increasing the activity of a CaMK that is different from a CaMK disclosed in the present disclosure, or that is different from a CaMK encoded by the genome of a subject, or that is different from a CaMK that would otherwise be expressed in one or more cells of a subject whose activity of CaMK is increased according to the methods disclosed herein by including, alone or in combination, a substitution, insertion, or deletion of one or more amino acids (about one or more, about two or more, about three or more, about four or more, about five or more, about ten or more, about fifteen or more, about twenty or more, about twenty-five or more, about thirty or more, about twenty-five or more, about forty or more, or about fifty or more amino acids) compared to said CaMK.

[0036] In other examples, increasing the activity of CaMKs can include increasing the activity of CaMKs that are constitutively active. Constitutively active refers to CaMKs whose activity, or increased activity, or sustained activity, is not otherwise dependent on or decreased by one or more other cell signaling events or is not generally required to increase or decrease the activity of CaMKs in a subject or one or more cells of a subject in which CaMK activity is increased according to the methods disclosed herein. As a non-limiting example, activation of CaMKII can generally be initiated by Ca2+ influx and subsequent Ca2+ / calmodulin binding, and the resulting conformational change of CaMKII allows its autophosphorylation at either threonine 286 (T286) for CaMKIIα or threonine 287 (T287) for CaMKIIβ, which can enhance the activity of such isoforms of both isoforms, whereby when autophosphorylation occurs, CaMKs can remain active even after Ca2+ concentration is reduced. In another example, a constitutively active CaMK can comprise a truncated N-terminal catalytic domain of CaMKIIα or a truncated N-terminal catalytic domain of CaMKIIβ, where these truncations are constitutively active.

[0037] In one example, the increase in activity of CaMK may include, but is not limited to, an increase in the activity of another CaMK, such as a constitutively active variant of CaMKIIα or CaMKIIβ, or any of the aforementioned variants. As a non-limiting example, the increase in activity of CaMK according to the present disclosure may include an increase in the level, expression, or activity of T286D-substituted CaMKIIα, which may simulate the active phosphorylation state of CaMK, without being limited to any particular mechanism of action. In another non-limiting example, the increase in level, expression, or activity of CaMK according to the present disclosure may include an increase in the activity of T287D-substituted CaMKIIβ, which may simulate the active phosphorylation state of CaMK, without being limited to any particular mechanism of action. In another example, the increase in activity of CaMK may include an increase in the level, expression, or activity of the N-terminal catalytic domain of CaMKIIα or the N-terminal catalytic domain of CaMKIIβ, which are known to be constitutively active.

[0038] In some examples, increasing the activity of CREB can include increasing the activity of a variant of CREB that has an amino acid sequence different from the CREB expressed by the subject or expressed by the subject in one or more cells in which CREB activity is increased according to the methods disclosed herein. For example, increasing the activity of CREB can include increasing the activity of a CREB different from the CREB disclosed in this disclosure, or increasing the activity of a CREB different from the CREB encoded by the genome of the subject, or increasing the activity of a CREB different from the CREB that would otherwise be expressed in one or more cells in the subject in which CREB activity is increased according to the methods disclosed herein by about 1% or more, or about 2% or more, or about 3% or more, or about 4% or more, It may include an increase of about 5% or more, or about 6% or more, or about 7% or more, or about 8% or more, or about 9% or more, or about 10% or more, or about 11% or more, or about 12% or more, or about 13% or more, or about 14% or more, or about 15% or more, or about 16% or more, or about 17% or more, or about 18% or more, or about 19% or more, or about 20% or more, or about 21% or more, or about 22% or more, or about 23% or more, or about 24% or more, or about 25% or more, or about 30% or more, or about 35% or more.

[0039] In other examples, increasing the activity of CREB may include increasing the activity of a CREB different from the CREB disclosed herein, or increasing the activity of a CREB different from the CREB encoded by the genome of the subject, or increasing the activity of a CREB different from the CREB that would otherwise be expressed in one or more cells of a subject whose activity of CREB is increased according to the methods disclosed herein by including, alone or in combination, a substitution, insertion, or deletion of one or more amino acids (about one or more, about two or more, about three or more, about four or more, about five or more, about ten or more, about fifteen or more, about twenty or more, about twenty-five or more, about thirty or more, about twenty-five or more, about forty or more, or about fifty or more amino acids) compared to the CREB described above.

[0040] In another example, increasing the activity of CREB can include increasing the activity of a constitutively active CREB. Constitutively active means a CREB whose activity, or increased activity, or sustained activity, is not otherwise dependent on or decreased by one or more other cell signaling events or is not generally required to increase or decrease the activity of CREB in a subject or one or more cells of a subject in which CREB activity is increased according to the methods disclosed herein. As a non-limiting example, increasing the activity of CREB can include increasing the expression of a CREB variant (known as VP16-CREB), which is a fusion between the activation domain of herpes simplex virus VP16 protein and the DNA binding domain of CREB, as disclosed in U.S. Patent No. 9,587,000 (incorporated herein by reference).

[0041] CREB activity can be increased by increasing the amount of CREB in a subject, such as in the retina of a subject that contains RGCs of the subject. CREB expression can be increased by transfecting a cell, such as an RGC, with CREB or a polynucleotide sequence encoding CREB to cause expression of CREB from the polynucleotide in a subject or one or more cells thereof. In some examples involving transfecting a cell with a polynucleotide encoding CREB, the polynucleotide can further comprise a cis-regulatory element that is operably associated with a portion of the polynucleotide encoding CREB to stimulate, promote, or enhance expression of CREB from the polynucleotide. Such a cis-regulatory element can include one or more of a promoter sequence and an enhancer sequence.

[0042] Cis-regulatory elements can include promoters, enhancers, or both. In some cases, the sequence of a cis-regulatory element is less than 10 nucleotides from the transcription start site, less than 20 nucleotides from the transcription start site, less than 30 nucleotides from the transcription start site, less than 40 nucleotides from the transcription start site, less than 50 nucleotides from the transcription start site, less than 60 nucleotides from the transcription start site, less than 70 nucleotides from the transcription start site, less than 80 nucleotides from the transcription start site, less than 90 nucleotides from the transcription start site, less than 100 nucleotides from the transcription start site, less than 125 nucleotides from the transcription start site, less than 150 nucleotides from the transcription start site, less than 175 nucleotides from the transcription start site, less than 20 nucleotides from the transcription start site, less than 25 nucleotides from the transcription start site, less than 30 nucleotides from the transcription start site, less than 40 nucleotides from the transcription start site, less than 50 nucleotides from the transcription start site, less than 60 nucleotides from the transcription start site, less than 70 nucleotides from the transcription start site, less than 80 nucleotides from the transcription start site, less than 90 nucleotides from the transcription start site, less than 100 nucleotides from the transcription start site, less than 125 nucleotides from the transcription start site, less than 150 nucleotides from the transcription start site, less than 175 nucleotides from the transcription start site, less than 20 nucleotides from the transcription start site, less than 30 nucleotides from the transcription start site, less than 30 nucleotides from the transcription start site, less than 40 nucleotides from the transcription start site, less than 50 nucleotides from the transcription start site, less than 60 nucleotides from the transcription start site, less than 70 nucleotides from the transcription start site, less than 80 nucleotide The transcription start site may be located less than 0 nucleotides, less than 225 nucleotides from the transcription start site, less than 250 nucleotides from the transcription start site, less than 275 nucleotides from the transcription start site, less than 300 nucleotides from the transcription start site, less than 325 nucleotides from the transcription start site, less than 35 nucleotides from the transcription start site, less than 375 nucleotides from the transcription start site, less than 400 nucleotides from the transcription start site, less than 425 nucleotides from the transcription start site, less than 450 nucleotides from the transcription start site, less than 475 nucleotides from the transcription start site, less than 500 nucleotides from the transcription start site, or within 500 to 1,000 nucleotides from the transcription start site.

[0043] A promoter is a nucleotide sequence to which an RNA polymerase binds to initiate transcription of a downstream gene sequence. Many genes that exhibit tissue or cell type specific expression contain a promoter upstream of a DNA sequence that encodes an RNA that is particularly active in the cell in which the gene is expressed. A promoter may be more active in some cells than in other cells, for example, only active in certain cell types or tissue types, or highly active in certain cell types or tissue types compared to others. A promoter includes a sequence at which transcription is initiated. Eukaryotic promoters may, and typically do, include features such as a TATA box, a transcription factor IIB recognition site, and a core promoter sequence (or initiator). Transcription factors bind and RNA polymerase binds to the promoter to initiate transcription.

[0044] Cis-regulatory elements may also include one or more enhancer sequences. Enhancers are parts of cis-regulatory elements that enhance transcription initiated at or by a promoter. Enhancers may function to facilitate the initiation of transcription at a promoter, such as by binding additional transcription factors to the enhancer, which facilitates or enhances the recruitment of other factors and transcriptional machinery to the promoter. As with promoters, many genes have enhancements that are responsible for cell- or tissue-specific or cell- or tissue-enhanced expression. In some instances, cis-regulatory elements may include other features, such as intronic sequences, splice sites, exonic sequences, or any combination thereof, that may affect the expression of the transcript in a given cellular environment. In one example, a cell-type specific cis-regulatory element may include features that suppress expression in cell types other than the cell type that the cell-type specific cis-regulatory element is intended to drive expression.

[0045] In one example, the cis-regulatory element may include a promiscuous cis-regulatory element. The promiscuous cis-regulatory element may include one or more polynucleotide sequences that may drive or may be designed to drive expression regardless of or minimally related to the cell type transfected by the polynucleotide. The promiscuous cis-regulatory element may promote expression of the polynucleotide encoding CaMK or CREB in different cell types, including cells of different tissues, lineages, ages, etc. Examples of promiscuous cis-regulatory elements include CMV early enhancer / chicken beta-actin (CAG) promoter cis-regulatory elements, human beta-actin promoter cis-regulatory elements, human elongation factor-1 alpha promoter cis-regulatory elements, cytomegalovirus (CMV) promoter cis-regulatory elements, simian virus 40 promoter cis-regulatory elements, and herpes simplex virus thymidine kinase. In another example, the cis-regulatory element may include a cell-specific cis-regulatory element.

[0046] A cell-specific cis-regulatory element may include one or more polynucleotide sequences that can drive or be designed to drive expression only, or mostly, or preferentially, or primarily, in one or more predetermined cell types. A cell-specific cis-regulatory element may include one or more polynucleotide sequences that can drive or be designed to drive expression only, or mostly, or preferentially, or primarily, in one or more predetermined cell types with no, minimal, negligible, or insubstantial expression in other cell types that may be transfected with the polynucleotide, or one or more polynucleotide sequences that do not increase CaMK or CREB activity in such cells, or increase it only to a minimal, negligible, or insubstantial extent compared to the activity induced in the one or more cell types that the cis-regulatory element is designed to drive expression.

[0047] A cell-specific cis-regulatory element, in accordance with the present disclosure, may reduce expression in a transfected cell type that the cell-specific cis-regulatory element is intended or designed to promote by about 0%, about 5% or less, about 10% or less, 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, compared to the expression level in a transfected cell type or types other than the cell type that the cell-specific cis-regulatory element is intended or designed to promote. The increase may be below about 50%, below about 55%, below about 60%, below about 65%, below about 70%, below about 75%, below about 80%, below about 85%, below about 90%, below about 95%, below about 100%, below about 150%, below about 200%, below about 250%, below about 300%, below about 350%, below about 400%, below about 450%, below about 500%, below about 550%, below about 600%, below about 650%, below about 700%, below about 750%, below about 800%, below about 850%, below about 900%, below about 1,000%, or more. In another example, the cell-specific cis-regulatory element may cause minimal, negligible, or undetectable levels of expression in one or more transfected cell types for which the cis-regulatory element is intended or designed to drive expression, but for which the cell-specific cis-regulatory element is not designed or intended to drive expression, in accordance with the present disclosure.

[0048] In one example, the cis-regulatory element can be a cis-regulatory element that drives the expression of a transcript in RGCs. For example, the cis-regulatory element can be a promoter (herein referred to as RGC promoter) that drives the expression of a transcript in RGCs. For example, the cis-regulatory element can be a promoter, enhancer, or both of a transcript known to be expressed in RGCs. The cis-regulatory element can be a promoter, enhancer, or both of a transcript known to be expressed in RGCs to a greater extent than the transcript is expressed in other cells of the retina or other cells of the eye. In one example, the RGC promoter can be a promoter of a transcript that is more highly expressed in RGCs compared to other cells of the ocular tissue or compared to other cells of the retina. In one example, the RGC promoter can drive the expression level of a transcript in RGCs where the expression level of the transcript in other cells of the ocular tissue or other cells of the retina is sufficiently high enough to allow the cell to be identified as an RGC based on a distinguishable expression level of the transcript in RGCs compared to other cells.

[0049] In one example, the RGC promoter may not drive expression of the transcript in cells of other tissues of the eye or other retinal cells other than RGCs. For example, the transcript may be detectable in RGCs (e.g., by in situ hybridization detection of the transcript's mRNA), but in one example, not detectable in cells of other tissues of the eye, or in another example, not detectable in other cell types of the retina. In another example, the RGC promoter exhibits at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 6-fold, or at least about 7-fold, or at least about 8-fold, or at least about 9-fold, or at least about 10-fold, or at least about 11-fold, or at least about 12-fold, or at least about 13-fold, or at least about 14-fold, or at least about 15-fold, or at least about 16-fold, or at least about 17-fold, or at least about 18-fold, or at least about 19-fold, or at least about 20-fold, or at least about 25-fold, or at least about 50-fold, or less, the expression level of the transcript in, in one example, a cell of another tissue of the eye, or in another example, a cell type of the retina. The expression level of the transgene may be determined by measuring the expression of the transgene by Western blot analysis in purified RGCs compared to other cell types, such as other purified cell types of cells from ocular tissues.

[0050] In one example, as disclosed herein, the RGC promoter can be a γ-synuclein promoter (e.g., a human γ-synuclein promoter, a mouse γ-synuclein promoter, or another γ-synuclein promoter that drives expression of a transcript in an RGC of a subject). In another example, the RGC promoter can be a synapsin 1 promoter (e.g., a human synapsin 1 promoter, a mouse synapsin 1 promoter, or another synapsin 1 promoter that drives expression of a transcript in an RGC of a subject). In another example, the RGC promoter can be a Thy-1 cell surface antigen promoter (e.g., a human Thy-1 cell surface antigen promoter, a mouse Thy-1 cell surface antigen promoter, or another Thy-1 cell surface antigen promoter that drives expression of a transcript in an RGC of a subject). In another example, the RGC promoter can be a neurofilament heavy chain promoter (e.g., a human neurofilament heavy chain promoter, a mouse neurofilament heavy chain promoter, or another neurofilament heavy chain promoter that drives expression of a transcript in an RGC of a subject). The neurofilament heavy chain promoter may be a long form neurofilament heavy chain promoter or a short form neurofilament heavy chain promoter. The subject in these examples may be a mammal, or a human, or a mouse, or a rat, or a dog, or a cat, or a horse, or a cow, or a sheep, or a pig. Examples of the nucleotide sequences of the aforementioned promoters are provided in Table 1, any of which is expressly included as a possible example of all examples disclosed herein. [Table 1] JPEG2024510911000003.jpg89135JPEG2024510911000004.jpg190135JPEG2024510911000005.jpg26132JPEG2024510911000006.jpg191135 JPEG2024510911000007.jpg191135JPEG2024510911000008.jpg162135JPEG2024510911000009.jpg190135JPEG2024510911000010.jpg34135

[0051] In one example, the RGC promoter can have less than 100% sequence homology to a promoter disclosed in Table 1. In one example, the RGC promoter can have at least about 60% sequence homology to a promoter disclosed in Table 1, at least about 65% sequence homology to a promoter disclosed in Table 1, at least about 70% sequence homology to a promoter disclosed in Table 1, at least about 75% sequence homology to a promoter disclosed in Table 1, at least about 80% sequence homology to a promoter disclosed in Table 1, at least about 85% sequence homology to a promoter disclosed in Table 1, at least about 90% sequence homology to a promoter disclosed in Table 1, at least about 95% sequence homology to a promoter disclosed in Table 1, at least about 97% sequence homology to a promoter disclosed in Table 1, or at least about 99% sequence homology to a promoter disclosed in Table 1.

[0052] As disclosed herein, the vector may include a polynucleotide comprising a cis-regulatory element and a sequence encoding any CaMK, including but not limited to any constitutively active CaMK disclosed herein. As disclosed herein, the vector may include a polynucleotide comprising a cis-regulatory element and a sequence encoding any CREB, including but not limited to any CREB disclosed herein, including any constitutively active CREB disclosed herein. In one example, any such cis-regulatory element may be a ubiquitous regulatory element, including one or more of one or more enhancers and one or more promoters. The cis-regulatory element may be ubiquitously active or promiscuous. This means that it can drive the expression of a transcript in multiple different cell types, including, for example, RGCs. In another example, any such cis-regulatory element may drive the expression of a transcript in RGBs but not in any other cell type or another cell type, or drive the expression of a transcript in RGCs but not in other cells of the retina, or drive the expression of a transcript in RGCs but not in cells of other tissues of the eye. Such a cis-regulatory element may be the RGC promoter.

[0053] Any combination of any of the aforementioned cis-regulatory elements with any of the polynucleotides encoding CaMK or CREB disclosed herein is expressly included in the present disclosure. Similarly, their use, including but not limited to those contained in vectors, in any and all methods disclosed herein is expressly included in the present disclosure. In one example, a polynucleotide comprising a cis-regulatory element disclosed herein and a sequence encoding CaMK or CREB can be recombinant. As used herein, recombinant means made by splicing together a sequence that does not occur in nature, a polynucleotide cis-regulatory element and a polynucleotide encoding CREB or CaMK. For example, any such example can include a polynucleotide comprising a cis-regulatory element and a sequence encoding CaMK or CREB, where the cis-regulatory element is or comprises one or more nucleotide sequences derived from a naturally occurring gene sequence other than CaMK or CREB. The Ant vector disclosed herein can include any of the aforementioned examples, and any such example comprising such a vector can be used in any of the methods disclosed herein, including but not limited to those disclosed herein.

[0054] In one example, the activity of CaMK or CREB can be increased by contacting a cell with a vector, which can introduce CaMK or CREB into the cell, or which can introduce a polynucleotide encoding CaMK or CREB into the cell, resulting in expression of the encoded CaMK or CREB. A vector refers to a macromolecule or complex of molecules that includes a protein, polypeptide, gene, or polynucleotide that is delivered to a cell. A vector can include, for example, a viral vector, such as a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, an alphaviral vector, a poxviral vector, or a herpes simplex viral vector. A vector can include liposomes and other lipid-containing complexes, as well as other macromolecular complexes that can mediate delivery of a polypeptide or polynucleotide to a target cell. Some embodiments of the present disclosure can include a vector that includes a recombinant nucleic acid.

[0055] In one example, a polynucleotide encoding CaMK or CREB can be encapsidated into a recombinant adeno-associated virus. In one example, the recombinant adeno-associated virus is a serotype selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. Other examples include viral vectors that are hybrids containing components from two or more of the aforementioned AAV serotypes.

[0056] Increasing activity in RGCs can include introducing CaMK or CREB into RGCs, such as by injecting a vector into a subject. Injection can be intraocular or systemic. Injection can be intramuscular, intracerebroventricular, intraperitoneal, subcutaneous, or by any other route that can bring RGCs into contact with the vector so as to increase the activity of CaMK or CREB in RGCs.

[0057] In one example, the method incorporating any one or more features disclosed herein, alone or in any combination, can include reducing the degeneration of RGCs. Reducing the degeneration of RGCs can include reducing one or more of the following: loss of number of RGCs, death of RGCs, loss of number of RGC soma, loss of number of RGC axons, and loss of RGC axon projections, including but not limited to in the brain. The degeneration of RGCs can include loss of vision, including increased blindness. In one example, reducing the degeneration of RGCs can include preventing or treating vision loss. In one example, increasing the activity of CaMK or CREB in RGVs prevents the loss of vision or visual ability, such as may be caused by excitotoxicity or other injury or damage that may cause RGC dysfunction or degeneration.

[0058] Degeneration of RGCs is the main cause of visual impairment and blindness in various pathological conditions. Some conditions damage the cell body of RGCs (including excitotoxicity and retinal ischemia), while other conditions damage the axons of RGCs (including optic nerve section, optic nerve compression, papilledema, and other cases of glaucoma or pathological or harmful increase in intraocular pressure). Preventing RGC degeneration can prevent, reduce, or reduce the visual ability or visual acuity loss or blindness of glaucoma patients, including but not limited to glaucoma patients who would otherwise progress to such impairment, blindness, even if other treatments such as treatments that reduce intraocular pressure are given. In another example, CaMK or CREB activity can be increased in the RGCs of a subject, where the subject has a genetic predisposition to developing or worsening glaucoma. In some examples, the methods can include increasing CaMK or CREB activity in RGCs before RGC degeneration has occurred or is believed to have occurred, or to prevent further RGC degeneration which may be believed to have already occurred to some extent, or where any degree of RGC degeneration is known or believed to have occurred but the subject is believed to be susceptible to developing such degeneration.

[0059] In some examples, the method may include increasing the activity of CaMK or CREB in RGCs in a subject, for example, to prevent, reduce, or alleviate the onset or worsening of any of the aforementioned visual disorders, where the subject has not experienced or been diagnosed with impaired or reduced visual ability or acuity or blindness (which may include partial or complete blindness), or has some impairment or reduction in visual ability or visual ability, or some degree of blindness has been detected, diagnosed, or experienced by the subject, and the subject is considered to be at risk of developing or worsening such impaired, reduced, or lost visual ability or acuity or blindness (which may include partial or complete blindness). In one example, such a subject may have been diagnosed with glaucoma, or diagnosed as susceptible to developing glaucoma, or diagnosed with an ischemic event or other trauma that is known, believed, expected, predicted, or likely to cause retinal degeneration, or reduced visual ability or acuity, or developmental blindness (which may include partial or complete blindness). Any one or more of the embodiments for increasing the activity of CaMK or CREB in RGCs disclosed herein may be included in any of the aforementioned methods for reducing or preventing degeneration of RGCs in a subject, or in the aforementioned methods for treating vision loss.

[0060] The pharmaceutical composition disclosed herein may include a polynucleotide and a vector, and the polynucleotide includes a retinal ganglion cell promoter and encodes CaMK or CREB. The CaMK or CREB encoded by such a polynucleotide may include any of the CaMKs or CREBs described above, including but not limited to CaMKI, CaMKII, CaMKIV, constitutively active CaMK, CaMKIIα, CaMKIIβ, CaMKIIγ, CaMKIIδ, T268D-substituted CaMKIIα, T287D-substituted CaMKIIβ, truncated N-terminal catalytic domain of CaMKIIα, truncated N-terminal catalytic domain of CaMKIIβ, CREB, constitutively active CREB, or VP16-CREB. The polynucleotide included in such a pharmaceutical composition may include a cis-regulatory element (e.g., a promiscuous cis-regulatory element or a cell-specific cis-regulatory element, an RGC promoter, a γ-synuclein promoter, or other cis-regulatory element).

[0061] Some examples include, but are not limited to, the polynucleotides disclosed herein that include sequences encoding any of the above CaMK or CREB, with or without any of the above cis-regulatory elements, including the RGC promoter, where such polynucleotides are not part of a pharmaceutical composition. In one example, a vector (including but not limited to any of the above vectors) can include such a polynucleotide, where such a vector is not part of a pharmaceutical composition.

[0062] In another example, compositions, compounds, drugs, pharmaceuticals, or other substances that can increase the activity of CaMK can be used to reduce the degeneration of RGCs according to the present disclosure. For example, oleic acid (CAS112-80-1) is known to stimulate CaMK activity. Given that increasing CaMK activity inhibits RGC degeneration and vision loss, oleic acid would be expected to prevent RGC degeneration and be used as a treatment for vision loss, as well as other methods of increasing CaMK disclosed herein. Expressly included in the present disclosure are methods of reducing the degeneration of retinal ganglion cells in a subject or methods of treating vision loss in a subject, comprising administering to the subject oleic acid or a pharma- ceutically acceptable salt thereof, or another stimulator or activator of CaMK activity or expression, or, including but not limited to, in place of a CaMK polynucleotide or vector in all of the embodiments of the method and in any subject disclosed herein.

[0063] The pharmaceutical composition may include a formulation for administration to a subject. Such formulations may include any of the formulations suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intraocular, intravenous, and intraarticular), rectal, and topical (including cutaneous, buccal, sublingual, and intraocular) administration. The most suitable route may depend on the condition and disorder of the recipient, or the intended purpose of the administration. The formulations may be conveniently presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The method may include the step of bringing into association CaMK or CREB, or a polynucleotide encoding CaMK or CREB, or a vector comprising any of the foregoing, including any of the embodiments disclosed herein ("active ingredient"), with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired formulation.

[0064] Formulations of the present disclosure suitable for administration to a subject may be presented as discrete units, such as capsules, cachets, or tablets, each containing a predetermined amount of the active ingredient, as a powder or granules, as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion. A composition containing the active ingredient may also be presented as a bolus, electuary, or paste. For oral or other administration, the active ingredient may be suspended in a solution or dissolved in a solvent (e.g., alcohol, DMSO, water, saline, or other solvent), which may be further diluted or dissolved in another solution or solvent, and in some instances may contain a carrier or other excipient.

[0065] In certain embodiments, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, lozenges, troches, capsules, elixirs, suspensions, syrups, wafers, etc. Tablets, troches, pills, capsules, etc. may also contain: binders (e.g., tragacanth gum, acacia, corn starch, gelatin, or combinations thereof), excipients (e.g., dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or combinations thereof), disintegrants (e.g., corn starch, potato starch, alginic acid, or combinations thereof), lubricants (e.g., magnesium stearate), sweeteners (e.g., sucrose, lactose, saccharin, or combinations thereof), flavoring agents (e.g., peppermint, wintergreen oil, cherry flavor, or orange flavor), etc. When the unit dosage form is a capsule, it may contain a liquid carrier in addition to the above types of materials. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain carriers such as liquid carriers in addition to the above types of materials. Gelatin capsules, tablets, or pills may be enteric coated. The enteric coating prevents the composition from denaturing in the stomach or upper intestine, where the pH is acidic. Upon reaching the small intestine, the basic pH therein dissolves the coating, allowing the composition to be released and absorbed by specialized cells (e.g., epithelial intestinal cells and Peyer's patch M cells). A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, dyes, and flavorings such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharma- ceutical pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained release preparations and formulations.

[0066] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricant, surface active agent, or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Tablets can be optionally coated or scored, and can be formulated to provide sustained, delayed, or controlled release of the active ingredient therein.

[0067] Formulations for parenteral or other administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient. Formulations for parenteral or other administration may also include aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier (such as, for example, saline, phosphate-buffered saline (PBS), or water for injection) immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0068] As used herein, the term "pharmaceutical acceptable carrier" refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions immediately prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending on the ratio of the compound of formula I to the polymer and the nature of the particular polymer used, the rate of release of the compound of formula I can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use. Suitable inert carriers can include sugars such as lactose.

[0069] The formulation of the compound of formula I may contain different types of carriers depending on whether it is administered in solid, liquid, or aerosol form and whether it needs to be sterile for administration routes such as injection.The present disclosure may be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, by inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, direct localized perfusion bath of target cells, via catheter, via lavage, in creams, in lipid compositions (e.g., liposomes), or by other methods or any combination of the above as known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990).

[0070] As used herein, the term "effective amount" refers to an amount of an active ingredient or pharmaceutical agent that can induce a biological or medical response in a cell, tissue, system, animal, or human that is desired, for example, by a researcher or clinician. The term "therapeutically effective amount" refers to any amount that results in the treatment, cure, prevention, or amelioration of a disease, disorder, or side effect, or results in a slowing of the rate of progression of a disease or disorder, compared to a corresponding subject not receiving such amount. The term also includes within its scope an amount effective to enhance normal physiological function. For use in therapy, a therapeutically effective amount of the active ingredient, as well as its salts, solvates, and physiologically functional derivatives, may be administered as raw chemicals. Additionally, the active ingredient may be presented as a pharmaceutical composition.

[0071] The pharmaceutical compositions of the present disclosure can include an effective amount of a compound of formula I and, optionally, one or more additional agents dissolved or dispersed in a pharma- ceutically acceptable carrier. The phrase "pharmaceutical acceptable or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other adverse reactions when administered to an animal, such as, for example, a human, as appropriate. The preparation of pharmaceutical compositions that contain an active ingredient and may optionally include one or more additional active ingredients will be known to those skilled in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Furthermore, it will be understood that for administration to animals (e.g., humans), preparations must meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards.

[0072] Further, in accordance with the present disclosure, compositions of the present disclosure suitable for administration may be provided in a pharma- ceutically acceptable carrier with or without an inert diluent. The carrier may be assimilable and may include liquid, semi-solid (i.e., paste), or solid carriers. The use of any conventional vehicle, agent, diluent, or carrier in an administrable composition for use in practicing the methods of the present disclosure is appropriate, except where such use is deleterious to the recipient or to the therapeutic effectiveness of the composition contained therein. Examples of carriers or diluents may include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers, and the like, or combinations thereof. The compositions may also include various antioxidants to retard oxidation of one or more components. In addition, prevention of the action of microorganisms may be provided by preservatives, such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0073] In accordance with the present disclosure, the compounds of formula I can be combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsion, mixing, encapsulation, absorption, etc. Such procedures are routine to those of ordinary skill in the art.

[0074] In one example, the disclosure may include the use of a pharmaceutical lipid vehicle or vector composition that includes an active ingredient and an aqueous solvent. As used herein, the term "lipid" is defined to include any of a wide range of substances that are characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds is well known to those of skill in the art, and the term "lipid" as used herein is not limited to any particular structure. Examples include compounds that contain long-chain aliphatic hydrocarbons and their derivatives. Lipids can be naturally occurring or synthetic (i.e., designed or manufactured by humans). However, lipids are typically biological substances. Biological lipids are well known in the art and include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids with ether- and ester-linked fatty acids, and polymerizable lipids, as well as combinations thereof. Of course, compounds other than those specifically described herein that are understood by those of skill in the art as lipids are also encompassed by the compositions and methods of the present invention.

[0075] Those skilled in the art will be familiar with a series of techniques that can be used to disperse composition in lipid vehicle.For example, active ingredient can be dispersed in a solution that contains lipid, dissolved in lipid, emulsified with lipid, mixed with lipid, combined with lipid, covalently bonded to lipid, contained in lipid as a suspension, contained in or complexed with micelle or liposome, or otherwise associated with lipid or lipid structure.Dispersion can or cannot result in the formation of liposome.

[0076] The actual dosage of the composition of the present disclosure administered to a subject (e.g., an animal or human patient) can be determined by physical and physiological factors such as body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic interventions, idiopathic nature of the patient, and route of administration, as well as the therapeutic objectives. Depending on the dosage and route of administration, the preferred dosage and / or the frequency of administration of an effective amount can vary depending on the subject's response or therapeutic objectives. The practitioner responsible for administration can, in any event, determine the concentration of active ingredient in the composition and the appropriate dose for the individual subject.

[0077] In certain embodiments, the pharmaceutical composition may, for example, contain at least about 0.1% of the active compound. In other embodiments, the active compound may, for example, contain about 2% to about 75% of the weight of the unit, or about 25% to about 60%, and any range derivable therein. Naturally, the amount of active ingredient in each therapeutically useful composition can be prepared in such a way that a suitable dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be contemplated by those skilled in the art of preparing such pharmaceutical formulations. Thus, various dosages and treatment regimes may be desirable.

[0078] For example, for parenteral administration in an aqueous solution, the solution containing the active ingredient can be suitably buffered, if necessary, and the liquid diluent can first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, a sterile aqueous medium can be used. For example, a dosage can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of hypodermoclysis fluid or injected at the proposed injection site (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, the formulation can meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.

[0079] Sterile injectable solutions can be prepared by incorporating active ingredients into a solvent containing various other ingredients listed above, followed by filtration sterilization.Generally, dispersions can be prepared by incorporating various sterilized active ingredients into a sterile vehicle, which contains a basic dispersion medium and other ingredients required from those listed above.In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method includes vacuum drying and freeze-drying technology, which obtains a powder of active ingredients plus any additional desired ingredients from a previously sterile-filtered solution.Powder compositions can be combined with liquid carriers, such as water or physiological saline solution, with or without stabilizers.

[0080] Exemplary amino acid sequences encoding examples of CaMK and CREB disclosed herein, along with exemplary nucleotide sequences which they encode, are provided in Table I. As will be understood, due to codon degeneracy, the amino acid sequences provided in Table I may be encoded by nucleotide sequences other than those provided in Table 2, and all such variations of nucleotide sequences which may encode the amino acid sequences provided in Table 2 are expressly included herein. The N-terminal catalytic domain of the CaMK protein lacks the regulatory region (located C-terminal to the catalytic N-terminal domain) of the complete CaMK protein, relieving the catalytic N-terminal domain from the inhibitory control that the regulatory region would otherwise exert over it. [Table 2] JPEG2024510911000012.jpg154135JPEG2024510911000013.jpg154135JPEG2024510911000014.jpg166135JPEG2024510911000015.jpg108135 JPEG2024510911000016.jpg190135JPEG2024510911000017.jpg191135JPEG2024510911000018.jpg168135JPEG2024510911000019.jpg166135 JPEG2024510911000020.jpg108135JPEG2024510911000021.jpg191135JPEG2024510911000022.jpg191135JPEG2024510911000023.jpg166135 JPEG2024510911000024.jpg166135JPEG2024510911000025.jpg187135JPEG2024510911000026.jpg137135JPEG2024510911000027.jpg133135

[0081] In one example, the CaMK or polynucleotide encoding CaMK may have less than 100% sequence homology to a CaMK or polynucleotide encoding CaMK disclosed in Table 2. In one example, the CaMK or polynucleotide encoding CaMK may have at least about 60% sequence homology to a CaMK or polynucleotide encoding CaMK disclosed in Table 2, at least about 65% sequence homology to a CaMK or polynucleotide encoding CaMK disclosed in Table 2, at least about 70% sequence homology to a CaMK or polynucleotide encoding CaMK disclosed in Table 2, at least about 75% sequence homology to a CaMK or polynucleotide encoding CaMK disclosed in Table 2, at least about 80% sequence homology to a CaMK or polynucleotide encoding CaMK disclosed in Table 2, at least about 85% sequence homology to a CaMK or polynucleotide encoding CaMK disclosed in Table 2, at least about 90% sequence homology to a CaMK or polynucleotide encoding CaMK disclosed in Table 2, at least about 95% sequence homology to a CaMK or polynucleotide encoding CaMK disclosed in Table 2, at least about 10 ... at least about 80% sequence homology to the CaMK or polynucleotide encoding CaMK disclosed in Table 2, at least about 85% sequence homology to the CaMK or polynucleotide encoding CaMK disclosed in Table 2, at least about 90% sequence homology to the CaMK or polynucleotide encoding CaMK disclosed in Table 2, at least about 95% sequence homology to the CaMK or polynucleotide encoding CaMK disclosed in Table 2, at least about 97% sequence homology to the CaMK or polynucleotide encoding CaMK disclosed in Table 2, or at least about 99% sequence homology to the CaMK or polynucleotide encoding CaMK disclosed in Table 2.

[0082] In one example, a polynucleotide encoding CREB or CREB may have less than 100% sequence identity to a polynucleotide encoding CREB or CREB disclosed in Table 2. In one example, a polynucleotide encoding CREB or CREB may have at least about 60% sequence identity to a polynucleotide encoding CREB or CREB disclosed in Table 2, at least about 65% sequence identity to a polynucleotide encoding CREB or CREB disclosed in Table 2, at least about 70% sequence identity to a polynucleotide encoding CREB or CREB disclosed in Table 2, at least about 75% sequence identity to a polynucleotide encoding CREB or CREB disclosed in Table 2, at least about 80% sequence identity to a polynucleotide encoding CREB or CREB disclosed in Table 2, at least about 85% sequence identity to a polynucleotide encoding CREB or CREB disclosed in Table 2, at least about 90% sequence identity to a polynucleotide encoding CREB or CREB disclosed in Table 2, at least about 95% sequence identity to a polynucleotide encoding CREB or CREB disclosed in Table 2, at least about 100% sequence identity to a polynucleotide encoding CREB or CREB disclosed in Table 2, at least about 110% sequence identity to a polynucleotide encoding CREB or CREB disclosed in Table 2, at least about 120% sequence identity to a polynucleotide encoding CREB or CREB disclosed in Table 2, at least about 130% sequence identity to a polynucleotide encoding CREB or CREB disclosed in Table 2, at least about 140% sequence identity to a polynucleotide encoding CREB or CREB disclosed in Table 2, at least about 150% sequence identity to a polynucleotide encoding CREB or CREB disclosed in Table 2, at least about 160% sequence identity to a polynucleotide encoding CREB or CREB disclosed in Table 2, at least about 170% sequence identity to a polynucleotide encoding CREB or CREB disclosed in Table 2, at least about 180% sequence identity at least about 80% sequence identity to CREB or a polynucleotide encoding CREB disclosed in Table 2, at least about 85% sequence identity to CREB or a polynucleotide encoding CREB disclosed in Table 2, at least about 90% sequence identity to CREB or a polynucleotide encoding CREB disclosed in Table 2, at least about 95% sequence identity to CREB or a polynucleotide encoding CREB disclosed in Table 2, at least about 97% sequence identity to CREB or a polynucleotide encoding CREB disclosed in Table 2, or at least about 99% sequence identity to CREB or a polynucleotide encoding CREB disclosed in Table 2.

[0083] Expressly disclosed herein are examples including, but not limited to, any of the promoters disclosed in Table 1, or any other RGC promoter driving expression of any of the CaMK or CREB promoters disclosed in the table. Also expressly included herein, but not limited to, is the use of a compound in the manufacture of a medicament for use in the prevention of RGCs or for the treatment of vision loss in any subject disclosed herein, where the compound comprises a CaMK or CREB protein or variant thereof, or a nucleotide sequence or variant thereof, and optionally comprises any of the RGC promoters or other promoters disclosed herein, and may include a vector comprising any combination of any of the foregoing for use in the treatment of RGC degeneration or vision loss herein.

[0084] Non-limiting examples The following examples are intended to illustrate certain embodiments of the present disclosure, but are not intended to limit its scope in any way.

[0085] It should be understood that all combinations of the foregoing concepts and additional concepts more particularly described herein (provided that such concepts are not mutually inconsistent) are contemplated as part of the inventive subject matter disclosed herein, and in particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as part of the inventive subject matter disclosed herein and can be used to achieve the benefits and advantages described herein.

[0086] Materials and Methods animal C57BL / 6 mice were obtained from The Jackson Laboratory (Bar Harbor, Maine). GLAST- / - mice were provided by Dr. Kohichi Tanaka at Tokyo Medical and Dental University (TMDU). All studies conformed to procedures consistent with animal protocols approved by the ICANH School of Medicine at Mount Sinai IACUC. Male mice were used for the magnetic microbead occlusion model, and mice of either sex were randomly assigned to different groups for other experiments.

[0087] Construction of AAV plasmids and preparation of AAV The pAAV-GFP plasmid was kindly provided by Dr. Kevin Park (University of Miami). For AAV plasmid construction, the protein coding region of pAAV-GFP was replaced with cDNA from the following plasmids: CaMKIIα WT (Addgene #21226), CaMKIIα K42R (Addgene #21221), CaMKIIα T286D (Addgene #16736), CaMKIIβ WT (Addgene #21227), CaMKIIβ K43R (Addgene #21225), CaMKIIβ T287D (Addgene #21223), and VP16-CREB and A-CREB (both from Dr. Hongbing Wang, Michigan State University). GenScript (Piscataway, NJ) generated CaMKIIα K42D and CaMKIIα T286A from CaMKIIα WT, CaMKIIα T286D / T305A / T306A and CaMKIIα T286D / T305D / T306D from CaMKIIα T286D, CaMKIIβ K43D and CaMKIIβ T287A from CaMKIIβ WT, and CaMKIIβ T287D / T306A / T307A and CaMKIIβ T287D / T306D / T307D from CaMKIIβ T287D. AAV-mSncg-GFP was kindly provided by Dr. Yang Hu (Stanford University) and was used to generate AAV-mSncg-EBFP and AAV-mSncg-CaMKIIα T286D. AAV Rap-Cap and Helper plasmids were used for co-transfection in AAVpro 293T cell line (Takara Bio, 632273). AAV was purified using discontinuous iodixanol gradient ultracentrifugation. AAV titers were determined by real-time PCR and ranged from 1 to 4 × 10 per milliliter. 13 The range was 100 copies of the genome.

[0088] Intravitreal injection and optic nerve crush Adult mice were anesthetized with a mixture of ketamine (100 mg / kg) and xylazine (10 mg / kg) by intraperitoneal injection. GLAST- / - mouse pups were anesthetized by cooling on ice, and a small incision was made in the eyelid with a 30-gauge needle to expose the eyeball. For intravitreal injections, a micropipette was inserted just behind the ora serrata and AAV or other solutions were injected intravitreally. N-methyl-D-aspartic acid (Millipore Sigma, 454575) and myristoylated Autocamtide-2 related inhibitory peptide (Millipore Sigma, 189482) were prepared in PBS. For optic nerve crush, the optic nerve was exposed and crushed intraorbitally for 5 seconds with jeweler's forceps, approximately 1 mm posterior to the eyeball. After surgery, ophthalmic ointment was applied to protect the cornea.

[0089] Induction of elevated IOP in mice To elevate IOP, 3-month-old mice were anesthetized with a mixture of ketamine and xylazine. Proparacaine hydrochloride eye drops were used preoperatively. Magnetic microbeads (Dynabeads® M-450 Epoxy, Thermo Fisher Scientific) were injected unilaterally into the anterior chamber and evenly distributed around the anterior chamber using a magnet, as recently described (Ito et al., 2016). IOP in both eyes was monitored using a TonoLab tonometer according to the manufacturer's instructions.

[0090] Histology and microscopy For immunohistochemistry, eyes with attached optic nerve segments surgically removed from perfused mice were post-fixed with 4% PFA. Retinas were dissected for whole mount staining. Retinal whole mounts were blocked for 1 h in staining buffer containing 5% normal donkey serum and 0.1% Triton X-100 in PBS. Retinas were incubated with primary antibodies overnight, washed with PBS (3×15 min), and then incubated with secondary antibodies for 2 h at room temperature. Retinas were washed again with PBS (3×15 min) and then mounted with Fluoromount-G. Primary antibodies used: Tuj1 (1:250, Biolegend, 801202), pCaMKII (1:100, Abcam, ab32678), pan-CaMKII (1:100, Abcam, ab52476), pCREB (1:100, Abcam, ab32096), pTrkB (1:100, Thermo Fisher Scientific, MA5-32207), DLK (1:100, Thermo Fisher Scientific, PA5-32173), pc-Jun (1:100, Cell Signaling Technology, 2361). Peptide used: CaMKIIα (phosphoT286) peptide (12.5 μg / ml, Abcam, ab115237).Secondary antibodies used: Alexa Fluor® 488 AffiniPure donkey anti-mouse IgG (1:500, Jackson ImmunoResearch Labs, 715-545-151), Alexa Fluor® 594 AffiniPure donkey anti-mouse IgG (1:500, Jackson ImmunoResearch Labs, 715-585-151), Cy™ 3 AffiniPure donkey anti-rabbit IgG (1:500, Jackson ImmunoResearch Labs, 711-165-152), Alexa Fluor® 594 AffiniPure donkey anti-rabbit IgG (1:500, Jackson ImmunoResearch Labs, 711-585-152), Alexa Fluor® 647 AffiniPure donkey anti-rabbit IgG (1:500, Jackson ImmunoResearch Labs, 711-585-152). Labs, 711-605-152). Confocal images were acquired using a Zeiss LSM800 microscope. For RGC counting and signaling pathway studies, squares (320×320 μm) were sampled around the peripheral region of each retinal whole mount for analysis (approximately 500 μm from the center of the square to the edge of the retina).

[0091] For visual pathway CTB tracing, 1.5 μl of cholera toxin subunit B (CTB, Alexa Fluor™ 488 conjugate, Thermo Fisher Scientific, C22841) (2 μg / μl in PBS) was injected into the vitreous. Three days after CTB injection, animals were perfused with 4% PFA. Optic nerves were dissected, fixed, and mounted for imaging. Brains were dissected, fixed, and mounted in 3% agarose. Brain slices (150 μm thick) were sectioned coronally for LGN or sagittally for superior colliculus using a vibratome (1000VT, Leica) and mounted for imaging as previously reported (Zhang et al., 2011). Confocal images were acquired using a Zeiss LSM800 microscope.

[0092] For visualization of microbead accumulation at the iridocorneal angle, eyes were harvested from perfused mice and post-fixed in 4% PFA. The anterior portion of each eye was dissected, embedded in OCT compound, sectioned using a cryostat, stained with H&E, and imaged with a Zeiss LSM800 microscope equipped with a color camera.

[0093] For examination of axonal viability in microbead-injected eyes, optic nerves were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer for 0.5 h at room temperature and then 2 h at 4° C. The optic nerve region 1 mm distal to the globe was embedded in resin. Semi-ultrathin sections of the optic nerve were stained with toluidine blue and imaged with a Zeiss LSM800 microscope equipped with a color camera through a 100x lens. Square areas (22×22 μm) were sampled for analysis around the peripheral region of each nerve section (approximately 50 μm from the center of the square to the nerve end).

[0094] For optic nerve head analysis, eyes with attached optic nerve segments surgically removed from perfused mice were post-fixed in 4% PFA. After removal of the cornea, iris, and lens, eyes were embedded in OCT compound for cryosectioning. Sections were taken across the optic nerve, stained with DAPI (Thermo Fisher Scientific, 62248), and imaged on a Zeiss LSM800 microscope.

[0095] Images were analyzed and organized using ImageJ (Schindelin et al., 2012) and Photoshop.

[0096] RGC purification and immunoblotting For RGC purification, dissected retinas were digested in papain and dissociated into single cells by gentle pipetting. Retinal cell suspensions were washed once in HBSS, resuspended in HBSS+4% BSA, and incubated with PE-Cyanine7 conjugated CD90.2 (Thy-1.2) antibody (1:2000, Thermo Fisher Scientific, 25-0902-81) for 15 min to label RGCs for cell sorting (Lu et al., 2020). After further washing with excess HBSS and resuspension in HBSS+4% BSA, DAPI (1mg / ml, 1:1000, Thermo Fisher Scientific, 62248) was added before filtration to label dead cells. Fluorescence-activated cell sorting (FACS) was performed on a BD FACSAriaII sorter (BD Biosciences) to recover RGCs.

[0097] For immunoblotting, purified RGCs were dissolved by heating at 95°C in Laemmli sample buffer. Proteins were separated by SDS-PAGE and electrotransferred onto PVDF membranes. Antibodies: pCaMKII (1:1000, Abcam, ab32678), WesternSure goat anti-rabbit HRP (1:50000, LI-COR Biosciences, 926-80011), recombinant HRP anti-GAPDH antibody (1:400000, Abcam, ab201822). SuperSignal™ West Atto Ultimate Sensitivity Substrate (Thermo Fisher Scientific, A38555) and ChemiDoc Touch Imaging System (Bio-Rad) were used for chemiluminescence detection. Images were analyzed using ImageJ (Schindelin et al., 2012) and Photoshop.

[0098] Pattern Electroretinography (PERG) PERG was recorded using the JORVEC PERG system as previously reported (Chou et al., 2014; Williams et al., 2017). Mice were anesthetized using a mixture of ketamine / xylazine. Animal body temperature was maintained at 37 °C with a feedback-controlled heating stage and monitored using a rectal probe. A small drop of balanced saline was applied topically as needed to prevent corneal drying. PERG signals in response to contrast inversion (0.05 cycles / degree, 100% contrast) of gratings generated at slightly different frequencies alternating on two LED tablets were recorded from a subcutaneous stainless steel needle placed in the nose. Reference and ground electrodes were similar needles, placed medially at the rear of the head and at the base of the tail, respectively. Electrical signals were amplified 10,000 times and band-pass (1–300 Hz) filtered. Independent PERG responses were acquired using the asynchronous averaging method. AAV injections and NMDA treatments were performed unilaterally in one eye for each mouse.

[0099] Pattern Visual Evoked Potentials (PVEP) For PVEP recordings, stainless steel microscrews (0.8 mm outer diameter, 120 threads per inch, NAS721CE80-120, Antrin Miniature Specialties) were implanted into the skull of mice 3 days after intravitreal NMDA injection, 2 mm rostral to the bregma (for the reference electrode) and 2 mm horizontally from the occipital suture (for the active electrode, over the contralateral primary visual cortex). The advancement of the microscrew was set to a cortical depth of 400 μm (because at this depth the amplitude of PVEPs is maximal) (Porciatti et al., 1999). PVEPs were recorded 1 week after the implantation surgery. Mice were anesthetized and kept warm during the recording process. A small drop of balanced saline was applied topically as needed to prevent corneal drying. An electrode with an alligator clip (ETL-36RSAF, The Electrode Store) was used to connect the skull screw. A ground electrode was placed at the base of the tail. A JORVEC system was used to display patterned stimuli (0.05 cycles / degree, 100% contrast gratings) and collect PVEP signals. Electrical signals were amplified 10,000 times and band-pass filtered (1-100 Hz). A typical PVEP response shows a major negative wave that peaks at approximately 100 ms (Porciatti et al., 1999). AAV injections and NMDA treatments were performed unilaterally in one eye for each mouse. The contralateral eye was light-deprived and its display screen was turned off to eliminate possible contributions.

[0100] Visual acuity measurement in the visual water task Visual acuity was measured using the Acumen system (Cerebral Mechanics) as previously reported (Ecker et al., 2010; Prusky et al., 2000). The task took place in a trapezoidal tank containing shallow water. There were two display screens on either side of the wide end of the tank. Stationary vertically oriented sinusoidal gratings were randomly displayed on one of the two screens, while a uniform gray image of the same mean luminance was displayed on the other screen. Animals were trained to swim from the narrow end to the wide end screen, and at a fixed distance, to select the screen displaying the grating and escape to a submerged platform hidden beneath it. The training gratings were set to a spatial frequency of 0.054 cycles / degree. During testing, the spatial frequency of the gratings was slowly increased at intervals of 0.018 cycles / degree until a pause, during which the animal made fewer than seven correct choices in 10 trials. At least four pauses were required to determine that the animal had reached its visual acuity (spatial frequency threshold). For the scatter plots, the cumulative percentage of correct choices at each spatial frequency was calculated. A best-fit trend line was generated and the point on the curve that intersected with 70% correct was taken as the acuity threshold. After training the mice and measuring their acuity, they were injected with NMDA to induce injury. From days 1 to 3 after NMDA injection, the mice were tested daily at a low (training) spatial frequency to maintain their training activity. From days 4 to 14 after NMDA injection, the spatial frequency was changed until a threshold (acuity) was determined for each mouse.

[0101] Visual Cliff Exam The visual cliff apparatus was purchased from Conduct Science (Boston, MA). The visual cliff test apparatus consisted of a clear Plexiglas box with dimensions of 62 × 62 × 19 cm, divided into two areas by a central platform (1.5 inches high, 2 inches wide). Directly below was a shallow side with a checkered pattern, and positioned 2 feet below was a deep side with the same checkered pattern to create the illusion of depth (Fox, 1965; Gu et al., 2018). Mice were placed on the central platform and their descent choice was recorded. Each mouse was subjected to one trial. The box and central platform were thoroughly cleaned after each test.

[0102] Rooming visual stimulus response test Testing for the looming visual stimulus response was performed in an enclosure with dimensions of 17 in. × 20 in. × 12 in., constructed with materials purchased from 80 / 20 Inc. (Columbia City, IN) as described (Koehler et al., 2019). A 5-in.-wide board was placed at one end of the enclosure at a height of 3 in. to serve as a hideout. Food was placed on the opposite side of the hideout to encourage the mouse to explore its environment and remain outside the hideout. A monitor was placed on top of the enclosure to display the looming stimulus. The looming stimulus was a video of a black disk expanding on a gray background and was created using Blender software. Stimulus parameters were taken from a previous study (Yilmaz and Meister, 2013) and consisted of a circle expanding from a radius of 2° to 20° in 250 ms and held for 250 ms. The stimulus was presented 15 times, with a 500-ms interval between presentations. Mice behavior was recorded with an overhead camera. Mice were placed in the enclosure for 10 min before stimulation began to allow time for acclimation. Three responses (freezing, fleeing, and tail shaking) were assessed during the rooming stimulation (Koehler et al., 2019; Lim et al., 2016; Salay et al., 2018; Yilmaz and Meister, 2013). Mice were counted as positive rooming responders if they exhibited at least one of these behaviors during the course of stimulation. Each mouse was subjected to one test. The enclosure was thoroughly cleaned after each test.

[0103] Quantification and statistical analysis Excel and GraphPad Prism 9 were used for statistical analysis. Full statistical details of each experiment are provided in the figure legends.

[0104] For behavioral tests, injections and injury procedures were performed bilaterally on both eyes of each mouse, and the "n" shown represents individual mice. For other tests, injections and injury procedures were performed unilaterally on one eye of each mouse, and the "n" shown represents individual eyes, therefore, only one eye per mouse was used for statistical analysis.

[0105] Unpaired t-tests were used to compare two groups. Multiple groups were compared using one-way analysis of variance with Tukey's multiple comparison test, with follow-up tests to compare each group with all other groups. Fisher's exact test was used to compare groups on the visual cliff test and the rooming visual stimulus response test. P values ​​≤ 0.05 were considered statistically significant.

[0106] result 1. Excitotoxicity and axonal injury lead to a loss of CaMKII activity in RGCs. CaMKII has four isoforms in mammals (α, β, γ, and δ), and each isoform is expressed from a different gene (Hudmon and Schulman, 2002b). CaMKIIα and CaMKIIβ are the two major isoforms that are highly expressed in the rodent retina (Terashima et al., 1994). Activation of CaMKII mediates Ca uptake through NMDARs. 2+ Influx and subsequent Ca 2+ / The resulting conformational change of CaMKII, initiated by calmodulin binding, allows autophosphorylation at either threonine 286 (T286) for CaMKIIα or threonine 287 (T287) for CaMKIIβ, which is important for sustained activation of both isoforms (Miller et al., 1988; Schworer et al., 1988; Thiel et al., 1988). 2+ Even when its concentration is reduced, CaMKII remains active ( Lisman et al., 2002 ).

[0107] We investigated CaMKII phosphorylation in RGCs after retinal injury using an antibody that specifically recognizes CaMKIIα / β autophosphorylation (Figures 2A-2F). First, we injected toxic levels of NMDA (20 mM, 1.5 μl) that damage RGCs into the vitreous cavity of 8-week-old C57BL / 6 mice, and then damaged RGC cell bodies using NMDA-induced excitotoxicity (Manabe and Lipton, 2003; Seitz and Tamm, 2013). In PBS-injected control retinas, CaMKII was highly phosphorylated in RGCs that were labeled by Tuj1 immunoreactivity in retinal flat-mount preparations (Figures 1A-1C). However, in NMDA-injected retinas, CaMKII was extensively dephosphorylated in Tuj1+ RGCs 2 h after injection (Figures 1D-1H). Immunoblots using purified RGCs confirmed a significant decrease in CaMKII phosphorylation after NMDA-induced excitotoxicity (Figures 2G and 2H). Loss of CaMKII activity correlated with a loss of over 80% of RGCs 1 week after NMDA injection (FIGS. 3A and 3E).

[0108] We next used an optic nerve crush (ONC) model to examine whether CaMKII activity changes after RGC axonal injury. Axotomy caused by ONC resulted in delayed death of RGCs, with nearly 80% loss of RGCs 2 weeks after injury (Berkelaar et al., 1994; Hu et al., 2012). ONC was performed in 8-week-old C57BL / 6 mice, and phosphorylation of CaMKII was analyzed at 5, 7, and 9 days post-crush (dpc) during the course of RGC degeneration. Compared with the uniform phosphorylation of CaMKII in all RGCs in uninjured retinas (Figures 1I-1K), at each time point after injury, a portion of RGCs (approximately 10%) lost CaMKII activity, consistent with the gradual loss of RGCs after axotomy (Figures 1L-1V). Overall, these results indicate that excitotoxicity to RGC somas or optic nerve injury to RGC axons resulted in loss of CaMKII activity prior to the loss of RGCs.

[0109] Because CaMKII activity is highly detectable in all RGCs in the uninjured retina (Figures 1B and 1J), we investigated whether CaMKII activity is required for normal RGC survival. CaMKII activity was inhibited by daily intravitreal injections (7 injections over 7 days) of myristoylated Autocamtide-2-related inhibitory peptide (AIP, 1 mM, 1.5 μl), a highly potent and specific substrate-competitive inhibitor of CaMKII (Goebel, 2009; Laabich and Cooper, 2000). One week after AIP injection, approximately half of the RGCs were lost compared to vehicle controls (Figures 1W-1Y). These results indicate that CaMKII activity is essential for RGC survival in the normal retina.

[0110] 2. Reactivation of CaMKII protects RGCs from excitotoxicity or axonal injury. To examine whether enhancing CaMKII activity is sufficient to protect RGCs from excitotoxicity or axonal injury, we performed intravitreal injections for AAV2-mediated gene transfer of CaMKII variants into RGCs in 8-week-old C57BL / 6 mice 2 weeks before the onset of NMDA injection or ONC-mediated injury. AAV2 was effective in transducing more than 95% of RGCs (Figures 4A-4D) (Park et al., 2008). The expression level of CaMKII variants in RGCs was approximately 60% of endogenous CaMKII based on the relative pan-CaMKII immunofluorescence intensity 2 weeks after AAV injection (Figures 4E-4K).

[0111] First, we investigated the protective effect of CaMKIIα variants on RGC cell body damage caused by NMDA-induced excitotoxicity. One week after NMDA administration, Tuj1 +The number of RGCs nearly doubled (~34%) in wild-type CaMKIIα-treated retinas compared to the control group injected with AAV2-EBFP. In this case, only ~15% of RGCs remained (Figure 3A, Figure 3B, and Figure 3E). To examine whether kinase activity is essential for CaMKIIα-mediated RGC protection, two kinase-dead mutants, CaMKIIα K42R and CaMKIIα K42D, were tested by replacing the conserved lysine residue in the catalytic core of the enzyme with arginine or aspartic acid, preventing ATP binding required for kinase activity (Hanson et al., 1994). No improvement in RGC survival was observed with either CaMKIIα K42R (Figure 3A, 3C, and 3E) or CaMKIIα K42D treatment (Figure 5A, 5B, and 5F). This indicated that activity of CaMKII kinase is required to protect RGCs from NMDA-induced excitotoxic RGC death.

[0112] To examine whether further enhancement of CaMKII activity might be more effective in countering excitotoxic injury, we tested CaMKIIα T286D, a constitutively active mutant of CaMKIIα that simulates its autophosphorylated state (Fong et al., 1989). Remarkably, CaMKIIα T286D robustly protected the majority of RGCs (~90%) compared with a small percentage of surviving RGCs (~15%) in the control group (Figures 3A, 3D, and 3E) 1 week after NMDA injection. However, the autophosphorylation-deficient mutant, CaMKIIα T286A (Fong et al., 1989), was much less effective than CaMKIIα T286D in protecting RGCs (Figures 5A and 5F).

[0113] In addition to autophosphorylation of CaMKIIα at T286, phosphorylation at T305 and T306 within the calmodulin-binding domain also regulates its kinase activity. 2+Following dissociation of / CaM, autophosphorylation of CaMKIIα at T305 and T306 abolishes its sensitivity to calmodulin binding, thus preventing its further activation (Coultrap et al., 2010; Patton et al., 1990). In hippocampal neurons, phosphorylation at T305 and T306 is involved in CaMKIIα T286D-mediated regulation of spine size and synaptic strength, as well as induction of long-term potentiation (LTP) or long-term depression (LTD) (Pi et al., 2010a; Pi et al., 2010b). To examine whether further phosphorylation of CaMKIIα at T305 and T306 affects RGC survival, we tested the therapeutic effects of CaMKIIα T286D / T305A / T306A (non-phosphorylated form of T305 / T306) and CaMKIIα T286D / T305D / T306D (pseudo-phosphorylated form of T305 / T306) after excitotoxic damage to RGC soma. CaMKIIα T286D / T305A / T306A protected approximately 90% of RGCs 1 week after NMDA injection (Figures 5D and 5F). CaMKIIα T286D / T305D / T306D protected approximately 80% of RGCs, but was slightly less effective at protecting RGCs compared to T286D / T305A / T306A (Figures 5E and 5F). These results suggest that phosphorylation at T305 and T306 may modestly prevent the maximal effect of CaMKIIα T286D in protecting RGCs.

[0114] The ubiquitous CAG promoter in our AAV2 expression system drives transgene expression in over 95% of RGCs, as well as several other non-RGC cells (Park et al., 2008). To examine whether CaMKII-mediated RGC protection is indeed due to transgene expression in a cell-autonomous manner, we restricted CaMKII expression to RGCs using the mouse γ-synuclein promoter (mSncg), a recently developed RGC-specific promoter that drives AAV2-mediated transgene expression in mouse RGCs (Wang et al., 2020). One week after NMDA injection, AAV2-mSncg-mediated expression of CaMKIIα T286D protected RGCs as effectively as that mediated by AAV2-CAG (Figures 5G-5I). This indicates that cell-autonomous expression of CaMKIIα T286D in RGCs is essential to protect them from NMDA-induced excitotoxicity.

[0115] Because CaMKIIβ is the other major isoform expressed in the mouse retina, we examined the protective effect of CaMKIIβ variants after NMDA-induced excitotoxicity. Similar to the protective effect obtained from CaMKIIα treatment, wild-type CaMKIIβ showed a modest protective effect, but the constitutively active mutant, CaMKIIβ T287D, was much more potent in protecting RGCs 1 week after NMDA injection (Figures 3F, 3G, 3I, and 3J). As expected, the autophosphorylation-deficient mutant, CaMKIIβ T287A, protected much fewer RGCs compared to CaMKIIβ T287D (Figures 5L and 5O). Phosphorylation at T306 and T307 within the calmodulin-binding domain also slightly reduced the protective effect of CaMKIIβ T287D, as evidenced by the effect of treatment with triple mutants CaMKIIβ T287D / T306A / T307A or CaMKIIβ T287D / T306D / T307D (Figures S3M-S3O). CaMKIIβ-mediated protection of RGCs apparently also depended on its kinase activity, since the kinase-dead mutants CaMKIIβ K43R (Figures 3H and 3J) or CaMKIIβ K43D (Figures 5K and 5O) were unable to protect RGCs under the same experimental conditions.

[0116] We next investigated the protective effect of CaMKIIα variants when RGC axons were injured by ONC. Two weeks after ONC, the number of surviving RGCs (about 49%) was more than doubled with wild-type CaMKIIα treatment compared with surviving RGCs (about 22%) in the control group injected with AAV-EBFP (Figures 3K, 3L, and 3O). However, the kinase-dead mutant CaMKIIα K42R showed no protection (Figures 3M and 3O). This indicates that the kinase activity of CaMKII is also required to protect RGCs from axotomy-induced cell death. Surprisingly, the constitutively active mutant CaMKIIα T286D showed the most effective protective effect, with about 90% of RGCs still surviving two weeks after injury (Figures 3N and 3O). As expected, CaMKIIβ variants showed similar protective effects as their CaMKIIα counterparts. This confirms the requirement for its kinase activity and the striking protective effect when activity of CaMKIIβ was further enhanced by the constitutively active mutant CaMKIIβ T287D (FIGS. 3P-3T).

[0117] Using two injury models, our results demonstrate that excitotoxic injury to RGC somas or ONC injury to RGC axons inevitably leads to inactivation of CaMKII prior to RGC death. Reactivation of CaMKII via gene transfer of a constitutively active CaMKII mutant robustly protects RGCs from both insults. CaMKIIα T286D showed the most robust protection for RGCs, so our subsequent studies focused on it.

[0118] 3. Reactivation of CaMKII provides post-injury and long-term protection to RGCs. To mimic a clinically relevant setting, we tested whether CaMKII could protect RGCs if reactivated after injury. Although the degeneration of RGCs in the excitotoxicity model is too fast for AAV-mediated gene expression to be effective, the relatively slow degeneration of RGCs in the optic nerve crush model could allow the time frame required for AAV-mediated gene therapy (Sun et al., 2011). Therefore, we performed intravitreal AAV injections immediately after ONC. After 2 weeks, approximately 70% of RGCs remained in the CaMKIIα T286D-treated group, tripling the survival rate compared to the control group that received AAV-mediated gene transfer of EBFP (Figures 6A-6C). These results indicate that delayed reactivation of CaMKII can robustly protect RGCs after injury initiation.

[0119] Reactivation of CaMKII protected the majority of RGCs 1 week after NMDA-induced excitotoxicity or 2 weeks after ONC. To evaluate the long-term protective effect of CaMKIIα T286D treatment, we assayed RGC survival at a later time point after excitotoxicity or axonal injury. After excitotoxic injury to RGC somata, the number of RGCs in the control group administered AAV-EBFP continued to decrease, from about 12% of surviving RGCs at 2 months (FIG. 6D) to about 8% at 12 months (FIG. 6E). Notably, with CaMKIIα T286D treatment, about 84% and about 77% of RGCs survived 2 and 12 months after injury, respectively (FIGS. 6F-6H). Next, we examined long-term RGC survival after ONC, which damages RGC axons. Only about 17%, 12%, and 7% of RGCs remained from the control group administered AAV-EBFP 1, 2, and 6 months after ONC (Figures 6I-6K and 6O), whereas CaMKIIα T286D treatment protected about 82%, 81%, and 77% of RGCs at the same time points (Figures 6L-6O). Collectively, these results demonstrate that reactivation of CaMKII can provide long-term protection of RGCs from excitotoxicity or axonal injury.

[0120] 4. CREB acts downstream of CaMKII in protecting RGCs. To demonstrate downstream signaling of CaMKII-mediated RGC protection, the stimulus-inducible transcription factor CREB (cAMP response element binding protein) was of particular interest because CREB plays an important role downstream of CaMKII in regulating synaptic plasticity and long-term memory formation (Deisseroth et al., 1996; Ma et al., 2014). CREB is also known to regulate brain neuron survival (Lonze and Ginty, 2002). However, the role of CREB in regulating retinal neuron survival remains to be determined. CREB is phosphorylated at Ser133 by CaMKII and other kinases, and the phosphorylation event is required for the transcriptional activity of CREB (Sheng et al., 1991).

[0121] First, we investigated whether CREB acts downstream of CaMKII to protect RGCs in the NMDA-induced excitotoxicity model. Using an antibody that recognizes phosphorylated CREB at Ser133, we found that CREB downregulates nearly all Tuj1 in the intact retina. + We found that CREB was highly phosphorylated in RGCs (Figures 7A, 7D, and 7E). However, a large loss of CREB phosphorylation occurred in RGCs 2 hours after NMDA injection (Figures 7B, 7D, and 7E). This indicates that impaired CREB activity may be downstream of CaMKII inactivation in RGCs after excitotoxic injury. We then examined whether reactivation of CaMKII could restore CREB activity after NMDA injection. Indeed, AAV2-mediated gene transfer of CaMKIIα T286D was sufficient to maintain CREB phosphorylation in nearly all RGCs exposed to NMDA insult (Figures 7C, 7D, and 7E).

[0122] To further investigate the role of CREB downstream of CaMKII in protecting RGCs, we performed AAV2-mediated gene transfer of CaMKIIα T286D together with A-CREB, a dominant-negative variant of CREB that binds to endogenous CREB protein and prevents CREB from binding to DNA (Ahn et al., 1998). AAV2 delivery was performed in 8-week-old C57BL / 6 mice 2 weeks before NMDA injection, and RGC viability was analyzed 1 week after excitotoxic injury. CaMKIIα T286D-mediated RGC protection was nearly neutralized by co-treatment of A-CREB (Figures 7F-7H). This indicates that activation of CREB is required for CaMKII-mediated RGC protection from excitotoxic injury. Indeed, co-treatment of A-CREB significantly impaired the phosphorylation of CREB by CaMKIIα T286D in RGCs 2 hours after NMDA injection (Figures 8A-8D). To test whether activation of CREB alone is sufficient to protect RGCs from excitotoxicity, independent of CaMKII activation, we performed AAV2-mediated gene transfer of a constitutively active variant of CREB, VP16-CREB (Barco et al., 2002), 2 weeks before NMDA injection and analyzed RGC viability 1 week after injury. Clearly, VP16-CREB treatment alone protected the majority (~65%) of RGCs (Figures 7I-7K). Consistent with a similar role in the hippocampus (Zhang et al., 2016), VP16-CREB maintained phosphorylation of CREB in RGCs 2 h after NMDA injection (Figures 8E-8H). The protective effect of VP16-CREB (188.7 ± 25.4 RGCs / 0.1 mm 2 ) was significantly higher than the protective effect of CaMKIIα T286D (251.2±16.9RGC / 0.1mm 2 ), suggesting that there may be other unidentified factors downstream of CaMKII in protecting RGCs from excitotoxicity.

[0123] BDNF (brain-derived neurotrophic factor) / TrkB (tropomyosin-related kinase receptor type B) signaling contributes to the neuroprotection of RGCs, and excitotoxicity downregulates this pathway (Chitranshi et al., 2019; Gomes et al., 2012). To examine whether CaMKIIα T286D treatment affects BDNF / TrkB signaling, we examined the phosphorylation of TrkB, which is essential for downstream signaling initiated by BDNF binding (Nagahara and Tuszynski, 2011). Phosphorylation of TrkB at Tyr817 was readily detectable in RGCs in uninjured retinas, and NMDA insult led to a significant decrease in phospho-TrkB in RGCs, which was rescued by CaMKIIα T286D treatment (Figures 8I and 8J). These results indicate that in addition to CREB, reactivation of CaMKII may affect other pathways, such as BDNF / TrkB signaling, that promote RGC survival after excitotoxic injury.

[0124] Next, we investigated whether CREB acts downstream of CaMKII to protect RGCs in the ONC-induced axonal injury model. 5, 7, and 9 days after ONC, CREB activity was analyzed using an antibody that recognizes CREB phosphorylation at Ser133. Compared with the uniform CREB phosphorylation in RGCs in uninjured retinas (Fig. 7L), a percentage (approximately 10%) of RGCs had lost CREB phosphorylation at each time point examined (Figs. 7M-7O, 7S, and 7T). The progressive loss of CREB activity coincided with the gradual loss of CaMKII activity after ONC (Figs. 1L-1V). This indicates that the impaired CREB activity may be downstream of CaMKII inactivation after RGC axonal injury. Next, we investigated whether reactivation of CaMKII could rescue CREB phosphorylation after ONC. Indeed, AAV-mediated gene transfer of CaMKIIα T286D reduced the phosphorylation of nearly all Tuj1 axons after injury. +ONC maintained CREB phosphorylation in RGCs (Figures 7P-7T). Similar to the observations in the excitotoxicity model, CREB transcriptional activity was required for CaMKII-mediated RGC protection 2 weeks after RGC axonal injury, as the dominant-negative variant A-CREB neutralized the protective effect mediated by CaMKIIα T286D (Figures 7U-7W). As expected, A-CREB cotreatment significantly impaired CaMKIIα T286D-mediated CREB phosphorylation in RGCs 5 days after ONC (Figures 8K-8N). Conversely, enhancing CREB activity alone by gene transfer of the constitutively active variant VP16-CREB maintained CREB phosphorylation in RGCs 5 days after ONC (Figures 8O-8R) and efficiently protected RGCs from axonal injury (Figures 7X-7Z).

[0125] The DLK (dual leucine zipper kinase) / c-Jun pathway is a prominent mediator of RGC cell death after optic nerve injury (Watkins et al., 2013; Welsbie et al., 2013). To examine whether CaMKIIα T286D treatment regulates the DLK / c-Jun pathway, we examined the expression of DLK and the phosphorylation of c-Jun at serine 63. As previously reported, the phosphorylation levels of DLK and c-Jun were very low in uninjured retina and dramatically upregulated 3 days after ONC. However, CaMKIIα T286D treatment did not affect these changes (Figures 8S-8V). This indicates that CaMKIIα-mediated RGC protection from optic nerve injury may not act by suppressing the DLK / c-Jun pathway.

[0126] Taken together, our results demonstrate that CREB is a critical downstream effector of CaMKII, as it is necessary and sufficient to protect RGCs from excitotoxicity to the RGC soma or compressive injury to the axons.

[0127] 5. CaMKII-mediated protection of RGCs in induced and genetic models of glaucoma. Glaucoma is characterized by the progressive degeneration of RGC axons and subsequent loss of their respective cell bodies and is the leading cause of irreversible blindness worldwide. The pathology of glaucoma is poorly understood but is typically associated with elevated intraocular pressure (IOP), which leads to damage to RGC axons and eventual death of RGCs (Calkins, 2012; Nickells et al., 2012; Weinreb et al., 2016).

[0128] We first tested whether gene transfer of CaMKIIα T286D protects RGCs in a mouse model of hypertension-dependent glaucoma. To induce high intraocular pressure, magnetic microbeads were injected into the anterior chamber to occlude aqueous humor outflow (Figures 9A and 9B) (Ito et al., 2016). As a result, IOP showed a sustained elevation over the following 8 weeks (Figure 9C). Two weeks before microbead injection, intravitreal injection of AAV was performed for treatment of either CaMKIIα T286D or EBFP as a control, and 8 weeks after induction of high intraocular pressure, RGC viability was analyzed. Two weeks after microbead injection, the expression level of CaMKII was increased by about 60% based on the relative pan-CaMKII immunofluorescence intensity (Figures 10A-10E). Compared to about 66% surviving RGCs in the control group, CaMKIIα T286D treatment protected about 82% of RGCs (Figures 9D-9F). This indicates that enhancing CaMKII is effective in protecting RGCs as they endure ongoing damage from elevated IOP. We also examined axonal survival in optic nerve sections collected 1 mm posterior to the eye (Yang et al., 2016) and found that CaMKIIα T286D treatment provided significant protection of RGC axons (Figures S10F-S10I).

[0129] Although elevated IOP is the best-known risk factor for glaucoma, a significant number of patients develop the disease despite normal intraocular pressure. GLAST- / - mice, a model of normal tension glaucoma (Harada et al., 2007), show characteristic degeneration of RGCs due to accumulation of glutamate in the extracellular fluid, as well as increased oxidative stress. We tested whether gene transfer of CaMKIIα T286D protects RGCs in the normal tension glaucoma model of GLAST- / - mice. The onset of RGC death in GLAST-deficient mice begins around postnatal day 7. Therefore, we performed intravitreal AAV injections for CaMKIIα T286D treatment on postnatal day 1 and analyzed the animals at 2 months of age, when RGC degeneration was stable (Harada et al., 2007). The expression level of CaMKII increased by about 50% based on the relative pan-CaMKII immunofluorescence intensity 3 weeks after AAV injection (Figures 10J-10N). Importantly, CaMKIIα T286D treatment protected over 90% of RGCs compared with about 65% remaining in the control group (Figures 9G-9I). Consistent with the loss of RGCs, optic nerve degeneration and cupping were evident in 8-month-old GLAST- / - mice (Harada et al., 2007). CaMKIIα T286D treatment also mitigated the optic disc depletion of RGC axons in these mice (Figures 10O and 10P).

[0130] Taken together, our results indicate that CaMKII may be a valuable therapeutic target for slowing disease progression in glaucoma.

[0131] 6. Reactivation of CaMKII protects RGC axons and their projections in the brain. RGC axons are the only pathway that transmits visual information from the retina to the brain. RGC axons rarely regenerate after injury, so degeneration of RGC axons leads to permanent vision loss (Tran et al., 2019). Therefore, protecting the integrity of RGC axons is important for maintaining vision. Although we did not expect that reactivation of CaMKII would render RGC axons resistant to severe mechanical injury such as optic nerve crush, it is crucial to examine whether CaMKII treatment protects RGC axons from pathophysiological insults such as excitotoxicity. Indeed, excitotoxic insults, in addition to damaging RGC cell bodies, lead to Waller-like degeneration of RGC axons in the optic nerve and loss of target innervation in the brain (Saggu et al., 2010).

[0132] To examine whether reactivation of CaMKII protects RGC axons and their axonal projections to the brain, Alexa Fluor488-conjugated cholera toxin subunit B (CTB) was injected intravitreally to trace RGC axons anterogradely to the lateral geniculate nucleus (LGN) and superior colliculus (SC), the two main projection targets of RGC axons in the brain (Figure 11A). One week after NMDA injection, RGC axons were severely damaged, and CTB labeling intensity was reduced to about 17% in the optic nerve compared to uninjured controls (Figures 11B, 11C, and 11E). In contrast, after CaMKIIα T286D treatment, the majority of axons were protected from excitotoxicity, and CTB labeling intensity was restored to about 84% (Figures 11D and 11E).

[0133] In mice, RGC axons project primarily to the contralateral hemisphere (Herrera and Mason, 2007). Severe loss of RGC axonal projections in brain targets was observed 1 week after NMDA injection, with only about 24% and about 9% of the CTB labeling intensity remaining in the contralateral LGN (Figures 11F, 11G, and 11I) and SC (Figures 11J, 11K, and 11M), respectively. In contrast, CaMKIIα T286D treatment preserved the majority of RGC axonal projections to the contralateral LGN (about 73%, Figures 6H and 6I) and SC (about 85%, Figures 11L and 11M). Similarly, RGC axonal projections to the ipsilateral LGN (Figures 12A-12D) and SC (Figures 12E-12H) were preserved. Taken together, our results demonstrated that reactivation of CaMKII not only protects RGC cell bodies but also robustly preserves the integrity of RGC axons in the optic nerve and their distal projection targets in the brain, which is ultimately necessary to maintain functional vision.

[0134] 7. Reactivation of CaMKII preserves visual function. To assess whether CaMKII-mediated protection of RGCs from excitotoxicity could preserve vision, we first tested whether treatment with CaMKIIα T286D preserved RGC function using pattern electroretinograms (PERGs), which measure RGC activity in response to contrast modulation of patterned visual stimuli (Porciatti, 2007). PERGs were induced simultaneously from each eye of mice using binocular stimulation and a common nose electrode (Chou et al., 2014). PERG responses (21.4 μV) were readily detectable in uninjured retinas (Figure 13A) and were significantly reduced 7 days after NMDA injection (4.0 μV) (Figures 13B and 13D). This reflects the severe loss of RGC function after NMDA-induced excitotoxicity. Importantly, reactivation of CaMKII via gene transfer of CaMKIIα T286D maintained PERG responses to levels similar to those recorded in uninjured retinas (Figures 13C and 13D).

[0135] After leaving the eye, visual information passes through several relay centers in the brain, such as the LGN and SC, before finally reaching the primary visual cortex. We then tested whether the preserved RGC responses could be transmitted to the primary visual cortex in vivo. We recorded pattern visual evoked potentials (PVEPs) in the primary visual cortex from uninjured, NMDA-lesioned, and CaMKIIα T286D-treated groups (Porciatti et al., 1999). Patterned visual stimulation elicited a prominent response (104.8 μV) in uninjured animals (FIG. 7E), which was significantly reduced (20.9 μV) after NMDA-lesion (FIG. 13F). Notably, CaMKIIα T286D treatment maintained the PVEP response at a level comparable to the uninjured condition (FIGS. 13G and 13H). Our results demonstrate that reactivation of CaMKII preserves function from excitotoxic injury throughout the entire visual pathway, from the retina to the primary visual cortex of the brain.

[0136] To test whether CaMKII-mediated protection of the visual pathway truly preserves vision, we performed several vision-based behavioral tests. First, we used the visual water task to quantitatively measure the visual acuity of mice after treatment with CaMKIIα T286D. The visual water task, a two alternative forced-choice visual behavioral test (Ecker et al., 2010; Prusky et al., 2000), utilizes the ability of rodents trained to swim to a submerged hidden platform, the location of which is associated with a visual stimulus displayed on a computer monitor (Figure 13I). Mice were trained to swim toward a grating with a low spatial frequency (0.05 cycles / degree). The spatial frequency was then gradually increased. Visual acuity (i.e., spatial frequency threshold) was determined when the animals made less than 70% correct choices. Visual acuity was measured at approximately 0.515 c / d in uninjured mice (FIGS. 13J and 13M) and decreased to approximately 0.128 c / d after NMDA injury (FIGS. 13K and 13M). Importantly, CaMKIIα T286D treatment significantly improved visual acuity to approximately 0.388 c / d (FIGS. 13L and 13M).

[0137] A visual cliff test was then performed to assess the maintenance of the ability to discriminate visual depth after CaMKIIα T286D treatment. This test is based on the innate tendency of mice to avoid the deep side of a visual cliff and step onto the shallow side (Fox, 1965; Gu et al., 2018). Mice were placed on a central platform between the deep and shallow sides of the cliff, and their choice to step towards either side was recorded (Figure 13N). In the uninjured group, 11 of 12 mice chose the shallow (safe) side, consistent with previous reports (Fox, 1965; Gu et al., 2018). Importantly, significantly worse performance was recorded after NMDA injury, with 7 of 12 mice choosing the shallow side. In contrast, all 12 CaMKIIα T286D-treated mice chose the shallow side (Figure 13O).

[0138] Finally, we evaluated the innate defensive response of mice to rooming visual stimuli representing environmental threats. Rooming experiments were conducted in an enclosure equipped with a shelter for the mice to hide in, a camera to record the mice's behavior, and an overhead monitor to display the rooming stimuli (Figure 13P) (Koehler et al., 2019, Lim et al., 2016, Yilmaz and Meister, 2013). In response to the rooming stimuli, mice with normal vision consistently exhibited one or more of the following behaviors: freezing, fleeing to the shelter, and tail shaking, consistent with previous studies (Koehler et al., 2019, Lim et al., 2016, Salay et al., 2018, Yilmaz and Meister, 2013). As a result, mice were scored as responders to the rooming stimuli if they responded to one or more of these behaviors. In the uninjured group, all 12 mice were responders. After NMDA lesioning, only 3 of 12 mice responded to the rooming stimulus. Notably, 11 of 12 CaMKIIα T286D-treated mice responded to the rooming stimulus (FIG. 13Q).

[0139] Taken together, our results provide the first in vivo evidence that CaMKII-mediated RGC protection can maintain functional vision.

[0140] While embodiments have been shown and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like, can be made without departing from the spirit of the disclosure, and therefore are deemed to be within the scope of the present disclosure as defined in the following claims.

[0141] References Ahn,S.,Olive,M.,Aggarwal,S.,Krylov,D.,Ginty,D.D.,and Vinson,C.(1998).A dominant-negative inhibitor of CREB reveals that it is a general mediator of stimulus-dependent transcription of c-fos.Mol Cell Biol 18,967-977. Almasieh,M.,and Levin,L.A.(2017).Neuroprotection in Glaucoma:Animal Models and Clinical Trials.Annu Rev Vis Sci 3,91-120. Ashpole,N.M.,and Hudmon,A.(2011).Excitotoxic neuroprotection and vulnerability with CaMKII inhibition.Mol Cell Neurosci 46,720-730. Ashpole,N.M.,Song,W.,Brustovetsky,T.,Engleman,E.A.,Brustovetsky,N.,Cummins,T.R.,and Hudmon,A.(2012).Calcium / calmodulin-dependent protein kinase II(CaMKII)inhibition induces neurotoxicity via dysregulation of glutamate / calcium signaling and hyperexcitability.J Biol Chem 287,8495-8506. Barco,A.,Alarcon,J.M.,and Kandel,E.R.(2002).Expression of constitutively active CREB protein facilitates the late phase of long-term potentiation by enhancing synaptic capture.Cell 108,689-703. Benowitz,L.I.,He,Z.,and Goldberg,J.L.(2017).Reaching the brain:Advances in optic nerve regeneration.Exp Neurol 287,365-373. Berkelaar,M.,Clarke,D.B.,Wang,Y.C.,Bray,G.M.,and Aguayo,A.J.(1994).Axotomy results in delayed death and apoptosis of retinal ganglion cells in adult rats.J Neurosci 14,4368-4374. Berridge,M.J.,Lipp,P.,and Bootman,M.D.(2000).The versatility and universality of calcium signalling.Nat Rev Mol Cell Bio 1,11-21. Berry,M.,Ahmed,Z.,and Logan,A.(2018).Return of function after CNS axon regeneration:Lessons from injury-responsive intrinsically photosensitive and alpha retinal ganglion cells.Prog Retin Eye Res. Calkins,D.J.(2012).Critical pathogenic events underlying progression of neurodegeneration in glaucoma.Prog Retin Eye Res 31,702-719. Chitranshi,N.,Dheer,Y.,Mirzaei,M.,Wu,Y.,Salekdeh,G.H.,Abbasi,M.,Gupta,V.,Vander Wall,R.,You,Y.,Graham,S.L.,et al.(2019).Loss of Shp2 Rescues BDNF / TrkB Signaling and Contributes to Improved Retinal Ganglion Cell Neuroprotection.Mol Ther 27,424-441. Chou,T.H.,Bohorquez,J.,Toft-Nielsen,J.,Ozdamar,O.,and Porciatti,V.(2014).Robust Mouse Pattern Electroretinograms Derived Simultaneously From Each Eye Using a Common Snout Electrode.Invest Ophth Vis Sci 55,2469-2475. Coultrap,S.J.,Buard,I.,Kulbe,J.R.,Dell’Acqua,M.L.,and Bayer,K.U.(2010).CaMKII autonomy is substrate-dependent and further stimulated by Ca2+ / calmodulin.J Biol Chem 285,17930-17937. Deisseroth,K.,Bito,H.,and Tsien,R.W.(1996).Signaling from synapse to nucleus:postsynaptic CREB phosphorylation during multiple forms of hippocampal synaptic plasticity.Neuron 16,89-101. Dhande,O.S.,Stafford,B.K.,Lim,J.H.A.,and Huberman,A.D.(2015).Contributions of Retinal Ganglion Cells to Subcortical Visual Processing and Behaviors.Annu Rev Vis Sc 1,291-328. Dong,Z.,Saikumar,P.,Weinberg,J.M.,and Venkatachalam,M.A.(2006).Calcium in cell injury and death.Annu Rev Pathol-Mech 1,405-434. Ecker,J.L.,Dumitrescu,O.N.,Wong,K.Y.,Alam,N.M.,Chen,S.K.,LeGates,T.,Renna,J.M.,Prusky,G.T.,Berson,D.M.,and Hattar,S.(2010).Melanopsin-expressing retinal ganglion-cell photoreceptors:cellular diversity and role in pattern vision.Neuron 67,49-60. Fong,Y.L.,Taylor,W.L.,Means,A.R.,and Soderling,T.R.(1989).Studies of the regulatory mechanism of Ca2+ / calmodulin-dependent protein kinase II.Mutation of threonine 286 to alanine and aspartate.J Biol Chem 264,16759-16763. Fox,M.W.(1965).The visual cliff test for the study of visual depth perception in the mouse.Anim Behav 13,232-233. Goebel,D.J.(2009).Selective blockade of CaMKII-alpha inhibits NMDA-induced caspase-3-dependent cell death but does not arrest PARP-1 activation or loss of plasma membrane selectivity in rat retinal neurons.Brain Res 1256,190-204. Gomes,J.R.,Costa,J.T.,Melo,C.V.,Felizzi,F.,Monteiro,P.,Pinto,M.J.,Inacio,A.R.,Wieloch,T.,Almeida,R.D.,Graos,M.,et al.(2012).Excitotoxicity downregulates TrkB.FL signaling and upregulates the neuroprotective truncated TrkB receptors in cultured hippocampal and striatal neurons.J Neurosci 32,4610-4622. Gu,L.,Bok,D.,Yu,F.,Caprioli,J.,and Piri,N.(2018).Downregulation of splicing regulator RBFOX1 compromises visual depth perception.PLoS One 13,e0200417. Hanson,P.I.,Meyer,T.,Stryer,L.,and Schulman,H.(1994).Dual Role of Calmodulin in Autophosphorylation of Multifunctional Cam Kinase May Underlie Decoding of Calcium Signals.Neuron 12,943-956. Harada,T.,Harada,C.,Nakamura,K.,Quah,H.M.,Okumura,A.,Namekata,K.,Saeki,T.,Aihara,M.,Yoshida,H.,Mitani,A.,et al.(2007).The potential role of glutamate transporters in the pathogenesis of normal tension glaucoma.J Clin Invest 117,1763-1770. Hardingham,G.E.,and Bading,H.(2010).Synaptic versus extrasynaptic NMDA receptor signalling:implications for neurodegenerative disorders.Nat Rev Neurosci 11,682-696. Hartwick,A.T.E.,Hamilton,C.M.,and Baldridge,W.H.(2008).Glutamatergic calcium dynamics and deregulation of rat retinal ganglion cells.J Physiol-London 586,3425-3446. He,Z.,and Jin,Y.(2016).Intrinsic Control of Axon Regeneration.Neuron 90,437-451. Herrera,E.,and Mason,C.A.(2007).3.23-The Evolution of Crossed and Uncrossed Retinal Pathways in Mammals.In Evolution of Nervous Systems,J.H.Kaas,ed.(Oxford:Academic Press),pp.307-317. Hu,Y.,Park,K.K.,Yang,L.,Wei,X.,Yang,Q.,Cho,K.S.,Thielen,P.,Lee,A.H.,Cartoni,R.,Glimcher,L.H.,et al.(2012).Differential effects of unfolded protein response pathways on axon injury-induced death of retinal ganglion cells.Neuron 73,445-452. Hudmon,A.,and Schulman,H.(2002a).Neuronal Ca2+ / calmodulin-dependent protein kinase II:The role of structure and autoregulation in cellular function.Annu Rev Biochem 71,473-510. Hudmon,A.,and Schulman,H.(2002b).Structure-function of the multifunctional Ca2+ / calmodulin-dependent protein kinase II.Biochem J 364,593-611. Ito,Y.A.,Belforte,N.,Cueva Vargas,J.L.,and Di Polo,A.(2016).A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice.J Vis Exp,e53731. Khatib,T.Z.,and Martin,K.R.(2017).Protecting retinal ganglion cells.Eye(Lond)31,218-224. Koehler,C.C.,Hall,L.M.,Hellmer,C.B.,and Ichinose,T.(2019).Using Looming Visual Stimuli to Evaluate Mouse Vision.J Vis Exp. Laabich,A.,and Cooper,N.G.(2000).Neuroprotective effect of AIP on N-methyl-D-aspartate-induced cell death in retinal neurons.Brain Res Mol Brain Res 85,32-40. Laabich,A.,Li,G.,and Cooper,N.G.(2000).Calcium / calmodulin-dependent protein kinase II containing a nuclear localizing signal is altered in retinal neurons exposed to N-methyl-D-aspartate.Brain Res Mol Brain Res 76,253-265. Lafuente,M.P.,Villegas-Perez,M.P.,Selles-Navarro,I.,Mayor-Torroglosa,S.,De Imperial,J.M.,and Vidal-Sanz,M.(2002).Retinal ganglion cell death after acute retinal ischemia is an ongoing process whose severity and duration depends on the duration of the insult.Neuroscience 109,157-168. Laha,B.,Stafford,B.K.,and Huberman,A.D.(2017).Regenerating optic pathways from the eye to the brain.Science 356,1031-1034. Levin,L.A.,and Gordon,L.K.(2002).Retinal ganglion cell disorders:types and treatments.Prog Retin Eye Res 21,465-484. Lim,J.H.,Stafford,B.K.,Nguyen,P.L.,Lien,B.V.,Wang,C.,Zukor,K.,He,Z.,and Huberman,A.D.(2016).Neural activity promotes long-distance,target-specific regeneration of adult retinal axons.Nat Neurosci 19,1073-1084. Lipton,S.A.,and Rosenberg,P.A.(1994).Excitatory amino acids as a final common pathway for neurologic disorders.N Engl J Med 330,613-622. Lisman,J.,Schulman,H.,and Cline,H.(2002).The molecular basis of CaMKII function in synaptic and behavioural memory.Nat Rev Neurosci 3,175-190. Lonze,B.E.,and Ginty,D.D.(2002).Function and regulation of CREB family transcription factors in the nervous system.Neuron 35,605-623. Lucas,D.R.,and Newhouse,J.P.(1957).The toxic effect of sodium L-glutamate on the inner layers of the retina.AMA Arch Ophthalmol 58,193-201. Ma,H.,Groth,R.D.,Cohen,S.M.,Emery,J.F.,Li,B.,Hoedt,E.,Zhang,G.,Neubert,T.A.,and Tsien,R.W.(2014).gammaCaMKII shuttles Ca(2)(+) / CaM to the nucleus to trigger CREB phosphorylation and gene expression.Cell 159,281-294. Malihi,M.,Moura Filho,E.R.,Hodge,D.O.,and Sit,A.J.(2014).Long-term trends in glaucoma-related blindness in Olmsted County,Minnesota.Ophthalmology 121,134-141. Manabe,S.,and Lipton,S.A.(2003).Divergent NMDA signals leading to proapoptotic and antiapoptotic pathways in the rat retina.Invest Ophth Vis Sci 44,385-392. Massey,S.C.,and Miller,R.F.(1987).Excitatory amino acid receptors of rod- and cone-driven horizontal cells in the rabbit retina.J Neurophysiol 57,645-659. Massey,S.C.,and Miller,R.F.(1990).N-methyl-D-aspartate receptors of ganglion cells in rabbit retina.J Neurophysiol 63,16-30. Miller,S.G.,Patton,B.L.,and Kennedy,M.B.(1988).Sequences of Autophosphorylation Sites in Neuronal Type-Ii Cam Kinase That Control Ca-2+-Independent Activity.Neuron 1,593-604. Nagahara,A.H.,and Tuszynski,M.H.(2011).Potential therapeutic uses of BDNF in neurological and psychiatric disorders.Nat Rev Drug Discov 10,209-219. Nickells,R.W.,Howell,G.R.,Soto,I.,and John,S.W.(2012).Under pressure:cellular and molecular responses during glaucoma,a common neurodegeneration with axonopathy.Annu Rev Neurosci 35,153-179. Olney,J.W.(1982).The toxic effects of glutamate and related compounds in the retina and the brain.Retina 2,341-359. Park,K.K.,Liu,K.,Hu,Y.,Smith,P.D.,Wang,C.,Cai,B.,Xu,B.,Connolly,L.,Kramvis,I.,Sahin,M.,et al.(2008).Promoting axon regeneration in the adult CNS by modulation of the PTEN / mTOR pathway.Science 322,963-966. Parsons,M.P.,and Raymond,L.A.(2014).Extrasynaptic NMDA receptor involvement in central nervous system disorders.Neuron 82,279-293. Patton,B.L.,Miller,S.G.,and Kennedy,M.B.(1990).Activation of type II calcium / calmodulin-dependent protein kinase by Ca2+ / calmodulin is inhibited by autophosphorylation of threonine within the calmodulin-binding domain.J Biol Chem 265,11204-11212. Pi,H.J.,Otmakhov,N.,El Gaamouch,F.,Lemelin,D.,De Koninck,P.,and Lisman,J.(2010a).CaMKII control of spine size and synaptic strength:role of phosphorylation states and nonenzymatic action.Proc Natl Acad Sci U S A 107,14437-14442. Pi,H.J.,Otmakhov,N.,Lemelin,D.,De Koninck,P.,and Lisman,J.(2010b).Autonomous CaMKII can promote either long-term potentiation or long-term depression,depending on the state of T305 / T306 phosphorylation.J Neurosci 30,8704-8709. Porciatti,V.(2007).The mouse pattern electroretinogram.Doc Ophthalmol 115,145-153. Porciatti,V.,Pizzorusso,T.,and Maffei,L.(1999).The visual physiology of the wild type mouse determined with pattern VEPs.Vision Res 39,3071-3081. Prilloff,S.,Noblejas,M.I.,Chedhomme,V.,and Sabel,B.A.(2007).Two faces of calcium activation after optic nerve trauma:life or death of retinal ganglion cells in vivo depends on calcium dynamics.Eur J Neurosci 25,3339-3346. Prusky,G.T.,West,P.W.R.,and Douglas,R.M.(2000).Behavioral assessment of visual acuity in mice and rats.Vision Research 40,2201-2209. Saggu,S.K.,Chotaliya,H.P.,Blumbergs,P.C.,and Casson,R.J.(2010).Wallerian-like axonal degeneration in the optic nerve after excitotoxic retinal insult:an ultrastructural study.BMC Neurosci 11,97. Salay,L.D.,Ishiko,N.,and Huberman,A.D.(2018).A midline thalamic circuit determines reactions to visual threat.Nature 557,183-189. Schindelin,J.,Arganda-Carreras,I.,Frise,E.,Kaynig,V.,Longair,M.,Pietzsch,T.,Preibisch,S.,Rueden,C.,Saalfeld,S.,Schmid,B.,et al.(2012).Fiji:an open-source platform for biological-image analysis.Nat Methods 9,676-682. Schworer,C.M.,Colbran,R.J.,Keefer,J.R.,and Soderling,T.R.(1988).Ca2+ / calmodulin-dependent protein kinase II.Identification of a regulatory autophosphorylation site adjacent to the inhibitory and calmodulin-binding domains.J Biol Chem 263,13486-13489. Seitz,R.,and Tamm,E.R.(2013).N-methyl-D-aspartate(NMDA)-mediated excitotoxic damage:a mouse model of acute retinal ganglion cell damage.Methods Mol Biol 935,99-109. Sheng,M.,Thompson,M.A.,and Greenberg,M.E.(1991).Creb-a Ca2+-Regulated Transcription Factor Phosphorylated by Calmodulin-Dependent Kinases.Science 252,1427-1430. Sisk,D.R.,and Kuwabara,T.(1985).Histologic changes in the inner retina of albino rats following intravitreal injection of monosodium L-glutamate.Graefes Arch Clin Exp Ophthalmol 223,250-258. Sun,F.,Park,K.K.,Belin,S.,Wang,D.,Lu,T.,Chen,G.,Zhang,K.,Yeung,C.,Feng,G.,Yankner,B.A.,et al.(2011).Sustained axon regeneration induced by co-deletion of PTEN and SOCS3.Nature 480,372-375. Terashima,T.,Ochiishi,T.,and Yamauchi,T.(1994).Immunocytochemical Localization of Calcium / Calmodulin-Dependent Protein-Kinase-Ii Isoforms in the Ganglion-Cells of the Rat Retina-Immunofluorescence Histochemistry Combined with a Fluorescent Retrograde Tracer.Brain Res 650,133-139. Tham,Y.C.,Li,X.,Wong,T.Y.,Quigley,H.A.,Aung,T.,and Cheng,C.Y.(2014).Global prevalence of glaucoma and projections of glaucoma burden through 2040:a systematic review and meta-analysis.Ophthalmology 121,2081-2090. Thiel,G.,Czernik,A.J.,Gorelick,F.,Nairn,A.C.,and Greengard,P.(1988).Ca2+ / calmodulin-dependent protein kinase II:identification of threonine-286 as the autophosphorylation site in the alpha subunit associated with the generation of Ca2+-independent activity.Proc Natl Acad Sci U S A 85,6337-6341. Thoreson,W.B.,and Witkovsky,P.(1999).Glutamate receptors and circuits in the vertebrate retina.Prog Retin Eye Res 18,765-810. Tran,N.M.,Shekhar,K.,Whitney,I.E.,Jacobi,A.,Benhar,I.,Hong,G.,Yan,W.,Adiconis,X.,Arnold,M.E.,Lee,J.M.,et al.(2019).Single-Cell Profiles of Retinal Ganglion Cells Differing in Resilience to Injury Reveal Neuroprotective Genes.Neuron 104,1039-1055 e1012. Vorwerk,C.K.,Lipton,S.A.,Zurakowski,D.,Hyman,B.T.,Sabel,B.A.,and Dreyer,E.B.(1996).Chronic low-dose glutamate is toxic to retinal ganglion cells.Toxicity blocked by memantine.Invest Ophthalmol Vis Sci 37,1618-1624. Wang,Q.,Zhuang,P.,Huang,H.,Li,L.,Liu,L.,Webber,H.C.,Dalal,R.,Siew,L.,Fligor,C.M.,Chang,K.C.,et al.(2020).Mouse gamma-Synuclein Promoter-Mediated Gene Expression and Editing in Mammalian Retinal Ganglion Cells.J Neurosci 40,3896-3914. Watkins,T.A.,Wang,B.,Huntwork-Rodriguez,S.,Yang,J.,Jiang,Z.,Eastham-Anderson,J.,Modrusan,Z.,Kaminker,J.S.,Tessier-Lavigne,M.,and Lewcock,J.W.(2013).DLK initiates a transcriptional program that couples apoptotic and regenerative responses to axonal injury.Proc Natl Acad Sci U S A 110,4039-4044. Weinreb,R.N.,Leung,C.K.,Crowston,J.G.,Medeiros,F.A.,Friedman,D.S.,Wiggs,J.L.,and Martin,K.R.(2016).Primary open-angle glaucoma.Nat Rev Dis Primers 2,16067. Welsbie,D.S.,Yang,Z.,Ge,Y.,Mitchell,K.L.,Zhou,X.,Martin,S.E.,Berlinicke,C.A.,Hackler,L.,Jr.,Fuller,J.,Fu,J.,et al.(2013).Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death.Proc Natl Acad Sci U S A 110,4045-4050. Williams,P.A.,Harder,J.M.,Foxworth,N.E.,Cochran,K.E.,Philip,V.M.,Porciatti,V.,Smithies,O.,and John,S.W.(2017).Vitamin B3 modulates mitochondrial vulnerability and prevents glaucoma in aged mice.Science 355,756-760. Yang,L.,Li,S.,Miao,L.,Huang,H.,Liang,F.,Teng,X.,Xu,L.,Wang,Q.,Xiao,W.,Ridder,W.H.,3rd,et al.(2016).Rescue of Glaucomatous Neurodegeneration by Differentially Modulating Neuronal Endoplasmic Reticulum Stress Molecules.J Neurosci 36,5891-5903. Yilmaz,M.,and Meister,M.(2013).Rapid innate defensive responses of mice to looming visual stimuli.Curr Biol 23,2011-2015. Zhang,J.,Ackman,J.B.,Xu,H.P.,and Crair,M.C.(2011).Visual map development depends on the temporal pattern of binocular activity in mice.Nat Neurosci 15,298-307. Zhang,J.,Cai,C.Y.,Wu,H.Y.,Zhu,L.J.,Luo,C.X.,and Zhu,D.Y.(2016).CREB-mediated synaptogenesis and neurogenesis is crucial for the role of 5-HT1a receptors in modulating anxiety behaviors.Sci Rep 6,29551.

Claims

1. 1. A pharmaceutical composition for use in reducing retinal ganglion cell degeneration in a subject, comprising: A pharmaceutical composition comprising calcium-calmodulin-dependent kinase (CaMK) or a polynucleotide encoding said CaMK.

2. The pharmaceutical composition of claim 1 , wherein the composition further comprises a viral vector.

3. 2. The pharmaceutical composition of claim 1, wherein the CaMK is selected from one or more of CaMKI, CaMKII, and CaMKIV, and the CaMK is constitutively active.

4. The pharmaceutical composition of claim 3 , wherein the CaMK is selected from one or both of CaMKIIα containing a T286D substitution and CaMKIIβ containing a T287D substitution.

5. The pharmaceutical composition of claim 1 , wherein the composition comprises a polynucleotide encoding the CaMK.

6. The pharmaceutical composition of claim 1 , wherein the composition comprises the CaMK.

7. the use comprises administering the pharmaceutical composition intraocularly or administering the pharmaceutical composition systemically; and The subject has or is at risk of having one or more of glaucoma, diabetic retinopathy, retinal ischemia, and optic nerve damage; and Reducing degeneration includes preventing the loss of one or both of retinal ganglion cell bodies and retinal ganglion cell axons. The pharmaceutical composition of claim 1.

8. 1. A pharmaceutical composition for use in treating vision loss in a subject, comprising: A pharmaceutical composition comprising calcium-calmodulin-dependent kinase (CaMK) or a polynucleotide encoding said CaMK.

9. The pharmaceutical composition of claim 8 , wherein the composition further comprises a viral vector.

10. 9. The pharmaceutical composition of claim 8, wherein the CaMK is selected from one or more of CaMKI, CaMKII, and CaMKIV, and the CaMK is constitutively active.

11. The pharmaceutical composition of claim 10 , wherein the CaMK is selected from one or both of CaMKIIα containing a T286D substitution and CaMKIIβ containing a T287D substitution.

12. The pharmaceutical composition of claim 8, wherein the composition comprises a polynucleotide encoding the CaMK, and the polynucleotide further comprises a gamma-synuclein promoter, a synapsin 1 promoter, a neurofilament heavy chain promoter, or a Thy-1 cell surface antigen promoter.

13. The pharmaceutical composition of claim 8 , wherein the composition comprises the CaMK.

14. said use comprising administering said pharmaceutical composition intraocularly or administering said pharmaceutical composition systemically; The subject has or is at risk of having one or more of glaucoma, diabetic retinopathy, retinal ischemia, and optic nerve damage; and Treating vision loss includes preventing the loss of one or both of retinal ganglion cell bodies and retinal ganglion cell axons; and Treating vision loss includes preventing vision loss. The pharmaceutical composition according to claim 8.

15. 1. A pharmaceutical composition comprising: A pharmaceutical composition comprising a polynucleotide and a vector, the polynucleotide comprising a retinal ganglion cell promoter and encoding calcium-calmodulin-dependent kinase (CaMK).

16. 16. The pharmaceutical composition of claim 15, wherein the retinal ganglion cell promoter comprises a gamma-synuclein promoter, a synapsin 1 promoter, a neurofilament heavy chain promoter, or a Thy-1 cell surface antigen promoter.

17. The pharmaceutical composition of claim 15 , wherein the vector comprises a viral vector.

18. The pharmaceutical composition of claim 15, wherein the CaMK is selected from one or more of CaMKI, CaMKII, and CaMKIV.

19. The pharmaceutical composition of claim 15, wherein the CaMK is constitutively active.

20. The pharmaceutical composition of claim 18 , wherein the CaMK is selected from one or both of CaMKIIα containing a T286D substitution and CaMKIIβ containing a T287D substitution.

Images