Methods and compositions for reducing gliaptosis: macroglia-induced neuronal cell death

Abstract

The present invention relates to compositions and methods for preventing neuronal cell death. Methods of the invention, in part, relate to reducing an abnormal function of a glial canal in a canal-forming glial cell adjacent to a neuronal cell.

Description

[0001] METHODS AND COMPOSITIONS FOR REDUCING GLIAPTOSIS: MACROGLIA-

[0002] INDUCED NEURONAL CELL DEATH

[0003] Related Applications

[0004] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional application serial number 63 / 612,602 filed December 20, 2023 and U.S. Provisional application serial number 63 / 644,708 filed May 9, 2024, the disclosure of each of which is incorporated by reference herein in its entirety.

[0005] Government Interest

[0006] This invention was made with Government support under Grant No. P20GM 103449 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0007] Field of the Invention

[0008] The invention, in some aspects, relates to methods and compositions for reducing neuronal cell death.

[0009] Background of the Invention

[0010] The prevalent causative hypothesis for Alzheimer disease is the proposed accumulation of cellular waste particularly in form of intracellular hyperphosphorylated tau tangles and extracellular amyloid-P plaques. These waste products are known to accumulate in the mammalian neocortex and limbic system and are thought to impair neuronal health leading to neurodegeneration [M. Askenazi et al., Nature Communications. 14. 1-15 (2023); E. Blas Gil, Recent Advances in Alzheimer Research, Bentham Science Publishers Ltd, Vol. 3 (2019); E. E. Congdon and E. M. Sigurdsson, Nature Reviews Neurology 14, 399-415 (2018); A. O. Fernando, Recent Advances in Alzheimer Research, Bentham Science Publishers Ltd. Vol. 2 (2018); L. Kiani, Nature Reviews Neurology 19, 459-459 (2023); G. Perry, A dvances in Alzheimer's Disease. I. O. S. Press (2013); G. Perry, Advances in Alzheimer's Disease, I. O. S. Press (2018); D. Philippe, Molecular Medicine and Medicinal Chemistry, Imperial College Press (2013)]. One mechanism by which waste may be cleared from the brain parenchyma in mammals is the ‘Glymphatic system' first proposed by Iliff et al. [J. J. Iliff et al., Science translational medicine 4, 147ral 11 (2012)]. This system is thought to facilitate waste clearance from the brain through: (a) the release of neuronal waste into interstitial spaces, (b) the existence of a convective (bulk) flow of subarachnoid cerebrospinal fluid (CFS) along para-arterial spaces first postulated by Cserr and Ostrach [H. F. Cserr and L. H. Ostrach, Experimental neurology 45, 50-60 (1974)], (c) The formation of an aquaporin4 water channel (AQP4)-mediated bulk flow of CSF into the brain parenchyma, that (d) is believed to flush cellular debris toward para-venous spaces for clearance from the brain [J. J. Iliff et al.. Science translational medicine 4, 147ral 11 (2012); J. J. Iliff and M. Nedergaard, Stroke 44, S93-S95 (2013); M. Nedergaard, Science 340, 1529-1530 (2013); M. Nedergaard and S. A. Goldman, Scientific American 314, 44-49 (201 )

[0011] ]. Impairment of this system that results in a reduced bulk flow would thus result in the failure to clear metabolites and explain waste accumulation within the brain. This proposed mechanism has been challenged, in particular regarding the proposed bulk flow [ S. B. Hladky and M. A. Barrand, Fluids and Barriers of the CNS 19, 1-33 (2022) ; A. J. Smith et al., eLife 6, (2017)], and the postulated mechanism by which AQP4-containing astrocytic end feet contribute to the formation of this convective flow [N. J. Abbott et al., Acta neuropathologica 135. 387-407 (2018)]. Experiments by Smith et al. [A. J. Smith et al., eLife 6, (2017) ] demonstrated the existence of this bulk flow in the absence of AQP4, suggesting that diffusion may be the underlying mechanism by which CSF enters the brain parenchyma. Abbott et al. [N. J. Abbott et al., Acta neuropathologica 135, 387-407 (2018)] provide an alternative purpose of CSF flow into interstitial spaces that focuses on the distribution of gases and nutrients to the brain parenchyma. Numerous ideas have been proposed regarding the cause of Alzheimer disease and other degenerative diseases, a lack of understanding remains and continues to inhibit progress toward prevention and / or treatment of such diseases.

[0012] Summary of the Invention

[0013] According to an aspect of the invention a method for preventing neuronal cell death is provided, the method including reducing an abnormal function of a glial canal in a canalforming glial cell adjacent to the neuronal cell, wherein the reduction in the abnormal function increases likelihood of survival of the neuronal cell compared to a control likelihood of survival. In some embodiments, the control likelihood of survival is the likelihood of survival of a neuronal cell adjacent to a canal-forming glial cell in which the abnormal function is not reduced. In certain embodiments, the abnormal function includes depleting the adjacent neuronal cell’s neuronal cytoplasm into the glial canal. In certain embodiments, the abnormally functioning canal-forming glial cell includes one or more glial canals including structural damage. In some embodiments, the glial canal structural damage statistically significantly increases flow of cytoplasm from the neuronal cell into the structurally damaged glial canal. In some embodiments, reducing an abnormal function includes maintaining a normal function of the canal-forming glial cell. In certain embodiments, the normal function of the canal-forming glial cell includes a controlled removal of neuronal waste from the adjacent neuronal cell into the glial canal of the canal-forming glial cell. In some embodiments, the neuronal waste includes one or more of lipid-based cellular waste, proteinbased cellular waste, and lipofuscin. In some embodiments, reducing the abnormal function of the canal-forming glial cell includes one or more of: increasing production of normal glial- canals by the canal-forming glial cell; maintaining a normal function of the glial-canals in the glial cell; and reducing damage to one or more glial-canals in the canal-forming glial cell. In some embodiments, the glial cell is in contact with the neuronal cell. In certain embodiments, the method includes contacting the canal-forming glial cell with a composition including an aquaporin inhibitor. In certain embodiments, the aquaporin inhibitor is an aquaporin 4 inhibitor. In some embodiments, the aquaporin 4 inhibitor is 2-(nicotinamide)-l,3,4- thiadiazole (TGN-020). In some embodiments, the aquaporin inhibitor is an aquaporin 7 inhibitor or is an aquaporin 9 inhibitor. In certain embodiments, the method additionally includes contacting the canal-forming glial cell with a composition including an agent that increases activity of a proteolytic enzyme. In certain embodiments, the enzyme is a caspase. In some embodiments, the caspase is a caspase 2 or a caspase 3. In some embodiments, the method includes contacting the canal-forming glial cell with a composition including an agent that reduces activity of a secretase or a BACE1. In some embodiments, the secretase is an alpha-secretase, a beta-secretase, or a gamma-s ecretas e. In certain embodiments, the neuronal cell is in a subject. In certain embodiments, the subject is a mammal, optionally is a human. In some embodiments, the neuronal cell is in culture. In some embodiments, the neuronal cell is an engineered neuronal cell. In certain embodiments, the neuronal cell is in or is obtained from a subject known to have, or suspected of having, a neurodegenerative disease or condition. In some embodiments, the neurodegenerative disease or condition is Alzheimer’s disease. Parkinson’s disease. Huntington’s disease, Chronic Traumatic Encephalopathy (CTE), Amyotrophic lateral sclerosis (ALS); or Motor neuron disease. In certain embodiments, the glial cell is a macroglial cell.

[0014] According to an aspect of the invention, a composition including an aquaporin inhibitor for treatment of a neurodegenerative disease or condition is provided. In some embodiments, the aquaporin inhibitor is an aquaporin 4 inhibitor, optionally wherein the aquaporin 4 inhibitor is 2-(nicotinamide)-l,3,4-thiadiazole (TGN-020). In some embodiments, the aquaporin inhibitor is an aquaporin 7 inhibitor or is an aquaporin 9 inhibitor.

[0015] According to an aspect of the invention, a composition including an agent that increases acti vity of a caspase for treatment of a neurodegenerative disease or condition is provided. In certain embodiments, the caspase is a caspase 2 or a caspase 3.

[0016] According to another aspect of the invention, a composition including an agent that decreases activity of a secretase for treatment of a neurodegenerative disease or condition is provided. In certain embodiments, the secretase is an alpha-secretase, a beta-secretase, or a gamma-secretase.

[0017] According to another aspect of the invention, a composition including an agent that decreases BACE1 activity for treatment of a neurodegenerative disease or condition is provided.

[0018] According to another aspect of the invention, a method of reducing neurodegeneration in a subject is provided, the method including administering to the subject an aquaporin inhibitor in an amount effective to maintain or increase a normal function of a canal-forming glial cell in the brain of the subject. In certain embodiments, the normal function of the canalforming glial cell includes a controlled removal of neuronal waste from a neuronal cell adjacent to a glial canal of the canal-forming glial cell. In some embodiments, the aquaporin inhibitor is an aquaporin 4 inhibitor, optionally wherein the aquaporin 4 inhibitor is 2- (nicotinamide)-l ,3,4-thiadiazole (TGN-020). In some embodiments, the aquaporin inhibitor is an aquaporin 7 inhibitor or is an aquaporin 9 inhibitor.

[0019] Brief Description of the Drawings

[0020] These and other features, aspects, and advantages of the embodiments of the present disclosure is better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawing, wherein:

[0021] Figure 1A-C is a schematic drawing of glial lobe formation in spider leg ganglia. Fig. 1 A shows spider oligodendroglia with two ‘forming lobes’ (FLs) characterized by the formation of circular membrane rings project toward neurons (N). Arrowheads indicate glial aqua canals. Fig. IB shows that the maturing lobes elongate and flatten, resulting in the linear alignment of glial membranes. Individual membranes are cleaved and anchored to microtubules (microtubule associated break points, MABs, indicated by black arrows). Fig. 1C shows that individual membranes detach from their microtubule attachment and project into the neuronal cytoplasm (white arrowheads). During this process glial aqua canals are translocated into the neuronal cytoplasm (neuronal aqua canals, white arrows) and create a cytoplasmic bulk flow that may cause the swelling of the neuronal aqua canals and the flow of cellular debris (black dots) into the lumina of the glial-canals. The schematic is not drawn to scale.

[0022] Figure 2A-B shows graphs indicating degeneration onset in the spider leg ganglion. Fig. 2A shows that the average maximum diameter of the GACs was significantly larger in degenerating (0.342 pm) compared to healthy tissue (0.240 pm; t(852) = 6.80, p = 1.925 x 10 ". unpaired / test). Fig.2B shows that the average area of the GACs is significantly larger in degenerating (0.084 pm2) compared to healthy tissue (0.033 pm2; t(852) = 5.33, / ? = 1.258 x 107. unpaired rtest).

[0023] Figure 3A-S provides photomicrographic images of Luxol Blue, Hematoxylin-Eosin (H&E), and toluidine blue-stained sections through the hippocampus of decedents with diagnosed AD. Fig. 3 A shows areas investigated including the alveus, stratum oriens and CA1-CA3 stratum pyramidale (Luxol H&E stain). Fig. 3B is a toluidine-blue de-stained vibratome section, which shows dense debris accumulations in the alveus, stratum oriens. and neuronal somata of the stratum pyramidale. Fig. 3C-D are images of H&E stained tanycytes (asterisks) in the alveus form apical projections (black arrowheads) that transected into the adjacent ventricle. The connectivity between adjacent somata and cell projections (Fig. 3D shown with lighter-colored arrowheads) indicated a syncytial network. Fig. 3E shows that Luxol blue stained hippocampal tanycyte processes formed varicosities (double arrowheads) that gave rise to lateral tanycyte projections (black arrow). Fig. 3F shows that tanycyte processes in the olivary nucleus (large white arrowhead) gave rise to numerous lateral projections that transected into adjacent tissue (small white arrowhead). Fig. 3G is a close up image showing pyramidal cell (P) contacted by tanycyte processes (white arrowheads). Fig. 3H-K shows different focal planes through a Luxol H&E stained tanycyte process that gave rise to circular protrusions (white arrows). Fig. 3L shows hypertrophic Luxol blue-stained tanycyte process that contained engorged translucent compartments (white arrowhead) consistent with excessive liquid intake. Adjacent tanycyte processes appeared less engorged (small arrowhead). Fig. 3M shows neuronal soma in the olivary nucleus of an AD-decedent densely obstructed with hypertrophic tanycyte receptacles (white arrow). Hypertrophic tanycyte processes contacted the neuronal cytoplasm and nucleus (large white arrowheads). Small white arrowhead: Tanycyte process transecting into the neuronal cytoplasm. Fig. 3N shows an H&E-stained hypertrophic tanycyte projection (white arrowheads) in the alveus in close proximity to brown cellular waste (grey arrow). Fig. 30 shows tanycyte soma with signs of hypertrophy onset in the soma (double arrowhead). Fig. 3P-S shows different focal planes through the soma of a hypertrophic tanycyte soma (double arrowhead) with attached tanycyte processes (arrow-head).

[0024] Figure 4A-D provides schematic diagrams and a photomicrographic image providing a schematic summary of the proposed waste removal system in the human hippocampus and observed histopathologies. Fig. 4A is a schematic diagram of myelinated aquaporin4-IR (AQP4-IR) tanycytes whose somata are located in the alveus send vast networks of tube-like processes that contain central canals into the stratum pyramidale. Tanycytes use adherence clamps to attach to surrounding cells and form intracellular receptacles that internalize catabolized neuronal w aste. This waste is transported to the ventricular lining and specialized glia canals where it may be removed from the brain via the Choroid plexus. Fig. 4B is a schematic diagram of a proposed mechanism of waste uptake into tanycyte receptacles. Receptacles are formed through the formation of myelin protrusions around the outer periphery of myelinated tanycytes. The central, AQP4-expressing canal branches into each receptacle-forming protrusion. Central canal and protrusion form a functional unit by which the functional significance of the receptacle is the filtering and catabolism of cellular waste to prevent obstruction by larger debris particles. The central canal creates a convective flow- tow ard the canal through activation of AQP4. The number of receptacles formed varies. Fig. 4C is an ultrastructural depiction of illustrated structures. Fig. 4D is a schematic diagraph of a proposed underlying histopathology is the AQP4-mediated hypertrophic sw-elling of entire tanycytes likely due to blockage of drainage canals. Swelling is observed in (1) adhesion clamps, (2) somata, (3) intracellular protrusions, and tanycyte processes.

[0025] Detailed Description

[0026] The invention, in part, includes methods of preventing neurodegenerative diseases and conditions. The instant disclosure describes studies performed to investigate waste disposal from neurons. Results show how neurons and glial cells in the invertebrate model system Cupiennius salei form a highly specialized glial-canal system by which cellular debris is cleared from neurons. Central American wandering spiders are exceptionally suitable for this study as they develop similar neurodegenerative pathologies to humans that are likely linked to impaired waste clearance from the brain [R. Fabian-Fine et al., Journal of Comparative Neurology! 531, 618-638 (2023)]. Advantages of spider brain are their large neuronal structures, easy accessibility, superior tissue preservation, and the ability to identify initial stages of neurodegeneration based on behavioral cues of affected animals [R. Fabian-Fine et al.. Journal of Comparative Neurology 531. 618-638 (2023)]. Based on results of studies, such as those disclosed herein, it has been determined that this glial-canal system relies on aquaporin-mediated cytoplasmic bulk flow to draw cellular waste from neuronal somata into the glia-canal system. Studies presented herein provide strong evidence that degeneration onset can be linked to the structural failure of the canal-forming glial cells, which have been identified as tanycytes. Studies presented herein demonstrate that this structural failure results in excessive depletion of neuronal cytoplasm into structurally impaired glial -canals.

[0027] It has now been shown that neurons in the human hippocampus are closely associated with AQP4-immunoreactive (AQP4-IR) myelinated tanycytes that form waste collection ‘receptacles’ within neurons, glial cells, and extracellular spaces. Studies disclosed herein provide ultrastructural evidence that the primary function of these receptacles is internalization and removal of cellular w aste from the brain into CSF. It has now been determined that neurons in AD-decedents are largely obstructed by hypertrophic tanycyte receptacles, supporting a conclusion that the structural and functional failure of tanycytes leads to uncontrolled depletion of cytoplasmic content from neurons and induce neuronal cell death. Studies provided herein support a conclusion that the catastrophic structural and functional failure of these canal-forming tanycytes may be due to obstruction of the tanycytes that leads to hypertrophic swelling followed by severe spongiform abnormalities and cell death in AD patients and to neuronal degeneration in other neurodegenerative diseases, nonlimiting example of which are Parkinson’s Disease and Spongiform Encephalitis.

[0028] Results of studies described herein provide strong evidence that this system is highly conserved in mammals, and the instant disclosure demonstrates spider neuronal and glial systems described herein as acceptable animal and cell model systems for mammalian neurodegenerative disease. It has now' been shown that there are striking similarities in the histopathology of both degenerating spider and human brains from Alzheimer patients with confirmed Alzheimer Disease neuropathologic change (ADNC). Based on results of studies set forth herein it has been identified that the structural failure of tanycytes may cause neuronal cell death in both the spider and human degenerative brain conditions. The studies disclosed herein provide evidence of (a) waste clearance mechanisms in the arachnid model system, (b) how the structural impairment of this system causes neurodegeneration, (c) the mammalian equivalent of this system, and (d) how hypertrophic abnormalities of this system in the brain of confirmed Alzheimer patients may induce neurodegeneration similar to the mechanism observed in spider nervous tissue.

[0029] Certain aspects of the invention comprise methods of preventing neuronal cell death in cells and subjects. In some embodiments, methods of the invention reduce an abnormal function of a canal-forming glial cell and prevent neuronal cell death. As used herein, the term glial-canal refers to a canal formed in a glial cell. Spider glial canals are formed through interaction of one or more spider oligodendrocytes with tanycyte-like macroglia. A mammalian canal-forming glial cell is a macroglial cell and an oligodendrocyte and is referred to herein as a tanycyte.

[0030] A role of canal-forming glial cells includes activities that maintain the health of adjacent neuronal cells. One activity' of a canal -forming glial cell is to produce one or more glial canals, into which neuronal waste is removed from an adjacent neuronal cell. The removal of neuronal waste in a controlled manner is a normal function of a glial canal, and a normal function of the canal-forming glial cell. A canal-forming glial cell that normally functions to maintain a neuronal cell, is considered to be “adjacent” to the neuronal cell, meaning the canal-forming glial cell and the neuronal cell it maintains are in close physical proximity to each other, which may include being in physical contact with each other. In some embodiments of methods of the invention, a canal-forming glial cell and the neuronal cell its normal function is to maintain are in physical contact with each other.

[0031] The invention, in part, includes methods that maintain a normal function of a canalforming glial cell in its maintenance of a neuronal cell. In some instances, maintaining a normal function means reducing an abnormal function of a canal-forming glial cell. Reducing an abnormal function of a canal-forming glial cell is also referred to herein as reducing an abnormal function of a glial canal in the canal-forming glial cell. As used herein, the term “abnormal” when used in reference to a function of a glial canal and / or a canalforming glial cell means a function that is different from a normal function of a glial canal and / or canal-forming glial cell, respectively. As a non-limiting example, a normal function of a glial canal and / or canal -forming glial cell comprises removing neuronal w aste from the neuronal cell into the glial canal. The term “neuronal waste” as used herein means materials that are normally removed from a neuronal cell in order to maintain the health of the neuronal cell. Non-limiting examples of materials that may be included in neuronal waste are lipid- based cellular waste, protein-based cellular waste, and lipofuscin.

[0032] The term '‘normal” as used herein in reference to removal of neuronal waste from a neuronal cell means removal of the neuronal cell waste in a controlled manner. Controlled removal of waste is important for maintaining the health of the neuronal cell and removal of such waste material is a normal function of glial canals and / or canal-forming glial cells. In contrast, an abnormal function of a glial canal and / or canal-forming glial cell comprises uncontrolled removal of cytoplasm from a neuronal cell adjacent to the canal-forming glial cell. In contrast to normal neuronal waste removal, abnormal function of a glial canal and / or canal-forming glial cell essentially evacuates cytoplasm from the neuronal cell in an uncontrolled manner, resulting in excess removal and / or depletion of the cytoplasm of the neuronal cell. An abnormally functioning glial canal can deplete the cytoplasm of a neuronal cell physically adjacent to the canal-forming cell comprising the glial-canal, resulting in death of both the neuronal cell and additional cells, such as surrounding oligodendroglia. Thus, an abnormal depletion of cytoplasm of a neuronal cell may result in abnormal removal of cytoplasm from a neuronal cell adjacent to a canal-forming glial cell results in death of the neuronal cell, the canal-forming glial cell, and additional oligodendroglia. A canal-forming glial cell is an oligodendroglial cell. Mammalian canal-forming glial cells are tanycytes.

[0033] Although not wishing to be bound to a particular theory, it is believed following the uncontrolled efflux of cytoplasm of a neuronal cell a chain-reaction response may occur resulting not only in the death of the neuronal cell and the canal-forming glial cell, but additional cells such as oligodendroglial cells may also die as a result of the release of enzymes and other materials by the neuronal cell, canal-forming glial cell, and / or other negatively impacted cells upon their death.

[0034] An abnormal function of one or more glial canals and / or canal-forming glial cells may result from structural damage to the one or more glial canals and / or canal-forming glial cells, respectively. In some embodiments, the presence of structural damage of a glial canal statistically significantly increases the level of flow of cytoplasm from a neuronal cell, such that a neuronal cell adjacent to a canal-forming glial cell comprising one or more structurally damaged glial canals experiences uncontrolled and lethal outflow7of its cytoplasm.

[0035] In certain embodiments the normal function of removing waste from a neuronal cell includes removing lipid-based cellular w aste, protein-based cellular waste, and / or lipofuscin cellular waste, which may be removed in conjunction an amount of neuronal cytoplasm. It will be understood that the amount of neuronal cytoplasm removed in the context of normal waste removal from a neuronal cell into a normally functioning glial canal is an amount that does not negatively impact the neuronal cell. In contrast to normal waste removal, an amount of neuronal cell cytoplasm removed into an abnormally functioning glial canal may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more than the amount of neuronal cell cytoplasm removed into a normally functioning glial canal.

[0036] As a non-limiting example of normal function of glial canals and canal-forming glial cells, a mammalian neuronal cell is contacted by one or more tanycytes that form varicose processes into the neuronal cell for removal of neuronal waste. Canals in the processes project membrane-cistemae into the neuronal cytoplasm permitting controlled removal of neuronal waste from the interior of the neuronal cell into the tanycyte. As a non-limiting example of an abnormal function of glial canals and canal-forming glial cells, a mammalian neuronal cell is contacted by one or more tanycytes that form varicose processes into the neuronal cell for removal of neuronal waste. Structurally damaged canals in the processes project membrane-cistemae into the neuronal cytoplasm and the damage results in uncontrolled draining of cytoplasm from the neuronal cell into the tanycyte, which may result in abnormal swelling varicosities in the tanycyte fdled with debris from the neuronal cell. The loss of cytoplasm results in death of the neuronal cell and the abnormal fdling of the tanycyte with debris result in death of the tanycyte. Cell damage and death may occur in additional cells negatively impacted by the local environment that results from released content (such as but not limited to released enzymes, etc.) of the damaged and dead neuronal and glial cells.

[0037] Treatment and Risk Reduction

[0038] Some embodiments of methods of the invention comprise reducing an abnormal function of a canal-forming glial cell, which, as indicated herein, reduces an abnormal function of one or more glial canals in the canal -forming glial cell. In some embodiments, methods of the invention include maintaining a normal function of the canal-forming glial cell, which as indicated herein, means maintaining a normal function of one or more glial canals of the canal-forming glial cell. Maintaining a normal function of a canal-forming glial cell may include one or more of (a) increasing production of normal glial canals by the canalforming glial cell, and (b) reducing damage, for example the occurrence of structural damage, to one or more glial-canals in the canal-forming glial cell. Aquaporins

[0039] It has been identified that an abnormal function of a glial canal can be reduced by contacting a canal-forming glial cell with a composition comprising an aquaporin inhibitor agent. Methods of the invention comprise contacting a canal-forming glial cell with a composition comprising one or more aquaporin inhibitor agents, non-limiting examples of which are aquaporin 4 inhibitor agents, aquaporin 7 inhibitor agents, and aquaporin 9 inhibitor agents. In certain embodiments of methods of the invention, a canal-forming glial cell is contacted with a composition comprising one or more aquaporin inhibitors in an amount effective to reduce an abnormal function of a glial canal in the contacted canalforming glial cell. The term “reduce” as used herein in reference to a change in an abnormal function of a glial canal and / or canal-forming glial cell means decreasing the abnormal function, which may include fully preventing, partially preventing, inhibiting, and / or eliminating the abnormal function.

[0040] In some embodiments, the composition comprising the aquaporin inhibitor comprises an aquaporin 4 inhibitor agent. Non-limiting examples of Aquaporin 4 inhibitor agents are 2- (nicotinamide)-l,3,4-thiadiazole (TGN-020), IMD 0354, zinc [see Yukutake Y, et al., Biochemistry. 2009 Dec 29;48(51): 12059-61], and an anti-aquaporin 4 antibody or functional fragment thereof. In certain embodiments of methods of the invention, a canal-forming glial cell is contacted with a composition comprising an aquaporin 7 inhibitor agent. Non-limiting examples of Aquaporin 7 inhibitor agents are. Z433927330 (for example: Cat. No.: HY- 126074, MCE Med Chem Express), monoacetin, monobutyrin and diacetin [Katano T, et al.. Drug Metab Pharmacokinet. 2014;29(4):348-51] and an anti-aquaporin 7 antibody or functional fragment thereof. In certain embodiments of methods of the invention, a canalforming glial cell is contacted with a composition comprising an aquaporin 9 inhibitor agent. Non-limiting examples of Aquaporin 9 inhibitor agents are RG100204 (see for example, U.S. Patent Publication US20190127360) and an anti-aquaporin 9 antibody or functional fragment thereof. (Each of the above publications is incorporated by reference herein in its entirety .)

[0041] It will be understood that a combination of aquaporin inhibitor agents may be selected and included in a composition that contacts a canal-forming glial cell in a method of the invention. As a non-limiting example, a canal-forming glial cell may be contacted with a composition comprising one, two, or more of aquaporin inhibitor agents, which may be independently selected from an aquaporin 4 inhibitor agent, an aquaporin 7 inhibitor agent, and an aquaporin 9 inhibitor agent. As used herein, the term “independently selected” used in reference to aquaporin inhibitor agents means each agent may be chosen to be administered for contacting a canal-forming glial cell independent of the selection of one or more other aquaporin inhibitor agents. As a non-limiting example, a subject may be administered three aquaporin inhibitor agents and the selected agents are: TGN-20, Z433927330, and an anti-aquaporin 4 antibody. In some embodiments, a composition comprising one or more aquaporin inhibitor agents comprise one or more components in addition to the aquaporin inhibitor agent(s), non-limiting examples of additional components that may be included in the composition are detectable labels, earners, delivery agents, etc.

[0042] In some aspects of the invention, contacting a neuronal cell or cells with a composition comprising an aquaporin inhibitor agent reduces an abnormal function and increases the likelihood of survival of one or more neuronal cells normally maintained by the canal-forming glial cell contacted with the composition, as compared to a control likelihood of survival. In some embodiments of the invention, a control likelihood of survival is a likelihood of survival of one or more neuronal cells adjacent to a canal -forming glial cell that is not contacted with the composition.

[0043] Certain methods of the invention include a treatment regimen comprising administering to a subject identified as having, or at risk of having, a neurodegenerative disease or condition, one or more aquaporin inhibitor agents. Certain embodiments of methods of the invention may include (1) identifying a subject who is at risk of having neuronal degeneration or a subject who has neuronal degeneration, (2) selecting a therapeutic regimen with which to treat the subject identified in step (1); and (3) administering the selected therapeutic regimen to the subject identified as in need of the selected therapeutic regimen.

[0044] It has now been determined that a reduced abnormal function of a glial canal of a canal-forming glial cell that normally functions to maintain a neuronal cell can be treated by contacting the canal-forming glial cell with one or more aquaporin inhibitor agents. Certain embodiments of methods of the invention can be used to prevent and / or treat a subject by administering a composition comprising one or more aquaporin inhibitor agents to the subject in an amount effective to reduce an abnormal glial-canal function and prevent and / or reduce the severity of neuronal death and neuronal degeneration in the subject. Certain methods of the invention include administering composition(s) comprising one or more aquaporin inhibitor agents to a subject identified as having a neuronal degenerative disease or condition or administering a composition comprising one or more aquaporin inhibitor agents prophy lactically to a subject identified as at risk of having a neuronal degenerative disease or condition. Methods that can be used to identify a subject as at risk of having or as having a neuronal degenerative disease or condition are known in the art, and may include but are not limited to imaging methods, behavioral assessment, genetic analysis, etc.

[0045] Enzymes

[0046] Enzy me Activity Increase

[0047] It has been identified that an abnormal function of a glial canal may be reduced by contacting a canal-forming glial cell with a composition comprising an agent that increases activity of an enzyme, a non-limiting example of which is a caspase. Although not wishing to be bound by a particular theory, increasing activity of an enzyme, such as a caspase may assist in the degradation of w aste products that are then removed into canal-forming glial cells. In some embodiments, methods of the invention comprise contacting a canal-forming glial cell with a composition comprising one or more agents that increase activity of a caspase, non-limiting examples of w hich caspase 2 and caspase 3. In certain embodiments of methods of the invention, a canal-forming glial cell is contacted with a composition comprising one or more agents that increase activity’ of a caspase 2. In certain embodiments of methods of the invention, a canal-forming glial cell is contacted with a composition comprising one or more agents that increase activity- of a caspase 3 in an amount effective to reduce an abnormal function of a glial canal in the contacted canal-forming glial cell. The term “reduce” as used herein in reference to a change in an abnormal function of a glial canal and / or canal-forming glial cell means decreasing the abnormal function, which may include fully preventing, partially preventing, inhibiting, and / or eliminating the abnormal function. In some embodiments an abnormal function is the failure of the glial canal and / or canal- formal glial cells to remove waste.

[0048] In some aspects of the invention, methods of treating a neurodegenerative disease or condition comprise increasing activity of an enzyme in a subject believed to have or to be at risk of having the neurodegenerative disease or condition. Caspase enzymes are non-limiting example of enzymes that methods of the invention may be used to increase to treat a neurodegenerative disease or condition. Caspase 2 and caspase 3 are non-limiting examples of caspase enzymes whose activity’ may be increased in a subject using a method of the invention. In some embodiments of the invention, activity of caspase 2 and / or caspase 3 is increased in an amount effective to treat a neurodegenerative disease or condition in a subject. Some embodiments of methods of the invention, comprise increasing a level of activity of a caspase 2 or caspase 3 enzyme. In some embodiments, a method of the invention increases activity of an enzyme in neuronal tissue of a subject by one or more of: increasing expression of the enzyme, reducing loss of the enzyme, increasing enzymatic functioning of the enzyme, and other means that results in a higher level of activity of the enzy e in the neuronal tissue of the subject. In some embodiments, one or more agents that increase a level of activity of an enzy me, such as but not limited to a caspase, are administered to a subject.

[0049] A level of activity of an enzyme may be determined before and / or after a treatment of the invention is administered to a subject. A level of activity of an enzyme, such as but not limited to a caspase in a subject being treated or to be treated with a method of the invention, may be compared to a control level of activity of the enzyme to identify efficacy of the treatment of the invention. In such instances, whether or not the treatment results in an increase in enzyme activity in the subject can be determined using such comparisons. In some embodiments, a control level of activity is a level of activity in a subject or subjects that do not have the degenerative disease or condition that is present in a subject or suspected to be present in the subject. In certain embodiments, a control level may be a level of enzyme activity in the subject prior to administering a treatment of the invention. In each case, the control level may be compared with a level of activity' of the enzyme following a treatment of the subject with a method of the invention as a determination of the efficacy of the treatment against the degenerative disease or condition in the subject. ft will be understood that determining a level of activity of an enzyme, such as but not limited to a caspase enzyme in a subject, may include but is not limited to one of more of imaging studies, symptom assessment, and other diagnostic methods with which to determine the status of the neurodegenerative disease or condition. A treatment method of the invention may result in a higher level of activity of an enzyme such as, but not limited to, a caspase following the treatment compared to a level of activity of the enzyme prior to the treatment. It will be understood that a “normal” level of activity may be used as a control level.

[0050] In some subjects, an activity level of an enzyme such as a caspase in a neuronal tissue of the subject may be at a level that is below a “normal” activity level and increasing activity in that subject may be an increase of activity in that subject to a level that is closer to, equal to, or greater than a normal level of activity of the enzyme. In certain embodiments of methods of the invention, a subject in need of treatment with a method of the invention has an essentially normal level of activity of an enzyme such as, but not limited to, a caspase in their neuronal tissues and increasing the enzyme activity in that subject may be an increase to a level greater than a normal level of activity. In such instances, the subject may be treated by providing a higher-than-normal level of activity of the enzyme, thereby treating the neurodegenerative disease or condition in the subject.

[0051] Enzyme Activity Reduction

[0052] In some aspects of the invention, methods of treating a neurodegenerative disease or condition comprise decreasing activity of an enzyme in a subject believed to have or to be at risk of having the neurodegenerative disease or condition. Although not wishing to be bound by any theory, alpha-, beta- and / or gamma- secretases may promote the growth of the tanycytes into neurons and the proliferation of receptacles. Results indicate that secretases may promote outgrow th and proliferation of blood vessels through interaction with amyloid precursor protein (APP) that is cleaved by an enzy me called "beta-site APP cleaving enzyme 1” (BACE1). Interaction between BACE1 and the secretases, for example beta and gamma secretases, may result in formation of amyloid beta. Some embodiments of methods of the invention comprise reducing activity of a secretase and / or a BACE1 enzyme reduces proliferation of excessive receptacles in the human AD brain.

[0053] BACE1 and secretase enzymes are non-limiting examples of enzy mes that methods of the invention may be used to decrease to treat a neurodegenerative disease or condition. Alpa-, beta-, and / or gamma-secretase enzymes are non-limiting examples of secretase enzymes whose activity may be decreased in a subject using a method of the invention. In some embodiments of the invention, activity’ of one or more of alpha-secretase, beta- secretase, and gamma-secretase is decreased in an amount effective to treat a neurodegenerative disease or condition in a subject. Some embodiments of methods of the invention, comprise decreasing a level of activity of one or more of alpha-secretase, beta- secretase, and gamma-secretase.

[0054] In some embodiments, a method of the invention decreases activity of an enzyme in neuronal tissue of a subject by' one or more of: decreasing expression of the enzyme, increasing elimination (for example increasing removal or degradation) of the enzy me, decreasing enzymatic functioning of the enzyme, and other means that results in a lower level of activity of the enzyme in the neuronal tissue of the subject. In some embodiments, one or more agents that decrease a level of activity of a secretase, such as but not limited to an anti- secretase antibody, a secretase-silencing RNA, are administered to a subject. In certain embodiments, one or more agents that decrease a level of activity of a BACE1. such as but not limited to an anti-BACEl antibody, a BACE1 -silencing RNA, are administered to a subject. A level of activity of an enzyme may be determined before and / or after a treatment of the invention is administered to a subject. Comparing a level of activity of an enzyme, such as but not limited to a secretase or BACE1, in a subject to a control level of activity of the enzyme may be used to identify efficacy of a treatment of the invention, by determining whether or not the treatment results in a decrease in secretase activity or BACE1 activity in the subject. In some embodiments, a control level of activity is a level of activity in a subject or subjects that do not have the degenerative disease or condition that is present in a subject or suspected to be present in the subject being treated or to be treated with a method of the invention. In certain embodiments, a control level may be a level of enzy me activity in the subject prior to administering a treatment of the invention. In each case, the control level maybe compared with a level of activity of the secretase enzyme and / or BACE1 activity following a treatment of the subject with a method of the invention as a determination of the efficacy of the treatment against the degenerative disease or condition in the subject.

[0055] It will be understood that determining a level of activity of an enzyme, such as but not limited to a secretase enzyme and a BACE1 enzyme in a subject, may include but is not limited to one of more of imaging studies, symptom assessment, and other diagnostic methods with which to determine the status of the neurodegenerative disease or condition. A treatment method of the invention may result in a lower level of activity7of an enzy me such as, but not limited to, a secretase and / or BACE1 following the treatment compared to a level of activity of the enzyme prior to the treatment. It will be understood that a “normal” level of activity7may be used as a control level.

[0056] In some subjects, an activity' level of an enzyme such as a secretase or BACE1 in a neuronal tissue of the subject may be at a level that is above a “normal"’ activity- level and decreasing activity in that subject may be a decrease of activity in that subject to a level that is closer to, equal to, or lower than a normal level of activity of the enzyme. In certain embodiments of methods of the invention, a subject in need of treatment with a method of the invention has an essentially normal level of activity of an enzy me such as, but not limited to, a secretase and / or BACE1, in their neuronal tissues and decreasing the enzyme activity- in that subject may be a decrease to a level less than a normal level of activity. In such instances, the subject may be treated by providing a lower-than-normal level of activity of the enzyme, thereby treating the neurodegenerative disease or condition in the subject.

[0057] Therapeutics and Risk Reduction In some embodiments, an agent administered to increase activity of an enzy me, or an agent administered to inhibit activity of an enzyme or aquaporin, (also referred to herein as an aquaporin inhibitor agent), is a protein, a polynucleotide encoding the enzyme, the enzyme, a functional variant of the enzyme, a functional fragment of the enzyme, a precursor of the enzy me, an siRNA, an antibody, or other agent that results in an increase in activity' of the enzyme or inhibits activity of the aquaporin, respectively. As used herein a “functional variant” means a molecule that differs from the natural enzyme or agent, for example in amino acid sequence, or due to the presence of one or more modified amino acids and retains at least a portion of the activity of the natural enzy me or agent. Similarly, as used herein a “functional fragment” of an enzy me or agent means a molecule that is a portion of the natural enzyme or agent or functional variant thereof that retains at least a portion of the activity of the natural enzyme or agent, respectively.

[0058] As used herein, the term “at least a portion” used in reference to activity means at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%. 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%. 22%, 23%. 24%. 25%. 26%. 27%. 28%. 29%. 30%. 31%. 32%.

[0059] 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,

[0060] 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,

[0061] 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,

[0062] 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%. 93%. 94%. 95%. 96%.

[0063] 97%. 98%. 99%. 100%, 105%, 110%. 115%, 120%, 130%. 135%, 140%, 145%. 150%, 160%, 170%, 180%, 190%, 200%, or more of the activity of the natural or “parent” agent. The term “parent” used herein in relation to an agent or molecule means the agent or molecule from which a functional variant or functional fragment is derived.

[0064] The invention, in part, includes methods of reducing a subject’s risk of developing a neurodegenerative disease or condition. Certain embodiments of risk reduction methods of the invention include administering one or more aquaporin inhibitor agents to a subject identified as at risk of developing a neurodegenerative disease or condition. In some embodiments of methods of the invention, an agent administered to the subject is an aquaporin inhibitor agent that is administered in an amount effective to reduce the subject’s risk of developing the neurodegenerative disease or condition. Some embodiments of risk reduction methods of the invention include administering one or more agents that increase activity of an enzyme, for example but not limited to a caspase, to a subject identified as at risk of developing a neurodegenerative disease or condition. Some embodiments of risk reduction methods of the invention include administering one or more agents that decrease activity of an enzyme, for example but not limited to a BACE1 enzy me, a secretase enzyme etc., to a subject identified as at risk of developing a neurodegenerative disease or condition.

[0065] In some embodiments of methods of the invention, an agent administered to the subject is an aquaporin inhibitor agent that is administered in an amount effective to reduce the subject’s risk of developing the neurodegenerative disease or condition. In certain embodiments of methods of the invention, an agent administered to the subject is an agent that increases activity of an enzyme, such as but not limited to caspase 2 or caspase 3 that is administered in an amount effective to reduce the subject’s risk of developing the neurodegenerative disease or condition. In certain embodiments of methods of the invention, an agent administered to the subj ect is an agent that decreases activity of an enzyme, such as but not limited to a secretase and / or BACE1 that is administered in an amount effective to reduce the subject’s risk of developing the neurodegenerative disease or condition.

[0066] Efficacy of a method of the invention to reduce a subject’s risk may be determined by comparing results of administering an aquaporin inhibitor agent to a subject with control results. In some embodiments of the invention, an aquaporin inhibitor agent administered to a subject reduces the subject’s risk of developing a neurodegenerative disease or condition compared to a control risk of developing the neurodegenerative disease or condition, wherein the control risk is a risk of a subject in essentially identical circumstances developing the neurodegenerative disease or condition in the absence of the administered aquaporin inhibitor agent.

[0067] Efficacy of a method of the invention to reduce a subject’s risk may be determined by comparing results of administering an agent that increases activity of an enzy me, such as but not limited to caspase 2 and / or caspase 3 to a subject with control results. In some embodiments of the invention, an agent that increases activity of a caspase is administered to a subject and reduces the subject’s risk of developing a neurodegenerative disease or condition compared to a control risk of developing the neurodegenerative disease or condition, wherein the control risk is a risk of a subject in essentially identical circumstances developing the neurodegenerative disease or condition in the absence of the administered agent that increases activity of caspase 2 and / or caspase 3.

[0068] Efficacy of a method of the invention to reduce a subject’s risk may be determined by comparing results of administering an agent that decreases activity of an enzyme, such as but not limited to a secretase and / or BACE1 to a subject with control results. In some embodiments of the invention, an agent that decreases activity of a secretase and / or BACE1 is administered to a subject and reduces the subject’s risk of developing a neurodegenerative disease or condition compared to a control risk of developing the neurodegenerative disease or condition, wherein the control risk is a risk of a subject in essentially identical circumstances developing the neurodegenerative disease or condition in the absence of the administered agent that decreases activity of a secretase and / or BACE1.

[0069] Identifying a subject at risk of developing a neurodegenerative disease or condition may be based, at least in part, on factors such as but not limited to medical history of the subject; genetic background of the subject; a prior, current, or future activity of the subject; and a prior, current, or future exposure of the subject to an agent or element believed to be a possible causative factor in the development of the neurodegenerative disease or condition.

[0070] In addition to activities or future activities that may indicate a level of risk of a subject developing a neurodegenerative disease or condition, the presence of an existing symptom or physiological indicator in a subject may indicate a risk of the subject developing a neurodegenerative disease or condition. For example, though not intended to be limiting, a subject’s age may assist in identifying the subject as being at risk for a neurodegenerative disease or condition. Additional examples of physiological conditions or events that may assist in identifying a subject at risk of having or as having a neurodegenerative disease or condition include but are not limited to CNS inflammation and / or infection, traumatic brain injury, repetitive brain injury, or an autoimmune condition.

[0071] Effective amounts

[0072] Certain embodiments of methods of the invention comprise administering an agent that reduces an abnormal function of a glial canal in a canal-forming glial cell in the subject (a non-limiting example of which is an aquaporin inhibitor agent) in an amount effective to treat a neurodegenerative disease or condition. Some embodiments of methods of the invention comprise administering an agent that increases activity of an enzyme, such as but not limited to a caspase, thereby reducing an abnormal function of a glial canal in a canalforming glial cell in the subject in an amount effective to treat a neurodegenerative disease or condition in the subject. Some embodiments of methods of the invention comprise administering an agent that decreases activity of an enzyme, such as but not limited to a secretase and / or BACE1, thereby reducing an abnormal function of a glial canal in a canalforming glial cell in the subject in an amount effective to treat a neurodegenerative disease or condition in the subject. An effective amount is a dosage of the agent sufficient to provide a medically desirable result. It should be understood that pharmacological agents of the invention are used to treat or prevent neurodegenerative diseases or conditions, that is, in some embodiments they may be used to treat an existing neurodegenerative disease or condition in a subject, and they may also be administered prophy lactically to a subject at risk of developing a neurodegenerative disease or condition. An effective amount is that amount that can lower a risk of, slow or perhaps prevent altogether the development of a neurodegenerative disease or condition in a subject.

[0073] Factors involved in determining an effective amount are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of a pharmacological agent of the invention be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a subject (also referred to herein as a patient) may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0074] The therapeutically effective amount of a pharmacological agent of the invention is that amount effective to treat the condition, such as a neurodegenerative disease or condition. In the case of s neurodegenerative diseases or conditions the desired response is inhibiting the progression of the neurodegenerative disease or condition and / or reducing the severity and / or the level of the neurodegenerative disease or condition. This may involve only slowing the progression of the neurodegenerative disease or condition temporarily, although it may include halting the progression of the neurodegenerative disease or condition permanently. This can be monitored by routine diagnostic methods known to those of ordinary skill in the art. A desired response to a method of the invention to treat a neurodegenerative disease or condition may in some embodiments, be preventing the onset of the neurodegenerative disease or condition.

[0075] Certain embodiments of methods of the invention include administering an agent (non-limiting examples of which are an aquaporin inhibitor agent, an agent that increases a level of caspase activity, and an agent that decreases a level of secretase activity and / or BACE1 activity ) that reduces an abnormal function of a glial canal in a canal-forming glial cell in the subject, wherein the agent is administered in an amount effective to reduce the subject's risk of developing a neurodegenerative disease or condition, and / or to reduce the severity of a neurodegenerative disease or condition present in the subject. In some embodiments a therapeutically effective amount refers to that amount of the agent being administered to a subject that is sufficient to prevent progression of a neurodegenerative disease or condition. Administration of the agent in an amount effective to reduce an abnormal function of a glial canal in a canal-forming glial cell in the subject may reduce the risk of a subject developing the neurodegenerative disease or condition by at least 1%, 2%, 3%, 4%, 5%. 6%, 7%, 8%. 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42,%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62,%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%. 76%. 77%. 78%. 79%. 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100%, compared to the percent control risk of the subject developing the neurodegenerative disease or condition. In a non-limiting example, if a subject’s risk of developing a neurodegenerative disease or condition based on medical history, genetic analysis, etc. is 20%. administering an effective amount of an aquaporin inhibitor agent to the subject may reduce the subject’s 20% risk down to less than 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% risk, or to 0% risk.

[0076] In certain embodiments of methods of the invention, administering an effective amount of an agent that reduces an abnormal function of a glial canal in a canal-forming glial cell in the subject (non-limiting examples of which are an aquaporin inhibitor agent and agent, an agent that increases a level of caspase activity, and an agent that decreases a level of secretase activity and / or BACE1 activity) to a subject believed to have or to be at risk of having a neurodegenerative disease or condition, increases the likelihood of survival of neuronal cells in the subject compared to a control likelihood of survival. In some instances, a control likelihood of survival of neuronal cells is a likelihood of survival of the neuronal cells in a subject in the absence of the administration of the effective amount of the agent. Administration of an effective amount of the agent to a subject in need of such treatment can increase the likelihood of survival neuronal cells in the subject to least 1, 2, 3, 4, 5. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 times higher than the control likelihood of survival of neuronal cells in the subject. Another way of expressing a change in likelihood of survival of neuronal cells is in reduction in the percent likelihood of neuronal cell death due to the neurodegenerative disease or condition. For example, as a result of treatment with an agent, that reduces an abnormal function of a glial canal in a canal-forming glial cell in the subject (non-limiting examples of which are an aquaporin inhibitor agent and agent, an agent that increases a level of caspase activity, and an agent that decreases a level of secretase activity and / or BACE1 activity), the subject may have a risk of neuronal cell death that is up to 1%. 2%, 3%, 4%, 5%, 6%. 7%, 8%, 9%. 10%. 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42,%,

[0077] 43%. 44%. 45%. 46%. 47%. 48%. 49%. 50%. 51%. 52%. 53%. 54%. 55%. 56%. 57%. 58%.

[0078] 59%, 60%, 61%, 62,%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,

[0079] 91%, 92%, 93%, 94%, or 95% of the risk of neuronal cell death in a control subject not treated with the agent. In a non-limiting example, if a control risk of neuronal cell death from a neurodegenerative disease or condition is 80%, administering an effective amount of an aquaporin inhibitor agent to a subject identified as having the neurodegenerative disease or condition may have a risk of neuronal cell death resulting from the neurodegenerative disease or condition that is reduced to less than 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%. 30%. 25%. 20%. 19%. 18%. 17%. 16%. 15%. 14%. 13%. 12%. 11%. 10%. 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% risk, or the risk of the neuronal cell death in the subject due to the neurodegenerative disease or condition may be reduced to 0%.

[0080] In some embodiments of methods of the invention, a therapeutic regimen comprising administration of one or more agents that reduce an abnormal function of a glial canal in a canal-forming glial cell in the subject (non-limiting examples of which are an aquaporin inhibitor agent and agent, an agent that increases a level of caspase activity, and an agent that decreases a level of secretase activity and / or BACE1 activity) to a subject determined to be in need such treatment may include administration of one or more agents once, or multiple times. Multiple administrations of an agent that reduces an abnormal function of a glial canal in a canal-forming glial cell in the subject means the agent is administered to a subject 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. It will be understood that administration of an agent that reduces an abnormal function of a glial canal in a canal-forming glial cell in the subject (nonlimiting examples of which are an aquaporin inhibitor agent and agent, an agent that increases a level of caspase activity, and an agent that decreases a level of secretase activity and / or BACE1 activity) may be done in combination with additional treatments for a neurodegenerative disease or condition.

[0081] Treatment Selection

[0082] As described herein, a therapeutic regimen (also referred to herein as a treatment) may be selected for a subject based at least in part on the identification that the subject is at risk of having or that the subject has neuronal degeneration. In some embodiments of the invention, selection of a treatment may be based, at least in part, on the severity of a neuronal degenerative disease or condition in a subject. In some embodiments of the invention, a selected treatment may include administering to the subject an effective amount of one or more agents that reduce an abnormal function of a glial canal in a canal-forming glial cell adjacent to a neuronal cell. In some embodiments, the administered agent is an aquaporin inhibitor agent.

[0083] Certain embodiments of methods of the invention include administering an agent that reduces an abnormal function of a glial canal and administering one or more additional treatments appropriate for the specific neurodegenerative disease or condition in the subject. As a non-limiting example, an additional treatment for a subject identified as having or at risk of having a neurodegenerative disease or condition includes one or more of physical therapy, surgery, administration of one or more additional therapeutic agents, dietary modification, etc. Upon a determination of the presence of, or a risk of. a neurodegenerative disease or condition in a subject, a practitioner will, without undue experimentation, be aware of and able to select one or more treatments that may be administered to a subject in addition to the administration of an agent that reduces an abnormal function of a glial canal, non-limiting examples of which are an aquaporin inhibitor agent, an agent that increases a level of caspase activity, and an agent that decreases a level of secretase activity and / or BACE1 activity.

[0084] Neurodegenerative Diseases and Conditions

[0085] Methods of the invention may be used to treat a neurodegenerative disease or condition in a subject. The terms “neuronal degenerative’7and “neurodegenerative” are used interchangeably herein in reference to diseases and conditions. Such diseases or conditions include, but are not limited to Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic lateral sclerosis (ALS); Chronic Traumatic Encephalopathy (CTE), and Motor Neuron Disease.

[0086] In some embodiments of methods of the invention, a neurodegenerative disease or condition is a disease or condition in which there is degeneration and death of neuronal cells. Some embodiments of methods of the invention may include selecting a therapeutic regimen for a subject, wherein the therapeutic regimen comprises administering to the subject an agent that reduces an abnormal function of a glial canal in a canal -forming glial cell in the subject. A non-limiting example of such an agent is an aquaporin inhibitor agent. A treatment method of the invention may also include one or more additional therapeutic actions or administered medicaments, depending on the specific neurodegenerative disease or condition, the severity of the neurodegenerative disease or condition, or other factors of which a practitioner will be aware as factors for consideration in selecting a treatment. Therapeutic Compositions and Methods and Monitoring Efficacy

[0087] Methods of the invention include producing in a subject in need of such treatment a therapeutic effect against neuronal cell death and / or a neurodegenerative disease or condition. The term “therapeutic effect” as used herein in reference to an agent that reduces an abnormal function of a glial canal in a canal-forming glial cell means a clinically beneficial effect of the agent against neuronal cell death and / or a neurodegenerative disease or condition when it is administered to a subject in need of such treatment. A therapeutic effect of an agent that reduces an abnormal function of a glial canal in a canal-forming glial cell in the subject (also referred to herein as a “response” to a treatment method of the invention) can be determined, for example, by detecting one or more physiological effects of the treatment, such as the decrease or lack of symptoms of the neurodegenerative disease or condition following administration of the treatment. Additional means of monitoring and assessing a neurodegenerative disease or condition in a subject, and ways to assess and determine one or more of a level, severity, change in severity, etc. of a neurodegenerative disease or condition in subject are known in the art and can be used to assess the neurodegenerative condition in a subject following a treatment method of the invention. Non-limiting examples of physiological symptoms of neurodegenerative diseases and conditions that may be assessed in certain embodiments of methods of the invention are provided elsewhere herein and will be known in the art.

[0088] Methods and compositions of the invention may be used to treat a neurodegenerative disease or condition. As used herein, the terms “treat”, “treated”, or “treating” when used in relation to a neurodegenerative disease or condition may refer to a prophylactic treatment that decreases the likelihood or risk of a subject developing the neurodegenerative disease or condition, and may be used to refer to a treatment after a subject has developed a neurodegenerative disease or condition in order to eliminate or ameliorate the neurodegenerative disease or condition, prevent the neurodegenerative disease or condition from becoming more advanced or severe, and / or to slow the progression of the neurodegenerative disease or condition compared to the progression of the neurodegenerative disease or condition in the absence of a therapeutic method of the invention.

[0089] Subjects and Samples

[0090] As used herein, a subject may be a vertebrate animal including but not limited to a human, mouse, rat, guinea pig, rabbit, cow, dog, cat, horse, goat, and non -human primate, e.g., monkey. A subject may be a mammal. In some embodiments, a subject is any human or non-human recipient of one or more of an aquaporin-inhibitor agents, a caspase-activity- increasing agent, a secretase-activity reducing agent, a BACE1 -activity reducing agent, a composition, or a pharmaceutical composition of the invention as described herein. In certain aspects of the invention, a subject may be a domesticated animal, a wild animal, or an agricultural animal. Thus, the invention can be used to treat a neurodegenerative diseases or conditions in human and non-human subjects. For instance, methods and compositions of the invention can be used in veterinary applications as well as in human treatment regimens. In some embodiments of the invention, a subject is a human. In some embodiments of the invention, a subject has or is at risk of having a neurodegenerative disease or condition and is in need of a treatment of the invention. In some embodiments, a subject does not have epilepsy or a seizure disorder. In some embodiments, a subject does not have cytotoxic brain swelling. In some embodiments, a subject does not have fluid- induced brain swelling. In some embodiments, a subject does not have brain oedema. In some embodiments, a subject does not have a brain tumor.

[0091] As used herein, a neuronal cell may be a cell obtained from a cell sample, tissue sample, blood sample, etc. A method of the invention may include contacting a cell with a composition of the invention in vitro, ex vivo, or in vivo. In some embodiments, a method of the invention comprises contacting a canal-forming neuronal cell that is in culture. In some embodiments of methods of the invention comprises contacting a canal-forming glial cell that is in a subject. Certain embodiments of methods of the invention include ex vivo treatment methods.

[0092] Assessments and Controls

[0093] A neurodegenerative disease or condition in a subject can be detected using an art- known method, certain of which are described elsewhere herein. Methods that may be used to detect a neurodegenerative disease or condition include, but are not limited to identifying the presence of one or more physiological characteristics or symptoms of the neurodegenerative disease or condition in the cell or subject, assessing genetic characteristics of a cell or subject, histological and / or imaging methods applied to a cell and / or a subject, etc. Characteristics of a neurodegenerative disease or condition detected in a subject can be compared to control values of the characteristics of the neurodegenerative disease or condition. A control value may be a predetermined value, which can take a variety of forms. It can be a single cut-off value, such as a median or mean. It can be established based upon comparative groups, such as in groups of individuals having the neurodegenerative disease or condition, groups of individuals who have been administered a treatment for the neurodegenerative disease or condition, groups of individuals who have not been administered a treatment for the neurodegenerative disease or condition, etc. Another example of comparative groups may be groups of subjects having one or more symptoms of or a diagnosis of the neurodegenerative disease or condition and groups of subjects without the one or more symptoms of or a diagnosis of the neurodegenerative disease or condition. A predetermined value will depend upon the particular population selected. Accordingly, the predetermined value selected may take into account a category in which an individual falls. Non-limiting examples of categories are a subject’s age, a subject’s genetic background, etc. Appropriate categories can be selected and utilized with no more than routine experimentation by those of ordinary skill in the art.

[0094] Controls can be used in methods of the invention to compare characteristics of different control groups, characteristics of a subject with those of a control group, etc. Comparisons between subjects and controls, one control with another control, etc. may be based on relative differences. For example, though not intended to be limiting, a physiological symptom in a subject treated with an agent that reduces an abnormal function of a glial canal in a canal-forming glial cell in the subject in a therapeutic method of the invention, can be compared to the physiological symptom of a control subject or subject group that has not been administered the agent. In some embodiments, a suitable control is a subject not treated with a treatment method of the invention. A comparison of a treated versus a control may include comparing disease severity differences between a treated subject and a selected control. In some instances, severity of symptoms or physiological effects of a neurodegenerative disease or condition in a subject treated with a method of the invention may be determined to be less relative to a selected control, with the comparison indicating up to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%,

[0095] 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%. 42,%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,

[0096] 50%. 51%. 52%. 53%. 54%. 55%. 56%. 57%. 58%. 59%. 60%. 61%. 62.%, 63%, 64%, 65%,

[0097] 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,

[0098] 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,

[0099] 98%, 99%, or 100% reduction in severity of one or more physiological effects and symptoms of the neurodegenerative disease or condition in the subject as compared to the control. In some embodiments, a level of severity' of a treated subject’s neurodegenerative disease or condition is less than 100% of a control severity level of the neurodegenerative disease or condition. In certain embodiments of the invention the severity of one or physiological symptoms of the neurodegenerative disease or condition in a subject treated according to a method of the invention is less than or equal to 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%. 86%. 85%. 84%. 83%. 82%. 81%. 80%. 79%.

[0100] 78%. 77%. 76%. 75%. 74%. 73%. 72%. 71%. 70%. 69%. 68%. 67%. 66%. 65%. 64%. 63%.

[0101] 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%,

[0102] 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%,

[0103] 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%. 15%.

[0104] 14%. 13%. 12%. 11%. 10%. 9%, 8%, 7%. 6%, 5%, 4%, 3%, 2%. 1%. 0.9%, 0.8%. 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the control level of severity of the one or more physiological symptoms, respectively, of the neurodegenerative disease or condition.

[0105] In another non-limiting example, a level of a neurodegenerative disease or condition in a subject and / or increase in a therapeutic effect of administering an aquaporin inhibitor agent to a subject may be determined by comparing a likelihood of survival of neuronal cells in the subject treated with aquaporin inhibitor agent with a control likelihood of survival of neuronal cells. A non-limiting example of a control likelihood of survival of neuronal cells is the likelihood of survival of neuronal cells in a subject with the neurodegenerative disease or condition who is not treated with a method of the invention. It will be understood that parameters such as behavioral change, CNS imaging changes, etc. can be used to assess neuronal cell survival and / or death and results obtained from a subject administered a composition of the invention can be compared to a control to determine a level of therapeutic effectiveness of the treatment of the invention.

[0106] It will be understood that controls may be, in addition to predetermined values, samples of materials tested in parallel with the experimental materials. Examples include samples from control populations or control samples generated through manufacture to be tested in parallel with the experimental samples; and a control may be a sample from a subject prior to, during, or after a treatment with an embodiment of a method or composition of the invention. Thus, one or more characteristics determined for a subj ect having a neurodegenerative disease or condition may be used as “control” values for those characteristics in that subject at a later time.

[0107] Preparation and administration of pharmacological agents The pharmacological agents used in the methods of the invention are preferably sterile and contain an effective amount of an aquaporin inhibitor agent, an effective amount of an agent that increases a level of caspase activity, or an agent that decreases a level of secretase activity and / or decreases a level of BACE1 activity, for producing the desired response in a unit of weight or volume suitable for administration to a subject. In some embodiments an agent that reduces an abnormal function of a glial canal in a canal-forming glial cell in the subject (non-limiting examples of which are an aquaporin inhibitor agent and an agent that increases a level of caspase activity, or an agent that decreases a level of secretase activity and / or decreases a level of BACE1 activity) is delivered in a composition formulated to cross into the brain, e.g., formulated to cross the blood-brain barrier. Doses of pharmacological agents administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. The dosage of a pharmacological agent may be adjusted by the individual healthcare provider or veterinarian, particularly in the event of any complication. A therapeutically effective amount typically varies from 0.01 mg / kg to about 1000 mg / kg, from about 0.1 mg / kg to about 200 mg / kg, or from about 0.2 mg / kg to about 20 mg / kg, in one or more dose administrations daily, for one or more days. An agent that reduces an abnormal function of a glial canal in a canal -forming glial cell in the subject (a non-limiting example of which is an aquaporin inhibitor agent) may also be referred to herein as a pharmacological agent.

[0108] The amount of a treatment of the invention administered to a subject may be varied for example by increasing or decreasing the amount of one or more agents administered to the subject to reduce an abnormal function of a glial canal in a canal -forming glial cell in the subject. Changes in a treatment of the invention may include one or more of changing the therapeutic composition administered, changing the route of administration, changing the dosage timing and so on. An effective amount of a composition of the invention, will vary with the particular neurodegenerative disease or condition being treated, the age and physical condition of the subject being treated, the severity of the neurodegenerative disease or condition, the duration of the treatment, the specific route of administration, and other art- known factors within the know ledge and expertise of a health practitioner.

[0109] Various modes of administration known to the skilled artisan can be used to effectively deliver a pharmaceutical composition of the invention comprising an aquaporin inhibitor agent to a subj ect in an amount effective to produce a desired therapeutic effect against a neurodegenerative disease or condition in the subject. Administration methods that may be used to deliver a composition or pharmaceutical compound of the invention to a subject include, but are not limited to topical, intravenous, oral, intracavity, intrathecal, intrasynovial, buccal, sublingual, intracranial, intraventricular, intranasal, transdermal, intravitreal, inhalation, subcutaneous, intramuscular, or intradermal administration.

[0110] The invention is not limited by the particular modes of administration disclosed herein. Standard references in the art (e g., Remington, The Science and Practice of Pharmacy, Editor: Adeboye Adejare, 23rdEdition, Elsevier, 2020) provide modes of administration and formulations for delivery of various pharmaceutical preparations and formulations in pharmaceutical carriers. Other protocols that are useful for the administration of pharmacological agents of the invention will be known to one of ordinary skill in the art, in which the dose amount, schedule of administration, sites of administration, mode of administration and the like vary from those presented herein.

[0111] Treatment compositions and treatment agents of the invention are also referred to herein as pharmaceutical agents, pharmaceutical compounds, and pharmaceutical compositions of the invention. Treatment compositions of the invention may be administered to mammals other than humans, e.g., for testing purposes or veterinary therapeutic purposes, and such administrations may be carried out under substantially the same conditions as described herein. It will be understood that methods and compositions of the invention are applicable to both human and animal diseases. Thus, this invention is intended to be used in husbandry and veterinary medicine as well as in human therapeutics.

[0112] When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically acceptable amounts and in pharmaceutically acceptable compositions. The term ■■pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.

[0113] A pharmacological agent or composition of the invention may be combined, if desired, with a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” as used herein means one or more compatible solid or liquid fillers, diluents, or encapsulating substances which are suitable for administration into a human. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the pharmacological agents of the invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.

[0114] The pharmaceutical compositions may contain suitable buffering agents, non-limiting examples of which are acetate, phosphate, citrate, glycine, borate, carbonate, bicarbonate, hydroxide (and other bases) and pharmaceutically acceptable salts of the foregoing compounds. The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.

[0115] The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier, which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0116] Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, pills, lozenges, each containing a predetermined amount of the active compound (e.g., an aquaporin inhibitor). Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir, an emulsion, or a gel. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Non-limiting examples of suitable excipients include fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers, i.e., EDTA for neutralizing internal acid conditions or may be administered without any carriers.

[0117] For a pharmacological agent, the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine. One skilled in the art has available formulations which will not dissolve in the stomach yet will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of the aquaporin inhibitor agent or by release of the biologically active material beyond the stomach environment, such as in the intestine.

[0118] The treatment agents of the invention, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions of the invention may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents.

[0119] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0120] Pharmacological agent(s), including but not limited to an aquaporin inhibitor agent may be provided in particles. The term ■■particles” as used herein means nano or microparticles (or in some instances larger particles) that can consist in whole or in part of an aquaporin agent as described herein. The particles may contain the pharmacological agent(s) in a core surrounded by a coating, including, but not limited to, an enteric coating. The pharmacological agent(s) also may be dispersed throughout the particles. The pharmacological agent(s) also may be adsorbed into the particles. The particles may be of any order release kinetics, including zero order release, first order release, second order release, delayed release, sustained release, immediate release, and any combination thereof, etc. The particle may include, in addition to the pharmacological agent(s), any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable material or combinations thereof. The particles may be microcapsules which contain the aquaporin inhibitor agent in a solution or in a semi-solid state. The particles may be of virtually any shape.

[0121] Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering the pharmacological agent(s). Such polymers may be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired. Bioadhesive polymers of particular interest include bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, (1993) 26:581-587, the teachings of which are incorporated herein. These include polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate). poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acry late), and poly(octadecyl acrylate).

[0122] A pharmacological agent of the invention may be contained in controlled-release systems. The term “controlled release7’ is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation are controlled. This refers to immediate as well as non-immediate release formulations, with non-immediate release formulations including but not limited to sustained-release and delayed-release formulations. The term “sustained release” (also referred to as “extended release”) is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, results in substantially constant blood levels and / or tissue levels of a drug over an extended time period. The term “delayed release” is used in its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of the drug therefrom. “Delayed release” may or may not involve gradual release of drug over an extended period of time, and thus may or may not be “sustained release.”

[0123] Use of a long-term sustained-release implant may be particularly suitable for treatment of chronic neurodegenerative diseases or conditions and / or chronic risk of developing a neurodegenerative disease or condition. “Long-term” release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the pharmacological agent(s) of the invention for at least 7 days, and / or from 30-60 days. Longterm sustained release implants are well-known to those of ordinary' skill in the art and include some of the release systems described above.

[0124] A treatment of the invention, may in some embodiments, comprise administering an agent such as, but not limited to an aquaporin inhibitor agent, an agent that increases a level of caspase activity, and / or an agent that decreases a level of secretase activity and / or decreases a level of BACE1 activity7, using fusion proteins, vectors, etc., to express the agent in the subject.

[0125] The invention also contemplates the use of kits. In some aspects of the invention, the kit can include one or more pharmaceutical preparation vial, a pharmaceutical preparation diluent vial, and an aquaporin inhibitor agent. A vial containing the diluent for the pharmaceutical preparation is optional. A diluent vial may contain a diluent such as physiological saline for diluting what could be a concentrated solution or lyophilized powder of the aquaporin inhibitor agent. The instructions can include instructions for mixing a particular amount of the diluent with a particular amount of the concentrated pharmaceutical preparation, whereby a final formulation for injection or infusion is prepared. The instructions may include instructions for treating a subject with effective amounts of the aquaporin inhibitor agent and / or instructions for treating a subject with effective amounts of an agent that increases a level of caspase activity or an agent that decreases a level of secretase activity7and / or decreases a level of BACE1 activity7. It also will be understood that the containers containing the preparations, whether the container is a bottle, a vial with a septum, an ampoule with a septum, an infusion bag. and the like, can contain indicia such as conventional markings that change color when the preparation has been autoclaved or otherwise sterilized.

[0126] Certain definitions

[0127] As used herein, the term “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and / or methods of the invention. It is contemplated that any method described herein can be implemented with respect to any other method described herein. As used herein, the term “or"’ in the claims is used to mean “and / or"’ unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.”

[0128] As used herein, “comprise” and its variations, such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated item, element or step or group of items, elements or steps but not the exclusion of any other item, element or step or group of items, elements or steps unless the context requires otherwise. Similarly, "another" or “other” may mean at least a second or more of the same or different claim element or components thereof.

[0129] As used herein, the term “contacting” refers to any suitable method of bringing an inhibitor agent, a composition, or a pharmaceutical composition into contact with a cell. For in vivo applications, any known method of administration is suitable as described herein.

[0130] The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

[0131] The following examples are provided to illustrate specific instances of the practice of the present invention and are not intended to limit the scope of the invention. As will be apparent to one of ordinary skill in the art. the present invention will find application in a variety of compositions and methods.

[0132] Examples

[0133] Example 1

[0134] Materials and Methods

[0135] Animals:

[0136] The spiders of the species Cupiennius salei used in aspects of this study were bred and raised under laboratory’ conditions as previously described [R. Fabian-Fine et al., Journal of Comparative Neurology 531, 618-638 (2023)]. Healthy animals were selected based on their age, their ability to maintain a perfectly aligned posture on a vertical surface, and appropriate hunting behavior that is typical for young healthy animals as described previously [R. Fabian-Fine et al., Journal of Comparative Neurology 531, 618-638 (2023)]. Old animals were 24+ months of age. showed an inability to maintain their vertical resting position and showed a decline in their hunting behavior. Brains of young animals had a healthy ivory color, whereas the brains of old animals showed brown discoloration.

[0137] Tissue Preparation for Histological and Electron-Microscopic Investigations:

[0138] Spider brain:

[0139] For the purpose of aspects of this work, the brains of two 6-month-old juvenile animals were examined, including two 13-month-old healthy adults and three 24 or 36- month-old spiders that showed mild (24 month), or severe (two 36-month-old animals) behavioral signs of degeneration. To achieve optimal tissue preservation at both light- and ultrastructural levels the animals were deeply anesthetized with CO2. After removal of the tarsal leg segments the animals were perfused with freshly prepared ice cold 4% paraformaldehyde (EMS 15710) and 2.5% glutaraldehyde (EMS 16019) in phosphate buffered saline (pH 7.4, 0. 1 M; PBS). All procedures were in accordance with IACUC protocols.

[0140] The peripheral structures used in this study are well-described mechanosensory neurons in the VS-3 slit sense organ that is located on the anterior part of the patella on all eight legs. Location and dissection procedure of this organ has been described previously [R. Fabian-Fine et al., The Journal of Neuroscience 19, 298-310 (1999)]. After dissection of the VS-3 organ and CNS in ice-cold fixative [R. Fabian-Fine et al., Cell and tissue research 362, 461-479 (2015)]. the preparations were washed in PBS and post-fixed for 20 min in 0.5-1.0% osmium tetroxide (Electron Microscopic Sciences, #19150). After washing in PBS (2x5 min), the specimens were dehydrated in a graded series of ethanol according to standard electron microscopy protocols. The preparations were transferred into propylene oxide (Electron Microscopic Sciences, #20401), slowly infiltrated with Araldite (Electron Microscopic Sciences, #13900), and polymerized overnight at 60 °C, according to manufacturer’s instructions.

[0141] Preparation of Human Brain Tissue:

[0142] The human brain samples used in the study were obtained from the brains of three decedents undergoing autopsy examination at the University of Vermont Medical Center and were fully consented for the purposes of diagnosis, research, and teaching. Samples were obtained promptly following brain removal.

[0143] Tissue from the decedents with a clinical history of Alzheimer dementia was obtained 14-43 hours postmortem. Preparation of human brain tissue for both light and electron microscopic approaches were carried out as described for spider tissue. Diagnostic neuropathologic examination of these brains revealed ADNC with an ABC score of A3B3C2, consistent with a high burden of ADNC. Significant amyloid plaques and phosphorylated tau tangles were present in the hippocampus.

[0144] Preparation of Rat Hippocampus:

[0145] For ethical reasons to avoid unnecessary animal sacrifices, the rat hippocampus used in this study originated from healthy adult control rats previously processed for electronmicroscopy during prior studies on rat hippocampus carried out by the first author [A. Ruiz et al., Neuron 39, 961-973 (2003)]. The tissue originated from healthy adult male Sprague Dawley rats and was fixed, dehydrated, and embedded in Araldite as described for spider tissue. All experiments were conducted in accordance with regulatory animal protocols.

[0146] Semi- and Ultrathin Sectioning:

[0147] Araldite-embedded tissue samples were cut with an 8 mm Diatome histo-knife altering between 1 pm semithin and 50 nm serial ultrathin sections using a Leica Ultracut E. Sections were stretched with chloroform (Electron Microscopic Sciences, #12540).

[0148] Semi-thin sections were collected in distilled water on glass slides and dried on a hot plate at 80 °C until completely dry. Staining was performed with 2% aqueous toluidine blue (Sigma 6586-04-5) stain for ~2 min at 80 °C and rinsed thoroughly with distilled water. For this staining to be successful it is essential that the tissue is not kept in fixative for more than 24 hours. It w as determined that the toluidine blue staining did not w ork on tissue that had been kept in paraformaldehyde / glutaraldehyde fixative for longer time periods. The sections were examined and imaged using a Zeiss Axiolmager MZ with apotome. Utrathin sections w ere cut at a thickness of 50 nm and collected on pioloform-coated single-slot copper or nickel grids (EMS# G2010CU). Grids were contrasted with aqueous 1.5% uranyl acetate (6 min) and Reynold’s lead citrate (2 min). Electron microscopic examination w as conducted using a JOEL 1400 electron microscope operated at 80 kV.

[0149] Light Microscopic Immunohistochemistry:

[0150] For anti-Aquaporin4 immunolabelling three healthy 19-month-old female spiders were anesthetized and perfused with 4% freshly made paraformaldehyde as described above. After ~1 min of perfusion the legs, pedipalps, cheliceres, and opisthosoma were removed, and the brain w as submerged under fixative overnight. Brain dissection, vibratome sectioning and GABA / Glutamate immunolabelling was conducted as described previously [R. Fabian- Fine et al., Cell and tissue research 362. 461-479 (2015)]. The sections were washed in PBS 4x5 min and unspecific binding sites were blocked with blocking medium containing 0.25% Bovine Serum Albumin (Sigma A4503) and 5% Normal goat serum (Sigma G9023) in 1% Triton-X / PBS for 20 min. The sections were incubated overnight at 6 °C within primary rabbit anti Aquaporin4 antiserum (BiCell #20104) at a dilution of 1: 100 in PBS containing 10% blocking medium. The next day sections were washed in PBS 3x2 min and 4x5 min and transferred into blocking medium for 20 min prior to incubation with the light sensitive secondary antibody (Cy3 goat anti rabbit, (Jackson ImmunoResearch Laboratories 111-165- 003); 1:600 in PBS containing 10% blocking medium overnight at 6 °C). The immunolabelled sections were washed in PBS (3x2 min), stained with Hoechst Blue (Sigma H 6024; 1 :3000 in PBS for 20 min). After washing in PBS (5x5 min) the sections were mounted on glass slides using Mowiol (Sigma# 81381). All steps were conducted under minimum illumination to avoid tissue damage and bleaching of the fluorochromes. The sections were analyzed using a confocal Zeiss Axiolmager with Apotome. The mouse antihuman vimentin antibody (Developmental Studies Hybridoma Bank IOWA # AMF-17b) was used at a dilution of 1 : 100. For the anti-Aquaporin4 pre-absorption controls the antiserum was pre-absorbed with an equal volume of blocking peptide for 1 h at room temperature prior to tissue incubation.

[0151] Western Blot Analysis

[0152] The CNS of a healthy 8-month-old spider was dissected in cold PBS and placed in a glass homogenizer containing 1 ml of RIPA lysis buffer (R0278 and 1 part protease inhibitor (Sigma S-8830) per 10 parts of RIPA buffer. The tissue was manually homogenized for ~2 min and sonicated for 2x5 sec to extract transmembrane proteins. The homogenate was centrifuged for 20 min at 6,000 x g at 4 °C. The supernatant was diluted with an equal volume of 2x Laemmli sample buffer (BioRad# 1 10737) and denatured at 95 °C for 5 min. Proteins were separated using a Mini-PROTEAN TGX precast polyacrylamide gel (# 456- 1083, BIO-RAD, USA). Wells were loaded with 30 pl of supernatant and 20 pl of protein standard (BioRad # 1610399) ranging from 5-250 kDa. The gel was run at 200 V for ~30 min. Subsequently the gel was washed in distilled water 3x5 min and incubated in blotting buffer for 5 min. Loading of the blotting chamber and transfer of the proteins into nitrocellulose was performed according to the manufacturer's instructions. For antibody staining the nitrocellulose membrane containing the transferred proteins was immersed in blocking solution (Tris buffered saline containing 1% Tween 20 (TBS-T), 0.2% bovine serum albumin and 5% normal goat serum) for 15 min. Incubation with the rabbit anti-Aquaporin4 antiserum was carried out overnight at a dilution of 1 : 10,000 in TBS containing 10% blocking solution at 4 °C. After subsequent washing in TBS-T (4x5 min) the secondary antibody (peroxidase conjugated goat anti-rabbit was used to detect the primary' anti- Aquaporin4 antibody BioRad# 1705046). The secondary antibody was applied for 1 h at a dilution of 1: 10,000 in the same blocking solution used for primary antibody dilution under gentle agitation, washing 4x5 min in TBS-T, immunoreactive protein bands were visualized using the BioRad Opti-4CN Substrate Kit #1708235. The nitrocellulose was submerged in a solution of 1 part diluent with 9 parts of purified distilled water. After adding 0.02% of substrate the blots were agitated under visual control for ~ 5 min. Upon visibility of the labelled protein bands the nitrocellulose was washed in TBS-T and photographed immediately.

[0153] Light Microscopic Image Acquisition

[0154] Toluidine blue-stained sections were examined and captured using a ZEISS Axiolmager. Images were exported using ZEN-Blue program. Figures were created using Adobe Photoshop and Illustrator.

[0155] Microscopic Image and Statistical Analysis

[0156] Collected images were analyzed in ImageJ / Fiji for all measurements [J. Schindelin et al., Nature Methods 9, 676-682 (2012)]. Briefly, images were appropriately scaled, median filtered to remove noise, contrast enhanced, aqua-canals were manually selected with the multi-point tool, and the interactive morphological reconstruction function in the MorphoLib ImageJ plugin was used to fill the area of the aqua canals [D. Legland et al., Bioinformatics 32, 3532-3534 (2016)]. Then, the canals’ areas were thresholded to remove non-canal areas and the binary' masks were cleaned up manually to match the original images, and the analyze particles ImageJ function was used to measure each canal’s area and longest axis. Finally, each canal was classified as being present in either degenerating or healthy tissue areas based on the intactness and regularity of the glial and neuronal membranes.

[0157] Unpaired, two-tailed Student’s / -tests were used to perform comparisons of the areas and maximum length of glial and neuronal aqua-canals across degenerating and healthy tissues in the same preparation. Statistics were calculated and graphs were created using Prism 10.0.2 (GraphPad Software). Results

[0158] The role of glial-canals for removal of cellular debris from spider neurons

[0159] Healthy neurons in the spider CNS are surrounded by macroglia with large chromatinrich nuclei that are up to 50 pm in diameter. Similar to mammalian oligodendrocytes, each of these glial cells surrounds several adjacent neurons; and were referred to as spideroligodendroglia (sODG). Results of studies examining neurons and spider oligodendroglia (sODG) in leg and opisthosomal ganglia of C. salei, demonstrated that sODG with large nuclei were closely associated with neuronal somata (N) and formed finger-like projections into the neuronal cytoplasm. Results also demonstrated the large size of the chromatin-rich glial nuclei in comparison to neuronal nuclei. Unstained glial membranes were observed around neurons, and results indicated that cellular waste accumulated near glial-canals. Electron micrographs showed large glial-canals in close proximity to each other.

[0160] A specialized characteristic of sODG is the formation of finger-like processes that project into the neuronal cytoplasm and form glial-canals of varying shapes and sizes that engulf cellular debris. Serial section analysis of images from these studies showed that these glial-canals project toward lymphatic canals that drain cellular debris from the brain.

[0161] Ultras truclural features of sODG that lead to glial-canal formation

[0162] Results of studies showed glial lobe formation in spider leg ganglia. Fig. 1 A-C provide a schematic diagram showing proposed glial lobe formation in spider leg ganglia. Studies demonstrated that sODG form two ‘lobe’ types. Studies demonstrated that sODG form two ‘lobe’ types. The first type called ‘forming lobes’ (FLs) consists of a circular central sODG-arm around which gradually expanding circular glial membranes form. These FLs were present in all spiders observed in this study, indicative of regenerative abilities of sODG. The second lobe type referred to as ‘mature lobes’ (MLs), consists of numerous glial rings that are compacted laterally and take on an elongated appearance whereby the membranes of individual glial rings are compacted laterally and appear linearly aligned. During this compaction, glial cytoplasm and organelles are pushed toward the lateral boundaries. Besides mitochondria, glial lobes contain two conspicuous structures.

[0163] Studies were performed that elucidated glial lobe formation in spider leg ganglia. Results of certain studies showed spider oligodendroglia with two ‘forming lobes’ (FLs) characterized by the formation of circular membrane rings project toward neurons. Next, the maturing lobes elongated and flattened, which resulted in the linear alignment of glial membranes. Individual membranes were cleaved and anchored to microtubules. Results showed that individual membranes detached from their microtubule attachment and projected into the neuronal cytoplasm. During this process glial aqua canals were translocated into the neuronal cytoplasm (neuronal aqua canals) and created a cytoplasmic bulk flow that caused swelling of the neuronal aqua canals and the flow of cellular debris into the lumina of the glial-canals.

[0164] Firstly, canal-like profiles of ~ 250 nm in diameter, were recognizable by their circular shape and clear, unstructured lumina. Individual canal profiles were continuous throughout serial semithin sections and were contained in the cytoplasm of sODG and were referred to as ‘glial aqua-canals' (GACs).

[0165] The second structural feature in the following referred to as ‘microtubule-associated- break points’ (MABs) consisted of varying numbers of cleaved glial membranes that were in close association with one or more microtubules. Linear glial membranes in MLs that bordered on neuronal cytoplasm partially detached along MABs and formed processes of diverse sizes and shapes that projected into the neuronal cytoplasm. These projections contributed to the formation of ‘glial-canals’ that could be observed around the neuronal perimeter at the neuron-sODG border. It was determined that this partial detachment of glial membranes from mature glial lobes caused the translocation of GACs into the neuronal cytoplasm. These translocated canals are referred to as ‘neuronal aqua-canals’ (NACs). Forming glial-canals were associated with both GACs and NACs and accumulated large waste clusters (up to > 2 pm in diameter) in or around their lumina. The internalization of waste into the glial cells was either through phagocytosis or through engulfment by one or more glial membranes. The distal tips of canal-forming glial membranes often appeared bulbous and contained GAC profiles that formed openings into the neuronal cytoplasm. Networks of membrane cistemae were located around these distal tips. The formation of debris-containing glial-canals was observed in animals of all developmental stages suggesting that their formation is a normal process by which macroglia ensure controlled waste removal from neurons.

[0166] Glial-canal systems show AQP4-like immunoreactivity (AQP4-LIR)

[0167] To investigate the hypothesis that the functional significance of NACs and GACs is to create a cytoplasmic bulk flow toward glial-canals to draw cellular waste into the canals, AQP4-immunolabeling was used. Strong AQP4-LIR was observed within myelinated glial- canals of varying diameters that are located alongside or within neuronal somata and were distinguishable by their unstructured lumina. These structures were referred to as (tanycyte- like) ‘glial-canals.’ AQP4-LIR profiles within the neuronal somata were consistent with the location, shape, and size of both NACs and GACs. Interestingly, the AQP4-LIR appeared restricted to the canal structures, whereas the cytoplasm of both sODG and neurons appeared unlabeled. However, degenerating neuronal profiles showed an increased AQP4-10 LIR signal. Western Blot analysis revealed a specific band around 25 kDa, which is within the range of reported molecular weights for AQP4 [P. Agre et al., Journal of Physiology 542, 3- 16 (2002)]. To identify the cells that give rise to the AQP4-LIR glial-canals serial coronal sections through the spider brain and surrounding tissue were investigated. AQP4-LIR was detected in (a) cells lining a tubular system that borders dorsally onto the CNS and is continuous with the gastrointestinal tract (b) tubular appearing glial cells within the brain parenchyma and leg nerves, and (c) in mesenchymal cells in the neural lamella.

[0168] Investigation of the cells located in the dorsal tubular system revealed a diverse population of AQP4-LIR cells. One conspicuous cell ty pe forms characteristic long tubular processes that contained varying numbers of punctate immunofluorescent particles and pulled into adjacent tissue. A second conspicuous cell type identified were epithelial cells lining the inner surface of this tubular system. The AQP4-LIR mesenchymal cells were identified as located in the outer surface of the neural lamella and represented a subpopulation of a mesenchymal cell mass outside of the neural lamella [M. Brightman et al., The Journal of cell biology 40. 648-677 (1969)].

[0169] Cellular hallmarks of progressive neurodegeneration

[0170] To identify cellular changes during neurodegeneration onset, studies were performed to investigate the CNS of adult spiders with mild behavioral signs of neurodegeneration [R. Fabian-Fine et al., Journal of Comparative Neurology 531, 618-638 (2023)]. The CNS parenchyma in these animals appeared predominantly7healthy, however, individual neurons showed localized areas with abnormal changes in sODG and adjacent neuronal cytoplasm. The underlying cause for localized neuronal abnormalities was the excessive unraveling of mature sODG lobes leading to increased formation of glial-canals each engulfing neuronal cytoplasmic content. This structural glial impairment explained the granular appearance of the adjacent neuronal cytoplasm. Membranes in healthy sODG were regularly aligned and contain numerous microtubules. In contrast, the glial membranes in degenerating sODG were determined to be less regularly aligned, to contain more cytoplasmic content, and to appear curved with few visible microtubules. Both diameter and area of individual GACs were significantly larger in degenerating sODG (0.342 pm and 0.084 gm2, respectively) compared to healthy sODG (0.240 pm and 0.033 pm2) diam.: 1(852) = 6.80. p = 1.925 x area: 1(852) = 5.33, p = 1.258 x | ()7. unpaired / -tests), (see Fig. 2A-B) which indicated swelling of the GACs consistent with the hypertrophic appearance of degenerating sODG. At the ultrastructural level, the neuronal perimeter showed large numbers of partially detached glial membranes that projected into the neuronal cytoplasm and engulfed neuronal cytoplasmic content. This process coincided with the formation of numerous swelling NACs that originated at the neuron glial border in areas where sODG unraveled.

[0171] Interestingly, degeneration onset most often appeared adjacent to tanycyte-like glial- canals where larger glial lobes detached, unraveled, and depleted the surrounding cytoplasmic content. Compared to healthy brain tissue, the hypertrophic and darker appearance of the toluidine-blue stained tanycyte-like glial -canals was consistent with increased uptake of hydrophilic cellular waste. Progressive swelling of NACs accompanied by depletion of neuronal cytoplasm explained the spongiform and increasingly ‘hollow’ appearance of affected neurons that originated around the neuronal perimeter. In advanced neurodegeneration, only centrally located cytoplasmic structures including the neuronal nucleus remained. Advanced degeneration resulted in the rupture of sODG lobes that separated the neuronal cytoplasm from adjacent tanycyte-like glial-canals facilitating the complete cytoplasmic evacuation from neurons. The term ‘gliaptosis' was used to describe this glial-induced neuronal cell death via evacuation of cytoplasmic content by adjacent macroglia. During this process, the dorsal tubular system was identified as taking on a brown discoloration consistent with this observed in nephrocytes that internalize cellular debris in degenerating brains. Neuronal cell death w as follow ed by the disintegration of surrounding sODG and gradual replacement with particulate matter as described previously [R. Fabian- Fine et al.. Journal of Comparative Neurology 531, 618-638 (2023)].

[0172] Histological and ultrastructural similarities of degenerating human and spider neurons

[0173] Investigation of human motor cortex, nucleus locus coeruleus and hippocampus from human decedents with diagnostically confirmed ADNC showed that cellular changes in degenerating neurons were strikingly similar to those in affected spider neurons. This included: (a) spongiform appearance of neuronal somata that originated at the outer perimeter of affected neurons, (b) gradual depletion of cytoplasm that progressed toward the center, and (c) observation of hollow profiles that resembled the shape of neuronal somata. Examination of human and rat brain show ed that the mammalian neurons investigated here lacked the myelination around their somata observed in spiders. However, both neuronal somata and neurites were closely associated with myelinated cell profiles that contained characteristically clear lumina consistent with those observed in spiders. AQP4-immunolabeling of hippocampus from an Alzheimer patient with ADNC showed that immuno-negative pyramidal cells were paralleled by varicose AQP4-LIR cell processes similar to those observed in spider tissue. Results of studies carried out indicated that these cell profiles in human hippocampus emanated from the ventricular lining and showed co-localization for the structural protein vimentin, which alluded to their ependymal origin. Experiments using Vimentin / AQP4 double labelling enabled differentiation of tanycyte profiles from AQP4- negative oligodendroglial and neuronal processes. These cell processes are referred to as tanycytes. Semithin sections showed the formation of swelling varicose projections by tanycytes that were often in close association with cellular waste. Interestingly, human AQP4-LIR tanycytes that project from the ventricular lining into hippocampal neurons form numerous circular fenestrated cell structures, which attach to adjacent cells, likely forming stabilizing ‘connective junctions’ between the cells. At the ultrastructural level, tanycytes have an inner lumen that is separated from the peri-luminal space. Varicose projections could be seen to emanate from this peri -luminal space projecting into adjacent cells. Such intraneuronal projections were often adjacent to or surrounded by a ‘penumbra’ of clear cytoplasm. To assess whether the function of these varicosities is to create an AQP4- mediated cytoplasmic bulk flow toward tanycytes and ensure waste removal from neurons, studies were conducted that included vimentin AQP4 double labeling. Tanycytes stained vimentin positive in the central lumen, whereas the peri-luminal space together with emanating varicose projections appeared strongly AQP4-LIR supporting the hypothesis. In a next step, studies were performed to assess whether the spongiform appearance of degenerating hippocampal neurons could be attributed to excessive water influx into AQP4- LIR varicosities resulting in the formation of hypertrophic varicosities within affected neurons. It was expected that degenerating spongiform neurons would show AQP4-LIR swelling varicosities either around the periphery of neurons during early stages of neurodegeneration or within the entire somata in advanced stages. Results showed that numerous neurons displayed irregular AQP4-LIR somata and around their perimeters consistent with this expectation.

[0174] These findings of studies performed were consistent with microscopic observations of healthy myelinated tanycytes in rat hippocampus that form small varicosities into adjacent neurons, whereas neurons with degeneration onset show the formation of swelling varicosities. It is expected that these projections may be the mammalian equivalent of the spider glial-canals described herein and play a vital role for waste clearance from neurons. Based on studies performed, some of which are presented herein, it is believed that these varicosities remove cellular waste from mammalian neurons via the AQP4-mediated cytoplasmic bulk flow toward membranous cistemae formed by tanycytes. It is expected, based on results of studies performed, that pathological changes in the tanycytes lead to increased formation and excessive swelling of these varicose projections causing the spongiform appearance of these neurons in human brains with high levels of ADNC, which results in gliaptosis similar to what has been demonstrated in spider neurons. Immunoreactivity' in pre-absorption control preparations revealed greatly diminished immunoreactivity supporting the specificity of antibody binding. Interestingly, tanycyte profiles were observed branching and forming canal-like projections into adjacent neuronal profiles including dendrites and axons. Similar glial projections could be observed projecting into axons of spider mechanosensory neurons. In single section analysis, these glial-canals may be mistaken for mitochondrial profiles if the connecting glial stalk is outside of the section plane. In spider sensory neurons, these glial projections formed prominent membrane cistemae that projected into the neuronal cytoplasm similar to this observed in central spider neurons. Results of studies support that a role of these membrane cistemae in endocytotic waste uptake from the neuronal cytoplasm into surrounding glial cells. At the ultrastructural level degenerating human tanycyte profiles form numerous swelling projections that transect from the peri-luminal space into adjacent cells. Interestingly, neuronal profiles adjacent to degenerating tanycyte profiles appeared to gradually clear of their cytoplasmic content.

[0175] Based on the findings presented here, it has been identified that both healthy spider and mammalian neurons receive close contact from AQP4-LIR tanycytes. It is believed that these contacts facilitate the controlled AQP4-mediated removal of cellular waste from healthy neurons. It is believed that the structural impairment and hypertrophy of tanycytes leads to an imbalance in the strength of the AQP4 water channel-medicated bulk flow toward the tanycytes and subsequent rupture resulting in excessive depletion of neuronal cytoplasm and cell, a process referred to herein as ‘gliaptosis’.

[0176] Discussion

[0177] Studies set forth herein demonstrate a newly discovered mechanism by which macroglia form an intricate canal system that removes cellular waste from neurons in the spider nervous system. Results of experiments described herein provide strong evidence that both the structural and functional integrity of this system depends on the interaction between (a) sODG that contain microtubule-associated break points, and (b) AQP4-LIR GACs and NACs likely responsible for the creation of a cytoplasmic bulk flow toward glial-canals. Results of experiments described herein demonstrate that the structural failure of this canal system caused the catastrophic depletion of cy toplasmic content from affected neurons leading to a previously undescribed type of macroglia-induced cell death, which is referred to herein as 'gliaptosis / Studies set forth herein demonstrated that the cellular characteristics of neuronal cell death in the brain of Alzheimer patients with ADNC resemble those described in spider neurons and provide compelling evidence that AQP4-LIR tanycyte profdes may form a similar AQP4 water channel -mediated waste clearance system as this proposed for spider neurons. Results show that tanycytes in degenerating human brain undergo hypertrophic changes and provide strong evidence for an AQP4 water channel -mediated catastrophic depletion of cytoplasmic content from affected neurons similar to the mechanism observed in spider brain. Based on evidence obtained from experiments described herein it is proposed that these pathological changes in tanycytes and the resulting impairment of the glial-canal system may be one of the underlying causes for neuronal cell death in the brain of Alzheimer patients with ADNC.

[0178] Vital importance of controlled waste clearance mechanisms from neurons

[0179] The current proposed mechanism by which cellular waste is cleared from neurons is the postulation that debris is released into interstitial spaces and cleared from the brain parenchyma via an AQP4-water channel mediated bulk flow toward para-venous spaces [M. Nedergaard, Science 340, 1529-1530 (2013); L. C. Walker et al., JAMA Neurology 70, 304- 310 (2013)]. This mechanism poses several problems for the functional integrity of neurons. As demonstrated herein, cellular waste consists of both protein-based (hydrophilic) and lipid- based (hydrophobic) compounds. Given the concept of hydrophobic exclusion, and hydrophilic interactions it would be expected that waste particles released by adjacent neurons would quickly interact with each other and form large waste aggregates. Given the narrow dimensions of interstitial spaces whose widths have been shown to measure between 10-20 nm [M. Brightman et al.. The Journal of cell biology 40, 648-677 (1969); A. Van Harreveld, The structure and function of nervous tissue 4, 447-511 (1972); E. Horstmann and H. Meves, Zeitschrift fur Zellforschung und Mikroskopische Anatomie 49, 569-604 (1959); A. Peters et al.. The fine structure of the nervous system : neurons and their supporting cells, Oxford University Press, Third edition (1991)] it is unclear how this waste would be effectively cleared. The accumulation of freely released hydrophobic waste would also pose the risk to rapidly obstruct and impair ion flow that is of critical importance for signal transduction in neurons. The proposed expansion of the glymphatic system during times of reduced neuronal activity [S. Gordleeva et al., Seminars in Immunopathology, Springer, vol. 42, pp. 647-665 (2020); N. L. Hauglund et al., Current Opinion in Physiology 15, 1-6 (2020); K. I. Voumvourakis et al., Biomedicines 11, 2092 (2023)] does not eliminate the risk of unspecific interactions between cellular waste and receptor binding sites, transport ATPases. and ion channels along the neuronal membrane. Even considering the higher affinity of endogenous ligands to specific receptor molecules, larger debris clusters would likely pose as physical barrier for endogenous ligands.

[0180] AQP4-LIR tanycytes and their role in waste clearance and neurodegeneration

[0181] In both the proposed arachnid and mammalian glial-canal systems, the two key components that form the structural and functional foundation for these canals are (a) myelin, and (b) AQP4-LIR glial cells that transect from the ventricular lining into the hippocampal formation.

[0182] It is believed that the role of myelin, is the formation of intraneuronal projections and membrane cistemae that are important for the uptake and removal of cellular waste via endocytosis, phagocytosis, or engulfment of debris by glial membranes. Studies presented herein, show that sODG myelinate somata of neurons in the spider CNS play an important role in waste clearance. Although myelination in invertebrate neurons is considered unusual [D. K. Hartline and D. R. Colman, Current Biology 17, R29-R35 (2007); A. I. Boulleme, Experimental Neurology 283, 431-445 (2016); B. I. Roots, The Node of Ranvier, 1-29 (1984)], the presence of myelinated neurons has previously been reported in a variety of invertebrate organisms [D. K. Hartline and D. R. Colman, Current Biology 17, R29-R35 (2007)] including crustaceans that are closely related to arachnids [A. D. Davis et al., Nature 398, 571 (1999)]. It is likely the AQP4-LIR component of this system that draws cellular waste toward the glial-canals. In the mammalian brain, AQP4 is predominantly localized in endfeet of astrocytes and ependymal cells including tanycytes, whereas it has been reported absent from neurons and oligodendroglia [M. Amiry-Moghaddam and O. P. Ottersen, Nature Reviews Neuroscience 4, 991-1001 (2003); E. Rodriguez et al., Journal of Neuroendocrinology 31, (2019)]. These findings are consistent with observations in both spider and human tissue as set forth herein and support a finding that the AQP4-LIR cell profiles may originate from tanycytes. Observations presented herein that these cells are located in the ventricular lining and show co-localization for the structural protein vimentin that is known to be expressed by tanycytes [V. Prevot et al.. Endocrine Reviews 39, 333-368 (2018)] supports a conclusion that these cells are of ependymal origin.

[0183] The glial-canal system described herein -although structurally altered- appears highly conserved between arachnids and mammals and alludes to the vital importance of this system for the functional integrity and survival of neurons. Compared to arachnids, the mammalian brain has evolved favoring increased numbers of (a) neurons, (b) dendritic arbors, and (c) synaptic connectivity in exchange for structurally smaller neurons and glial cells. It is likely due to this evolutionary trend and spatial limitations that waste clearance mechanisms in mammals have been reduced to myelinated tanycyte projections along neuronal somata and neurites compared to the space-occupying peri-somatic myelination in spiders.

[0184] Studies presented herein support a conclusion that comparatively narrow mammalian tanycytes internalize catabolized cell waste that is likely broken down by lytic enzymes contained in and around tanycyte waste receptacles . As shown here, mammalian tanycyte projections into neurons are often adjacent to or surrounded by a penumbra in which the cytoplasm appears clear and less structured. In addition, structurally impaired tanycytes whose outer myelin sheath unravels and opens to the extracellular space show a similar penumbra in which cells that border on impaired tanycytes appear to gradually lyse. As demonstrated here in the invertebrate system, cellular debris such as membrane aggregates can grow to sizes that are larger than 2 pm in diameter. In the comparatively gigantic spider neurons that are up to five times the size of mammalian neurons these aggregates will not obstruct the larger glial canals that can measure >10 pm in diameter. However, particularly in the human brain tany cyte diameters are in the range of 0.5 to ~3 pm. Larger debris clusters would thus pose the risk to block and impair glial canals. Results suggest that mammalian tanycytes may contain lytic enzymes within their membranous cistemae to catabolize cellular debris for removal from neurons. The observation that AQP4-LIR varicosities swell is consistent with a mechanism of AQP4-mediated water influx into their lumina. Aquaporin- mediated swelling has been demonstrated in Xenopus laevis oocytes that were injected with AQP1 -water channel cRNA leading to significant swelling and ultimate bursting of the eggs [G. M Preston et al., Science 256, 385-387 (1992)].

[0185] These observations demonstrate vulnerabilities of aquaporin water channel mediated mechanisms. To ensure that intracellular waste is effectively drawn toward the glial-canals the appropriate regulation of flow strength is critical. Decreased flow strength may lead to incomplete waste removal and thus lead to accumulation of cellular waste within neurons, whereas excessive flow strength may lead to the uncontrolled depletion of neuronal content suggested here. Results presented herein support a conclusion that both cases may exist in neurodegenerative diseases and that the structural impairment and resulting hypertrophy of tanycytes may be one of the main causes for neurodegeneration.

[0186] Current knowledge regarding the functional significance, types, causes for degeneration, and regenerative potential of tanycytes is fragmentary’. Mammalian tanycytes are located in the lining of the third ventricle and have been shown to send long slender processes into surrounding brain parenchyma. Among the proposed roles of these ependymal cells are energy and nutrient supply to neurons, regulator}' functions at the brain-blood- barrier, and neurohormonal secretion [R. Dali et al., Physiology & Behavior 263, 114108 (2023)]. Electrophysiological investigations on cultured tanycytes indicate that their long cell projections do not propagate electrical impulses [P. N. De Francesco et al., Cell and tissue research 369, 369-380 (2017)]. However, the activation of AMPA and Kainate-type glutamate receptors expressed by tanycytes has been linked to increases in intracellular Camlevels within these ependymal cells alluding to the modulation of Ca2+-dependent signaling pathways in response to synaptic activity [E. Farkas et al., IScience 23, (2020)]. Based on their location, four distinct types of tanycytes are distinguished, alpha-1 and -2, 13-1 and -2 [F. J. Ebling and J. E. Lewis, Glia 66, 1176-1184 (2018)]. However, immunohistochemical and ultrastructural investigations have revealed numerous morphological differences leading Pasquettaz et al. [R. Pasquettaz et al., The Journal of Comparative Neurology 529. 553 (2021)] to propose that additional types may exist [V. Prevot et al., Endocrine Reviews 39, 333-368 (2018); R. Dali et al., Physiology & Behavior 263, 114108 (2023); R. Pasquettaz et al., The Journal of Comparative Neurology 529, 553 (2021); M. Bolborea and N. Dale, Trends in neurosciences 36, 91-100 (2013); F. Doetsch et al., Journal of Neuroscience 17, 5046-5061 (1997)]. Consistent with results set forth herein, it has now been demonstrated that mammalian tanycytes interact with surrounding neurons via numerous morphologically diverse processes, including circular swellings similar to the fenestrated structures on tanycyte projections shown here. It is expected that the function of these structures is the formation of "connective junctions’ between long, slender cell processes to provide secure attachment.

[0187] It is believed that the disclosure herein provides the first description of spider tanycytes and it is unclear whether they can be subdivided into distinct types. Based on experimentation observations, it is expected that spider tanycytes send their processes from the anterior and medial sections of the dorsal tubular system into the spider CNS where they transect into both oligodendroglia and neuronal profiles. The posterior part of this tubular system forms the gastrointestinal tract of the animals. Based on experimentation results provided herein, it is proposed that anterior and medial parts of the spider dorsal tubular system may present the phylogenetically more ancient spider version of the mammalian ventricular system that channels cellular waste toward the posterior gastrointestinal division.

[0188] A conclusion that AQP4-LIR spider-tanycytes may play a role in waste clearance from the brain parenchyma via direct uptake of cell debus is supported by two observations. Firstly, the presence of fluorescent particles within the cell processes of spider-tanycytes that closely resemble those described in cultured mammalian tanycytes [P. N. De Francesco et al., Cell and tissue research 369, 369-380 (2017)]. Based on results presented herein, it is believed that these particles represent autofluorescent lipofuscin particles that are taken up by these cells either through rupture of the glial boundaries in degenerating neurons, or through forming glial-canals that connect tanycyte profiles with the neuronal cytoplasm shown here. The autofluorescent nature of lipofuscin in neurons has been demonstrated in both spider and mammalian neurons [P. A. Adeniyi et al., ASN Neuro (Sage Publications, Ltd.) 14. 1-13 (2022); R. Fabian and E.-A. Seyfarth, Cell and tissue research 287, 413-423 (1997); A. Moreno-Garcia et al.. Front Neurosci 12, 464 (2018); T. M. O'Herron et al., Neurohiology of Aging 21, 145-145 (2000); N. Sakr et al., International Journal of Molecular Sciences 24, 3432 (2023)]. The second observation is that the tubular system that contains the somata of these cells appears brown in spiders with advanced neurodegeneration similar to the brown discoloration of degenerating spider brain that is caused by accumulating cellular waste [R. Fabian-Fine et al., Journal of Comparative Neurology 531, 618-638 (2023)]. In degenerating brains, the discolored tubular system is thus easily distinguished from surrounding ivorycolored muscle tissue, a distinction that is absent in healthy spiders. It is expected that AQP4 water channel expressing spider-tanycytes transect into brain ganglia and give rise to both GACs and NACs as described herein. The increased diameters of NACs, and GACs that border on the neuronal cytoplasm suggests water influx into the lumina of these canals explaining the formation of a cytoplasmic flow toward glial canals. Results of studies suggest that the activation of this system may be controlled by synaptic contacts onto glial cells, which have been reported in the nervous system of C. salei [R. Fabian-Fine et al.. The Journal of comparative neurology 420, 195-210 (2000)]. It is thus conceivable that waste removal from neurons is controlled by neuronal activity and may be activated in a circadian manner similar to the proposed activation of the glymphatic system [J. J. Iliff et al., Science translational medicine 4, 147ral 11 (2012); J. J. Iliff and M. Nedergaard, Stroke 44, S93-S95 (2013); M. Nedergaard, Science 340, 1529-1530 (2013); M. Nedergaard and S. A. Goldman.

[0189] Scientific American 314. 44-49 (2016)].

[0190] The role of microtubules and microtubule-associated break points in glial-canal formation, waste clearance and neurodegeneration in C. salei

[0191] Microtubule-associated break points (MABs) have not been described previously and it is unclear whether they exist in the mammalian system. Studies presented herein support a conclusion that MABs provide a sustainable mechanism for controlled glial-canal formation by anchoring cleaved glial membranes to glial-derived microtubules. Based on findings presented herein, it is believed that selective severance of these micro tubule / membrane anchors by regulatory proteins ensures the controlled release of glial membranes into the neuronal cytoplasm and provides a tightly regulated and sustainable formation of glial canals for waste clearance in healthy spider brain. This would explain the chromatin-rich appearance of sODG as this mechanism would require the continued formation of glial lobes and thus coincide with high levels of gene expression. The dynamic instability of microtubules that consist of heteromeric alpha and beta tubulin dimers [H. V. Goodson and E. M. Jonasson, Cold Spring Harbor perspectives in biology 10, a022608 (2018); M. Kirschner and T. Mitchison, Cell 45, 329-342 (1986)] make them uniquely suitable for such regulatory processes. Three main characteristics of microtubules provide the basis for this conclusion: (a) the interactions of microtubules with a large variety of proteins and other cellular structures, (b) the existence of numerous regulatory proteins that govern the dynamic instability of microtubules, and (c) the stabi 1 i ty of polymerized microtubules and their ability to securely anchor cellular components that critically rely on their structural integrity for survival of the system.

[0192] Close interactions of microtubules with a variety of cellular structures are largely mediated through microtubule associated proteins [H. V. Goodson and E. M. Jonasson, Cold Spring Harbor perspectives in biology 10, a022608 (2018)]. Such interactions include motor proteins [H. L. Sweeney and E. L. Holzbaur, Cold Spring Harbor Perspectives in Biology 10, a021931 (2018)], membrane-bound organelles [G. Kanfer et al., Molecular biolog / of the cell 28, 2400-2409 (2017); A. J. Lomakin et d ., Molecular biology of the cell 22, 4029-4037 (2011)], chromosomes [N. B. Gudimchuk and J. R. McIntosh, Nature reviews Molecular cell biology 22. 777-795 (2021)], syncytial nuclei [S. Eckerle et al., Developmental Biology / 434, 249-266 (2018); L. Solnica-Krezel and W. Driever, Development 120, 2443-2455 (1994)]. and cell adhesion molecules [L. A. Ligon et al., Nature Cell Biology 3, 913-917 (2001)]. Particularly considering the ultrastructural evidence set forth herein, it is thus reasonable to postulate that microtubules anchor cleaved glial membranes to provide a readily available membrane pool for the formation of glial-canals to ensure reliable waste removal. A large variety of known microtubule regulating proteins have been described to modulate the dynamic of microtubules, including controlled formation, stabilization, and dissociation [H. V. Goodson and E. M. Jonasson, Cold Spring Harbor perspectives in biology 10, a022608 (2018); P. W. Baas et al., Cytoskeleton 73, 442-460 (2016)]. Among the large variety of regulatory proteins are severases, a family of microtubule cleaving AAA-ATPases including Spastin, Katanin, and Fidgetin [J. J. Hartman and R. D. Vale, Science 286, 782-785 (1999); Y.-W. Kuo and J. Howard, Trends in cell biology 31, 50-61 (2021)]. It is feasible that the abnormal upregulation of severases in sODG may lead to uncontrolled severance of MAB’s explaining the catastrophic unraveling of glial membranes described here. Another hypothesis to explain the catastrophic dissociation of microtubules builds on a possible dow nregulation or post-translational modification of microtubule stabilizing proteins such as tau-protein, which has long been implicated as underlying cause for multiple neurodegenerative conditions [M. Askenazi et al.. Nature Communications. 14, 1-15 (2023); E. Blas Gil, Recent Advances in Alzheimer Research, Bentham Science Publishers Ltd, Vol. 3 (2019); E. E. Congdon and E. M. Sigurdsson, Nature Reviews Neurology) 14, 399-415 (2018); A. O. Fernando, Recent Advances in Alzheimer Research, Bentham Science Publishers Ltd. Vol. 2 (2018); L. Kiani, Nature Reviews Neurology 19. 459-459 (2023); G. Perry, Advances in Alzheimer's Disease, I. O. S. Press (2013); G. Perry, Advances in Alzheimer's Disease, I. O. S. Press (2018); D. Philippe, Molecular Medicine and Medicinal Chemistry, Imperial College Press (2013)] . Results and observations presented herein, that sODG around degenerating neurons contain fewer visible microtubules and appear less regularly aligned supports a conclusion that the underlying cause for the catastrophic unraveling of glial membranes from affected sODG may be due to abnormal concentrations of microtubule regulating proteins. Because this dissociation predominantly originates around spider-tanycytes. it supports a conclusion that these regulatory processes may be controlled by spider tanycytes, and their structural impairment may trigger the abnormal activation of the pathological processes described here.

[0193] Example 2

[0194] Materials and Methods

[0195] Preparation of Human Brain Tissue. The human brain tissue used here was obtained from four decedents undergoing autopsy examination at the University of Vermont Medical Center with full consent for biomedical research, diagnosis, and teaching purposes according to Vermont State law. The samples were fixed immediately after removal of the brains.

[0196] Tissue from three decedents with a clinical history of Alzheimer dementia (one age 79, two ages 86) was obtained 14- and 15-50 hours post-mortem. Diagnostic examination for neuropathology showed significant accumulation of amyloid plaque and phosphorylated tau tangles in the hippocampus consistent with Alzheimer disease neuropathologic change (ADNC). The ABC scores of A3B3C1 and A3B3C2, indicating intermediate and high burdens of ADNC, respectively. Tissue from three 76-, 40-. and 35-year-old decedents were negative for ADNC. Tissue collection for these patients was 45- and 14-hours post-mortem, respectively. All procedures were conducted in accordance with state laws.

[0197] Preparation of Rat and Spider Brain Tissue.

[0198] Fixation, labeling and embedding methods used for spider and rat brain were identical to those used in human tissue with the exception that the animals were deeply anesthetized prior to perfusion with fixative.

[0199] Immunohistochemical procedures.

[0200] All steps described here were conducted on ice to keep the tissue cold and maximize the quality of tissue preservation. After fixation in 4% paraformaldehyde in phosphate buffered saline (pH 7.4, 0. 1 M; PBS) overnight the preparations were washed in PBS and embedded in 4% Agarose (Sigma A9539). Vibratome sections (70-mm) were cut using a Leica VT 1000S vibratome. Sections were washed in PBS (4x5 min) to remove excess aldehyde. To block unspecific binding sites the sections were kept in blocking medium consisting of 0.25% Bovine Serum Albumin (Sigma A4503) and 5% Normal Goat Serum (Sigma G9023) in 1% Triton-X / PBS for 20 min. Incubation with the primary goat anti-rabbit aquaporin-4 antiserum (BiCell #20104) and goat-anti mouse anti-Vimentin (#AMF-17b-s) at a dilution of 1 : 100 in PBS containing 10% blocking medium overnight. Both antibodies are routinely used in human tissue. Subsequently the sections were washed for seven wash cycles in PBS prior to an additional 20-min blocking step in blocking medium. The secondary' fluorochrome-coupled antibodies (Cy3 goat anti-rabbit, Jackson ImmunoResearch Laboratories 111-165-003. FITC goat anti-mouse Jackson ImmunoResearch Laboratories 115-096-072) were used at a dilution of 1:600 in PBS containing 10% blocking medium overnight in the fridge. The sections were washed in PBS for three washing cycles prior to staining with Hoechst Blue nuclear stain (Sigma H 6024; 1 :3000 in PBS) for 20 min. After washing in PBS for five wash cycles the sections were mounted on glass slides and embedded in Mowiol (Sigma# 81381). Due to the light sensitivity of the secondary antibodies all steps were conducted under minimum light conditions to prevent bleaching of the fluorochromes. The sections were analyzed using a confocal Zeiss Axiolmager MZ with Apotome.

[0201] Antibody characterization.

[0202] For control purposes and to establish the background fluorescence level of brain sections control preparations were processed under the omission of the primary antibody incubation. All samples examined were void of the fluorescent signals observed in sections treated with the primary antibodies. The specificity of antibody binding in human tissue has been established previously for both primary’ antibodies used here [Verma N et al., Nature communications 6.1 (2015): 6064],

[0203] Tissue preparation for electron microscopy.

[0204] Preparations processed for ultrastructural analysis were fixed in a mixture of 4% paraformaldehyde and 2.5% glutaraldehyde (EMS 16019) in PBS overnight in the fridge. It is important to note that incubation in this fixative for longer time periods as described here are detrimental to histological staining procedures as semithin sections of the tissue fixed for extended time periods does not stain sufficiently for histological dyes. After three wash cycles in PBS the preparations were post-fixed in 1.0% osmium tetroxide (Electron Microscopic Sciences, #19150) for approximately 1 hour. After two wash cycles in PBS, the tissue was dehydrated in a graded series of molecular grade ethanol. The preparations were promptly transferred into propylene oxide (Electron Microscopic Sciences, #20401) and slowly infiltrated with Araldite (Electron Microscopic Sciences, #13900). It is important to note that the extended exposure (1-3 days) of the osmicated tissue to ethanol and propylene oxide may result in the removal of osmium from the tissue and compromise tissue preservation. It is thus important not to leave the samples in these media overnight. The tissue was embedded in pure Araldite and polymerized overnight at 60 °C, according to manufacturer's instructions. Semi- and Ultrathin Sectioning.

[0205] Using a Leica Ultracut E Araldite-embedded hippocampal specimens were cut with an 8 mm Diatome histo-knife altering between 1-pm semithin and 65-nm serial ultrathin sections. Chloroform (Electron Microscopic Sciences, #12540) was used to stretch individual sections. Semi-thin sections were collected with a plastic rod in distilled water on glass slides. The sections were dried on a hot plate at 80 °C and stained with 2% aqueous toluidine blue (Sigma 6586-04-5) stain for ~2 min at 80 °C and rinsed thoroughly with distilled water. The sections were examined and imaged using an Olympus microscope with digital image acquisition capabilities and a Zeiss Axiolmager MZ.

[0206] Electron microscopy.

[0207] Ultrathin sections were collected on pioloform-coated single-slot copper or nickel grids (EMS# G2010CU). Grids were contrasted with aqueous 1.5% uranyl acetate (6 min) and Reynold’s lead citrate (5 min). Electron microscopic examination was conducted using a JOEL 1400 electron microscope operated at 80 kV.

[0208] Toluidine-blue stain of vibratome sections for light microscopic investigations.

[0209] Vibratome sections from 4% paraformaldehyde-fixed tissue were washed in PBS for four wash cycles. Staining with toluidine blue was conducted under visual control by slowly dripping a 2% toluidine blue solution into a petri dish containing brain sections in PBS. The staining was terminated when the neurons appeared appropriately stained for light microscopic investigation without overstaining the preparations. Overstaining will result in the inability to distinguish individual structures in the section. The sections were embedded in Mowiol and examined promptly using an Olympus microscope with differential phase contrast and digital image capturing capabilities. The embedding medium will slowly de-stain the cells, however cellular debris within the cells remains clearly visible in form of brown deposits.

[0210] Luxol H&E staining of Autopsy Tissue.

[0211] Brains were emersed in 10% neutral buffered formalin and incubated for at least 1 week at room temperature. Hippocampal tissue was removed from the brains, dehydrated in a graded series of ethanol, infiltrated with 100% xylene (3x 40 min), and embedded in paraffin (25 min). Sections were cut at 10 mm thickness and mounted on glass slides. Slides were rinsed in xylene (2x), 100% and 95% EtOH (lx each) prior to immersion in 0.1% Luxol fast blue solution (Leica #) at 60 °C overnight at 60 degrees C. Excess stain was removed with 95% EtOH. Slides were rinsed in distilled water and placed in a 0.05% lithium carbonate solution (2 min) followed by 70% EtOH (2 min). After additional incubation in 0.05% lithium carbonate (1 min) preparations were rinsed in 70% EtOH (1 min) and distilled water. Slides were then stained with hematoxylin (Leica 3801571) for 1 min. Sections were washed (1 min) in water, immersed in defining solution (1 min, Leica 3803598), and rinsed in water (1 min). To enhance staining quality preparations were suspended in blue buffer (1 min, Leica 3802918), washed in distilled water (1 min) and 95% EtOH (1 min). Sections were immersed in Eosin (2 min, Leica 3801619) and rinsed in 95% EtOH (1x30 sec and lx 1 min). For permanent storage water was removed by rinsing in sections in 100% EtOH and xylene (2x1.5 min each). Sections were permanently mounted using Permaslip Mounting Medium and Liquid Coverslip (Alban Scientific, Inc.).

[0212] Image Processing.

[0213] Confocal images were exported from the ZEN Blue program using the ‘Image export’ function. Figures from confocal and histological and ultrastructural preparations were created using Adobe Photoshop.

[0214] RESULTS

[0215] Tanycytes form varicose protrusions into hippocampal neurons and glial cells

[0216] Light microscopic analysis of toluidine-blue stained vibratome sections through the hippocampal formation of patients with diagnosed Alzheimer disease neuropathologic change (referred to in the following as ‘ AD-decedents’) reveals that neuronal somata and their initial axon segments are contacted by numerous varicose cell processes that form large numbers of bulbous intraneuronal protrusions that are associated with cellular debris. These structures are commonly assumed to represent lipofuscin. Double labeling for AQP4 and vimentin indicate an ependymal origin of these cell processes . Pyramidal cells from decedents negative for Alzheimer disease neuropathologic change (in the following referred to as non-AD decedents) showed fewer and smaller protrusions within the cytoplasm and less debris accumulation around intraneuronal protrusions. Investigation of both human hippocampus and olivary nucleus using Luxol blue stain show vast numbers of myelinated tanycyte processes transecting from the ventricular lining into the brain parenchyma. Immunolabeling for AQP4 shows strong AQP4-immunoreactivity (AQP4-IR) within these ependymal processes. Higher magnification and ultrastructural investigations show that these myelinated processes transect into neuronal somata where they give rise to numerous intraneuronal protrusions. Circular myelin-derived protrusions that transect into the neuronal cytoplasm mature into specialized receptacles that internalize electron dense cellular debris into a centrally located myelinated canal.

[0217] AQP4-IR tanycytes form ‘adhesion-clamps ’ and protrusions into adjacent cell profiles

[0218] Tanycyte processes form AQP4-IR circular clamp-like structures whose diameters range between ~1 and 10 pm. Individual clamps surround cellular processes of adjacent cells. These circular structures are referred to herein as "adhesion clamps’ (ACs). In AD-affected tissue ACs form numerous protrusions and could be seen swelling to large diameters of >10 pm. Immunolabeling for AQP4 and vimentin demonstrated that these clamps formed along the processes of ependymal tanycytes whose somata reside in the ventricular lining. AQP4-IR tanycytes were observed projecting into the stratum pyramidale forming numerous protrusions alongside and within neuronal somata and astrocytes consistent with the protrusions observed in toluidine-blue stained vibratome sections. Interestingly, tanycytes were observed forming numerous projections along astrocytes whose somata appeared unstained for AQP4 compared to the somata of tanycytes. H&E-stained tanycyte processes in the alveus further show their varicose and inter-connected nature consistent with the ultrastructural observations. Vimentin / AQP4-double labeling demonstrated that AQP4-IR protrusions that transect toward cell nuclei emanate from vimentin-IR fiber profiles consistent with the observations that AQP4-expressing tanycytes form cellular protrusions into somata.

[0219] Cytoplasmic depletion of cells in the stratum pyramidale and olivary nucleus ofAD- decedents.

[0220] Examination of Luxol H&E-stained preparations reveals the progressive cytoplasmic depletion in neurons and glial cells throughout the brain parenchyma. Early stages of neurodegeneration are characterized by cytoplasmic obstruction with hypertrophic tanycyte receptacles and onset of spongiform vacuolization around the cell cortex. Tanycytes that transect into affected cells show7hypertrophic sw elling. In advanced stages of degeneration, the cytoplasm of affected cells is largely depleted with only the nucleus, tanycytes and their receptacles remaining. In the final stage the cell somata appear depleted with only the nucleus and hypertrophic tanycyte projections remaining. Light- and ultrastructural characteristics of rat and human tanycytes.

[0221] Due to the limitations of tissue preservation in human brain, studies have been performed to investigate rat tanycytes to determine, whether the varicose nature of human tanycytes may be due to fixation artifacts. Somata of rat-tanycytes adjacent to the CA3 area show striking resemblance to those in human brain. In rat tissue well-preserved ependymal cells form multinucleated syncytia that form a vast reticulum of myelinated processes consistent with the observations in human brain. Adjacent to the ventricle myelinated tanycyte processes appear circular in shape and form few protrusions. Interestingly, with increasing proximity to the stratum oriens, the processes elongate and form numerous protrusions. Serial section analysis reveals that tanycytes project into the stratum pyramidale, where they are in close association with pyramidal and glial cells. In human tissue AQP4-IR was observed along the outside of astrocytes, whereas the somata did not display typical immunoreactivity'. Close interactions of varicose, myelinated tanycyte profiles with astrocytes and neurons was consistent in rat tissue at the ultrastructural level. In rat, fibrillary’ canal-like structures were observed that emanated from tanycytes into adjacent cells. These structures often shoyv electron-dense material along the outside and resemble glial fibrillary acidic protein (GFAP, see discussion). Interestingly, numerous tanycyte profiles could be observed transecting into the cytoplasm of both neurons and astrocytes. Myelin protrusions were observed forming on the outside of rat tanycytes, some of which contained electron dense granular matter. Rat tanycytes contained membranous structures with electron lucent lumina that formed close associations yvith microtubules and clearly differed from mitochondria.

[0222] Ultrastructure of tanycyte protrusions in human stratum pyramidale.

[0223] To investigate the ultrastructure of tanycyte protrusions in neuronal somata semithin sections and their following ultrathin sections through CAI and CA3 neurons yvere examined. In semithin sections, tanycyte protrusions appeared as characteristic, light brown circular structures, commonly assumed to be lipofuscin. The myelinated and highly varicose nature of tanycyte processes was apparent at the ultrastructural level. Cross sections showed that the cytoplasm of tanycytes consisted of (1) electron lucent, and (2) cytoplasm-containing compartments. Within both compartments varying numbers of distinct canal profiles were observed that appeared to branch into the myelin sheath. As a result, varicose protrusions that bud around the outer surface of tanycytes consisted of an electron lucent circular component that contained myelinated ‘central canals.' Ultrastructural observations showed electron dense accumulations within some central canals that may have appeared engorged as a result.

[0224] In non-AD tissue tanycyte processes were observed in which the myelin sheath appeared less tightly compacted and gave rise to numerous peripheral protrusions. This was consistent with the proposed formation of bulbous protrusions that ‘bud’ along the outer surface of tanycyte projections within neuronal somata. Similar varicose structures were observed along tanycyte processes in Luxol blue-stained hippocampal tissue. The myelin around tanycytes in AD-decedents appeared hypertrophic compared to non-AD tissue whereby electron-lucent canals within the myelin sheath appeared engorged.

[0225] Ultrastructural examination of human tanycyte protrusions revealed at least two different types. Type-A protrusions were predominantly located within the neuronal cytoplasm and consisted of the central canal that appeared electron dense that was surrounded by several electron lucent bulbous structures (in the following referred to as ‘receptacles’) that have small openings to the neuronal cytoplasm (in the following referred to as ‘outer gates’). The receptacles were separated from the central canal via perforated membranes, referred to herein as ’inner diaphragm.’ Interestingly, numerous vesicular structures approx. 20-50 nm in diameter were observed (a) near the outer gates, (b) within the receptacles, and (c) within the central canal. Shape and size of these vesicles w ere consistent with those of transport and synaptic vesicles. Vesicular structures could be seen both entering the central canal through specialized circular openings and within the central canal. Mature receptacles w ere determined to be interconnected with each other. A noticeable darker coloration of the receptacle lumen w as observed in receptacles that internalized debris compared to forming receptacles. Interestingly, the outer gates were surrounded by membranous structures that resembled rough endoplasmic reticulum (rER) but appeared round or oval.

[0226] The second protrusion type (Type-B) was predominantly observed in extracellular spaces. This type consisted of a central canal that contained numerous membrane folds within its lumen. In this type, debris-filled central canals were observed around the receptacle. Characteristic circular openings resembling those in Type A receptacles were observed. Interestingly, Type-B protrusions contained numerous distinct circular structures that were near electron-dense cellular debris.

[0227] Myelin mediated clearance of lipofuscin versus myelin-derived tanycyte receptacles.

[0228] The above-described tanycyte projections into human neurons are frequently referred to as ’lipofuscin’ (see discussion). However, classical lipofuscin granules are found in phylogenetically more ancient invertebrates and vertebrates where they appear as granular accumulations in toluidine-blue stained neurons as demonstrated on the example of Cupiennius salei where lipofuscin is engulfed by myelinated glial cells that form canals through which cellular waste is removed from the brain parenchyma. Results of studies comprising immunolabeling for AQP4 showed punctate immunofluorescence along neuronal profiles consistent with size and distribution of these channels around and within neurons. At the ultrastructural level, the diverse appearance of lipofuscin granules that were engulfed by detaching myelin sheaths that formed discrete canals became apparent. Interestingly, spider tissue was found to contain GF AP -like canals that emanated from the myelinated glial cells. Discrete myelin canals that contained electron-dense granular material similar to those observed in spider neurons were observed forming on the outer surface of rat tanycytes. In human tissue cellular waste is taken up into myelin-derived receptacles and lacks the granular appearance of typical lipofuscin granules but has a brown, unstructured appearance at the light microscopic level and appeared as electron dense mass at the ultrastructural level consistent with catabolized debris (see discussion).

[0229] Hypertrophic appearance of tanycytes in AD patients.

[0230] Neurons from non- AD decedents consistently contained moderate numbers of intracellular tanycyte protrusions. Adjacent tanycyte projections formed characteristic varicose protrusions. The cytoplasm of these neurons appeared overall well preserved and contained organelles typical for healthy neurons. In contrast, the cytoplasm in AD-affected neurons appeared obstructed with numerous hypertrophic tanycyte receptacles and waste accumulations were consistent with the light microscopic observations. A similar hypertrophic appearance was observed in adjacent tanycyte processes. The brain tissue surrounding obstructed neurons appeared spongiform due to the excessive formation of hypertrophic tanycyte protrusions. The formation of electron-lucent protrusions by both healthy and AD-affected tanycytes was seen at the ultrastructural level. The cytoplasm of hypertrophic tanycytes appeared dark blue stained compared to healthy tanycytes. Remaining neuronal cytoplasm in AD-affected neurons showed dark blue stained patches around the cellular cortex similar in coloration to the tanycyte lumina. In healthy neurons these areas contain electron lucent tanycyte protrusions; (see discussion). Severe hypertrophic swelling was frequently observed in associated ACs whose diameters may exceed 10 pm, resulting in their donut-shaped appearance. Interestingly, a honey-comb-shaped appearance of neuronal somata in vibratome sections appeared similar to the staining pattern in the Amyloid immunolabeled vibratome sections from the same AD decedent. Immunolabeling in smaller amyloid 0 immunoreactive structures commonly described as ‘plaques’ does not appear random but was arranged around unlabeled circular structures consistent with the ultrastructural appearance of electron-lucent tanycyte receptacles adjacent to debriscontaining central canals; (see discussion). Due to the hypertrophic appearance of both tanycyte processes and their intraneuronal protrusions in the brains of AD decedents, studies were performed to investigate whether the somata of tanycytes within the alveus showed similar hypertrophic abnormalities.

[0231] Fig. 3A-S shows results of studies in which sections through the hippocampus of decedents with diagnosed AD were assessed. De-stained hippocampal sections of AD patients (in which cellular debris appeared brown stained) showed abundant brown coloration within most brain areas (Fig. 3). Particularly dense accumulations were observed (1) within neuronal somata in the stratum pyramidale, (2) the stratum oriens, and (3) the alveus. Fig. 3A provides photomicrographic image showing areas investigated, which included the alveus, stratum oriens and CAI -C A3 stratum pyramidale (Luxol H&E stain). Fig. 3B is an image of a toluidine-blue de-stained vibratome section, showing dense debris accumulations in the alveus, stratum oriens, and neuronal somata of the stratum pyramidale. Dark discoloration was also observed around blood vessels. Tanycytes that bordered directly on the ventricular lining appeared interconnected and send short apical ‘drainage canals’ toward the ventricle (Fig. 3C-D). These apical areas were often in close proximity to cell-like receptacles (Fig. 3C). In both hippocampus and olivary nucleus tanycytes were observed to form collateral projections that gave rise to circular protrusions (Fig. 3E-G). Hypertroph tanycytes were frequently observed in the brains of AD-decedents. Close examination of the ventricular lining revealed engorged tanycyte processes in close association with brown accumulations in addition to tanycyte somata with hypertrophic abnormalities. Fig. 3H-K provides images of different focal planes of a stained tanycyte process that gave rise to circular protrusions. The image in Fig. 3L shows hypertrophic Luxol blue-stained tanycyte process that contained engorged translucent compartments consistent with excessive liquid intake. Adjacent tanycyte processes appeared less engorged. Fig. 3M is an image showing neuronal soma in the olivary nucleus of an AD-decedent densely obstructed with hypertrophic tanycyte receptacles. Results demonstrated that hypertrophic tanycyte processes contacted the neuronal cytoplasm and nucleus and showed tanycyte processes transecting into the neuronal cytoplasm. The Fig. 3N image shows an H&E-stained hypertrophic tanycyte projection in the alveus in close proximity to brown cellular waste and Fig. 30 shows tanycyte soma with signs of hypertrophy onset in the soma. Images at different focal planes through the soma of a hypertrophic tanycyte soma with attached tanycyte processes are shown in Fig. 3P-S.

[0232] A summary of the findings presented in Example 2 is provided in Fig. 4. Fig.4A-D provides schematic diagrams and photomicrographic images and give a schematic “summary” of elements of the waste removal system in the human hippocampus and observed histopathologies. Fig. 4A is a schematic diagram of myelinated aquaporin4-IR (AQP4-IR) tanycytes whose somata are located in the alveus send vast networks of tube-like processes that contain central canals into the stratum pyramidale. Tanycytes use adherence clamps to attach to surrounding cells and form intracellular receptacles that internalize catabolized neuronal waste. The waste is transported to the ventricular lining and specialized glia canals where it is removed from the brain via the Choroid plexus. Fig. 4B provides a schematic diagram of a mechanism of waste uptake into tanycyte receptacles. Receptacles are formed through the formation of myelin protrusions around the outer periphery' of myelinated tanycytes. The central, AQP4-expressing canal branches into each receptacle-forming protrusion. Central canal and protrusion form a functional unit by which the functional significance of the receptacle is the filtering and catabolism of cellular waste to prevent obstruction by larger debris particles. The central canal creates a convective flow toward the canal through activation of AQP4. The number of receptacles formed varies. Fig. 4C shows an ultrastructural depiction of illustrated structures. Fig. 4D is a schematic diagram of underlying histopathology in the AQP4-mediated hypertrophic swelling of entire tanycytes is due to blockage of drainage canals. Swelling is observed in (1) adhesion clamps, (2) somata, (3) intracellular protrusions, and tanycyte processes.

[0233] Results of the studies set forth herein demonstrated that tanycytes interacted with neurons, glial cells, and extracellular spaces via specialized receptacles, likely to ensure controlled waste removal from the brain. The results support a conclusion that hypertrophic abnormalities result in functionally compromised tanycytes. The resulting obstruction of neurons by hypertrophic tanycyte receptacles may cause neuronal cell death in affected neurons. Based on observations such as those disclosed herein, one cause for hypertrophic abnormalities in affected tanycytes appears to be the physical obstruction of apical drainage canals due to abnormal waste aggregation.

[0234] Discussion

[0235] The experiments and results presented herein demonstrate that myelinated, AQP4-IR tanycytes emanate from the temporal horn of the lateral ventricle and form a vast interconnected glial-canal system that is likely responsible for waste removal from the brain parenchyma. The results support a conclusion that an underlying mechanism by which tanycytes internalize debris from adjacent cells is the formation of specialized myelin-derived receptacles that transect into the cytoplasm of adjacent cells. Results indicate: (a) debris is flushed toward these receptacles via the formation of an AQP4-mediated bulk flow, (b) internalized into central canals, (c) flushed toward the ventricular lining as well as paravascular spaces and (d) drained into CSF. Results suggest that obstruction of this canal system in apical drainage canals may cause an imbalance whereby waste uptake exceeds waste drainage. The resulting increase in turgor within affected tanycytes explains the hypertrophic swelling of myelin protrusions, and spongiform pathologies in the brains of AD- decedents described here. Results indicate that the large number of affected brain cells may be explained by the reticular, interconnected nature of these ependymal cells and their processes.

[0236] Myelinated tanycytes and their proposed role in waste clearance and neurodegeneration

[0237] Tanycytes are ependymal glial cells located in the ventricular lining that send long slender processes into the brain parenchyma. Prior knowledge regarding these glial cells is only fragmentary' and the significance of the myelinated processes they form within the brain parenchyma has not been recognized. Currently four different tanycyte types are distinguished based on their location in the third ventricle [R. Dali et al., Physiol. Behav. 263, 1 14108 (2023); R. Pasquettaz et al., J. Comp. Neurol. 529, 553 (2021 )]. However, it has been proposed that more types may exist [Z. Gong et al., Nature Communications 14, 2902 (2023)], which is supported by this study. Observations in both the hippocampal formation and the olivary nucleus presented herein support a finding that numerous diverse types of tanycytes emanate from the ventricular lining throughout the nervous system and that these cells play a paramount role for neuronal health through controlled waste removal from the brain among other proposed metabolic functions [N. J. Abbott et al., ActaNeuropathol. 135, 387-407 (2018); R. Dali et al., Physiol. Behav. 263, 114108 (2023); R. Pasquettaz et al., J. Comp. Neurol. 529, 553 (2021)]. The observation of tanycyte-like processes that did not stain for Luxol blue, a histological marker for myelin [K. Weber et al., Eur. J. Cell Biol. 101, 151218 (2022)] poses the question whether these processes originate from other cell ty pes or whether they appear unmyelinated due to the gradual loss of myelin through continued formation of waste receptacles. Results showing AQP4-negative cell profiles in the ventricular lining suggests that these processes may originate from different ependymal cells whose functional significance may be related to metabolic processes.

[0238] Cellular interactions between tanycytes and neurons

[0239] Results presented herein demonstrate close cell interactions between tanycytes with neurons and astrocytes. Results support a conclusion that the primary purpose of these interactions may be waste removal from metabolically active cells. Results of studies disclosed herein indicate that a functional foundation for tanycyte-mediated waste clearance in the human brain depends on: (a) the process by which myelinated tanycytes transect into adjacent cells, (b) the ability of myelinated tanycytes to form serial bulbous protrusions, that (c) differentiate into specialized receptacles for waste uptake, (d) the proposed AQP4- mediated flow toward tanycyte receptacles, and (e) proteolytic enzy mes that catabolize larger debris particles, thus preventing blockage of the long, narrow tanycyte processes.

[0240] Myelin-mediated waste removal from the nervous system

[0241] Until now myelination in the CNS was thought to be associated with oligodendrocytes and its main purposes are attributed to signal transduction along axons and metabolic support for neurons [G. Seifert and C. Steinhauser, Cell and tissue research 373, 653-670 (2018); M. Simons and K.-A. Nave, Cold Spring Harb. Perspect. Biol. 8, a020479 (2015); C. Stadelmann et al.. Physiol. Rev. 99, 1381-1431 (2019)]. Studies and results presented herein provide the first description of the identity and significance of vast numbers of heavily myelinated tanycyte processes within the hippocampal formation and their functional significance regarding waste removal from the human and rodent brain. Results presented herein relating to spider brain support the proposed role of myelinated glial cells that transect into the neuronal cytoplasm to engulf and remove cellular waste from the healthy brain. Like humans, C. salei also shows progressive neurodegeneration and degeneration onset has similarly been linked to hypertrophic abnormalities of myelinated glial cells [R. Fabian-Fine et al., J. Comp. Neurol. 531, 618-638 (2023)].

[0242] The findings presented here demonstrate that (1) myelination may not be a sufficient criterion to identify axonal processes in the absence of other identifying criteria or serial section analysis, and (2) that imperfections in myelin may not be due to fixation artifacts or disease [W. Mobius et al., Methods Cell Biol. (Elsevier, 2010), vol. 96. pp. 475-512; W. Mobius et al., Brain Res. 1641, 92-100 (2016)], but may indeed be due to functional characteristics as demonstrated here. To investigate myelination patterns around the axons of CA3 pyramidal cells and tanycytes studies presented herein included investigation of serial sections. Surprisingly, significant myelination was not observed around axonal processes and additional studies are underway.

[0243] Aqiiaporin4-mediated bulk flow

[0244] Results of studies presented herein demonstrated that an aquaporin-mediated bulk flow may cause the accumulation of cellular debris within the AQP-IR tanycyte protrusions. Studies presented herein support a conclusion that hypertrophic tanycyte protrusions in AD affected neurons that cause the obstruction and likely depletion of their cytoplasmic content demonstrate vulnerabilities of AQP4 mediated bulk flow. To ensure that intracellular waste is effectively drawn toward the receptacles appropriate regulation of flow strength is critical. Insufficient flow strength may result in the accumulation of cellular waste within neurons. In contrast, excessive flow strength may lead to the uncontrolled obstruction and depletion of neuronal somata shown here. Results presented herein provide evidence supporting a conclusion that structural impairment and resulting hypertrophy of tanycytes may be one of the main causes for neurodegeneration. This type of glia-mediated cell death characterized by the depletion of cytoplasmic content into adjacent glial cells is referred to herein as “gliaptosis.” Interestingly, structurally impaired glia cells in spiders showed similar hypertrophic swelling, discoloration and depletion of neuronal content shown here in AD affected brain [R. Fabian-Fine et al.. J. Comp. Neurol. 531, 618-638 (2023)].

[0245] Alpha synuclein, tau tangles and Amyloid beta plaques

[0246] The observed hypertrophy of tanycytes and affected neuronal somata reported here is consistent with the reported increase in cell surface and vacuole lumina in AD-affected brains compared to non-AD brain tissue [M. Askenazi et al.. Nature Communications 14, 1-15 (2023)]. Results provided herein support a conclusion that reported ghost tangles [S. Pockes et al., Translational Research 254, 34-40 (2023)] may be explained by the remaining hypertrophic tanycyte projections after cytoplasmic depletion as shown herein. Results of studies disclosed herein demonstrate that these tanycyte projections likely catabolize and internalize neuronal proteins. As demonstrated here hypertrophic tanycyte projections are still visible after cytoplasmic depletion of affected cells.

[0247] Interestingly, results of studies set forth herein indicated that the majority of tanycyte receptacles were located in the axon-hillock facing side of neurons. These observations are consistent with the location where retrograde transport vesicles first enter, and trans-Golgi vesicles exit neuronal somata.

[0248] Regarding the observed accumulation of amyloid beta plaques in AD affected brain tissue, studies are performed to investigate whether the accumulation of this protein may be associated with reported secretase-mediated cell sprouting [M. Oklinski et al., J. Histochem. Cytochem. 62, 598-611 (2014)]. Such a pathway may promote the continued formation of waste receptacles within healthy neurons.

[0249] AQP4 expression in astrocytes

[0250] The prevalence of AQP4 in the brain has mainly been attributed to their expression in the end feet of astrocytes [S. Wang et al., Aquaporins, Springer, pp. 317-330 (2023); K. Oshio et al.. Neuroscience 127, 685-693 (2004); Q. Lu et al., Int. J. Biol. Sci. 18, 441-458 (2022)]. Interestingly, astrocytes are abundant in the stratum oriens, adjacent to the stratum radiatum from which large numbers of AQP4-IR tanycyte processes emerge. If astrocytes indeed synthesize large numbers of AQP4 one would expect strongly immunoreactive somata, reflective of the place of protein synthesis. However, as shown in results herein, the somata of astrocytes did not show significant immunolabeling for AQP4.

[0251] The appearance of AQP4 labelling pattern along the outer surface of astrocytesin results presented herein is consistent with observations of AQP4-IR tanycytes that form close contacts with astrocytes. Results presented herein support an explanation that these two cell types interact with each other and that the AQP4-immunoreactivity originates from tanycytes, like the observations along neurons as presented herein. Results presented herein suggest these two cell types eliminate potentially harmful pathogens at the brain blood barrier, which are vulnerable entry gates to the brain. Based on the ultrastructural observations in experiments presented herein, it appears that astrocytes internalize pathogens via their end feet and that tanycytes catabolize and remove these pathogens from astrocytes.

[0252] Mechanisms that may lead to hypertrophic abnormalities in tanycytes

[0253] The findings presented here pose certain question regarding the underlying causes that result in these pathological changes. Results presented herein support a conclusion that the obstruction of apical drainage canals should be considered, and that tanycytes whose apical waste-drainage canals are blocked lose the ability to effectively drain debris that is taken up at their basal ends. As waste and fluid intake exceeds drainage capacity the intracellular turgor increases which explains the hypertrophic appearance of (a) tanycyte somata in the ventricular lining, (b) ACs that are formed by affected tanycytes, (c) spongiform abnormalities within and around tanycyte processes, and (d) the excessive formation and swelling of both intra- and extracellular tanycyte receptacles in neurons that are connected to affected tanycytes.

[0254] Possible therapeutic approaches

[0255] Results presented herein support a conclusion that cellular debris is enzymatically catabolized in human brain to avoid blockage of the comparatively narrow tanycyte processes by larger debris particles. Receptacle formation in AD-affected patients may result in upregulated enzymatic activity to catabolize cellular content that is flushed toward the activated receptacles. Based on data presented herein, methods are proposed comprising downregulation of caspase activity for therapeutic purposes, and upregulation of secretase and / or BACE1, to support and increase catabolic processes in neurons. Results presented herein support a conclusion that the controlled downregulation of AQP4 channels and / or increase activity of secretases and / or BACE1, would allow affected tanycytes to restore balance between water uptake and drainage and reduce hypertrophy of both affected tanycytes and neurons whose somata are obstructed.

[0256] Lipofuscin versus tanycyte receptacles

[0257] The presence of tanycyte protrusions in mammalian and human neurons has been described previously, however these structures have been identified as "lipofuscin’ rather than tanycyte receptacles [M. Schweighauser et al., Nature 585, 464-469 (2020); K. A. Jellinger and A. D. Korczyn, BMC Med. 16, 34 (2018)]. Based on results presented herein, it is proposed that most (but not all) lipofuscin described in invertebrate organisms are different structures compared to those in the mammalian system and should therefore not be used interchangeably. The structural appearance of the receptacles in human brain described herein is likely specialized to prevent larger debris particles from blocking the long and narrow mammalian tanycyte processes. The narrow openings of the receptacles are lined by membranous structures that resemble enzyme complexes. The combination of narrow openings surrounded by proteolytic enzymes may prevent larger particles from entering the receptacles. This conclusion is supported by results of studies presented herein indicating that receptacles with waste containing central canal appeared darker compared to forming receptacles suggestive of catabolized debris. At least for this reason, it is believed that Type- A receptacles described herein are formed within the cytoplasm of cells and may be triggered by intracellular signals inherent to catabolic processes. Results of studies presented herein, indicated that Type-B receptacles were predominantly found in extracellular spaces, supporting a conclusion that they play an important role in extracellular waste clearance.

[0258] Concluding remarks

[0259] Studies comprising immunohistochemical, ultrastructural. and light microscopic methods have been done to investigate the structural foundation of neurodegeneration in human brain. The results and evidence presented herein demonstrate the presence and proposed significance of myelinated tanycyte protrusions in human neurons in health and disease. The fact that both spiders and rodents show similar waste removal mechanisms supports a conclusion that waste removal from the brain is similarly conserved as in the intestinal tract and nephridial systems. The significance of these findings is profound and opens new approaches to investigate the underlying causes for neurodegeneration. The studies presented herein demonstrate the importance or re-visiting anatomical characteristics of the nervous system using serial section analysis.

[0260] Equivalents

[0261] Although several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto; the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, if such features, systems, articles, materials, and / or methods are not mutually inconsistent, is included within the scope of the present invention.

[0262] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0263] The indefinite articles “a” and “an.” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0264] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified, unless clearly indicated to the contrary.

[0265] All references, patents and patent applications and publications that are cited or referred to in this application are incorporated herein in their entirety herein by reference.

[0266] What is claimed is:

Claims

Claims1. A method for preventing neuronal cell death, comprising reducing an abnormal function of a glial canal in a canal-forming glial cell adjacent to the neuronal cell, wherein the reduction in the abnormal function increases likelihood of survival of the neuronal cell compared to a control likelihood of survival.

2. The method of claim 1 , wherein the control likelihood of survival is the likelihood of survival of a neuronal cell adj cent to a canal -forming glial cell in which the abnormal function is not reduced.

3. The method of claim 1, wherein the abnormal function comprises depleting the adjacent neuronal cell’s neuronal cytoplasm into the glial canal.

4. The method of claim 1 , wherein the abnormally functioning canal-forming glial cell comprises one or more glial canals comprising structural damage.

5. The method of claim 4, wherein the glial canal structural damage statistically significantly increases flow of cytoplasm from the neuronal cell into the structurally damaged glial canal.

6. The method of claim 1 , wherein reducing an abnormal function comprises maintaining a normal function of the canal-forming glial cell.

7. The method of claim 6, wherein the normal function of the canal-forming glial cell comprises a controlled removal of neuronal waste from the adjacent neuronal cell into the glial canal of the canal-forming glial cell.

8. The method of claim 7, wherein the neuronal waste comprises one or more of lipid- based cellular waste, protein-based cellular waste, and lipofuscin.

9. The method of claim 1, wherein reducing the abnormal function of the canal-forming glial cell comprises one or more of: increasing production of normal glial-canals by the canalforming glial cell; maintaining a normal function of the glial-canals in the glial cell; and reducing damage to one or more glial-canals in the canal-forming glial cell.

10. The method of claim 1, wherein the glial cell is in contact with the neuronal cell.

11. The method of claim 1, wherein the method comprises contacting the canal-forming glial cell with a composition comprising an aquaporin inhibitor.

12. The method of claim 11. wherein the aquaporin inhibitor is an aquaporin 4 inhibitor.

13. The method of claim 12, wherein the aquaporin 4 inhibitor is 2-(ni cotinamide)-!, 3,4- thiadiazole (TGN-020).

14. The method of claim 11, wherein the aquaporin inhibitor is an aquaporin 7 inhibitor or is an aquaporin 9 inhibitor.

15. The method of claim 1, wherein the method comprises contacting the canal-forming glial cell with a composition comprising an agent that increases activity of a proteolytic enzyme.

16. The method of claim 15, wherein the enzyme is a caspase.

17. The method of claim 16, wherein the caspase is a caspase 2 or a caspase 3.

18. The method of claim 1, wherein the method comprises contacting the canal-forming glial cell with a composition comprising an agent that reduces activity of a secretase or a BACE1.

19. The method of claim 18, wherein the secretase is an alpha-secretase, a beta-secretase, or a gamma-secretase.

20. The method of claim 1 , wherein the neuronal cell is in a subject.

21. The method of claim 20, wherein the subject is a mammal, optionally is a human.

22. The method of claim 1, wherein the neuronal cell is in culture.

23. The method of claim 1, wherein the neuronal cell is an engineered neuronal cell.

24. The method of claim 1, wherein the neuronal cell is in or is obtained from a subject known to have, or suspected of having, a neurodegenerative disease or condition.

25. The method of claim 24. wherein the neurodegenerative disease or condition is Alzheimer’s disease, Parkinson’s disease, Huntington’s disease. Chronic Traumatic Encephalopathy (CTE), Amyotrophic lateral sclerosis (ALS); or Motor neuron disease.

26. The method of any one of claims 1-25, wherein the glial cell is a macroglial cell.

27. A composition comprising an aquaporin inhibitor for treatment of a neurodegenerative disease or condition.

28. The composition of claim 27, wherein the aquaporin inhibitor is an aquaporin 4 inhibitor, optionally wherein the aquaporin 4 inhibitor is 2-(nicotinamide)-l,3,4-thiadiazole (TGN-020).

29. The composition of claim 27, wherein the aquaporin inhibitor is an aquaporin 7 inhibitor or is an aquaporin 9 inhibitor.

30. A composition comprising an agent that increases activity of a caspase for treatment of a neurodegenerative disease or condition.

31. The composition of claim 30, wherein the caspase is a caspase 2 or a caspase 3.

32. A composition comprising an agent that decreases activity of a secretase for treatment of a neurodegenerative disease or condition.

33. The composition of claim 32, wherein the secretase is an alpha-secretase, a beta- secretase, or a gamma-secretase.

34. A composition comprising an agent that decreases BACE1 activity for treatment of a neurodegenerative disease or condition.

35. A method of reducing neurodegeneration in a subject, the method comprising administering to the subject an aquaporin inhibitor in an amount effective to maintain or increase a normal function of a canal -forming glial cell in the brain of the subject.

36. The method of claim 35, wherein the normal function of the canal-forming glial cell comprises a controlled removal of neuronal waste from a neuronal cell adjacent to a glial canal of the canal-forming glial cell.

37. The method of claim 35, wherein the aquaporin inhibitor is an aquaporin 4 inhibitor, optionally wherein the aquaporin 4 inhibitor is 2-(nicotinamide)-l,3,4-thiadiazole (TGN- 020).

38. The method of claim 35, wherein the aquaporin inhibitor is an aquaporin 7 inhibitor or is an aquaporin 9 inhibitor.

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