EP4203987A2

EP4203987A2 - Compositions and methods for the treatment of ocular neuroinflammation

Info

Publication number: EP4203987A2
Application number: EP21862665.3A
Authority: EP
Inventors: Yury Miller; Soo-Ho Choi; Wonkyu JU
Original assignee: University of California
Current assignee: University of California
Priority date: 2020-08-25
Filing date: 2021-08-25
Publication date: 2023-07-05
Also published as: WO2022046919A3; CA3190597A1; EP4203987A4; WO2022046919A2

Abstract

Provided herein are products of manufacture, kits, and methods, for: treating, ameliorating, protecting against, reversing or decreasing the severity or duration of neuroinflammation in the eye, as for example, during glaucomatous neurodegeneration by administration of a polypeptide composition or a nucleic acid composition encoding a polypeptide composition, wherein the polypeptide composition is, or is comprised of, an ApoA-I Binding Protein.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF OCULAR NEUROINFLAMMATION

RELATED APPLICATIONS

This Patent Convention Treaty (PCT) International Application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/070,145, filed August 25, 2020. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes. All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH This invention was made with government support under HL135737, EY018658, NS102432, awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

TECHNICAL FIELD

The present invention generally relates to neuroinflammation in the eye, including neuroinflammation in the eye during glaucomatous neurodegeneration in glaucoma. In alternative embodiments, provided are products of manufacture, kits, and methods, for: treating, ameliorating, protecting against, reversing or decreasing the severity or duration of a glaucoma, which typically results from mitochondrial dysfunction in retinal ganglion cells (RGCs), Muller glia or microglia. In alternative embodiments, provided are products of manufacture, kits, and methods, for: treating, ameliorating, protecting against, reversing or decreasing neuroinflammation in the eye during glaucomatous neurodegeneration; treating, ameliorating, protecting against, reversing or decreasing the severity or duration of mitochondrial dysfunction in retinal ganglion cells (RGCs), Muller glia or microglia during glaucomatous neurodegeneration in the eye; and/or decreasing or slowing the rate of induced RGC death in response to elevated intraocular pressure (IOP). In alternative embodiments, methods as provided herein treat glaucoma by intraocular or intravitreal administration of a composition comprising ApoA-I Binding Protein (APOA1BP, AIBP, or ALBP) or a protein related thereto, or nucleic acids encoding AIBP or these related proteins. BACKGROUND

ApoSipoprotein A-I Binding Protein, or ApoA-I binding protein (AIBP), also known as NAXE, NAD(P)HX epimerase, was discovered in a screen of proteins that physically associate with apoA-I f The human AIBP gene (APOA1BP) is located at lq22. AIBP is localized in cytosol and mitochondria or in some instances is secreted. It has a presumed N-terminal signal peptide, and is therefore likely cleaved from the protein during protein secretion from the cell ². Alternatively, this N-terminal peptide is considered to serve as a mitochondria localization signal ⁴.

Glaucoma is a leading cause of irreversible blindness worldwide in individuals 60 years of age and older. Despite the high prevalence of glaucoma, the factors contributing to its progressive worsening are currently not well characterized. To date, intraocular pressure (IOP) is the only proven treatable risk factor. Eye drops or systemic administration of medications are employed to lower IOP. However, lowering IOP often is insufficient for preventing disease progression caused by neuroinflammation, which was initiated by the prolonged high IOP.

Neuroinflammation is defined as immune responses in the central nervous system, and it is of great interest to better understanding the role of glia-mediated neuroinflammation in glaucoma (1, 2). However, the interplay between glia-mediated neuroinflammation and mitochondrial dysfunction in glaucomatous neurodegeneration is poorly understood.

Therefore, there is a need in the art for compositions and methods useful for reducing ocular neuroinflammation, in particular, ocular neuroinflammation resulting from glaucoma.

SUMMARY

In alternative embodiments provided are methods for treating, ameliorating, protecting against, reversing or decreasing the severity or duration of a glaucoma, wherein optionally the glaucoma is open-angle glaucoma or closed angle glaucoma, treating, ameliorating, protecting against, reversing or decreasing the severity or duration of neuroinflammation in an eye during glaucomatous neurodegeneration, treating, ameliorating, protecting against, reversing or decreasing the severity or duration of mitochondrial dysfunction in retinal ganglion cells (RGCs), microglia or Muller glia during glaucomatous neurodegeneration in an eye, or decreasing or slowing the rate of induced RGC death in response to elevated intraocular pressure (IOP), wherein the method comprises the step of: administering a pharmaceutically acceptable formulation to a subject in need thereof, wherein the pharmaceutically acceptable formulation is comprised of:

(1) a polypeptide composition, wherein the polypeptide composition is, or is comprised of, an ApoA-I Binding Protein) polypeptide, wherein the polypeptide composition has, or is capable of providing for, an ApoA-I Binding Protein polypeptide activity, or

(2) a nucleic acid composition that increases expression or activity of, or encodes for, a polypeptide composition, wherein the polypeptide composition is, or is comprised, of an ApoA-I Binding Protein polypeptide, wherein the polypeptide composition has, or is capable of providing, an ApoA-I Binding Protein polypeptide activity, or

(3) an ApoA-I Binding Protein polypeptide-inducing compound or composition.

In alternative embodiments, provided are methods for:

- treating, ameliorating, protecting against, reversing or decreasing the severity or duration of a glaucoma, wherein optionally the glaucoma is open-angle glaucoma or closed angle glaucoma,

- treating, ameliorating, protecting against, reversing or decreasing the severity or duration of neuroinflammation in an eye during glaucomatous neurodegenerati on,

- treating, ameliorating, protecting against, reversing or decreasing the severity or duration of mitochondrial dysfunction in retinal ganglion cells (RGCs) or Muller glia during glaucomatous neurodegeneration in an eye, or

- decreasing or slowing the rate of induced RGC death in response to elevated intraocular pressure (IOP), wherein the method comprises:

(a) (i) providing or having provided a formulation or a pharmaceutical composition comprising:

(1) an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP) polypeptide compound or composition, or a compound that increases expression or activity of, or encodes, an APOA1BP polypeptide or nucleic acid, or a polypeptide or peptide having an APOA1BP activity, or an APOAIBP-stimulating compound or composition;

(2) the formulation or pharmaceutical composition of (1), wherein the compound that increases expression or activity of, or encodes, a APOA1BP polypeptide is a nucleic acid that expresses or encodes a APOA1BP polypeptide or a polypeptide having a APOA1BP polypeptide activity; or

(3) an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP)-inducing compound or composition; and

(ii) administering or having administered the formulation or the pharmaceutical composition of (a) to a subject in need thereof, or

(b) administering a formulation or the pharmaceutical composition comprising: an ApoA-I Binding Protein (APOA1BP) polypeptide compound or composition, or a compound that increases expression or activity of, or encodes, an APOA1BP polypeptide or nucleic acid, or a polypeptide or peptide having an APOA1BP activity, or an APOAIBP-stimulating compound or composition, to a subject in need thereof, thereby:

- treating, ameliorating, protecting against, reversing or decreasing the severity or duration of a glaucoma, wherein optionally the glaucoma is openangle glaucoma or closed angle glaucoma,

- treating, ameliorating, protecting against, reversing or decreasing the severity or duration of mitochondrial dysfunction in a retinal ganglion cell (RGC) or a Muller glia during glaucomatous neurodegeneration in an eye, or

- decreasing or slowing the rate of induced RGC death in response to elevated intraocular pressure (IOP).

In alternative embodiments, of the methods:

- the APOAIBP-stimulating compound or composition increases or stimulates (activates) the activity of a APOA1BP promoter or transcriptional regulatory sequence or motif; - the nucleic acid that expresses or encodes a APOA1BP polypeptide or a polypeptide having a APOA1BP polypeptide activity is contained in an expression vehicle, vector, recombinant virus, or equivalent, and optionally the expression vector or virus is or comprises an adenovirus vector or an adeno-associated virus (AAV) vector, a retrovirus, a lentiviral vector, a herpes simplex virus, a human immunodeficiency virus (HIV), or a synthetic vector, wherein optionally the AAV vector comprises or is: an adeno-associated virus (AAV), or an adenovirus vector, an AAV serotype or variant AAV5, AAV6, AAV8 or AAV9, AAV-DJ or AAV- DJ/8™ (Cell Biolabs, Inc., San Diego, CA), a rhesus-derived AAV vector, wherein optionally the rhesus-derived AAV AAVrh. l0hCLN2, an AAV capsid mutant or AAV hybrid serotype, wherein optionally the AAV variant is engineered to increase efficiency in targeting a specific cell type that is non -permissive to a wild type (wt) AAV vector and/or to improve efficacy in infecting only a cell type of interest, and wherein optionally the hybrid AAV variant is retargeted or engineered as the AAV hybrid serotype by one or more modifications comprising: 1) a transcapsidation, 2) adsorption of a bi-specific antibody to a capsid surface, 3) engineering a mosaic capsid, and/or 4) engineering a chimeric capsid;

- the ApoA-I Binding Protein (APOA1BP) polypeptide or protein is a mammalian APOA1BP polypeptide or peptide;

- the ApoA-I Binding Protein (APOA1BP) polypeptide or protein is a human APOA1BP polypeptide or peptide;

- the subject is a human, or the subject is an animal;

- the APOA1BP polypeptide or peptide is a recombinant APO A IBP polypeptide or peptide having an APOA1BP activity;

- the APOA1BP polypeptide or peptide is a synthetic APOA1BP polypeptide or peptide;

- the APOA1BP polypeptide or peptide is a APOA1BP activity-stimulating compound or composition; - the formulation or pharmaceutical composition is formulated for administration in vivo or is formulated for intraocular or intravitreal administration, or is administered in vivo by intrathecal injection;

- the formulation or pharmaceutical composition is administered by in vivo intrathecal, intravitreal, or intraocular injection;

- the formulation or pharmaceutical composition is formulated for intravenous (IV) administration;

- the APOA1BP polypeptide or peptide or the formulation or pharmaceutical composition, is formulated in or with a nanoparticle, a particle, a micelle or a liposome or lipoplex, a polymersome, a polyplex or a dendrimer;

- the APOA1BP polypeptide or peptide or the formulation or pharmaceutical composition, is formulated in or as a nanoparticle, a liposome, a liquid, an emulsion, an aqueous or a sterile or an injectable solution, or an implant, wherein optionally the implant is an intraocular implant; and/or

- the nanoparticle, particle, micelle or liposome or lipoplex, polymersome, polyplex or dendrimer further comprise or express a cell or CNS penetrating moiety or peptide or a CNS targeting moiety or peptide.

In alternative embodiment, provided are uses of a formulation or a pharmaceutical composition comprising:

(2) the formulation or pharmaceutical composition of (1), wherein the compound that expresses or increases expression or activity of, or encodes, a APOA1BP polypeptide or a polypeptide having a APOA1BP polypeptide is a nucleic acid, optionally wherein the nucleic acid expresses or encodes a APOA1BP polypeptide or a polypeptide having the APOA1BP polypeptide activity; or

(3) an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP)-inducing compound or composition, in the manufacture of a medicament for: - treating, ameliorating, protecting against, reversing or decreasing the severity or duration of a glaucoma, wherein optionally the glaucoma is open-angle glaucoma or closed angle glaucoma,

In alternative embodiment, provided are formulations or pharmaceutical compositions comprising:

(2) the formulation or pharmaceutical composition of (1), wherein the compound that expresses or increases expression or activity of, or encodes, a APOA1BP polypeptide is a nucleic acid, optionally wherein the nucleic acid expresses or encodes the APOA1BP polypeptide or the polypeptide having the APOA1BP polypeptide activity; or

(3) an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP)-inducing compound or composition for use in:

- treating, ameliorating, protecting against, reversing or decreasing the severity or duration of neuroinflammation in an eye during glaucomatous neurodegenerati on, - treating, ameliorating, protecting against, reversing or decreasing the severity or duration of mitochondrial dysfunction in a retinal ganglion cell (RGC) or a Muller glia during glaucomatous neurodegeneration in an eye,

The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

All publications, patents, patent applications cited herein are hereby expressly incorporated by reference in their entireties for all purposes.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

The drawings set forth herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims.

Figures are described in detail herein.

FIG. 1 A-I illustrate data from studies to determine whether elevated pressure alters expression level of AIBP in murine RGCs by using transiently inducing acute intraocular pressure (IOP) elevation in the eye of normal C57BL/6I mice by the cannulation of the anterior chamber of the eye:

FIG. 1 A-B graphically illustrate data showing that elevated IOP significantly reduced Apoalbp gene and AIBP protein expression in the retina at 24 h compared with sham control retina, and that AIBP expression is decreased in glaucomatous retinas and pressure-induced RGCs: FIG. 1 A graphically illustrates Apoalbp gene expression in control and injured retina at 1 day after acute IOP elevation; FIG. IB graphically illustrates AIBP protein expression in control and injured retina at 1 day after acute IOP elevation;

FIG. 1C illustrates representative images showing AIBP (green) and TU 1 (red) immunoreactivities at 1 day after acute IOP elevation, where arrows indicate AIBP immunoreactivity co-labeled with TUJ 1 in RGC somas and arrowhead indicates AIBP co-labeled with TUJ 1 in RGC axon bundle;

FIG. ID graphically illustrates data showing that AIBP protein expression in control and injured RGCs at 3 day after elevated HP;

FIG. IE illustrates representative images showing AIBP (green), TUJ1 (red) and Brn3a (yellow) immunoreactivities;

FIG. IF illustrates FIG. IE representative images at higher magnification images, arrows indicate AIBP immunoreactivity co-labeled with TUJ 1 in RGC somas and arrowhead indicates AIBP co-labeled with TUJ1 in RGC axon bundle;

FIG. 1G graphically illustrates data showing that quantitative fluorescent intensity showed a significant decrease in AIBP immunoreactivity in the inner retina of glaucomatous DBA/2J mice;

FIG. 1H illustrates representative images showing ABCA1 (green), AIBP (red) and Brn3a (yellow) immunoreactivities, where concave arrowheads indicate ABC Al -positive RGCs co-labeled with AIBP and Brn3a; and

FIG II graphically illustrates data showing quantitative fluorescent intensity showed a significant decrease in ABCA1 immunoreactivity in the GCL of glaucomatous DBA/2J mice; as described in detail in Example 1, below.

FIG. 2A-H illustrate data showing that AIBP deficiency exacerbates vulnerability to elevated intraocular pressure (IOP) in RGCs and triggers visual dysfunction:

FIG. 2A graphically illustrates data where RGC loss was measured in the retina of 4 month-old wild type (WT) and age-matched Apoalbp~ ~ mice at 4 weeks after acute IOP elevation, and visual function were measured in 4-month-old Apoalbp~ ~ mice, and the average of IOP elevation in WT mice is shown;

FIG. 2B illustrates representative images from whole-mount immunohistochemistry showed Brn3a-positive RGCs in WT and Apoalbp~ ~ following acute IOP elevation;

FIG. 2C graphically illustrates data from a quantitative analysis by RGC counting using whole-mount immunohistochemistry for Bm3a in WT and Apoalbp~ ~ following acute IOP elevation, where each retinal quadrant was divided into three zones by central, middle, and peripheral retina, and the central, middle, peripheral and total retina amounts are graphically shown;

FIG. 2D graphically illustrates data of a visual function test in WT and naive Apoalbp~ ~ mice by OKT analyses, showing male, female and total counts;

FIG. 2E graphically illustrates data of a visual function test in WT and naive Apoalbp~ ~ mice by VEP analyses, where VEP Pl-Nl potentials and latency was measured in naive Apoalbp~ ~ mice compared with WT mice;

FIG. 2F graphically illustrates total recordings of VEP responses, Left: total recordings of the VEP response of WT mice, and Right: total recordings of the VEP response of naive Apoalbp~ ~ mice, or the VEP analyses shown in FIG. 2E;

FIG. 2G illustrates representative images of CTB (red) labeling in the SCs of in WT (left image) and naive Apoalbp~ ~ mice (right image);

FIG. 2H graphically illustrates a quantitative analysis of CTB fluorescence density in the SCs (superior colliculi) of WT and naive Apoal bp~ ~ mice; as described in detail in Example 1, below.

FIG. 3A-D illustrate data showing that glaucomatous and Apoalbp~ ~ Muller glia endfeet upregulate TLR4 and IL- 10 expression, where immunohistochemical analyses for TLR4 and IL-10 were conducted on retina wax sections in glaucomatous and Apoal bp~ ~ retina:

FIG. 3A-B illustrate representative images showing TLR4 and IL- 10 immunoreactivities in Muller glia of the inner retinas from human patient with POAG, and glaucomatous DBA/2J and naive Apoalbp~ ~ mice;

FIG. 3C-D graphically illustrate quantitative fluorescent intensity showed a significant increase in TLR4 and IL- 10 immunoreactivities in Muller glia endfeets from human patient with POAG, and glaucomatous DBA/2J and naive Apoalbp~ ~ mice compared with control groups, GCL is ganglion cell layer; INL is inner nuclear layer; IPL is inner plexiform layer; NFL is nerve fiber layer; as described in detail in Example 1, below.

FIG. 4A-R illustrate data showing that AIBP deficiency induces mitochondrial fragmentation, outer membrane onion-like swirls, lower crista density, and reduces ATP production in Muller glia endfeet:

FIG. 4A illustrates images of SBEM WT volume showing typical cytoplasmic structures; mitochondria (yellow trace) highlighted; FIG. 4B illustrates images of SBEM Apoalbp~ ~ volume showing mitochondria (yellow trace) with lower crista density (red arrowheads) and dark outer membrane onion-like swirls (blue trace);

FIG. 4C illustrates images of WT surface rendering highlighting long tubular mitochondria (yellow), cytoplasmic membrane is blue;

FIG. 4D illustrates images of surface rendering showing short fragmented mitochondria (yellow) in Apoalbp~ ~

FIG. 4E-G illustrate images of expedited and accurate segmentation and analysis of mitochondria:

FIG. 4E-F illustrate images of cross image planes (see inset cross image sections labeled a, b, c and d) showing the need for 3DEM. 357 electron micrographs, which were serially collected to follow many mitochondria through the large volume;

FIG. 4G schematically illustrates an exemplary approach to determine mitochondrial length (red) and variable shapes;

FIG. 4H schematically illustrates a surface rendering showing long tubular forms of mitochondria in WT;

FIG. 41 schematically illustrates a surface rendering showing smaller, round forms of mitochondria in Apoalbp~ ~

FIG. 4J graphically illustrates the volume of mitochondria was not significantly different in the Apoal bp~ ~

FIG. 4K graphically illustrates the mitochondrial volume density in the Apoalbp~ ~ was almost identical to the WT;

FIG. 4L graphically illustrates data showing no significant difference in the number of mitochondria between WT and Apoal bp~ ~

FIG. 4M graphically illustrates data showing that the form factor for Apoal bp~ ^/_ mitochondria was significantly lower, confirming less elongation;

FIG. 4N graphically illustrates data showing that mitochondrial length was significantly lower in the Apoal bp~ ~

FIG. 40 graphically illustrates data showing that the crista density was significantly lower in the Apoal bp~ ~

FIG. 4P graphically illustrates data showing that the rate of ATP production per mitochondrial volume was lower in the Apoal bp~ ~ FIG. 4Q graphically illustrates data showing that the modeled rate of ATP production per mitochondrion was no different in the Apoal bp~ ~

FIG. 4Q graphically illustrates data showing that there was a significant lowering of ATP availability per unit cellular volume in the Apoal bp~ ~ as described in detail in Example 1, below.

FIG. 5A-F illustrate data showing that AIBP deficiency impairs mitochondrial dynamics and OXPHOS activity in the retina, where mitochondrial AIBP expression was assessed in the retina of a mouse model of acute IOP elevation and alteration of mitochondrial dynamics and OXPHOS were assessed in the retina of WT and Apoalbp~ ~ mice:

FIG. 5A illustrates (left image) fractionation of cytosolic and mitochondrial extracts, mitochondrial AIBP protein expression in control and injured retina at 1 day after acute IOP elevation, and graphically illustrates data from the fractionation of the AIBP (right image);

FIG. 5B illustrates gel fractionation of OPA1 and MFN2 protein expression in the retina of WT and Apoalbp~ ~ mice, and graphically illustrates data from the fractionation of OPA1 and MFN2;

FIG. 5C illustrates representative images showing OP Al (green), cytochrome c (red) and Brn3a (yellow) immunoreactivities in the wax sections from WT and Apoalbp~ ~ retinas, where arrowheads indicate accumulation of OPA1 co-labeled with cytochrome c in RGC somas in WT mice and arrows indicate OPA1 -labeled Muller glia endfeet;

FIG. 5D illustrates an image of a gel showing DRP1 and pDRPl S637 expression in the retina of WT and Apoalbp~ ~ mice, and graphically illustrates the DRP1 and pDRPl S637 expression;

FIG. 5E illustrates representative images showing DRP1 (green) and Brn3a (red) immunoreactivities in the wax sections from WT and Apoalbp~ ~ retinas, where arrowheads indicate accumulation of DRP1 co-labeled with Brn3a in RGC somas in WT and Apoalbp~ ~ mice;

FIG. 5F illustrates images of gels showing OXPHOS Cxs protein expression in the retina of WT and Apoalbp~ ~ mice; For FIG. 5A-F: CNT, control; Cx, complex; GCL, ganglion cell layer; HIOP, high intraocular pressure; HP, hydrostatic pressure; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; as described in detail in Example 1, below.

FIG. 6A-J illustrate data showing that AIBP deficiency triggers mitochondrial fragmentation, swelling and rounding, and ER swelling in RGC somas:

FIG. 6 A illustrates an image showing tomographic volume of WT RGC showing typical mitochondrial and ER structures, note 2x inset showing well-formed mitochondria and ER (white arrowheads);

FIG. 6B illustrates an image showing tomographic volume of Apoal bp~ ~ RGC showing rounded mitochondria with lower crista density and swollen ER, note 2x inset showing swollen ER (white arrowheads), including one contacting a mitochondrion;

FIG. 6C illustrates an image showing Apoalbp~ ~ volume showing two adjacent mitochondria and ER sandwiched at their fission site;

FIG. 6D illustrates an image showing mitochondrial outer membrane (blue trace), IBM (yellow trace) and ER (green fill), partly dilated (arrow), noting the bulge in top mitochondrion (white arrowhead) caused by expansion of both outer and IBM, and inward bulge in bottom mitochondrion (black arrowhead) caused by expansion of the IBM only;

FIG. 6E illustrates an image showing three dimensional (3D) surfacerendering overlaid on a Apoalbp~ ~ volume;

FIG. 6F illustrates a 3D image showing that even though many ER strands are dilated, mitochondrial fission can proceed in the Apoal bp~ ~.-

FIG. 6G illustrates an image showing abnormal mitochondria with onion-like swirling membrane (white arrow) were common in the Apoal bp~ ~

FIG. 6H illustrates an image showing onion-like swirl (blue) not part of the IBM (yellow);

FIG. 61 schematically illustrates an image showing a surface rendering of SBEM sub-volume showing cytoplasmic membrane (green), neurites (green), nucleus (blue) long tubular form (yellow) and branched mitochondria (red) in the WT; FIG. 6J schematically illustrates an image showing surface rendering of the cytoplasmic membrane, nucleus, dendrites and axons, and smaller round form (yellow) and branched (red) mitochondria in Apoal bp~ ~

FIG. 6K-0 graphically illustrate data from measurements of structural features of mitochondria:

FIG. 6K shows that volume of mitochondria was significantly greater in Apoalbp- -'.

FIG. 6L shows that mitochondrial volume density was higher in Apoalbp'^/_:

FIG. 6M shows that no significant difference in the number of mitochondria:

FIG. 6N shows that form factor was significantly lower in the Apoa l bp". _j confirming rounded mitochondria:

FIG. 60 shows that mitochondria lengths were significantly decreased in the Apoal bp" as described in detail in Example 1, below.

FIG. 7A-L illustrate data showing AIBP deficiency reduces cristae density, ATP production and mitofilin protein expression in RGC mitochondria:

FIG. 7A graphically illustrates data showing the mean crista density was significantly lower in the mitochondria of Apoal bp" RGC somas;

FIG. 7B graphically illustrates data showing the mean modeled rate of ATP production per mitochondrion was higher in the Apoalbp~ ~ RGC soma;

FIG. 7C graphically illustrates data showing the mean rate of ATP production per unit mitochondrial volume was lower in the Apoal bp" RGC soma;

FIG. 7D graphically illustrates data showing that there was no significant lowering of modeled availability of ATP per unit cellular volume (P = 0.39);

FIG. 7E-J illustrate data showing that the crista density was lower in the Apoal bp" mitochondria due in part to onion-like outer membrane protuberances, where mitochondria-associated ER strands were often dilated:

FIG. 7E illustrates an image of a 4.2 nm-thick slice from a WT tomographic volume of RGC showing typical cristae, a well-formed ER strand is nearby (arrowhead);

FIG. 7F schematically illustrates an image of a surface rendering of the segmented volume emphasizes the density of the cristae (shades of brown), where the mitochondrial outer membrane is shown in translucent maroon; FIG. 7G schematically illustrates an image of a 4.2 nm-thick slice from an Apoalbp~ ~ tomographic volume of RGC showing cristae that is less densely packed and an onion-like protuberance, and an adjacent ER strand is dilated;

FIG. 7H schematically illustrates an image of a surface rendering of a segmented volume that emphasizes the less-dense cristae packing and the 3 protuberances (black) that occupy part of the volume that would normally have been occupied by cristae;

FIG. 71 schematically illustrates an image of a surface rendering that shows that without the protuberances emphasizes the part of the volume not occupied by cristae (arrow points to one of these volumes);

FIG. 7J schematically illustrates an image of a surface rendering showing only the protuberances to highlight their size relative to the mitochondrial volume;

FIG. 7K illustrates an image of a mitofilin protein expression as assessed by Western blot analysis in WT and Apoalbp~ ~ retinas, and graphically illustrates data from the Western blot analysis;

FIG. 7L graphically illustrates Mitofilin gene expression as assessed by quantitative PCR analysis in WT and Apoalbp~ ~ retinas, n = 3 mice; as described in detail in Example 1, below.

FIG. 8A-H illustrate data showing that AIBP deficiency induces oxidative stress and activates MAPK signaling in the retina, where oxidative stress and MAPKs signaling were assessed in the retina of WT and Apoalbp~ ~ mice;

FIG. 8A-B illustrate images of gels (left images) showing SIRT3 (FIG. 8A) and SOD2 (FIG. 8B) protein expression in the retina of WT and Apoal bp~ ~ mice, and graphically illustrate data from these gels (right images);

FIG. 8C-D illustrate representative images of SIRT3 (green), SOD2 (green) and Brn3a (red) immunoreactivities in the wax sections from WT and Apoalbp~ ~ retinas, arrowheads indicate accumulation of SIRT3 or SOD2 co-labeled with Brn3a in RGC somas in WT and Apoalbp~ ~ mice;

FIG. 8E-F illustrate images of gels (left images) showing phospho-p38 (pp38) and phospho-ERKl/2 (pERKl/2) protein expression in the retina of WT and Apoalbp~ ~ mice, and graphically illustrate data from these gels (right images);

FIG. 8G-H illustrate representative images showed pp38 (green), pERKl/2 (green) and Brn3a (red) immunoreactivities in WT and Apoalbp~ ~ retinas; arrowheads indicate accumulation of phospho-p38 co-labeled with Bm3a in RGC somas in WT and Apoalbp~ ~ mice;

In FIG. 8A-H GCL is ganglion cell layer; INL is inner nuclear layer; IPL is inner plexiform layer; ONL is outer nuclear layer; OPL is outer plexiform layer; as described in detail in Example 1, below.

FIG. 9A-D illustrate data showing that AIBP promotes RGC survival and prevents glia-mediated inflammatory responses against elevated pressure, where apoptotic cell death was assessed in a mouse model of acute IOP elevation, and inflammatory responses and/or cytokine production was assessed in retinal Muller glia or cultured BV-2 microglia exposed to EHP:

FIG. 9A-B illustrate images where recombinant AIBP protein or BSA (1 L, 0.5 mg/ml) was intravitreally injected at 2 days before acute IOP elevation and assessed TUNEL-positive cells in the retina of WT mice at 1 day after acute IOP elevation, and following RBPMS (green) immunohistochemistry, TUNEL (red) staining was conducted:

FIG. 9A illustrates representative images showing RBPMS-positive RGCs in the GCL and TUNEL-positive cells in the retinas;

FIG. 9B graphically illustrates a quantitative analysis by TUNEL-positive cell counting;

FIG. 9C illustrates representative images showing IL- 10 immunoreactivity in the inner retina;

FIG. 9B graphically illustrates a quantitative analyses for fluorescent intensity showing that AIBP treatment significantly decreased in IL-ip immunoreactivity in Muller glia endfeets against elevated IOP. n = 4 mice;

In FIG. 9A-D, error bars represent SEM, BSA is bovine serum albumin; CNT is control; GCL is ganglion cell layer; EHP is elevated hydrostatic pressure; HIOP is high intraocular pressure; INL is inner nuclear layer; IPL is inner plexiform layer; NP is no pressure; ONL is outer nuclear layer; OPL is outer plexiform layer; as described in detail in Example 1, below.

FIG. 10A-E illustrate images showing that AIBP deficiency induces abnormal structures of mitochondria and rough ER, and mitophagosome formation in Muller glia endfeet: FIG. 10A illustrates an image where color is added to an additional slice to highlight the WT mitochondria (yellow trace);

FIG. 10B illustrates an image where color added to an additional slice to not only identify the mitochondria (yellow trace), but also to point to the mitochondria with lower cristae density (red arrowheads) compared to the WT traced and a dark outer membrane onion-like swirl (blue trace);

FIG. 10C-E illustrate serial slice images through the tomographic volume from WT and Apoal bp~ ~ Muller glia endfeets:

FIG. 10C illustrates serial slice images from WT Muller glia endfeet showing a long tubular form of mitochondrion with abundant rough ER;

FIG. 10D illustrates serial slice images from Apoal bp~ ~ Muller glia endfeet to point to the mitochondria with lower cristae density and dark outer membrane onionlike swirls (blue arrow);

FIG. 10E illustrates serial slice images from Apoal bp~ ~ Muller glia endfeet showing an abnormal mitochondrion with a vesicular inclusion (arrowhead) as well as lower rough ER density and dilated rough ER strands (arrows); as described in detail in Example 1, below.

FIG. 11 A-B illustrates images showing that AIBP deficiency impairs mitochondrial dynamics and OXPHOS activity in the retina:

FIG. 11 A illustrates representative images from immunohistochemical analyses showed OPA1 (green) and GS (red) immunoreactivities in the wax sections from WT and Apoal bp~ retinas;

FIG. 1 IB illustrates representative images from immunohistochemical analyses showed DRP1 (green) and GS (red) immunoreactivities in the wax sections from WT and Apoal bp~ ~ retinas;

For FIG. 11 A-B, blue is Hoechst 33342 staining for nucleus; GCL is ganglion cell layer; INL is inner nuclear layer; IPL is inner plexiform layer; ONL is outer nuclear layer; OPL is outer plexiform layer; as described in detail in Example 1, below.

FIG. 12 A-B graphically illustrate data showing that AIBP deficiency does not affect mitochondrial dynamics- and oxidative stress- related gene expression in the retina, and illustrates Opal, Nfn2, and Drpl, as well as Sirt3 and Sod2 gene expression was assessed by quantitative PCR analysis in WT and Apoal bp~ ~ retina:

FIG. 12A graphically illustrates Opal, Mfn2, and Drpl gene expression;

FIG. 12B graphically illustrates Sirt3 and Sod2 (B) gene expression; as described in detail in Example 1, below.

FIG. 13A-D illustrate images showing that AIBP deficiency induces abnormal structure of mitochondria and ER, and mitophagosome formation in RGC soma, where serial slice images through the tomographic volume from WT and Apoal bp~ ~ RGC somas are shown:

FIG. 13 A illustrates serial slice images from WT Muller glia endfeet showing a long tubular form of mitochondria with normal structure of ER strands (arrowheads);

FIG. 13B illustrates serial slice images from Apoal bp~ ~ RGC soma to point to the dark outer membrane onion-like swirls (arrows) and dilated ER strands (arrowheads);

FIG. 13C illustrates serial slice images from Apoal bp~ ~ RGC soma showing a ring-shaped mitochondrion (arrow);

FIG. 13D illustrates serial slice images from Apoal bp~ ~ RGC soma showing two ongoing autophagosome formation; as described in detail in Example 1, below.

FIG. 14 illustrates an exemplary list of antibodies (also called “supplementary table 1”;

FIG. 15 illustrates an exemplary list of nucleic acid primers (also called

“supplementary table 2”:

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

In alternative embodiments, provided are methods for: treating, ameliorating, protecting against, reversing or decreasing the severity or duration of a glaucoma or neuroinflammation in an eye during glaucomatous neurodegeneration; treating, ameliorating, protecting against, reversing or decreasing the severity or duration of mitochondrial dysfunction in retinal ganglion cells (RGCs) or Muller glia during glaucomatous neurodegeneration in the eye; and/or decreasing or slowing the rate of induced RGC death in response to elevated intraocular pressure (IOP). In alternative embodiments, methods as provided herein treat glaucoma by intraocular or intravitreal administration of ApoA-I Binding Protein (APOA1BP, AIBP, or ALBP) protein or nucleic acids encoding AIBP.

Definitions

As used herein unless explicitly stated or indicated otherwise by context, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

As used herein, unless specifically stated or indicated otherwise by context, the term “or” is understood to be inclusive and covers both “or” and “and”.

As used herein, unless specifically stated or indicated otherwise by context, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About (use of the term “about”) can be understood as within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12% 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

As used herein, unless specifically stated or indicated otherwise by context, the terms “substantially all”, “substantially most of’, “substantially all of’ or “majority of’ encompass at least about 90%, 95%, 97%, 98%, 99% or 99.5%, or more of a referenced amount of a composition.

Embodiments In some embodiments, provided are methods for:

- treating, ameliorating, protecting against, reversing or decreasing the severity or duration of neuroinflammation in an eye during glaucomatous neurodegeneration,

- decreasing or slowing the rate of induced RGC death in response to elevated intraocular pressure (IOP), wherein the method comprises the step of: administering a pharmaceutically acceptable formulation to a subject in need thereof wherein the pharmaceutically acceptable formulation is comprised of:

(3) an ApoA-I Binding Protein polypeptide-inducing compound or composition.

In some of those embodiments:

- the APOAIBP-stimulating compound or composition increases or stimulates (activates) the activity of an APOA1BP promoter or transcriptional regulatory sequence or motif;

- the nucleic acid sequence that expresses or encodes the APOA1BP polypeptide or the related protein having the APOA1BP polypeptide activity is contained within an expression vehicle, vector, recombinant virus, or equivalent thereof, wherein in some instances; or -the vector or virus for expressing the APOA1BP polypeptide or related protein is or comprises an adenovirus vector or an adeno-associated virus (AAV) vector, a retrovirus, a lentiviral vector, a herpes simplex virus, a human immunodeficiency virus (HIV), or a synthetic vector, wherein in some aspects of the invention the AAV vector is or is comprised of: an adeno-associated virus (AAV), an AAV serotype or variant AAV5, AAV6, AAV8 or AAV9, AAV-DJ or AAV- DJ/8™ (Cell Biolabs, Inc., San Diego, CA), a rhesus-derived AAV vector, wherein optionally the rhesus-derived AAV vector is AAVrh.10hCLN2, or an AAV capsid mutant, or an AAV hybrid serotype.

In other embodiments, the AAV is engineered so as to increase efficiency in targeting a specific cell type that is non -permissive to a wild-type (wt) AAV and/or to improve its efficacy in infecting only the cell type of interest. In some of those embodiments the AAV vector is retargeted or engineered as an AAV hybrid serotype by one or more modifications including: 1) a transcapsidation, 2) adsorption of a bispecific antibody to a capsid surface, 3) engineering a mosaic capsid, and/or 4) engineering a chimeric capsid;

In still other aspects as provided herein:

- the ApoA-I Binding Protein (APOA1BP) polypeptide is a mammalian APOA1BP polypeptide or a polypeptide composition comprised of a mammalian APOA1BP polypeptide, wherein the polypeptide composition has, or is capable of providing, a mammalian APOA1BP polypeptide activity,

- the ApoA-I Binding Protein (APOA1BP) polypeptide is a human APOA1BP polypeptide or a polypeptide composition comprised of the APOA1BP polypeptide, wherein the polypeptide composition has, or is capable of providing, a human APOA1BP polypeptide activity,

- the subject is a human, or the subject is a mammal, including a non-human primate,

- the APOA1BP polypeptide is a recombinant APOA1BP polypeptide or a polypeptide composition comprised of the recombinant APOA1BP polypeptide wherein the polypeptide composition has, or is capable of providing, an APOA1BP polypeptide activity, or

- the APOA1BP polypeptide is a synthetic APOA1BP polypeptide or a polypeptide composition comprised of the synthetic APOA1BP polypeptide, wherein the polypeptide composition has, or is capable of providing, an APOA1BP polypeptide activity.

In any one of the above embodiments the pharmaceutically acceptable formulation is for intraocular or intravitreal administration, or administration by intrathecal injection, or the pharmaceutically acceptable formulation is for intravenous (IV) administration.

In any one of above embodiments the APOA1BP polypeptide or the polypeptide composition comprised of the APOA1BP polypeptide is within or on a particle, such as a nanoparticle, a micelle, a liposome, a lipoplex, a polymersome, a polyplex or a dendrimer. In some of those embodiments the particle is further comprised of a cell or CNS penetrating moiety or peptide or a CNS targeting moiety or peptide.

In any one of the above embodiments the polypeptide comprised of the APOA1BP polypeptide further comprises a cell or CNS penetrating moiety or peptide or a CNS targeting moiety or peptide.

In any one of the above embodiments, formulation of the APOA1BP polypeptide or the polypeptide comprised of the APOA1BP polypeptide is in the form of a liquid, a sterile injectable solution, or an implant, typically an intraocular implant.

In alternative embodiments, provided are uses of a pharmaceutically acceptable formulation comprising:

(1) an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP) polypeptide compound or composition, or a compound that increases expression or activity of, or a nucleic acid sequence that encodes the APOA1BP polypeptide related protein having an APOA1BP activity, or an APOAIBP-stimulating compound or composition thereof;

(2) the formulation or pharmaceutical composition of (1), wherein the compound that increases expression or activity of, or encodes, a APOA1BP polypeptide is a nucleic acid that expresses or encodes a APOA1BP polypeptide or a polypeptide having a APOA1BP polypeptide activity; or (3) an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP)-inducing compound or composition, in the manufacture of a medicament for:

Formulations and pharmaceutical compositions

In alternative embodiments, provided are pharmaceutical formulations or compositions comprising nucleic acids and polypeptides for practicing methods and uses as provided herein to treat, ameliorate, protect against, reverse or decrease the severity or duration of glaucoma, or neuroinflammation in the eye during glaucomatous neurodegeneration, the methods comprising upregulating or increasing the expression of ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP) in the eye. In alternative embodiments, provided are pharmaceutical formulations or compositions for use in in vivo, in vitro or ex vivo methods to treat, prevent, reverse and/or ameliorate glaucoma. In alternative embodiments, pharmaceutical compositions and formulations used to practice methods and uses as provided herein comprise APOA1BP nucleic acids and polypeptides or result in an increase in expression or activity of APOA1BP nucleic acids and polypeptides are administered to an individual in need thereof in an amount sufficient to treat, prevent, reverse and/or ameliorate, for example, a glaucoma. In alternative embodiments, pharmaceutical compositions and formulations used to practice methods and uses as provided herein comprise APOA1BP nucleic acids and polypeptides or result in an increase in expression or activity of APOA1BP nucleic acids and polypeptides are administered to an individual in need thereof in an amount sufficient to treat, ameliorate, protect against, reverse or decrease the severity or duration of glaucoma, or neuroinflammation in the eye during glaucomatous neurodegeneration,.

In alternative embodiments, the pharmaceutical compositions used to practice methods and uses as provided herein can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally, or intravitreal injection. The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, for example, the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co., Easton PA (“Remington’s”).

For example, in alternative embodiments, these compositions used to practice methods and uses as provided herein are formulated in a buffer, in a saline solution, in a powder, an emulsion, in a vesicle, in a liposome, in a nanoparticle, in a nanolipoparticle and the like. In alternative embodiments, the compositions can be formulated in any way and can be applied in a variety of concentrations and forms depending on the desired in vivo, in vitro or ex vivo conditions, a desired in vivo, in vitro or ex vivo method of administration and the like. Details on techniques for in vivo, in vitro or ex vivo formulations and administrations are well described in the scientific and patent literature. Formulations and/or carriers used to practice methods or uses as provided herein can be in forms such as tablets, pills, powders, capsules, liquids, gels, syrups, slurries, suspensions, etc., suitable for in vivo, in vitro or ex vivo applications.

In alternative embodiments, formulations and pharmaceutical compositions used to practice methods and uses as provided herein can comprise a solution of compositions (which include peptidomimetics, racemic mixtures or racemates, isomers, stereoisomers, derivatives and/or analogs of compounds) disposed in or dissolved in a pharmaceutically acceptable carrier, for example, acceptable vehicles and solvents that can be employed include water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose any fixed oil can be employed including synthetic mono- or diglycerides, or fatty acids such as oleic acid. In one embodiment, solutions and formulations used to practice methods and uses as provided herein are sterile and can be manufactured to be generally free of undesirable matter. In one embodiment, these solutions and formulations are sterilized by conventional, well known sterilization techniques.

The solutions and formulations used to practice methods and uses as provided herein can comprise auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and can be selected primarily based on fluid volumes, viscosities and the like, in accordance with the particular mode of in vivo, in vitro or ex vivo administration selected and the desired results.

The compositions and formulations used to practice methods and uses as provided herein can be delivered by the use of liposomes. By using liposomes, particularly where the liposome surface carries ligands specific for target cells (for example, an injured or diseased neuronal cell or CNS tissue), or are otherwise preferentially directed to a specific tissue or organ type, one can focus the delivery of the active agent into a target cells in an in vivo, in vitro or ex vivo application. Nanoparticles, Nanolipoparticles and Liposomes

Also provided are nanoparticles, nanolipoparticles, vesicles and liposomal membranes comprising compounds used to practice methods and uses as provided herein, for example, to deliver compositions comprising APOA1BP nucleic acids and polypeptides in vivo, for example, to the eye. In alternative embodiments, these compositions are designed to target specific molecules, including biologic molecules, such as polypeptides, including cell surface polypeptides, for example, for targeting a desired cell type or organ, for example, a nerve cell or the CNS, and the like.

Provided are multilayered liposomes comprising compounds used to practice methods and uses as provided herein, for example, as described in Park, et al., U.S. Pat. Pub. No. 20070082042. The multilayered liposomes can be prepared using a mixture of oil-phase components comprising squalane, sterols, ceramides, neutral lipids or oils, fatty acids and lecithins, to about 200 to 5000 nm in particle size, to entrap a composition used to practice methods and uses as provided herein.

Liposomes can be made using any method, for example, as described in Park, et al., U.S. Pat. Pub. No. 20070042031, including method of producing a liposome by encapsulating an active agent (for example, APOA1BP nucleic acids and polypeptides), the method comprising providing an aqueous solution in a first reservoir; providing an organic lipid solution in a second reservoir, and then mixing the aqueous solution with the organic lipid solution in a first mixing region to produce a liposome solution, where the organic lipid solution mixes with the aqueous solution to substantially instantaneously produce a liposome encapsulating the active agent; and immediately then mixing the liposome solution with a buffer solution to produce a diluted liposome solution. In one embodiment, liposome compositions used to practice methods and uses as provided herein comprise a substituted ammonium and/or polyanions, for example, for targeting delivery of a compound (for example, a APOA1BP nucleic acid and polypeptide) to a desired cell type (for example, a retinal cell), as described for example, in U.S. Pat. Pub. No. 20070110798.

Provided are nanoparticles comprising compounds (for example, APOA1BP nucleic acids and polypeptides used to practice methods provided herein) in the form of active agent-containing nanoparticles (for example, a secondary nanoparticle), as described, for example, in U.S. Pat. Pub. No. 20070077286. In one embodiment, provided are nanoparticles comprising a fat-soluble active agent or a fat-solubilized water-soluble active agent to act with a bivalent or trivalent metal salt.

In one embodiment, solid lipid suspensions can be used to formulate and to deliver compositions used to practice methods and uses as provided herein to mammalian cells in vivo, for example, to the CNS, as described, for example, in U.S. Pat. Pub. No. 20050136121.

Delivery vehicle modifications and modification of AIBP

In alternative embodiments, AIBP peptides or polypeptides, or AIBP- comprising nanoparticles, liposomes and the like (for example, comprising or having contained therein APOA1BP nucleic acids or polypeptides used to practice methods provided herein) are modified to facilitate intravitreal injections. For example, in alternative embodiments, AIBP peptides or polypeptides, or AIBP-comprising nanoparticles, liposomes and the like, are engineered to comprise a moiety that allows the AIBP peptides or polypeptides, or AIBP-comprising nanoparticles, liposomes and the like, to bind to a receptor or cell membrane structure that facilitates delivery into the eye, CNS or brain, for example, where the moiety can comprise a mannose-6- phosphate receptor, a melanotransferrin receptor, a LRP receptor or any other receptor that is ubiquitously expressed on the surface of any CNS or brain cell. For example, conjugation of mannose-6-phosphate moieties allows the AIBP peptides or polypeptides, or AIBP-comprising nanoparticles, liposomes and the like, to be taken up by a CNS cell that expresses a mannose-6-phosphate receptor. In alternative embodiments, any protocol or modification of the AIBP peptides or polypeptides, or AIBP-comprising nanoparticles, liposomes and the like, that facilitates entry or delivery into the CNS or brain in vivo can be used, for example, as described in USPN 9,089,566.

Delivery cells and delivery vehicles

In alternative embodiments, any delivery vehicle can be used to practice the methods or uses as provided herein, for example, to deliver compositions (for example, APOA1BP nucleic acids and/or polypeptides) into an eye in vivo. For example, delivery vehicles comprising polycations, cationic polymers and/or cationic peptides, such as polyethyleneimine derivatives, can be used for example as described, for example, in U.S. Pat. Pub. No. 20060083737. In one embodiment, a delivery vehicle is a transduced cell engineered to express or overexpress and then secrete an endogenous or exogenous AIBP.

In one embodiment, a dried polypeptide-surfactant complex is used to formulate a composition used to practice methods as provided herein, for example as described, for example, in U.S. Pat. Pub. No. 20040151766.

In one embodiment, a composition used to practice methods and uses as provided herein can be applied to cells using vehicles with cell membrane-permeant peptide conjugates, for example, as described in U.S. Patent Nos. 7,306,783; 6,589,503. In one aspect, the composition to be delivered is conjugated to a cell membrane-permeant peptide. In one embodiment, the composition to be delivered and/or the delivery vehicle are conjugated to a transport-mediating peptide, for example, as described in U.S. Patent No. 5,846,743, describing transport-mediating peptides that are highly basic and bind to poly-phosphoinositides.

In one embodiment, cells that will be subsequently delivered into an eye are transfected or transduced with an AIBP-expressing nucleic acid, for example, a vector, for example, by electro-permeabilization, which can be used as a primary or adjunctive means to deliver the composition to a cell, for example, using any electroporation system as described for example in U.S. Patent Nos. 7,109,034; 6,261,815; 5,874,268.

In alternative embodiments, APOA1BP nucleic acids used to practice embodiments as provided herein comprise or are comprised of human APOA1BP cDNA sequence:

GGGCCGGGCCGGGCCGGGGGCGCGCGCTCTGCGAGCTGGATGTCC

AGGCTGCGGGCGCTGCTGGGCCTCGGGCTGCTGGTTGCGGGCTCGC GCGTGCCGCGGATCAAAAGCCAGACCATCGCCTGTCGCTCGGGAC CCACCTGGTGGGGACCGCAGCGGCTGAACTCGGGTGGCCGCTGGG ACTCAGAGGTCATGGCGAGCACGGTGGTGAAGTACCTGAGCCAGG AGGAGGCCCAGGCCGTGGACCAGGAGCTATTTAACGAATACCAGT TCAGCGTGGACCAACTTATGGAACTGGCCGGGCTGAGCTGTGCTAC AGCCATCGCCAAGGCATATCCCCCCACGTCCATGTCCAGGAGCCCC CCTACTGTCCTGGTCATCTGTGGCCCGGGGAATAATGGAGGAGATG GTCTGGTCTGTGCTCGACACCTCAAACTCTTTGGCTACGAGCCAAC CATCTATTACCCCAAAAGGCCTAACAAGCCCCTCTTCACTGCATTG GTGACCCAGTGTCAGAAAATGGACATCCCTTTCCTTGGGGAAATGC CCGCAGAGCCCATGACGATTGATGAACTGTATGAGCTGGTGGTGGA TGCCATCTTTGGCTTCAGCTTCAAGGGCGATGTTCGGGAACCGTTC CACAGCATCCTGAGTGTCCTGAAGGGACTCACTGTGCCCATTGCCA GCATCGACATTCCCTCAGGATGGGACGTGGAGAAGGGAAATGCTG GAGGGATCCAGCCAGACTTGCTCATATCCCTCACAGCCCCCAAAAA ATCTGCAACCCAGTTTACCGGTCGCTACCATTACCTGGGGGGTCGT TTTGTGCCACCTGCTCTGGAGAAGAAGTACCAGCTGAACCTGCCAC CCTACCCTGACACCGAGTGTGTCTATCGTCTGCAGTGAGGGAAGGT GGGTGGGTATTCTTCCCAATAAAGACTTAGAGCCCCTCTCTTCCAG AACTGTGGATTCCTGGGAGCTCCTCTGGCAATAAAAGTCAGTGAAT GGTGGAAGTCAGAGACCAACCCTGGGGATTGGGTGCCATCTCTCTA GGGGTAACACAAAGGGCAAGAGGTTGCTATGGTATTTGGAAACAA

TGAAAATGGACTGTTAGATGCCAA (SEQ ID NO: 17)

In alternative embodiments, human APOA1BP polypeptides used to practice embodiments as provided herein comprise or are comprised of the amino acid sequence:

MSRLRALLGLGLLVAGSRVPRIKSQTIACRSGPTWWGPQRLNSGGRW DSEVMASTVVKYLSQEEAQAVDQELFNEYQFSVDQLMELAGLSCATA lAKAYPPTSMSRSPPTVLVICGPGNNGGDGLVCARHLKLFGYEPTIYYP KRPNKPLFTALVTQCQKMDIPFLGEMPAEPMTIDELYELVVDAIFGFSF KGDVREPFHSILSVLKGLTVPIASIDIPSGWDVEKGNAGGIQPDLLISLT APKKSATQFTGRYHYLGGRFVPPALEKKYQLNLPPYPDTECVYRLQ (SEQ ID NO: 18) In vivo delivery of AIBP-encoding nucleic

In alternative embodiments, provided are compositions and methods for delivering nucleic acids encoding AIBP peptides or polypeptides, or nucleic acids encoding peptides or polypeptides having AIBP activity, or vectors or recombinant viruses having contained therein these nucleic acids. In alternative embodiments, the nucleic acids, vectors or recombinant viruses are designed for in vivo or CNS delivery and expression.

In alternative embodiments, provided are compositions and methods for the delivery and controlled expression of an AIBP-encoding nucleic acid or gene, or an expression vehicle (for example, vector, recombinant virus, and the like) comprising (having contained therein) an AIBP encoding nucleic acid or gene, that results in an AIBP protein being released into the bloodstream or general circulation where it can have a beneficial effect on in the body, for example, such as the CNS, brain or other targets.

In alternative embodiments, the provided are methods for being able to turn on and turn off AIBP-expressing nucleic acid or gene expression easily and efficiently for tailored treatments and insurance of optimal safety.

In alternative embodiments, AIBP protein or proteins expressed by the AIBP- expressing nucleic acid(s) or gene(s) have a beneficial or favorable effects (for example, therapeutic or prophylactic) on a tissue or an organ, for example, the eye, or other targets, even though secreted into the blood or general circulation at a distance (for example, anatomically remote) from their site or sites of action.

In alternative embodiments, provided are expression vehicles, vectors, recombinant viruses and the like for in vivo expression of an AIBP-encoding nucleic acid or gene to practice the methods as provide herein. In alternative embodiments, the AIBP-encoding nucleic acids (such as RNA or DNA), expression vehicles, vectors, recombinant viruses and the like expressing the an AIBP nucleic acid or gene can be delivered by intravitreal injection or intramuscular (IM) injection (using for example, AIBP-encoding RNA in liposomes), by intravenous (IV) injection, by subcutaneous injection, by inhalation, by a biolistic particle delivery system (for example, a so-called “gene gun”), and the like, for example, as an outpatient, for example, during an office visit. In alternative embodiments, this “peripheral” mode of delivery, for example, expression vehicles, vectors, recombinant viruses and the like injected intravitreal, IM or IV, can circumvent problems encountered when genes or nucleic acids are expressed directly in an organ (for example, an eye, the brain or into the CNS) itself. Sustained secretion of an AIBP in the bloodstream or general circulation also circumvents the difficulties and expense of administering proteins by infusion.

In alternative embodiments a recombinant virus (for example, a long-term virus or viral vector), or a vector, or an expression vector, and the like, can be injected, for example, in a systemic vein (for example, IV), or by intravitreal, intramuscular (IM) injection, by inhalation, or by a biolistic particle delivery system (for example, a so-called “gene gun”), for example, as an outpatient, for example, in a physician's office. In alternative embodiments, days or weeks later (for example, four weeks later), the individual, patient or subject is administered (for example, inhales, is injected or swallows), a chemical or pharmaceutical that induces expression of the AIBP-expressing nucleic acids or genes; for example, an oral antibiotic (for example, doxycycline or rapamycin) is administered once daily (or more or less often), which will activate the expression of the gene. In alternative embodiments, after the “activation”, or inducement of expression (for example, by an inducible promoter) of the nucleic acid or gene, an AIBP protein is synthesized and released into the subject's circulation (for example, into the blood), and subsequently has favorable physiological effects, for example, therapeutic or prophylactic, that benefit the individual or patient (for example, benefit heart, kidney or lung function). When the physician or subject desires discontinuation of the AIBP treatment, the subject simply stops taking the activating chemical or pharmaceutical, for example, antibiotic.

Alternative embodiments comprise use of "expression cassettes" comprising or having contained therein a nucleotide sequence used to practice methods provided herein, for example, an AIBP-expressing nucleic acid, which can be capable of affecting expression of the nucleic acid, for example, as a structural gene or a transcript (for example, encoding an AIBP protein) in a host compatible with such sequences. Expression cassettes can include at least a promoter operably linked with the polypeptide coding sequence or inhibitory sequence; and, in one aspect, with other sequences, for example, transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used, for example, enhancers. In alternative aspects, expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant “naked DNA” vector, and the like. In alternative aspects, a "vector" can comprise a nucleic acid that can infect, transfect, transiently or permanently transduce a cell. In alternative aspects, a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. In alternative aspects, vectors can comprise viral or bacterial nucleic acids and/or proteins, and/or membranes (for example, a cell membrane, a viral lipid envelope, etc.). In alternative aspects, vectors can include, but are not limited to replicons (for example, RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (for example, plasmids, viruses, and the like, see, for example, U.S. Patent No. 5,217,879), and can include both the expression and non-expression plasmids. In alternative aspects, a vector can be stably replicated by the cells during mitosis as an autonomous structure, or can be incorporated within the host's genome.

In alternative aspects, “promoters” include all sequences capable of driving transcription of a coding sequence in a cell, for example, a mammalian cell such as a retinal cell. Promoters used in the constructs provided herein include c/.s-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a nucleic acid, for example, an AIBP-encoding nucleic acid. For example, a promoter can be a exacting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3’ untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) transcription.

In alternative embodiments, “constitutive” promoters can be those that drive expression continuously under most environmental conditions and states of development or cell differentiation. In alternative embodiments, “inducible” or “regulatable” promoters can direct expression of a nucleic acid, for example, an AIBP-encoding nucleic acid, under the influence of environmental conditions, administered chemical agents, or developmental conditions. Gene Therapy and Gene Delivery Vehicles In alternative embodiments, methods of the invention comprise use of nucleic acid (for example, an AIBP gene or any AIBP-encoding nucleic acid) delivery systems to deliver a payload of the nucleic acid or gene, or AIBP-expressing nucleic acid, transcript or message, to a cell or cells in vitro, ex vivo, or in vivo, for example, as gene therapy delivery vehicles.

In alternative embodiments, expression vehicle, vector, recombinant virus, or equivalents used to practice methods provided herein are or comprise: an adeno- associated virus (AAV), a lentiviral vector or an adenovirus vector; an AAV serotype AAV5, AAV6, AAV8 or AAV9; a rhesus-derived AAV, or the rhesus-derived AAV

AAVrh.l0hCLN2; an organ-tropic AAV, or a neurotropic AAV; and/or an AAV capsid mutant or AAV hybrid serotype. In alternative embodiments, the AAV is engineered to increase efficiency in targeting a specific cell type that is non- permissive to a wild type (wt) AAV and/or to improve efficacy in infecting only a cell type of interest. In alternative embodiments, the hybrid AAV is retargeted or engineered as a hybrid serotype by one or more modifications comprising: 1) a transcapsidation, 2) adsorption of a bi-specific antibody to a capsid surface, 3) engineering a mosaic capsid, and/or 4) engineering a chimeric capsid. It is well known in the art how to engineer an adeno-associated virus (AAV) capsid in order to increase efficiency in targeting specific cell types that are non-permissive to wild type (wt) viruses and to improve efficacy in infecting only the cell type of interest; see for example, Wu et al., Mol. Ther. 2006 Sep; 14(3):316-27. Epub 2006 Jul 7; Choi, et al., Curr. Gene Ther. 2005 Jun;5(3):299-310.

For example, in alternative embodiments, serotypes AAV-8, AAV-9, AAV-DJ or AAV-DJ/8™ (Cell Biolabs, Inc., San Diego, CA), which have increased uptake in brain tissue in vivo, are used to deliver an AIBP-encoding nucleic acid payload for expression in the CNS. In alternative embodiments, the following serotypes, or variants thereof, are used for targeting a specific tissue: Tissue Optimal Serotype

CNS AAVI, AAV2, AAV4, AAV5, A.AV8, A V9

Photoreceptor Cells AAV2, A.AV5, AAV8

RPE (Retinal Pigment . . . _{A r} . . . , „ _A > . ...

... . AAV I, AAV2. AAV4, AAVx AA 8

Epithelium) Skeletal Muscle AAV1, AAV6, AAV7, AAV8, AAV9 In alternative embodiments, the rhesus-derived AAV AAVrh.l0hCLN2 or equivalents thereof can be used, wherein the rhesus-derived AAV may not be inhibited by any pre-existing immunity in a human; see for example, Sondhi, et al., Hum Gene Ther. Methods. 2012 Oct;23(5):324-35, Epub 2012 Nov 6; Sondhi, et al., Hum Gene Ther. Methods. 2012 Oct 17; teaching that direct administration of AAVrh.l0hCLN2 to the CNS of rats and non-human primates at doses scalable to humans has an acceptable safety profile and mediates significant payload expression in the CNS.

Because adeno-associated viruses (AAVs) are common infective agents of primates, and as such, healthy primates carry a large pool of AAV-specific neutralizing antibodies (NAbs) which inhibit AAV-mediated gene transfer therapeutic strategies, methods provided herein can comprise screening of patient candidates for AAV-specific NAbs prior to treatment, especially with the frequently used AAV8 capsid component, to facilitate individualized treatment design and enhance therapeutic efficacy; see, for example, Sun, et al., J. Immunol. Methods. 2013 Jan 31 ; 387( 1 -2) : 114-20, Epub 2012 Oct 11.

In alternative embodiments, the AIBP gene or other AIBP-encoding nucleic acid as delivered in vivo using methods as provided herein can be in the form of, or comprise, an RNA, for example, mRNA, which can be formulated in a lipid formulation or a liposome and injected for example intramuscularly (IM), for example using formulations and methods as described in U.S. patent application no. US 20210046173 Al, which describes delivering to a subject (for example, via intramuscular administration) the AIBP gene or other AIBP-encoding nucleic acid that comprises a RNA (for example, mRNA) that comprises an open reading frame (ORF) that comprises (or consists of, or consists essentially of) or encodes for the AIBP gene or other AIBP-encoding nucleic acid; wherein optionally the RNA (or the DNA-carrying expression vehicle) is formulated in a liposome, or a lipid nanoparticle (LNP), or nanoliposome, that comprises: non-cationic lipids comprise a mixture of cholesterol and DSPC, or a PEG-lipid, or PEG-modified lipid, or LNP, or an ionizable cationic lipid; or a mixture of (13Z,16Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien- 1 -amine, cholesterol, DSPC, and PEG-2000 DMG. In alternative embodiments, the PEG-lipid is 1,2-Dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1, 2-dimyristyloxlpropyl-3-amine (PEG-c-DMA), or, the PEG- lipid is PEG coupled to dimyristoylglycerol (PEG-DMG). In alternative embodiments, the LNP comprises 20-99.8 mole % ionizable cationic lipids, 0.1-65 mole % non-cationic lipids, and 0.1-20 mole % PEG-lipid. In alternative embodiments, the LNP comprises an ionizable cationic lipid selected from the group consisting of (2S)-l-({6-[(3))-cholest-5-en-3-yloxy]hexyl}oxy)-N,N-dimethyl-3-[(9 Z)-octadec-9-en-l-yloxy]propan-2-amine; (13Z,16Z)-N,N-dimethyl-3-nonyldocosa- 13,16-dien- 1 -amine; and N,N-dimethyl- 1 -[( 1 S,2R)-2-octylcyclopropyl]heptadecan-8- amine; or a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing. In alternative embodiments, the PEG modified lipid comprises a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG- modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In alternative embodiments, the ionizable cationic lipid comprises: 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin- MC3-DMA), di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy) heptadecanedioate (L319), (13Z, 16Z)-N,N-dimethyl-3 -nonyldocosa- 13 , 16-dien- 1 - amine, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-l-amine, and N,N- dimethyl-l-[(lS,2R)-2-octylcyclopropyl]heptadecan-8-amine. In one embodiment, the lipid is (13Z,16Z)-N,N-dimethyl-3 -nonyldocosa- 13, 16-dien-l -amine or N,N- dimethyl-l-[(lS,2R)-2-octylcyclopropyl]heptadecan-8-amine, each of which are described in PCT/US2011/052328, the entire contents of which are hereby incorporated by reference. In some embodiments, a non-cationic lipid of the disclosure comprises l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), 1,2- dioleoyl-sn-glycero-3 -phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-diundecanoyl-sn-gly cero-phosphocholine (DUPC), 1- palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), 1,2-di-O-octadecenyl-sn- glycero-3 -phosphocholine (18:0 Diether PC), l-oleoyl-2 cholesterylhemisuccinoyl-sn- glycero-3 -phosphocholine (OChemsPC), 1 -hexadecyl-sn-glycero-3 -phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3 -phosphocholine, 1,2-diarachidonoyl- sn-glycero-3 -phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3 -phosphocholine,

1.2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn- glycero-3 -phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3 -phosphoethanolamine,

1.2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero- 3 -phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine,

1.2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG), sphingomyelin, or mixtures thereof.

Dosaging

The pharmaceutical compositions and formulations used to practice methods and uses as provided herein can be administered for prophylactic and/or therapeutic treatments, for example, to treat, ameliorate, protect against, reverse or decrease the severity or duration of glaucoma, or neuroinflammation in an eye during glaucomatous neurodegeneration. In therapeutic applications, compositions are administered to a subject already suffering from a disease, condition, infection or defect in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the disease, condition, infection or disease and its complications (a “therapeutically effective amount”), including for example, glaucoma. For example, in alternative embodiments, APOA1BP nucleic acid- or polypeptide- comprising pharmaceutical compositions and formulations as provided herein are administered to an individual in need thereof in an amount sufficient to treat, ameliorate, protect against, reverse or decrease the severity or duration of glaucoma, or neuroinflammation in an eye during glaucomatous neurodegeneration,.

The amount of pharmaceutical composition adequate to accomplish this is defined as a "therapeutically effective dose." The dosage schedule and amounts effective for this use, i.e., the “dosing regimen,” will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient’s physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.

In alternative embodiments, viral vectors such as adenovirus or AAV vectors are administered to an individual in need therein, and in alternative embodiment the dosage administered to a human comprises: a dose of about 2 x io¹² vector genomes per kg body weight (vg/kg), or between about 10¹⁰ and 10¹⁴ vector genomes per kg body weight (vg/kg), or about 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, or more vg/kg, which can be administered as a single dosage or in multiple dosages, as needed. In alternative embodiments, these dosages are administered intravitreally, orally, IM, IV, or intrathecally. In alternative embodiments, the vectors are delivered as formulations or pharmaceutical preparations, for example, where the vectors are contained in a nanoparticle, a particle, a micelle or a liposome or lipoplex, a polymersome, a polyplex or a dendrimer. In alternative embodiments, these dosages are administered once a day, once a week, or any variation thereof as needed to maintain in vivo expression levels of AIBP, which can be monitored by measuring actually expression of AIBP or by monitoring of therapeutic effect, for example, to treat, ameliorate, protect against, reverse or decrease the severity or duration of glaucoma, or neuroinflammation in an eye during glaucomatous neurodegeneration,. The dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents’ rate of absorption, bioavailability, metabolism, clearance, and the like (see, for example, Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51 :337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84: 1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24: 103-108; the latest Remington’s, supra). The state of the art allows the clinician to determine the dosage regimen for each individual patient, active agent and disease or condition treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, i.e., dose schedule and dosage levels, administered practicing the methods as provided herein are correct and appropriate.

Single or multiple administrations of formulations can be given depending on the dosage and frequency as required and tolerated by the patient. The formulations should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate a conditions, diseases or symptoms as described herein. For example, alternative exemplary pharmaceutical formulations for oral administration of compositions used to practice methods as provided herein are in a daily amount of between about 0.1 to 0.5 to about 20, 50, 100 or 1000 or more z/g per kilogram of body weight per day. In an alternative embodiment, dosages are from about 1 mg to about 4 mg per kg of body weight per patient per day are used. Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ. Substantially higher dosages can be used in topical or oral administration or administering by powders, spray or inhalation. Actual methods for preparing parenterally or non-parenterally administrable formulations will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's, supra.

The methods as provided herein can further comprise co-administration with other drugs or pharmaceuticals, for example, compositions for treating any neurological or neuromuscular disease, condition, infection or injury, including related inflammatory and autoimmune diseases and conditions, and the like. For example, the methods and/or compositions and formulations as provided herein can be co-formulated with and/or co-administered with, fluids, antibiotics, cytokines, immunoregulatory agents, anti-inflammatory agents, pain alleviating compounds, complement activating agents, such as peptides or proteins comprising collagen-like domains or fibrinogen-like domains (for example, a ficolin), carbohydrate-binding domains, and the like and combinations thereof.

Bioisosteres of compounds

In alternative embodiment, also provided are bioisosteres of compounds used to practice the methods provided herein, for example, polypeptides having a APOA1BP activity. Bioisosteres used to practice methods as provided herein include bioisosteres of, for example, APOA1BP nucleic acids and polypeptides, which in alternative embodiments can comprise one or more substituent and/or group replacements with a substituent and/or group having substantially similar physical or chemical properties which produce substantially similar biological properties to compounds used to practice methods or uses as provided herein. In one embodiment, the purpose of exchanging one bioisostere for another is to enhance the desired biological or physical properties of a compound without making significant changes in chemical structures.

For example, in one embodiment, one or more hydrogen atom(s) is replaced with one or more fluorine atom(s), for example, at a site of metabolic oxidation; this may prevent metabolism (catabolism) from taking place. Because the fluorine atom is similar in size to the hydrogen atom the overall topology of the molecule is not significantly affected, leaving the desired biological activity unaffected. However, with a blocked pathway for metabolism, the molecule may have a longer half-life or be less toxic, and the like.

Products of Manufacture, Kits

Also provided are products of manufacture such as implants or pumps, kits and pharmaceuticals for practicing the methods as provided herein. In alternative embodiments, provided are products of manufacture, kits and/or pharmaceuticals comprising all the components needed to practice a method as provided herein. In alternative embodiments, kits also comprise instructions for practicing a method as provided herein.

The following numbered embodiments are illustrative of the invention and are not intended to limit the invention in any manner.

1. A method for treating, ameliorating, protecting against, reversing or decreasing the severity or duration of a glaucoma, wherein optionally the glaucoma is open-angle glaucoma or closed angle glaucoma, treating, ameliorating, protecting against, reversing or decreasing the severity or duration of neuroinflammation in an eye during glaucomatous neurodegeneration, treating, ameliorating, protecting against, reversing or decreasing the severity or duration of mitochondrial dysfunction in retinal ganglion cells (RGCs), microglia or Muller glia during glaucomatous neurodegeneration in an eye, or decreasing or slowing the rate of induced RGC death in response to elevated intraocular pressure (IOP), wherein the method comprises the step of: administering a pharmaceutically acceptable formulation to a subject in need thereof, wherein the pharmaceutically acceptable formulation is comprised of;

(1) a polypeptide composition, wherein the polypeptide composition is, or is comprised, of an ApoA-I Binding Protein) polypeptide, wherein the polypeptide composition has, or is capable of providing, an ApoA-I Binding Protein polypeptide activity, or

(2) a nucleic acid composition that increases expression or activity of, or encodes for, a polypeptide composition, wherein the polypeptide composition is or is comprised of an ApoA-I Binding Protein polypeptide, wherein the polypeptide composition has, or is capable of providing, an ApoA-I Binding Protein polypeptide activity, or (3) an ApoA-I Binding Protein polypeptide-inducing compound or composition. The method of embodiment 1, wherein the ApoA-I Binding Protein polypeptide- inducing compound or composition increases, stimulates, or activates the activity of an APOA1BP promoter or transcriptional regulatory sequence or motif for expression of the polypeptide composition.

3. The method of embodiment 1, wherein the nucleic acid that expresses or encodes for the polypeptide composition is contained within an expression vehicle, vector, recombinant virus, or equivalent thereof.

4. The method of embodiment 3, wherein the vector or virus is, or comprised of an adenovirus vector or an adeno-associated virus (AAV) vector, a retrovirus, a lentiviral vector, a herpes simplex virus, a human immunodeficiency virus (HIV), or a synthetic vector.

5. The method of embodiment 4, wherein the AAV vector is or is comprised of; an adeno-associated virus (AAV), an AAV serotype, an AAV variant, wherein the AAV variant is AAV5, AAV6, AAV8 or AAV9, AAV-DJ or AAV-DJ/8™ (Cell Biolabs, Inc., San Diego, CA), a rhesus-derived AAV, wherein the rhesus-derived AAV is AAVrh.10hCLN2, or an AAV capsid mutant or AAV hybrid serotype.

6. The method of embodiment 5, wherein the AAV vector is engineered to increase efficiency in targeting a specific cell type that is non -permissive to a wild type (wt) AAV and/or to improve efficacy in infecting only a cell type of interest.

7. The method of embodiment 6, wherein, the AAV serotype is retargeted or engineered as a hybrid serotype by one or more modifications selected from the group consisting of 1) a transcapsidation, 2) adsorption of a bi-specific antibody to a capsid surface, 3) engineering a mosaic capsid, and 4) engineering a chimeric capsid.

8. The method of any one of embodiments 1-7, wherein the polypeptide composition is a mammalian APOA1BP polypeptide.

9. The method of embodiment 8, wherein the mammalian ApoA-I Binding Protein (APOA1BP) polypeptide is a human APOA1BP polypeptide.

10. The method of any one of embodiments 1-7 and 9, wherein the subject is a human.

11. The method of any one of embodiments 1-8, wherein the subject is a mammal. 12. The method of any one of embodiments 1-11, wherein the polypeptide composition is comprised of a recombinant APO A IBP polypeptide having an APOA1BP activity.

13. The method of any one of embodiments 1-11, wherein the polypeptide composition is comprised of a synthetic APOA1BP polypeptide.

14. The method of embodiment 1, wherein pharmaceutically acceptable formulation is comprised of an ApoA-I Binding Protein polypeptide-inducing compound or composition.

15. The method of any one of embodiments 1-14, wherein the pharmaceutically acceptable formulation is suitable for intraocular or intravitreal administration.

16. The method of any one of embodiments 1-14, wherein said administration is by intrathecal injection.

17. The method of any one of embodiments 1-141, wherein said administration is by intraocular injection.

18. The method of any one of embodiments 1-14, wherein said administration is by intravenous (IV) administration.

19. The method of any one of embodiments 1-18, wherein the polypeptide composition is on or within a particle, wherein the particle is a nanoparticle, a micelle, a liposome, a lipoplex, a polymersome, a polyplex or a dendrimer.

20. The method of any one of embodiments 1-18, wherein the pharmaceutically acceptable formulation is in the form of a sterile injectable solution, or an implant.

21. The method of embodiment 20, wherein the implant is an intraocular implant.

22. The method of any one of embodiments 1-21, wherein the polypeptide composition, further comprises a CNS penetrating peptide.

23. The method of embodiment 19, wherein the particle further comprises a CNS targeting moiety.

24. Use of a formulation composition in preparation of a medicant for treating, ameliorating, protecting against, reversing or decreasing the severity or duration of a glaucoma, wherein optionally the glaucoma is open-angle glaucoma or closed angle glaucoma, treating, ameliorating, protecting against, reversing or decreasing the severity or duration of neuroinflammation in an eye during glaucomatous neurodegeneration, treating, ameliorating, protecting against, reversing or decreasing the severity or duration of mitochondrial dysfunction in retinal ganglion cells (RGCs), microglia or Muller glia during glaucomatous neurodegeneration in an eye, or decreasing or slowing the rate of induced RGC death in response to elevated intraocular pressure (IOP), wherein formulation composition is comprised of:

(2) a nucleic acid composition that increases expression or activity of, or encodes for, a polypeptide composition, wherein the polypeptide composition is or is comprised of an ApoA-I Binding Protein polypeptide, wherein the polypeptide composition has, or is capable of providing, an ApoA-I Binding Protein polypeptide activity, or

(3) an ApoA-I Binding Protein polypeptide-inducing compound or composition.

25. The use according to embodiment 24, wherein the ApoA-I Binding Protein polypeptide-inducing compound or composition increases, stimulates, or activates the activity of an APOA1BP promoter or transcriptional regulatory sequence or motif for expression of the polypeptide composition.

26. The use according to embodiment 24, wherein the nucleic acid that expresses or encodes for the polypeptide composition is contained within an expression vehicle, vector, recombinant virus, or equivalent thereof.

27. The use according to embodiment 26, wherein the vector or virus is, or comprised of an adenovirus vector or an adeno-associated virus (AAV) vector, a retrovirus, a lentiviral vector, a herpes simplex virus, a human immunodeficiency virus (HIV), or a synthetic vector.

28. The use according to embodiment 27, wherein the AAV vector is or is comprised of an adeno-associated virus (AAV), an AAV serotype, an AAV variant, wherein the AAV variant is AAV5, AAV6, AAV8 or AAV9, AAV-DJ or AAV-DJ/8™ (Cell Biolabs, Inc., San Diego, CA), a rhesus-derived AAV, wherein the rhesus-derived AAV is AAVrh.10hCLN2, or an AAV capsid mutant or AAV hybrid serotype. 29. The use according to embodiment 27, wherein the AAV vector is engineered to increase efficiency in targeting a specific cell type that is non-permissive to a wild type (wt) AAV and/or to improve efficacy in infecting only a cell type of interest.

30. The use according to embodiment 28, wherein, the AAV serotype is retargeted or engineered as a hybrid serotype by one or more modifications selected from the group consisting of 1) a transcapsidation, 2) adsorption of a bi-specific antibody to a capsid surface, 3) engineering a mosaic capsid, and 4) engineering a chimeric capsid.

31. The use according to any one of embodiments 24-30, wherein the polypeptide composition is a mammalian APOA1BP polypeptide.

32. The use according to embodiment 31, wherein the mammalian ApoA-I Binding Protein (APOA1BP) polypeptide is a human APOA1BP polypeptide.

33. The use according to any one of embodiments 24-30 and 32, wherein the subject is a human.

34. The use according to any one of embodiments 24-31, wherein the subject is a mammal.

35. The use according to any one of embodiments 24-34, wherein the polypeptide composition is comprised of a recombinant APO A IBP polypeptide having an APOA1BP activity.

36. The use according to any one of embodiments 24-34, wherein the polypeptide composition is comprised of a synthetic APOA1BP polypeptide.

37. The use according to embodiment 24, wherein pharmaceutically acceptable formulation is comprised of an ApoA-I Binding Protein polypeptide-inducing compound or composition.

38. The use according to any one of embodiments 1-14, wherein the pharmaceutical composition is suitable for intraocular or intravitreal administration.

39. The use according to any one of embodiments 1-14, wherein the pharmaceutical composition is suitable for administration by intrathecal injection.

40. The use according to of any one of embodiments 1-141, wherein the pharmaceutical composition is suitable for intraocular injection.

41. The use according to of any one of embodiments 1-14, wherein the pharmaceutically acceptable is suitable for intravenous (IV) administration. 42. The use according to any one of embodiments 24-41, wherein the polypeptide composition is on or within a particle, wherein the particle is a nanoparticle, a micelle, a liposome, a lipoplex, a polymersome, a polyplex or a dendrimer.

43. The use according to any one of embodiments 24-41, wherein the pharmaceutical composition is in the form of a sterile injectable solution or an implant.

44. The use according to embodiment 43, wherein the implant is an intraocular implant.

45. The use according to any one of embodiments 24-44, wherein the polypeptide composition, further comprises a CNS penetrating peptide.

46. The use according to embodiment 42, wherein the particle further comprises a CNS targeting moiety.

Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed here in the Summary, Figures and/or Detailed Description sections.

The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Incorporation by reference of these documents, standing alone, should not be construed as an assertion or admission that any portion of the contents of any document is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the right is reserved for relying upon any of such documents, where appropriate, for providing material deemed essential to the claimed subject matter by an examining authority or court.

Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of, and "consisting of may be replaced with either of the other two terms. Thus, the terms and expressions which have been employed are used as terms of description and not of limitation, equivalents of the features shown and described, or portions thereof, are not excluded, and it is recognized that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims.

The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.

EXAMPLES

Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols, for example, as described in Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

Example 1 : AIBP protects retinal ganglion cells against neuroinflammation and mitochondrial dysfunction in glaucomatous neurodegeneration

This example demonstrates that AIBP plays a critical role in protection against neuroinflammation and mitochondrial dysfunction during glaucomatous neurodegeneration. Using systemic AIBP knockout (Apoal bp~ ~) mice, we show that AIBP deficiency triggers mitochondrial dysfunction in both retinal ganglion cells (RGCs) and Muller glia. It also increases TLR4 and IL-ip expression in Muller glia endfeet, leading to oxidative stress, RGC death and visual dysfunction. Moreover, AIBP deficiency exacerbates vulnerability to elevated intraocular pressure (IOP)- induced RGC death. In particular, AIBP treatment inhibits inflammatory responses in Muller glia and protects RGCs against elevated IOP. These results suggest that AIBP has therapeutic potential for restraining excessive mitochondrial dysfunction and neuroinflammation in glaucomatous neurodegeneration.

In particular, this study demonstrated that:

• Loss of endogenous AIBP induces glial activation and impairs visual function.

• Loss of endogenous AIBP mediates glia-driven neuroinflammation and mitochondrial dysfunction in glaucomatous neurodegeneration.

• Administration of recombinant AIBP protects RGCs via inhibiting inflammatory responses in Muller glia and has a therapeutic potential for treating glaucoma.

• This finding connects for the first time AIBP with protecting mechanisms controlling mitochondrial pathogenic mechanisms, neuroinflammation and RGC death.

2, Materials and methods

2.1. Human tissue samples

Human retina tissue sections were obtained from a normal (age 81 years) donor and a patient with glaucoma (age 91 years) (San Diego Eye Bank, CA, USA) with a protocol approved by the University of California, San Diego Human Research Protection Program. The normal patient has no history of eye disease, diabetes, or chronic central nervous system disease.

2.2. Animals

Adult male and female DBA/2J and age-matched DBA/2J-G/wmZ>⁺ (D2- Gpnmb ) mice (The Jackson Laboratory, ME, USA), and WT and Apoalbp'^/_ mice were housed in covered cages, fed with a standard rodent diet ad libitum, and kept on al2 h light/12 h dark cycle. C57BL/6J mice were initially purchased from the Jackson Laboratory, bred in-house for experiments and used as wild-type (WT) mice. Apoalbp~ ~ mice on a C57BL/6J background were generated in our group as previously reported (8, 23). Animals were assigned randomly to experimental and control groups. To investigate the effect of IOP elevation and/or AIBP deficiency, 10 month-old DBA/2J and age-matched D2-Gpnmb mice, and 4 month-old WT and age-matched Apoalbp~ ~ mice were used. Behavioral responses and visual function were studied with 3-4 month old male and female mice. All procedures concerning animals were in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic Vision Research and under protocols approved by Institutional Animal Care and Use Committee at the University of California, San Diego.

2.3. Induction of acute IOP elevation

Mice were anesthetized by an intraperitoneal (IP) injection of a cocktail of ketamine (100 mg/kg, Ketaset; Fort Dodge Animal Health, IA, USA) and xylazine (9 mg/kg, TRANQUIVED™; VEDCO Inc., MO, USA). Eyes were also treated with 1% proparacaine drops. Induction of acute IOP elevation was performed as previously described (24). Briefly, a 30-gauge needle was inserted into the anterior chamber of right eye that was connected by flexible tubing to a saline reservoir. By raising the reservoir, IOP was elevated to 70-80 mmHg for 50 min. Sham treatment was performed in the contralateral eyes by the insertion of a needle in the anterior chamber without saline injection. Retinal ischemia was confirmed by observing whitening of the iris and loss of the retina red reflex. IOP was measured with a tonometer (icare TONOVET, Vantaa, Finland) during IOP elevation. Non-IOP elevation contralateral control retinas were used as sham control.

2.4. IOP measurement

IOP elevation onset typically occurs between 5 and 7 months of age, and by 9 to 10 months of age, lOP-linked optic nerve axon loss is well advanced (16, 25). IOP measurement was performed as previously described (16, 25). Each of the 10 month- old DBA/2J mice used in this study had a single IOP measurement (to confirm development of spontaneous IOP elevation exceeding 20 mmHg) (n = 5 for selected DBA/2J mice). Also, each of the non-glaucomatous control C57BL/6 or 2-Gpnmb mice (n = 5) used in this study had a single IOP measurement. For WT and Apoalbp~ ~ mice, IOP was measured with a tonometer (icare TONOVET) as described above.

2.5. Cell culture and hydrostatic pressure (HP) system in vitro.

RGCs from postnatal 5 days of Sprague-Dawley rat were purified by immune- panning and were cultured in serum-free defined growth medium as previously described (16). Approximately 2 x 10⁵ purified cells were seeded on 60 mm dishes coated first with poly-D-lysine (70 kDa, 10 pg/ml; Sigma, MO, USA) and then with laminin (10 pg/ml; Sigma) in neurobasal medium. RGCs were cultured in serum -free defined growth medium containing BDNF (50 pg/ml; Sigma), CNTF (10 pg/ml; Sigma), insulin (5 pg/ml; Sigma), and forskolin (10 pg/ml; Sigma). A pressurized incubator was used to expose the cells to elevated HP as previously described (16). The plexiglass pressure chamber was connected via a low-pressure two-stage regulator (Gilmont Instruments, Barnant Company, IL, USA) to a certified source of 5% CO2 /95% air (Airgas Inc., CA, USA).

2.6. Recombinant AIBP

His-tagged AIBP was produced in a baculovirus/insect cell expression system to allow for post-translational modification and to ensure endotoxin-free preparation as previously described (9, 11). AIBP protein was purified using a Ni-NTA agarose column (Qiagen, CA, USA) eluted with imidazole. Purified AIBP was dialyzed against phosphate buffered saline (PBS, Sigma), and concentration was measured. Aliquots were stored at -80°C.

2. 7. Tissue preparation

Mice were anesthetized by an IP injection of a cocktail of ketamine/xylazine as described above prior cervical dislocation. For immunohistochemistry, the retinas and superior colliculus (SC) tissues were dissected from the choroids and fixed with 4% paraformaldehyde (Sigma) in PBS (pH 7.4) for 2 h at 4 °C. Retinas and SCs were washed several times with PBS then dehydrated through graded levels of ethanol and embedded in polyester wax. For electron microscopy (EM), the eyes were fixed via cardiac perfusion with 2% paraformaldehyde, 2.5% glutaraldehyde (Ted Pella, CA, USA) in 0.15 M sodium cacodylate (pH 7.4, Sigma) solution at 37 °C and placed in pre-cooled fixative of the same composition on ice for 1 h. As described below, the procedure was used to optimize mitochondria structural preservation and membrane contrast. For Western blot and PCR analyses, extracted retinas were immediately used.

2. 7. Western blot analyses

Harvested retinas were homogenized for 1 min on ice with a modified RIPA lysis buffer (#9806, Cell Signaling Technology, MA, USA), containing complete protease inhibitor cocktail (#HY-K0010, MedChemExpress, NJ, USA). The lysates were then centrifuged at 15,000 g for 15 min and protein amounts in the supernatants were measured by Bradford assay. Proteins (10-20 pg) were run on a NuPAGE™ BisTris gel (Invitrogen, CA, USA) and transferred to polyvinylidene difluoride membranes (GE Healthcare Bio-Science, NJ, USA). The membranes were blocked with 5% non-fat dry milk and PBS/0.1% Tween-20 (PBS-T) for 1 hour (h) at room temperature and incubated with primary antibodies (FIG. 14, or supplementary (supp.) Table 1) for overnight at 4 °C. Membrane were washed three times with PBS- T then incubated with horseradish peroxidase-conjugated secondary antibodies (BioRad, CA, USA) for 1 h at room temperature. Membranes were developed using enhanced chemiluminescence substrate system. The images were captured using a UVP imaging system (UVP LLC, CA, USA).

2.8. Immunohistochemistry

Immunohistochemical staining of 7 pm wax sections of full thickness retina were performed. Sections from wax blocks from each group (n = 4 retinas/group) were used for immunohistochemical analysis. To prevent non-specific background, tissues were incubated in 1% bovine serum albumin (BSA, Sigma)/PBS for 1 h at room temperature before incubation with the primary antibodies for 16 h at 4°C. After several wash steps, the tissues were incubated with the secondary antibodies (FIG. 14, or supp. Table 1) for 4 h at 4°C and subsequently washed with PBS. The sections were counterstained with the nucleic acid stain Hoechst 33342 (1 pg/ml; Invitrogen) in PBS. Images were acquired with FLUOVIEW1000™ (Olympus) confocal microscopy (Olympus, Tokyo, Japan) or Leica SPE-II confocal microscope (Leica, Wetzlar, Germany). Each target protein fluorescent integrated intensity in pixel per area was measured using the ImageJ software. All imaging parameters remained the same and were corrected with the background subtraction.

2.9. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining

TUNEL staining was performed using In Situ Cell Detection Kit (TMR red, Roche Biochemicals, IN, USA) as previously described (26, 27). After rinsing in PBS, the sections were incubated with TUNEL mixture in reaction buffer for 60 minutes at 37°C. To count TUNEL-positive cells, the areas were divided into three layers by ganglion cell layer (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL). To determine whether TUNEL-positive cells are RGCs, we performed immunohistochemistry before TUNEL staining using RNA-binding protein with multiple splicing (RBPMS, Cat# NBP2-20112, Novus Biologicals, CO, USA) antibody as described above. The sections were counterstained with the Hoechst 33342 (1 pg/ml; Invitrogen) in PBS as described above. TUNEL-positive cells were counted in 5 microscopic fields (20x) per condition (n = 5 retinas) by two investigators in a masked fashion, and the scores were averaged. Images were acquired with a FLUOVIEWIOOO™ confocal microscopy (Olympus).

2.10. Whole-Mount Immunohistochemistry and RGC Counting

Retinas from enucleated eyes were dissected as flattened whole-mounts from WT and Apoal bp~ ~ mice. Retinas were immersed in PBS containing 30% sucrose for 24 h at 4°C. The retinas were blocked in PBS containing 3% donkey serum, 1% bovine serum albumin, 1% fish gelatin and 0.1% triton X-100, and incubated with primary antibodies (FIG. 14, or supp. Table 1) for 3 days at 4°C. After several wash steps, the tissues were incubated with the secondary antibodies (FIG. 14, or supp. Table 1) for 24 h, and subsequently washed with PBS. Images were captured under fluorescence microscopy using a Nikon ECLIPSE microscope (E800; Nikon Instruments Inc., NY, USA) equipped with digital camera (SPOT Imaging, MI, USA) or Olympus FluoViewlOOO confocal microscopy (Olympus). Image exposures were the same for all tissue sections and were acquired using Simple PCI version 6.0 software. To count RGCs labeled with Bm3a, each retinal quadrant was divided into three zones by central, middle, and peripheral retina (one sixth, three sixths, and five sixths of the retinal radius). RGC densities were measured in 12 distinct areas (one area at central, middle, and peripheral per retinal quadrant) per condition by two investigators in a masked fashion, and the scores were averaged.

2.11. Serial block-face scanning electron microscopy (SBEM)

Retina tissues were washed with cacodylate buffer for 2 h at 4 °C and then placed into cacodylate buffer containing 2 mM CaCh and 2% OsO4/1.5% potassium ferrocyanide as previously described (16). The tissues were left for 2 h at room temperature. After thorough washing in double distilled water, the tissues were placed into 0.05% thiocarbohydrazide for 30 min. The tissues were again washed and then stained with 2% aqueous OsCU for 1 h. The tissues were washed and then placed into 2% aqueous uranyl acetate overnight at 4°C. The tissues were washed with water at room temp and then stained with en bloc lead aspartate for 30 min at 60°C. The tissues were washed with water and then dehydrated on ice in 50%, 70%, 90%, 100%, 100% ethanol solutions for 10 min at each step. The tissues were then washed twice in dry acetone and then placed into 50:50 DURCUP AN ACM™:acetone overnight. The tissues were transferred to 100% DURCUP AN™ resin overnight. The tissues were then embedded and left in an oven at 60°C for 72 h. BEM was performed on Merlin scanning electron microscopy (ZEISS™, Oberkochen, Germany) equipped with a 3view2XP and OnPoint backscatter detector (Gatan, CA, USA). The retina volumes were collected at 2.5 kV accelerating voltages, with pixel dwell time of 0.5ps. The raster size was 20k x 5k, with 3.5 nm pixels and 50 nm z step size. Once a volume was collected, the histograms for the tissues throughout the volume stack were normalized to correct for drift in image intensity during acquisition. Digital micrograph files (,dm4) were normalized using Digital Micrograph and then converted to MRC format. The stacks were converted to eight bit and volumes were manually traced for reconstruction and analysis using IMOD software (http : //bi o3 d . col orado . edu/imod/) .

2.12. 3D EM Tomography

EM tomography experiments were conducted on a FEI TITAN HALO™ operating in the Scanning Transmission Electron Microscope mode at 300kV, with the possibility to resolve micrometer thick plastic embedded specimen down to nanoscale spatial resolution as described previously. Vertical sections of retina tissues from each group were cut at a thickness of 750 nm and electron tomography was performed following a 4-tilt series scheme described in, with the specimen tilted from -60° to +60° every 0.5° at four evenly distributed azimuthal angle positions. The magnification was 28,500*and the pixel resolution was 4.2 nm. The IMOD package was used for alignment, reconstruction and volume segmentation. Volume segmentation was performed by manual tracing of membranes in the planes of highest resolution with the Drawing Tools and Interpolator plug-ins (16, 25, 28). The reconstructions and surface-rendered volumes were visualized using 3DM0D. Measurements of mitochondrial outer, inner boundary (IBM), and cristae membrane surface areas and volumes were made within segmented volumes using IMODinfo. These were used to determine the cristae density, defined as the ratio: sum of the cristae membrane surface areas divided by the mitochondrial outer membrane surface area.

2.13. Quantitative PCR analyses

Total RNA from the retina was isolated using NUCLEOSPIN™ RNA columns (Clontech, CA, USA). Isolated RNA was reverse transcribed using RNA to cDNA ECODRY™ (Clontech) following the manufacturer’s instructions. Quantitative PCR (qPCR) was performed using KAPA SYBR FAST™ Universal qPCR kit (KAPA Biosystems, KK4602™, Roche Diagnostics, IN, USA), with primers ordered from Integrated DNA Technologies (IDT, CA, USA), and a ROTOR GENE Q™ thermocycler (Qiagen). The qPCR was performed with cDNAs synthesized from 1 pg of the total RNA of each group as a template and specific primers (see FIG. 15, or supplementary (or supp.) Table 2).

2.14. Optokinetic (OKT) analysis

Spatial visual function was performed on a virtual OKT system (OPTOMOTRY™ (or OptoMotry™); CerebralMechanics Inc., AB, Canada) (29). Unanesthetized mice were placed on an unrestricted platform in the center of a virtual cylinder comprised of four monitors arranged in a square (arena) that project a sinusoidal grating (i.e., white versus black vertical bars) rotating at 12 deg/sec. Mice were monitored by a camera mounted at the top of the arena while a cursor placed on the forehead centers the rotation of the cylinder at the animal’s viewing position. To assess visual acuity, tracking was determined when the mouse stops moving its body and only head-tracking movement is observed. Spatial frequency threshold, a measure of visual acuity, was determined automatically with accompanying OKT software, which uses a step-wise paradigm based upon head-tracking movements at 100% contrast. Spatial frequency began at 0.042 cyc/deg, which gradually increased until head movement was no longer observed.

2.15. Visual evoked potential (VEP) analysis

VEP was measured as previously described (30, 31). Mice were dark adapted in the procedure room at vivarium for less than 12 h in a dark room. Mice were prepared for recording under dim red light and anesthetized with IP injection of a mixture of ketamine/xylazine as described above. Pupils were dilated using equal parts of topical phenylephrine (2.5%) and tropicamide (1%). Proparacaine (0.5%) was used as a topical anesthetic to avoid blinking and a drop of lubricant is frequently applied on the cornea to prevent dehydration and allow electrical contact with the recording electrode (a gold wire loop, disposable). The top of the mouse's head was cleaned with an antiseptic solution. A scalpel was used to incise the scalp skin, and a metal electrode was inserted into the primary visual cortex through the skull, 0.8 mm deep from the cranial surface, 2.3 mm lateral to the lambda. A platinum subdermal needle (Grass Telefactor) was inserted through the animal's mouth as a reference and through the tail as ground. The measurements commenced when the baseline waveform became stable, 10-15 s after attaching the electrodes. Flashes of light at 2 log cd.s/m2 were delivered through a full-field Ganzfeld bowl at 2 Hz. Signal was amplified, digitally processed by the software (Veris Instruments, OR, USA), then exported, and peak-to-peak responses were analyzed in Excel (Microsoft). To isolate VEP of the measured eye from the crossed signal originating in the contralateral eye, a black aluminum foil eyepatch was placed over the eye not undergoing measurement. For each eye, peak-to-peak response amplitude of the major component Pl -N1 in IOP eyes was compared to that of their contralateral non-IOP controls. The All the recordings are carried out with the same stimulus intensity. The average signals for each group were compared with respect to both amplitude and latency.

2.16. Cholera toxin-B (CTB) labeling

Mice were anesthetized with IP injection of a mixture of ketamine/xylazine as described above and topical 1% proparacaine eye drops. A Hamilton syringe was used to inject a 1 pL of Alexa Fluor 594-conjugated CTB (Invitrogen), into the vitreous humor. Injections were given slowly over 1 min and the needle was maintained in position for an additional 5 min to minimize CTB loss through the injection tract. At 3 days after injection, the mice were fixed via cardiac perfusion with 4% paraformaldehyde (Ted Pella) following an IP injection of a mixture of ketamine/xylazine. After perfusion, the SC tissues were dissected and immersed in PBS containing 30% sucrose for 24 h at 4°C. The SC tissues were coronally sectioned at 50 pm using a Leica Cryostat (Wetzlar, Germany). The 30 representative sections were mounted on slides and images were acquired with Olympus FluoViewlOOO (Olympus). The area densities from the images were analyzed using Imaged (http://rsb.info.nih.gov/ij/; provided in the public domain by the National Institutes of Health, MD, USA) and Imaris software (Bitplane Inc., MA, USA).

2.17. Statistical analysis

For comparison between two groups, statistical analysis was performed using a two-tailed Student's /-test. For multiple group comparisons, we used either one-way ANOVA or two-way ANOVA, using GraphPad Prism (GraphPad, CA, USA). A P value less than 0.05 was considered statistically significant.

3 , Results

3.1. Reduced AIBP expression in RGCs in glaucomatous retinas In neonatal mice, Apoalbp mRNA is expressed in RGCs (8). To determine whether elevated pressure alters expression level of AIBP in murine RGCs, we first transiently induced acute IOP elevation in the eye of normal C57BL/6J mice by the cannulation of the anterior chamber of the eye, which was elevated to maintain an IOP of 70-80 mmHg for 50 min (24). This model is also widely used by others to determine RGC death and survival in degenerative retinal diseases including acute glaucoma (14, 32). We found that elevated IOP significantly reduced Apoalbp gene and AIBP protein expression in the retina at 24 h compared with sham control retina (Fig. 1 A and B). Immunohistochemical analysis showed that AIBP immunoreactivity was localized in the outer plexiform layer (OPL), INL, inner plexiform layer (IPL), and GCL of control mice. In the GCL, AIBP immunoreactivity was present in RGC somas and axons, which were labeled with neuron-specific 0-111 tubulin (TUJ1), a marker for RGCs. Consistently, elevated IOP decreased AIBP immunoreactivity in the OPL and inner retinal layer (Fig. 1C). We further cultured primary RGCs and exposed cells to elevated HP (30 mmHg) for 3 days (16). Notably, elevated HP exposure significantly reduced AIBP protein expression in RGCs (Fig. ID).

We next examined whether a chronic IOP elevation alters AIBP protein expression in the retina using glaucomatous DBA/2J mice, which spontaneously develop elevated IOP and glaucomatous damage with age, and age-matched control \yi-Gprimb mice (33, 34). Interestingly, we found that glaucomatous DBA/2J retina showed a similar pattern of reduced AIBP immunoreactivity as in our acute model of IOP elevation (Fig. IE). In 2-Gpnmb mice, AIBP immunoreactivity was present not only in RGC soma in the GCL but also in RGC dendrites in the IPL (Fig. IF). In contrast, AIBP immunoreactivity was significantly decreased in the inner retina of glaucomatous DBA/2J mice (Fig. IF and G). Since AIBP mediates the stabilization of ATP -binding cassette transporter Al (ABCA1) by facilitating apoA-1 binding to ABCA1 and prevents ABC Al degradation via the ubiquitination pathway (35), we further tested whether a chronic IOP elevation also alters expression level of ABCA1 protein. In 2-Gpnmb mice, ABCA1 immunoreactivity was present in Brn3a- positive RGCs in the GCL and OPL (Fig. 1H). In contrast, ABCA1 immunoreactivity was highly diminished in the neurons of the GCL, including Brn3a-positive RGCs, of glaucomatous DBA/2J retina (Fig. 1H and I). 3.2. AIBP deficiency exacerbates RGC vulnerability to elevated IOP and triggers visual dysfunction

To test the hypothesis that AIBP deficiency plays an important role in glaucomatous RGCs, we used Apoalbp~ ~ mice that are viable and fertile, and have no apparent morphological defects compared with control mice under naive conditions (8). As shown in Figure 2, we induced IOP elevation in WT and Apoalbp~ ~ mice and assessed RGC loss at 4 weeks after IOP elevation. There was no statistically significant difference in lOPs between WT and naive Apoalbp~ ~ mice. The mean IOP of contralateral control eyes was 9-10 mmHg and IOP was elevated in ipsilateral eyes to 70-75 mmHg in WT and Apoal bp~ ~ mice (n = 15 mice; Fig. 2A). Remarkably, we found that elevated IOP significantly enhanced RGC loss in all retinal areas of Apoalbp~ ~ mice compared with sham control WT or naive Apoal bp~ ~ retinas (Fig. 2B and C). In addition, we observed that no statistically significant differences in RGC number were detected in the retinas between sham control WT and naive Apoalbp~ ~ mice (Fig. 2B and C).

To test the effect of AIBP deficiency on visual function, we next measured 1) the maximum spatial frequency that could elicit head tracking (“acuity”) in a virtual- reality optomotor system by OKT test and 2) central visual function using VEP, a measurement of the electrical signal recorded at the scalp over the occipital cortex in response to light stimulus. In the absence of AIBP, we found a significant reduction of visual acuity by decreasing spatial frequency in both male and female naive Apoal bp~ ^/_ mice (Fig. 2D). However, there were no statistically significant differences in VEP Pl-Nl potentials and latency in naive Apoal bp~ ~ mice compared with WT mice (Fig. 2E and F). Because VEP is considered to be valid in analyzing and predicting visual properties in glaucoma patients with severe visual impairments (36), our results suggest the possibility that while AIBP deficiency triggers spatial vision dysfunction in the eye, it may not be sufficient to induce severe progression of optic nerve damage. Additionally, we further determined whether AIBP deficiency alters axon transport from retina to SC by intravitreal injection of an anterograde tracer, CTB, into the eyes of WT and naive Apoal bp~ ~ mice. At 3 days after injection, we measured anterograde tracing of CTB to SC. We found that there was no statistically significant difference in the density of CTB labeling in the SC between WT and naive Apoalbp~ ~ mice (Fig. 2G and H). 3.3. Increased TLR4 and IL- 1 /3 expression in glaucomatous and Apoal bp~ ~ Muller glia endfeet

AIBP plays a unique role of targeting cholesterol efflux machinery to TLR4- occupied inflammarafts (10, 11). Evidence from clinical and animal studies indicates that TLR4-dependent signaling is an important factor in the pathogenesis of POAG and that this signaling is associated with activated glial cells and contributes to inflammatory responses in experimental glaucoma (37-39). First, we determined the expression level and distribution of TLR4 and IL- 10 proteins in glaucomatous retinas from human patients with POAG and DBA/2J mice. Remarkably, we observed significantly increased patterns of TLR4 and IL- 10 immunoreactivity in glutamine synthase (GS)-positive Muller glia in both glaucomatous human and DBA/2J mouse retinas compared with control retinas (Fig. 3 A and B). In glaucomatous human retina, we found that TLR4 immunoreactivity was increased in the endfeet of Muller glia of the GCL and nerve fiber layer (NFL) compared with normal retina, while IL-10 immunoreactivity was increased in both processes and endfeet of Muller glia of the IPL, GCL and NFL (Fig. 3 A and B). In glaucomatous DBA/2J mouse retina, both TLR4 and IL-10 immunoreactivities were significantly increased in the endfeet of Muller glia of the GCL but depleted in the processes of Muller glia compared with age-matched control 'l-Gpnmb mouse retina (Fig. 3 A and B). Consistent with these results, glaucomatous retinas displayed significantly increased relative fluorescence intensity of both TLR4 and IL- 10 proteins in the endfeet of Muller glia in the GCL compared with control Muller glia (Fig. 3C and D). Second, we further determined whether AIBP deficiency alters TLR4 and IL- 10 protein expression in Muller glia using Apoalbp~ ~ mice. We found that AIBP deficiency not only significantly increased TLR4 and IL- 10 immunoreactivities in the endfeet of Muller glia in the GCL but also showed a characteristic pattern of increased TLR4 and IL- 10 expression in the processes of Muller glia in the IPL observed in DBA/2J mouse and human glaucomatous retinas (Fig. 3A-D).

3.4. AIBP deficiency induces mitochondrial fragmentation and reduces ATP production in Muller glia

TLR4 is associated with mitochondrial damage caused by intracellular ROS and defective mitochondrial dynamics (20, 21). Using naive Apoalbp~ ~ mice, we further investigated whether AIBP contributes to the regulation of mitochondrial structure and function in the endfeet of Muller glia. 3D EM (Fig. 4A and B, and sFig. 1) demonstrated lower crista density and dark outer membrane onion-like swirls in Apoalbp~ ~ mitochondria (Fig. 4B and sFig. ID), although fewer in number than found in the RGC. Interestingly, we also found ring-shaped mitochondria, a hallmark of mitochondrial stress (sFig. IE) (40), as well as lower rough endoplasmic reticulum (ER) density and dilated ER strands (sFig. ID and E). The mitochondria were traced in yellow to make it easier to identify them and those with lower crista density pointed to in Apoalbp~ ~ (Fig. 4B and sFig. 1). Mitochondria in the Muller glia have rarely been studied via EM at high resolution or in 3D in a quantitative manner (41). Reconstructions showed examples of long tubular mitochondria in WT but small rounded mitochondria in Apoalbp~ ~ (Fig. 4C and D, and sMovie 1 and 2). Because each mitochondrion covered multiple image planes at variable cutting angles (Fig. 4E and F), to perform more accurate length measurement, mitochondria were segmented by drawing a series of connected spheres centered along the length of each mitochondrion using IMOD open contour (Fig. 4G-I). Measurements of mitochondria showed that there were no significant changes in mitochondrial volume (Fig. 4J), volume density (Fig. 4K), or mitochondrial number (Fig. 4L) in the Apoal bp~ ~ . The form factor for the Apoalbp~ ~ mitochondria was significantly lower than for the WT (Fig. 4M), meaning more mitochondrial rounding in the Apoalbp~ ~. Interestingly, mitochondrial length was significantly decreased in Apoalbp~ ~ (Fig. 4N). Similarly, the crista density (Fig. 40) and the modeled rate of ATP production per mitochondrial volume (Fig. 4P) were lower in the Apoalbp~ ~. In contrast, the modeled rate of ATP production per mitochondrion was not lower in the Apoalbp~ ~ (Fig. 4Q), yet there was a significant decrease in cellular ATP production via mitochondria in the Apoalbp~ ~ Muller glia endfeet (Fig. 4R). Together, these results suggest that AIBP deficiency triggers mitochondrial fragmentation, depletes mitochondrial cristae, and compromises energy production that may lead to dysfunction of Muller glia and activation of inflammatory responses.

3.5. AIBP deficiency impairs mitochondrial dynamics and OXPHOS activity in the retina

AIBP in transfected cells was shown to localize to mitochondria (22). At 24 h after acute IOP elevation, we found AIBP protein expression was significantly decreased in the mitochondrial fraction (Fig. 5A), indicating that elevated IOP alters mitochondrial AIBP expression. Thus, we further determined the effect of AIBP deficiency on mitochondrial dynamics and function in the retina. We first found significant decreases of the mitochondrial fusion proteins optic atrophy type 1 (OPA1) and mitofusin 2 (MFN2) in naive Apoalbp~ ~ retina compared with WT retina (Fig. 5B). In WT retina, we observed that OPA1 immunoreactivity was highly present in Brn3a-positive RGCs, colocalizing with cytochrome c immunoreactivity (Fig. 5C). In contrast, AIBP deficiency diminished OPA1 immunoreactivity in the OPL and INL, as well as RGCs of the GCL (Fig. 5C). Interestingly, we also observed that AIBP deficiency induced an increase of OPA1 immunoreactivity in GS-positive Muller glia (sFig. 2A). In the absence of AIBP, we next found significant decreases of the levels of total mitochondrial fission protein, dynamin-related protein 1 (DRP1), and phosphorylated DRP1 at serine 637 in the retina (Fig. 5D). Immunohistochemical analysis showed that DRP1 immunoreactivity was highly present in the dendrites of the IPL and Brn3a-positive RGCs of the GCL in WT retina (Fig. 5E). In the absence of AIBP, we consistently observed that total DRP1 immunoreactivity was diminished in the OPL and inner retinal layer, especially in RGC somas and dendrites (Fig. 5E). In contrast to Figure s2A, DRP1 immunoreactivity was not detectable in Apoalbp~ ~ Muller glia (sFig. 2B). Additionally, there were no statistically significant differences in mitochondrial dynamics-related gene expression (Opal, Mfn2 and Drpl between WT and naive Apoalbp~ ~ retinas (sFig. 3 A). We further determined whether AIBP is involved in mitochondrial OXPHOS in the retina and found that OXPHOS complexes (Cxs) protein expression were significantly decreased in Apoalbp~ ~ retina (Fig. 5F).

3.6. AIBP deficiency triggers mitochondrial fragmentation and reduces ATP production in RGCs

To determine whether AIBP deficiency directly affects mitochondrial structure and function in RGCs, we assessed the structural and functional changes of mitochondria in Apoalbp~ ~ RGC somas. Applying 3DEM, we found that AIBP deficiency principally caused swelling and rounding of mitochondria and altered ER structure in RGC somas (Fig. 6A and B and sFig. 4A and B). Even though many of the ER strands were dilated, a hallmark of ER stress, ER-facilitated mitochondrial fission did not appear to be impaired (Fig. 6C-F and sFig. 4B). As with the Muller glia endfeet, abnormal mitochondria with localized structural perturbation of the outer membrane, usually manifest as onion-like swirling membrane, were commonly seen in the Apoalbp~ ~ RGC soma (Fig. 6G and H). Long extended axons distinguish RGCs from displaced amacrine cells in the GCL. Variable RGC mitochondrial structures were rendered and analyzed (Inserts in Fig. 61 and J; sMovie 3 and 4). 3D volumes showed long tubular forms of mitochondria and branched mitochondria in WT RGC somas (Fig. 61), whereas small and rounded forms of mitochondria and sometimes branched mitochondria were observed in Apoalbp~ ~ RGC somas (Fig. 6J). We also found ring-shaped mitochondria and mitophagosome formation in Apoalbp~ ~ RGC somas (sFig. 4C and D). Unlike in the Apoalbp~ ~ Muller glia endfeet, it was verified that Apoalbp~ ~ RGC mitochondria were larger (Fig. 6K) and occupied more of the cytoplasmic volume (Fig. 6L) likely due to their largeness, yet they did not have increased numbers (Fig. 6M). The form factor for mitochondria was significantly lower, indicating more rounded mitochondria caused by volume dilation in the Apoalbp~ ~ RGC compared to the WT (Fig. 6N). As with the Muller glia endfeet, the lengths of Apoalbp~ ~ mitochondria were significantly decreased in RGC somas (Fig. 60); their greater volume comes from their rounding.

As implied in Figure 6, the crista density was significantly lower in the Apoalbp~ ~ RGC mitochondria (Fig. 7A) leading to a lower modeled rate of ATP production per mitochondrial volume. Yet, because mitochondria were larger in the Apoalbp~ ~ RGC soma, each mitochondrion, on average, was modeled to produce more ATP per second (Fig. 7B). However, unlike in the Apoalbp~ ~ Muller glia endfeet, the model for the rate of ATP production, which is based on 3D cristae surface area, predicts that there is not much decrease in cellular ATP production via mitochondria in the Apoalbp~ ~ RGC soma (Fig. 7B) even though the rate of ATP production per mitochondrial volume had significantly decreased (Fig. 7C); this decrease was simply offset by larger mitochondria. Tomographic volumes of WT RGC soma mitochondria show typical cristae (Fig. 7E and F). In contrast, Apoal bp~ ^/_ RGC soma mitochondria typically show cristae that are less densely packed and commonly observed onion-like protuberances (Fig. 7G-J; sMovie 5 and 6). Also, adjacent ER strands are often dilated (Fig. 7G). The crista density was lower in the Apoalbp~ ~ RGC soma mitochondria due in part to these onion-like outer membrane protuberances. In summary, the Apoal bp~ ~ RGC somas have mitochondria that are structurally perturbed by dilation and rounding with some localized structural perturbation of the outer membrane and some loss of cristae membrane. We next measured expression levels of mitofilin in naive Apoal bp~ ~ and WT mice. Mitofilin is a mitochondrial inner membrane protein that controls cristae architecture (42). In the absence of AIBP, we found a significant reduction of mitofilin protein expression in the retina (Fig. 7K), suggesting that AIBP deficiency may be the underlying factor for the loss of cristae membrane. However, there was no significant difference in mitofilin gene expression between WT and naive Apoal bp~ ~ mice (Fig. 7L).

3. 7. AIBP deficiency induces oxidative stress and MAPK signaling activation in RGCs

Under oxidative stress conditions, sirtuin 3 (SIRT3) impairment reduces the activity of superoxide dismutase 2 (SOD2) and increases ROS production (43, 44), and multiple mitogen-activated protein kinases (MAPKs) signaling pathways such as p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) are activated (45, 46). Thus, we tested whether AIBP regulates expression levels of SIRT3 and SOD2, as well as p38 and ERK1/2 activation in WT and naive Apoalbp'^/_ mice. In the absence of AIBP, we found that SIRT3 and SOD2 protein expression were significantly decreased in the retina (Fig. 8A and B). However, there were no statistically significant differences in Sirl3 and Sod2 gene expression (sFig. 3B). Also, we found that SIRT3 and SOD2 immunoreactivity was dramatically diminished in the IPL and GCL, especially in the in Brn3a-positive RGCs (Fig. 8C and D). Next, we found that AIBP deficiency significantly increased phosphorylation of p38 and ERK1/2 in the retina (Fig. 8E and F). Consistently, we also observed that phospho-p38 and phospho- ERK1/2 immunoreactivities were increased in the inner retinal layer in naive Apoalbp~ ~ mice (Fig. 8G and H). We noted that AIBP deficiency showed an increased pattern of phospho-p38 immunoreactivity in Brn3a-positive RGCs (Fig. 8G and H).

3.8. Administration of AIBP promotes RGC survival and inhibits inflammatory responses in IOP mouse model

Since AIBP deficiency was associated with a neuroinflammatory and RGC death phenotype, next we tested the hypothesis that injections of recombinant AIBP will be protective. We intravitreally injected recombinant AIBP protein or BSA (1 pL, 0.5 mg/ml) into C57BL/6J mice at 2 days before the induction of acute IOP elevation as described above. At 24 h after IOP elevation, we performed TUNEL staining and RBPMS immunohistochemistry. In BSA-injected animals, elevated IOP significantly increased the number of TUNEL-positive cells in all retinal layers compared with control mice (Fig. 9A and B). In the GCL, RBPMS -positive RGCs were co-stained with TUNEL, indicating apoptotic RGC death. In contrast, we remarkably observed that AIBP treatment significantly reduced the number of TUNEL-positive cells in the retina in response to elevated IOP, whereas there were no TUNEL-positive cells in both sham controls (BSA and AIBP) (Fig. 9A and B). To determine the effect of AIBP administration on inflammatory responses in activated Muller glia in response to elevated IOP, we quantified expression levels of IL- 10 protein in Muller glia endfeet by measuring relative fluorescence intensity of IL- 10 immunoreactivity in the GCL. Consistent with the data from glaucomatous retinas (Fig. 3), we found that elevated IOP significantly increased IL- 10 immunoreactivity in the endfeet of Muller glia of BSA-treated control retina compared with BSA- treated sham control (Fig. 9C and D). In AIBP-treated animals, however, we found that IL-10 immunoreactivity was significantly decreased in the endfeet of Muller glia against elevated IOP (Fig. 9C and D). There was no significant difference in IL-10 immunoreactivity between BSA- and AIBP-treated sham control groups (Fig. 9C and D).

Discussion

Factors contributing to neuroinflammation, the process that plays a critical role in glaucomatous neurodegeneration, are poorly understood. In the present study, we identified AIBP as an important neuroprotective protein in the retina. We demonstrated that elevated IOP reduced AIBP expression in glaucomatous retina and that Apoal bp~ ~ Muller glia had an upregulated TLR4-mediated inflammatory response via increasing IL- 10 expression that is accompanied by compromised mitochondrial dynamics and energy depletion. In parallel, we found that AB IP deficiency contributed to dysfunctional RGC mitochondria, oxidative stress and visual dysfunction. Also, AIBP deficiency exacerbated RGC death in response to elevated IOP. Remarkably, recombinant AIBP administration prevented apoptotic RGC cell death and inflammatory responses in Muller glia in vivo. Here we propose for the first time that AIBP could be a therapeutic target for treating neuroinflammation, mitochondrial dysfunction and RGC death in glaucoma progression.

AIBP has been known to accelerate cholesterol efflux from endothelial cells and macrophages (4, 7-9, 23, 35). Accumulating evidence indicates that cholesterol is considered as a risk factor for POAG (47-51). Indeed, epidemiological studies indicate that POAG is linked to single-nucleotide polymorphisms of ABCA1 (47-49). Interestingly, ABCA1 is expressed in human RGCs (47, 48) and significantly decreased in RGCs in response to elevated IOP (52). In the current study, both AIBP and ABCA1 protein expression were found to be reduced in glaucomatous retina. A previous study has demonstrated that AIBP mediates the stabilization of ABCA1 by facilitating apoA-1 binding to ABCA1 and prevents ABCA1 degradation via the ubiquitination pathway (35). Although the relationship between AIBP and ABC Al, particularly in RGCs needs to be elucidated, it is likely that AIBP stabilizes ABCA1 and regulate cholesterol efflux in glaucomatous RGCs. Moreover, loss of AIBP induced by elevated IOP may contribute to deregulation of ABCA1 in glaucomatous neurodegenerati on .

Epidemiological clinical studies indicate that POAG is linked to single- nucleotide polymorphisms of TLR4 (37, 39) and recent evidence from animal studies further suggests that TLR4-dependent signaling is an important factor in the pathogenesis of POAG (14, 38). In the current study, we found that glaucomatous retina significantly increased Tlr4 gene expression. Moreover, both glaucomatous and Apoalbp~ ~ Muller glia endfeet, which are in close contact with RGCs, were characterized by increased TLR4 protein expression. Since our previous study demonstrated that AIBP bound to activated microglia via TLR4, augmented cholesterol efflux and also the disruption of lipid rafts in LPS-stimulated cells, as well as reduced TLR4 dimerization (7, 11), the current results strongly suggest that AIBP mediates inhibition of TLR4 activity in Muller glia and may have a critical role in protection against glaucomatous neuroinflammation. Recent evidence suggests that acute IOP elevation induces TLR4-mediated inflammasome activation, including pyrin domain containing 1 (NLRP1) and NLRP3, and activates the IL-ip cascade in the retina (14, 15). Further, genetic deletion or pharmacological inhibition of TLR4 significantly reduces RGC death and proinflammatory responses in experimental glaucoma (38, 53, 54). Thus, we propose that loss of AIBP and activation of TLR4 signaling in glaucomatous Muller glia are critical to inflammatory response-mediated glaucomatous RGC degeneration. Indeed, this notion is strongly supported by our results that show a significant increase in IL-ip protein expression in both glaucomatous and Apoalbp~ ~ Muller glia endfeet.

In transfected cells, AIBP was localized to mitochondria (22), but the role of AIBP in regulation of mitochondrial structure and function in mammalian cells remained unknown. Interestingly, activated TLR4 signaling is associated with mitochondrial damage in microglia caused by intracellular ROS and defective mitochondrial dynamics (20, 21). In the current study, we demonstrated for the first time that the loss of AIBP impaired mitochondrial network and function in Muller glia endfeet via induction of mitochondrial fragmentation and reduction of ATP production. Recent evidence suggests that Muller glia-induced neuroinflammation is linked with RGC death (55) and that Muller glia-mediated lactate is a critical source in maintaining RGC energy metabolism and survival (56). Given our findings and those of others, it is conceivable that loss of AIBP augments the TLR4 signaling in glaucomatous Muller glia that might compromise mitochondrial network, increase ROS production and deplete energy production, leading to dysfunction of Muller glia, activation of inflammatory responses and RGC death. Another possible mechanism of a protective role of AIBP in glaucoma is related to angiogenesis. Muller glia are associated with the regulation of angiogenesis that is linked to a severe form of secondary glaucoma commonly associated with proliferative diabetic retinopathy, ischemic central retinal vein occlusion, and ocular ischemic syndrome (57). Further, Muller glia activation is increased with age in glaucomatous DBA/2J mice, showing abnormal neovascularization (58). Since previous studies have demonstrated that loss of AIBP results in dysregulated sprouting/branching angiogenesis and that enhanced AIBP expression inhibits angiogenesis (4, 8), it is possible that Muller glia dysfunction induced by loss of AIBP may contribute to abnormal angiogenesis in secondary glaucoma. In addition, microglial activation is a common inflammatory response to elevated IOP -induced retinal injury and microglia-mediated TLR4 activation is involved in retinal degeneration (14, 59). Our findings collectively suggest the possibility that loss of AIBP exacerbates vulnerability to elevated IOP- induced RGC death through TLR4 signaling activation, mitochondrial dysfunction and inflammatory response by activated Muller glia and microglia.

We also provide the first evidence that AIBP regulates structural and functional integrity of mitochondria in RGCs. Our study intriguingly demonstrated a significant loss of mitochondrial AIBP in the retina in response to elevated IOP. Moreover, loss of AIBP significantly impaired not only the OXPHOS system in the retina but also mitochondrial dynamics and ATP production in RGCs, resulting in extensive mitochondrial fragmentation, energy depletion and mitophagosome formation. A separate study from our group demonstrated that AIBP associated with MFN1 and MFN2 and regulated mitophagy and thus contributed to mitochondrial quality control (Choi et al., Submitted). Since we have demonstrated that impairment of mitochondrial dynamics and function was strongly linked to RGC death in glaucomatous neurodegeneration (16, 19, 25, 60-62), it is likely that AIBP might play a critical role in mitochondrial quality control to maintain cellular homeostasis by preserving mitochondrial dynamics and bioenergetics in RGCs against glaucomatous insults such as elevated IOP and oxidative stress. Since degenerative pruning of RGC dendrites and their dysfunctional synapse, as well as mitochondrial degeneration have been implicated as early features of glaucomatous neurodegeneration (63, 64), combined, these and our results with decreased AIBP in the inner retina and Muller glia activation suggest an intriguing possibility that loss of AIBP in the inner retina may affect not only RGC soma and axon in the GCL, but also RGC dendrites and their synapses in the IPL via an autocrine/paracrine manner during glaucomatous neurodegenerati on .

SIRT3, a mitochondrial NAD⁺-dependent deacetylase, has protective roles against oxidative stress, neuroinflammation and neurodegeneration (65, 66). SIRT3- mediated SOD2 activation and deacetylation reduces ROS levels, leading to the enhancement of resistance against oxidative stress (67, 68). Our study demonstrated that loss of AIBP significantly reduced the expression levels of SIRT3 and SOD2 proteins in the inner retina including RGCs. Recent evidence indicates that the SIRT3-SOD2 pathway is linked to inflammation and oxidative stress (69, 70). In line with these and our findings, it is possible that mitochondrial AIBP may contribute to the stabilization of the SIRT3-SOD2 axis, rescuing RGC mitochondria from neuroinflammation and/or oxidative stress. Under oxidative stress conditions, multiple MAPK signaling pathways, including p38 and ERK1/2, are activated (45, 46). Our study demonstrated that loss of AIBP persistently increased phosphorylation of p38 and ERK1/2 in the retina. p38 is phosphorylated in response to cytokines and oxidative stress (71, 72) and activation of the p38 signaling pathway leads to mitochondrial dysfunction and inflammatory responses (73-76). Because a p38 inhibitor blocks mitochondrial dysfunction and inhibits cytochrome c release (77), it is likely that retinal AIBP not only plays a role in the stabilization of mitochondrial proteins, but also inhibits stress-activated intracellular signaling responses, such as p38 activation. On the other hand, ERK1/2 is also activated in response to cytokines, free radicals and inflammatory factors in neurodegenerative diseases (78, 79). In experimental glaucoma, ERK1/2 activation has a neuroprotective effect on RGC survival (80-83). Since phosphorylation of p38 and ERK1/2 induced by lipopolysaccharide (LPS) in alveolar macrophages is inhibited in the presence of AIBP (9), our study suggests that AIBP may contribute to a differential regulation of MAPK signaling pathways in RGCs against inflammatory responses and/or oxidative stress.

Our recent studies have demonstrated that recombinant AIBP protein administration reduced not only spinal myeloid cell lipid rafts, TLR4 dimerization, neuroinflammation, and glial activation in facilitated pain states, but also LPS- induced airspace neutrophilia, alveolar capillary leak, and secretion of IL-6 in acute lung inflammation (9, 11). These results demonstrated a mechanism by which AIBP regulates neuroinflammation and suggested the therapeutic potential of AIBP in treating preexisting pain states and lung inflammation.

In the current study, we found that intravitreal administration of AIBP protected RGCs against apoptotic cell death by a significant reduction of IL-ip- mediated inflammatory responses in activated Muller glia in response to elevated IOP. Thus, this data demonstrates that AIBP has therapeutic value for treating glaucoma via blocking glia-driven neuroinflammation.

Conclusion

This study connects for the first time AIBP with protecting mechanisms controlling mitochondrial pathogenic mechanisms, neuroinflammation and RGC death as illustrated in Fig. S5. Here, we demonstrate that combined therapeutic strategies that block glia-driven inflammatory TLR4/IL-1P axis and mitochondrial dysfunction in glaucomatous neurodegeneration are therapeutically effective.

Figure Legends

Fig, 1. AIBP expression is decreased in glaucomatous retinas and pressure-induced RGCs. (A) Apoalbp gene expression in control and injured retina at 1 day after acute IOP elevation, n = 4 mice. (B) AIBP protein expression in control and injured retina at 1 day after acute IOP elevation, n = 3 mice. (C) Representative images showed AIBP (green) and TUJ1 (red) immunoreactivities at 1 day after acute IOP elevation. Arrows indicate AIBP immunoreactivity co-labeled with TUJ 1 in RGC somas and arrowhead indicates AIBP co-labeled with TUJ1 in RGC axon bundle. (D) AIBP protein expression in control and injured RGCs at 3 day after elevated HP. n = 3 independent experiments with cultures. (E) Representative images showed AIBP (green), TUJ1 (red) and Bm3a (yellow) immunoreactivities. (F) In higher magnification images, arrows indicate AIBP immunoreactivity co-labeled with TUJ1 in RGC somas and arrowhead indicates AIBP co-labeled with TUJ1 in RGC axon bundle. (G) Quantitative fluorescent intensity showed a significant decrease in AIBP immunoreactivity in the inner retina of glaucomatous DBA/2J mice, n = 4 mice. (H) Representative images showed ABCA1 (green), AIBP (red) and Bm3a (yellow) immunoreactivities. Concave arrowheads indicate ABC Al -positive RGCs co-labeled with AIBP and Brn3a. (I) Quantitative fluorescent intensity showed a significant decrease in ABCA1 immunoreactivity in the GCL of glaucomatous DBA/2J mice, n = 4 mice. Error bars represent SEM. Statistical significance determined using Student’s / test. * < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Blue is Hoechst 33342 staining. Scale bar: 20 pm. CNT, control; GCL, ganglion cell layer; HIOP, high intraocular pressure; EHP, elevated hydrostatic pressure; INL, inner nuclear layer; IPL, inner plexiform layer; NP, no pressure; ONL, outer nuclear layer; OPL, outer plexiform layer.

Fig, 2. AIBP deficiency exacerbates vulnerability to elevated IOP in RGCs and triggers visual dysfunction. RGC loss was measured in the retina of 4 month-old WT and age-matched Apoalbp~ ~ mice at 4 weeks after acute IOP elevation, and visual function were measured in 4-month-old Apoalbp~ ~ mice. (A) The average of IOP elevation in WT mice, n = 5-7 mice. (B) Representative images from whole-mount immunohistochemistry showed Brn3a-positive RGCs in WT and Apoalbp~ ~ following acute IOP elevation. (C) Quantitative analysis by RGC counting using whole-mount immunohistochemistry for Bm3a in WT and Apoalbp~ ~ following acute IOP elevation, n = 5-7 mice. (D) Visual function test in WT and naive Apoalbp~ ~ mice by OKT analyses, n = 10-15 mice. (E) Visual function test in WT and naive Apoal bp~ ~ mice by VEP analyses. Note that there were no changes of VEP responses and latency in WT and naive Apoal bp~ ~ mice, n = 15 mice. (F) Total recordings of VEP responses. Left: total recordings of the VEP response of WT mice. Right: total recordings of the VEP response of naive Apoalbp~ ~ mice. (G) Representative images of CTB (red) labeling in the SCs of in WT and naive Apoalbp~ ~ mice, n = 3 mice. (H) Quantitative analysis of CTB fluorescence density in the SCs of WT and naive Apoalbp~ ~ mice, n = 3 mice. Error bars represent SEM. Statistical significance determined using one-way ANOVA or Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Scale bar: 100 pm (B and I); 20 pm (D). CNT, control; HIOP, high intraocular pressure; ns, not significant; SC, superior colliculus.

Fig, 3. Glaucomatous and Apoalbp~ ~ Muller glia endfeet upregulate TLR4 and IL- 10 expression. Immunohistochemical analyses for TLR4 and IL- 10 were conducted on retina wax sections in glaucomatous and Apoalbp~ ~ retina. (A and B) Representative images showed TLR4 and IL- 10 immunoreactivities in Muller glia of the inner retinas from human patient with POAG, and glaucomatous DBA/2J and naive Apoalbp~ ~ mice. (C and D) Quantitative fluorescent intensity showed a significant increase in TLR4 and IL-10 immunoreactivities in Muller glia endfeets from human patient with POAG (n = 5 retina sections per group), and glaucomatous DBA/2J and naive Apoalbp~ ~ mice compared with control groups (n = 4 mice per group). Error bars represent SEM. Statistical significance determined using Student’s / test. **P < 0.01; *** < 0.001; ****P < 0.0001. Blue is Hoechst 33342 staining. Scale bar: 20 pm (A and B). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; NFL, nerve fiber layer.

Fig, 4. AIBP deficiency induces mitochondrial fragmentation, outer membrane onionlike swirls, lower crista density, and reduces ATP production in Muller glia endfeet. (A) SBEM WT volume showing typical cytoplasmic structures; mitochondria (yellow trace) highlighted. (B) SBEM Apoalbp~ ~ volume showing mitochondria (yellow trace) with lower crista density (red arrowheads) and dark outer membrane onion-like swirls (blue trace). (C) WT surface rendering (Volume: 4,830 pm³ 30 pm x 23 pm x 7 pm; voxel size: 3 nm x 3 nm x 50 nm) highlighting long tubular mitochondria (yellow) (cytoplasmic membrane— blue). (D) Surface rendering (Volume: 4,408 pm³, 29 pm x 19 pm x 8 pm; voxel size 3 nm x 3 nm x 50 nm) showing short fragmented mitochondria (yellow) in Apoal bp~ ~. (E-G) Expedited and accurate segmentation and analysis of mitochondria. (E-F) Cross image planes (a-d) showing the need for 3DEM. 357 electron micrographs (50-nm step) were serially collected to follow many mitochondria through the large volume. (G) Approach to determine mitochondrial length (red) and variable shapes. (H) Surface rendering showing long tubular forms of mitochondria in WT. (I) Surface rendering showing smaller, round forms of mitochondria in Apoalbp~ ~ . (J) The volume of mitochondria was not significantly different in the Apoalbp~ ~. (K) The mitochondrial volume density in the Apoalbp~ ~ was almost identical to the WT. (L) No significant difference in the number of mitochondria between WT and Apoalbp~ ~. (M) The form factor for Apoalbp~ ~ mitochondria was significantly lower, confirming less elongation. (N) The mitochondrial length was significantly lower in the Apoal bp~ ~. (O) The crista density was significantly lower in the Apoal bp~ ~. (P) The rate of ATP production per mitochondrial volume was lower in the Apoalbp~ ~. (Q) The modeled rate of ATP production per mitochondrion was no different in the Apoal bp~ ~. (R) There was a significant lowering of ATP availability per unit cellular volume in the Apoalbp~ ~. n = 3 Muller glia from 2 mice per group. Scale bars: 1 pm (A and B), 50 nm (C-I). FIG. 5. AIBP deficiency impairs mitochondrial dynamics and OXPHOS activity in the retina. Mitochondrial AIBP expression was assessed in the retina of a mouse model of acute IOP elevation and alteration of mitochondrial dynamics and OXPHOS were assessed in the retina of WT and Apoalbp~ ~ mice. (A) Using fractionation of cytosolic and mitochondrial extracts, mitochondrial AIBP protein expression in control and injured retina at 1 day after acute IOP elevation, n = 4 mice. (B) OPA1 and MFN2 protein expression in the retina of WT and Apoalbp~ ~ mice, n = 3 mice.

(C) Representative images showed OPA1 (green), cytochrome c (red) and Bm3a (yellow) immunoreactivities in the wax sections from WT and Apoalbp~ ~ retinas. Arrowheads indicate accumulation of OPA1 co-labeled with cytochrome c in RGC somas in WT mice and arrows indicate OPA1 -labeled Muller glia endfeet, n = 3 mice.

(D) DRP1 and pDRPl S637 expression in the retina of WT and Apoal bp~ ~ mice, n = 3 mice. (E) Representative images showed DRP1 (green) and Brn3a (red) immunoreactivities in the wax sections from WT and Apoalbp~ ~ retinas. Arrowheads indicate accumulation of DRP1 co-labeled with Brn3a in RGC somas in WT and Apoalbp~ ~ mice, n = 3 mice. (F) OXPHOS Cxs protein expression in the retina of WT and Apoal bp~ ~ mice, n = 3 mice. Error bars represent SEM. Statistical significance determined using Student’s / test. * < 0.05; **P < 0.01; **** < 0.0001. Blue is Hoechst 33342 staining. Scale bar: 20 pm. CNT, control; Cx, complex; GCL, ganglion cell layer; HIOP, high intraocular pressure; HP, hydrostatic pressure; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer.

FIG. 6. AIBP deficiency triggers mitochondrial fragmentation, swelling and rounding, and ER swelling in RGC somas. (A) Tomographic volume of WT RGC showing typical mitochondrial and ER structures. 2xinset displays well-formed mitochondria and ER (white arrowheads). (B) Tomographic volume of Apoalbp~ ~ RGC showing rounded mitochondria with lower crista density and swollen ER. 2xinset displays swollen ER (white arrowheads), including one contacting a mitochondrion. (C) Apoalbp~ ~ volume showing two adjacent mitochondria and ER sandwiched at their fission site. (D) Mitochondrial outer membrane (blue trace), IBM (yellow trace) and ER (green fill), partly dilated (arrow). Bulge in top mitochondrion (white arrowhead) caused by expansion of both outer and IBM. Inward bulge in bottom mitochondrion (black arrowhead) caused by expansion of the IBM only. (E) 3D Surface-rendering overlaid on a Apoalbp~ ~ volume. (F) Even though many ER strands are dilated, mitochondrial fission can proceed in the Apoalbp~ ~. (G) Abnormal mitochondria with onion-like swirling membrane (white arrow) were common in the Apoalbp~ ~. (H) Onion-like swirl (blue) not part of the IBM (yellow). (I) Surface rendering of SBEM sub-volume (volume: 5,814 pm³, 34 pm x 19 pm x 9 pm, voxel size:3nm x 3nm x 50nm) showing cytoplasmic membrane (green), neurites (green), nucleus (blue) long tubular form (yellow) and branched mitochondria (red) in the WT. (J) Surface rendering (volume: 7,752 pm³, 34 pm x 19 pm x 12 pm, voxel size: 3 nm x 3 nm x 50 nm) showing the cytoplasmic membrane, nucleus, dendrites and axons, and smaller round form (yellow) and branched (red) mitochondria in Apoalbp~ ~. (K-O) Measurements of structural features of mitochondria. (K) Volume of mitochondria was significantly greater in Apoal bp~ ~. (L) Mitochondrial volume density was higher in Apoalbp'^/_. (M) No significant difference in the number of mitochondria. (N) Form factor was significantly lower in the Apoal bp~ ~. confirming rounded mitochondria. (O) Mitochondria lengths were significantly decreased in the Apoal bp~ ~. n = 3 RGCs from 2 mice per group. Error bars represent SEM. Statistical significance determined using Student’s / test. *P < 0.05; ****P < 0.0001. Scale bars: 500 nm (A-H), 2 pm (I- J).

FIG. 7. AIBP deficiency reduces cristae density, ATP production and mitofilin protein expression in RGC mitochondria. (A) The mean crista density was significantly lower in the mitochondria of Apoal hp~ ~ RGC somas. (B) The mean modeled rate of ATP production per mitochondrion was higher in the Apoalbp~ ~ RGC soma. (C) The mean rate of ATP production per unit mitochondrial volume was lower in the Apoalbp~ ~ RGC soma. (D) There was no significant lowering of modeled availability of ATP per unit cellular volume (P = 0.39). (E-J) The crista density was lower in the Apoal bp~ ' mitochondria due in part to onion-like outer membrane protuberances.

Mitochondria-associated ER strands were often dilated. (E) A 4.2 nm-thick slice from a WT tomographic volume of RGC showing typical cristae. A well-formed ER strand is nearby (arrowhead). (F) Surface rendering of the segmented volume emphasizes the density of the cristae (shades of brown). The mitochondrial outer membrane is shown in translucent maroon. (G) A 4.2 nm-thick slice from an Apoalbp~ ~ tomographic volume of RGC showing cristae that is less densely packed and an onionlike protuberance. An adjacent ER strand is dilated. (H) Surface rendering of the segmented volume emphasizes the less-dense cristae packing and the 3 protuberances (black) that occupy part of the volume that would normally have been occupied by cristae. (I) Surface rendering without the protuberances emphasizes the part of the volume not occupied by cristae (arrow points to one of these volumes). (J) Surface rendering showing only the protuberances to highlight their size relative to the mitochondrial volume. (K) Mitofilin protein expression was assessed by Western blot analysis in WT and Apoal bp~ ~ retinas, n = 4 mice. (L) Mitofilin gene expression was assessed by quantitative PCR analysis in WT and Apoalbp~ ~ retinas, n = 3 mice. Error bars represent SEM. Statistical significance determined using Student’s / test. *P < 0.05. Scale bars: 500nm.

FIG. 8. AIBP deficiency induces oxidative stress and activates MAPK signaling in the retina. Oxidative stress and MAPKs signaling were assessed in the retina of WT and Apoalbp~ ~ mice. (A and B) SIRT3 and SOD2 protein expression in the retina of WT and Apoalbp~ ~ mice, n = 3-7 mice. (C and D) Representative images showed SIRT3 (green), SOD2 (green) and Bm3a (red) immunoreactivities in the wax sections from WT and Apoalbp~ ~ retinas. Arrowheads indicate accumulation of SIRT3 or SOD2 colabeled with Brn3a in RGC somas in WT and Apoalbp~ ~ mice, n = 3 mice. (E and F) Phospho-p38 (pp38) and phospho-ERKl/2 (pERKl/2) protein expression in the retina of WT and Apoal bp~ ~ mice, n = 3-6 mice. (G and H) Representative images showed pp38 (green), pERKl/2 (green) and Bm3a (red) immunoreactivities in WT and Apoalbp~ ~ retinas. Arrowheads indicate accumulation of phospho-p38 co-labeled with Bm3a in RGC somas in WT and Apoalbp~ ~ mice, n = 3 mice. Error bars represent SEM. Statistical significance determined using Student’s t test. *P < 0.05; **P < 0.01. Blue is Hoechst 33342 staining. Scale bar: 20 pm. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer.

FIG. 9. AIBP promotes RGC survival and prevents glia-mediated inflammatory responses against elevated pressure. Apoptotic cell death was assessed in a mouse model of acute IOP elevation, and inflammatory responses and/or cytokine production was assessed in retinal Muller glia or cultured BV-2 microglia exposed to EHP. (A and B) Recombinant AIBP protein or BSA (1 L, 0.5 mg/ml) was intravitreally injected at 2 days before acute IOP elevation and assessed TUNEL-positive cells in the retina of WT mice at 1 day after acute IOP elevation. Following RBPMS (green) immunohistochemistry, TUNEL (red) staining was conducted. (A) Representative images showed RBPMS -positive RGCs in the GCL and TUNEL-positive cells in the retinas. (B) Quantitative analysis by TUNEL-positive cell counting, n = 5 mice. (C) Representative images showed IL- 10 immunoreactivity in the inner retina. (D) Quantitative analyses for fluorescent intensity showed that AIBP treatment significantly decreased in IL-ip immunoreactivity in Muller glia endfeets against elevated IOP. n = 4 mice. Error bars represent SEM. Statistical significance determined using one-way ANOVA or Student’s t test. *P < 0.05; **P < 0.01; ****P < 0.0001. Blue is Hoechst 33342 staining. Scale bar: 20 pm. BSA, bovine serum albumin; CNT, control; GCL, ganglion cell layer; EHP, elevated hydrostatic pressure; HIOP, high intraocular pressure; INL, inner nuclear layer; IPL, inner plexiform layer; NP, no pressure; ONL, outer nuclear layer; OPL, outer plexiform layer.

FIG. 10: AIBP deficiency induces abnormal structures of mitochondria and rough ER, and mitophagosome formation in Muller glia endfeet. (A) Color added to an additional slice to highlight the WT mitochondria (yellow trace). (B) Color added to an additional slice to not only identify the mitochondria (yellow trace), but also to point to the mitochondria with lower cristae density (red arrowheads) compared to the WT traced and a dark outer membrane onion-like swirl (blue trace). (C-E) Serial slice images through the tomographic volume from WT and Apoal bp~ Muller glia endfeets. (C) Serial slice images from WT Muller glia endfeet showing a long tubular form of mitochondrion with abundant rough ER. (D) Serial slice images from Apoal bp~ Muller glia endfeet to point to the mitochondria with lower cristae density and dark outer membrane onion-like swirls (blue arrow). (£) Serial slice images from Apoal bp~ Muller glia endfeet showing an abnormal mitochondrion with a vesicular inclusion (arrowhead) as well as lower rough ER density and dilated rough ER strands (arrows). Scale bars: 1 pm (A-D'y

FIG. 11 : AIBP deficiency impairs mitochondrial dynamics and OXPHOS activity in the retina. (A) Representative images from immunohistochemical analyses showed OPA1 (green) and GS (red) immunoreactivities in the wax sections from WT and Apoal bp~ ~ retinas. Note that OPA1 immunoreactivity was decreased in the inner retinal layer but increases in Muller glia of Apoal bp~ ~ retina. 8) Representative images from immunohistochemical analyses showed DRP1 (green) and GS (red) immunoreactivities in the wax sections from WT and Apoal bp~ ~ retinas. Note that DRP1 immunoreactivity was decreased in the inner retinal layer but did not detected in Muller glia of Apoal bp~ ~ retina. Blue is Hoechst 33342 staining for nucleus. Scale bar: 20 pm. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer.

FIG. 12: AIBP deficiency does not affect mitochondrial dynamics- and oxidative stress- related gene expression in the retina. (A and 8) Opal, Nfn2, and Drpl, as well as Sirt3 and Sod2 gene expression was assessed by quantitative PCR analysis in WT and Apoal bp~ ~ retinas. Note that there were no statistically significant differences in Opal, Mfri2, and Drpl (A), as well as Sirt3 and Sod2 (B) gene expression, n = 3 mice. Error bars represent SEM.

FIG. 13: AIBP deficiency induces abnormal structure of mitochondria and ER, and mitophagosome formation in RGC soma. (A-D) Serial slice images through the tomographic volume from WT and Apoal bp~ RGC somas. (A) Serial slice images from WT Muller glia endfeet showing a long tubular form of mitochondria with normal structure of ER strands (arrowheads). (8) Serial slice images from Apoalbp~ RGC soma to point to the dark outer membrane onion-like swirls (arrows) and dilated ER strands (arrowheads). (C) Serial slice images from Apoal bp~ ~ RGC soma showing a ring-shaped mitochondrion (arrow). (£>) Serial slice images from Apoal hp~ RGC soma showing two ongoing autophagosome formation. Scale bars: 1 pm (A- ).

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Claims

WHAT IS CLAIMED IS:

1. A method for:

- decreasing or slowing the rate of induced RGC death in response to elevated intraocular pressure (IOP), wherein the method comprises: administering a pharmaceutically acceptable formulation or a pharmaceutical composition to a subject in need thereof, wherein the pharmaceutically acceptable formulation or pharmaceutical composition is comprised of:

(1) a polypeptide composition, wherein the polypeptide composition is or is comprised of an ApoA-I Binding Protein wherein the polypeptide composition has, or is capable of providing, an ApoA-I Binding Protein polypeptide activity, or

(2) a nucleic acid composition that increases expression or activity of, or encodes for, a polypeptide composition, wherein the polypeptide composition is or is comprised of an ApoA-I Binding Protein polypeptide, wherein the polypeptide composition has, or is capable of providing, an ApoA-I Binding Protein polypeptide activity; or

(3) an ApoA-I Binding Protein-inducing compound or composition.

2. The method of claim 1, wherein the APOA1 BP-inducing compound or composition increases or stimulates or activates the activity of a APOA1BP promoter or transcriptional regulatory sequence or motif for expression of the polypeptide composition.

3. The method of claim 1, wherein the nucleic acid that expresses or encodes for the polypeptide composition is contained within an expression vehicle, vector, recombinant virus, or equivalent thereof.

4. The method of claim 3, wherein the vector or virus is comprised of an adenovirus vector or an adeno-associated virus (AAV) vector, a retrovirus, a lentiviral vector, a herpes simplex virus, a human immunodeficiency virus (HIV), or a synthetic vector.

5. The method of claim 4, wherein the AAV vector is comprised of: an adeno-associated virus (AAV), an AAV serotype, an AAV variant, wherein optionally the AAV variant is AAV5, AAV6, AAV8 or AAV9, AAV-DJ or AAV-DJ/8™ (Cell Biolabs, Inc., San Diego, CA), a rhesus-derived AAV, wherein the rhesus-derived AAV is AAVrh.10hCLN2, or an AAV capsid mutant or AAV hybrid serotype.

6. The method of claim 5, wherein the AAV is engineered to increase efficiency in targeting a specific cell type that is non -permissive to a wild type (wt) AAV and/or to improve efficacy in infecting only a cell type of interest.

7. The method of claim 5, wherein the AAV serotype is retargeted or engineered as a hybrid serotype by one or more modifications selected from the group consisting of: 1) a transcapsidation, 2) adsorption of a bi-specific antibody to a capsid surface, 3) engineering a mosaic capsid, and 4) engineering a chimeric capsid.

8. The method of claim 1, wherein the nucleic acid that expresses or encodes the APOA1BP polypeptide is comprised of an RNA or an DNA.

9. The method of claim 1, wherein the RNA or the DNA are formulated in a lipid formulation or a liposome or equivalents, and optionally the lipid formulation or equivalent is injected, optionally is injected intramuscularly (IM). and optionally the RNA or the DNA are formulated in a lipid formulation as described in U.S. patent application no. US 20210046173 Al, and optionally the RNA or the DNA are formulated in a lipid formulation comprising or formulated as a liposome, or a lipid nanoparticle (LNP), or nanoliposome, that comprises: non-cationic lipids comprise a mixture of cholesterol and DSPC, or a PEG-lipid, or PEG-modified lipid, or LNP, or an ionizable cationic lipid.

10. The method of claim 1, wherein the polypeptide composition is a mammalian APOA1BP polypeptide.

11. The method of claim 10, wherein the polypeptide is a human APOA1BP polypeptide.

12. The method of claim 1, wherein the subject is a human.

13. The method of claim 1, wherein the subject is a mammal.

14. The method of claim 1, wherein the polypeptide composition is comprised of a recombinant APOA1BP polypeptide having an APOA1BP activity.

15. The method of claim 1, wherein the polypeptide composition is comprised of a synthetic APOA1BP polypeptide.

16. The method of claim 1, wherein the pharmaceutically acceptable formulation is comprised of an ApoA-I Binding Protein polypeptide-inducing compound or composition or is a APOA1BP activity-stimulating compound or composition.

17. The method of claim 1, wherein the said administration is by intraocular or intravitreal administration.

18. The method of claim 1, wherein said administration is by intrathecal injection.

19. The method of claim 1, wherein said administration is by intraocular injection.

20. The method of claim 1, wherein the formulation or pharmaceutical composition is formulated for intravenous (IV) administration.

21. The method of claim 1, wherein the pharmaceutically acceptable formulation is formulated in the form of or with a nanoparticle, a particle, a micelle, a liposome, a lipoplex, a polymersome, a polyplex or a dendrimer.

22. The method of claim 1, wherein the pharmaceutically acceptable formulation is formulated in or is in the form of a tablet, a pill, a capsule, a gel, a geltab, a liquid, a powder, an emulsion, a lotion, an aerosol, a spray, a lozenge, an aqueous or a sterile or an injectable solution, or an implant.

23. The method of claim 22, wherein the implant is an intraocular implant.

24. The method of claim 21, wherein the nanoparticle, particle, micelle or liposome or lipoplex, polymersome, polyplex or dendrimer further comprise or express a cell or CNS penetrating moiety or peptide or a CNS targeting moiety or peptide.

25. The method of claim 24, wherein the polypeptide composition or particle further comprises a CNS penetrating peptide.