WO2022046919A2 - Compositions and methods for the treatment of ocular neuroinflammation - Google Patents
Compositions and methods for the treatment of ocular neuroinflammation Download PDFInfo
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- WO2022046919A2 WO2022046919A2 PCT/US2021/047569 US2021047569W WO2022046919A2 WO 2022046919 A2 WO2022046919 A2 WO 2022046919A2 US 2021047569 W US2021047569 W US 2021047569W WO 2022046919 A2 WO2022046919 A2 WO 2022046919A2
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- aibp
- aav
- apoa1bp
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0048—Eye, e.g. artificial tears
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0306—Animal model for genetic diseases
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0368—Animal model for inflammation
Definitions
- the present invention generally relates to neuroinflammation in the eye, including neuroinflammation in the eye during glaucomatous neurodegeneration in glaucoma.
- 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.
- 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).
- IOP intraocular pressure
- 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.
- APOA1BP ApoA-I Binding Protein
- AIBP AIBP
- ALBP a protein related thereto
- nucleic acids encoding AIBP or these related proteins BACKGROUND
- AIBP ApoSipoprotein A-I Binding Protein
- NAXE NAD(P)HX epimerase
- APOA1BP human AIBP gene
- 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.
- IOP intraocular pressure
- 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.
- compositions and methods useful for reducing ocular neuroinflammation in particular, ocular neuroinflammation resulting from glaucoma.
- 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)
- 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:
- 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
- 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
- glaucoma is open-angle glaucoma or closed angle glaucoma
- IOP intraocular pressure
- an ApoA-I Binding Protein APOA1BP, AIBP, or AI-BP
- APOA1BP ApoA-I Binding Protein
- AIBP AIBP
- AI-BP ApoA-I Binding Protein
- a APOA1BP polypeptide is a nucleic acid that expresses or encodes a APOA1BP polypeptide or a polypeptide having a APOA1BP polypeptide activity;
- an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP)-inducing compound or composition;
- APOA1BP ApoA-I Binding Protein
- glaucoma is openangle glaucoma or closed angle glaucoma
- IOP intraocular pressure
- 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/8TM (Cell Biolabs, Inc., San
- 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;
- APOA1BP ApoA-I Binding Protein
- APOA1BP ApoA-I Binding Protein
- 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.
- a formulation or a pharmaceutical composition comprising:
- an ApoA-I Binding Protein APOA1BP, AIBP, or AI-BP
- APOA1BP ApoA-I Binding Protein
- AIBP AIBP
- AI-BP ApoA-I Binding Protein
- 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
- an ApoA-I Binding Protein APOA1BP, AIBP, or AI-BP
- APOA1BP ApoA-I Binding Protein
- AIBP AIBP
- AI-BP ApoA-I Binding Protein
- IOP intraocular pressure
- formulations or pharmaceutical compositions comprising:
- an ApoA-I Binding Protein APOA1BP, AIBP, or AI-BP
- APOA1BP ApoA-I Binding Protein
- AIBP AIBP
- AI-BP ApoA-I Binding Protein
- an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP)-inducing compound or composition for use in:
- glaucoma is open-angle glaucoma or closed angle glaucoma
- IOP intraocular pressure
- 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:
- IOP acute intraocular pressure
- 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;
- 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;
- 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. 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.
- 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. 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. 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;
- 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
- error bars represent SEM
- 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. 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;
- 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
- 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.
- APOA1BP ApoA-I Binding Protein
- 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.”
- 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.
- glaucoma is open-angle glaucoma or closed angle glaucoma
- 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:
- 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
- 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
- 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/8TM (Cell Biolabs, Inc., San Diego, CA), a rhesus-derived AAV vector, wherein optionally the rhes
- 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.
- 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;
- APOA1BP ApoA-I Binding Protein
- APOA1BP 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,
- APOA1BP ApoA-I Binding Protein
- 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.
- 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.
- 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.
- the particle is further comprised of a cell or CNS penetrating moiety or peptide or a CNS targeting moiety or peptide.
- polypeptide comprised of the APOA1BP polypeptide further comprises a cell or CNS penetrating moiety or peptide or a CNS targeting moiety or peptide.
- 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.
- a pharmaceutically acceptable formulation comprising:
- an ApoA-I Binding Protein APOA1BP, AIBP, or AI-BP
- APOA1BP, AIBP, or AI-BP ApoA-I Binding Protein
- 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:
- glaucoma is open-angle glaucoma or closed angle glaucoma
- IOP intraocular pressure
- formulations or pharmaceutical compositions comprising:
- an ApoA-I Binding Protein APOA1BP, AIBP, or AI-BP
- APOA1BP ApoA-I Binding Protein
- AIBP AIBP
- AI-BP ApoA-I Binding Protein
- a APOA1BP polypeptide is a nucleic acid that expresses or encodes a APOA1BP polypeptide or a polypeptide having a APOA1BP polypeptide activity;
- an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP)-inducing compound or composition for use in:
- glaucoma is open-angle glaucoma or closed angle glaucoma
- IOP intraocular pressure
- 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.
- APOA1BP, AIBP, or AI-BP ApoA-I Binding Protein
- 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.
- 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,.
- 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”).
- 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.
- 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.
- 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.
- acceptable vehicles and solvents that can be employed include water and Ringer's solution, an isotonic sodium chloride.
- 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.
- 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.
- 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.
- 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.
- compositions and formulations used to practice methods and uses as provided herein can be delivered by the use of liposomes.
- 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.
- target cells for example, an injured or diseased neuronal cell or CNS tissue
- 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.
- 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.
- 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.
- an active agent for example, APOA1BP nucleic acids and polypeptides
- 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.
- a compound for example, a APOA1BP nucleic acid and polypeptide
- a desired cell type for example, a retinal cell
- 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.
- active agent-containing nanoparticles for example, a secondary nanoparticle
- nanoparticles comprising a fat-soluble active agent or a fat-solubilized water-soluble active agent to act with a bivalent or trivalent metal salt.
- 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.
- AIBP peptides or polypeptides, or AIBP- comprising nanoparticles, liposomes and the like are modified to facilitate intravitreal injections.
- 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.
- 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.
- 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.
- 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.
- 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.
- a delivery vehicle is a transduced cell engineered to express or overexpress and then secrete an endogenous or exogenous AIBP.
- 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.
- 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.
- the composition to be delivered is conjugated to a cell membrane-permeant peptide.
- 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.
- 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.
- APOA1BP nucleic acids used to practice embodiments as provided herein comprise or are comprised of human APOA1BP cDNA sequence:
- human APOA1BP polypeptides used to practice embodiments as provided herein comprise or are comprised of the amino acid sequence:
- the nucleic acids, vectors or recombinant viruses are designed for in vivo or CNS delivery and expression.
- 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.
- 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.
- 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.
- 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.
- IM intramuscular
- IV intravenous
- biolistic particle delivery system for example, a so-called “gene gun”
- 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.
- a recombinant virus for example, a long-term virus or viral vector
- 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.
- a systemic vein for example, IV
- IM intramuscular
- a biolistic particle delivery system for example, a so-called “gene gun”
- 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.
- 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.
- 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).
- 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.
- expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant “naked DNA” vector, and the like.
- a "vector" can comprise a nucleic acid that can infect, transfect, transiently or permanently transduce a cell.
- a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid.
- vectors can comprise viral or bacterial nucleic acids and/or proteins, and/or membranes (for example, a cell membrane, a viral lipid envelope, etc.).
- 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.
- a vector can be stably replicated by the cells during mitosis as an autonomous structure, or can be incorporated within the host's genome.
- 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.
- 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.
- “constitutive” promoters can be those that drive expression continuously under most environmental conditions and states of development or cell differentiation.
- “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.
- 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.
- nucleic acid for example, an AIBP gene or any AIBP-encoding nucleic acid
- 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.
- 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
- 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.
- 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.
- AAV adeno-associated virus
- serotypes AAV-8, AAV-9, AAV-DJ or AAV-DJ/8TM which have increased uptake in brain tissue in vivo, are used to deliver an AIBP-encoding nucleic acid payload for expression in the CNS.
- serotypes, or variants thereof are used for targeting a specific tissue: Tissue Optimal Serotype
- RPE Retinal Pigment . . . A r . . . , mecanic A > . ...
- AAV I AAV2.
- AAV4 AAVx AA 8
- 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.
- AAVs adeno-associated viruses
- NAbs neutralizing antibodies
- 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.
- 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.
- RNA for example, mRNA
- IM intramuscularly
- RNA for example, mRNA
- ORF open reading frame
- the RNA or the DNA-carrying expression vehicle
- the RNA 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 D
- 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).
- PEG-DMG 1,2-Dimyristoyl-sn-glycerol methoxypolyethylene glycol
- PEG-DSG PEG-disteryl glycerol
- PEG-dipalmetoleyl PEG-dioleyl,
- the LNP comprises 20-99.8 mole % ionizable cationic lipids, 0.1-65 mole % non-cationic lipids, and 0.1-20 mole % PEG-lipid.
- 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 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.
- 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]hepta
- 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.
- 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-s
- DOPC
- DOPG 1.2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt
- 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.
- 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.
- 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.
- 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 io 12 vector genomes per kg body weight (vg/kg), or between about 10 10 and 10 14 vector genomes per kg body weight (vg/kg), or about 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 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.
- 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.
- 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 active agents rate of absorption, bioavailability, metabolism, clearance, and the like
- 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.
- 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.
- 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.
- 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 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.
- 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.
- 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.
- 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.
- kits for practicing the methods as provided herein.
- products of manufacture such as implants or pumps, kits and pharmaceuticals for practicing the methods as provided herein.
- products of manufacture, kits and/or pharmaceuticals comprising all the components needed to practice a method as provided herein.
- kits also comprise instructions for practicing a method as provided herein.
- 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;
- 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
- nucleic acid composition that increases expression or activity of, or encodes for, a polypeptide composition
- 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
- 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.
- nucleic acid that expresses or encodes for the polypeptide composition is contained within an expression vehicle, vector, recombinant virus, or equivalent thereof.
- 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.
- AAV adeno-associated virus
- 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/8TM (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.
- AAV adeno-associated virus
- 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.
- polypeptide composition is a mammalian APOA1BP polypeptide.
- APOA1BP mammalian ApoA-I Binding Protein
- polypeptide composition is comprised of a synthetic APOA1BP polypeptide.
- composition 14. The method of embodiment 1, wherein pharmaceutically acceptable formulation is comprised of an ApoA-I Binding Protein polypeptide-inducing compound or composition.
- 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.
- 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:
- 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
- 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
- nucleic acid that expresses or encodes for the polypeptide composition is contained within an expression vehicle, vector, recombinant virus, or equivalent thereof.
- 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.
- AAV adeno-associated virus
- 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/8TM (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.
- 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.
- 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.
- polypeptide composition is a mammalian APOA1BP polypeptide.
- APOA1BP mammalian ApoA-I Binding Protein
- polypeptide composition is comprised of a recombinant APO A IBP polypeptide having an APOA1BP activity.
- polypeptide composition is comprised of a synthetic APOA1BP polypeptide.
- composition is comprised of an ApoA-I Binding Protein polypeptide-inducing compound or composition.
- the pharmaceutically acceptable is suitable for intravenous (IV) administration.
- 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.
- polypeptide composition further comprises a CNS penetrating peptide.
- Example 1 AIBP protects retinal ganglion cells against neuroinflammation and mitochondrial dysfunction in glaucomatous neurodegeneration
- AIBP plays a critical role in protection against neuroinflammation and mitochondrial dysfunction during glaucomatous neurodegeneration.
- systemic AIBP knockout Apoal bp ⁇ ⁇ mice
- 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.
- AIBP deficiency exacerbates vulnerability to elevated intraocular pressure (IOP)- induced RGC death.
- AIBP treatment inhibits inflammatory responses in Muller glia and protects RGCs against elevated IOP.
- 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.
- 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, TRANQUIVEDTM; 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.
- IP intraperitoneal
- 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.
- 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 10 5 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).
- BDNF 50 pg/ml
- CNTF 10 pg/ml
- insulin 5 pg/ml
- 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).
- 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.
- mice were anesthetized by an IP injection of a cocktail of ketamine/xylazine as described above prior cervical dislocation.
- 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.
- EM electron microscopy
- 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.
- EM electron microscopy
- the procedure was used to optimize mitochondria structural preservation and membrane contrast.
- Western blot and PCR analyses extracted retinas were immediately used.
- 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.
- Primary antibodies FIG. 14, or supplementary (supp.) Table 1
- 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).
- TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling
- 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.
- RGCs RNA-binding protein with multiple splicing
- 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.
- 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 ACMTM:acetone overnight.
- the tissues were transferred to 100% DURCUP ANTM 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 (ZEISSTM, 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.
- EM tomography experiments were conducted on a FEI TITAN HALOTM 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.
- IBM inner boundary
- 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.
- RNA from the retina was isolated using NUCLEOSPINTM RNA columns (Clontech, CA, USA). Isolated RNA was reverse transcribed using RNA to cDNA ECODRYTM (Clontech) following the manufacturer’s instructions. Quantitative PCR (qPCR) was performed using KAPA SYBR FASTTM Universal qPCR kit (KAPA Biosystems, KK4602TM, Roche Diagnostics, IN, USA), with primers ordered from Integrated DNA Technologies (IDT, CA, USA), and a ROTOR GENE QTM 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).
- mice Spatial visual function was performed on a virtual OKT system (OPTOMOTRYTM (or OptoMotryTM); 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.
- 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).
- 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.
- the mice were fixed via cardiac perfusion with 4% paraformaldehyde (Ted Pella) following an IP injection of a mixture of ketamine/xylazine.
- 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).
- AIBP plays a unique role of targeting cholesterol efflux machinery to TLR4- occupied inflammarafts (10, 11).
- 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).
- TLR4 and IL- 10 proteins were determined in human patients with POAG and DBA/2J mice.
- TLR4 and IL- 10 immunoreactivity were significantly increased 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).
- GS glutamine synthase
- IL-10 immunoreactivity was increased in both processes and endfeet of Muller glia of the IPL, GCL and NFL (Fig. 3 A and B).
- 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).
- 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).
- 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.
- ring-shaped mitochondria a hallmark of mitochondrial stress (sFig.
- 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 ⁇ ⁇ .
- mitochondrial length was significantly decreased in Apoalbp ⁇ ⁇ (Fig. 4N).
- the crista density Fig.
- AIBP deficiency impairs mitochondrial dynamics and OXPHOS activity in the retina
- AIBP deficiency triggers mitochondrial fragmentation and reduces ATP production in RGCs
- 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.
- 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.
- Mitofilin is a mitochondrial inner membrane protein that controls cristae architecture (42).
- Fig. 7K mitochondrial inner membrane protein that controls cristae architecture (42).
- SIRT3 sirtuin 3
- SOD2 superoxide dismutase 2
- MAPKs mitogen-activated protein kinases
- ERK1/2 extracellular signal-regulated kinase 1/2
- AIBP an important neuroprotective protein in the retina.
- IOP 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.
- AB IP deficiency contributed to dysfunctional RGC mitochondria, oxidative stress and visual dysfunction.
- AIBP deficiency exacerbated RGC death in response to elevated IOP.
- 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.
- AIBP mediates the stabilization of ABCA1 by facilitating apoA-1 binding to ABCA1 and prevents ABCA1 degradation via the ubiquitination pathway (35).
- ABC Al particularly in RGCs needs to be elucidated, it is likely that AIBP stabilizes ABCA1 and regulate cholesterol efflux in glaucomatous RGCs.
- loss of AIBP induced by elevated IOP may contribute to deregulation of ABCA1 in glaucomatous neurodegenerati on .
- TLR4 significantly reduces RGC death and proinflammatory responses in experimental glaucoma (38, 53, 54).
- 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.
- 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.
- 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.
- SIRT3 a mitochondrial NAD + -dependent deacetylase
- SIRT3- mediated SOD2 activation and deacetylation reduces ROS levels, leading to the enhancement of resistance against oxidative stress (67, 68).
- AIBP significantly reduced the expression levels of SIRT3 and SOD2 proteins in the inner retina including RGCs.
- SIRT3-SOD2 pathway is linked to inflammation and oxidative stress (69, 70).
- mitochondrial AIBP may contribute to the stabilization of the SIRT3-SOD2 axis, rescuing RGC mitochondria from neuroinflammation and/or oxidative stress.
- 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).
- 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.
- 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).
- AIBP lipopolysaccharide
- AIBP expression is decreased in glaucomatous retinas and pressure-induced RGCs.
- 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.
- 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.
- 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. 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.
- 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).
- 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.
- OPA1 and MFN2 protein expression in the retina of WT and Apoalbp ⁇ ⁇ mice, n 3 mice.
- 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.
- 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.
- E-J The crista density was lower in the Apoal bp ⁇ ' mitochondria due in part to onion-like outer membrane protuberances.
- Oxidative stress and MAPKs signaling were assessed in the retina of WT and Apoalbp ⁇ ⁇ mice.
- 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.
- 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.
- 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.
- 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.
- 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- ).
- Prusky GT Alam NM, Beekman S and Douglas RM. Rapid quantification of adult and developing mouse spatial vision using a virtual optomotor system. Invest Ophthalmol Vis Sci. 2004;45:4611-6.
- Nakano Y, et al. Toll-like receptor 4 inhibitor protects against retinal ganglion cell damage induced by optic nerve crush in mice. J Pharmacol Sci. 2017; 133: 176- 183.
- Tumour suppressor SIRT3 deacetylates and activates manganese superoxide dismutase to scavenge ROS. EMBO Rep. 2011;12:534-41.
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