WO2019183282A1 - Agents sénolytiques pour le traitement de tauopathies - Google Patents

Agents sénolytiques pour le traitement de tauopathies Download PDF

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WO2019183282A1
WO2019183282A1 PCT/US2019/023259 US2019023259W WO2019183282A1 WO 2019183282 A1 WO2019183282 A1 WO 2019183282A1 US 2019023259 W US2019023259 W US 2019023259W WO 2019183282 A1 WO2019183282 A1 WO 2019183282A1
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bcl
inhibitor
subject
family inhibitor
tau
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Darren J. BAKER
Jan M.A. VAN DEURSEN
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Mayo Foundation For Medical Education And Research
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine

Definitions

  • Tauopathies including but not limited to Alzheimer’s disease, progressive supranuclear palsy, corticobasal syndrome, some frontotemporal dementias, and chronic traumatic
  • encephalopathy are a class of neurodegenerative diseases associated with the pathological aggregation of tau protein in neurofibrillary or gliofibrillary tangles in the human brain.
  • Neurofibrillary tangle depositions are a major pathological hallmark of human Alzheimer’s disease. It has been discovered that tauopathy pathology is associated with cellular senescence. There are limited therapeutic options for subjects diagnosed with tauopathies, such as Alzheimer’s, and related symptoms, including cognitive decline.
  • Age is the main risk factor for most chronic human diseases, including many neurodegenerative diseases.
  • Senescent cells which are characterized by a permanent cell cycle arrest, have been shown to accumulate with age and at sites of age-related diseases where they are believed to contribute to pathology through the production and secretion of various pro- inflammatory cytokines, chemokines, growth factors and matrix metalloproteinases, collectively referred to as the senescence-associated secretory phenotype or SASP.
  • SASP senescence-associated secretory phenotype
  • the Bcl-2 family inhibitor is a Bcl-2 inhibitor, a Bcl-XL inhibitor, a Bcl-w inhibitor, a Bcl-2 and Bcl-XL inhibitor, a Bcl-2 and Bcl-w inhibitor, a Bcl-XL and Bcl-w inhibitor, a pan-Bcl-2 family inhibitor.
  • the Bcl-2 family inhibitor is a Bcl-2 inhibitor but not a Bcl-xL or Bcl-w inhibitor.
  • the Bcl-2 family inhibitor is WEHI-539, A-l 155463, ABT-737, ABT-199, Obatoclax, BX1-61, BX1-72, 2,3- DCPE, ((R)-4-(4-chlorophenyl)-3-(3-(4-(4-(4-((4-(dimethylamino)-l-(phenylthio)butan-2- yl)amino)-3-nitrophenylsulfonamido)phenyl)piperazin-l -yl)phenyl)-5-ethyl-l -methyl- lH-pyrrole- 2-carboxylic acid (“Compound 21”), (R)-5-(4-chlorophenyl)-4-(3-(4-(4-(4-((4-(dimethylamino)-l- (phenylthio)butan-2-yl)amino)-3-nitrophenyl
  • the Bcl-2 family inhibitor is a BH3 mimetic.
  • cognitive function is measured by assessment of a subject’s executive function, working memory, attention, short-term memory, intermediate-term memory, or long-term memory using one or more cognitive behavioral tasks.
  • the Bcl-2 family inhibitor attenuates cognitive decline.
  • the Bcl-2 family inhibitor improves short term memory in the subject.
  • the subject has an elevated level of a senescence marker relative to a baseline senescence marker level.
  • the Bcl-2 family inhibitor reduces expression of the senescence marker.
  • the senescence marker is selected from the group consisting of pl6, p2l, PAI1, GFAP, SlOOb, and Cdl lb.
  • the Bcl-2 family inhibitor eliminates or reduces levels of senescent cells in the subject.
  • the senescent cells are senescent microglia.
  • the senescent cells are senescent astrocytes.
  • the Bcl-2 family inhibitor reduces neurofibrillary tangle (NFT) deposition in the subject. In some embodiments, the reduction of NFT is measured by positron emission tomography (PET) imaging.
  • the Bcl-2 family inhibitor reduces tau protein levels in the subject.
  • tau protein levels are measured by quantification of levels of total Tau (T-Tau) or phosphorylated Tau (P-Tau) in cerebrospinal fluid (CSF) or blood plasma obtained from a subject.
  • the tau protein is phosphorylated tau.
  • the Bcl-2 family inhibitor attenuates tau aggregation.
  • the Bcl-2 family inhibitor attenuates gliosis in the subject.
  • the Bcl-2 family inhibitor reduces expression of a marker of gliosis.
  • the marker of gliosis is selected from the group consisting of Gfap , SI 00b, and Cdllb.
  • the Bcl-2 family inhibitor reduces neuroinflammation.
  • neuroinflammation is measured by the reduction of Senescence Associated Secretory Phenotype (SASP) factors.
  • the Bcl-2 family inhibitor is a Bcl-2 inhibitor, a Bcl-XL inhibitor, a Bcl-w inhibitor, a Bcl-2 and Bcl-XL inhibitor, a Bcl-2 and Bcl-w inhibitor, a Bcl-XL and Bcl-w inhibitor, a pan-Bcl-2 family inhibitor.
  • the Bcl-2 family inhibitor is a Bcl-2 inhibitor but not a Bcl-xL or Bcl-w inhibitor.
  • the Bcl-2 family inhibitor is WEHI-539, A-l 155463, ABT-737, ABT-199, Obatoclax, BX1-61, BX1-72, 2,3-DCPE, ((R)-4-(4- chlorophenyl)-3-(3-(4-(4-(4-((4-(dimethylamino)-l-(phenylthio)butan-2-yl)amino)-3- nitrophenylsulfonamido)phenyl)piperazin-l-yl)phenyl)-5-ethyl-l-methyl-lH-pyrrole-2-carboxylic acid ("Compound 21”), (R)-5-(4-chlorophenyl)-4-(3-(4-(4-(4-((4-(dimethylamino)-l-
  • the Bcl-2 family inhibitor is a BH3 mimetic. In some embodiments, the Bcl-2 family inhibitor attenuates cognitive decline. In some embodiments, cognitive decline is measured by assessment of a subject’s executive function, working memory, attention, short-term memory, intermediate-term memory, or long-term memory using one or more cognitive behavioral tasks. In some embodiments, the Bcl-2 family inhibitor improves short term memory in the subject. In some embodiments, the subject has an elevated level of a senescence marker relative to a baseline senescence marker level. In some embodiments, the Bcl-2 family inhibitor reduces expression of the senescence marker.
  • the senescence marker is selected from the group consisting of pl6, p2l, PAI1, GFAP, SlOOb, and Cdl lb.
  • the Bcl-2 family inhibitor eliminates or reduces levels of senescent cells in the subject.
  • the senescent cells are senescent microglia.
  • the senescent cells are senescent astrocytes.
  • the Bcl-2 family inhibitor reduces neurofibrillary tangle (NFT) deposition in the subject. In some embodiments, the reduction of NFT is measured by positron emission tomography (PET) imaging.
  • the Bcl-2 family inhibitor reduces tau protein levels in the subject.
  • tau protein levels are measured by quantification of levels of total Tau (T-Tau) or phosphorylated Tau (P-Tau) in cerebrospinal fluid (CSF) or blood plasma obtained from a subject.
  • the tau protein is phosphorylated tau.
  • the Bcl-2 family inhibitor attenuates tau aggregation.
  • the Bcl-2 family inhibitor attenuates gliosis in the subject.
  • the Bcl-2 family inhibitor reduces expression of a marker of gliosis.
  • the marker of gliosis is selected from the group consisting of Gfap , SI 00b, and Cdllb.
  • the Bcl-2 family inhibitor reduces neuroinflammation.
  • neuroinflammation is measured by the reduction of Senescence Associated Secretory Phenotype (SASP) factors.
  • the Bcl-2 family inhibitor is a Bcl-2 inhibitor, a Bcl-XL inhibitor, a Bcl-w inhibitor, a Bcl-2 and Bcl-XL inhibitor, a Bcl-2 and Bcl-w inhibitor, a Bcl-XL and Bcl-w inhibitor, a pan-Bcl-2 family inhibitor.
  • the Bcl-2 family inhibitor is a Bcl-2 inhibitor but not a Bcl-xL or Bcl-w inhibitor.
  • the Bcl-2 family inhibitor is WEHI-539, A-l 155463, ABT-737, ABT-199, Obatoclax, BX1-61, BX1-72, 2,3-DCPE, ((R)-4-(4-chlorophenyl)-3-(3-(4-(4-(4-((4-(dimethylamino)-l-(phenylthio)butan-2-yl)amino)-3- nitrophenylsulfonamido)phenyl)piperazin-l-yl)phenyl)-5-ethyl-l-methyl-lH-pyrrole-2-carboxylic acid ("Compound 21”), (R)-5-(4-chlorophenyl)-4-(3-(4-(4-(4-((4-(dimethylamino)-l-
  • the Bcl-2 family inhibitor is a BH3 mimetic. In some embodiments, the Bcl-2 family inhibitor attenuates cognitive decline. In some embodiments, cognitive decline is measured by assessment of a subject’s executive function, working memory, attention, short-term memory, intermediate-term memory, or long-term memory using one or more cognitive behavioral tasks. In some embodiments, the Bcl-2 family inhibitor improves short term memory in the subject. In some embodiments, the subject has an elevated level of a senescence marker relative to a baseline senescence marker level. In some embodiments, the Bel -2 family inhibitor reduces expression of the senescence marker.
  • the senescence marker is selected from the group consisting of pl6, p2l, PAI1, GFAP, SlOOb, and Cdl lb.
  • the Bel -2 family inhibitor eliminates or reduces levels of senescent cells in the subject.
  • the senescent cells are senescent microglia.
  • the senescent cells are senescent astrocytes.
  • the Bel -2 family inhibitor reduces neurofibrillary tangle (NFT) deposition in the subject.
  • the reduction of NFT is measured by positron emission tomography (PET) imaging.
  • the Bel -2 family inhibitor reduces tau protein levels in the subject.
  • tau protein levels are measured by quantification of levels of total Tau (T-Tau) or phosphorylated Tau (P-Tau) in cerebrospinal fluid (CSF) or blood plasma obtained from a subject.
  • the tau protein is phosphorylated tau.
  • the Bel -2 family inhibitor attenuates tau aggregation.
  • the Bel -2 family inhibitor attenuates gliosis in the subject.
  • the Bel -2 family inhibitor reduces expression of a marker of gliosis.
  • the marker of gliosis is selected from the group consisting of Gfap , SI 00b, and Cdllb.
  • the Bel -2 family inhibitor reduces neuroinflammation.
  • neuroinflammation is measured by the reduction of Senescence Associated Secretory Phenotype (SASP) factors.
  • SASP Senescence Associated Secretory Phenotype
  • FIGURES 1A-1D show the effect of the removal of senescent cells in adipose tissue.
  • FIG. 1A schematic oiATTAC transgene.
  • FIG. 1B single cell FACS profile of inguinal white adipose tissue (iWAT) for GFP.
  • FIGURES 2A-2D show the effect of the removal of senescent cells on lifespan.
  • FIG. 2A study design for clearance of senescent cells in mixed and BL/6 mouse cohorts.
  • FIG. 2B and FIG. 2C survival curves for vehicle- (-AP) and AP -treated (+AP) mixed (FIG. 2B) and BL/6 mice (FIG. 2C).
  • FIG. 2D representative images of aged mice with and without senescent cell clearance.
  • FIGURES 3A-3C show the effect of the removal of senescent cells on locomotory and exploratory behavior.
  • FIG. 3C Summary table of healthspan improvements observed in l8-month-old AP treated mice. *p ⁇ 0.05, **r ⁇ 0.01, ***p ⁇ 0.00l; unpaired t-test.
  • FIGURES 4A-4C show the effect of the removal of senescent cells in kidney.
  • FIG. 4A SA-P-Gal-stained kidney sections. In the upper panel, the arrowheads indicate senescence- associated b-galactosidase activities.
  • FIG. 4B electron micrograph showing X-Gal crystal containing renal epithelial cell with brush border membrane (arrowheads) from an 18-month-old vehicle treated ATTAC male. Insets show X-Gal crystals.
  • FIGURE 5 illustrates different cell types (with their distinguishing markers) for senescence cell localization in the brain.
  • FIGURE 6 illustrates pl6 expression at different ages in the brain.
  • FIGURE 7 shows the effect of the removal of senescent cells on short-term memory.
  • Experimental setup is as described in FIG.11D.
  • FIGURE 8 illustrates gene expression in single mouse embryonic fibroblasts.
  • FC fold change for GFP+ vs. GFP- cells.
  • FIGURES 9A-9E show senescent astrocytes and microglia that accumulate in brains of P301S (MAPT) PS 19 mice can be removed using the INK-ATTAC transgene.
  • FIG. 9A RT-qPCR analysis for pl6 lnk4a expression in hippocampus (left) and cortex (right) from Wildtype and P301S (MAPT) PS19 mice (animal numbers for each column are indicated in parentheses of hippocampus graph, 2 independent experiments; normalized to 3 m Wildtype group).
  • FIG. 9B Study design for clearance of senescent cells in PS19;ATTAC mice. Abbreviations: AP - AP20187; Veh. - vehicle.
  • FIG. 9A RT-qPCR analysis for pl6 lnk4a expression in hippocampus (left) and cortex (right) from Wildtype and P301S (MAPT) PS19 mice (animal numbers for each column are indicated in parentheses of hippocampus graph, 2 independent experiments; normalized to
  • p21 is also known as Cdkn I a Pail is also known as Serpinel.
  • FIG. 9D Electron micrograph showing an X-Gal-positive astrocyte (left) and microglia (right) following SA-P-Gal staining from a 6-month-old vehicle-treated PS19;ATTAC male.
  • FIG. 9D Electron micrograph showing an X-Gal-positive astrocyte (left) and microglia (right) following SA-P-Gal staining from a 6-month-old vehicle-treated PS19;ATTAC male.
  • FIGURES 10A-10C show senescent cells promote insoluble tau aggregates.
  • FIG. 10A Representative western blot (> 3 independent experiments) analysis of 6-month-old whole brain for soluble tau (top), soluble phosphorylated tau (S202/T205; middle), and insoluble phosphorylated tau (S202/T205; bottom).
  • the upper right panel labled with PS 19; ATTAC-AP shows more yellow staining of phosphorylated tau.
  • the middle bar of the bar graph corresponds with the upper right photo labeled as PS 19; ATTAC-AP.
  • FIG. 10A Representative western blot (> 3 independent experiments) analysis of 6-month-old whole brain for soluble tau (top), soluble phosphorylated tau (S202/T205; middle), and insoluble phosphorylated tau (S202/T205; bottom
  • the middle bar of the bar graph corresponds with the middle panel of the photo labeled as PS 19; ATTAC-AP. Key is as indicated in FIG. 10B.
  • Scale bars 100 pm (10B) and 50 pm (10C). Data are mean ⁇ s.e.m. **P ⁇ 0.01; ***P ⁇ 0.001 (one-way ANOVA with Tukey’s multiple comparisons test).
  • FIGURES 11A-11D show senescent cells drive neurodegenerative disease.
  • FIG. 11B Nissl stains of the dentate gyrus from 8-month-old mice.
  • the left bar corresponds with the top photo labeled as ATTAC-AP; the middle with the middle photo labeled as PS 19; ATTAC-AP; and the right bar with the bottom photo labeled as PS 19; ATTAC-AP.
  • FIGURES 12A-12B show ABT263 can modulate senescent cells and attenuate tau phosphorylation.
  • FIG. 12A Expression of senescence markers from 6-month-old hippocampus (left) and cortex (cortex) either vehicle (-ABT263) or ABT263 (+ABT263) treated assessed by RT- qPCR (animal numbers indicated in the legend; normalized to WT -ABT263 group). p21 is also known as Cdknla ; Pail is also known as Serpinel.
  • FIGURE 13 shows senescent cells accumulate in PS19 mice.
  • RT-qPCR analysis for senescence-associated genes in hippocampi (left) and cortices (right) of 3- and lO-month-old male mice (animal numbers indicated in the legend, 2 independent experiments; normalized to 3 m Wildtype group).
  • Data are mean ⁇ s.e.m. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001 (one-way ANOVA with Tukey’s multiple comparisons test).
  • FIGURE 14 shows AP-mediated clearance selectively removes senescent cells that accumulate in brains of PS19;ATTAC mice.
  • p21 is also known as Cdknla ; Pail is also known as Serpinel.
  • Data are mean ⁇ s.e.m. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001 (one-way ANOVA with Tukey’s multiple comparisons test).
  • FIGURES 16A-16E show increased senescence-associated gene expression is observed in astrocytes and microglia isolated from PS19 mice.
  • FIGs 16A-E Gating strategy (FIG. 16A) for FACS isolation of living astrocytes (FIG. 16B), microglia (FIG. 16C), oligodendrocytes (FIG. 16D), and neuron-enriched Cd56 + cells (FIG. 8E) from cortices from 6-month-old WT and PS19 mice.
  • FIG. 16B Astrocyte (Cdl lb _ ,Cd45 _ ,Ol _ ,GLAST + ,Cd56 ) fraction (left) and RT-qPCR analysis (right).
  • FIG. 16A Gating strategy for FACS isolation of living astrocytes (FIG. 16B), microglia (FIG. 16C), oligodendrocytes (FIG. 16D), and neuron-enriched Cd56 + cells
  • FIG. 16C Microglia (Cdl lb + ,Cd45 + ,Ol,GLAST + ,Cd56 ) fraction (left) and RT- qPCR analysis (right).
  • FIG. 16D Oligodendrocyte (Cdl lb _ ,Cd45 _ ,Ol + ,GLAST _ ,Cd56 ) fraction (left) and RT-qPCR analysis (right).
  • FIG. 16E Neuron-enriched Cd56 + (Cdl lb _ ,Cd45 _ ,Ol ,GLAST ,Cd56 + ) fraction (left) and RT-qPCR analysis (right).
  • FIGURES 17A-17D show the cell identity verification of cell populations isolated by FACS. RT-qPCR analysis for cell identity markers from cell populations isolated from 6-month- old Wildtype and PS 19 mice for Aqp4 expression enriched in astrocytes (FIG. 17A), Cx3crl expression enriched in microglia (FIG.
  • FIG. 17B Olig expression enriched in oligodendrocytes (FIG. 17C), and Nefl expression enriched in neurons (FIG. 17D).
  • FIGURES 18A-18D show AP administration does not precociously eliminate non- senescent glial cells isolated from ATTAC mice.
  • FIGURES 19A-19B show AP-admini strati on does not broadly eliminate cells or increase proliferation of microglia.
  • FIGURES 20A-20D show senescent cells promote gliosis.
  • FIG. 20B RT-qPCR analysis as in (FIG. 20A) in hippocampi of 6-month-old female mice (animal number indicated in legend; normalized to ATTAC -AP group).
  • FIG. 20B
  • FIGURES 21A-21C show AP treatment attenuates tau phosphorylation.
  • FIG. 13A Ponceau S loading controls for western blot lysates of 6-month-old whole brain total -tau (left) and phosphorylated tau (S202/T205; right) shown in FIG. 4A.
  • FIG. 13B Quantification of westerns blot analysis from 6-month-old whole brain for soluble tau (left), soluble phosphorylated tau (S202/T205; middle), and insoluble phosphorylated tau (S202/T205; right). Biologically independent animal numbers are indicated, data are from > 3 independent experiments.
  • FIG. 13A Ponceau S loading controls for western blot lysates of 6-month-old whole brain total -tau (left) and phosphorylated tau (S202/T205; right) shown in FIG. 4A.
  • FIG. 13B Quantification of westerns blot analysis from 6-month-old whole brain for soluble tau (left), soluble phosphorylated tau (S
  • FIGURE 22 shows vision-based novel object discrimination remains intact in AP- treated PS19;ATTAC mice.
  • Objects used for novel object recognition during the training and testing phase for visual discrimination left
  • Data are mean ⁇ s.e.m. **P ⁇ 0.01; ***P ⁇ 0.001 (two-way ANOVA with Tukey’s multiple comparisons test).
  • FIGURES 23A-23D show senolytic treatment attenuates established tau-dependent disease.
  • the panel labeled with P301S; P19S +ABT shows the most brown staining.
  • FIG. 23A nissl staining of phosphorylation of tau proteins of the coronal sections of the brains from lO-month-old wild-type (WT) and PS 19 male and female mice with either vehicle or 50 mg/kg navitoclax (ABT-263) daily for 5 consecutive days by
  • FIG. 23B immunofluorescence (IF) staining for Gfap to illustrate astrocyte activation of the same samples obtained in FIG. 23 A.
  • the panel labeled with P301S; P19S +ABT shows the most green staining.
  • FIG. 23C immunofluorescence (IF) staining for Iba-l to illustrate microglial activation of the same samples obtained in FIG. 23A.
  • the panel labeled with P301S; P19S +ABT shows the most red staining.
  • FIG. 23D nissl staining of the same samples obtained in FIG. 23 A to show the density of neurons.
  • the panel labeled with P301S; P19S +ABT shows the least blue staining.
  • Age is the main risk factor for most chronic human diseases, including Alzheimer’s disease (AD).
  • Senescent cells which are characterized by a permanent cell cycle arrest, have been shown to accumulate with age and at sites of age-related diseases where they are believed to contribute to pathology through the production and secretion of various pro-inflammatory cytokines, chemokines, growth factors and matrix metalloproteinases, collectively referred to as the senescence-associated secretory phenotype or SASP.
  • SASP matrix metalloproteinases
  • senescent cell clearance as a late-life therapeutic strategy for AD, as well as a variety of other age-related diseases.
  • pl6 Ink4a hereafter pl6
  • INK-ATTAC novel transgene
  • Our data show that preventing senescent cell accumulation in the MAPT (P301S) AD taupathy mouse model prior to neurofibrillary tangle (NFT) formation and neuronal degeneration markedly delays memory loss, NFT deposition, and histological alterations associated with AD.
  • NFT neurofibrillary tangle
  • senescence occurs within the brain during the early stages of AD and other tauopathies and promotes disease severity. Knowing that senescent cells impact neighboring cells through cell non-autonomous mechanisms, AD and other tauopathies progression is accelerated by the SASP from senescent cells. Based on the data, removal of senescent cells has attenuating effects on established AD and other tauopathies progression. Such removal has an important positive impact, because the identification, contribution and elimination of senescent cells in AD and other tauopathies provides new targets for preventive and therapeutic interventions in this currently untreatable disease.
  • Astrogliosis describes how astrocytes respond to CNS damage, CNS insult, or neurological disease with a variety of potential changes in gene expression, cellular structure, and function. This response can include, for example, senescence of astroglia or astrocytes and/or inflammation of astroglia or astrocytes.
  • A“Bcl-2 family inhibitor”, as that term is used herein, may refer to, for example, a Bcl-2 inhibitor, a Bcl-XL inhibitor, a Bcl-w inhibitor, a Bcl-2 and Bcl-XL inhibitor, a Bcl-2 and Bcl-w inhibitor, a Bcl-XL and Bcl-w inhibitor, or a pan-Bcl-2 family inhibitor.
  • a Bcl-2 family inhibitor may include, for example, WEH1-539, A-l 155463, ABT-737, ABT-199, Obatoclax, BX1- 61, BX1-72, 2,3-DCPE, ((R)-4-(4-chlorophenyl)-3-(3-(4-(4-(4-(dimethylamino)-l-
  • Cognitive function means those brain functions that are primarily affected by neurodegenerative diseases and/or aging, such as, for example, attention, working memory, perception, language processing, decision making, executive function, short-term memory, intermediate-term memory, or long-term memory.
  • Cognitive function and cognitive decline may be measured by any standard clinical means known in the art using one or more cognitive behavioral tasks, such as, for example, the Montreal Cognitive Assessment (MoCA), used as a rapid screening instrument for mild cognitive dysfunction, the Cambridge Neuropsychological Test Automated Battery (CANTAB), the Corsi block-tapping test or the Eriksen flanker task, and the like.
  • MoCA Montreal Cognitive Assessment
  • CANTAB Cambridge Neuropsychological Test Automated Battery
  • Microgliosis is the response of the brain to disease or injury by the activation of resident microglial cells. This response can include, for example, senescence of microglia and/or inflammation of microglia.
  • Neurofibrillary tangle are insoluble twisted fibers found inside the brain's cells. These tangles consist primarily of a protein called tow, which forms part of a structure called a microtubule. Neurofibrillary tangles are formed due to the hyperphosphorylation of tau protein. It has been reported that the number of neurofibrillary tangles is linked to the degree of dementia, suggesting that the formation of neurofibrillary tangles more directly correlates with neuronal dysfunction.
  • NFTs can be detected in a subject’s brain by positron emission tomography (PET) imaging and using a radiotracer, such as, for example, [l8F]THK5l 17, [l8F]THK53 l7, [l8F]THK535l, [l8F]AV-l45l, [HC]PBB3, [l8F]MK-6240, or [l8F]FDDNP and the like.
  • PET positron emission tomography
  • Cellular senescence is a natural biological state in which a cell permanently halts division. As senescent cells accumulate with age, they begin secreting large quantities of more than 100 proteins, including inflammatory factors, proteases, fibrotic factors, and growth factors that disturb the tissue micro-environment, such as, but are not limited to, inflammatory cytokines, such as the interleukins IL-lB, IL-6 or IL-8; matrix metallopeptidases, such as MMP-l, MMP-3 and -13; tumor necrosis factor alpha (TNF-a) and TNF B; and prostanoids, such as prostaglandin E2, as well as PAI1 or Serpine 1, VEGF, PDGF and CTGF, for example.
  • inflammatory cytokines such as the interleukins IL-lB, IL-6 or IL-8
  • matrix metallopeptidases such as MMP-l, MMP-3 and -13
  • TNF-a tumor necrosis factor alpha
  • prostanoids
  • SASP Session Associated Secretory Phenotype
  • the SASP contains factors that induce senescence in neighboring cells, setting off a cascade of events that culminates in the formation of the functionally aged and/or diseased tissue that underlies a variety of age-associated diseases.
  • Senolytic medicines can selectively eliminate senescent cells and stop the production of the SASP at its source.
  • Senescent or“Senescence” or“Senescent cell”, as those terms are used herein, means a cell that is generally thought to be derived from a cell type that typically replicates, but as a result of aging or other event that causes a change in cell state, can no longer replicate. It remains metabolically active and commonly adopts a senescence associated secretory phenotype (SASP) that includes chemokines, cytokines and extracellular matrix and fibrosis modifying proteins and enzymes.
  • SASP senescence associated secretory phenotype
  • the nucleus of senescent cells is often characterized by senescence-associated heterochromatin foci and DNA segments with chromatin alterations reinforcing senescence.
  • senescent cells can be identified as expressing at least one marker selected from pl6, senescence-associated b-galactosidase, and lipofuscin; sometimes two or more of these markers, and other markers of SASP such as, but not limited to, interleukin 6 (IL-6), and inflammatory, angiogenic and extracellular matrix modifying proteins.
  • pl6, senescence-associated b-galactosidase, and lipofuscin sometimes two or more of these markers, and other markers of SASP such as, but not limited to, interleukin 6 (IL-6), and inflammatory, angiogenic and extracellular matrix modifying proteins.
  • IL-6 interleukin 6
  • a “senescence associated” disease, disorder, or condition is a physiological condition that presents with one or more symptoms or signs, wherein a subject having the condition needs or would benefit from a lessening of such symptoms or signs.
  • the condition is senescence associated if it is caused or mediated in part by senescent cells, which may be induced by multiple etiologic factors including age, DNA damage, oxidative stress, genetic defects, etc. Lists of senescence associated disorders that can potentially be treated or managed using the methods and products taught in this disclosure include those discussed in this disclosure and the previous disclosures to which this application claims priority.
  • a compound is typically referred to as“senolytic” if it eliminates senescent cells, compared with replicative cells of the same tissue type, or quiescent cells lacking SASP markers.
  • a compound or combination may effectively be used according to this invention if it decreases the release of pathological soluble factors or mediators as part of the senescence associated secretory phenotype (SASP) that play a role in the initial presentation or ongoing pathology of a condition, or inhibit its resolution.
  • SASP senescence associated secretory phenotype
  • Tau protein(s) are proteins that stabilize microtubules and are found in neurons, astrocytes and oligodendrocytes of the CNS. Through its isoforms and phosphorylation, tau protein interacts with tubulin to stabilize microtubule assembly. All of the six tau isoforms are present in an often hyperphosphorylated state in certain neurodegenerative disorders, such as, for example, tauopathies.
  • Phosphorylated tau protein can be detected and measured in cerebrospinal fluid (CSF) or blood plasma by any standard protein detection means, such as, for example, a specific enzyme-linked immunosorbent assay test, using a commercially available kit (Innotest PHOSPHO-TAU Antigen, Innogenetics, Belgium).
  • the attenuation or removal of tau protein in a subject can be measured by, for example, quantification of levels of total tau (T-Tau) or phosphorylated tau (P-Tau) in CSF or blood plasma obtained from the subject.
  • Tauopathy or“tauopathies”, as that term is used herein, defines a group of neurodegenerative diseases characterized by abnormal hyperphosphorylation of microtubule- associated protein Tau that leads to the formation of neurofibrillary tangles.
  • Tauopathies may include, for example, is Alzheimer’s Disease (AD), primary age-related tauopathy (PART), chronic traumatic encephalopathy (CTE), dementia pugilistica, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia, frontotemporal dementias with parkinsonism linked to chromosome 17 (FTDP-17), Lytico-Bodig disease, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing pan encephalitis, Pick’s Disease, corticobasal degeneration, traumatic brain injury (TBI), argyrophilic grains disease, postencephalic parkinsonism (PEP), parkinsonism dementia complex of Guam (PDCG), tangle-dominant dementia, and globular glial tauopathies (GGTs).
  • AD Alzheimer’s Disease
  • PART primary age-related tauopathy
  • CTE chronic traumatic encephalopathy
  • dementia pugilistica dementia pugilistic
  • AD Alzheimer's disease
  • AD is characterized by the neuropathological accumulation of Ab peptide- containing plaques and aggregates of hyper-phosphorylated or misfolded tau.
  • Cellular senescence a permanent cell cycle arrest triggered in response to a variety of stresses, increases in a variety of human tissues with advancing age and cells with features of senescence have been observed in AD. ETnlike a normally programmed terminal differentiation process (i.e. neurons), senescence is a distinct fate where cells acquire a distinctive secretome of cytokines, chemokines, proteases and growth factors known as the SASP. Elevated markers of DNA damage, which are also a
  • Microglia a replication competent inflammatory cell of the brain, exhibit telomere shortening with natural age and AD patients have shorter telomeres than aged-matched controls.
  • Neurons with NFTs from AD patients have increased expression of the cyclin-dependent kinase inhibitor pl6.
  • This biomarker is especially relevant, as its expression increases in a variety of tissues with age and we have shown that senescent cells that express high amounts of pl6 drive age-related pathologies. Together, these data demonstrate that several distinct cell types accumulate indicators of senescence in AD. Clearance of senescent cells during normal aging
  • FIG. 1A In order to evaluate the therapeutic benefits of senescent cell clearance on AD, the expression of th e ATTAC transgene (FIG. 1A) coincides with endogenous pl6 in the context of chronological aging was first demonstrated. To do this, we first collected inguinal white adipose tissue (iWAT) from 12 -month-old A TTAC mice, produced single cells by collagenase treatment, and used fluorescence activated cell sorting (FACS) to separate and collect GFP+ and GFP- cells (FIG. IB) We then analyzed the RNA extracted from these two cell populations for expression of ATTAC , endogenous pl6 and various senescence markers by qRT-PCR. As shown in FIG.
  • iWAT inguinal white adipose tissue
  • FACS fluorescence activated cell sorting
  • transcript levels of FKBP-Casp8,pl6 , and other senescence markers were significantly elevated in GFP + cells compared with GFP- cells, demonstrating that expression of the ATTAC transgene tightly correlates with senescence in vivo.
  • mice were treated with AP or vehicle until they became moribund or died of natural causes (FIG. 2A).
  • Median lifespan of AP -treated animals was markedly extended in both sexes and genetic backgrounds (FIGS. 2B and 2C).
  • AP treatment had no beneficial impact on lifespan in mice lacking the ATTAC transgene (C57 +AP; FIG. 2C).
  • the longest-living 10% of the cohorts were almost exclusively AP -treated mice, indicating that clearance also increases maximum lifespan.
  • Vehicle- and AP -treated mice were overtly indistinguishable at 18 months of age. By 22 months, however, AP -treated animals typically had a healthier appearance than vehicle-treated littermates (FIG. 2D).
  • AP treated mice were analyzed for a broad spectrum of age- related healthspan alterations. Despite a lack of overt difference at 18 months, AP -treated mice of both sexes and backgrounds showed more locomotor activity and exploratory behavior than vehicle-treated mice in an open field assay (FIGS. 3A and 3B). In both cohorts, AP treatment had no significant impact on the incidence or spectrum of macroscopically detectable tumors at autopsy, although tumor latency was significantly increased (FIG. 3C).
  • the first group contained the ATTAC transgene alone and the second was P 30 IS; ATTAC double transgenic.
  • the latter group was subsequently split into two groups; one treated with vehicle and the other with AP (at a dose of 2mg/kg) beginning at weaning age twice per week (FIG. 9B).
  • AP at a dose of 2mg/kg
  • this region is relevant to human AD, as it is one of the first areas to be impacted in patients, where it results in loss of short and long-term memory.
  • AP treatment when initiated prior to disease onset, reduced the accumulation of senescent cells in the model (FIG. 9C). Collectively, these results demonstrate that senescent cells are induced in the hippocampus of P301S mice and that these cells are effectively eliminated with AP treatment.
  • microglia The first cell type in the hippocampus that responds to over-expression of the P301S tau mutant are microglia. Active microglia exhibit several distinguishing characteristics, including increased expression of Cdl lb and pro-inflammatory cytokines such as 116 and II lb. Not surprisingly, we find that these markers of microglial activation are induced in 6-month-old P 30 IS; ATTAC hippocampi (FIG. 9C). Importantly, AP treatment prevented this induction, implying that microglial activation is blunted with the removal of senescent cells or that microglia themselves are the target of the senescence program. Furthermore, the results obtained indicate that microgliosis is a key step in the initiating stages of AD in this particular mouse model.
  • NFTs form in a number of brain regions of P301S mice, including the neocortex and brain stem. Having demonstrated that senescent cell
  • AD is characterized by neurodegeneration in a variety of brain regions.
  • brain atrophy occurs between 6 to 9 months in the hippocampus.
  • To determine whether neurodegeneration was blunted with AP treatment we compared the dentate gyrus of the hippocampus at 8-months of age in the cohorts described in FIG. 9B. Consistent with the published reports, we found that the dentate gyrus of P301S;ATTAC mice had a reduction in the thickness using Nissl staining (FIG. 11B).
  • AP treatment to prevent senescent cell accumulation resulted in a dentate gyrus that was thicker and had an increased density of neurons (FIG. 11B).
  • AD negatively impacts both short- and long-term memory.
  • a novel object is introduced during the testing phase, normal mice exhibit an increased exploration of this new object, which was readily observable in ATTAC vehicle-treated mice, with the number of investigations of this object twice that of the familiar object.
  • senescent cell types of the brain include endothelial cells, microglia, oligodendrocyte precursors, astrocytes and neuronal stem cells. From our studies using both progeroid and naturally aged mice, we find that senescence in adult tissues occurs primarily in cells that are proliferation competent, including various adult stem/progenitor cell populations, epithelial cells and fibroblasts. In some embodiments, the senescent cell types of the brain are oligodendrocyte precursors, microglia or astrocytes.
  • AD is generally a late-life disease, with incidence increasing dramatically above age 60, which coincides with the increased detection of senescent cells.
  • Our data demonstrate that senescence is evident in 6-month-old P301S hippocampi when pathology is present.
  • Treatment to remove senescent cells beginning at weaning age prevents both the accumulation of senescent cells and pathology, demonstrating that clearance of senescent cells is quite efficient in the diseased brain.
  • senescent cells arise at earlier ages and continue to accumulate until the end of life due to the progressive nature of the disease.
  • Senescent cells contribute to AD through a variety of mechanisms, including disturbance of the stem cell niche, disruption of normal tissue architecture/function, and elevated inflammation, all of which are potentially attributable to the cell non-autonomous effects of SASP production.
  • components expressed and secreted from senescent cells promote AD and the deposition of NFTs.
  • Most studies of senescent phenotypes use cultured cells, primarily human fibroblasts. From these studies, a subset of SASP components are fairly conserved irrespective of whether senescence is induced by serial passage, ionizing irradiation (IR) or mitogenic stress.
  • cell-surface markers are determined based upon single-cell transcriptomics studies we have performed (FIG. 8). Non-limiting examples of these cell-surface markers include Ly6a and Cxcr2. [00065] We have shown that lifelong treatment of P301S;ATTAC mice with AP20187 prior to disease onset attenuates several parameters of AD, including tangle deposition and
  • clearance of senescent cells from subjects with diseased tissue delays further AD progression and development of AD symptoms, such as reduced neuron density, NFT deposition, and memory loss, such as short-term memory loss.
  • AD symptoms such as reduced neuron density, NFT deposition, and memory loss, such as short-term memory loss.
  • AD is attenuated by selectively eliminating senescent cells using two distinct genetic models if initiated prior to disease onset.
  • First generation senolytic agents that eliminate senescent cells are beginning to be elucidated.
  • Non-limiting examples of senolytics include Navitoclax (hereafter Navi), that functions to eliminate senescent cells through inhibition of the anti-apoptotic Bcl-2 family members Bcl-2, BC1-XL and Bcl-w. These proteins are induced in senescent cells, which could explain the apoptosis resistance that is thought to occur with senescence. Navi crosses the blood brain barrier, which makes it a suitable first agent to test for impacts on AD initiation and progression. In some embodiments, Navi treatment is administered to attenuate AD symptoms prior to disease onset. The senolytic activity of Navi has been established both in vitro and in vivo , in support of this as a senolytic agent.
  • Embodiment 1 is a method of treating a tauopathy, comprising administering to a subject in need thereof a therapeutically effective amount of a Bcl-2 family inhibitor.
  • Embodiment 2 is the method of Embodiment 1, wherein the tauopathy is is Alzheimer’s Disease (AD), primary age-related tauopathy (PART), chronic traumatic encephalopathy (CTE), dementia pugilistica, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia, frontotemporal dementias with parkinsonism linked to chromosome 17 (FTDP-17), Lytico-Bodig disease, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing pan encephalitis, Pick’s Disease, corticobasal degeneration, traumatic brain injury (TBI), argyrophilic grains disease, postencephalic parkinsonism (PEP), parkinsonism dementia complex of Guam (PDCG), tangle-dominant dementia, and globular glial tauopathies (GGTs).
  • AD Alzheimer’s Disease
  • PART primary age-related tauopathy
  • CTE chronic traumatic encephalopathy
  • Embodiment 3 is a method of improving cognitive function, comprising administering to a subject in need thereof a therapeutically effective amount of a Bcl-2 family inhibitor.
  • Embodiment 4 is the method of Embodiment 3, wherein the subject is suffering from a tauopathy.
  • Embodiment 5 is a method of reducing or reversing cognitive decline, comprising administering to a subject in need thereof a therapeutically effective amount of a Bel -2 family inhibitor.
  • Embodiment 6 is the method of Embodiment 5, wherein the subject is suffering from a tauopathy.
  • Embodiment 7 is the method of Embodiments 1-6, wherein the Bcl-2 family inhibitor is a Bcl-2 inhibitor, a Bcl-XL inhibitor, a Bcl-w inhibitor, a Bcl-2 and Bcl-XL inhibitor, a Bcl-2 and Bcl-w inhibitor, a Bcl-XL and Bcl-w inhibitor, a pan-Bcl-2 family inhibitor.
  • the Bcl-2 family inhibitor is a Bcl-2 inhibitor, a Bcl-XL inhibitor, a Bcl-w inhibitor, a Bcl-2 and Bcl-XL inhibitor, a pan-Bcl-2 family inhibitor.
  • Embodiment 8 is the method of Embodiments 1-7, wherein the Bcl-2 family inhibitor is WEH1-539, A-l 155463, ABT-737, ABT-199, Obatoclax, BX1-61, BX1-72, 2,3-DCPE, ((R)-4-(4-chlorophenyl)-3-(3-(4-(4-(4-((4-(dimethylamino)-l-(phenylthio)butan-2-yl)amino)-3- nitrophenylsulfonamido)phenyl)piperazin-l-yl)phenyl)-5-ethyl-l-methyl-lH-pyrrole-2-carboxylic acid ("Compound 21”), (R)-5-(4-chlorophenyl)-4-(3-(4-(4-(4-((4-(dimethylamino)-l-
  • Embodiment 9 is the method of Embodiments 1-8, wherein the Bcl-2 family inhibitor reduces the number of or eliminates senescent astrocytes.
  • Embodiment 10 is the method of Embodiments 1-8, wherein the Bcl-2 family inhibitor reduces the number of or eliminates senescent microglia.
  • Embodiment 11 is the method of Embodiment 10, wherein the Bcl-2 family inhibitor further reduces the number of or eliminates senescent astrocytes.
  • Embodiment 12 is the method of Embodiments 1-11, wherein the Bcl-2 family inhibitor reduces neurofibrillary tangle (NFT) deposition in the subject.
  • NFT neurofibrillary tangle
  • Embodiment 13 is the method of Embodiments 1-12, wherein the Bcl-2 family inhibitor attenuates or removes tau protein in the subject.
  • Embodiment 14 is the method of Embodiment 13, wherein the tau protein is hyper- phosphorylated.
  • Embodiment 15 is the method of Embodiments 1-14, wherein the administration of the Bcl-2 family inhibitor lowers neuro-inflammation in the subject.
  • Embodiment 16 is the method of Embodiment 15, wherein the neuro-inflammation is measured by the reduction of Senescence Associated Secretory Phenotype (SASP) factors.
  • SASP Senescence Associated Secretory Phenotype
  • Embodiment 17 is the method of Embodiment 3, wherein cognitive function is measured by assessment of a subject’s executive function, working memory, attention, short-term memory, intermediate-term memory, or long-term memory using one or more cognitive behavioral tasks.
  • Embodiment 18 is the method of Embodiment 5, wherein cognitive decline is measured by quantifying a subject’s change in performance in one or more cognitive behavioral tasks.
  • Embodiment 19 is the method of Embodiment 12, wherein the reduction of NFT is measured by positron emission tomography (PET) imaging.
  • PET positron emission tomography
  • Embodiment 20 is the method of Embodiment 13, wherein the attenuation or removal of tau protein is measured by quantification of levels of total Tau (T-Tau) or phosphorylated Tau (P-Tau) in CSF or blood plasma obtained from a subject.
  • T-Tau total Tau
  • P-Tau phosphorylated Tau
  • Embodiment 21 is a method of delaying, stopping, or reversing the course of neurodegeneration, comprising administering to a subject in need thereof a therapeutically effective amount of a Bcl-2 family inhibitor.
  • Embodiment 22 provides a method of improving cognitive function in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a Bcl-2 family inhibitor.
  • Embodiment 23 is the method of Embodiment 22, wherein the Bcl-2 family inhibitor is a Bcl-2 inhibitor, a Bcl-XL inhibitor, a Bcl-w inhibitor, a Bcl-2 and Bcl-XL inhibitor, a Bcl-2 and Bcl-w inhibitor, a Bcl-XL and Bcl-w inhibitor, a pan-Bcl-2 family inhibitor.
  • the Bcl-2 family inhibitor is a Bcl-2 inhibitor, a Bcl-XL inhibitor, a Bcl-w inhibitor, a Bcl-2 and Bcl-XL inhibitor, a Bcl-2 and Bcl-w inhibitor, a pan-Bcl-2 family inhibitor.
  • Embodiment 24 is the method of Embodiments 22 and 23, wherein the Bcl-2 family inhibitor is a Bcl-2 inhibitor but not a Bcl-xL or Bcl-w inhibitor.
  • Embodiment 25 is the method of Embodiments 22-24, wherein the Bcl-2 family inhibitor is WEHI-539, A-l 155463, ABT-737, ABT-199, Obatoclax, BX1-61, BX1-72, 2,3-DCPE, ((R)-4-(4-chlorophenyl)-3-(3-(4-(4-(4-((4-(dimethylamino)-l-(phenylthio)butan-2-yl)amino)-3- nitrophenylsulfonamido)phenyl)piperazin-l-yl)phenyl)-5-ethyl-l-methyl-lH-pyrrole-2-carboxylic acid ("Compound 21”), (R)-5-(4-chlorophenyl)-4-(3-(4-(4-(4-((4-(dimethylamino)-l- (phenylthio)butan-2-y
  • Embodiment 26 is the method of Embodiments 22-25, wherein the Bel -2 family inhibitor is a BH3 mimetic.
  • Embodiment 27 is the method of Embodiments 22-26, wherein cognitive function is measured by assessment of a subject’s executive function, working memory, attention, short-term memory, intermediate-term memory, or long-term memory using one or more cognitive behavioral tasks.
  • Embodiment 28 is the method of Embodiments 22-27, wherein the Bel -2 family inhibitor attenuates cognitive decline.
  • Embodiment 29 is the method of Embodiments 22-28, wherein the Bel -2 family inhibitor improves short term memory in the subject.
  • Embodiment 30 is the method of Embodiments 22-29, wherein the subject has an elevated level of a senescence marker relative to a baseline senescence marker level.
  • Embodiment 31 is the method of Embodiment 30, wherein the Bcl-2 family inhibitor reduces expression of the senescence marker.
  • Embodiment 32 is the method of Embodiments 31 and 30, wherein the senescence marker is selected from the group consisting of pl6, p2l, PAI1, GFAP, SlOOb, and Cdl lb.
  • Embodiment 33 is the method of Embodiments 22-32, wherein the Bcl-2 family inhibitor eliminates or reduces levels of senescent cells in the subject.
  • Embodiment 34 is the method of Embodiment 33, wherein the senescent cells are senescent microglia.
  • Embodiment 35 is the method of Embodiment 33, wherein the senescent cells are senescent astrocytes.
  • Embodiment 36 is the method of Embodiments 22-35, wherein the Bcl-2 family inhibitor reduces neurofibrillary tangle (NFT) deposition in the subject.
  • Embodiment 37 is the method of Embodiment 36, wherein the reduction of NFT is measured by positron emission tomography (PET) imaging.
  • PET positron emission tomography
  • Embodiment 38 is the method of Embodiments 22-37, wherein the Bcl-2 family inhibitor reduces tau protein levels in the subject.
  • Embodiment 39 is the method of Embodiment 38, wherein tau protein levels are measured by quantification of levels of total Tau (T-Tau) or phosphorylated Tau (P-Tau) in cerebrospinal fluid (CSF) or blood plasma obtained from a subject.
  • T-Tau total Tau
  • P-Tau phosphorylated Tau
  • Embodiment 40 is the method of Embodiments 38 or 39, wherein the tau protein is phosphorylated tau.
  • Embodiment 41 is the method of Embodiments 22-40, wherein the Bcl-2 family inhibitor attenuates tau aggregation.
  • Embodiment 42 is the method of Embodiments 22-41, wherein the Bcl-2 family inhibitor attenuates gliosis in the subject.
  • Embodiment 43 is the method of Embodiments 22-42, wherein the Bcl-2 family inhibitor reduces expression of a marker of gliosis.
  • Embodiment 44 is the method of Embodiment 43, wherein the marker of gliosis is selected from the group consisting of Gfap , SI 00b, and Cdllb.
  • Embodiment 45 is the method of Embodiments 22-44, wherein the Bcl-2 family inhibitor reduces neuroinflammation.
  • Embodiment 46 is the method of Embodiment 45, wherein neuroinflammation is measured by the reduction of Senescence Associated Secretory Phenotype (SASP) factors.
  • SASP Senescence Associated Secretory Phenotype
  • Embodiment 47 provides a method of treating frontotemporal dementia in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a Bcl- 2 family inhibitor.
  • Embodiment 48 is the method of Embodiment 47, wherein the Bcl-2 family inhibitor is a Bcl-2 inhibitor, a Bcl-XL inhibitor, a Bcl-w inhibitor, a Bcl-2 and Bcl-XL inhibitor, a Bcl-2 and Bcl-w inhibitor, a Bcl-XL and Bcl-w inhibitor, a pan-Bcl-2 family inhibitor.
  • Embodiment 49 is the method of Embodiments 47 and 48, wherein the Bel -2 family inhibitor is a Bcl-2 inhibitor but not a Bcl-xL or Bcl-w inhibitor.
  • Embodiment 50 is the method of Embodiments 47-49, wherein the Bcl-2 family inhibitor is WEHI-539, A-l 155463, ABT-737, ABT-199, Obatoclax, BX1-61, BX1-72, 2,3-DCPE, ((R)-4-(4-chlorophenyl)-3-(3-(4-(4-(4-((4-(dimethylamino)-l-(phenylthio)butan-2-yl)amino)-3- nitrophenylsulfonamido)phenyl)piperazin-l-yl)phenyl)-5-ethyl-l-methyl-lH-pyrrole-2-carboxylic acid ("Compound 21”), (R)-5-(4-chlorophenyl)-4-(3-(4-(4-(4-((4-(dimethylamino)-l- (phenylthio)butan-2-
  • Embodiment 51 is the method of Embodiments 47-50, wherein the Bcl-2 family inhibitor is a BH3 mimetic.
  • Embodiment 52 is the method of Embodiments 47-51, wherein the Bcl-2 family inhibitor attenuates cognitive decline.
  • Embodiment 53 is the method of Embodiment 52, wherein cognitive decline is measured by assessment of a subject’s executive function, working memory, attention, short-term memory, intermediate-term memory, or long-term memory using one or more cognitive behavioral tasks.
  • Embodiment 54 is the method of Embodiments 47-53, wherein the Bcl-2 family inhibitor improves short term memory in the subject.
  • Embodiment 55 is the method of Embodiments 47-54, wherein the subject has an elevated level of a senescence marker relative to a baseline senescence marker level.
  • Embodiment 56 is the method of Embodiment 55, wherein the Bcl-2 family inhibitor reduces expression of the senescence marker.
  • Embodiment 57 is the method of Embodiment 55, wherein the senescence marker is selected from the group consisting of pl6, p2l, PAI1, GFAP, SlOOb, and Cdl lb.
  • Embodiment 58 is the method of Embodiments 47-57, wherein the Bcl-2 family inhibitor eliminates or reduces levels of senescent cells in the subject.
  • Embodiment 59 is the method of Embodiment 58, wherein the senescent cells are senescent microglia.
  • Embodiment 60 is the method of Embodiment 58, wherein the senescent cells are senescent astrocytes.
  • Embodiment 61 is the method of Embodiments 47-60, the Bcl-2 family inhibitor reduces neurofibrillary tangle (NFT) deposition in the subject.
  • NFT neurofibrillary tangle
  • Embodiment 62 is the method of Embodiment 61, wherein the reduction of NFT is measured by positron emission tomography (PET) imaging.
  • PET positron emission tomography
  • Embodiment 63 is the method of Embodiments 47-62, wherein the Bcl-2 family inhibitor reduces tau protein levels in the subject.
  • Embodiment 64 is the method of Embodiment 63, wherein tau protein levels are measured by quantification of levels of total Tau (T-Tau) or phosphorylated Tau (P-Tau) in cerebrospinal fluid (CSF) or blood plasma obtained from a subject.
  • T-Tau total Tau
  • P-Tau phosphorylated Tau
  • Embodiment 65 is the method of Embodiments 63 and 64, wherein the tau protein is phosphorylated tau.
  • Embodiment 66 is the method of Embodiments 47-65, wherein the Bcl-2 family inhibitor attenuates tau aggregation.
  • Embodiment 67 is the method of Embodiments 47-66, wherein the Bcl-2 family inhibitor attenuates gliosis in the subject.
  • Embodiment 68 is the method of Embodiments 47-67, wherein the Bcl-2 family inhibitor reduces expression of a marker of gliosis.
  • Embodiment 69 is the method of Embodiment 68, wherein the marker of gliosis is selected from the group consisting of Gfap , SI 00b, and Cdllb.
  • Embodiment 70 is the method of Embodiments 47-69, wherein the Bcl-2 family inhibitor reduces neuroinflammation.
  • Embodiment 71 is the method of Embodiment 70, wherein neuroinflammation is measured by the reduction of Senescence Associated Secretory Phenotype (SASP) factors.
  • SASP Senescence Associated Secretory Phenotype
  • Embodiment 72 provides a method of treating traumatic brain injury in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a Bel -2 family member inhibitor.
  • Embodiment 73 is the method of Embodiment 72, wherein the Bcl-2 family inhibitor is a Bcl-2 inhibitor, a Bcl-XL inhibitor, a Bcl-w inhibitor, a Bcl-2 and Bcl-XL inhibitor, a Bcl-2 and Bcl-w inhibitor, a Bcl-XL and Bcl-w inhibitor, a pan-Bcl-2 family inhibitor.
  • the Bcl-2 family inhibitor is a Bcl-2 inhibitor, a Bcl-XL inhibitor, a Bcl-w inhibitor, a Bcl-2 and Bcl-XL inhibitor, a pan-Bcl-2 family inhibitor.
  • Embodiment 74 is the method of Embodiments 72 and 73, wherein the Bcl-2 family inhibitor is a Bcl-2 inhibitor but not a Bcl-xL or Bcl-w inhibitor.
  • Embodiment 75 is the method of Embodiments 72-74, wherein the Bcl-2 family inhibitor is WEHI-539, A-l 155463, ABT-737, ABT-199, Obatoclax, BX1-61, BX1-72, 2,3-DCPE, ((R)-4-(4-chlorophenyl)-3-(3-(4-(4-(4-((4-(dimethylamino)-l-(phenylthio)butan-2-yl)amino)-3- nitrophenylsulfonamido)phenyl)piperazin-l-yl)phenyl)-5-ethyl-l-methyl-lH-pyrrole-2-carboxylic acid ("Compound 21”), (R)-5-(4-chlorophenyl)-4-(3-(4-(4-(4-((4-(dimethylamino)-l- (phenylthio)butan-2-
  • Embodiment 76 is the method of Embodiments 72-75, wherein the Bcl-2 family inhibitor is a BH3 mimetic.
  • Embodiment 77 is the method of Embodiments 72-76, wherein Bcl-2 family inhibitor attenuates cognitive decline.
  • Embodiment 78 is the method of Embodiments 72-77, wherein cognitive decline is measured by assessment of a subject’s executive function, working memory, attention, short-term memory, intermediate-term memory, or long-term memory using one or more cognitive behavioral tasks.
  • Embodiment 79 is the method of Embodiments 72-78, wherein the Bcl-2 family inhibitor improves short term memory in the subject.
  • Embodiment 80 is the method of Embodiments 72-79, wherein the subject has an elevated level of a senescence marker relative to a baseline senescence marker level.
  • Embodiment 81 is the method of Embodiment 80, wherein the Bcl-2 family inhibitor reduces expression of the senescence marker.
  • Embodiment 81 is the method of Embodiment 80 or 81, wherein the senescence marker is selected from the group consisting of pl6, p2l, PAI1, GFAP, SlOOb, and Cdl lb.
  • Embodiment 83 is the method of Embodiments 72-82, wherein the Bcl-2 family inhibitor eliminates or reduces levels of senescent cells in the subject.
  • Embodiment 84 is the method of Embodiment 83, wherein the senescent cells are senescent microglia.
  • Embodiment 85 is the method of Embodiment 83, wherein the senescent cells are senescent astrocytes.
  • Embodiment 86 is the method of Embodiments 72-85, wherein the Bcl-2 family inhibitor reduces neurofibrillary tangle (NFT) deposition in the subject.
  • NFT neurofibrillary tangle
  • Embodiment 87 is the method of Embodiment 86, wherein the reduction of NFT is measured by positron emission tomography (PET) imaging.
  • PET positron emission tomography
  • Embodiment 88 is the method of Embodiment 72-87, wherein the Bcl-2 family inhibitor reduces tau protein levels in the subject.
  • Embodiment 89 is the method of Embodiment 88, wherein tau protein levels are measured by quantification of levels of total Tau (T-Tau) or phosphorylated Tau (P-Tau) in cerebrospinal fluid (CSF) or blood plasma obtained from a subject.
  • T-Tau total Tau
  • P-Tau phosphorylated Tau
  • Embodiment 90 is the method of Embodiments 88 and 89, wherein the tau protein is phosphorylated tau.
  • Embodiment 91 is the method of Embodiment 72-90, wherein the Bcl-2 family inhibitor attenuates tau aggregation.
  • Embodiment 92 is the method of Embodiments 72-91, wherein the Bcl-2 family inhibitor attenuates gliosis in the subject.
  • Embodiment 93 is the method of Embodiments 72-92, wherein the Bel -2 family inhibitor reduces expression of a marker of gliosis.
  • Embodiment 94 is the method of Embodiment 93, wherein the marker of gliosis is selected from the group consisting of Gfap , SI 00 p and Cdllb.
  • Embodiment 95 is the method of Embodiments 72-94, wherein the Bel -2 family inhibitor reduces neuroinflammation.
  • Embodiment 96 is the method of Embodiment 95, wherein neuroinflammation is measured by the reduction of Senescence Associated Secretory Phenotype (SASP) factors.
  • SASP Senescence Associated Secretory Phenotype
  • MAPT P301S PS19 ( PS19 ) mice were purchased from The Jackson Laboratory (stock #008169) and bred to C57BL/6 for three generations. C57BL/6 ATTAC transgenic mice are as described. Male PS19 mice were bred to ATTAC females to generate cohorts of ATTAC and PS19;ATTAC mice. All mice were on a pure C57BL/6 genetic background. Mice from this cohort were randomly assigned to receive AP20187 (AP; B/B homodimerizer; Clontech ® ) or vehicle twice a week beginning at weaning age (3 weeks). Dosing of AP was 2.0 mg kg -1 body weight. 6-month- old short-term AP pulse treated animals (FIG.
  • mice 18A received a dose of 10 mg kg -1 body weight for 5 consecutive days prior to tissue collection. Senolytic intervention was performed in C57BL/6 WT and PS 19 animals. At weaning, mice were assigned to receive either ABT263 (Cayman, 923564- 51-6) or vehicle (Phosal 50 PG, Lipoid NC0130871 - 60%; PEG400, Sigma 91893 - 30%, EtOH - 10%). ABT263 was administered by oral gavage at a dose of 50 mg kg -1 body on a repeating regiment of five consecutive days of treatment followed by 16 days of rest. Animals were housed in a l2h L/D cycle environment in pathogen-free barrier conditions as described in detail. Compliance with relevant ethical regulations and all animal procedures were reviewed and approved by the Mayo Clinic Institutional Animal Care and Lise Committee.
  • Detection of X-Gal crystals by transmission electron microscopy (TEM) after senescence-associated b-galactosidase (SA-P-Gal) staining was performed as described with the following alterations to accommodate central nervous tissue.
  • Mice were transcardially perfused with ice-cold Dulbecco’s phosphate-buffered saline (DPBS; pH 7.4) until fluid runoff was clear. This was followed by perfusion with 4% paraformaldehyde (PFA) for 10 minutes at a rate of 3 ml per minute, and then ice-cold DPBS was perfused again for 2 minutes at the same rate to remove the remaining fixative.
  • DPBS phosphate-buffered saline
  • astrocytes - circular nucleus with spattered electron density pattern 1) astrocytes - circular nucleus with spattered electron density pattern; 2) microglia - abnormally shaped nucleus with a much darker, often phagosome containing cytoplasm; and 3) neuron - large circular nucleus with less electron density and periodically denoted by an offshooting axon. Only cells with morphology consistent of astrocytes or microglia were clearly X-gal crystal positive.
  • the supernatant (Sl -soluble protein fraction) was transferred to a new tube and the pellet homogenized in 3x volume of sucrose buffer (10 mM Tris [pH7.4], 0.8M NaCl, 10% sucrose, lmM EGTA, 1 mM PMSF) before being ultracentrifuged at l50,000g for 15 minutes at 4°C.
  • sucrose buffer 10 mM Tris [pH7.4], 0.8M NaCl, 10% sucrose, lmM EGTA, 1 mM PMSF
  • the pellet was discarded and the supernatant incubated with sarkosyl (Sodium lauroyl sarcosinate) at a final concentration of 1% for 1 hour at 37°C. Following incubation, the samples were ultracentrifuged at l50,000g for 30 minutes at 4°C.
  • sarkosyl Sodium lauroyl sarcosinate
  • the supernatant was removed and added to equal parts 2x laemmli buffer with 5% b-mercaptoethanol and boiled at l00°C for 15 minutes to prepare the total protein lysate.
  • Western blotting was performed as previously described 23 . Blots were probed with antibodies for total tau (ThermoFisher; MN1000, 1 :5000) and phospho-tow S202/T205 (Therm oFisher; MN1020, 1 : 1000). Ponceau S staining was performed to normalize lysate loading for the total and Sl fraction lysates. Quantification was performed using ImageJ as described.
  • RNA extraction, cDNA synthesis, and RT-qPCR analysis were performed on hippocampi and cortical samples from mouse brains as previously described. Primers used to amplify Casp8, GFP, r19 A ⁇ , p2l, Pail, 11-6, Il-lb and Cdllb were as previously described.
  • GFAP forward 5’- CCTTCTGACACGGATTTGGT-3' SEQ ID NO: l
  • reverse 5’-TAAGCTAGCCCTGGACATCG- 3' SEQ ID NO:2
  • S100p forward 5’ -CCGGAGTACTGGTGGAAGAC-3 1 SEQ ID NO:3
  • reverse 5’ -GGAC ACTGAAGCC AGAGAGG-3 1 SEQ ID NO:4
  • TGAGCTCCAC ATC AGGAC AG-3’ (SEQ ID NO:5), reverse 5’ -TCC AGCTCGATCTTTTGGAC- 3’ (SEQ ID NO:6); Cx3crl forward 5’ -GTT C C A A AGGC C AC A AT GT C -3’ (SEQ ID NO:7), reverse 5’-TGAGTGACTGGCACTTCCTG-3’ (SEQ ID NO: 8); Olig forward 5’-
  • CCCC AGGGATGATCTAAGC-3’ (SEQ ID NO: 9), reverse 5’-CAGAGCCAGGTTCTCCTCC-3’ (SEQ ID NO: 10); NeFL forward 5’ -AGGCC ATCTTGAC ATTGAGG-3’ (SEQ ID NO: 11), reverse 5’ -GC AGAATGCAGAC ATTAGCG-3’ (SEQ ID NO: 12); TBP forward 5’-
  • GGCCTCTCAGAAGCATCACTA-3' (SEQ ID NO: 13), reverse 5’-GCCAAGCCCTGAGCATAA- 3' (SEQ ID NO: 14). Expression for all experiments was normalized first to TBP.
  • mice were transcardially perfused as described above. Brains were stored in 4% PFA overnight at 4°C and then cryoprotected by incubating in a 30% sucrose solution for 48 hours at 4°C. Samples were sectioned into 30 mM thick coronal sections and stored in antifreeze solution (300g Sucrose, 300 mL Ethylene Glycol, 500 mL PBS) at -20°C.
  • Nissl staining (Bregma -2.1 to - 2.4 mm), thioflavin S staining (Bregma -1.4 to -1.6 mm), and phospho-tow S202/S205 (ThermoFisher, MN1020; 1 :500), phospho-tow T231 (ThermoFisher, MN1040; 1 :500), phospho- tau S396 (Abeam, 109390; 1 :500), and Gfap (Dako, Z0334; 1 :500) and Ibal (Novus, NB100-1028; 1 : 100) IHC staining (Bregma 1.6 to 1.0 mm and Lateral 2.0 to 2.7 mm) was performed on free- floating sections as described.
  • TUNEL staining (Lateral 0.75 to 1.25 mm) was performed according to the manufacturer’s instructions (Roche In Situ Cell Death Detection Kit, Fluorescein: 11684795910). Thioflavin S, EdU/Ibal colocalization, and in vivo TUNEL stained images were acquired on a Zeiss LSM 780 confocal system using multi -track configuration.
  • Dissociation of cerebral tissue was performed using the Adult Brain Dissociation kit from Miltenyi (MACS, 130-107-677) according to the manufacturer’s instructions. Samples were then incubated with a viability dye, LIVE/DEAD Aqua (Invitrogen, L34966; 1 :250) followed by incubation with Cdl lb eFluor 450 (eBioscience, 48-0112-80, 1 : 100), Cd45 APC eFluor 780 (eBioscience, 47-0451-82; 1 :200), Glastl PE (Miltenyi Biotec, 130-095-821; 1 : 100), 01 AF 700 (R&D Systems, FAB1327N-100UG; 1 : 100), and Cd56 APC (R&D Systems, FAB7820A; 1 : 100).
  • a viability dye LIVE/DEAD Aqua (Invitrogen, L34966; 1 :250)
  • Novel object recognition testing was performed as previously described. Briefly, mice from each cohort were acclimated to a 50 cm x 50 cm testing environment for a period of two minutes. After acclimation, the mice were removed, the testing area was cleaned with 70% EtOH, and two identical scented candles were placed in either corner of the testing area approximately 5 cm from either wall. Mice were reintroduced, and the ratio of both the number of visits and time spent at each candle was recorded for a period of ten minutes. Recording was performed from above (Panasonic WV-CP294) and all video files were analyzed with TopScan Version 3.00 (Clever Sys Inc.).
  • mice were removed, the testing area cleaned with 70% EtOH, and one candle was replaced with a novel scent.
  • the mice were reintroduced and the number of visits and total time per candle was recorded as before. Testing also was performed with visual stimuli by placing identical toy brick towers at either corner and then replacing with a different toy brick tower in the testing phase using the same experimental paradigm monitoring for the number of investigations.
  • Astrocyte and microglia primary cultures were prepared in tandem from mixed glial cultures as previously described 29 .
  • C57BL/6 WT and A llA ' C pups (p0-p3) were sacrificed, and the cerebellum was discarded. The remaining tissue had its meninges removed using forceps and a dissection scope.
  • GCM glial cell culture media
  • FBS FBS
  • C 3 H 3 Na0 3 lmM
  • Pen/Strep 500 ug/ml
  • InvivoGen Primocin Cultures were grown for 14 days (37°C ambient 0 2 ) with media changes every 4 days.
  • Microglia were isolated as previously described 30 using the EasySep Mouse CD1 lb Positive Selection Kit from Stem Cell (cat #: 18970).
  • Microglia were collected and plated on 10-well glass slides (5,000 cells/well) and cultured for 6 days in GCM with LADMAC-conditioned media (20%, generously provided by the Howe Laboratory) before further experimentation. This conditioned media aids in the proliferation and maintenance of microglia cultures through the secretion of M-CSF by the LADMAC cells 31 . Microglia were allowed to proliferate for 6 days prior to experimentation. The mixed glial culture flow-through from the EasySep CDl lb kit was replated in GCM on a PDL-coated T75 dish (10 million cells/flask). These cultures then underwent purification for astrocytes as previously described 29 .
  • Microglia samples were exposed to media containing IFNy (R&D Systems, 285-IF; 200ng/ml), LPS (Sigma, L2654; lOOng/ml) or a combination of both for a period of 24 hours to induce an inflammatory response 32 .
  • Cells were then processed for immunofluorescence to determine inflammation state as previously described 33 .
  • Anti-Cdl lb antibody BioRad, MCA711G; 1 :500
  • goat-anti-rat AlexaFluor 594 Invitrogen, A-11007; 1 :500 staining was counterstained with DAPI (Invitrogen, D1306; 1 : 1000).
  • AP20187 (Clontech, 635059; 10hM or 100hM) for a period of 24 hours.
  • TUNEL staining was then performed according to manufacturer’s instructions (Roche In Situ Cell Death Detection Kit, Fluorescein: 11684795910). All imaging was performed using an Olympus BX53 Fluorescence microscope and DP80 digital camera. Analysis was performed using the Fiji distribution of ImageJ (version l.5ln) 34 .
  • a region of interest was defined using Li Auto Thresholding of the DAPI channel, and the colocalization percentage was calculated using the colocalization threshold plugin bound by that region.
  • astrocytes were plated in a 48 well culture plate (10,000 cells/well) and placed into the IncuCyte S3 Live-Cell Analysis System.
  • the IncuCyte System is a time-lapse imaging system that records cell culture changes through photographic capture of the culture well within the incubator. Cultures were acclimated to the system for a period of 6 hours, then exposed to media containing IFNy (R&D Systems, 285-IF; 200ng/ml), LPS (Sigma, L2654; lOOng/ml) or a combination of both for a period of 24 hours to induce an inflammatory response 35 .
  • Example 1 Determination of which cells in the mouse brain become senescent
  • pl6 lnk4a expression was significantly increased beginning at 4 months of age in the hippocampus and at 6 months in the cortex (FIG. 9A), which precedes the onset of NFT deposition.
  • increased p!6 lnk4a expression correlated with expression of widely established senescence markers (FIG. 13), indicating that senescent cells accumulate at sites of pathology in the PS19 model.
  • Example 2 Elimination of senescent cells in the PS19;ATTAC mouse model
  • Senescence indicators including the cell cycle regulators p!6 lnk4a , pl9 kt ⁇ /i2/ Cipl/Wai l and the pro-inflammatory genes Pail (also called Serpinel ), 11-6 , and II-Ib, were also elevated (FIG. 9C and FIG. 14).
  • AP administration in PS19;ATTAC mice maintained the expression of these genes at a level comparable to control mice (FIG. 9C and FIG. 14).
  • AP treatment of ATTAC mice lacking the PS 19 transgene had no impact on the expression of these markers (FIG. 14).
  • AP administration effectively and selectively cleared senescent cells in the hippocampus and cortex of PS19;ATTAC mice.
  • FIGS. 17A- 17D supporting the conclusion that senescence occurs in astrocytes and microglia of PS19 mice.
  • AP administration selectively targeted senescent cells
  • in vitro cultures were made of primary microglia and astrocytes isolated from ATT AC mice. These cultures were not sensitive to AP-mediated elimination in the absence of senescence-inducing stimuli (FIGS. 19A-19D).
  • short-term AP administration did not promote excessive cellular death (FIGS. 19A) or increased proliferation of microglia with extended treatment of ATTAC transgenic mice in vivo (FIGS. 19B).
  • PS19 mice present progressive gliosis with disease progression.
  • RT-qPCR was performed on 6-month-old hippocampi for markers of astrocytes ( GFAP and 8100b) and microglia ( Cdllb ).
  • Vehicle-treated PS19;ATTAC mice had an ⁇ 2-3 fold induction in these markers, whereas AP -treated PS19;ATTAC mice expressed these markers at a similar level to control mice (FIGS. 20A-20B).
  • Immunohistochemistry (IHC) for GFAP and Ibal confirmed these observations (FIGS. 20C-20D).
  • a distinguishing characteristic of PS19 mice is the development of aggregates consisting of hyperphosphorylated tau protein by 6 months of age.
  • the levels of soluble total and phosphorylated tau (Ser202/Thr205) was probed in addition to the level of insoluble phosphorylated tau in vehicle- treated PS 19; A TTAC and AP -treated ATTAC and PS19;ATTAC mice.
  • Vehicle-treated PS19;ATTAC mice displayed increased soluble total and phosphorylated tau and insoluble phosphorylated tau (FIGS. 10A and FIGS. 21A-21B).
  • AP -treated PS19;ATTAC mice showed identical levels of soluble total tau protein to vehicle-treated PS 19; ATTAC mice (FIG. 10A), indicating that tau over- expression from the transgene was maintained.
  • AP treatment of PS19;ATTAC mice significantly reduced the amount of phosphorylated tau in both the soluble and insoluble fraction (FIG. 10A and FIG. 21B).
  • IHC staining for phospho-tow modifications at S202/T205, T231, and S396 confirmed that senescent cell clearance attenuated tau phosphorylation at a number of residues relevant for tau aggregation (FIG. 10B and FIG. 21C).
  • mice were transcardially perfused with PBS.
  • Half of the brain was microdissected for qRT-PCR analysis.
  • the other half brains were stored in 4% PFA overnight at 4°C and then cryoprotected by incubating in a 30% sucrose solution for 48 hours at 4°C.
  • Brain samples were sectioned into 30 mM thick coronal sections and stored in antifreeze solution (300g Sucrose, 300 mL Ethylene Glycol, 500 mL PBS) at -20°C.
  • Nissl staining (Bregma -2.1 to -2.4 mm) was performed to show the density of neurons and phospho-tau S202/S205 (ThermoFisher, MN1020; 1 :500) (FIGS. 23A and 23D) was investigated by immunohistochemistry (IHC) for tangle deposition.
  • Immunofluorescence (IF) staining (Bregma 1.6 to 1.0 mm and Lateral 2.0 to 2.7 mm) were all performed on free-floating coronal sections for Gfap (Dako, Z0334; 1 :500) (FIG. 23B) to show reactive astrocytes and Iba-l (Novus, NB100-1028; 1 : 100) (FIG. 23C) to show reactive microglia.
  • FIG. 24 shows qRT-PCR analysis for various senescence markers and the analysis was performed on isolated hippocampi.
  • qRT-PCR analysis on hippocampi from each group revealed ABT-263 administration downregulated indicators of senescence presence (pl6 , p21, and PAI1 ) and markers of gliosis ( GFAP , SlOOb, Cdllb).
  • PS19 mice show neurodegeneration by 8 months of age. As NFT deposition was attenuated with AP treatment in both the cortex and hippocampus of PS19;ATTAC mice, assessments for degeneration were performed in these areas. Overt brain size of vehicle-treated PS19;ATTAC mice was reduced compared to both ATTAC and AP -treated PS19;ATTAC mice (FIG. 11 A). In addition, localized neurodegeneration was observed in the dentate gyrus of the hippocampus through Nissl staining in vehicle-treated PS19;ATTAC mice (FIG. 11B). AP administration prevented thinning of the dentate gyrus and increased neuron density. Sequential coronal sectioning and NeuN staining revealed that the dentate gyrus was significantly reduced in vehicle-treated PS19;ATTAC mice (FIG. 11C), further demonstrating that senescent cells promote neurodegeneration in PS19 mice.
  • Example 7 Evaluating Loss of Cognition in PS19; ATTAC mice
  • Example 9 Determination of which cells in the mouse brain become senescent during AD
  • mice were produced a cohort of 120 P301S;ATTAC and 60 ATTAC mice (see Vertebrate Animals section for cohort size calculations). Half of the P301S;ATTAC cohort are treated with vehicle beginning at weaning age, the other half receive AP administration biweekly at a dose of 2 mg/kg. AP -treated mice are used to compare elimination efficiency. Twenty mice of each cohort are sacrificed at 3, 6 and 10 months to establish the cell populations that are senescent in the early, middle and late stages of disease for the P301S model. 10 animals of each group are used for immunostaining (see below). The other 10 animals of each group are sacrificed by decapitation and their brains are quickly removed and prepared for SA-P-Gal activity.
  • the brain is placed in ice-cold Krebs-Ringer Bicarbonate (KRB) buffer (pH 7.3) and subsequently embedded in 2% agar to provide the needed support to prevent tissue deformation.
  • KRB Krebs-Ringer Bicarbonate
  • the tissue is subsequently cut coronally by vibratome into 250 pm-thick slices, followed by removal of the agar.
  • the advantage of using tissue slices is that this procedure allows for both maximal penetration of staining solution and maintenance of tissue architecture.
  • SA-P-Gal staining we microdissect all regions that exhibit increased activity from the groups for TEM to localize the specific cell types that have inclusion of X-Gal crystals (Gal -TEM) in a manner similar to as we have done previously (see
  • FIGS. 4A-4C Detection of X-Gal crystals by transmission electron microscopy is performed as previously described. The specificity of crystal formation is verified using tissue that was not stained for SA-P-Gal. Using distinguishing morphological characteristics of astrocytes, microglia, neural stem cells and neurons observable via TEM, we define the particular cell types that contain X-Gal crystals. Additionally, we measure the percentage of X-Gal-crystal-containing cells in vehicle and AP -treated mice. We screen 5 grids (>1500 cells/grid) per treatment group for cells X- Gal-positive cells at 3000x magnification. Cells with one or more crystal and the total number of cells are counted. Furthermore, NFTs are distinguishable via TEM. We determine whether X-Gal positive cells also contain NFTs, whether NFTs occur in close proximity to senescent cells or whether these two features are mutually exclusive, as it is currently unclear what the relationship is.
  • Example 10 Determination of the kinetics of senescent cell accumulation in AD
  • mice were produced a cohort of 100 P301S;ATTAC and 100 ATTAC mice (see Vertebrate Animals section for cohort size calculations). Beginning at 1 month of age, 10 animals of each group are sacrificed monthly (last mice are sacrificed at 10 months of age) and their brains are characterized for the presence of senescent cells. All mice are sacrificed by decapitation and their brains are quickly removed. The right hemisphere are separated into the following regions and snap frozen: olfactory bulb, brain stem, cerebellum, cerebral cortex, entorhinal cortex, and hippocampus. RNA is isolated and cDNA is synthesized from all mice.
  • Example 11 Identification of SASP components from AD brains
  • Example 12 Evaluation of transcriptional alterations of senescent cells triggered by AD
  • Example 13 Evaluation of elimination of senescent cells on AD progression
  • ATTAC and 120 P301S;ATTAC mice are created.
  • 2 pg/g body weight of AP or vehicle the same dose as was used for the treatment regiment when initiated at weaning age
  • ATTAC mice are administered AP to serve as a control for drug administration.
  • stains include, but are not limited to, Congo red, GFAP and thioflavin S. We evaluate both the number and area of the plaques that are present throughout the brain.
  • Example 14 Evaluation of senolvtics for treatment of AD
  • Example 15 Phase 2 Study of a Senolvtic Agent in Alzheimer’s Patients
  • the senolytic agent is given in doses 800-2000 mg per day, or in doses that correspond to efficacy in pre-clinical animal models.
  • ADCS-CCGIC Alzheimer's Disease Cooperative Study Clinician's Global Impression of Change
  • ADCS-ADL Alzheimer's Disease Cooperative Study Activities of Daily Living Inventory
  • the ADCS-ADL scale discriminates well between normal controls and mild AD patients. It has good test-retest reliability.
  • the ADCS-ADL includes some items from traditional basic ADL scales (e.g., grooming, dressing, walking, bathing, feeding, toileting) as well as items from instrumental activities of daily living scales (e.g., shopping, preparing meals, using household appliances, keeping appointments, reading).
  • NPI Neuropsychiatric Inventory
  • Plasma beta-amyloid proteins are collected from blood samples obtained at visit 2 (week 2), visit 6 (week 12), and visit 8 (week 24).
  • APO-E genotyping [ Time Frame: Collected at visit 2 (week 2) ] [ Designated as safety issue: No ]
  • APOe e4 is an important genetic risk factor for AD. In this trial, as in many studies of AD and memory and cognition in aging, the APOe e4 allele is analyzed as a predictor of clinical change over time.
  • Inclusion Criteria NINCDS/ADRDA criteria for probable; AD MMSE between 12- 27; Treatment with a cholinesterase inhibitor or an NMDA (N-methyl-D-asparate) antagonist with stable dose for at least 12 weeks; Home monitoring available for supervision of medications; Caregiver available to accompany patient to all visits and willing to participate in study as informant Fluent in English or Spanish; Medical stability for this study as confirmed by review of records, internist's physical exam, neurological exam, and laboratory tests Stable doses of non- excluded medication; No evidence of hepatic insufficiency; Able to swallow oral medications; Ability to participate in the informed consent process.
  • Exclusion Criteria History of Diabetes Mellitus (OGTT criteria) requiring treatment with an excluded antidiabetic medication or history of hypoglycemia; Active hepatic or renal disease; Cardiac disease including history of congestive heart failure or current treatment for CHF; history of recent myocardial infarction; Use of another investigational drug within the past two months; History of clinically significant stroke; History of seizure or head trauma with disturbance of consciousness within the past two years; Major mental illness including psychotic disorders, bipolar disorder, or major depressive episode within the past two years; Medication Exclusion Current use of oral hypoglycemic agents including sulfonylureas and meglintinides; Current or past treatment with insulin for longer than two weeks; Current use of drugs with significant anticholinergic or antihistaminic properties.
  • OGTT criteria History of Diabetes Mellitus (OGTT criteria) requiring treatment with an excluded antidiabetic medication or history of hypoglycemia; Active hepatic or renal disease; Cardiac disease including history of
  • mice are examined for alterations in cognitive behaviors using both novel object recognition experiments as described in Bussian TJ, et al. Clearance of senescent glial cells prevents tau-dependent pathology and cognitive decline. Nature 562, pages578-582 (2016) and contextual fear conditioning (CFC) before and after senolytic intervention in lO-month-old mice as described in Example 5.
  • CFC contextual fear conditioning
  • tone duration is 30 s
  • level is 70 dB, 2 kHz
  • shock duration is 2 s
  • intensity is 0.6 mA. This intensity is not painful and can easily be tolerated but generates an unpleasant feeling. More specifically, on day 1 each mouse is placed in a fear-conditioning chamber and allowed to explore for 2 min before delivery of a 30-s tone (70 dB) and light cue ending with a 2-s foot shock (0.6 mA).
  • a second CS-US pair is delivered.
  • each mouse is first placed in the fear-conditioning chamber containing the same exact context, but with no CS or foot shock. Freezing is analyzed for 1-3 min.
  • the mice are placed in a new context containing a different cleaning solution, floor texture, chamber walls and shape. Animals are allowed to explore for 2 min before being re-exposed to the CS alone, then again 2 min later. Freezing is analyzed for 0-120 seconds (No-Stimulus 1 (NS-l)), 120-150 seconds (CS-l), 150-170 seconds (NS-2), and 170-200 seconds (CS-2). Freezing is measured using a FreezeScan video tracking system and software (Cleversys, Inc).

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Abstract

La tauopathie décrit une classe de maladies neurodégénératives associées à l'agrégation pathologique de protéine tau dans des enchevêtrements neurofibrillaires ou gliofibrillaires dans le cerveau humain, qui s'est avérée coïncider avec une accumulation de cellules sénescentes dans le cerveau. La clairance de cellules sénescentes par administration de sénolytiques chez des mammifères adultes s'est avérée améliorer la fonction cognitive. En outre, la présente invention concerne des procédés de clairance de cellules sénescentes chez des sujets pour traiter la maladie d'Alzheimer, comprenant l'administration d'agents sénolytiques. Les procédés de l'invention ont le potentiel à la fois de réduire la probabilité de développer la maladie d'Alzheimer, de retarder la progression de la maladie d'Alzheimer ou d'inverser le développement de pathologies de la maladie d'Alzheimer.
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CN113694071A (zh) * 2021-08-31 2021-11-26 上海交通大学医学院附属瑞金医院 清除和/或溶解衰老细胞和/或抑制细胞衰老的化合物用于治疗精神障碍

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US20170056421A1 (en) * 2014-05-05 2017-03-02 Board Of Trustees Of The University Of Arkansas COMPOSITIONS AND METHODS FOR INHIBITING ANTIAPOPTOTIC Bcl-2 PROTEINS AS ANTI-AGING AGENTS
WO2017065837A1 (fr) * 2015-10-13 2017-04-20 Siwa Corporation Anticorps anti-age et procédés d'utilisation correspondants
US20170209435A1 (en) * 2014-01-28 2017-07-27 Buck Institute For Research On Aging Methods and compositions for killing senescent cells and for treating senescence-associated diseases and disorders

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US20170209435A1 (en) * 2014-01-28 2017-07-27 Buck Institute For Research On Aging Methods and compositions for killing senescent cells and for treating senescence-associated diseases and disorders
US20170056421A1 (en) * 2014-05-05 2017-03-02 Board Of Trustees Of The University Of Arkansas COMPOSITIONS AND METHODS FOR INHIBITING ANTIAPOPTOTIC Bcl-2 PROTEINS AS ANTI-AGING AGENTS
WO2017065837A1 (fr) * 2015-10-13 2017-04-20 Siwa Corporation Anticorps anti-age et procédés d'utilisation correspondants

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113694071A (zh) * 2021-08-31 2021-11-26 上海交通大学医学院附属瑞金医院 清除和/或溶解衰老细胞和/或抑制细胞衰老的化合物用于治疗精神障碍

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