[0001] This application claims priority benefit of U.S. Provisional Application No.

62/190,644, filed July 9, 2015, which application is herein incorporated by reference for all purposes. BACKGROUND OF THE INVENTION

[0002] Brain aging and neurodegenerative diseases remain major medical challenges of the 21st century. Alzheimer’s disease, the most common form of dementia, is estimated to have cost the USA $172 billion and the world $604 billion in 2010 alone (Wimo and Prince, 2010). Amyotrophic lateral sclerosis is characterized by the loss of upper and lower motor neurons and life expectancy is 2-5 years following diagnosis. It is estimated to affect about 500,000 individuals in the USA and costs are $256-$433 million per year (Larkindale et al., 2014). Despite continued scientific effort, few therapies exist for these neurodegenerative diseases.

[0003] Astrocytes are the most abundant cell type in the brain and play multiple key roles in providing structural, functional, and metabolic support to neurons (Barres, et al., 2008). Although Alzheimer’s disease and amyotrophic lateral sclerosis possess different etiologies, a commonality between these two diseases is the role of astrocytes in neurodegeneration (Verkhratsky, et al., 2012; Furman, et al., 2012). Dysfunctional neuron-astrocyte crosstalk is known to be a central feature of neurodegenerative diseases (Das, et al., 2014; Haidet- Phillips, et al., 2011). Astrocytes exert both toxic and protective effects on neurons in a context-dependent manner. The neurotoxic effects of astrocytes are mediated in part through pro-inflammatory cytokines, such as interleukin-6 (IL-6). Overproduction of these factors is associated with human neurodegeneration (Jia, et al., 2005) and neurodegeneration in murine models (Campbell, et al., 1993). The neuroprotective effects of astrocytes are mediated through nerve growth factor (NGF) and insulin-like growth factor-1 (IGF-1) (Farina, et al., 2007), which are deficient in neurodegenerative diseases (Nagatsu, et al., 2000; Tuszynski, et al., 2007) and, to a lesser extent, during physiological brain aging (Erickson, et al., 2010). Alexander disease, a rare astrocyte disease involving a mutation in the glial fibrillary acidic protein (GFAP), results in neurodegeneration (Brenner, et al., 2001) and highlights the role of astrocytes in neurotoxicity. Riluzole, a FDA (Food and Drug Administration)-approved drug for amyotrophic lateral sclerosis that provides modest therapeutic benefit, targets a glutamate transporter, EAAT2, in astrocytes (Miller, et al., 2007). Thus, development of therapies targeting astrocytes is a subject of intense research for neurodegenerative diseases (Furman, et al., 2012; Verkhratsky, et al., 2012).

[0004] Cellular senescence is characterized by proliferation arrest after extensive cell divisions or upon cellular stresses such as oxidative damage (Campisi, et al., 2013). The characteristics of cellular senescence include enlarged and flattened cell morphology, upregulation of cell cycle inhibitors p16 ^INK4 (Rayess, et al., 2012) and p21 ^WAF1 (Roninson, et al., 2002), senescence-associated β-galactosidase (SA-β-Gal) activity (Debacq-Chainiaux et al., 2009), elevated nitric oxide synthase 2 (NOS2) associated with oxidative stress (Sohn, et al.2012), and DNA double-strand breaks marked by phosphorylated H2AX (ȖH2AX) (Wang, et al., 2009). A growing body of evidence suggests that senescent cells secrete various cytokines, growth factors, and proteases that alter tissue microenvironment, which is collectively termed the senescence-associated secretory phenotype (SASP) (Coppé, et al., 2010). A major outcome of SASP is tissue inflammation and degeneration mainly through the activity of pro-inflammatory cytokines such as IL-6 and IL-1β, as represented by neurodegeneration in which these cytokines are secreted from astrocytes (as described above). These pro-inflammatory cytokines may also promote carcinogenesis (Campisi, et al., 2013). In addition to these unfavorable effects, senescent cells and SASP can play beneficial and physiological roles as well. Senescent cells play an essential role in wound healing through the secretion of platelet-derived growth factor AA (Demaria et al., 2014; Serrano, 2014). Programmed senescence with SASP is essential to mammalian embryonic development (Banito and Lowe, 2013; Muñoz-Espín, et al., 2013; Storer, et al., 2013). In the brain, cellular senescence with SASP is mostly observed in proliferation-competent cell types such as astrocytes (Tan et al., 2014) and it increases in neurodegenerative diseases and during physiological aging (Chinta et al., 2014; López-Otín et al., 2013; Muñoz-Espín and Serrano, 2014).

[0005] A main regulator of cellular stress responses and senescence is the TP53 gene (Zilfou and Lowe, 2009). The human TP53 encodes, in addition to full-length p53 protein, at least 12 natural protein isoforms through alternative mRNA splicing or alternative promoter usage (Bourdon et al., 2005). Δ133p53, an N-terminal truncated isoform, functions as a negative regulator of cellular senescence by dominant-negatively inhibiting full-length p53 (Fujita et al., 2009; Mondal et al., 2013) and, unlike full-length p53, is degraded via selective autophagy (Horikawa et al., 2014). p53β, a C-terminal modified isoform, functions as a co- activator of full-length p53 to promote senescence (Fujita et al., 2009; Mondal et al., 2013), and its expression level is regulated via alternative mRNA splicing involving splicing factor SRSF3 (Tang et al., 2013). The roles of Δ133p53 and p53β in cellular senescence have been demonstrated in multiple cell types, including fibroblasts of mesenchymal origin (Fujita et al., 2009), colon adenoma of epithelial origin (Fujita et al., 2009) and CD8+ T-lymphocytes of hematopoietic origin (Mondal et al., 2013). BRIEF SUMMARY OF CERTAIN ASPECTS OF THE INVENTION

[0006] In certain aspects, this invention is based, in part, on the discovery that Δ133p53 and p53β regulate the neuroprotective and neurotoxic functions of human astrocytes.

[0007] In one aspect, the invention provides a method of enhancing the neuroprotective function of an astrocyte of a subject that has a neurodegenerative disease involving astrocyte dysfunction, the method comprising contacting a cell of a subject that has a disease selected from the group consisting of Alzheimer’s Disease, amyotrophic lateral sclerosis, Parkinson’s Disease, hepatic encephalopathy and Alexander’s disease with an agent that activates the function or expression of Δ133p53, thereby enhancing the neuroprotective function of the astrocyte. In some embodiments, the neurodegenerative disease is Alzheimer’s Disease. In some embodiments, the neurodegenerative disease is amyotrophic lateral sclerosis. In some embodiments, the neurodegenerative disease is Parkinson’s Disease. In some embodiments, the neurodegenerative disease is hepatic encephalopathy. In some embodiments, the neurodegenerative disease is Alexander’s disease. In some embodiments, the agent comprises a polynucleotide sequence encoding Δ133p53, such as an expression cassette comprising a polynucleotide sequence encoding Δ133p53. In some embodiments, the agent is a small molecule.

[0008] In a further aspect, the invention provides a method of enhancing the

neuroprotective function of an astrocyte of a subject that has a neurodegenerative disease involving astrocyte dysfunction, the method comprising contacting a cell of a subject that has a disease selected from the group consisting of Alzheimer’s Disease, amyotrophic lateral sclerosis, Parkinson’s Disease, hepatic encephalopathy and Alexander’s disease with an agent that inhibits the function or expression of p53β, thereby enhancing the neuroprotective function of the astrocyte. In some embodiments, the neurodegenerative disease is

Alzheimer’s Disease. In some embodiments, the neurodegenerative disease is amyotrophic lateral sclerosis. In some embodiments, the neurodegenerative disease is Parkinson’s Disease. In some embodiments, the neurodegenerative disease is hepatic encephalopathy. In some embodiments, the neurodegenerative disease is Alexander’s disease. In some embodiments, the agent comprises a polynucleotide sequence that inhibits p53β, such as an expression cassette comprising a polynucleotide sequence that inhibits p53β. In some embodiments, the agent is a small molecule. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1: Astrocytes express p53 isoforms, Δ133p53 and p53β, and their expression is regulated during cellular senescence. (A) Immunofluorescence staining showing p53β and Δ133p53 expression in GFAP-positive astrocytes (arrows). No expression of p53β and Δ133p53 was observed in GFAP-negative cells (arrow heads). Top panel, Non-disease (ND) tissue case 1; bottom panel, ND case 2. Scale bars = 10 μm. (B) SA-β-gal staining in primary human astrocytes at passage number 5 (P5) and P10. These astrocytes became completely growth arrested at P15. Scale bars = 20 μm. (C) Western blot analysis of full-length p53, p53 isoforms and senescence marker p16INK4A in early passage (P5) and aged (P13) primary human astrocytes. Densitometric values are normalized to β-actin.

[0010] Figure 2: Autophagic degradation of Δ133p53 and SRSF3-mediated regulation of p53β. (A) qRT-PCR analysis of Δ133p53 and p53β in early-passage (P5) and aged (P13) primary astrocytes. (B) Δ133p53 expression is restored by bafilomycin A1 treatment (100 nM for 4 hours) in senescent human astrocytes (P15). The activity of bafilomycin A1 was confirmed by increased LC3-II. (C) Knockdown of p62/SQSTM1 stabilizes Δ133p53.

Immortalized astrocytes were transfected with p62/SQSTM1 siRNA (+) or control siRNA (-). (D) SRSF3 knockdown induces p53β mRNA. Immortalized astrocytes were transfected with SRSF3 siRNA or control siRNA and examined by qRT-PCR for SRSF3 and p53β expression. Immortalized astrocytes, which can be transfected with siRNA at a higher efficiency than primary astrocytes, were used in C and D. Data are presented as mean ± SEM. NS indicates p > 0.05, * p≤ 0.05, **p≤ 0.01, *** p≤ 0.001 by unpaired two-tailed Student’s t test. [0011] Figure 3: Knockdown of Δ133p53 induces senescent phenotype in astrocytes. Immortalized astrocytes were transfected with Δ133p53 siRNA or control siRNA and analyzed after 3 days. (A-B) Confirmation of Δ133p53 knockdown by Western blot (A) and immunofluorescence (B). Scale bars = 10 μm. (C) Phase-contrast images of siRNA- transfected astrocytes (upper panel, 20X magnification; lower panel, 40X magnification). Arrows show vacuolization. Scale bars = 20 μm. (D-E) An increase in SA-β-gal staining by Δ133p53 knockdown. Representative images, Scale bars = 20 μm (D) and quantitative summary from triplicated experiments (E). (F) Increase in IL-6, IL-1β and p21WAF1 mRNA expression by Δ133p53 knockdown. qRT-PCR was performed in triplicate. Data are presented as mean ± SEM. * p≤ 0.05, *** p≤ 0.001 by unpaired two-tailed Student’s t test.

[0012] Figure 4: Overexpression of p53β induces senescent phenotype in astrocytes.

Immortalized astrocytes were transduced with p53β lentiviral vector or control vector and analyzed after 3 days. (A) Confirmation of p53β overexpression by Western blot. (B) Phase- contrast images of transduced astrocytes. Scale bars = 20 μm. (C-D) An increase in SA-β- gal staining by p53β overexpression. Representative images, Scale bars = 20 μm (C) and quantitative summary from triplicated experiments (D). (E) Elevated mRNA expression of IL-8, IL-6, IL-1β and p21 ^WAF1 by p53β overexpression. qRT-PCR was performed in triplicate. Data are presented as mean ± SEM. * p≤ 0.05, *** p≤ 0.001 by unpaired two- tailed Student’s t test.

[0013] Figure 5: Increased neuronal death upon co-culture with Δ133p53-knocked-down or p53β-overexpressing astrocytes. Early-passage primary astrocytes (P5) with Δ133p53 siRNA and control siRNA (generated as in Fig 12) (A, B, E and F) and those with p53β - overexpression or control vector (generated as in Fig 13) (C, D, G and H) were used in co- culture for 48 hours with motor neurons (Grunseich, 2014) or less specialized neurons (as generated as in Fig 14C-F). (A and C) Immunofluorescence staining of MAP2 (neuronal marker) and cleaved caspase-3 (apoptosis marker). Astrocytes are marked by GFAP (A) or fluorescent signals derived from the lentiviral vectors (C). Scale bars = 10 μm. (B and D) Quantification of neuronal apoptosis. Cleaved caspase-3-positive neurons per total number of MAP2-positive neurons were counted in triplicate experiments in 5 microscopic fields (40X magnification). (E and G) Immunofluorescence staining of NeuN (neuronal marker). Scale bars = 10 μm. (F and H) Quantification of number of neurons. Total number of NeuN- positive neurons were counted in at least 5 microscopic fields (40X magnification) in triplicate experiments. Data are presented as mean ± SEM. * p≤ 0.05, **p≤ 0.01, *** p≤ 0.001 by unpaired two-tailed Student’s t test.

[0014] Figure 6: Δ133p53 protects astrocytes from senescence and enhances

neuroprotective function. Aged primary astrocytes (P12) were transduced with a lentiviral vector driving Δ133p53 or its control vector and analyzed after 3 days. (A) Confirmation of lentiviral transduction by RFP (vector control) or GFP (Δ133p53). Scale bars = 10 μm. (B) Decreased SA-β-gal staining by reconstituted expression of Δ133p53. Representative images of SA-β-gal staining (top panels, scale bars = 20 μm) and enlarged images of the insets (bottom panels). (C) Quantitative summary of SA-β-gal staining from triplicated experiments. (D) Decreased expression of IL-1β and IL-6 and increased expression of NGF and IGF-1. qRT-PCR analysis was performed in triplicate. (E-H) Δ133p53-reconstituted astrocytes and control astrocytes were used in co-culture with motor neurons (E and F) or less specialized neurons (G and H), as performed in Figure 5. (E) Decrease in cleaved caspse-3-positive motor neurons upon co-culture with Δ133p53-reconstituted aged (P12) astrocytes.

Immunofluorescence staining of cleaved-caspase 3 and MAP2 was performed. Astrocytes were detected by the vector-derived fluorescence. Scale bars = 10 μm. (F) Quantification of neuronal apoptosis. Data were achieved by counting the number of cleaved caspase 3- positive neurons per total number of MAP2-positive neurons in triplicate experiments in 5 microscopic fields (40X magnification). (G) Immunofluorescence staining of NeuN. Scale bars = 10 μm. (H) Quantification of number of NeuN-positive neurons. Total number of NeuN-positive neurons were counted in at least 5 microscopic fields (40X magnification) in triplicate experiments. (I) Quantification of neuronal apoptosis in co-culture experiments with IL-6 or NGF neutralizing antibodies. Data were from the experiment shown in Figure 15E and achieved by counting the number of cleaved caspase 3-positive neurons per total number of NeuN-positive neurons in triplicate experiments in 5 microscopic fields (40X

magnification). Data are presented as mean ± SEM. NS indicates p > 0.05, * p≤ 0.05, **p≤ 0.01, *** p≤ 0.001 by unpaired two-tailed Student’s t test.

[0015] Figure 7: Increased senescent astrocytes in brain tissues from neurodegenerative disease patients. (A) Increased expression of senescence-associated proteins, ȖH2AX, NOS2, and p16 ^INK4A in Alzheimer’s disease (AD) (Case 5), and amyotrophic lateral sclerosis (ALS) (Case 4). Non-disease (ND) age-matched control tissue was case 5. Scale bars = 50 μm. Insets in AD and ALS show enlarged positive cells. (B) Quantification of p16 ^INK4A -positive cells in AD (Cases 1-4) and ALS (Cases 1-3) compared to ND (Cases 1-4). (C) Elevated mRNA expression of senescence-associated genes in AD (Cases 1-4) and ALS (Cases 1-3) compared to ND (Cases 1-4). qRT-PCR experiments were performed in triplicate. Data are presented as mean ± SEM. (D) Increase in p16 ^INK4A - and GFAP-positive cells in AD (Case 1) compared to ND (Case 1). Scale bars = 10 μm. NS indicates p > 0.05, **p≤ 0.01, *** p≤ 0.001 by unpaired two-tailed Student’s t test.

[0016] Figure 8. p53β is upregulated and Δ133p53 is downregulated in neurodegenerative diseases. (A) Representative Western blot showing elevated expression of full-length p53 and p53β and decreased Δ133p53 expression in Alzheimer’s disease (AD) (Case 3) and amyotrophic lateral sclerosis (ALS) (Case 3) compared to non-disease (ND) age-matched control brain tissue (Case 2). Densitometric values are normalized to β-actin. (B) Summary of densitometric analyses of Western blots from AD (Cases 1-4), ALS (Cases 1-3), and ND (Cases 1-4). (C-D) qRT-PCR analysis of Δ133p53 (C) and p53β (D) in pediatric brain tissue (Cases 1-4), non-disease (ND) control (Cases 1-4), Alzheimer’s disease (AD) (Cases 1-4), and amyotrophic lateral sclerosis (ALS) (Cases 1-3). Expression levels are shown relative to ND. Densitometric values are normalized to β-actin. Data are presented as mean ± SEM. * p ≤ 0.05, **p≤ 0.01, *** p≤ 0.001 by unpaired two-tailed Student’s t test.

[0017] Figure 9. Proposed models of the p53 isoform regulation of astrocyte-mediated neuroprotection and neurodegeneration. (A) The p53 isoforms regulate astrocyte-mediated neuroprotection and neurodegeneration. Δ133p53, which is regulated by selective autophagy- mediated degradation32, is abundant in early-passage astrocytes in vitro and astrocytes in non-disease brain tissues (here collectively termed“young”) and contributes to the neuroprotective function of these“young” astrocytes. p53β, which is generated via alternative mRNA splicing 33, is induced in senescent astrocytes in vitro and in

neurodegenerative disease brain tissues and contributes to the neurotoxic effect of these senescent astrocytes. (B) IL-6 is a therapeutic target in astrocytes to prevent neurotoxicity. DNA damage and oxidative stress activate full length p53 (FL-p53), which leads to transcriptional activation of IL-6 (Lowe et al., 2014) and neurotoxicity. The modulation of FL-p53 activity by the p53 isoforms, as well as IL-6 neutralizing antibody, prevents neurotoxicity.

[0018] Figure 10 (supplementary to Figure 1). Immunofluorescence staining of p53 isoforms and their expression in human brain tissue. (A) Immunofluorescence staining with anti-Δ133p53 antibody MAP4 or anti-full-length (FL)-p53 antibody DO-1 in H358 cells with (+Dox) and without (-Dox) induction of Δ133p53 (left) or FL-p53 (right). MAP4 does not cross-react with FL-53. Scale bars = 10 μm. (B) Immunofluorescence staining with anti-p53β antibody TLQ40 and anti-FL-p53 antibody DO-1 in H358 cells with or without constitutive p53β expression (left) or inducible FL-p53 expression (right).TLQ40 does not cross-react with FL-p53. Scale bars = 10 μm. (C) p53β and Δ133p53 in GFAP-positive astrocytes (arrows). No expression of p53β and Δ133p53 was observed in GFAP-negative cells (arrow heads). ND tissue was case 2. Part of these images are also shown in Figure 1A at higher magnification. (D) Full blots of Δ133p53 and (E) p53β in early passage (P5) and aged (P13) primary human astrocytes as shown in Figure 1. The major bands are Δ133p53α (35 kDa) in the MAP4 blot and p53β (47 kDa) in the TLQ40 blot. The β-actin signal in (D) was carry- over from the previous experiment. Solid arrows indicate the positions of all isoforms that can be detected by the MAP4 or TLQ40 antibody. Open arrows indicate the position of the full-length p53, which is not cross-detected by MAP4 or TLQ40.

[0019] Figure 11 (supplementary to Figure 2). (A) Δ133p53 is stabilized by autophagy Inhibition. Immortalized human astrocytes were treated with 100 nM of bafilomycin A1 for 4 hours. (B) Full-length p53 is stabilized and Δ133p53 is decreased by treatment with proteosome inhibitor, MG132 (15μM for 8 hours) in primary astrocytes (P5 ).

[0020] Figure 12 (supplementary to Figure 3). Δ133p53 knockdown induces senescent phenotypes in primary human astrocytes. (A) Full Western blot confirming Δ133p53 knockdown. Primary human astrocytes at early-passage (P5) were transfected with Δ133p53 siRNA and control siRNA as Δ133p53 as performed in Figure 3. The 35 kDa band corresponding to Δ133p53α is shown as Δ133p53 in Figure 3A. The 29 kDa band corresponding to Δ133p53β and/or Δ133p53Ȗ was also diminished by Δ133p53 siRNA. (B) Confirmation of Δ133p53 knockdown via immunofluorescence. Scale bars = 10 μm. (C-D) Loss of Δ133p53 induces an increase in SA-β-gal-positive cells. Representative images (C) and quantitative summary from triplicated experiments (D) are shown. Scale bars = 20 μm. (E) An increase in IL-6 and IL-1β mRNA expression by Δ133p53 knockdown. (F) Δ133p53 knockdown increases expression of p21 in immunofluorescence. Scale bars = 10 μm. Data are presented as mean ± SEM. * p≤ 0.05, *** p≤ 0.001 by unpaired two-tailed Student’s t test.

[0021] Figure 13 (supplementary to Figure 4). p53β overexpression induces senescent phenotype in primary human astrocytes. (A) Full Western blot confirming p53ȕ overexpression as shown in Figure 4A. Arrows indicate the positions of all β isoforms that can be detected by the TLQ40 antibody. Primary human astrocytes at early passage (P5) were transduced with a p53β-overexpressing lentiviral vector or control vector as performed in Figure 4. (B) Confirmation of p53β overexpression via immunofluorescence. Scale bars = 10 μm. (C) Phase-contrast images of transduced astrocytes. Scale bars = 20 μm. (D-E) Increased SA-β-gal staining in p53β-overexpressing cells. Representative images (D) and quantitative summary from triplicated experiments (E) are shown. Scale bars = 20 μm. (F) Elevated mRNA expression of IL-6 and IL-1β in p53β-overexpressing astrocytes. (G) p53β overexpression in primary human astrocytes had no significant effect on cell viability, which was assessed by dye exclusion assay using Trypan blue. (H) p53β overexpression in primary human astrocytes increased expression of p21 but not Bax mRNA. (I) Expression of p16 was increased following p53β overexpression. Data are presented as mean ± SEM. *p≤ 0.05, **p ≤ 0.01, *** p≤ 0.001 by unpaired two-tailed Student’s t test.

[0022] Figure 14 (supplementary to Figure 5). Generation of neurons from induced pluripotent stem cells (iPSC) and their co-culture with astrocytes. (A) Experimental scheme of co-culture system in which primary human astrocytes (red) are co-cultured with iPSC- derived neurons (green) for 48 hours. (B) Immunofluorescence staining of astrocytes (GFAP, red) and neurons (NeuN, green) in co-culture. Enlarged image of the inset is shown rightmost. (C-F) Neuronal differentiation time course. Sequential differentiation of iPSC was confirmed through the expression of TRA-1-81 (D), Sox1 and Nestin (E), and NeuN (F). Scale bars = 10 μm. (G) Motor neurons were characterized by the expression of SMI-32. Scale bars = 10 μm. (H) Co-culture of iPSC-derived neurons and astrocytes. For quadruple- immunofluorescence in this experiment, a non-fluorescence-carrying retroviral vector was used for p53β overexpression in astrocytes. Antibodies used were for GFAP (astrocyte- specific marker), NeuN (neuron specific marker), and cleaved caspase-3 (apoptotic marker). Scale bars = 10 μm. (I) Quantification of neuronal apoptosis was achieved by counting the number of cleaved caspase-3-positive neurons per total number of NeuN-positive neurons in triplicate experiments in 5 microscopic fields (40X magnification). Data are presented as mean ±SEM. * p≤ 0.05 by unpaired two-tailed Student’s t test.

[0023] Figure 15 (supplementary to Figure 6). Δ133p53 upregulates NGF. (A)

Immunofluorescence staining showing increased NGF protein upon Δ133p53 reconstitution in aged (P12) astrocytes. Transduced astrocytes were marked by RFP (control) or GFP (Δ133p53; pseudocolored in red). Scale bars = 10 μm. (B) Immunofluorescence staining of NGF in early-passage (P5) and aged (P11) astrocytes. Merged images with DAPI are shown. Scale bars = 10 μm. (C) Immunofluorescence staining of astrocytes treated with 5 ng/mL recombinant IL-6 either alone or in combination with 5 μg/mL IL-6 neutralizing antibody (IL-6-N-Ab). The assay after 24 hours of treatment showed increased cleaved caspase-3 upon IL-6 treatment and reduced levels back to untreated control by co-incubation with IL-6-Nab. Scale bars = 10 μm. (D) Quantification of number of NeuN-positive neurons in the experiment shown in C. (E) Immunofluorescence staining of cleaved-caspase 3 and NeuN. Co-culture experiments were performed using aged astrocytes (P12) and motor neurons, as in Figure 6E and G, with IL-6 or NGF neutralizing antibodies at concentrations of 5 μg/mL or 500 ng/mL, respectively . Quantitative data from this experiment are shown in Figure 6I. Scale bars = 10 μm.

[0024] Figure 16 (supplementary Figure 7). Increased senescent astrocytes in brain tissues from neurodegenerative disease patients. (A) Increased expression of IL-6 and p21WAF1 mRNA in aged non-disease (ND) tissues (Cases 1-4) compared to pediatric non-disease brain tissues (Cases 1-4). (B) GFAP immunohistochemistry staining of ND (Case 5), Alzheimer’s disease (AD) (Case 5), and amyotrophic lateral sclerosis (ALS) (Case 4) tissues. Scale bar = 20 μm. (C) GFAP immunofluorescence staining of ND (Case: 1) and AD (Case 1) tissues showing increased ramified astrocytes (arrows) in AD tissues. Scale bar = 10 μm. Data are presented as mean ±SEM.* p≤ 0.05 by unpaired two-tailed Student’s t test.

[0025] Figure 17 (supplementary to Figure 8). Δ133p53 is downregulated and p53β is upregulated in neurodegenerative diseases. (A) Full scan of Western blot of MAP4 antibody of non-disease (ND) tissue (Case 2) compared to Alzheimer’s disease (AD) (Case 3) and amyotrophic lateral sclerosis (ALS) (Case 3) as in Figure 8A. Single bands of 35 kDa corresponding to Δ133p53α, but not a 29 KDa band, were detected. MAP4 does not cross- react with full-length p53. (B) Full scan of Western blot of TLQ40 antibody of ND (Case 2), AD (Case 3), and ALS (Case 3) tissues as in Figure 8A. The major bands were 47 kDa corresponding to p53β. TLQ40 does not cross-react with full-length p53. Positions of other β isoforms detected by TLQ40 are also indicated. The quantitative data from these blots are shown in Figure 8A.

[0026] Figure 18. Only primates have the methionine (M) codon at the amino acid position corresponding to human codon 133. Amino acid sequences of p53 orthologs in various organisms (p53 Knowledgebase at the http address p53.bii.a-star.edu.sg) were compared using ClustalW2 (http address www.ebi.ac.uk/Tools/msa/clustalw2). Full names of organisms are as follows: African clawed frog, Barbel, Beluga whale, Blind subterranean mole rat, Cat, Channel catfish, Chicken, Chinese hamster, Common tree shrew, Congo puffer, Cow, Crab eating macaque, Dog, European flounder, Golden hamster, Green monkey, Guinea pig, Human, Japanese macaque, Mongolian jird, Mouse, Pig, Rabbit, Rainbow trout, Rat, Rhesus macaque, Sheep, Southern platyfish, Swordtail, Western clawed frog,

Woodchuck, Zebrafish, Zebu. DETAILED DESCRIPTION OF THE INVENTION

[0027] The term“p53” refers generally to a protein of a molecular weight of about 53kDa on SDS PAGE that functions as a transcription factor and plays a role in cell cycle control and DNA damage repair as well as other essential pathways in cell growth. The protein and nucleic sequences of the p53 protein from a variety of organisms from humans to Drosophila are known and are available in public databases, such as in accession numbers, NM_000546, NP_000537, NM_011640, and NP_035770, for the human and mouse sequences.

Mammalian p53 sequences are highly conserved between species. Mouse and human p53 proteins are 85% identical. In humans, p53 is encoded by the TP53 gene located on the short arm of chromosome 17 (17p13.1). A p53 protein in the context of this invention includes allelic variants and other functional variants and orthologs. In some embodiments, variants have at least 85%, at least 90%, or at least 95%, or greater, amino acid sequence identity across their whole sequence compared to a naturally occurring p53 family member, such as the polypeptide sequence listed under accession number NP_000537 (human p53).

[0028] The term“Δ133p53” refers generally to the isoform of p53 that arises from initiation of transcription of the p53 gene from an alternative promoter in intron 4 of human p53, which results in an N-terminally truncated p53 protein translated from codon 133. A Δ133p53 isoform for use in this invention comprises the following p53 protein domains: the majority of the DNA binding domain, the NLS, and the C-terminal oligomerization domain (see Bourdon, Brit. J. Cancer, 97: 277-282 (2007)). An illustrative human Δ133p53 protein sequence is listed under accession number ABA29755.1.

[0029] The term“p53β” refers generally to the isoform of p53 that arises from alternative splicing of intron 9 to provide a p53 isoform comprising the following p53 protein domains: TAD1, TAD2, prD, the DNA binding domain, the NLS, and the C-terminal sequence DQTSFQKENC (see Bourdon, Brit. J. Cancer, 97: 277-282 (2007)). [0030] The term“cell senescence” refers generally to the phenomenon where normal diploid differentiated cells lose the ability to divide after undergoing a finite number of cell divisions characteristic of a particular type of cell. Cellular senescence is also associated with other phenotypic changes, such as senescence-associated secretory phenotype (SASP).

[0031] The term“enhancing the neuroprotective function” as used herein in the context of astrocytes refers to delaying or otherwise inhibiting cell senescence, including delaying or otherwise inhibiting cellular changes that occur with senescence, including, but not limited to, the senescence-associated secretory phenotype (SASP). A neuroprotective effect may be assessed, e.g., by measuring the level of neuroprotective factors such as NGF and IGF-1. Neuron apoptosis and survival in co-culture may also be evaluated to determine

neuroprotective function.

[0032] The term "inhibiting" as used, for example in the context of "inhibiting senescence" or“inhibiting SASP”, refers generally to conditions or agents that reduce or down-regulate, or otherwise decrease, cell senescence and SASP compared to a corresponding cell that is not treated with the agent.

[0033] “Senescence-associated secretory phenotype (SASP) in the context of astrocytes typically refers to age-related cellular changes that occur, which include increased expression of proinflammatory cytokines such as IL-6 and IL-1β. SASP can be assessed by any number of endpoints, including level of inflammatory cytokines, e.g., IL-6 and IL-1β, [0034] As used herein, the term“expression" or“increasing expression” of Δ133p53 in the context of this invention includes, but is not limited to, introducing a Δ133p53 nucleic acid into a cell. In some embodiments, the level of expression of Δ133p53 may be increased using another agent that upregulates expression. An "increase" in Δ133p53 expression is generally determined relative to control cells to which an agent, e.g., a nucleic acid encoding Δ133p53 or other agent that increases levels of Δ133p53, has not been added. Expression is typically considered to be increased when levels of Δ133p53 protein or RNA, are increased by at least 20%, typically by at least 50%, or 100% or more. In some embodiments, expression levels may be two-fold higher than the control, or five-fold higher than the control, or greater than five-fold higher than the control. In some embodiments, an increase of Δ133p53 may be determined by measuring the level of expression or activity of a protein that undergoes increased expression upon up-regulation of Δ133p53, e.g., the level of expression or activity of a protein such as NGF or IGF-1 that exhibits a Δ133p53-mediated increase in expression when Δ133p53 is expressed in astrocytes from a patient that has a neurodegenerative disease as described herein. Similarly, proteins that exhibit a Δ133p53 -mediated decrease in expression, e.g., inflammatory cytokines such as IL-6 or IL-1β, may be assessed as an endpoint in measuring increased Δ133p53 expression. [0035] As used herein, the term“decreasing” or“inhibiting” expression of p53β in the context of this invention refers to decreasing the level of p53β or activity in a cell. In some embodiments, the level of expression of p53β may be decreased using a polynucleotide inhibitor of p53β. In some embodiments, another agent that down regulates expression is employed. A "decrease" in p53β expression is generally determined relative to control cells to which an agent, e.g., a nucleic acid encoding a polynucleotide inhibitor of p53β, has not been added. Expression is typically considered to be decreased when levels of p53β protein or RNA, are decreased by at least 10% or at least 20%, typically by at least 50%, or 75% or more. In some embodiments, expression levels may be two-fold lower than the control, or five-fold lower than the control, or greater than five-fold lower than the control. As noted above, in some embodiments, inhibited expression of p53β can be assessed by evaluating levels of inflammatory cytokines, such as IL-6 or IL-1β, to determine if they are decreased relative to control astrocytes, or by measuring levels of protective growth factors such as NGF or IGF-1, to determine if protective growth factor levels are increased.

[0036] A polynucleotide sequence is“heterologous to” a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified by human action from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is different from any naturally occurring allelic variants.

[0037] The term "exogenous" refers to a substance present in a cell or organism other than its native source. For example, the terms "exogenous nucleic acid" or "exogenous protein" refer to a nucleic acid or protein that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found or in which it is found in lower amounts. A substance will be considered exogenous if it is introduced into a cell or an ancestor of the cell that inherits the substance. In contrast, the term "endogenous" refers to a substance that is native to the biological system.

[0038] The term "identity" as used herein refers to the extent to which the sequence of two or more nucleic acids or polypeptides is the same. The percent identity between a sequence of interest and a second sequence over a window of evaluation, e.g., over the length of the sequence of interest, may be computed by aligning the sequences, determining the number of residues (nucleotides or amino acids) within the window of evaluation that are opposite an identical residue allowing the introduction of gaps to maximize identity, dividing by the total number of residues of the sequence of interest or the second sequence (whichever is greater) that fall within the window, and multiplying by 100. When computing the number of identical residues needed to achieve a particular percent identity, fractions are to be rounded to the nearest whole number. Percent identity can be calculated with the use of a variety of computer programs. For example, computer programs such as BLAST2, BLASTN, BLASTP, Gapped BLAST, etc., generate alignments and provide percent identity between sequences of interest. The algorithm of Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. ScL USA 87:22264-2268, 1990) modified as in Karlin and Altschul, Proc. Natl. Acad. ScL USA 90:5873-5877, 1993 is incorporated into the NBLAST and XBLAST programs of Altschul et al. (Altschul, et al., J. MoI. Biol.215:403-410, 1990). To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (Altschul, et al. Nucleic Acids Res.25: 3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs may be used. A PAM250 or BLOSUM62 matrix may be used. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI). See the Web site having URL world-wide web address of: "ncbi.nlm.nih.gov" for these programs. In a specific embodiment, percent identity is calculated using BLAST2 with default parameters as provided by the NCBI.

[0039] The term "isolated" or "partially purified" as used herein refers, in the case of a nucleic acid or polypeptide, to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide as found in its natural source and/or that would be present with the nucleic acid or polypeptide when expressed by a cell, or secreted in the case of secreted polypeptides. A chemically synthesized nucleic acid or polypeptide or one synthesized using in vitro transcription/translation is considered "isolated". [0040] The term "isolated cell" as used herein refers to a cell that has been removed from an organism in which it was originally found or a descendant of such a cell. An“isolated” cell may be cultured in vitro in the presence of other cells. Optionally, the cell is later introduced into a second organism or re-introduced into the organism from which it (or the cell from which it is descended) was isolated.

[0041] The term "vector" refers to a carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host cell. In some embodiments, vectors of use in the invention are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". Thus, an "expression vector" is a specialized vector that contains the necessary regulatory regions needed for expression of a gene of interest in a host cell. In some embodiments the gene of interest is operably linked to another sequence in the vector, e.g., a promoter. Vectors include non-viral vectors such as plasmids and viral vectors.

[0042] The term“operably linked” refers to a functional linkage between a first nucleic acid sequence and a second nucleic acid sequence, such that the first and second nucleic acid sequences are transcribed into a single nucleic acid sequence. Operably linked nucleic acid sequences need not be physically adjacent to each other. The term“operably linked” also refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a transcribable nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the transcribable sequence.

[0043] The term "viral vectors" refers to the use of viruses, or virus-associated vectors as carriers of a nucleic acid construct into a cell. Constructs may be integrated and packaged into non-replicating, defective viral genomes like Adenovirus, Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others, including retroviral and lentiviral vectors, for infection or transduction into cells. The vector may or may not be incorporated into the cell's genome. The constructs may include viral sequences for transfection, if desired.

Alternatively, the construct may be incorporated into vectors capable of episomal replication, e.g., EPV and EBV vectors. [0044] In the context of this invention,“transfection” is used to refer to any method of introducing nucleic acid molecules into a cell including both viral and non-viral techniques. The term as used here thus includes techniques such as transduction with a virus.

[0045] The terms "regulatory sequence" and "promoter" are used interchangeably herein, and refer to nucleic acid sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operatively linked. In some examples, transcription of a recombinant gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell- type in which expression is intended. It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring form of a protein. In some instances the promoter sequence is recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required for initiating transcription of a specific gene.

[0046] As used herein, "proliferating" and "proliferation" refer to an increase in the number of cells in a population (growth) by means of cell division. Cell proliferation is generally understood to result from the coordinated activation of multiple signal transduction pathways in response to the environment, including growth factors and other mitogens. Cell proliferation may also be promoted by release from the actions of intra- or extracellular signals and mechanisms that block or negatively affect cell proliferation.

[0047] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0048] Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J. Biol. Chem.260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0049] Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes.

[0050] “Conservatively modified variants” as used herein applies to amino acid sequences. One of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

Introduction

[0051] Astrocytes exert both neuroprotective and neurotoxic roles. Their neurotoxic effect is mediated via senescence-associated secretory phenotype (SASP), which increases with aging and neurodegenerative diseases. This invention is based, in part, on the discovery that human astrocytes express p53 isoforms, Δ133p53 and p53β, and that these isoforms regulate SASP in astrocytes and their protective and toxic effects on neurons. Amyotrophic lateral sclerosis and Alzheimer’s disease brains, as well as senescent astrocytes after in vitro passaging, show decreased Δ133p53 and increased p53β, which may be attributed to selective autophagy-mediated degradation and SRSF3-mediated alternative mRNA splicing, respectively, conserved across different cell types. Early-passage astrocytes with Δ133p53 knockdown or p53β overexpression are induced to show SASP and to exert neurotoxicity in co-culture with neurons. Restored expression of Δ133p53 in near-senescent, neurotoxic astrocytes results in repressed SASP, elevated neurotrophic growth factors and enhanced neuroprotection. The p53 isoforms and their regulatory mechanisms in human astrocytes are targets for therapeutic intervention in neurodegenerative diseases.

[0052] Thus, in one aspect, the invention provides methods of manipulating p53 isoforms Δ133p53 and/or p53β to enhance astrocyte function in neuroprotection. In some embodiments, the invention provides methods of enhancing astrocyte function in neuroprotection by increasing the level or activity of Δ133p53 in cells of such patients. Increasing the level or activity of Δ133p53 may be achieved using any number of methods, including, but not limited to, introducing a polynucleotide encoding Δ133p53 into cells, e.g., an expression vector that expresses Δ133p53, and/or using an agent that upregulates Δ133p53 expression. In some embodiments, the invention provides methods of enhancing astrocyte function in neuroprotection by decreasing the level or activity of p53β in cells of such patients. Decreasing the level or activity of p53β may be achieved using any number of methods, including, but not limited to, introducing a polynucleotide inhibitor of p53β into cells, e.g., an expression vector that expresses a polynucleotide inhibitor of p53β, and/or using an agent that down regulates p53β expression

[0053] In some embodiments, the patient may be treated with an agent that up-regulates Δ133p53 expression and/or activity. In some embodiments, such an agent targets a cellular autophagy degradation pathway.

[0054] In further embodiments, the invention provides methods of identifying agents that increase Δ133p53 levels and/or activity in cells of patients having a neurodegenerative disease involving astrocyte dysfunction, where the method comprises screening candidate small molecule compounds for the ability to up-regulate Δ133p53 expression or activity in astrocytes.

[0055] In further embodiments, the invention provides methods of identifying agents that decrease p53β levels and/or activity in cells of patients having a neurodegenerative disease involving astrocyte dysfunction, where the method comprises screening candidate small molecule compounds for the ability to down-regulate p53β expression or activity in astrocytes. Methods for expressing Δ133p53

[0056] Nucleic acid sequences encoding Δ133p53 and related nucleic acid sequence homologues can be cloned from cDNA libraries or are typically isolated using amplification techniques with oligonucleotide primers.

[0057] In typical embodiments, the cloning of Δ133p53, or other p53 isoforms, or desired genes, can employ the use of synthetic oligonucleotide primers and amplification of an RNA or DNA template (see U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)). Methods such as polymerase chain reaction (PCR) and ligase chain reaction (LCR) can be used to amplify nucleic acid sequences of Δ133p53 directly from mRNA, from cDNA, from genomic libraries or cDNA libraries. Genes obtained via an amplification reaction can be purified and cloned into an appropriate vector.

[0058] The nucleic acids encoding Δ133p53 are typically cloned into intermediate vectors before transformation into eukaryotic cells for replication and/or expression. These intermediate vectors are typically prokaryote vectors, e.g., plasmids, or shuttle vectors. The isolated nucleic acids encoding Δ133p53 or other p53 isoforms may also encode interspecies homologues, alleles and polymorphic variants of the illustrative Δ133p53 described herein.

[0059] Expression of Δ133p53 (or of other proteins that it may be desirable to express as described herein) is performed using a variety of techniques. Basic texts disclosing general methods that can be employed in this invention include Green and Sambrook (2012) Molecular Cloning: A laboratory manual 4th ed. Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology and supplements through supplement 110, 2015) John Wiley and Sons.

Expression vectors

[0060] Any number of vectors may be employed, including plasmid and viral vectors, to introduce a nucleic acid encoding Δ133p53 into cells. [0061] In some embodiments, a nucleic acid construct encoding Δ133p53 is a plasmid- based vector (e.g.,“naked” DNA). [0062] In some embodiments a nucleic acid construct encoding Δ133p53 is contained within a viral vector and administered as a virus. Viral delivery systems include adenovirus vectors, adeno-associated viral vectors, herpes simplex viral vectors, retroviral vectors, pox viral vectors, lentiviral vectors, alphavirus vectors, poliovirus vectors, and other positive and negative stranded RNA viruses, viroids, and virusoids, or portions thereof. One of skill in the arts understands that any number of methods of constructing and using such vectors can be employed.

[0063] For example, recombinant viruses in the pox family of viruses can be used for delivering the nucleic acid molecules encoding Δ133p53. These include vaccinia viruses and avian poxviruses, such as the fowlpox and canarypox viruses. Illustrative methods for generating these viruses using genetic recombination can be found, e.g, in WO 91/12882; WO 89/03429; WO 92/03545; and n US Patent No.5,863,542. Representative examples of recombinant pox viruses include ALVAC, TROVAC, and NYVAC.

[0064] A number of adenovirus vectors, including, for example, Ad2, Ad5, and Ad7, can also be used to deliver a nucleic acid that encodes Δ133p53 (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human Gene Therapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al., Gene Therapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988) 6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476). Additionally, various adeno-associated virus (AAV) vector systems have been developed for gene delivery. These vectors can be readily constructed. See, e.g., U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published 23 Jan.1992) and WO 93/03769 (published 4 Mar.1993); Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B. J. Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. Current Topics in Microbiol. and Immunol. (1992) 158:97- 129; Kotin, R. M. Human Gene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994) 1:165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875.

[0065] Retroviruses also provide a platform for gene delivery systems. A number of retroviral systems have been described (U.S. Pat. No.5,219,740; Miller and Rosman, BioTechniques (1989) 7:980-990; Miller, A. D., Human Gene Therapy (1990) 1:5-14; Scarpa et al., Virology (1991) 180:849-852; Burns et al., Proc. Natl. Acad. Sci. USA (1993) 90:8033-8037; and Boris-Lawrie and Temin, Cur. Opin. Genet. Develop. (1993) 3:102-109. Additional gene delivery systems include lentiviral vectors that employ lentiviral vector backbones. Thus, in some embodiments, a nucleic acid encoding Δ133p53 may be introduced into cells from a patient having a disease of pre-mature aging using a lentiviral vector.

[0066] Members of the Alphavirus genus, such as, but not limited to, vectors derived from the Sindbis, Semliki Forest, and Venezuelan Equine Encephalitis viruses, can also be used as viral vectors to deliver a nucleic acid encoding Δ133p53. For a description of illustrative Sindbis-virus derived vectors useful for the practice of the instant methods, see, Dubensky et al., J. Virol. (1996) 70:508-519; and International Publication Nos. WO 95/07995 and WO 96/17072; as well as, Dubensky, Jr., T. W., et al., U.S. Pat. No.5,843,723, issued Dec.1, 1998, and Dubensky, Jr., T. W., U.S. Pat. No.5,789,245, issued Aug.4, 1998).

[0067] As appreciated by one of skill in the art, the techniques used to obtain Δ133p53 nucleic acids and introduce them into cells may also be used for other nucleic acids as described herein that are employed to extend the neuroprotective function of astrocytes of patients with a neurodegenerative disease in which astrocytes are dysfunctional. Agents that up-regulate Δ133p53 [0068] In some embodiments, an agent that up-regulates Δ133p53, i.e., that increases the level and/or activity of Δ133p53 in a cell, is administered to a subject that has a

neurodegenerative disease in which astrocytes play a significant role, e.g., Alzheimer’s disease, amyotrophic lateral sclerosis, Parkinson’s disease, hepatic encephalopathy, or Alexander’s disease. Such agents are often small organic molecules. As used herein, a “small organic molecule” refers to an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 daltons and less than about 2500 daltons, preferably less than about 2000 daltons, preferably between about 100 to about 1000 daltons, more preferably between about 200 to about 500 daltons.

[0069] In some embodiments, the agent may be an agent that targets selective autophagy, the mechanism by which Δ133p53 is degraded in astrocytes, thereby increasing cellular Δ133p53 levels. [0070] In some embodiments, an agent that up-regulates Δ133p53 in astrocytes from a subject that has a neurodegenerative disease, e.g., Alzheimer’s disease, amyotrophic lateral sclerosis, Parkinson’s disease, hepatic encephalopathy, or Alexander’s disease, may be an agent that increases the level or activity of a protein that interacts with Δ133p53, such as STUB1 (STIP1 homology and U-Box-containing protein 1, also referred to as CHIP, carboxy terminus of Hsp70-interacting protein). STUB1 has been mapped to 16p33 on human chromosome 16. Protein and nucleic acid sequences for human CHIP are known, as are structural features of the protein that are important to activity. For example, it has been shown that STUB1 has intrinsic E3 ubiquitin ligase activity and promotes ubiquitylation in an in vitro ubiquitylation assay with recombinant proteins ( Jiang et al., J. Biol. Chem.

276:42938-42944, 2001). This activity was dependent on the C-terminal U box.

Inhibition of endogenous p53β expression

[0071] In some aspects of the invention, it is desirable to disrupt endogenous expression of p53β. Inhibitory nucleic acids to p53β such as siRNA, shRNA, ribozymes, or antisense molecules, can be synthesized and introduced into cells using methods known in the art. Molecules can be synthesized chemically or enzymatically in vitro (Micura, Agnes Chem. Int. Ed. Emgl.41: 2265–9 (2002); Paddison et al., Proc. Natl. Acad. Sci. USA, 99: 1443–82002) or endogenously expressed inside the cells in the form of shRNAs (Yu et al., Proc. Natl. Acad. Sci. USA, 99: 6047–52 (2002); McManus et al., RNA 8, 842–50 (2002)). Plasmid- based expression systems using RNA polymerase III U6 or H1, or RNA polymerase II U1, small nuclear RNA promoters, have been used for endogenous expression of shRNAs (Brummelkamp et al., Science, 296: 550–3 (2002); Sui et al., Proc. Natl. Acad. Sci. USA, 99: 5515–20 (2002); Novarino et al., J. Neurosci., 24: 5322–30 (2004)). Synthetic siRNAs can be delivered by electroporation or by using lipophilic agents (McManus et al., RNA 8, 842– 50 (2002); Kishida et al., J. Gene Med., 6: 105–10 (2004)). Alternatively, plasmid systems can be used to stably express small hairpin RNAs for the suppression of target genes (Dykxhoorn et al., Nat. Rev. Mol. Biol., 4: 457–67 (2003)). Various viral delivery systems have been developed to deliver shRNA-expressing cassettes into cells that are difficult to transfect (Brummelkamp et al., Cancer Cell, 2: 243–7 (2002); Rubinson et al., Nat. Genet., 33: 401-62003). Furthermore, siRNAs can also be delivered into live animals. (Hasuwa et al., FEBS Lett., 532, 227–30 (2002); Carmell et al., Nat. Struct. Biol., 10: 91–2 (2003);

Kobayashi et al., J. Pharmacol. Exp. Ther., 308: 688–93 (2004)).

[0072] Methods for the design of siRNA or shRNA target sequences have been described in the art. Among the factors to be considered include: siRNA target sequences should be specific to the gene of interest and have ~20–50% GC content (Henshel et al., Nucl. Acids Res., 32: 113–20 (2004); G/C at the 5ƍ end of the sense strand; A/U at the 5ƍ end of the antisense strand; at least 5 A/U residues in the first 7 bases of the 5ƍ terminal of the antisense strand; and no runs of more than 9 G/C residues (Ui-Tei et al., Nucl. Acids Res., 3: 936–48 (2004)). Additionally, primer design rules specific to the RNA polymerase will apply. For example, for RNA polymerase III, the polymerase that transcribes from the U6 promoter, the preferred target sequence is 5ƍ-GN18-3ƍ. Runs of 4 or more Ts (or As on the other strand) will serve as terminator sequences for RNA polymerase III and should be avoided. In addition, regions with a run of any single base should be avoided (Czauderna et al., Nucl. Acids Res., 31: 2705–16 (2003)). It has also been generally recommended that the mRNA target site be at least 50–200 bases downstream of the start codon (Sui et al., Proc. Natl. Acad. Sci. USA, 99: 5515–20 (2002); Elbashir et al., Methods, 26: 199–213 (2002); Duxbury and Whang, J. Surg. Res., 117: 339–44 (2004) to avoid regions in which regulatory proteins might bind. Additionally, a number of computer programs are available to aid in the design of suitable siRNA and shRNAs for use in the practice of this invention.

[0073] Ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy target mRNAs, particularly through the use of hammerhead ribozymes. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. Preferably, the target mRNA has the following sequence of two bases: 5'-UG-3'. The construction and production of hammerhead ribozymes is well known in the art.

[0074] Gene targeting ribozymes necessarily contain a hybridizing region complementary to two regions, each of at least 5 and preferably each 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleotides in length of a target mRNA, e.g., a p53β mRNA. In addition, ribozymes possess highly specific endoribonuclease activity, which

autocatalytically cleaves the target sense mRNA.

[0075] With regard to antisense, siRNA or ribozyme oligonucleotides, phosphorothioate oligonucleotides can be used. Modifications of the phosphodiester linkage as well as of the heterocycle or the sugar may provide an increase in efficiency. Phophorothioate is used to modify the phosphodiester linkage. An N3'-P5' phosphoramidate linkage has been described as stabilizing oligonucleotides to nucleases and increasing the binding to RNA. Peptide nucleic acid (PNA) linkage is a complete replacement of the ribose and phosphodiester backbone and is stable to nucleases, increases the binding affinity to RNA, and does not allow cleavage by RNAse H. Its basic structure is also amenable to modifications that may allow its optimization as an antisense component. With respect to modifications of the heterocycle, certain heterocycle modifications have proven to augment antisense effects without interfering with RNAse H activity. An example of such modification is C-5 thiazole modification. Finally, modification of the sugar may also be considered.2'-O-propyl and 2'- methoxyethoxy ribose modifications stabilize oligonucleotides to nucleases in cell culture and in vivo.

[0076] One of skill understands that inhibitory nucleic acids that target p53β can be introduced into cells using principles and methods as described above for the introducing of Δ133p53 nucleic acids into cells.

[0077] Inhibitory oligonucleotides can be delivered to a cell by direct transfection or transfection and expression via an expression vector. Appropriate expression vectors include mammalian expression vectors and viral vectors, into which has been cloned an inhibitory oligonucleotide with the appropriate regulatory sequences including a promoter to result in expression of the antisense RNA in a host cell. Suitable promoters can be constitutive or development-specific promoters. Transfection delivery can be achieved by liposomal transfection reagents, known in the art (e.g., Xtreme transfection reagent, Roche, Alameda, CA; Lipofectamine formulations, Invitrogen, Carlsbad, CA). Delivery mediated by cationic liposomes, by retroviral vectors and direct delivery are efficient. Another possible delivery mode is targeting using antibody to cell surface markers for the target cells.

Agents that down-regulate p53β [0078] In some embodiments, an agent that down-regulates p53β, i.e., that decreases the level and/or activity of p53β in a cell, is administered to a subject that has a

neurodegenerative disease in which astrocytes play a significant role, e.g., Alzheimer’s disease, amyotrophic lateral sclerosis, Parkinson’s disease, hepatic encephalopathy, or Alexander’s disease. Such agents are often small organic molecules. As used herein, a “small organic molecule” refers to an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 daltons and less than about 2500 daltons, preferably less than about 2000 daltons, preferably between about 100 to about 1000 daltons, more preferably between about 200 to about 500 daltons.

Assays to test activity of candidate agents [0079] Agents are tested for the ability to up-regulate Δ133p53 isoform and/or down- regulate p53β isoform using any measure of RNA or protein expression or protein activity for that isofrom. In some embodiments, other endpoints may be measured to assess the functional effect of a candidate agent.“Functional effects” include in vitro, in vivo, and ex vivo activities. For example, any of a number of methods for the determination and measurement of cell senescence or cell proliferation can be used. In some embodiments, direct measurements of cell proliferation by counting cells may be employed. Other markers of cellular proliferation, e.g., cell markers that are expressed in proliferating cells, such PCNA, or a marker for cellular metabolism such as MTT (see, e.g., Hughes, D., Cell proliferation and apoptosis, Taylor & Francis Ltd, UK (2003), may also be used to assess cell proliferation and replicative lifespan).

[0080] In some embodiments, the level of proinflammatory cytokines produced by astrocytes and/or the level of neuroprotective factors, e.g., NGF or IGF-1 is measured.

[0081] A number of markers for cell senescence may also be used to monitor this process in the practice of this invention. A common marker is senescence-associated-β-galactoside (Dimri, G. P. et al., Proc. Natl. Acad. Sci. USA 92:9363 (1995)), although others markers of cell senescence may also be employed. For example senescence can be measured by direct measurement of telomere length by in situ hybridization or by measurement of age-dependent cellular accumulation of lipofucin in cells (Coates, J. Pathol., 196: 371-3 (2002)). Other markers of senescence include SAHF senescence-associated heterochromatin foci (SAHF) (see, e.g., Methods Mol Biol.965:185–196, 2013).

[0082] Up-regulation of Δ133p53 is achieved when the activity value relative to the control (untreated with activators) is 110%, typically 150%, or 200-500% (i.e., two to five fold higher relative to the control) in those assays that measure Δ133p53 protein or RNA levels, or that measure an endpoint that is increased when Δ133p53 is increased, e.g., replicative lifespan or production of neuroprotective factors by astrocytes. For assays that measure endpoint markers that are decreased upon increased activity or levels of Δ133p53, e.g., markers of astrocyte senescence, including, but not limited to, production of proinflammatory cytokines, up-regulation of Δ133p53 is achieved when the value relative to the control is decreased by at least 10% or at least 20%, typically at least 50%, or 75% or 90% or more. An illustrative control astrocyte for such assays may be an astrocyte cultured in vitro until it undergoes senescence. Levels of Δ133p53 protein or RNA, or of other proteins that exhibit modulated expression when Δ133p53 is expressed may then be determined relative to such a control. [0083] Similarly, an agent that down-regulates p53β may also be assessed for functional activity using cell senescence assays, assays for replicative lifespan, or other endpoints as described herein to identify agents that decrease p53β. Down-regulation of p53β is achieved when the activity value relative to the control (untreated with a p53β inhibitor) is typically reduced by at least 10%, more often at least 20%, 50%, 75%, 80%, or greater, in those assays that measure p53β protein or RNA levels. Assays that measure an endpoint that is increased when p53β is inhibited, e.g., replicative lifespan, may also be employed. For example, down- regulation of p53β may be achieve when the activity value for replicative lifespan is increased by at least 10%, 20%, 50%, 100%, 200%, or greater. For assays that measure endpoint markers that are decreased upon down-regulation of p53β, e.g., markers of astrocyte senescence, including, but not limited to, production of proinflammatory cytokines, inhibition of p53β is achieved when the value relative to the control is decreased by at least 10% or at least 20%, typically at least 50%, or 75% or more. An illustrative control astrocyte for such assays may be an astrocyte cultured in vitro until it undergoes senescence. Levels of p53β protein or RNA, or of other proteins that exhibit modulated expression when p53β is inhibited may then be determined relative to such a control.

[0084] The term“test compound” or“drug candidate” or grammatical equivalents as used herein describes any molecule, either naturally occurring or synthetic, e.g., protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, peptide, circular peptide, lipid, fatty acid, inhibitory RNAs, polynucleotide, oligonucleotide, etc., to be tested for the capacity to directly or indirectly up-regulate Δ133p53 and/or down-regulate p53β. The test compound can be in the form of a library of test compounds, such as a combinatorial or randomized library that provides a sufficient range of diversity. Test compounds are optionally linked to a fusion partner, e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties. Conventionally, new chemical entities with useful properties are generated by identifying a test compound with some desirable property or activity, e.g., increasing Δ133 levels and/or activity or decreasing p53β levels and/or activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis. Such assay formats are well-known in the art. Administration of agents

[0085] Cells that are targeted in a patient in accordance of the invention are astrocytes of a patient that has a neurodegenerative disease in which astrocytes are dysfunctional, e.g., Alzheimer’s Disease, amyotrophic lateral sclerosis, Parkinson’s Disease, hepatic encephalopathy or Alexander’s disease. For example, in some embodiments, astrocytes of a patient that has Alzheimer’s Disease, amyotrophic lateral sclerosis, Parkinson’s Disease, hepatic encephalopathy or Alexander’s disease may be treated with an agent or otherwise modified to overexpress Δ133p53.

Vector Delivery and Cell Transformation

[0086] Any suitable method for nucleic acid delivery for transformation of a cell or a tissue of a subject that has a neurodegenerative disease in which astrocytes are dysfunctional, can be used in the current invention. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection (Wilson et al., Science, 244:1344-1346, 1989, Nabel and Baltimore, Nature 326:711-713, 1987), optionally with Fugene6 (Roche) or

Lipofectamine (Invitrogen), by injection (U.S. Pat. Nos.5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection and gene gun injection (Harland and

Weintraub, J. Cell Biol., 101:1094-1099, 1985; U.S. Pat. No.5,789,215, incorporated herein by reference); by electroporation (U.S. Pat. No.5,384,253, incorporated herein by reference; Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986; Potter et al., Proc. Nat'l Acad. Sci. USA, 81:7161-7165, 1984); by calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52:456-467, 1973; Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987; Rippe et al., Mol. Cell Biol., 10:689-695, 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, Mol. Cell Biol., 5:1188-1190, 1985); by direct sonic loading (Fechheimer et al., Proc. Nat'l Acad. Sci. USA, 84:8463-8467, 1987); by liposome mediated transfection (Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982; Fraley et al., Proc. Nat'l Acad. Sci. USA, 76:3348-3352, 1979; Nicolau et al., Methods Enzymol., 149:157- 176, 1987; Wong et al., Gene, 10:87-94, 1980; Kaneda et al., Science, 243:375-378, 1989; Kato et al., J Biol. Chem., 266:3361-3364, 1991) and receptor-mediated transfection (Wu and Wu, Biochemistry, 27:887-892, 1988; Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987); each incorporated herein by reference). Further, any combination of such methods may be employed. As explained above, nucleic acids may also be delivered using viral vector systems in which the nucleic acids are delivered in viral particles that infect the desired cells.

[0087] For transfection, a composition comprising one or more nucleic acid molecules (within or without vectors) can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations. Methods for the delivery of nucleic acid molecules are described, for example, in Gilmore, et al., Curr Drug Delivery (2006) 3:147-5 and Patil, et al., AAPS Journal (2005) 7:E61-E77, each of which are incorporated herein by reference. Delivery of inhibitory RNA molecules is also described in several U.S. Patent Publications, including for example, 2006/0019912; 2006/0014289; 2005/0239687;

2005/0222064; and 2004/0204377, the disclosures of each of which are hereby incorporated herein by reference. Nucleic acid molecules can be administered to cells by a variety of methods, including, but not restricted to, encapsulation in liposomes, by iontophoresis, by electroporation, or by incorporation into other vehicles, including biodegradable polymers, hydrogels, cyclodextrins (see, for example Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCT publication Nos. WO 03/47518 and WO

03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see for example U.S. Pat. No.6,447,796 and US Patent Application Publication No.2002/130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722). In another embodiment, the nucleic acid molecules of the invention can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine- polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine- polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives.

[0088] Examples of liposomal transfection reagents of use with this invention include, for example: CellFectin, 1:1.5 (M/M) liposome formulation of the cationic lipid N,NI,NII,NIII- tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine and dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); Cytofectin GSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen Research); DOTAP (N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl- ammoniummethylsulfate) (Boehringer Manheim); Lipofectamine, 3:1 (M/M) liposome formulation of the polycationic lipid DOSPA and the neutral lipid DOPE (GIBCO BRL); and (5) siPORT (Ambion); HiPerfect (Qiagen); X-treme GENE (Roche); RNAicarrier (Epoch Biolabs) and TransPass (New England Biolabs). [0089] Nucleic acids for administration to a subject are formulated for pharmaceutical administration. While any suitable carrier known may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. For parenteral administration, including intranasal, intradermal, subcutaneous or intramuscular injection or electroporation, the carrier preferably comprises water, saline, and optionally an alcohol, a fat, a polymer, a wax, one or more stabilizing amino acids or a buffer. General formulation technologies are known to those of skill in the art (see, for example, Remington: The Science and Practice of Pharmacy (20th edition), Gennaro, ed., 2000, Lippincott Williams & Wilkins; Injectable Dispersed Systems: Formulation,

Processing And Performance, Burgess, ed., 2005, CRC Press; and Pharmaceutical

Formulation Development of Peptides and Proteins, Frkjr et al., eds., 2000, Taylor &

Francis).

[0090] Nucleic acid compositions encoding Δ133p53 or encoding a polynucleotide inhibitor of p53β can be administered once or multiple times. Multiple administrations can be administered, for example, bi-weekly, weekly, bi-monthly, monthly, or more or less often, as needed, for a time period sufficient to achieve the desired response.

[0091] The nucleic acid constructs in accordance with the invention are administered to a mammalian host. The mammalian host usually is a human or a primate. In some embodiments, the mammalian host can be a domestic animal, for example, canine, feline, lagomorpha, rodentia, rattus, hamster, murine. In other embodiment, the mammalian host is an agricultural animal, for example, bovine, ovine, porcine, equine, etc. [0092] Compositions comprising nucleic acids that encode Δ133p53, or a nucleic acid that inhibits p53β, can be formulated in accordance with standard techniques well known to those skilled in the pharmaceutical art. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the route of administration.

[0093] In therapeutic applications, the nucleic acids may be administered in an amount sufficient to elicit a therapeutic effect that at least partially arrests or slows one or more symptoms and/or complications of a neurodegenerative disease in which astrocytes are dysfunctional, e.g., Alzheimer’s Disease, amyotrophic lateral sclerosis, Parkinson’s Disease, hepatic encephalopathy or Alexander’s disease. An amount adequate to accomplish this is defined as "therapeutically effective dose." Amounts effective for this use will depend on, e.g., the particular composition of the vaccine regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.

[0094] The amounts and formulations depend on various factors, including delivery methods. In embodiments that employ a naked nucleic acid composition, the dose of a naked nucleic acid composition is from about 1.0 ng to about 10 mg for a patient. Subcutaneous or intramuscular doses for naked nucleic acid (typically DNA encoding a fusion protein) may range from 0.1 ug to 100 ug for a subject. For example, naked DNA or polynucleotide in an aqueous carrier can be injected into tissue, e.g., intramuscularly or intradermally, in amounts of from 10 μl per site to about 1 ml per site. The concentration of polynucleotide in the formulation is usually from about 0.1 μg/ml to about 5 mg/ml. In some embodiments, the DNA may be administered in ng amounts, for example at a level of 1 to 100 ng. For example, the dose is 0.1 μg, 0.5 μg, 1 μg, 1.5 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, or 60 μg of nucleic acids per dose. In a specific embodiment, the dose is in the range of 10 ng to 100 mg, or 50 ng to 100 mg, or 100 ng to 100 mg of nucleic acids per dose. In some specific embodiments, the dose is in the range of 10 pg to 100 mg, or 50 pg to 100 mg, or 100 pg to 100 mg, or 100 pg to 100 ng of nucleic acids per dose.

[0095] In some embodiments, a nucleic acid encoding Δ133p53, or an inhibitory nucleic acid that targets p53β is delivered using a viral delivery system in which virus particles that comprise the nucleic acid are introduced into the recipient. [0096] For other agents that increase expression of Δ133p53 and/or inhibit p53β, the amount of the therapeutic agent that will be effective in the prevention, treatment and/or management of a neurodegenerative disease, e.g., Alzheimer’s Disease, amyotrophic lateral sclerosis, Parkinson’s Disease, hepatic encephalopathy or Alexander’s disease, can be determined by standard clinical techniques. In vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed will also depend, e.g., on the route of administration, the type of symptoms, and the seriousness of the symptoms, and should be decided according to the judgment of the practitioner and each patient's or subject's circumstances. EXAMPLES

[0097] In illustrative experiments described in this section, it was demonstrated using a neuron-astrocyute co-culture system that downregulation of Δ133p53 or upregulation of p53β in astrocytes promoted SASP and non-cell autonomous neurotoxicity. Furthermore, reconstituted expression of Δ133p53 in neurotoxic astrocytes prevents SASP and reverses them to neuroprotective astrocytes.

Example 1. Astrocytes express p53 isoforms, Δ133p53 and p53β, and their expression is regulated during cellular senescence

[0098] To examine whether p53 isoforms Δ133p53 and p53β are expressed in the human brain, immunofluorescence staining using ¨133p53-specific antibody MAP4 and p53β- specific antibody TLQ40 (Fujita, et al., 2009; Mondal, et al., 2013) was performed in non- disease human brain tissues (Table 1). The specificity of the isoform-specific antibodies in immunofluorescence staining was confirmed using a p53-null cell line H358 with a Δ133p53, full-length p53, or p53β expression vector (Figure 10A-B). As astrocytes were reported to be a major cell type showing p53 immunoreactivity in non-neoplastic human brain tissues (Mendrysa, et al., 2011; Kurtkaya-Yapicier, et al., 2002) co-staining with astrocyte-specific marker glial fibrillary acidic protein (GFAP) (Figure 1A and 10C) was performed to investigated whether the p53 isoforms would also be expressed in astrocytes.

[0099] In vitro cultured astrocytes were also evaluated to determine whether they xpress the p53 isoforms. Primary human astrocytes (Bitto, et al., 2010) were serially passaged with monitoring by SA-β-gal staining and p16 ^INK4A 16 expression (Figure 1B-C) and those at early passage (passage number 5) and approaching senescence (passage number 13) were examined in Western blot analysis (Figure 1C; 10D-E). Full-length p53 was upregulated during senescence. The Δ133p53-specific antibody detected a single band of 35 kDa corresponding to Δ133p53α35 (i.e., Δ133p53, as used in the context of this disclosure) in early-passage astrocytes, which was diminished upon senescence. In contrast, p53β (47 kDa) was also upregulated upon senescence. These results indicated that human astrocytes express the p53 isoforms, Δ133p53 and p53β and that astrocytes are the major cell type in the human brain that expressed Δ133p53 and p53β.

Example 2. Autophagic degradation of Δ133p53 and SRSF3-mediated regulation of p53β in human astrocytes [0100] To investigate whether increased p53β protein and decreased ¨133p53 protein in senescent astrocytes are due to regulation at the mRNA level, qRT-PCR was performed using RNA samples extracted from serially passaged astrocytes in vitro (passage numbers 5 and 13, as above). Similar levels of Δ133p53 mRNA were expressed at both passage numbers (Figure 2A). p53β, by contrast, significantly increased at the mRNA level in astrocytes approaching senescence (Figure 2A). These data indicated that Δ133p53 expression is mainly regulated at the protein level, while p53β is regulated at the mRNA level, as was previously reported in human fibroblasts and CD8+ T-lymphocytes (Fujita, et al., 2009; Mondal, et al., 2013).

[0101] It was next determined whether Δ133p53 is degraded via selective autophagy in astrocytes, as had previously reported in senescent fibroblasts and CD8+T-lymphocytes (Mondal, 20113; Horikawa, 2014). Senescent astrocytes (passage number 15) with low levels of ¨133p53 were treated with a pharmacological inhibitor of autophagy, bafilomycin A137, whose action was confirmed by increased LC3-II (Tanida et al., 2008) (Figure 2B). Treatment with bafilomycin A1 resulted in the upregulation of Δ133p53 in senescent astrocytes (Figure 2B), indicating that autophagic degradation of ¨133p53 contributes to its downregulation in these cells. The stabilization of ¨133p53 by bafilomycin A1 was also observed in an immortalized human astrocyte cell line (Ferenczy, et al., 2013; Major, et al., 1985) (Figure 11A). Knockdown of p62/SQSTM1, which is a ubiquitin binding adaptor specifically functioning in the selective autophagy pathway (Johansen, et al., 2011), also stabilized ¨133p53 (Figure 2C), further supporting the degradation of ¨133p53 via selective autophagy. Additionally, while proteasome inhibitor MG132 stabilized full-length p53, ¨133p53 was not stabilized but rather decreased in astrocytes (Figure 11B), as previously observed in fibroblasts (Fujuita, et al., 2009), likely due to induction of autophagy by proteasome inhibition (Wu, et al., 2008, Pandey, et al., 2007). Because it has been observed that a splicing factor SRSF3 inhibited the alternative mRNA splicing generating p53β in human fibroblasts and CD8+ T-lymphocytes (Mondal, et al., 2013; Tang, et al., 2013), it was determined whether SRSF3 regulates p53β in astrocytes. Knockdown of SRSF3 through small interfering RNA (siRNA) in immortalized astrocytes resulted in a significant increase in p53β mRNA (Figure 2D), consistent with its negative regulation of p53β at the transcript level. These results indicated that the regulatory mechanisms for p53 isoform expression (autophagic degradation of Δ133p53 and SRSF3-mediated regulation of p53β splicing) were also present in astrocytes. Example 3. Δ133p53 knockdown or p53β overexpression induces cellular senescence and SASP in human astrocytes

[0102] Immortalized astrocytes were transfected with siRNA specifically targeting Δ133p5345 or scramble siRNA control, followed by Western blot analysis and

immunofluorescence staining confirming Δ133p53 knockdown using Δ133p53-specific antibody MAP4 (Fujita, et al., 2009; Mondal, et al., 2013) (Figure 3A-B; 12A). Three days following Δ133p53 knockdown, cells became growth-arrested (Figure 3C, top panel) with vacuolization (Figure 3C, bottom panel) and increased SA-β-gal staining (Figure 3D-E), which are characteristics of cellular senescence. Increased mRNA expression of pro- inflammatory cytokines IL-6 and IL-1β, as well as p21 ^WAF1 , was also observed in these senescent cells (Figure 3F), suggesting that loss of ¨133p53 induces SASP. Knockdown of ¨133p53 in early-passage primary astrocytes (confirmed by immunofluorescence; Figure 12B) also resulted in increased SA-β-gal staining (Figure 12C-D), the induction of SASP cytokines (Figure 12E), and p21 ^WAF1 protein expression (Figure 12F).

[0103] A lentiviral vector driving p53β expression or a vector control was transduced to immortalized astrocytes and its overexpression was confirmed by western blot analysis using a p53β-specific antibody (Fujita, et al., 2009; Mondal, et al., 2013) (Figure 4A; 13A). It was observed that p53β overexpression inhibited cell proliferation (Figure 4B), increased SA-β- gal-positive cells (Figure 4C-D), and induced pro-inflammatory SASP cytokines IL-6, IL-8, IL-1β and p21 ^WAF1 (Figure 4E). Early-passage primary astrocytes with p53β overexpression (Figure 13B) were also growth-inhibited (Figure 13C), had increased SA-β-gal staining (Figure 13D-E), and induction of SASP (Figure 13F). As p53β has been shown to promote apoptosis and senescence by differentially regulating gene expression in different cell types (Bourdon, et al., 2005; Marcel, et al., 2014; Marcel, et al., 2011), cell viability as well as expression of Bax, p16 and p21 was also assessed (Figure 13G-I). Percent cell viability was not significantly affected by overexpression of p53β (Figure 13G). Consistently, there was no difference in expression of the apoptosis regulator Bax (Figure 13H). In contrast, p21 and p16 expression were increased following p53β overexpression (13H-I). These results indicate that ¨133p53 downregulation and p53β upregulation play causative roles in inducing cellular senescence and SASP in astrocytes.

Example 4. Increased neuronal death upon co-culture with Δ133p53-knocked-down or p53β- overexpressing astrocytes [0104] To examine whether a phenotypic change in astrocytes (such as cellular senescence and SASP via ¨133p53 knockdown or p53β overexpression) alters astrocyte-to-neuron interaction, a co-culture system was employed in which human primary astrocytes were co- plated with induced pluripotent stem cell (iPSC)-derived mature neurons (Haidet-Phillips, et al., 2012), followed by assays for neuronal survival and death (Figure 14A-B). Mature neurons were derived from iPSC (Figure 14C) and confirmation of neuronal phenotypes was achieved through expression of sequential markers of differentiation: iPSCs expressed TRA- 1-81 demonstrating pluripotency (Figure 14D); neural stem cells (NSC) expressed Nestin and Sox1 (Figure 14E); and neurons expressed neuronal nuclei marker (NeuN) (Figure 14F). Motor neurons, a mature neuronal subtype that is primarily lost in amyotrophic lateral sclerosis, were also derived from iPSC (Grunseich, et al., 2014) and their differentiation was confirmed by expression of non-phosphorylated neurofilament marker, SMI-32 (Figure 14G).

[0105] When early-passage astrocytes with ¨133p53 siRNA or control siRNA (same cells as used in Figure 12) were co-cultured with motor neurons for 48 hours, ¨133p53-knocked- down, senescent astrocytes induced a higher number of neurons positive for cleaved caspase- 3 (15.0 ± 0.2%), a final effector of neuronal apoptosis (Zhao, et al., 2003), than control astrocytes (1.3 ± 0.3%) (Figure 5A-B). Similarly, p53β-overexpressing astrocytes (same cells as used in Figure 13) also resulted in an increase in cleaved caspase-3-positive motor neurons (18.8 ± 1.0%) compared to vector control transduced cells (0.4 ± 0.3%) (Figure 5C- D). To examine the effects of astrocytes on neurons in general and to obtain another quantitative measure, the co-culture experiment was performed using less specialized neurons (generated as in Figure 14C) and the number of surviving neurons (NeuN-positive) was counted after 48 hours co-culture with astrocytes. A significant decrease in the number of surviving neurons was observed following co-culture with Δ133p53-knocked-down astrocytes (Figure 5E-F) or p53β-overexpressing astrocytes (Figure 5G-H). An increase in neuronal apoptosis by p53β-overexpressing astrocytes was also confirmed in this experiment (Figure 14H-I). These findings indicated that the senescence-associated p53 isoform expression signature conferred astrocytes with neurotoxic activity.

Example 5. Δ133p53 protects astrocytes from senescence and enhances their neuroprotective function

[0106] This example investigated whether manipulated expression of the p53 isoforms leads to senescence inhibition and neuroprotective function in astrocytes. To test this, Δ133p53 expression was reconstituted via lentiviral vector transduction in primary astrocytes approaching senescence (passage number 12) (Figure 6A). Reconstituted expression of Δ133p53 restored cell proliferation (Figure 6B), decreased SA-β-gal positive cells (Figure 6B-C), and reduced the levels of pro-inflammatory SASP cytokines IL-6 and IL-1β (Figure 6D). Significantly, two neurotrophic growth factors, NGF and IGF-1, out of the three examined were 2- to 3-fold upregulated by ¨133p53 reconstitution (Figure 6D).

Immunofluorescence staining of NGF confirmed its increased expression in ¨133p53- reconstituted astrocytes (Figure 15A), which was comparable to that in early-passage astrocytes (Figure 15B).

[0107] The ¨133p53-reconstituted astrocytes were then used in a co-culture experiment (as performed in Figure 5). Vector control-transduced astrocytes (derived from passage number 12) resulted in a much larger number of motor neurons positive for cleaved caspase-3 (35.0 ± 2.0%; Figure 6E-F) than those derived from passage number 5 (1.3 ± 0.3%; Figure 5A-D), indicating a senescence-associated progression of neurotoxicity in astrocytes. The reconstitution of ¨133p53 in these near-senescent astrocytes significantly reduced the number of cleaved caspase-3-positive motor neurons (~12%; Figure 6E-F), indicating that ¨133p53 in astrocytes suppressed neuronal apoptosis. Counting of NeuN-positive neurons following co-culture (Figure 6G-H) also showed that aged astrocytes were less neuroprotective than early-passage astrocytes (compare controls in Figure 6H versus 5F and H), and that reconstituted expression of Δ133p53 restored the number of surviving neurons back to the level exerted by early-passage astrocytes (¨133p53 in Figure 6H; compare with controls in Figure 5F and H).

[0108] To confirm the roles of NGF and IL-6 in Δ133p53-mediated astrocyte

neuroprotective and neurotoxic function, neutralizing antibodies were used in co-culture experiments. As a positive control for IL-6 neutralizing antibody, immunofluorescence staining of untreated neurons and neurons treated with IL-6 alone or both IL-6 and IL-6 neutralizing antibody (IL-6-NAb) was first performed. Cleaved caspase-3

immunofluorescence staining was increased upon IL-6 treatment and reduced back to the control level upon co-incubation with IL-6-NAb (Figure 15C). Consistently, the number of NeuN-positive surviving neurons was significantly reduced upon IL-6 treatment and restored by IL-6-NAb (Figure 15C-D). These results confirmed that IL-6 promoted neurotoxicity and that IL-6-NAb was effective at neutralizing its function. Next, IL-6-Nab was used in co- culture experiments with neurons and aged astrocytes (P12) (Figure 6I, 15E). In these co- culture experiments, incubation with IL-6-NAb reduced neuronal apoptosis (indicated by cleaved caspase-3-positive neurons) to the level of Δ133p53-reconstituted astrocytes, but there was no combinatorial effect of Δ133p53 and IL-6-NAb over either alone (Figure 6I, 15E). In co-culture experiments with NGF neutralizing antibody (NGF-NAb), the reduction in neuronal apoptosis by Δ133p53 reconstitution was abolished by NGF-NAb, and thus the level of neuronal apoptosis in Δ133p53-reconstited astrocytes with NGF-NAb was similar to that in control astrocytes with or without NGF-NAb (Figure 6I, 15E). These results indicated that Δ133p53 in astrocytes promotes neuroprotection through upregulation of NGF and downregulation of IL-6.

Example 6. Increased senescent astrocytes in brain tissues from neurodegenerative disease patients

[0109] To examine whether neurodegenerative disease tissues have increased features of cellular senescence, Alzheimer’s disease, amyotrophic lateral sclerosis, age-matched non- disease, and non-disease pediatric tissues (Table 1) were obtained. Immunohistochemical staining was performed using antibodies to proteins known to be associated with cellular senescence, such as p16 ^INK4A (Rayess, et al., 2012), NOS2 (Sohn, et al., 2012), and ȖH2AX21 (Figure 7A). An increase in the senescence-associated biomarkers was prominent in all neurodegenerative samples examined compared to controls (Figure 7A). Quantification of the number of p16 ^INK4A -expressing cells revealed a significant increase in this senescence- associated gene in Alzheimer’s disease and amyotrophic lateral sclerosis tissues (Figure 7B). Although both Alzheimer’s disease and amyotrophic lateral sclerosis are associated with increased cellular senescence, qRT-PCR using brain tissue RNA samples showed that SASP cytokine IL-6 and a p53-inducible senescence regulator p21 ^WAF1 were upregulated more markedly in amyotrophic lateral sclerosis, while NOS2 upregulation was more evident in Alzheimer’s disease (Figure 7C). IL-6 and p21WAF1 expression levels were much lower in pediatric brain tissues versus aged brain tissues (Figure 16A), indicating that these senescent changes not only are associated with neurodegenerative diseases, but also may occur during physiological brain aging. In agreement with astrocyte-like morphology of the senescent cells (Figure 7A), immunofluorescence co-staining of p16 ^INK4A and GFAP as an astrocyte marker showed that the senescent cells were astrocytes (Figure 7D). Furthermore, both immunohistochemical (Figure 16B) and immunofluorescence staining (Figure 16C) of GFAP showed the presence of astrocytes with enlarged and flattened cytoplasms, which is characteristic of senescent cells (Kuilman, et al., 2010), in Alzheimer’s disease and amyotrophic lateral sclerosis brain tissues, but not in non-disease control tissues.

Example 7. p53β is upregulated and Δ133p53 is downregulated in neurodegenerative disease brains

[0110] To examine the expression of p53β and Δ133p53 in Alzheimer’s disease, amyotrophic lateral sclerosis and age-matched non-disease brain tissues, Western blot analysis was performed using the ¨133p53-specific antibody MAP4 and the p53β-specific antibody TLQ4030,31, along with detection of full-length p53. Only the 35 kDa ¨133p53 bands corresponding to Δ133p53α, but not smaller-size bands corresponding to Δ133p53β and Δ133p53γ, were detected by MAP4 in these brain tissues (Figure 17A). The major bands detected by TLQ40 were 47 kDa in size, which corresponds to p53β that starts at the same methionine as full-length p53, with smaller amounts of N-terminally truncated β isoforms (Figure 17B). These detection patterns were similar to those observed in primary human astrocytes (Figure 1C). It was found that full-length p53 (53 kDa) and p53β (47 kDa) were upregulated, while Δ133p53 (35 kDa) was downregulated, in Alzheimer’s disease and amyotrophic lateral sclerosis tissues compared to non-disease tissues (Figure 8A).

Quantitative densitometric analysis determined that the upregulation of full-length p53 and p53β was 2-3-fold and the downregulation of Δ133p53 was 0.5-0.6-fold (Figure 8B). The upregulation of p53β and the downregulation of ¨133p53 in neurodegenerative diseases were consistent with the expression profiles of these p53 isoforms observed in senescent astrocytes in vitro (Figure 1C). The expression levels of Δ133p53 mRNA were not significantly changed between non-disease and Alzheimer’s disease or amyotrophic lateral sclerosis tissues (Figure 8C), while p53β mRNA was increased in Alzheimer’s disease and amyotrophic lateral sclerosis compared to non-disease tissues (Figure 8D). A higher level of expression of ¨133p53 mRNA in non-disease pediatric tissues compared to non-disease aged brain tissues (Figure 8C) also suggest a transcriptional control of ¨133p53 during physiological brain development and aging. Thus, Alzheimer’s disease and amyotrophic lateral sclerosis are associated with increased astrocyte senescence and the senescence- associated p53 isoform expression signature, validating that the in vitro experiments using serially passaged human astrocytes recapitulated part of in vivo pathology of these neurodegenerative diseases.

Discussion of illustrative data presented in the Examples section [0111] The results demonstrated that p53 isoforms are endogenous regulators of cellular senescence in the brain. The senescence-associated p53 isoform signature (upregulation of p53β and downregulation of ¨133p53) was identified in neurodegenerative diseases and aged astrocytes in vitro, and was shown to induce astrocyte SASP and neurotoxic effect.

Reconstitution of Δ133p53 expression rescued astrocyte senescence and enhanced neuroprotection through increased expression of neurotrophic factors.

[0112] The mechanisms of p53 isoform regulation in astrocytes (autophagic degradation of Δ133p53 and SRSF3-mediated splicing regulation of p53β) (Figure 3) are consistent with other previously examined human cell types including aged fibroblasts and senescent CD8+ T-lymphocytes (Fujita et al., 2009; Horikawa et al., 2014; Mondal et al., 2013; Tang et al., 2013), possibly suggesting a general aging program that is found in a variety of age-related diseases, including Alzheimer’s disease and amyotrophic lateral sclerosis. As observed in age-related disorders in general (Rubinsztein et al., 2011), neurodegeneration is associated with impaired activity of bulk autophagy (Nixon, et al., 2013), which would stabilize autophagy substrates. However, ¨133p53, which is downregulated rather than stabilized in neurodegenerative diseases (Figure 2), does not appear to be degraded via bulk autophagy, highlighting the importance of specific degredation of ¨133p53 via selective autophagy that is dependent on p62/SQSTM1 (Horikawa et al., 2014; Johansen and Lamark, 2011).

[0113] Astrocytes exert both neuroprotective and neurodegenerative effects in a context- dependent manner, which are associated with either repression or induction of SASP, respectively(Pertusa, et al., 2007). Not to be bound by theory, the data presented in the examples are consistent with the notion that the astrocyte-mediated effects of the p53 isoforms on neurons are exerted through their regulatory roles for SASP. One main feature of astrocyte SASP driving neuronal loss is the release of pro-inflammatory cytokines such as IL-6 (Jiang, et al., 2014). Consistent with the neurodegenerative role of these SASP cytokines, the induction of SASP in astrocytes either by ¨133p53 knockdown (Figure 3F) or p53β overexpression (Figure 4E) leads to increased neuronal apoptosis (Figure 5A-D), while the repression of astrocyte SASP by ¨133p53 restoration (Figure 6D) leads to increased neuronal survival (Figure 6E-F). These data suggest that IL-6 is a therapeutic target in astrocytes to prevent neurotoxicity (Figure 9B).

[0114] The data also suggest that the upregulation of neurotrophic growth factors such as NGF and IGF-1 in astrocytes (Figure 6D, 15A) mediates Δ133p53-induced neuroprotection (Figure 6G-I, 15E). NGF and IGF-1 are known to promote neuronal survival (Hefti, et al., 1986; Wine, et al., 2009) and are decreased in Alzheimer’s and Parkinson’s disease brain tissues (Nagatsu, et al., 2000; Tuszynski, 2007). Furthermore, astrocytes utilize IGF-1 to protect neurons from oxidative stress (Genis, et al., 2014). While this study focused on astrocyte-to-neuron signaling, possible roles of the p53 isoforms in neurons and neuron-to- astrocyte signaling in neurodegenerative disease deserve further investigation.

[0115] Full-length p53 is known to transactivate pro-inflammatory cytokine genes such as IL-6 and IL-8 (Lowe, 2014). Again not to be bound by theory, it is thus likely that the effect of p53β or ¨133p53 on SASP and neurodegeneration is at least in part through cooperation with or dominant-negative inhibition of full-length p53 (Fujita, et al., 2009; Mondal, et al., 2013). Since full-length p53 functions to inhibit the IGF-1 signaling pathway (Levine, et al., 2006), the dominant-negative inhibition by ¨133p53 of full-length p53 activity30 may increase IGF-1 signaling towards neuroprotection. Another possibility is that ¨133p53 may directly upregulate the neurotrophic factors NGF and IGF-1 through its gain-of-function activity, which was recently reported to activate a set of genes for DNA-damage repair (Gong, et al., 2015). Although in silico analysis of the NGF and IGF-1 gene promoters did not identify a perfect match to the predicted Δ133p53-binding response element (Gong, et al., 2015), further studies will elucidate the regulation of these neurotrophic factors by Δ133p53 in dominant-negative and gain-of-function manners.

[0116] A phenotypic shift from neurotoxic SASP astrocytes to neuroprotective astrocytes may represent a promising therapeutic approach for inhibiting or delaying the progression of neurodegenerative diseases. In the proposed model (Figure 9), enhancement of Δ133p53 activity and/or inhibition of p53β activity could lead to such phenotypic shift of astrocytes. Because increased neurotoxicity, which was exerted by in vitro aged astrocytes and reverted by reconstituted expression of ¨133p53 in this study, is also characteristic of astrocytes derived from patients with neurodegenerative diseases (Ilieva, et al., 2009; Marchetto, et al., 2008), our findings have implications in developing therapeutic interventions. This study provides a rationale for exploration of small molecules that can modulate the expression level or activity of the p53 isoforms to inhibit the progression of neurodegeneration. Finally, only humans and other primates have the equivalent of ¨133p53 (Figure 18). It is interesting whether this primate-specificity of ¨133p53 is related to primate-specific accumulation of β- amyloid (Heuer, et al., 2012) and other physiological or pathological processes specific or preferential to humans and other primates. Experimental procedures

Patient samples

[0117] Alzheimer’s disease, amyotrophic lateral sclerosis, age- and region- matched non- disease control tissues were obtained from the Georgetown Brain Bank. Pediatric control tissues were obtained from the Human Brain Collection Core, NIMH. All tissues obtained are listed in Table 1. All human post-mortem tissues are de-identified and deemed exempt by Georgetown University Institutional Review Board. All cases were fully worked up neuropathologically. Use of tissues for research was approved by informed consent of next- of-kin.

Table 1. Human brain tissues

revatons– Forman- xe para n em e e FFPE, zemers sease D, myotrop c lateral sclerosis (ALS). Primary cells and cell lines

[0118] Primary human astrocytes were obtained from Sciencell (Carlsbad, CA, USA) (Bitto et al., 2010). They were maintained in Astrocyte Medium supplemented with 2% fetal bovine serum,1% astrocyte growth supplement from Sciencell (Carlsbad, CA, USA), and 1% penicillin/streptomycin solution. When confluent, cells were split at a ratio of 1:4 (earlier passages) to 1:2 (later passages) until they reached replicative senescence. Immortalized human astrocytes were prepared as described (Ferenczy et al., 2013; Major et al., 1985). After 3 weeks, cells were transfected using calcium phosphate precipitation with a plasmid DNA containing the SV40 mutant with a deletion of its origin of replication (pMK16)/ H358 cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Neuronal differentiation of induced pluripotent stem cells (iPSC)

[0119] An iPSC line, i20 (NIH stem cell bank) was differentiated to neurons using Gibco® Pluripotent Stem Cell Neural Induction (Life Technologies). For motor neuron

differentiation iPSCs were grown to 80% confluency, then digested with collagenase IV (Invitrogen) for 8 minutes. Cells were scraped off of the dish, and after settling the supernatant was aspirated, and cells were re-plated into low adherence dishes (Corning, Corning, NY) in KSR (Invitrogen) based media with 20 ng/ml FGF (R + D Systems, Minneapolis, MN), 20≤M ROCK-I (Tocris, Bristol, UK), 10 μM SB431542 (Tocris), and 0.2 μM LDN193189 (Stemgent). Embryoid bodies (EBs) were transitioned to a KSR free medium after 3 days. Retinoic acid was added to the media after 5 days to direct the cells towards a rostral spinal cord phenotype, with additional patterning using 1 μM smoothened agonist (Calbiochem, Billerica, MA) and 0.5 μM purmorphamine (Stemgent) after 7 days to ventralize the differentiating population. After 14–16 days in suspension, the EBs were dissociated and plated on dishes coated with polyornithine or poly-D-lysine and laminin for an additional 7–14 days. Neuronal cultures were maintained in neurobasal media

(Invitrogen) with 25 μM glutamate (Sigma, St. Louis, MO), 0.4 μg/ml ascorbic acid (Sigma), 10 ng/ml GDNF (Sigma), 10 ng/ml CNTF (Sigma), 1 μg/ml laminin (BD Bioscience, Franklin Lakes, NJ). B-27, N2, non-essential amino acids, and pen/strep/glutamine were all from Invitrogen. Two days after plating 10 nM dihydrotestosterone (DHT) was added, and the cultures were maintained for an additional 7–14 days.

Co-culture system [0120] Following differentiation of iPSC to neural stem cells (NSC), NSCs were plated at a density of 3 × 10 ⁴ cells/cm ² in a 4-well chamber slide coated with 20 μg/mL poly-L-ornithine and 10 μg/mL laminin. Media was changed to neural differentiation media (1X Neurobasal Medium, 2% B-27 Serum-Free Supplement, 2 mM GlutaMAX-1 Supplement, Life

Technologies) after 2 days. Media was changed every 3 days and NSCs were allowed to mature neurons for 1 month. Primary human astrocytes were plated on top of the mature neurons at a density of 3 × 104 cells/cm2. Media was changed to a 1:1 ratio of astrocyte (Sciencell) and neuron differentiation medium (Brennand et al., 2011; Haidet-Phillips et al., 2012; Muratore et al., 2014). After 48 hours cells were fixed with 4% paraformaldehyde for immunocytochemistry staining. Percent apoptosis was calculated by counting number of cleaved caspase-3-positive, MAP2- or NeuN-positive neurons in triplicate experiments in 5 microscopic fields (40X magnification) for 3 biological replicates. Additionally, the number of neurons remaining after the 48 hour co-culture period was also quantified with 5 frames per condition for 3 biological replicates and counting the number of NeuN-positive neurons. Plasmid constructs

[0121] To generate retroviral expression vectors of human p53 isoforms, Flag-tagged p53β was PCR-amplified using pSVp53β and pSVDNp53 (Bourdon et al., 2005), respectively, as templates and then inserted into NotI and EcoRI sites of a pQCXIN vector (BD Biosciences). These constructs were verified by DNA sequencing. The Precision LentiORF RFP control vector (which drives RFP as an ORF insert, as well as IRES-translated GFP from the pLOC lentiviral vector backbone) was purchased from Open Biosystems/GE Dharmacon (Lafayette, CO). For overexpression of p53β and Δ133p53, the RFP insert was replaced with cDNA inserts of p53β and Δ133p53, respectively.

Retroviral and lentiviral transduction

[0122] Retroviral constructs were transfected into Phoenix packaging cells (Orbigen, Inc.) using Lipofectamine 2000 (Invitrogen). Lentiviral supernatants were obtained using the Trans-lentiviral packaging system (Open Biosystems). Retro- and lentiviral vector supernatants were collected 48 hours after transfection and used to infect cells in the presence of polybrene (8 μg/mL; Sigma-Aldrich). Two days after infection, the cells were selected with G418 (600 μg/ml; Sigma-Aldrich), puromycin (2 μg/mL; Sigma-Aldrich) or zeocin (1 mg/mL; Invitrogen).

Transfection of siRNA oligonucleotides [0123] siRNA oligonucleotides were transfected at a final concentration of 12 nM using Lipofectamine RNAiMAX (Invitrogen). The following oligonucleotides were from

Invitrogen Stealth Select siRNA targeting Δ133p53: 5’- GGAGGUGCUUACACAUGUU-3’ (Bernard et al., 2013), p62/SQSTM1: 5’-AGAAGUGGACCCGUCUACAGGUGAA-3’ (Horikawa et al., 2014), SRSF3: 5ƍ-AGAGCUAGAUGGAAGAACATT-3ƍ (Tang et al., 2013), and Stealth nonspecific RNAi negative control (no.12,935-100).

SA-ȕ-Gal staining and cell treatments

[0124] SA-β-Gal staining was performed with the Senescence-β-Galactosidase Staining Kit (Cell Signaling Technology, Danvers, MA, USA). Bafilomycin A1 was obtained from Sigma-Aldrich (St. Louis, MO, USA) and incubated for 4 hours at a concentration of 100nM. MG132 was obtained from LC Laboratories (Woburn, MA, USA) and incubated for 8 hours at a concentration of 15μM.

Cell Viability Assay

[0125] Cell pellets were resuspended in 20 uL of Trypan blue and cell viability was calculated using a TC20™ Automated Cell Counter (Biorad).

Recombinant IL-6 and neutralizing antibody treatments

[0126] Recombinant IL-6 (Invivogen) was incubated for 24 hours at a concentration of 5 ng/mL. In experiments with neutralizing antibodies, co-culture was performed as above with NGF neutralizing antibody (Alomone labs) at a concentration of 500 ng/mL or IL-6 neutralizing antibody (Invivogen) at a concentration of 5 μg/mL. After 48 hours cells were fixed and stained as described above.

Immunohistochemistry (IHC) and Immunofluorescence (IF)

[0127] Frozen and formalin-fixed paraffin embedded human tissue sections were washed in PBS before blocking for 1 hour in PBS containing 0.1% Triton X and 10% donkey serum (Sigma-Aldrich). Donkey serum is used to block non-specific binding sites before incubation with primary antibody overnight at 4°C. Antigens were detected using the antibodies listed in Table 2. After overnight incubation they were washed in PBS 3 times for 10 minutes, before incubation with the appropriate conjugated secondary antibodies for 1 hour at room temperature (RT). The secondary antibody was conjugated to fluorophores: Alexa-488, -568 or -647 (Invitrogen, Paisley, Renfrewshire, UK; 1:400). After washing in PBS 3 times for 10 minutes, sections were incubated for 10 minutes in 4’,6-diamidino-2-phenylindole (DAPI, 10ug/mL, Sigma-Aldrich) to counterstain the cell nuclei, and rinsed 3 times for 10 minutes in 0.1 M phosphate buffer (PB). Sections were mounted and slides coverslipped with FluorSave mounting medium (Chemicon). Omission of primary antibody was used as a negative control in all IF experiments. For IHC on paraffin sections, slides were heated to 65°C before immersion in histoclear and rehydration with graded alcohols. Sections were blocked in 1% H2O2 in PBS-Tween 20 (PBS-T) and then in 5% normal goat serum in PBS-T. Antigens were detected using the antibodies listed in Table 2. Binding of the primary antibodies was detected using secondary biotinylated secondary antibodies with an ABC standard kit (Vector Laboratories). Visualization was enabled using a 0.05% diaminobenzene hydrochloride solution (DAB; Sigma-Aldrich). Omission of primary antibody was used as negative controls in all IHC experiments.

Table 2. Primary antibodies. Abbreviations–Immunohistochemistry (IHC),

Immunocytochemistry (ICC), Western blot (WB), Immunofluorescence (IF).

Immunocytochemistry

[0128] Cells were washed with PBS and fixed for 10 minutes with 4% paraformaldehyde. Cells were permeabilized with 0.01% Triton-X for 4 minutes, washed with PBS and then blocked in 5% FBS for 1 hour at room temperature. Primary antibodies listed in Table 2 were applied overnight at 4°C. Cells were washed with PBS before incubation with a secondary antibody conjugated to fluorophores: Alexa-488, 568 and 647 at a dilution of 1:400 (Life Technologies) and DAPI for 1 hour. Coverslips were mounted on to slides with FluorSave mounting medium (Chemicon). Omission of primary antibody was used as a negative control in all immunocytochemistry experiments.

Immunoblotting

[0129] Cells and tissues were lysed in RIPA buffer. Lysates were kept on ice for 30 minutes prior to sonication. Protein concentration was measured using the Bradford assay method. NuPAGE 4X loading buffer was added to all lysates and then boiled for 5 minutes. Then, 40 μg of protein was loaded onto a Tris-glycine gel (Novex) for electrophoresis. Proteins were then transferred onto a PVDF membrane. Membranes were blocked in 1:1 mixture of Superblock and Tris Buffered Saline (TBS, 125 mM Tris and 200 mM NaCl), containing 0.1% Tween-20. Membranes were incubated in the primary antibodies listed in Table 2 overnight at 4°C, and washed 3 times in TBS-Tween-20. Membranes were then incubated in secondary for 1 hour at RT and the signal visualized SuperSignal developing reagent and visualized using X-ray films (Fujifilm).

RNA extraction and cDNA preparation

[0130] mRNA was extracted using the RNeasy Mini Kit (Qiagen, Crawley, UK) according to the manufacturer’s instructions. For mouse brain tissue, 10-20 mg of frozen cortical tissue was added to 300 μL lysis buffer containing 0.001% β-mercaptoethanol. Tissues or cells were homogenized and lysate mixed 1:1 with 70% ethanol and centrifuged through an RNeasy Mini Spin column. The column was washed and treated with DNAse 1 for 15 minutes, before washing again to remove contaminants. RNA was eluted with RNase-free water. The abundance and quality of the resulting RNA was assessed using a Nanodrop ND- 1000 spectrophotometer (Nanodrop Technologies). RNA samples were diluted so that 200 ng total RNA could be used for a 25 μl- reverse-transcription reaction. cDNA was synthesized using SuperScript II Reverse Transcriptase (Invitrogen).

Quantitative Real-Time Polymerase Chain reaction (qRT-PCR)

[0131] For the quantitative analysis of mRNA expression, the Tecan Sunrise 7500 real time PCR system (Applied Biosystem) was employed, with the DNA binding dye SYBR Green (Qiagen) or Taqman (Life Technologies) primers for the detection of PCR products. Each reaction was performed in triplicate using 2 μl of cDNA in a final volume of 20 μl. The following thermal cycle was used for all samples: 10 minutes-95°C; 40 cycles of 30 seconds- 95°C, 40 seconds-primer specific annealing temperatures, 40 seconds-72°C. The melting points, optimal conditions, and specificities of the reactions were first determined using a standard procedure. The expression level of each target gene was analyzed based on the ΔΔCt method and the results expressed as relative expression normalized to 18S or β-actin. Primers for Δ133p53, p53β, and 18S were purchased from Invitrogen and their sequences are as follows: Δ133p53: forward 5’-TGACTTTCAACTCTGTCTCCTTCCT-3’; reverse 5’- GGCCAGACCATCGCTATCTG-3.’ p53β: forward 5’- GCGAGCACTGCCCAACA-3’; reverse 5’- GAAAGCTGGTCTGGTCCTGA-3.’ 18S : forward 5’- GTAACCCGTTGAACCCCATT-3’; reverse 5’- CCATCCAATCGGTAGTAGCG-3.’ Taqman primer assays for IL-6, p21, NOS2, IL1-β, β-actin were purchased from Life Technologies (sequences available from Life Technologies).

Statistical analysis

[0132] Data are presented as mean ± standard error of the mean (SEM) of an n=3 independent experiments unless otherwise stated. Statistical comparisons were made using unpaired two-tailed Student’s t test. Differences were considered significant at a value of * p ≤ 0.05, **p≤ 0.01, *** p≤ 0.001. ImageJ software was used to quantify gel bands from immunoblots using densitometry.

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[0133] The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. [0134] All publications, patents, accession numbers, and patent applications cited in this specification are hereby incorporated herein by reference in their entirety for their disclosures of the subject matter in whose connection they are cited herein.