WO2014089449A1 - Composition and method for treating neuronal ceroid lipofuscinosis - Google Patents

Abstract

Provided herein are methods for treatment of a neurodegenerative disease, such as neuronal ceroid lipofuscinosis including administering to a subject in need of such treatment a composition comprising a therapeutically effective amount of an agent that mediates upregulation of TPP 1.

Description

COMPOSITION AND METHOD FOR TREATING NEURONAL CEROID

LIPOFUSCINOSIS

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 61/734,679 filed December 7, 2012, which is incorporated by reference herein in its entirety.

SUPPORT

[0002] This invention was made with government support under contract numbers AT6681 , NS64564 and NS71479 awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

[0003] The present invention relates to a composition and method for treating a

neurodegenerative disease, such as neuronal ceroid lipofuscinosis.

BACKGROUND

[0004] Neuronal ceroid lipofuscinosis (NCL) is a group of neurodegenerative diseases mainly composed of typical autosomal recessive lysosomal storage disorders. The NCLs can be characterized by clinical manifestations like progressive mental deterioration, cognitive impairment, visual failures, seizures and deteriorating motor function accompanied by histological findings such as the accumulation of autofluorescent storage material in neurons or other cell types (1). The NCLs have been subdivided into several groups (Typel-10) based on the age of onset, ultrastructural variations in accumulated storage materials, and genetic alterations unique to each specific disease type (2,3).

[0005] Late infantile neuronal ceroid lipofuscinosis (Jansky-Bielschowsky disease, LINCL, Type 2) typically produces symptoms at the age of 2-4 years, progresses rapidly and ends in death between ages 8 to 15 as a result of a dramatic decrease in the number of neurons and other cells (2,4). LI CL is associated with mutations in the Cln2 gene, a 13 exon and 12 intron gene of total length of 6.65 kb mapped to chromosome 1 lpl5.5. The Cln2 gene encodes lysosomal tripeptidyl tripeptidase I (TPP-I or pepstin insensitive protease), a 46 KD protein that function in the acidic environment of the lysosomal compartment to remove tripeptides from the amino terminus of proteins (5,6). This mutation in the Cln2 gene results in a deficiency and/or loss of function of the TPP 1 protein that leads to intralysosomal accumulation of autofluoroscent lipopigments known as ceroid-lipofuscin (5). Currently there is no established treatment or drugs available for this disease; all approaches are merely supportive or symptomatic, indicating a need for novel therapeutic approaches (7). However, there are different variants of Cln2 mutations and there have been reports that residual TPP-I activity can be found in patients with LINCL, indicating that there must be a few copies of normal Cln2 gene remaining in patients affected with LINCL (8,9).

SUMMARY

[0006] Provided herein is a method for treatment of a neurodegenerative disease. The neurodegenerative disease may be neuronal ceroid lipofuscinosis, Alzheimer's disease,

Huntington's disease, Amyotrophic lateral sclerosis (ALS), Parkinson's disease, including Parkinson's plus diseases such as multiple system atrophy (MSA), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) or dementia with Lewy bodies (DLB). The neurodegenerative disorder may be characterized by defective autophage. Such disorders include Alzheimer's, Parkinson's disease, and Huntington's disease. The neurodegenerative disease may be a lysosomal storage disorder. The lysosomal storage disorder may be, for example, Tay-Sach's disease, Fabry disease, Niemann-Pick disease, Gaucher disease, Hunter Syndrome, Alpha-mannosidosis, Aspartylglucosaminuria, Cholesteryl ester storage disease, Chronic Hexosaminidase A Deficiency, Cystinosis, Danon disease, Farber disease, Fucosidosis, or Galactosialidosis.

[0007] The method may comprise administering to a subject in need of such treatment a composition comprising a therapeutically effective amount of an agent that mediates

upregulation of TPP 1. The agent may be a lipid-lowering drug. The lipid-lowering drug may be a fibrate. The fibrate may be gemfibrozil or fenofibrate. The neuronal ceroid lipofuscinosis may be late infantile neuronal ceroid lipofuscinosis or Batten disease. TPPl may be upregulated by increasing TPP1 mRNA levels, increasing TPP1 protein levels, increasing TPP1 enzymatic activity, or activating a PPAR -RXR heterodimer.

[0008] The composition may further comprise a therapeutically effective amount of all-trans retinoic acid. The agent may be a fibrate. Administering all-trans retinoic acid and the fibrate may provide a greater therapeutic effect in the subject than administration of all-trans retinoic acid or the fibrate alone.

[0009] Also provided herein is a method for treatment of neuronal ceroid lipofuscinosis, comprising administering to a subject in need of such treatment a composition comprising a therapeutically effective amount of an agent, wherein the agent restores TPP 1 activity. The agent may be a fibrate. The fibrate may be gemfibrozil or fenofibrate. The therapeutically effective amount of the fibrate may be lower when the fibrate is administered in combination with all-trans retinoic acid.

[0010] Further provided herein is a method for treatment of neuronal ceroid lipofuscinosis, comprising administering to a subject in need of such treatment a composition comprising a therapeutically effective amount of an agent that mediates upregulation of a gene selected from the group of Clnl, Cln2, Cln3, and any combination thereof. The agent may be a fibrate. The fibrate may be gemfibrozil or fenofibrate.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 shows gemfibrozil and fenofibrate upregulating TPP1 mRNA and

functionally active protein in mouse brain cells. (A, B) Mouse primary astrocytes were treated with 25μΜ gemfibrozil in serum-free DMEM/F12 for 2, 6, 12, and 24 hrs followed by monitoring the mRNA expression of Clnl, Cln2 and Cln3 by semi-quantitative RT-PCR (A) and qPCR (B) (for Clnl). (C, D) Mouse astrocytes were treated with different concentrations of gemfibrozil for 24hrs under the same culture conditions followed by monitoring the mRNA expression of Cln2 by semi-quantitative RT-PCR (C) and real-time PCR (D). (E) Mouse primary astrocytes were treated with 25 μΜ gemfibrozil and 10 μΜ fenofibrate for 24hrs under the same culture conditions followed by Western blot for TPP 1. (F) Densitometric analysis of TPP 1 expression (relative to β-Actin) by gemfibrozil and fenofibrate treatment. (G) Mouse primary astrocytes were treated with different concentrations of gemfibrozil and fenofibrate under similar culture conditions and were double-labeled for TPP1 (red) and GFAP (green). Scale bar = 10μΜ. (H, J, L) Mouse primary neurons were isolated from different parts of the brain and were treated with 25 μΜ gemfibrozil and Ι ΟμΜ fenofibrate in neurobasal media containing B27-AO for 24hrs and were double-labeled for TPP1 (red) and β-tubulin (green) (H-for cortical neurons, J-for hippocampal neurons, L-for striatal neurons). DAPI was used to stain nuclei. Scale bar = 20μΜ. (I, K, M) Mouse neurons were treated with 25μΜ gemfibrozil under same culture conditions for 24hrs followed by Western blot for TPP1 {l-for cortical neurons, K-for hippocampal neurons, M-for striatal neurons). Graphs represent the densitometric analysis of TPP1 level (relative to β- Actin). (N) Mouse primary neurons were treated with different concentrations of gemfibrozil and fenofibrate in B27-AO containing Neurobasal media for 24hrs followed by the activity assay using cell extract containing 5μg of total protein. (O) Mouse primary astrocytes were treated with different concentrations of gemfibrozil and fenofibrate in serum free DMEM/F 12 medium for 24hrs followed by the activity assay using cell extract containing 5μg of total protein. All results are mean ± SEM of at least three independent experiments, p * < 0.05 vs control;

p**<0.01 vs control.

[0012] Figure 2 shows gemfibrozil and fenofibrate upregulating TPP 1 mRNA and protein in human primary astrocytes and SHSY5Y neuronal cells. (A, B) Human primary astrocytes were treated with 25μΜ gemfibrozil in serum-free DMEM/F12 for 2, 6, 12 and 24 hrs followed by monitoring the mRNA expression of Cln2 by semi-quantitative RT-PCR (A) and real-time PCR (B). (C-F) Human primary astrocytes were treated with different concentrations of gemfibrozil and fenofibrate for 12hrs under the same culture conditions followed by monitoring the mRNA expression of Cln2 by semi-quantitative RT-PCR (C-for gemfibrozil, E-for fenofibrate) and realtime PCR (D- for gemfibrozil, F -for fenofibrate) . (G) Human primary astrocytes were treated with 25μΜ of gemfibrozil and ΙΟμΜ fenofibrate for 24hrs under similar culture conditions and were double-labeled for TPP1 (red) and GFAP (green). (H) SH-SY5Y cells were treated with 25μΜ of gemfibrozil and Ι ΟμΜ fenofibrate in B27-AO containing Neurobasal media for 24hrs and were double-labeled for TPP1 (red) and β-tubulin (green). DAPI was used to stain nuclei. All results are mean ± SEM of at least three independent experiments. ?* < 0.05 vs control. UN- Untreated. Scale bar = ΙΟμΜ.

[0013] Figure 3 shows involvement of PPAR in fibrate drug-mediated upregulation of TPP1 mRNA and protein. (A-E) Mouse primary astrocytes isolated from PPARa^"7" and PPAR ^{" "} and wild type mouse were treated with different concentrations of gemfibrozil and fenofibrate in serum free DMEM/F12 for 24hrs followed by monitoring the mRNA expression of Cln2 by semi-quantitative RT-PCR (A, C) and real-time PCR (B, D) and protein level of TPPl by Western blot (E). (F) Densitometric analysis of TPPl levels (relative to β-Actin) in PPARa^"7" and PPAR ^7" and wild type astrocytes by gemfibrozil and fenofibrate treatment. p^a<0.05 vs WT control; p^h<0.05 vs PPAR ^"7" control; ns- not significant w.r.t PPARa^"7" control (G, H) Mouse primary astrocytes isolated from WT mice were pre -treated with GW9662 for 30 min followed by treatment with 25 Μ gemfibrozil under similar culture conditions. The mRNA expression of Cln2 was monitored by semi-quantitative RT-PCR (G) real time PCR (H). p^c<0.05 vs control; p^d<0.05 vs only GW treated. (I-K) Mouse primary astrocytes isolated from PPARa^"7" and PPAR and WT mice were treated with 25 μΜ gemfibrozil and Ι ΟμΜ fenofibrate in serum free DMEM/F12 for 24hrs and double-labeled for TPPl (red) and GFAP (green) (I- for WT, J- for PPARa '^' and K- for PPARf> '^~ astrocytes). DAPI was used to stain nuclei. UN- No treatment. Scale bar = ΙΟμΜ. (L-N) Mouse primary astrocytes isolated from PPARa^"7" and PPARP^"7" and WT mice were treated with 25 μΜ gemfibrozil and ΙΟμΜ fenofibrate in serum free DMEM/F12 for 24hrs. Whole cell extracts containing 5μg total protein was incubated at 37°C with 250μΜ 7- amido-4-methylcoumarin in 96 well plates and readings were taken at an interval of 30, 45, 60, 90 and 120 mins. Mean values were taken and plotted in graphical format (L-for Wild type cells, M- for PPARa cells and ' N- for PPARp'^' cells). All results are mean ± SEM of at least three independent experiments.

[0014] Figure 4 shows oral administration of gemfibrozil upregulates TPPl in vivo in cortical and nigral astrocytes and neurons of WT and PPARP^{" "}, but not PPARa^"' mice. WT, PPAR ^" and PPARP^"7" mice (n=4 in each group) were treated with 7.5mg/kg body wt/day of gemfibrozil (dissolved in 0.1% methylcellulose) or vehicle (0.1 % methylcellulose) via gavage. After 21 d of treatment, mice were killed and cortical and nigral sections were double labeled for TPP 1 (red) along with either GFAP (green) or NeuN (green) or TH (green). DAPI was used to visualize nucleus. (A1-A3, B1-B3) TPPl levels were compared between the astrocytes and neurons of the cortical sections of vehicle treated and gemfibrozil treated (Al, Bl) WT, (A2, B2) PPARa^"7", & (A3, B3) PPARP^"7" mice. (A1-A3: GFAP and TPPl; B1-B3: NeuN and TPPl). Scale bar = 20μΜ. (A4, B4) Higher magnification images showing co-localization of TPPl and GFAP in the (A4) cortical astroglia and co-localization of NeuN and TPPl in (B4) cortical neurons of gemfibrozil treated mice (WT and PPARP^"7"). Scale bar = 10μΜ. (C1-C3, D1-D3) TPP l levels were compared between the astrocytes and neurons of the nigral sections of vehicle treated and gemfibrozil treated (CI, Dl) WT, (C2, D2) PPARa^{" "} & (C3, D3) PPAR ^7" mice. Scale bar = 20μΜ. (C1-C3: GFAP and TPPl; D1-D3: TH and TPPl). (C4, D4) Higher magnification images showing co-localization of TPPl and GFAP in the (C4) nigral astroglia and co- localization of TH and TPPl in (D4) nigral TH neurons of gemfibrozil treated mice (WT and PPAR ^{" "}). Scale bar = ΙΟμΜ. All results represent analysis of each of three cortical and nigral sections of each of four different mice per group.

[0015] Figure 5 shows oral administration of gemfibrozil upregulates TPPl in vivo in the hippocampus of WT and ΡΡΑΣφ^7", but not PPARa ^" mice. WT, PPARa^{" "} and PPARp^7" mice (n=4 in each group) were treated with 7.5mg/kg body wt/day of gemfibrozil (dissolved in 0.1% methylcellulose) or vehicle (0.1 % methylcellulose) via gavage. After 21 d of treatment, mice were killed and hippocampal (CA1 and dentate gyrus) sections were double labeled for TPPl (red) and GFAP (green). DAPI was used to visualize nucleus. (A-C) TPPl levels were compared between the dentate gyrus region of vehicle treated and gemfibrozil treated (A) WT, (B) PPARa^" '^", & (C) PPARP^"7" mice. Scale bar = 20μΜ. (D) Higher magnification images showing co localization of TPPl and GFAP in the dentate gyrus of gemfibrozil treated mice (WT, PPARa^{" "} and PPAR ^"7"). Scale bar = 10μΜ. (E-G) TPPl levels were compared between the CA1 region of vehicle treated and gemfibrozil treated (E) WT, (F) PPAR & (G) PPAR ^"' ^" mice. Scale bar = 20μΜ. (H) Higher magnification images showing co-localization of TPPl and GFAP in the CA1 region of gemfibrozil treated mice (WT, PPARa^{" "} and PPAR ^"7"). Scale bar = 10μΜ. All results represent analysis of each of three hippocampal sections of each of four different mice per group.

[0016] Figure 6 shows upregulation of TPPl by fibrate drugs involves both PPARa and RXRa. (A-C) Mouse primary astrocytes were treated with different concentrations of all-trans retinoic acid (RA) and gemfibrozil and the combination of the two in serum- free DMEM/F12 medium for 24hrs followed by monitoring of mRNA expression of Cln2 by quantitative RT-PCR (A) and protein expression of TPPl by Western blot (B). (C) Denistometric analysis of TPPl (relative to β-actin) with RA and gemfibrozil treatment. p^a<0.05 vs WT control; p^h<0.05 vs 0.5μΜ RA only treatment; p^c<0.05 vs 10μΜ gemfibrozil only treatment. (D, E) Mouse primary astrocytes were untransfected, transfected with scrambled siRNA (l ^g) or RXRa siRNA (l ^g) for 36hrs followed by treatment with RA (0.5μΜ) and gemfibrozil (10μΜ) alone and in combination for 24hrs serum free DMEM/F12 medium followed by RT-PCR for Rxra, χτβ, Rxry (D) and quantitative real time PCR for Cln2 (E). p * < 0.05 vs untransfected control; p** < 0.05 vs scrambled siRNA transfected control; p^d < 0.05 vs untransfected-gemfibrozil treated sample; p^e < 0.05 vs untransfected-RA treated sample; p ^ < 0.05 vs scrambled siRNA

transfected-gemfibrozil treated sample; p⁸ < 0.05 vs scrambled siRNA transfected-RA treated sample; ns- not significant w.r.t. RXR- siRNA transfected control. (F) Mouse primary astrocytes were transfected with scrambled siRNA (l .(^g) or RXRa siRNA (l .(^g) and treated with gemfibrozil (ΙΟμΜ) and RA (0.5μΜ) alone and in combination under similar culture conditions and the protein expression of TPP1 were estimated by Western blot. (G)

Denistometric analysis of TPP1 (relative to β-actin) with RA and gemfibrozil treatment, p** < 0.05 vs scrambled siRNA transfected control; p^h <0.05 vs only gemfibrozil treatment; p^l <0.05 vs only RA treatment; ns- not significant w.r.t. RXR-a siRNA transfected control. (H) Mouse primary astrocytes isolated from wild type, PPARa^"7" and PPAR ^"7" mice were treated with RA (0.5 μΜ) alone and in combination with gemfibrozil (ΙΟμΜ) under similar culture conditions and cells were subjected to Western blot for TPP1. (I) Densitometric analysis of TPP1 levels (relative to β-Actin) in PPARa^"7" and PPAR ^"7" and wild type astrocytes after RA and

gemfibrozil+RA treatment. p^† <0.05 vs WT control; p^†† <0.05 vs PPAR ^"7" control; ρί<0.05 vs only RA treatment in WT cells; p^k<0.05 vs only RA treatment in PPAR ^"7" cells; ns- not significant w.r.t. PPARa^"7" control. All results are mean ± SEM of at least three independent experiments.

[0017] Figure 7 shows fibrate drugs upregulating TPP1 via activation of PPARa/RXRa heterodimer. (A) Mouse primary astrocytes were treated with gemfibrozil (10μΜ) and RA (0.5 μΜ) alone and in combination in serum free DMEM/F12 for 6hrs and the nuclear extract was subjected to (i) immunoprecipitation by PPARa Ab followed by immunoblot for both RXRa and PPARa, (ii) immunoprecipitation by RXRa Ab followed by immunoblot for both PPARa and RXRa, (iii) immunoprecipitation by control IgG followed by immunoblot for both PPARa and RXRa, (iv) nuclear extract was subjected to immunoblot for PPARa, RXRa and Histone3 (H3). (B) Schematic diagram for RXR binding site on the Cln2 promoter with the core sequence and amplicon length. (C) Mouse astrocytes were treated with the combination of gemfibrozil (10μΜ) and RA (0.5 μΜ) for 6hrs and recruitment of PPARa and RXRa on the RXR binding site of Cln2 promoter was monitored by ChIP analysis as described under "Materials and Methods". Normal IgG was used as control. All results are representative of at least there independent experiments. [0018] Figure 8 is a schematic representation of the mechanism of upregulation of TPP1 by fibrate drugs via PPARa/RXRa pathway.

[0019] Figure 9 shows the upregulation of lysosomal proliferation in primary human astrocytes by gemfibrozil and fenofibrate. Cells were treated with 25μΜ gembibrozil and 10μΜ fenofibrate for 24 hrs followed by labeling lysosomes with LysoTracker. DAPI was used to visualize nuclei. Results represent 3 independent experiments.

[0020] Figure 10 shows the upregulation of lysosomal proliferation in primary human neurons by gemfibrozil and fenofibrate. Cells were treated with 25μΜ gemfibrozil and 10 μΜ

fenofibrate for 24 hours followed by labeling lysosomes with LysoTracker. DAPI was used to visualize nuclei. Results represent 3 independent experiments.

[0021] Figure 1 1 shows the upregulation of the lysosomal marker protein tripeptidyl peptidase 1 (TPP1) by gemfibrozil and fenofibrate. Cells were treated with 25μΜ gemfibrozil and 10 μΜ fenofibrate for 24 hours followed by double-labeling for TPP1 and β-tubulin (neuronal marker). DAPI was used to visualize nuclei. Results represent 3 independent experiments.

[0022] Figure 12 shows the upregulation of lysosome-associated membrane protein 2

(LAMP2) in human neurons by gemfibrozil and fenofibrate. Cells were treated with 25μΜ gemfibrozil and 10 μΜ fenofibrate for 24 hours followed by double-labeling for LAMP2 and β- tubulin (neuronal marker). DAPI was used to visualize nuclei. Results represent 3 independent experiments.

[0023] Figure 13 shows the upregulation of lysosomal genes in brain cells by gemfibrozil and fenofibrate. (A) Quantitative real-time PCR for lysosomal genes in mouse astrocytes treated with 25 μΜ gemfibrozil for different time points under serum free conditions. Results are mean ± SD of three different experiments. ^ap < 0.001 vs control. (B) RT-PCR for lysosomal genes in human astrocytes treated under same condition. (C) Human neurons were treated with different concentrations of fenofibrate for 24 hrs under serum free conditions and RT-PCR was done lysosomal genes. Results represent 3 independent experiments.

[0024] Figure 14 illustrates that gemfibrozil prolongs the life span of CLN2 (-/-) mouse: CLN2(-/-) animals were orally administered with gemfibrozil dissolved in 0.1% methyl-cellulose at a dosage of 7.5mg/ kg body weight/ day. Treatment was started from 4 weeks of age for all groups. Methyl cellulose was used as vehicle. Top panel: Schematic representation of experiments performed. Bottom panel: Kaplan-Meier plot for percentage survival of treated vs. vehicle or untreated animals.

[0025] Figure 15 illustrates that gemfibrozil treatment delays the loss of motor activity in CLN2 (-/-) mice: Treated and untreated CLN2 (-/-) animals from the previous experiment (Fig.1) were monitored after 2 weeks (Left panel) and 6 weeks (right panel) of gemfibrozil treatment for various motor activity parameters. Each animal was allowed 5mins of adaptation time followed by monitoring for 5min. *p-value <0.05. n=15 (untreated), n=20 (treated)

[0026] Figure 16 illustrates that gemfibrozil treatment reduces peroxide production in CLN2 knockdown mouse astrocytes. Mouse astrocytes were transfected with CLN2 siRNA (l .(^g) for 36hrs and treated with gemfibrozil (25μΜ) for 6hrs. Cells were then harvested and subjected to peroxide assay. Results are mean ± SD for three independent experiments. * &**p-value < 0.05

DETAILED DESCRIPTION

[0027] The present invention relates to methods of treatment of neuronal ceroid lipofuscinosis ( CL). Neuronal ceroid lipofuscinosis includes a group of diseases such as late infantile neuronal ceroid lipofuscinosis (LINCL) and Batten disease that are neurodegenerative lysosomal storage diseases. Many forms of NCL occur via mutations in the Cln genes. LINCL is associated with mutations in the Cln2 gene, which encodes tripeptidyl peptidase 1 (TPP1). The genes Clnl and Cln3 are associated with infantile NCL (INCL) and juvenile NCL (JNCL), respectively.

[0028] The methods of the present invention include administering to a subject suffering from NCL an agent that upregulates or enhances expression from a gene such as Clnl, Cln2, Cln3, and/or any combination thereof. Upregulation may include increasing mRNA levels for Clnl, Cln2, Cln3, and/or any combination thereof.

[0029] The methods of the present invention also include administering to a subject suffering from NCL an agent that upregulates TPP 1 or restores TPP 1 activity. Upregulation may include increasing TPP1 mRNA levels, increasing TPP1 proteins levels, or increasing TPP1 enzymatic activity. The inventors have also discovered that activating a PPARa/RXRa heterodimer results in upregulation of TPP 1. The inventors have also surprisingly shown that TPP 1 is upregulated through the activity or involvement of PP ARa, but not PP AR or PP ARy. [0030] The agent may be a lipid-lowering drug such as a fibrate. The fibrate may be gemfibrozil, fenofibrate, or clofibrate. Alternatively, the agent may be all-trans retinoic acid. Surprisingly and unexpectedly, administration of the fibrate in combination with all-trans retinoic acid to the subject upregulates TPPl more than administration of the fibrate or all-trans retinoic acid alone. The fibrate and all-trans retinoic acid, when administered together to the subject, cooperatively enhance upregulation of TPPl . In other words, a lower dose of the fibrate is needed in the presence of all-trans retinoic acid to achieve the same degree of TPP 1 upregulation as occurs when only a higher dose of the fibrate is administered to the subject.

[0031] Both gemfibrozil and fenofibrate are capable of enhancing TPPl in cultured neurons and glial cells and in vivo in the brain. In addition, PPARa, but not ΡΡΑΡνβ and PPARy, is involved in gemfibrozil- and fenofibrate-mediated upregulation of TPP 1. Furthermore, fibrate drugs upregulate TPP 1 via activation of PPARa- RXR heterodimer. Collectively, gemfibrozil and fenofibrate, FDA-approved drugs for hyperlipidemia, are of therapeutic value in the treatment of LI CL.

1. Definitions

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0033] The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments "comprising," "consisting of and "consisting essentially of," the embodiments or elements presented herein, whether explicitly set forth or not. [0034] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

2. Methods of Treating a Neurodegenerative Disease

[0035] Provided herein is a method of treating a neurodegenerative disease. The

neurodegenerative disease may be, for example, neuronal ceroid lipofuscinosis, Alzheimer's disease, Huntington's disease, Amyotrophic lateral sclerosis (ALS), Parkinson's disease, including Parkinson's plus diseases such as multiple system atrophy (MSA), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) or dementia with Lewy bodies (DLB). The neurodegenerative disorder may be characterized by defective autophage. Such disorders include Alzheimer's, Parkinson's disease, and Huntington's disease. The

neurodegenerative disease may be a lysosomal storage disorder. The lysosomal storage disorder may be, for example, Tay-Sach's disease, Fabry disease, Niemann-Pick disease, Gaucher disease, Hunter Syndrome, Alpha-mannosidosis, Aspartylglucosaminuria, Cholesteryl ester storage disease, Chronic Hexosaminidase A Deficiency, Cystinosis, Danon disease, Farber disease, Fucosidosis, or Galactosialidosis.

[0036] The neuronal ceroid lipofuscinosis may be LINCL or Batten disease, for example. The method may include administering to a subject suffering from NCL an agent that upregulates TPP 1 or restores TPP 1 activity. The method may also include administering to a subject suffering from NCL an agent that upregulates or enhances expression from a gene such as Clnl, Cln2, Cln3, and/or any combination thereof.

[0037] NCL is a group of neurodegenerative diseases comprising typical autosomal recessive lysosomal storage disorders. NCLs may include clinical manifestations such as progressive mental deterioration, cognitive impairment, visual failure, seizures, and deteriorating motor function. NCLs may be associated with accumulation of autofluoroscent storage materials in neurons and/or other types of cells. NCLs may be divided into several types including Types 1 to 10, and the types may be based on the age of onset, ultra structural variations in accumulated storage materials, and genetic alterations. Presently, no established treatment and/or drugs are available for treatment of NCLs. Rather, present treatment is merely supportive of disease symptoms. [0038] Many types of NCL may be associated with mutations in the Cln genes. These mutations may be associated with a deficiency or loss of function. For example, late infantile neuronal ceroid lipofuscinosis (LINCL) may be associated with mutations in the gene Cln2, while infantile NCL (INCL) and juvenile (JNCL) may be associated with mutations in the genes Clnl and Cln3, respectively. The gene Cln2 encodes lysosomal tripeptidyl tripeptidase 1 (TPP1), which is a 46 kilodalton protein that functions in the acidic environment of the lysosomal compartment of a cell to remove tripeptides from the amino termini of proteins.

Deficiency and/or loss of function of TPP 1 may lead to intralysosomal accumulation of autofluoroscent lipopigments known as ceroid-lipofuscins. Some subjects suffering from NCL may have residual activity of TPP 1.

a. Agent

[0039] The agent may be a lipid-lowering drug, all-trans retinoic acid, or a combination of the lipid-lowering drug and all-trans retinoic acid. Administration of the agent to a subject suffering from NCL may upregulate or enhance expression from a gene such as Clnl, Cln2, Cln3, and/or any combination thereof. Upregulation may include increasing mRNA levels for Clnl, Cln2, Cln3, and/or any combination thereof.

[0040] Administration of the agent to a subject suffering from NCL may also upregulate TPP 1 or restore TPP 1 activity. Upregulation of TPP 1 may include increasing the levels of TPP 1 mRNA, increasing the levels of TPP 1 protein, or increasing the enzymatic activity of TPP 1. Additionally, upregulation of TPP 1 may include activating the PPAR /RXR heterodimer, which is recruited to the promoter of the Cln2 gene to affect TPP 1 upregulation.

(1) Lipid-lowering drug

[0041] The agent mediating upregulation of TPP 1 may be a lipid-lowering drug. Lipid- lowering drugs may be drugs that reduce the level of triglycerides circulating in the blood of the subject. Additionally, lipid-lowering drugs may be drugs that decrease the risk of

hyperlipidemia. Such lipid-lowering drugs may include fibrates such as gemfibrozil, fenofibrate, and clofibrate.

[0042] The fibrate may mediate upregulation of TPP 1 via PPAR , but not PPAR and PPARy. During upregulation of TPP 1 , PPARa forms a heterodimer with RXR-a and the RXR- a/PPAR-a heterodimer is recruited to the promoter of the Cln2 gene via a RXR binding site. (2) All-trans retinoic acid

[0043] The agent mediating upregulation of TPP1 may be all- trans retinoic acid. All- trans retinoic acid may also be known as ATRA, retinoic acid, tretinoin, and vitamin A acid. All-trans retinoic acid may mediate upregulation of TPP1 via the retinoid X receptor- (RXR- ). During upregulation of TPP1, RXR-a forms a heterodimer with peroxisome proliferator-activated receptor-a (PPAR-a) and the RXR-a/PPAR-a heterodimer is recruited to the promoter of the Cln2 gene via a RXR binding site.

(3) Combination of lipid-lowering drug and all-trans retinoic acid

[0044] The agent mediating upregulation of TPP 1 may comprise a combination of the lipid- lowering drug and all-trans retinoic acid. Such a combination may cooperatively mediate or enhance upregulation of TPP 1 as compared to administration of the lipid-lowering drug or all- trans retinoic acid alone. The combination may cooperatively enhance upregulation of TPP 1 about 2-fold, about 3-fold, about 4-fold, about 5-fold, or about 10-fold as compared to administration of the lipid-lowering drug or all-trans retinoic acid alone. Particularly, the combination may cooperatively enhance upregulation of TPP 1 about 3-fold as compared to administration of the lipid-lowering drug or all-trans retinoic acid alone.

b. Pharmaceutical Compositions

[0045] The agent may be incorporated into pharmaceutical compositions suitable for administration to a subject (such as a patient, which may be a human or non-human).

[0046] The pharmaceutical compositions may include a "therapeutically effective amount" or a "prophylactically effective amount" of the agent. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the composition may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. [0047] For example, a therapeutically effective amount of gemfibrozil may be about 5 mg to about 2000 mg, about 10 mg to about 1900 mg, about 15 mg to about 1800 mg, about 20 mg to about 1700 mg, about 25 mg to about 1600 mg, about 30 mg to about 1500 mg, about 35 mg to about 1400mg, about 40 mg to about 1300 mg, about 45 mg to about 1200 mg, about 50 mg to about 1 100 mg, about 55 mg to about 1000 mg, about 60 mg to about 900 mg, about 65 mg to about 800 mg, about 70 mg to about 700 mg, about 75 mg to about 600 mg, about 80 mg to about 500 mg, about 85 mg to about 400 mg, about 90 mg to about 300 mg, about 95 mg to about 200 mg, or about 100 mg to about 175 mg. In another example, the therapeutically effective amount of gemfibrozil may be about 600 mg or about 1200 mg.

[0048] For example, a therapeutically effective amount of fenofibrate may be about 5 mg to about 2000 mg, about 10 mg to about 1900 mg, about 15 mg to about 1800 mg, about 20 mg to about 1700 mg, about 25 mg to about 1600 mg, about 30 mg to about 1500 mg, about 35 mg to about 1400mg, about 40 mg to about 1300 mg, about 45 mg to about 1200 mg, about 50 mg to about 1 100 mg, about 55 mg to about 1000 mg, about 60 mg to about 900 mg, about 65 mg to about 800 mg, about 70 mg to about 700 mg, about 75 mg to about 600 mg, about 80 mg to about 500 mg, about 85 mg to about 400 mg, about 90 mg to about 300 mg, about 95 mg to about 200 mg, or about 100 mg to about 175 mg. In another example, the therapeutically effective amount of fenofibrate may be about 40 mg, about 48 mg, about 54 mg, about 67 mg, about lOOmg, about 120 mg, about 134 mg, about 145 mg, about 160 mg, or about 200 mg.

[0049] For example, a therapeutically effective amount of clofibrate may be about 5 mg to about 2000 mg, about 10 mg to about 1900 mg, about 15 mg to about 1800 mg, about 20 mg to about 1700 mg, about 25 mg to about 1600 mg, about 30 mg to about 1500 mg, about 35 mg to about 1400mg, about 40 mg to about 1300 mg, about 45 mg to about 1200 mg, about 50 mg to about 1 100 mg, about 55 mg to about 1000 mg, about 60 mg to about 900 mg, about 65 mg to about 800 mg, about 70 mg to about 700 mg, about 75 mg to about 600 mg, about 80 mg to about 500 mg, about 85 mg to about 400 mg, about 90 mg to about 300 mg, about 95 mg to about 200 mg, or about 100 mg to about 175 mg. In another example, the therapeutically effective amount of clofibrate may be about 500 mg.

[0050] The pharmaceutical compositions may include pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier," as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt;

gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

c. Modes of Administration

[0051] Methods of treating neuronal ceroid lipofuscinosis may include any number of modes of administering the agent or pharmaceutical compositions of the agent. Modes of

administration may include tablets, pills, dragees, hard and soft gel capsules, granules, pellets, aqueous, lipid, oily or other solutions, emulsions such as oil-in-water emulsions, liposomes, aqueous or oily suspensions, syrups, elixiers, solid emulsions, solid dispersions or dispersible powders. For the preparation of pharmaceutical compositions for oral administration, the agent may be admixed with commonly known and used adjuvants and excipients such as for example, gum arabic, talcum, starch, sugars (such as, e.g., mannitose, methyl cellulose, lactose), gelatin, surface-active agents, magnesium stearate, aqueous or non-aqueous solvents, paraffin derivatives, cross-linking agents, dispersants, emulsifiers, lubricants, conserving agents, flavoring agents (e.g., ethereal oils), solubility enhancers (e.g., benzyl benzoate or benzyl alcohol) or bioavailability enhancers (e.g. Gelucire.TM.). In the pharmaceutical composition, the agent may also be dispersed in a microparticle, e.g. a nanoparticulate, composition.

[0052] For parenteral administration, the agent or pharmaceutical compositions of the agent can be dissolved or suspended in a physiologically acceptable diluent, such as, e.g., water, buffer, oils with or without solubilizers, surface-active agents, dispersants or emulsifiers. As oils for example and without limitation, olive oil, peanut oil, cottonseed oil, soybean oil, castor oil and sesame oil may be used. More generally spoken, for parenteral administration the agent or pharmaceutical compositions of the agent can be in the form of an aqueous, lipid, oily or other kind of solution or suspension or even administered in the form of liposomes or nano- suspensions.

[0053] The term "parenterally," as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0054] The present invention has multiple aspects, illustrated by the following non-limiting examples.

3. Examples

Example 1

Materials and Methods for Examples 2-9

[0055] Reagents: DMEM/F-12 50/50 lx, Hank's balanced salt solution (HBSS) and 0.05% trypsin were purchased from Mediatech (Washington, DC). Fetal bovine serum (FBS) was obtained from Atlas Biologicals (Fort Collins, CO). Antibiotic-antimycotic, gemfibrozil and Akt- inhibitor (Akt-i) were obtained from Sigma-Aldrich (St. Louis, MO). Wortmannin and

LY294002 were purchased from Calbiochem (Darmstadt, Germany).

[0056] Isolation of mouse primary astroglia: Astroglia were isolated from mixed glial cultures as described (17,18) according to the procedure of Giulian and Baker (19). Briefly, on day 9, the mixed glial cultures were washed three times with Dulbecco's modified Eagle's medium/F-12 and subjected to shaking at 240 rpm for 2 h at 37°C on a rotary shaker to remove microglia. After 2 days, the shaking was repeated for 24 h for the removal of oligodendroglia and to ensure the complete removal of all nonastroglial cells. The attached cells were seeded onto new plates for further studies.

[0057] Isolation of primary human astroglia: Primary human astroglia were prepared as described (20,21). All experimental protocols were reviewed and approved by the Institutional Review Board of the Rush University Medical Center. Briefly, 11- to 17-week-old fetal brains obtained from the Human Embryology Laboratory (University of Washington, Seattle, WA, USA) were dissociated by trituration and trypsinization. On 9th day, these mixed glial cultures were placed on a rotary shaker at 240 rpm at 37°C for 2 h to remove loosely attached microglia. On 11^th day, the flasks were shaken again at 190 rpm at 37°C for 18 h to remove oligodendroglia. The attached cells remaining were primarily astrocytes. These cells were trypsinized and subcultured in complete media at 37°C with 5% C0₂ in air to yield more viable and healthy cells. By immunofluorescence assay, these cultures homogeneously expressed GFAP, a marker for astrocytes (22).

[0058] Isolation of neurons from different brain regions: Fetal (El 8-E16) mouse neurons were prepared as previously described (23) with modifications. Whole brains were removed and cortical, hippocampal, striatal and cerebellar fractions were dissected in serum free Neurobasal media. The cells were washed by centrifugation three times at 1200 rpm for 10 min, the pellet dissociated and the cells plated at 10% confluence in 8-well chamber slides pre-treated for >2 hr with Poly-D-Lysine (Sigma, St. Louis, MO). After 4 min, the non-adherent cell suspension was aspirated and 500ml complete Neurobasal media (Invitrogen) supplemented with 2% B27 was added to each well. The cells were incubated for 4 days prior to experimentation. Double-label immunofluorescence with β-tubulin and either GFAP or CD1 lb revealed that neurons were more than 98% pure (data not shown). The cells were stimulated with gemfibrozil in Neurobasal media supplemented with 2% B27 minus antioxidants (Invitrogen) for 24 hr prior to methanol fixation and immunostaining.

[0059] Semi-Quantitative Reverse Transcriptase-Coupled Polymerase Chain Reaction (RT- PCR): Total RNA was isolated from mouse primary astrocytes and human primary astrocytes using RNA-Easy Qiagen (Valencia, CA) kit following manufactures protocol. Semi-quantitative RT-PCR was carried out as described earlier (24) using oligo (dT) 12-18 as primer and moloney murine leukemia virus reverse transcriptase (MMLV-RT, Invitrogen) in a 20μ1 reaction mixture. The resulting cDNA was appropriately amplified using Promega Master Mix (Madison, WI) and the following primers (Invitrogen) for murine genes:

Mouse CM: Sense, 5 -ACACAGAGGACCGCCTGGGG-3 ' (SEQ ID NO: 1)

Antisense, 5 -TCATGCACGGCCCACACAGC-3 '(SEQ ID NO: 2)

Mouse Cln2: Sense, 5 '-CACCATCCAGTTACTTCAATGC-3 ' (SEQ ID NO: 3)

Antisense, 5 '- CTGACCCTCCACTTCTTCATTC-3 ' (SEQ ID NO: 4)

Mouse Cln3: Sense, 5 -TGCTGCCCTGCCATCGAGTG-3 ' (SEQ ID NO: 5)

Antisense, 5 - GGCAGCGCTCAGCATCACCA-3 ' (SEQ ID NO: 6)

Mouse Gapdh. Sense, 5'- GCACAGTCAAGGCCGAGAAT-3 ' (SEQ ID NO: 7)

Antisense, 5 '- GCCTTCTCCATGGTGGTGAA-3 ' (SEQ ID NO: 8) Amplified products were electrophoresed on 2% agarose (Invitrogen) gels and visualized by ethidium bromide (Invitrogen) staining. Glyceraldehyde-3 -phosphate dehydrogenase (Gapdh) mRNA was used as a loading control to ascertain that an equivalent amount of cDNA was synthesized from each sample.

[0060] Quantitative Real-Time PCR: The mRNA quantification was performed using the ABI-Prism7700 sequence detection system (Applied Biosystems, Foster City, CA) using iTaq™ Fast Supermix With ROX (Bio-Rad, Hercules, CA) and the following 6-FAM/ZEN/IBFQ- labeled primers for murine genes: Cln2 and Gapdh (Integrated DNA Technologies Coralville, IA). The mRNA expression of the targeted genes was normalized to the level of Gapdh mRNA and data was processed by the ABI Sequence Detection System 1.6 software.

[0061] Immunostaining of cells: Immunocytochemistry was performed as described earlier (25). Briefly, 8 well chamber slides containing mouse primary astrocytes, mouse neurons, human primary astrocytes or SH-SY5Y cells were cultured to 70-80% confluence were fixed with chilled Methanol (Fisher Scientific, Waltham, MA) overnight, followed by two brief rinses with filtered PBS. Samples were blocked with 2% BSA (Fisher Scientific) in PBS containing Tween 20 (Sigma) and Triton X-100 (Sigma) for 30 min and incubated at room temperature under shaking conditions for 2 hr in PBS containing the following anti-mouse primary antibodies: TPPl (1 :200; Santa Cruz Biotech, Santa Cruz, CA), GFAP, (1 : 100; Santa Cruz), and β-tubulin (1 :5000; Millipore). After four 15 min washes in filtered PBS, the slides were further incubated with Cy2 or Cy5-labeled secondary antibodies (all 1 :200; Jackson ImmunoResearch, West Grove, PA) for 1 hr under similar shaking conditions. Following four 15 minute washes with filtered PBS, cells were incubated for 4-5 min with 4', 6-diamidino-2-phenylindole (DAPI, 1 : 10,000; Sigma). The samples were run in an EtOH and Xylene (Fisher) gradient, mounted and observed under Olympus BX41 fluorescence microscope.

[0062] Immunostaining of tissue sections: After 21 days of treatment, mice were sacrificed and their brains fixed, embedded, and processed. Sections were made from different brain regions and for immunofluorescence staining on fresh frozen sections, anti-mouse TPPl (1 :200), goat anti mouse GFAP (1 : 100) were used. The samples were mounted and observed under Olympus BX41 fluorescence microscope (26).

[0063] Immunoblotting: Western blotting was conducted as described earlier (27,28) with modifications. Briefly, cells were scraped in double-distilled H₂0 and sodium dodecyl sulfate (SDS), electrophoresed on NuPAGE® Novex® 4- 12% Bis-Tris gels (Invitrogen) and proteins transferred onto a nitrocellulose membrane (Bio-Rad) using the Thermo-Pierce Fast Semi-Dry Blotter. The membrane was then washed for 15 min in TBS plus Tween 20 (TBST) and blocked for 1 hr in TBST containing BSA. Next, membranes were incubated overnight at 4°C under shaking conditions with the following 1° antibodies; TPP1 (1 :250, Santa Cruz), β-actin (1 :800; Abeam, Cambridge, MA. The next day, membranes were washed in TBST for 1 hr, incubated in 2° antibodies against 1° antibody hosts (all 1 : 10,000; Jackson ImmunoResearch) for 1 hr at room temperature, washed for one more hour and visualized under the Odyssey® Infrared Imaging System (Li-COR, Lincoln, NE).

[0064] TPP1 activity assay: TPP-I activity was assayed in 96-well format plates using the following modification of the method described by Vines and Warburton (6). Briefly, samples and substrate (40 μΕ) were mixed in individual wells of polystyrene 96-well plate (Nalge Nunc International). The substrate solution consisted of 250 μπιοι/L Ala-Ala-Phe 7- amido-4-methylcoumarin (cat. no. A3401 ; Sigma; diluted freshly from a 25 mmol/L stock solution in dimethyl sulfoxide stored at -20 °C) in 0.15 mol/L NaCl-1 g/L Triton X- 100-0.1 mol/L sodium acetate, adjusted to pH 4.0 at 20 °C. Plates were centrifuged briefly to dispel bubbles and placed in a 37 °C. Plates were mixed for 10s before each reading. The plates were read from the bottom using 360/20 nm excitation and 460/25 nm emission filters. Prior to the assay, the optimum substrate concentration and total protein in cell extract that can used to get best results were determined in the same manner described above, using different substrate concentrations and protein concentrations in the cell extract.

[0065] Immunoprecipitation from nuclear extract: After treatment, cells were washed with PBS, scraped into 1.5 mL tubes and centrifuged in 4°C for 5 min at 500 rpm. The supernatant was aspirated and the pellet was resuspended in a membrane lysis buffer comprised of (4-(2- hydroxyethyl)-l-piperazineethanesulfonic acid) (HEPES, pH 8.0), MgCl₂, KC1, dithiothreitol (DTT) and protease/phosphatase inhibitors, vortexed and centrifuged in 4°C at 15,000 rpm for 3 minutes. Again, the supernatant was aspirated and the pellet was resuspended in a high salt, nuclear envelope lysis buffer comprised of HEPES (pH 8.0), MgCl₂, glycerol, NaCl, ethylenediaminetetraacetic acid (EDTA), DTT and protease/phosphatase inhibitors, rotated vigorously in 4°C for 30 min and centrifuged in 4°C at 15,000 rpm for 15 minutes. The resultant nuclear pellet was resuspended in IP buffer and a fraction was kept separately as lysate. The remaining nuclear extract was then precleared with 25 ul of protein A-agarose (50%, v/v). The supernatants were immunoprecipitated with 5ug of anti-RXRa or anti-PPAR or normal IgG (Santa Cruz Biotechnology, Inc.) overnight at 4C, followed by incubation with protein A-agarose for 4hrs at 4 °C. Protein A-agarose-antigen-antibody complexes were collected by centrifugation at 12,000 rpm for 60 s at 4 °C. The pellets were washed five times with 1 ml of IP buffer (20 Mm Tris-HCl, pH 8.0, 137 mM NaCl, 2 mM EDTA, 1 % Nonidet P-40, 10% glycerol, 0.1 mM phenylmethylsulfonyl fluoride) for 20 min each time at 4 °C. Bound proteins were resolved by SDS-PAGE, followed by Western blotting with the anti-RXRa (1 :2000, Santa Cruz) and/or anti- PPARa (1 :250, Santa Cruz). The lysate was resolved by SDS-PAGE followed by immunoblot for PPARa, RXRa and H3.

[0066] Chromatin Immunoprecipitation Assay: ChIP assays were performed using method described by Nelson et al (29), with certain modifications. Briefly, mouse primary astrocytes were stimulated by l OuM gemfibrozil and 0.5 μΜ RA together for 6hrs followed by fixing with formaldehyde (1.42% final volume) and quenching with 125mM Glycine. The cells were pelleted and lysed in IP buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, NP-40 (0.5% vol/vol), Triton X-100 (1.0% vol/vol). For 500 ml, add 4.383 g NaCl, 25 ml of 100 mM EDTA (pH 8.0), 25 ml of 1 M Tris-HCl (pH 7.5), 25 ml of 10% (vol/vol) NP-40 and 50 ml of 10% (vol/vol) Triton X- 100 containing the following inhibitors; 10 μg/ml leupeptin, 0.5 mM phenylmethlysulfonyl fluoride (PMSF), 30 mM p-nitrophenyl phosphate, 10 mM NaF, 0.1 mM Na₃V0₄, 0.1 mM Na₂Mo0₄ and 10 mM β-glycerophosphate. After one wash with 1.0 ml IP buffer the pellet was resuspended in 1 ml IP buffer (containing all inhibitors) and sonicated and sheared chromatin was split into two fractions (one to be used as Input). The remaining fraction was incubated overnight under rotation at 4°C with 5-7μg of anti-PPARa or anti-RXRa Abs or normal IgG (Santa Cruz) followed by incubation with Protein G- Agarose (Santa Cruz) for 2hrs at 4°C under rotation. Beads were then washed five times with cold IP buffer and a total of 100 μΐ of 10% Chelex (10 g/ 100 ml H₂0) is added directly to the washed protein G beads and vortexed. After 10 min boiling, the Chelex protein G bead suspension is allowed to cool to room temperature. Proteinase K (100 μg/ml) is then added and beads are incubated for 30 min at 55°C while shaking, followed by another round of boiling for 10 min. Suspension is centrifuged and supernatant is collected. The Chelex/protein G beads fraction is vortexed with another 100 μΐ water, centrifuged again, and the first and the second supernatants are combined. Eluate is used directly as a template in PCR. The following primers were used to amplify fragments flanking RXR binding element in the mouse Cln2 promoter: Setl : sense: 5'- CAG CTG CCA TGT CCC CCA GC-3', (SEQ ID NO: 9) antisense: 5'- TGC GCA GCT CTG TGT CAT CCG -3' (SEQ ID NO: 10); Set2: sense: 5'- GCT CCC TCT CCT CAG CTG CCA -3' (SEQ ID NO: 1 1), antisense: 5'- CAT CCG GAG GCT CCA GGC CA -3' (SEQ ID NO: 12). The PCRs were repeated by using varying cycle numbers and different amounts of templates to ensure that results were in the linear range of PCR.

[0067] Densitometric Analysis: Protein blots were analyzed using ImageJ (NIH, Bethesda, MD) and bands were normalized to their respective β-actin loading controls. Data are representative of the average fold change with respect to control for three independent experiments.

[0068] Statistics: Values are expressed as means ± SEM of at least three independent experiments. Statistical analyses for differences were performed via Student's T-test. This criterion for statistical significance was p < 0.05.

Example 2

Fibrate drugs upregulate TPP1 mRNA and protein in mouse primary astrocytes

[0069] There have been reports that residual TPP-I activity can be found in patients indicating that a few copies of normal Cln2 gene are left in patients affected with LINCL (8,9,30,31). We examined if FDA-approved lipid- lowering drugs like gemfibrozil and fenofibrate were capable of upregulating the expression of TPP1 in brain cells. Mouse primary astrocytes were treated in serum free media with gemfibrozil with different doses and for different time points. Both RT- PCR and real-time quantitative PCR (qPCR) analyses clearly indicated that gemfibrozil upregulated Cln2 mRNA levels in mouse primary astrocytes in a time and dose dependant manner with maximum increase at 24hrs of 25μΜ gemfibrozil treatment (Fig. 1A, IB, 1C & ID). Other lysosomal genes like Clnl and Cln3 which are responsible for Infantile NCL (INCL) and Juvenile NCL (JNCL) respectively were also found to increase within 12 to 24 h (Fig. 1 A). The mRNA data was validated by Western blot where 3-4 fold increase in TPP1 protein level was found with 25μΜ gemfibrozil and 10μΜ fenofibrate treatment for 24hrs. (Fig. IE and IF) Immunofluorescence of primary mouse astrocytes stimulated with gemfibrozil and fenofibrate also revealed a dose dependant increase in TPP1 protein (Fig. 1G). Example 3

Gemfibrozil and fenofibrate upregulate TPP 1 in neurons from different parts of the mouse brain

[0070] Lack of TPP 1 enzyme causes accumulation of lipofuscines in neurons leading to loss of neurons in brain causing the disease to progress (5). Hence we examined the effect of the fibrate drugs in neurons and determined whether the induction of TPP 1 occurs throughout the brain. Mouse primary neurons were isolated from different brain regions viz. cortex,

hippocampus, and striatum and cultured and treated with gemfibrozil and fenofibrate. The immunofluorescence showed a significant increase in the mouse neurons from all three brain regions (Fig. 1H, 1 J & 1L). Further, the neurons from those there brain regions were treated with gemfibrozil for 24hrs followed by Western blotting for TPP 1 which showed about 2-3 fold increase in TPP1 protein, as determined by densitometric quantification (Figs. II, IK & 1M).

Example 4

TPP 1 proteins unregulated by the fibrate drugs are functionally active

[0071] Since the functional activity of the TPP1 protein is of critical importance in the clinical setting for LINCL (6), activity of the enzyme was measured. The cells were homogenized and the cell extracts were subjected to TPP1 activity assay. Prior to that, the optimal substrate concentration and optimal amount of extract for the assay was determined by using different concentrations of substrate and sample respectively (Supplementary Fig. 1A & IB). TPP1 activity was measured (as described in the methods section) in mouse primary neurons and mouse primary astrocytes. The product formation increased with increasing doses of treatment indicating an increase in activity of the protein in the cell extracts (Fig. IN & 10). This can be attributed to the increase in the levels of proteins in the cells observed in the earlier experiments. Collectively, these data strongly suggest that fibrate drugs can enhance both the mRNA and protein levels resulting in an increased activity of the protein in the cell.

Example 5

Fibrate drugs upregulate TPP1 mRNA and protein in human brain cells

[0072] We further examined whether a similar increase in Cln2 mRNA and protein was obtained upon treatment of human cells with gemfibrozil and fenofibrate. Human astrocytes were treated in the same way as the mouse cells and the mRNA levels were quantified. Again, both RT and qPCR data indicated an increase in Cln2 mRNA levels in human astrocytes in a dose and time dependant manner with maximum at a dose of 25uM gemfibrozil (~15 fold) and at 12 hrs (~10 fold) (Fig. 2A, 2B, 2C & 2D). However, fenofibrate was seen to increase the mRNA levels at a relatively lower dose (lOuM) but at same time point (12hrs) as that of gemfibrozil (Fig.2E & 2F). Once again, the protein levels were assessed in human astrocytes and SH-SY5Y cell lines by immunofluorescence and in both the cell types a considerable increase in the level of TPP1 protein was observed. (Fig. 2G & 2H).

Example 6

PPARq is involved in fibrate drug mediated upregulation of TPP 1

[0073] Since it is known that PPARs are activated by fibrate drugs, the role of these receptors in mediating upregulation of TPP 1 protein was examined (15). Astrocytes isolated from PPARa^" and PPAR ^"'^" and wild-type (WT) mice were treated with gemfibrozil and fenofibrate and Cln2 mRNA levels measured. The data from semi-quantitative RT-PCR and qRT-PCR showed that WT and PPAR ^"7" cells showed similar patterns of upregulation, whereas PPARa^"7" cells showed little or no effect on the upregulation of Cln2 mRNA expression upon gemfibrozil treatment (Fig. 3A and 3B) and fenofibrate treatment (Fig. 3C and 3D). When mouse primary astrocytes were treated with GW9662, a PPARy antagonist, followed by gemfibrozil or fenofibrate treatment, there was increased expression of Cln2 mRNA, even in presence of the antagonist (Fig. 3G and 3H).

[0074] To confirm the mRNA measurements the WT and PPARa^{" "} and PPAR ^"7" astrocytes were processed for protein analysis. The cells were treated similarly with gemfibrozil and fenofibrate and immunofluorescence and Western blotting were performed. The immunoblot and densitometric analysis of the blots showed no significant increase in TPP1 levels in PPARa^"7" cells, but about 4-5 fold increase in WT and PPAR ^"7" cells (Fig. 3E and 3F). The data from the Western blot was confirmed by immunofluorescence where a similar effect of the drugs on WT and KO astrocytes was observed, i.e. little or no increase in TPP1 in PPARa^"7" cells, compared to WT and PPAR ^"7" cells (Fig. 31, 3J & 3K).

[0075] Furthermore, the presence of active TPP1 enzyme was also confirmed by

measurement of TPP 1 activity in WT and KO cell types. The enzymatic activity was drastically increased in WT and ΡΡΑΡνβ^{" "} cells upon treatment with gemfibrozil or fenofibrate (Fig. 3L and 3N), while PPARa^{" "} cell extracts showed no significant increase in TPPl enzymatic activity (Fig. 3M). Collectively, these data indicate that PPAR , but neither ΡΡΑΡνβ nor PPARy, is involved in the gemfibrozil- and fenofibrate-mediated upregulation of TPP 1.

Example 7

TPPl is upregulated by fibrate drugs in vivo in the CNS of

WT and PPARB^{" "}, but not in PPAR ^{" "}. mice

[0076] Once we confirmed the involvement of PPARa in the fibrate mediated upregulation of TPPl protein, we further checked whether the same results could be replicated in in vivo settings. WT, PPARa^{" "} and PPARB^"7" mice from same background were treated orally for 21 days with 7.5mg/kg body wt/day gemfibrozil dissolved in 0.1% methylcellulose, which was also used as vehicle. At the end of the treatment, the mice were killed and different regions of their brain, viz. Substantia nigra pars compacta, cortex, hippocampus, and dentate gyrus were sectioned, and immunofluorescence was performed for the presence of TPP 1. Gemfibrozil treatment markedly increased the level of TPPl both in GFAP -positive cortical astrocytes (Fig. 4 Al , A2, A3 & A4) and NeuN-positive cortical neurons (Fig. 4 Bl , B2, B3 & B4) in WT and PPARB^{" "}, but not PPARa^{" "}, mice. Similarly, gemfibrozil treatment also increased the level of TPP l in GFAP- positive astrocytes (Fig. 4 CI , C2, C3 & C4) and tyrosine hydroxylase-positive neurons (Fig. 4 Dl , D2, D3 & D4) in the substantia nigra of WT and PPARB^{" "}, but not PPARa^{" "}, mice.

Gemfibrozil also increased TPPl mostly in the non-neuronal cells in the dentate gyrus (Fig. 5 A, 5B, 5C & 5D) and CA1 region of the hippocampus (Fig. 5E, 5F, 5G & 5H) of WT and PPARB^{" "}, but not PPARa^"7", mice. These data clearly indicate that gemfibrozil increases TPPl in vivo in the CNS via PPARa.

Example 8

Upregulation of TPP 1 by fibrate drugs involve both PPARa and RXRq

[0077] Next we investigated the mechanism of this upregulation. We observed that Cln2 gene promoter lacked PPAR binding site but contained a RXR binding site, instead. Due to the facts that RXRa is abundant in the brain and astrocytes (32,33) and that PPARa and RXRa form heterodimer, we thought the mechanism of upregulation of TPP 1 may involve co-operative action of both PPARa and RXRa and not PPARa alone. To verify our hypothesis, we performed an array of experiments. First, we checked whether activating RXR by all-trans retinoic acid (RA), a known activator of RXRs, caused any change in the mR A or protein levels of TPP1. Interestingly, quantitative real time PCR data showed that even RA alone enhanced the mRNA levels of Cln2 (Fig. 6A). RA at a concentration of 0.5μΜ caused about 3.5 fold increase in Cln2 mRNA levels which is comparable to the effect of ΙΟμΜ gemfibrozil treatment (~4 fold) (Fig. 6 A). Moreover, when cells were treated with low dose of gemfibrozil (ΙΟμΜ) together with RA (at different concentrations), there was a profound increase in the Cln2 levels with optimum concentration of the combination being at ΙΟμΜ gemfibrozil and 0.5 μΜ RA (about 12 fold increase) (Fig 6A). These mRNA data was validated by Western blot performed in mouse astrocytes using the similar treatments (Fig. 6B). The densitometry analysis showed similar pattern of increase in the protein levels of TPP1 as observed from the mRNA data (Fig. 6C). In both real-time PCR and immunoblot experiments the increase of TPP 1 expression with the combinatorial treatment (ΙΟμΜ gemfibrozil and 0.5μΜ RA) was found to be statistically significant when compared to either gemfibrozil (ΙΟμΜ) or RA (0.5μΜ) treatment alone. This finding clearly indicates the possible involvement of RXR in the upregulation of the Cln2 gene. Secondly, to confirm the involvement of RXRa, we knocked down RXRa in astrocytes by RXRa siRNA followed by treatment with gemfibrozil and RA. The siRNA was found to specifically knockdown RXRa, but neither RXR nor RXRy (Fig. 6D). The effect of gemfibrozil and RA was found to be abrogated in the absence of RXRa, as observed from the quantitative real time PCR data (Fig. 6E). There was almost 8-10 fold increase in the Cln2 level in both untransfected cells as well as cells transfected with scrambled siRNA, whereas cells with RXRa knockdown were almost unresponsive to the treatment of gemfibrozil or RA alone as well as the combination (Fig. 6E). Similar results were obtained with the protein analysis. The denistometric analysis for the TPP 1 Western blot showed almost no enhancement of TPP 1 levels in RXRa siRNA transfected cells (Fig. 6F & 6G). Finally, to validate our hypothesis that both PPARa and RXRa are involved in the upregulation process, we checked whether activation of RXRa alone (in the absence of PPARa) can induce the expression of Cln2. Mouse astrocytes from wild type (WT), PPARa and PPAR ^"' ^" mice were treated with RA (0.5μΜ) and the combination of gemfibrozil (ΙΟμΜ) and RA (0.5 μΜ) followed by immunoblot analysis for TPP1. It was observed that neither RA alone nor the combination could induce TPP1 in PPARa^{" "} cells, whereas WT and ΡΡΑΡνβ^{" "} cells were responsive to the treatment (about 5-6 fold induction of TPP1) (Fig. 6H & 61). These data suggest that either PPARa or RXRa alone is not sufficient for the upregulation of TPP1.

Example 9

Fibrate drugs upregulate TPP 1 via activation of PPARq/RXRq heterodimer

[0078] After confirming the involvement of both PPARa and RXRa, we were interested to find out the actual role of the two factors. First, we examined whether there was any actual physical interaction between PPARa and RXRa. Mouse astrocytes were treated with gemfibrozil and RA separately as well as in combination and the nuclear extract was subjected to Co-IP for both PPARa and RXR. Immunoprecipitation with PPARa Ab showed increased presence of RXR in the immunoblot for the treated samples compared to control (Fig. 7A-i). Similarly, increased abundance of PPARa was also observed when the nuclear extracts were

immunoprecipitated with RXR Ab (Fig. 7A-ii). These results demonstrate the presence of PPARa/RXRa heterodimer in the nucleus of cells stimulated with gemfibrozil and RA. These results are specific as we did not find any bands with IgG (Fig. 7A-iii). Levels of PPARa and RXRa and Histone3 (H3) have been shown as loading controls (Fig. 7A-iv). Next, we performed ChIP studies to show the recruitment of the PPARa and RXRa on the RXR binding site on the Cln2 gene (Fig. 7B). Chromatin fragments from cells treated with gemfibrozil and RA were immunoprecipitated with both PPARa Ab and RXRa Ab and the DNA obtained was amplified by PCR with primers spanning the RXR binding site on the Cln2 gene promoter. In both cases, we were able to amplify 200 bp fragments flanking the RXR binding site (Fig. 7C). In contrast, no amplification product was observed in any of the immunoprecipitates obtained with control IgG (Fig. 7C), suggesting the specificity of these interactions. These results suggest that gemfibrozil and RA are capable of recruiting both PPARa and RXRa to the RXR binding site of the Cln2 gene promoter (Fig 7C).

Example 10

Summary of Examples 2-9

[0079] The classical late infantile neuronal ceroid lipofuscinosis (LINCLs) is an autosomal recessive disease, where the defective gene is Cln2, encoding tripeptidyl peptidase I (TPP1). At the molecular level LI CL is caused by accumulation of autofluorescent storage materials in neurons and other cell types. Currently there is no established treatment for this fatal disease. This study reveals a novel use of gemfibrozil and fenofibrate, FDA-approved lipid-lowering drugs, in upregulating TPP1 in brain cells. Both gemfibrozil and fenofibrate upregulated mR A, protein and enzymatic activity of TPP 1 in primary mouse neurons and astrocytes as well as human astrocytes and neuronal cells. Since gemfibrozil and fenofibrate are known to activate peroxisome proliferator-activated receptor-a (PPARa), the role of PPARa in gemfibrozil- and fenofibrate-mediated upregulation of TPP 1 was investigated revealing that both drugs upregulated TPP 1 mRNA, protein and enzymatic activity both in vitro and in vivo in wild type (WT) and PPARβ^"/", but not PPARa-/-, mice. In an attempt to delineate the mechanism of TPP 1 upregulation, it was found that the effects of the fibrate drugs were abrogated in the absence of retinoid X receptor-a (RXRa), a molecule known to form heterodimer with PPARa.

Accordingly, all-trans retinoic acid, alone or together with gemfibrozil, upregulated TPP 1. Co- immunoprecipitation and ChIP studies revealed the formation of PPARa-RXRa heterodimer and binding of the heterodimer to RXR binding site on the Cln2 promoter. Together, this study demonstrates a unique mechanism for the upregulation of TPP 1 by fibrate drugs via PPARa: RXRa pathway.

Example 1 1

Discussion of Examples 2-9

[0080] The NCL family of disease can be considered to be one of the most important hereditary neurodegenerative lysosomal storage diseases (LSD) in children (34). Mutations in the Cln2 gene result in deficiency or loss of function of the TPP1 enzyme (9,30,35). There have been reports of over 68 missense mutations in the Cln2 gene including 35 single amino acid substitutions. Studies with 14 different naturally occurring disease associated mutations showed alteration of lysosomal transport, increased half-life of the proenzyme and improper folding, resulting to loss of function of the enzyme (9). Currently there is no established drug mediated therapy for LINCL, a classic subtype of the NCLs. Studies using adeno-associated virus (AAV) and other viral vectors expressing recombinant TPP 1 demonstrate widespread expression of TPP 1 and treatment of Cln2- targeted mice with these recombinant vectors shows slowing of disease associated pathology and increase in survival in mutant mice (36-38). However, levels of TPPI activity achievable by AAV-mediated gene therapy can vary and depend on various critical parameters and there is considerable doubt whether similar effects can be achieved in humans (36,39)

[0081] On the other hand, restoration of activity at even low levels could prove helpful for most LSDs, where restoration of even <10% of normal activity may have therapeutic benefits (40). Studies with hypomorphs of Cln2 mutant mice, expressing different levels of TPPI enzyme indicate that even 3% of normal TPPI activity is capable of delaying the onset of the disease and 6% of the normal activity attenuates the disease and increases the lifespan of mice (40). Also two specific variants of mutated TPP 1 were responsive to molecular chaperone treatment, indicating that folding improvement strategies can be used to restore the enzymatic activity (9). Recent studies suggest that some misfolded variants or misprocessed proteins may also be rescued by treatment in permissive temperatures under suitable condition (9). There have also been reports of some mutations in TPPI (Arg447His), which apparently may not have any pathogenic effect (31). Moreover, a sensitive enzyme activity assay detected residual levels of TPPI activity in various biological samples from patients who were confirmed to have LINCL by genetic analysis (30). This study also showed the presence of enzyme activity in various animals having NCL- like neurodegenerative symptoms rendering them unsuitable for being a model for classical LINCL (30). Furthermore, using highly sensitive capillary electrophoresis technique, Vigilio et al, reported that lymphocytes from patients affected with LINCL exhibited TPP 1 activity, although at low levels (in a range between 0.1 and 0.8 mU/mg) (8). These findings about the presence of residual enzymatic activity in LINCL patients are very interesting as it indicate the presence of at least a few copies of the functional gene in the system. Therefore, identifying specific drugs and understanding the mechanisms by which these drugs can upregulate the endogenous normal copies of the gene may be a critical step for LINCL therapy.

[0082] Gemfibrozil, marketed as 'Lopid', and fenofibrate, known as 'Tricor', are FDA- approved drugs prescribed for hyperlipidemia (10,12). Here we delineate for the first time that, these drugs are capable of upregulating TPPI in brain cells. This finding was confirmed by both mRNA and protein studies in both mouse and human cells. The increase in protein levels was throughout the brain as neurons isolated from different brain regions of mouse showed increased TPP 1 expression upon treatment with gemfibrozil. In case of LINCL, the presence of functionally active TPP 1 enzyme is critical for therapy, as we have to rely on the upregulation of residual enzyme activity in patients. The TPP1 activity assay, performed in different cell types, clearly showed that there was significant increase in the activity of the enzyme which is a result of increased levels of the protein. Considering the possibility of treatment by upregulation of the endogenous Cln2 gene, this finding could be of importance in the therapy of LI CL.

[0083] Over the last few years, a number of studies emphasized the role of PPARs in different regulatory and modulatory pathways. It is also well known that PPAR is activated by polyunsaturated fatty acids and oxidized derivatives and by lipid-modifying drugs of the fibrate family, including feno fibrate and gemfibrozil (41 ,42). PPARa is present in the cytoplasm as an inactive complex with heat-shock protein 90 (HSP-90) and hepatitis virus B-X-associated protein-2 (XAP-2), which act as an inhibitor of PPARa. Fibrate drugs replace the HSP90 repressor complex and help to rescue the transcriptional activity of PPARa (15). Therefore, we investigated the role of the PPAR group of receptors in this phenomenon. We examined all three PPARs, viz PPARa, PPAR , PPARy for their involvement in upregulation of TPP1. These studies clearly indicate the involvement of PPARa, but not PPAR and PPARy, in this process. In astrocytes from WT and PPARa^{" "} and PPAR ^{" "} mice, both the TPP 1 mR A and protein analysis showed the involvement of only PPARa. Involvement of PPARy was ruled out as studies using known antagonist of PPARy revealed no effect. TPP 1 enzyme activity in the cell extracts was also increased in WT and PPAR ^{" "}, but not PPARa^"7", cells. The in vitro studies were further validated by in vivo studies, where we used the knockout mice for PPARa and PPAR . Our in vivo results also supported the cell culture data.

[0084] In order to delineate the mechanism of fibrate drug-mediated upregulation of TPP 1 , we analyzed the promoter region of the Cln2 gene. Surprisingly, no PPAR binding site was found in the mouse Cln2 promoter, but further analysis of the promoter revealed a RXR binding site. It is well known that in order to bind to DNA and activate transcription, PPAR requires the formation of heterodimer with the retinoid X receptor (RXR) (43). Together the PPAR RXR heterodimer regulates the transcription of genes for which products are involved in lipid homeostasis, cell growth, and differentiation (44,45). This led us to think whether the pathway of TPP 1 upregulation requires a co-operative effect of both PPAR and RXR. It was observed that the activation of RXR by low doses of RA alone (0.5μΜ) was capable of upregulating TPP1 to a comparable level of that of gemfibrozil (10μΜ). Also, when cells were treated with both gemfibrozil and RA together, they co- operative ly enhanced the expression of TPP 1 by almost more than 3 fold compared to the levels achieved by either gemfibrozil or RA alone, which implies that a combinatorial therapy could be more useful than using the compounds separately for treatment. Furthermore, the effect of both gemfibrozil and RA were abrogated in the absence of either RXR or PPAR . The Co-IP studies performed with the nuclear extracts on astrocytes stimulated with gemfibrozil and RA demonstrated physical interaction between PPARa and RXR. These data clearly suggest that the treatment with gemfibrozil and RA activates both PPARa and RXRa which forms heterodimer in the nucleus. The ChIP data indicated the recruitment of the PPARa and RXRa on the RXR binding site of the Cln2 promoter, hence validating our hypothesis. Collectively, these data outlines a unique mechanism where gemfibrozil, a known activator of PPARa, and RA, an agonist of RXRa, together can upregulate TPP 1 in brain cells via PPARa/RXRa heterodimer.

[0085] Gemfibrozil and other fibrate drugs are known to reduce superoxide, lipid

peroxidation products. It also strengthens the cellular defense by stimulating the activity of antioxidant proteins such as paraxonase and is associated with free radical scavenging ability as well as metal ion chelation. Therefore, apart from its lipid lowering effects, these drugs also have antiinflammatory, immunomodulatory and anti-oxidative properties(14,27,46-48). In the NCL cases, predominantly in LINCL, different brain regions have been shown to be immunoreactive for 4- hydroxynonenal (4-FfNE) or 8-hydroxydeoxyguanosine (8-OHdG), popular markers for evaluation of oxidative stress which may be caused due to accumulation of lipofuscines and elevated cytokine response (1). Therefore, treatment with these drugs will not only lead to the upregulation of endogenous normal TPP 1 leading to clearance of lipofuscines but also can be beneficial for the peripheral immune system by downregulating the inflammatory pathways generated due to accumulation of lipofuscines.

[0086] In summary, we have delineated that gemfibrozil and fenofibrate, FDA-approved lipid-lowering drugs, upregulate TPP1 in cultured mouse and human brain cells and in vivo in mouse brain via PPARa/RXRa pathway. Although the in vitro situation of mouse and human brain cells in culture and its treatment with gemfibrozil and fenofibrate may not truly resemble the in vivo situation of the CNS of patients with late infantile neuronal ceroid lipofuscinosis (LINCLs), our results clearly identify these two drugs as possible therapeutic agents for LINCL that can be immediately taken to clinical trials for testing. [0087] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents.

[0088] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.

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Claims

CLAIMS What is claimed is:

1. A method for treatment of a neurodegenerative disease, comprising administering to a subject in need of such treatment a composition comprising a therapeutically effective amount of an agent that mediates upregulation of TPP 1.

2. The method of claim 1 , wherein the neurodegenerative disease is selected from the group consisting of neuronal ceroid lipofuscinosis, Alzheimer's disease, Huntington's disease, Amyotrophic lateral sclerosis (ALS), Parkinson's disease, including Parkinson's plus diseases such as multiple system atrophy (MSA), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and dementia with Lewy bodies (DLB).

3. The method of claim 1 , wherein the neurodegenerative disease is a lysosomal storage disorder.

4. The method of claim 3 , wherein the lysosomal storage disorder is selected from the group consisting of Tay-Sach's disease, Fabry disease, Niemann-Pick disease, Gaucher disease, Hunter Syndrome, Alpha-mannosidosis, Aspartylglucosaminuria, Cholesteryl ester storage disease, Chronic Hexosaminidase A Deficiency, Cystinosis, Danon disease, Farber disease, Fucosidosis, and Galactosialidosis.

5. The method of claim 2, wherein the neurodegenerative disease is neuronal ceroid

lipofuscinosis.

6. The method of any of claims 1-5, wherein the agent is a lipid- lowering drug.

7. The method of claim 6, wherein the lipid-lowering drug is a fibrate.

8. The method of claim 7, wherein the fibrate is gemfibrozil or fenofibrate.

9. The method of claim 5, wherein the neuronal ceroid lipofuscinosis is late infantile

neuronal ceroid lipofuscinosis or Batten disease.

10. The method of any of claims 1-9, wherein TPP1 is upregulated by increasing TPP1

mRNA levels, increasing TPP1 protein levels, increasing TPP1 enzymatic activity, or activating a PPAR -RXR heterodimer.

11. The method of any of claims 1-10, wherein the composition further comprises a therapeutically effective amount of all-trans retinoic acid.

12. The method of claim 1 1, wherein the agent is a fibrate.

13. The method of claim 12, wherein administering all-trans retinoic acid and the fibrate provides a greater therapeutic effect in the subject than administration of all-trans retinoic acid or the fibrate alone.

14. A method for treatment of a neurodegenerative disease, comprising administering to a subject in need of such treatment a composition comprising a therapeutically effective amount of an agent, wherein the agent restores TPP 1 activity.

15. The method of claim 14, wherein the neurodegenerative disease is selected from the group consisting of neuronal ceroid lipofuscinosis, Alzheimer's disease, Huntington's disease, Amyotrophic lateral sclerosis (ALS), Parkinson's disease, including Parkinson's plus diseases such as multiple system atrophy (MSA), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and dementia with Lewy bodies (DLB).

16. The method of claim 14, wherein the neurodegenerative disease is neuronal ceroid

lipofuscinosis.

17. The method of claim 14, wherein the agent is a fibrate

18. The method of claim 17, wherein the fibrate is gemfibrozil or feno fibrate.

19. The method of claim 17, wherein the therapeutically effective amount of the fibrate is lower when the fibrate is administered in combination with all-trans retinoic acid.

20. A method for treatment of neurodegenerative disease, comprising administering to a subject in need of such treatment a composition comprising a therapeutically effective amount of an agent that mediates upregulation of a gene selected from the group of Clnl , Cln2, Cln3, and any combination thereof.

21. The method of claim 20, wherein the neurodegenerative disease is selected from the group consisting of neuronal ceroid lipofuscinosis, Alzheimer's disease, Huntington's disease, Amyotrophic lateral sclerosis (ALS), Parkinson's disease, including Parkinson's plus diseases such as multiple system atrophy (MSA), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and dementia with Lewy bodies (DLB).

22. The method of claim 21 , wherein the neurodegenerative disease is neuronal ceroid

lipofuscinosis.

23. The method of claim 20, wherein the agent is a fibrate.

24. The method of claim 23, wherein the fibrate is gemfibrozil or fenofibrate.