WO2012170657A1 - Targeting gsk-3beta for the treatment of parkinson's disease - Google Patents

Targeting gsk-3beta for the treatment of parkinson's disease Download PDF

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WO2012170657A1
WO2012170657A1 PCT/US2012/041292 US2012041292W WO2012170657A1 WO 2012170657 A1 WO2012170657 A1 WO 2012170657A1 US 2012041292 W US2012041292 W US 2012041292W WO 2012170657 A1 WO2012170657 A1 WO 2012170657A1
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lithium
disease
mice
parkinson
gsk
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Anita Sidhu
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Georgetown University
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/433—Thidiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A61K38/063—Glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • a method for treating Parkinson's Disease involves the use of Lithium (Li) to inhibit GSK-3P, and simultaneously stimulate autophagy to clear protein aggregates of a-synuclein (a-Syn) and hyperphosphorylated Tau.
  • Li Lithium
  • a-Syn a-synuclein
  • Parkinson's Disease is the second-most common
  • PMCID PMC2922478; Haggerty T, Credle J, Rodriguez O, Wills J, Duka V, Oaks AW, Masliah E, Sidhu A. Hyperphosphorylated Tau in an a- synuclein overexpressing transgenic model of Parkinson's disease. 33(9) Eur. J. Neurosci. 1598-610 (201 1 May); Wills J, Credle J, Haggerty T, Lee J- H, Oaks AW, Sidhu A. Tauopathic changes in the striatum of A53T a- synuclein mutant mouse model of Parkinson's disease.
  • Treatment of PD primarily consists of l-dopa therapy to replace dopamine [Tomlinson CL, Stowe R, Patel S, Rick C, Gray R, Clarke CE. Systematic review of levodopa dose equivalency reporting in Parkinson's disease.Mov Disord. 2010 Nov 15;25(15):2649-53. Review; Perez-Lloret S, Rascol O. Dopamine receptor agonists for the treatment of early or advanced Parkinson's disease. CNS Drugs. 2010 Nov 1 ;24(1 1):941 - 68. Review.].
  • Lithium is already FDA-approved for treatment of bipolar disorder, and it is safe for long-term use in humans [Kwok JB, Hallupp M, Loy CT, Chan DK, Woo J, Mellick GD, Buchanan DD, Silburn PA, Halliday GM, Schofield PR. GSK3B polymorphisms alter transcription and splicing in Parkinson's disease. Ann Neurol. 2005 Dec;58(6):829-39]. The method described herein demonstrates that lithium treats and prevents PD-like pathology in vivo, and can be immediately used in clinical trials.
  • Lithium is inexpensive and acts in a manner different from l-dopa, and will not have the same side-effects as L-DOPA, such as dyskinesias and desensitization.
  • the present invention envisions the development of an entirely new family of novel, non-dopaminergic therapeutic tools to treat PD.
  • Lithium is useful in the treatment of Alzheimer's disease, multiple system atrophy and Lewy body disease with dementia, which are all characterized by high levels of a-Syn, p-Tau and activated GSK-3p.
  • the discovery of the signaling cascade comprising a-Syn/GSK-3p/p- Tau in the genesis of PD is a highly innovative and important finding.
  • a-Syn is mandatory for observing tauopathy— in a-Syn-/- mice or cells lacking a-Syn, tauopathy and GSK-3 activation is not observed upon treatment with the potent neurotoxin MPTP/MPP+; a-Syn recruits and activates GSK-3p through its hyperphosphorylation at Tyr216; GSK-3 is the primary kinase involved in hyperphosphorylation of Tau in PD; protein phosphatase levels and activity are unchanged in postmortem PD striata and in mice; blockade of GSK-3P activation with lithium or with selective inhibitors (TDZD-8 and methylmaleimide compounds) prevents synucleinopathy and tauopathy in cells and neurons [Alan P.
  • PD Parkinson's disease
  • Drugs used for treatment of PD are not curative, and moreover, are of limited long-term use due to development of resistance and/or adverse side-effects in patients. While the genesis of PD is not known, a-synuclein (a-Syn) overexpression through gene duplication and multiplication is causal in sporadic PD.
  • a-Syn a-synuclein
  • p-Tau hyperphosphorylated Tau
  • SNCA a-Syn gene
  • MAT Tau gene
  • Li overexpressing a-Syn
  • Li is approved by the FDA for treatment of bipolar disorders.
  • the off-label use of Li to treat PD is demonstrated by data showing that treatment of a- Syn overexpressing Tg mice with low levels of Li reverses synucleinopathy and tauopathy of dopaminergic neurons enervating the striatum (see
  • Li suppresses the overexpression of a-Syn and reduces aggregates of both a-Syn and p-Tau to levels seen in non-Tg wild type mice. This is the first evidence of a small molecule directly preventing the overexpression and accumulation of a-Syn.
  • Li The actions of Li are two-fold: Inhibition of the Tau kinase, GSK-3p, whose activation is entirely dependent on a-Syn, and stimulation of autophagy, a pathway central for clearing aggregates of a-Syn and p-Tau.
  • Example 1 extensive studies in vivo using different doses of Li in aged mice are performed to estimate efficacy of treatment in reversing PD- like pathology. The lowest dose of Li that can be used to reverse both synucleinopathy and tauopathy is identified.
  • Example 2 pre-symptomatic mice are treated with different doses of Li to determine if long-term use of low levels of Li will prevent onset of PD-like pathology. This allows the development of the first therapy for the prevention of sporadic PD, and is useful in individuals who carry multiplications of SNCA and are at high risk for developing PD.
  • Detailed analyses measuring the efficacy of each treatment is performed, including biochemical, behavioral and immunohistochemical analyses. Since Li is already FDA approved, and its toxicity and side-effects are well documented in both humans and mice, the drug can be readily used in other PD animal models, and ultimately, in clinical trials in humans.
  • Figure 1 shows coexpression of a-Syn/pTau in a-Syn overexpressing mice (OEM) mice and reinforces the choice of PDGF-a-Syn as a model system to analyze lithium treatments.
  • FIG 2 details blood plasma levels of lithium in wild-type (WT) and transgenic (Tg) mice following the development and execution of the lithium treatment paradigm.
  • Figure 3 quantifies the treatment of a-Syn overexpressing mice (OEM) following lithium treatment and shows a reversal of motor impairment seen in PDGF-a-Syn transgenic mice.
  • OEM a-Syn overexpressing mice
  • Figure 4 illustrates that lithium reduces high levels of a-Syn and activated p-GSK-3p in striatum of PDGF-a-Syn overexpressing mice and has no effect on wild-type mice fed lithium.
  • Sal saline
  • X lithium
  • Tg transgenic
  • WT wild-type
  • +Cpd.X lithium.
  • FIG. 5 illustrates that lithium reverses tauopathy in striatum of PDGF-a-Syn transgenic mice.
  • Sal saline
  • X lithium
  • Tg transgenic
  • WT wild-type
  • +Cpd.X lithium.
  • a novel method for treating Parkinson's Disease involves the administration of therapeutically effective amounts of Lithium (Li) to inhibit GSK-3 and simultaneously stimulate autophagy to clear protein aggregates of a-synuclein (a-Syn) and hyperphosphorylated Tau.
  • Li Lithium
  • a-Syn a-synuclein
  • Genomics Consortium Nails MA, Plagnol V, Hernandez DG, Sharma M, Sheerin UM, Saad M, Simon-Sanchez J, Schulte C, Lesage S,
  • Alpha-synuclein locus triplication causes Parkinson's disease. Science. 2003; 302(5646): 841. Crews L, Tsigelny I, Hashimoto M, Masliah E. Role of Synucleins in Alzheimer's Disease. Neurotox Res. 2009; 16(3): 306-317].
  • a-Syn is misfolded into oligomers, aggregates and accumulates into neuronal inclusion bodies, Lewy bodies (LBs), that are the pathological hallmarks of PD and other synucleinopathies [Forno LS. Neuropathology of Parkinson's disease. J. Neuropathol Exp Neurol.
  • PMCID PMC2922478; Haggerty T, Credle J, Rodriguez O, Wills J, Duka V, Oaks AW, Masliah E, Sidhu A. Hyperphosphorylated Tau in an a- synuclein overexpressing transgenic model of Parkinson's disease. 33(9) Eur. J. Neurosci. 1598-610 (201 1 May); Wills J, Credle J, Haggerty T, Lee J- H, Oaks AW, Sidhu A. Tauopathic changes in the striatum of A53T a- synuclein mutant mouse model of Parkinson's disease.
  • tauopathy in dopaminergic neurons in a manner strictly dependent on a-Syn has been shown.
  • a-Syn also recruits and activates the Tau kinase, ⁇ -3 ⁇ (p-GSK- 3 ⁇ , hyperphosphorylated at Tyr216), to induce the hyperphosphorylation of Tau (p-Tau) at multiple sites, leading to a toxic gain of function and microtubule destabilization, with eventual cell death.
  • GSK-3P can be targeted with lithium, providing for a novel therapeutic approach in the treatment of PD.
  • lithium is FDA-approved and has been long used in the treatment of bipolar disorders. Moreover, its use is associated with very few side effects, with no psychotropic effects in humans; it is not a sedative, depressant, or euphoriant [Marcus WL. Lithium: a review of its pharmacokinetics, health effects, and toxicology. J Environ Pathol Toxicol Oncol. 1994 -13(2):73-9].
  • Inhibitors of GSK-3 such as lithium, are a novel group of drugs for the treatment of PD.
  • tauopathies is used to refer to a class of neurodegenerative diseases associated with the pathological aggregation of tau protein in the human brain.
  • synucleinopathies is used to name a group of neurodegenerative disorders characterized by fibrillary aggregates of alpha- synuclein protein in the cytoplasm of selective populations of neurons and glia. These disorders include Parkinson's disease (PD), dementia with Lewy bodies (DLB), pure autonomic failure (PAF), and multiple system atrophy (MSA).
  • PD Parkinson's disease
  • DLB dementia with Lewy bodies
  • PAF pure autonomic failure
  • MSA multiple system atrophy
  • Parkinsonism is the predominant symptom of PD, but it can be indistinguishable from the parkinsonism of DLB and MSA.
  • Autonomic dysfunction which is an isolated finding in PAF, may be present in PD and DLB, but is usually more prominent and appears earlier in MSA.
  • DLB could be the same disease as PD but with widespread cortical pathological states, leading to dementia, fluctuating cognition, and the characteristic visual hallucinations.
  • the deposition of aggregates of synuclein in neurons and glia suggests that a common pathogenic mechanism may exist for these disorders.
  • a method of treating a mammal suffering from a malady associated with synucleinopathy and/or tauopathy involves administering to said mammal a therapeutically effective amount of lithium and/or a lithium salt.
  • the malady is Parkinson's Disease (PD)
  • the method involves the administration to a mammal suffering from a malady associated with aggregates of both a-Syn and p-Tau, and involves the administration of an aggregate-reducing or - eliminating amount of lithium.
  • the method involves the prevention and/or treatment of a mammal suffering from overproduction of GSK-3P, or from the activation of p-GSK-3p.
  • the lithium may be co-administered, complexed or covalently linked with other carriers or active agents.
  • the term “lithium” includes elemental lithium, lithium ion, lithium salts, and lithium complexed with, or covalently bonded to, other carriers or active agents.
  • lithium is co-administered (or complexed or covalently linked) with TDZD-8 or with methylmaleimide.
  • the mammal is a human, murine, porcine, bovine, ovine, equine, canine, feline, or gibbon.
  • the mammal is human, the malady is Parkinson's Disease, and the lithium is administered as a lithium salt (e.g., lithium carbonate, lithium citrate, lithium sulfonates, lithium orotate).
  • a lithium salt e.g., lithium carbonate, lithium citrate, lithium sulfonates, lithium orotate.
  • Other lithium salts are known, and may also be used in these methods.
  • the lithium-containing active agent may be administered orally, parenterally (e.g., intravenous, intramuscular, etc.), subcutaneously, transdermally, or by sublingual, intranasal, or suppository, or by any means commonly known and accepted in the art for the administration of lithium or lithium salts.
  • the lithium agent is administered orally.
  • the therapeutically effective amount for treating, reversing, or preventing these maladies can be readily determined by those of ordinary skill in the art, and will vary based upon the patient and the seventy of his or her condition. The severity of the patient's condition will be affected by a constellation of factors including genetic predisposition, environmental exposure, and other factors. One skilled in the art will be able to assess the therapeutic efficacy of a particular dosage regimen based upon routine screening and diagnostic testing.
  • the lithium agent can be administered orally, with blood levels reaching therapeutic blood levels in the patient of about 0.01 - 3 mEq/L.
  • lithium is administered so as to achieve therapeutically effective blood levels of about 0.05 - 2.0 mEq/L.
  • lithium is administered to achieve patient blood levels of about 0.1-1.5 mEq/L.
  • the method is employed to administer lithium to patient blood levels of about 0.6 to about 0.8 mEq/L.
  • Patient treatment according to these methods can be achieved by repeated and routine administration of lithium.
  • the lithium may be administered daily for periods of six months or more.
  • Treatment regimens will generally be about one month or more.
  • lithium when administering lithium orally according to these methods, lithium can be combined with food for ease of administration. Dosage regimens of about 1-20 g lithium per kg food can be used. In other embodiments, about
  • lithium per kg food can be used. And in still other embodiments, about 3-6 g lithium per kg food can be used.
  • mice overexpressing human a-Syn were obtained [Masliah E, Rockenstein E, Veinbergs I, Mallory M, Hashimoto M, Takeda A, Sagara Y, Sisk A, Mucke L. Dopaminergic loss and inclusion body formation in alpha- synuclein mice: implications for neurodegenerative disorders. Science. 2000 Feb 18;287(5456): 1265-9] and bred. These mice have been used in several studies and both synucleinopathy and tauopathy are well- characterized in these animals [Haggerty T, Credle J, Rodriguez O, Wills J, Duka V, Oaks AW, Masliah E, Sidhu A.
  • Hyperphosphorylated Tau in an a- synuclein overexpressing transgenic model of Parkinson's disease 33(9) Eur. J. Neurosci. 1598-610 (2011 May); Kaul T, Credle J, Haggerty T, Oaks A, Masliah E, Sidhu A (20 1 ) Region-specific tauopathic changes in brain of the alpha-synuclein overexpressing mouse model of Parkinson's disease. 12 BMC Neurosci. 79 (201 1 Aug 3)]. Accumulation of a-Syn and p-Tau develops in these mice at >8 months of age; younger mice do not show such pathology.
  • mice From IHC, it is shown that these mice have large intraneuronal aggregates of a-Syn and p-Tau in striatum, which resemble Lewy bodies, with proteins co-localizing with one another (Figure 1) and with p-GSK-3 [Haggerty T, Credle J, Rodriguez O, Wills J, Duka V, Oaks AW, Masliah E, Sidhu A. Hyperphosphorylated Tau in an a-synuclein overexpressing transgenic model of Parkinson's disease. 33(9) Eur. J. Neurosci. 1598-610 (201 1 May)]. Moreover, it has been shown that activation of p-GSK-3 (phosphorylation at Tyr216) occurs in these mice, resulting in
  • a-Syn overexpressing mice also display abnormal motor behavior, when compared to WT mice, as indexed by reduced latency to fall and latency to passive rotation (see Figure 3).
  • lithium may act to not only inhibit GSK-3P activation but also to remove existing aggregates of a-Syn and p-Tau through increase in proteasomal activity.
  • lithium is also a known activator of autophagy [Sarkar S, Rubinsztein DC. Inositol and IP3 levels regulate autophagy: biology and therapeutic speculations. Autophagy. 2006 Apr- Jun;2(2):132-4. Epub 2006 Apr 6. Review; Pasquali L, Longone P, Isidoro C, Ruggieri S, Paparelli A, Fornai F. Autophagy, lithium, and amyotrophic lateral sclerosis. Muscle Nerve. 2009 Aug;40(2): 173-94.
  • Example 1 To test whether long-term treatment of aged Tg mice, who have both synucleinopathy and tauopathy, with lithium will reverse PD- like pathology. PD is most commonly seen in the elderly population. In aged mice (18 months) PDGF-a-Syn is administered at different doses of lithium in chow for 2 months. Improvement in biochemical,
  • GSK-3 is central to the tauopathic process seen in PD, and that its inhibition in neurons and transfected neuronal cells with lithium, TDZD-8 or with methylmaleimide inhibitors of GSK-3 , rescues cells from MPP+-mediated cell death, with abolition of synucleinopathy and tauopathy [Duka T, Sidhu A. (2006) The neurotoxin, MPP+, induces hyperphosphorylation of Tau, in the presence of alpha- Synuclein, in SH-SY5Y neuroblastoma cells. Neurotox Res. 10: 1-10; Alan P. Kozikowski, Irina N. Gaisina, Pavel A.
  • the PDGF-a-Syn mouse is an ideal model for our studies, as it mimics the vast majority of sporadic PD, which is caused by multiplications of the SNCA [Hofer A, Berg D, Asmus F, Niwar M, Ransmayr G, et al (2005). The role of alpha-synuclein in gene multiplications in early-onset Parkinson's disease and dementia with Lewy bodies. J Neural Transm.
  • PD pathology has key features of PD pathology seen in humans: (1 ) elevation of a-Syn to levels similar to those seen in PD; (2) hyperphosphorylation of Tau at sites identical to those seen in PD striatum, namely, at Ser202, Ser262 and Ser396/404; (3) increases in p-Tau to levels similar to those seen in PD; (4) increases in p-GSK-3 to levels similar to those seen in PD [Wills J, Jones J,Haggerty T, Duka V, Joyce JN, Sidhu A (2010). Elevated taupathy and alpha-synuclein pathology in postmortem Parkinson's disease brains with and without dementia. Exp Neurol. 2010 Sep;225(1 ):210-8.
  • mice Treatment of mice. WT and a-Syn Tg mice at age 16 months are used, since this is the age which most closely models the onset of PD in humans. Mice are treated with low, medium and high levels of lithium in chow in order to obtain serum levels of ⁇ 0.2, 0.5 and 0.8 mEq/L.
  • the treatment groups are: WT + Tg mice fed normal chow; WT + Tg mice fed low levels of lithium chow; WT + Tg mice fed medium levels of lithium; WT + Tg mice fed high levels of lithium. There are 12 mice per group. After 2 months of treatments, all mice are sacrificed. Results from lithium-fed mice are compared to Tg and WT mice fed normal chow.
  • mice are induced to grip the wire screen cover from a rodent housing cage and the screen will be inverted and suspended 45 cm above an empty housing cage [Mata IF, Yearout D, Alvarez V, Coto E, de Mena L, Ribacoba R, Lorenzo-Betancor O, Samaranch L, Pastor P, Cervantes S, Infante J, Garcia-Gorostiaga I, Sierra M, Combarros O, Snapinn KW, Edwards KL, Zabetian CP. Replication of MAPT and SNCA, but not PARK16-18, as susceptibility genes for
  • Dopamine metabolites are measured by HPLC, as previously described [Duka T, Sidhu A. (2006) The neurotoxin, MPP+, induces hyperphosphorylation of Tau, in the presence of alpha-
  • brains are sectioned in a 1 :2 series at 40 pm and IHC performed for TH and counterstained with a Nissl stain. Counts are made at regular predetermined intervals (x, 140 pm, y 140 pm). Systematic samples of the area occupied by the nuclei are made from a random starting point. An unbiased counting frame of known area (45 pm x 35 pm) is superimposed on the image of the tissue sections, using a 63 times oil objective NA 1 .36.
  • Active p-GSK-3 is phosphorylated at Tyr216, while inactive GSK-3p is phosphorylated at Ser9.
  • levels of p-GSK-3p and GSK-3p-Ser9 are determined by conducting Western blots. Total GSK-3 levels are
  • a-Syn levels are measured in striata of mice after lithium treatments, by Western blots.
  • p-a-Syn levels are also measured.
  • p-Tau levels are measured by Western blots.
  • the epitopes examined are: Ser202, Ser262 and Ser396/404. Assessment of these sites is sufficient to determine if tauopathy is reduced. Comparison of results obtained with the lithium treated Tg and WT groups is made to controls fed normal chow.
  • Example 2 To test the ability of lithium to prevent onset of PD-like pathology and behavioral deficits in pre-symptomatic PDGF-a-Syn
  • PD the most common form of PD.
  • Pre-symptomatic adult mice (6 months of age) are continuously fed very low doses of lithium for 4 months to test the ability of lithium to prevent onset of PD-like pathology. Improvement in biochemical, neuropathological and behavioral correlates is assessed.
  • Gene multiplications of a-Syn are causal in sporadic PD, the most common form of PD. Genotyping can identify individuals carrying SNCA multiplications; as a preventive measure, administration of low levels of lithium is useful in preventing onset of PD. Moreover, other individuals identified at high risk for developing PD, particularly through work-related exposure to environmental toxins and agrichemicals, are administered low levels of lithium as a precautionary measure.
  • the a-Syn overexpressing Tg mice develop synucleinopathy and tauopathy at 8 months of age. Treatment of 6 month old pre-symptomatic mice with low levels of lithium prevents synucleinopathy and tauopathy at a later age in mice. This approach is very useful in prevention strategies aimed at individuals who carry multiplications of the a-Syn gene, and who are at the highest risk of developing PD.
  • mice with lithium Treatment of mice with lithium for 4 months. Mice 6 months of age (12/group) are treated with lithium in their chow as described above, and treatment will be continued for 4 months. As a control, a group of Tg mice will be fed normal chow and allowed to age to 10 months. Another group of WT mice will be fed normal chow and a group of WT mice will be fed lithium chow. The purpose of this study is to determine if long-term treatment of mice will prevent onset of PD-like pathology.

Abstract

A method for treating Parkinson's Disease (PD) uses Lithium (Li) to inhibit GSK-3 beta and simultaneously stimulate autophagy to clear protein aggregates of a-synuclein (a-Syn) and nyperphosphorylated Tau. The method reverses synucleinopathy and tauopathy associated with neurodegenerative diseases, especially Parkinson's disease. Lithium is useful in the treatment of Alzheimer's disease, multiple system atrophy and Lewy body disease with dementia, which are all characterized by high levels of a-Syn, p-Tau and activated GSK-3 beta.

Description

TARGETING GSK-3beta FOR THE TREATMENT OF
PARKINSON'S DISEASE
A method for treating Parkinson's Disease (PD) involves the use of Lithium (Li) to inhibit GSK-3P, and simultaneously stimulate autophagy to clear protein aggregates of a-synuclein (a-Syn) and hyperphosphorylated Tau.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
This invention was made with Government support of Grant No.
R01AGO28108, awarded by the National Institutes of Health. The
Government has certain rights in this invention.
BACKGROUND OF THE INVENTION
Parkinson's Disease (PD) is the second-most common
neurodegenerative disorder, occurring primarily in the elderly. It affects 7-10 million people worldwide and 1 million in the United States alone; the cost of treatment for this disease has been estimated at $25 billion in the United States. With an aging population, the prevalence of PD is expected to double in the U.S. by 2030 and to be even higher worldwide. The known risk factors for PD are: genetics, age and agrichemicals. Several genome wide studies have identified the a-Syn and Tau gene [SNCA and MAPT, respectively] as risk factors in the development of PD [Mata IF, Yearout D, Alvarez V, Coto E, de Mena L, Ribacoba R, Lorenzo-Betancor O,
Samaranch L, Pastor P, Cervantes S, Infante J, Garcia-Gorostiaga I, Sierra M, Combarros O, Snapinn KW, Edwards KL, Zabetian CP. Replication of MAPT and SNCA, but not PARK16- 8, as susceptibility genes for
Parkinson's disease. 26(5) Mov Disord. 819-23. Epub 201 1 Mar 21 ;
International Parkinson Disease Genomics Consortium, Nails MA, Plagnol V, Hernandez DG, Sharma M, Sheerin UM, Saad M, Simon-Sanchez J, Schulte C, Lesage S, Sveinbjornsdottir S, Stefansson K, Martinez M, Hardy J, Heutink P, Brice A, Gasser T, Singleton AB, Wood NW. Imputation of sequence variants for identification of genetic risks for Parkinson's disease: a meta-analysis of genome-wide association studies. Lancet. 201 1 Feb 19;377(9766):641 -9. Epub 2011 Feb 1 ; Edwards TL, Scott WK, Almonte C, Burt A, Powell EH, Beecham GW, Wang L, Zuchner S, Konidari I, Wang G, Singer C, Nahab F, Scott B, Stajich JM, Pericak-Vance M, Haines J, Vance JM, Martin ER. Genome-wide association study confirms SNPs in SNCA and the MAPT region as common risk factors for Parkinson disease. Ann Hum Genet. 2010 Mar;74(2):97-109. Epub 2010 Jan 8; Simon-Sanchez J, Schulte C, Bras JM, Sharma M, Gibbs JR, Berg D, Paisan-Ruiz C, Lichtner P, Scholz SW, Hernandez DG, Kruger R, Federoff M, Klein C, Goate A, Perlmutter J, Bonin M, Nails MA, lllig T, Gieger C, Houlden H, Steffens M, Okun MS, Racette BA, Cookson MR, Foote KD, Fernandez HH, Traynor BJ, Schreiber S, Arepalli S, Zonozi R, Gwinn K, van der Brug M, Lopez G, Chanock SJ, Schatzkin A, Park Y, Hollenbeck A, Gao J, Huang X, Wood NW, Lorenz D, Deuschl G, Chen H, Riess O, Hardy JA, Singleton AB, Gasser T. Genome-wide association study reveals genetic risk underlying Parkinson's disease. Nat Genet. 2009 Dec;41 (12):1308-12. Epub 2009 Nov 15]. Other studies have shown the existence of polymorphisms in GSK-3 genes in PD [Kwok JB, Hallupp M, Loy CT, Chan DK, Woo J, Mellick GD, Buchanan DD, Silburn PA, Halliday GM, Schofield PR. GSK3B
polymorphisms alter transcription and splicing in Parkinson's disease. Ann Neurol. 2005 Dec;58(6):829-39]. PD is identified as a tauopathic disease [Duka T, Sidhu A. (2006) The neurotoxin, MPP+, induces
hyperphosphorylation of Tau, in the presence of alpha-Synuclein, in SH- SY5Y neuroblastoma cells. Neurotox Res. 10:1-10; Alan P. Kozikowski, Irina N. Gaisina, Pavel A. Petukhov, Jayalakshmi Sridhar, LaShaunda T. King, Sylvie Y. Blond, Tetyana Duka, Milan Rusnak, Anita Sidhu (2006). Highly Potent and Specific GSK-3beta Inhibitors That Block Tau Phosphorylation. ChemMedChem. 1 :256-66; Duka T, Duka V, Joyce JN, Sidhu (2009) Alpha- Synuclein contributes to GSK-3beta-catalyzed Tau phosphorylation in Parkinson's disease models. A FASEB J;23(9):2820-30. PMID: 19369384 [PubMed - indexed for MEDLINEJRelated articles PMCID: PMC2796901 ; Wills J, Jones J, Haggerty T, Duka V, Joyce JN, Sidhu A (2010). Elevated taupathy and alpha-synuclein pathology in postmortem Parkinson's disease brains with and without dementia. Exp Neurol. 2010 Sep;225(1 ):210-8. Epub 2010. PMCID: PMC2922478; Haggerty T, Credle J, Rodriguez O, Wills J, Duka V, Oaks AW, Masliah E, Sidhu A. Hyperphosphorylated Tau in an a- synuclein overexpressing transgenic model of Parkinson's disease. 33(9) Eur. J. Neurosci. 1598-610 (201 1 May); Wills J, Credle J, Haggerty T, Lee J- H, Oaks AW, Sidhu A. Tauopathic changes in the striatum of A53T a- synuclein mutant mouse model of Parkinson's disease. 6(3) Plos One, e17953 (201 1 Mar 21 ); Smolinsky C, Wills J, Duka V, Oaks A, Sidhu A. (201 1 ) Taupathic changes in brain of alpha-synuclein A53T mutant mouse model of Parkinson's disease is restricted to the straitum and is dependent on oxidative stress. In Press; Kaul T, Credle J, Haggerty T, Oaks A, Masliah E, Sidhu A (201 1 ) Region-specific tauopathic changes in brain of the alpha- synuclein overexpressing mouse model of Parkinson's disease. 12 BMC Neurosci. 79 (201 1 Aug 3)], through a-Syn-mediated activation of GSK-3P leading to increases in p-Tau, hyperphosphorylated at multiple toxic epitopes.
In recent publications, it is shown that a tight regulation exists between a-Syn, GSK-3p and Tau in the genesis of PD. In cells lacking SNCA or in a-Syn-/-, the parkinsonism-inducing toxin, MPTP, failed to result in activation of GSK-3P and formation of p-Tau [Duka T, Sidhu A. (2006) The neurotoxin, MPP+, induces hyperphosphorylation of Tau, in the presence of alpha-Synuclein, in SH-SY5Y neuroblastoma cells. Neurotox Res. 10:1-10; Alan P. Kozikowski, Irina N. Gaisina, Pavel A. Petukhov, Jayalakshmi Sridhar, LaShaunda T. King, Sylvie Y. Blond, Tetyana Duka, Milan Rusnak, Anita Sidhu (2006). Highly Potent and Specific GSK-3beta Inhibitors That Block Tau Phosphorylation. ChemMedChem. 1 :256-66; Duka T, Duka V, Joyce JN, Sidhu (2009) Alpha-Synuclein contributes to GSK-3beta-catalyzed Tau phosphorylation in Parkinson's disease models, A FASEB J;23(9):2820- 30. PMID: 19369384 [PubMed - indexed for MEDLINE]Related articles PMCID: PMC2796901 ; Wills J, Jones J.Haggerty T, Duka V, Joyce JN, Sidhu A (2010). Elevated taupathy and alpha-synuclein pathology in postmortem Parkinson's disease brains with and without dementia. Exp Neurol. 2010 Sep;225(1):210-8. Epub 2010. PMCID: PMC2922478], indicating that a-Syn triggers these events and is central to generation of p- Tau. It has been shown that a-Syn recruits and binds to GSK-3P, causing its activation [hyperphosphorylation at Tyr216]. Blockade of GSK-3p in either cells or neurons with chemical inhibitors [lithium or TDZD-8, or other highly selective methylmaleimide inhibitors], causes reversal of both tauopathy and synucleinopathy.
There are few therapies available for treating PD and none that can prevent onset of the disease. Treatment of PD primarily consists of l-dopa therapy to replace dopamine [Tomlinson CL, Stowe R, Patel S, Rick C, Gray R, Clarke CE. Systematic review of levodopa dose equivalency reporting in Parkinson's disease.Mov Disord. 2010 Nov 15;25(15):2649-53. Review; Perez-Lloret S, Rascol O. Dopamine receptor agonists for the treatment of early or advanced Parkinson's disease. CNS Drugs. 2010 Nov 1 ;24(1 1):941 - 68. Review.]. However, prolonged use of l-dopa causes insensitivity to the drug and increased dosage results in psychotic side-effects [Tomlinson CL, Stowe R, Patel S, Rick C, Gray R, Clarke CE. Systematic review of levodopa dose equivalency reporting in Parkinson's disease.Mov Disord. 2010 Nov 15;25(15):2649-53. Review; Perez-Lloret S, Rascol O. Dopamine receptor agonists for the treatment of early or advanced Parkinson's disease. CNS Drugs. 2010 Nov 1 ;24(1 1 ):94 -68. Review.]. Stimulation of the globus pallidus is effective in some, but not all, patients [Bronstein JM, Tagliati M, Alterman RL, Lozano AM, Volkmann J, Stefani A, Horak FB, Okun MS,
Foote KD, Krack P, Pahwa R, Henderson JM, Hariz Ml, Bakay RA, Rezai A, Marks WJ Jr, Moro E, Vitek JL, Weaver FM, Gross RE, DeLong MR. Deep brain stimulation for Parkinson disease: an expert consensus and review of key issues. Arch Neurol. 201 1 Feb;68(2):165. Epub 2010 Oct 1 1. Review], and it is also an invasive procedure. There is an urgent need to develop new drugs for PD.
SUMMARY OF THE INVENTION
Research both in vitro and in vivo has shown that blockade of GSK- 3β might lead to a highly effective and novel therapeutic approach [Alan P. Kozikowski, Irina N. Gaisina, Pavel A. Petukhov, Jayalakshmi Sridhar, LaShaunda T. King, Sylvie Y. Blond, Tetyana Duka, Milan Rusnak, Anita Sidhu (2006). Highly Potent and Specific GSK-3beta Inhibitors That Block Tau Phosphorylation. ChemMedChem. 1 :256-66; Duka T, Duka V, Joyce JN, Sidhu (2009) Alpha-Synuclein contributes to GSK-3beta-catalyzed Tau phosphorylation in Parkinson's disease models. A FASEB J;23(9):2820- 30. PMID: 19369384 [PubMed - indexed for MEDLINEJRelated articles PMCID: PMC2796901]. In particular, the use of lithium shows enormous promise. Lithium is already FDA-approved for treatment of bipolar disorder, and it is safe for long-term use in humans [Kwok JB, Hallupp M, Loy CT, Chan DK, Woo J, Mellick GD, Buchanan DD, Silburn PA, Halliday GM, Schofield PR. GSK3B polymorphisms alter transcription and splicing in Parkinson's disease. Ann Neurol. 2005 Dec;58(6):829-39]. The method described herein demonstrates that lithium treats and prevents PD-like pathology in vivo, and can be immediately used in clinical trials. Lithium is inexpensive and acts in a manner different from l-dopa, and will not have the same side-effects as L-DOPA, such as dyskinesias and desensitization. Thus, the present invention envisions the development of an entirely new family of novel, non-dopaminergic therapeutic tools to treat PD. Lithium is useful in the treatment of Alzheimer's disease, multiple system atrophy and Lewy body disease with dementia, which are all characterized by high levels of a-Syn, p-Tau and activated GSK-3p. The discovery of the signaling cascade comprising a-Syn/GSK-3p/p- Tau in the genesis of PD is a highly innovative and important finding. It has been shown that: a-Syn is mandatory for observing tauopathy— in a-Syn-/- mice or cells lacking a-Syn, tauopathy and GSK-3 activation is not observed upon treatment with the potent neurotoxin MPTP/MPP+; a-Syn recruits and activates GSK-3p through its hyperphosphorylation at Tyr216; GSK-3 is the primary kinase involved in hyperphosphorylation of Tau in PD; protein phosphatase levels and activity are unchanged in postmortem PD striata and in mice; blockade of GSK-3P activation with lithium or with selective inhibitors (TDZD-8 and methylmaleimide compounds) prevents synucleinopathy and tauopathy in cells and neurons [Alan P. Kozikowski, Irina N. Gaisina, Pavel A. Petukhov, Jayalakshmi Sridhar, LaShaunda T. King, Sylvie Y. Blond, Tetyana Duka, Milan Rusnak, Anita Sidhu (2006). Highly Potent and Specific GSK-3beta Inhibitors That Block Tau
Phosphorylation. ChemMedChem. 1 :256-66; Duka T, Duka V, Joyce JN, Sidhu (2009) Alpha-Synuclein contributes to GSK-3beta-catalyzed Tau phosphorylation in Parkinson's disease models. A FASEB J;23(9):2820-30. PMID: 19369384 [PubMed - indexed for MEDLINE]Related articles PMCID: PMC2796901]; and blockade of GSK-3p activation is sufficient and necessary to reverse synucleinopathy and tauopathy in primary neurons, in cultured neuronal cells [Alan P. Kozikowski, Irina N. Gaisina, Pavel A.
Petukhov, Jayalakshmi Sridhar, LaShaunda T. King, Sylvie Y. Blond, Tetyana Duka, Milan Rusnak, Anita Sidhu (2006). Highly Potent and Specific GSK-3beta Inhibitors That Block Tau Phosphorylation. ChemMedChem. 1 :256-66; Duka T, Duka V, Joyce JN, Sidhu (2009) Alpha-Synuclein contributes to GSK-3beta-catalyzed Tau phosphorylation in Parkinson's disease models. A FASEB J;23(9):2820-30. PMID: 19369384 [PubMed - indexed for MEDLINE]Related articles PMCID: PMC2796901] and in vivo in a widely used Tg model of PD (see Examples).
There are no drugs that can reverse synucleinopathy and tauopathy associated with neurodegenerative diseases, especially Parkinson's disease (PD), the second most common degenerative disease. Drugs used for treatment of PD are not curative, and moreover, are of limited long-term use due to development of resistance and/or adverse side-effects in patients. While the genesis of PD is not known, a-synuclein (a-Syn) overexpression through gene duplication and multiplication is causal in sporadic PD.
Evidence shows that hyperphosphorylated Tau (p-Tau) also has a central role in the genesis of PD; and several studies have identified the a-Syn gene (SNCA) and Tau gene (MAPT) to be important risk factors in this disease. Pre-clinical studies in a PD-like transgenic (Tg) mouse model
overexpressing a-Syn, has been performed using Li as a therapeutic agent. Li is approved by the FDA for treatment of bipolar disorders. The off-label use of Li to treat PD is demonstrated by data showing that treatment of a- Syn overexpressing Tg mice with low levels of Li reverses synucleinopathy and tauopathy of dopaminergic neurons enervating the striatum (see
Examples). Thus, Li suppresses the overexpression of a-Syn and reduces aggregates of both a-Syn and p-Tau to levels seen in non-Tg wild type mice. This is the first evidence of a small molecule directly preventing the overexpression and accumulation of a-Syn.
The actions of Li are two-fold: Inhibition of the Tau kinase, GSK-3p, whose activation is entirely dependent on a-Syn, and stimulation of autophagy, a pathway central for clearing aggregates of a-Syn and p-Tau.
In Example 1 , extensive studies in vivo using different doses of Li in aged mice are performed to estimate efficacy of treatment in reversing PD- like pathology. The lowest dose of Li that can be used to reverse both synucleinopathy and tauopathy is identified. In Example 2, pre-symptomatic mice are treated with different doses of Li to determine if long-term use of low levels of Li will prevent onset of PD-like pathology. This allows the development of the first therapy for the prevention of sporadic PD, and is useful in individuals who carry multiplications of SNCA and are at high risk for developing PD. Detailed analyses measuring the efficacy of each treatment is performed, including biochemical, behavioral and immunohistochemical analyses. Since Li is already FDA approved, and its toxicity and side-effects are well documented in both humans and mice, the drug can be readily used in other PD animal models, and ultimately, in clinical trials in humans. These and other embodiments are more fully described in connection with the detailed description.
Moderate levels of lithium completely reverse parkinsonism-like motor deficits in PDGF-a-Syn mice, with abolition of elevated levels of a-Syn and p-Tau, through blockade of p-GSK-3p. These results provide strong and compelling evidence for the utility of lithium in the treatment of PD.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows coexpression of a-Syn/pTau in a-Syn overexpressing mice (OEM) mice and reinforces the choice of PDGF-a-Syn as a model system to analyze lithium treatments.
Figure 2 details blood plasma levels of lithium in wild-type (WT) and transgenic (Tg) mice following the development and execution of the lithium treatment paradigm.
Figure 3 quantifies the treatment of a-Syn overexpressing mice (OEM) following lithium treatment and shows a reversal of motor impairment seen in PDGF-a-Syn transgenic mice. Sal = saline; X = lithium; Tg = transgenic; WT = wild-type.
Figure 4 illustrates that lithium reduces high levels of a-Syn and activated p-GSK-3p in striatum of PDGF-a-Syn overexpressing mice and has no effect on wild-type mice fed lithium. Sal = saline; X = lithium; Tg = transgenic; WT = wild-type; +Cpd.X = lithium.
Figure 5 illustrates that lithium reverses tauopathy in striatum of PDGF-a-Syn transgenic mice. Sal = saline; X = lithium; Tg = transgenic; WT = wild-type; +Cpd.X = lithium. DETAILED DESCRIPTION OF THE INVENTION
A novel method for treating Parkinson's Disease (PD) involves the administration of therapeutically effective amounts of Lithium (Li) to inhibit GSK-3 and simultaneously stimulate autophagy to clear protein aggregates of a-synuclein (a-Syn) and hyperphosphorylated Tau.
There are several highly innovative features of this method: (1 ) the off-target use of a FDA-approved drug, lithium, in the treatment of PD; (2) targeting GSK-3 , a major kinase in the development of tauopathy, as it has the ability to hyperphosphorylate Tau at most of the epitopes, which in turn promotes aggregation of Tau and tauopathy; (3) through both in vitro and in vivo studies, showing that targeting ΘδΚ-3β is both neuroprotective and neurorestorative to dopaminergic neurons; (4) reducing the levels of two of the most important genes that have been identified to be risk factors for PD, SNCA and MAPT, and studying the combined effect of these genes in PD; (5) lithium is already FDA-approved, its long-term toxicity is already well described, in general, lithium is well tolerated in patients, with few side- effects and it should be similarly well tolerated in PD patients; and (6) lithium can be immediately used in clinical trials in humans.
Several genome-wide studies recently identified the a-synuclein and Tau gene (SNCA and MAPT, respectively) as important risk factors in the development of PD [Mata IF, Yearout D, Alvarez V, Coto E, de Mena L, Ribacoba R, Lorenzo-Betancor O, Samaranch L, Pastor P, Cervantes S, Infante J, Garcia-Gorostiaga I, Sierra M, Combarros O, Snapinn KW, Edwards KL, Zabetian CP. Replication of MAPT and SNCA, but not
PARK16-18, as susceptibility genes for parkinson's disease. 26(5) Mov Disord. 819-23. Epub 201 1 Mar 21 ; International Parkinson Disease
Genomics Consortium, Nails MA, Plagnol V, Hernandez DG, Sharma M, Sheerin UM, Saad M, Simon-Sanchez J, Schulte C, Lesage S,
Sveinbjornsdottir S, Stefansson K, Martinez M, Hardy J, Heutink P, Brice A, Gasser T, Singleton AB, Wood NW. Imputation of sequence variants for identification of genetic risks for Parkinson's disease: a meta-analysis of genome-wide association studies. Lancet. 201 1 Feb 19;377(9766):641-9. Epub 201 1 Feb 1 ; Edwards TL, Scott WK, Almonte C, Burt A, Powell EH, Beecham GW, Wang L, Zuchner S, Konidari I, Wang G, Singer C, Nahab F, Scott B, Stajich JM, Pericak-Vance M, Haines J, Vance JM, Martin ER.
Genome-wide association study confirms SNPs in SNCA and the MAPT region as common risk factors for Parkinson disease. Ann Hum Genet. 2010 Mar;74(2):97-109. Epub 2010 Jan 8; Simon-Sanchez J, Schulte C, Bras JM, Sharma M, Gibbs JR, Berg D, Paisan-Ruiz C, Lichtner P, Scholz SW, Hernandez DG, Kruger R, Federoff M, Klein C, Goate A, Perlmutter J, Bonin M, Nails MA, lllig T, Gieger C, Houlden H, Steffens M, Okun MS, Racette BA, Cookson MR, Foote KD, Fernandez HH, Traynor BJ, Schreiber S, Arepalli S, Zonozi R, Gwinn K, van der Brug M, Lopez G, Chanock SJ, Schatzkin A, Park Y, Hollenbeck A, Gao J, Huang X, Wood NW, Lorenz D, Deuschl G, Chen H, Riess O, Hardy JA, Singleton AB, Gasser T. Genome- wide association study reveals genetic risk underlying Parkinson's disease. Nat Genet. 2009 Dec;41 (12):1308-12. Epub 2009 Nov 15]. While the genesis of PD is unknown, overexpression of a-synuclein (a-Syn) through SNCA multiplications is causal in development of sporadic PD [Singleton AB, Farrer M, Johnson J, Singleton A, Hague S, Kachergus J, Hulihan M, Peuralinna T, Dutra A, Nussbaum R, Lincoln S, Crawley A, Hanson M, Maraganore D, Adler C, Cookson MR, Muenter M, Baptista M, Miller D, Blancato J, Hardy J, Gwinn-Hardy K. Alpha-synuclein locus triplication causes Parkinson's disease. Science. 2003; 302(5646): 841. Crews L, Tsigelny I, Hashimoto M, Masliah E. Role of Synucleins in Alzheimer's Disease. Neurotox Res. 2009; 16(3): 306-317]. In pathological states, a-Syn is misfolded into oligomers, aggregates and accumulates into neuronal inclusion bodies, Lewy bodies (LBs), that are the pathological hallmarks of PD and other synucleinopathies [Forno LS. Neuropathology of Parkinson's disease. J. Neuropathol Exp Neurol. 1996; 55(3): 259-272; Spillantini MG, Crowther RA, Jakes R, Hasegawa M, Goedert M. a-synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with Lewy bodies, Proc Natl Acad Sci USA. 1998; 95(1 1); 6469-6473; Duka T, Rusnak M, Drolet RE, Duka V, Wersinger C, Goudreau JL, Sidhu A. (2006) Alpha- synuclein induces hyperphosphorylation of Tau in the MPTP model of parkinsonism. FASEB J. 20:2302-12], Evidence indicates that PD is also a tauopathic disease [Duka T, Sidhu A. (2006) The neurotoxin, MPP+, induces hyperphosphorylation of Tau, in the presence of alpha-Synuclein, in SH- SY5Y neuroblastoma cells. Neurotox Res. 10:1 -10; Alan P. Kozikowski, Irina N. Gaisina, Pavel A. Petukhov, Jayalakshmi Sridhar, LaShaunda T. King, Sylvie Y. Blond, Tetyana Duka, Milan Rusnak, Anita Sidhu (2006). Highly Potent and Specific GSK-3beta Inhibitors That Block Tau Phosphorylation. ChemMedChem. 1 :256-66; Duka T, Duka V, Joyce JN, Sidhu (2009) Alpha- Synuclein contributes to GSK-3beta-catalyzed Tau phosphorylation in Parkinson's disease models. A FASEB J;23(9):2820-30. PMID: 19369384 [PubMed - indexed for MEDLINE]Related articles PMCID: PMC2796901 ; Wills J, Jones J.Haggerty T, Duka V, Joyce JN, Sidhu A (2010). Elevated taupathy and alpha-synuclein pathology in postmortem Parkinson's disease brains with and without dementia. Exp Neurol. 2010 Sep;225(1 ):210-8. Epub 2010. PMCID: PMC2922478; Haggerty T, Credle J, Rodriguez O, Wills J, Duka V, Oaks AW, Masliah E, Sidhu A. Hyperphosphorylated Tau in an a- synuclein overexpressing transgenic model of Parkinson's disease. 33(9) Eur. J. Neurosci. 1598-610 (201 1 May); Wills J, Credle J, Haggerty T, Lee J- H, Oaks AW, Sidhu A. Tauopathic changes in the striatum of A53T a- synuclein mutant mouse model of Parkinson's disease. 6(3) Plos One, e17953 (2011 Mar 21 ); Smolinsky C, Wills J, Duka V, Oaks A, Sidhu A. (201 1 ) Taupathic changes in brain of alpha-synuclein A53T mutant mouse model of Parkinson's disease is restricted to the straitum and is dependent on oxidative stress. In Press; Kaul T, Credle J, Haggerty T, Oaks A, Masliah E, Sidhu A (201 1 ) Region-specific tauopathic changes in brain of the alpha- synuclein overexpressing mouse model of Parkinson's disease. 12 BMC Neurosci. 79 (201 1 Aug 3)]. Using diverse in vitro and in vivo models of PD, as well as postmortem PD tissues, the presence of tauopathy in dopaminergic neurons in a manner strictly dependent on a-Syn has been shown. a-Syn also recruits and activates the Tau kinase, ΘβΚ-3β (p-GSK- 3β, hyperphosphorylated at Tyr216), to induce the hyperphosphorylation of Tau (p-Tau) at multiple sites, leading to a toxic gain of function and microtubule destabilization, with eventual cell death. In cells and neurons, blockade of GSK-3p with lithium, or with highly selective and specific inhibitors of GSK-3P, reversed increases in a-Syn accumulation and reversed p-Tau accumulation and formation [Duka T, Duka V, Joyce JN, Sidhu (2009) Alpha-Synuclein contributes to GSK-3beta-catalyzed Tau phosphorylation in Parkinson's disease models. A FASEB J;23(9):2820-30. PMID: 19369384 [PubMed - indexed for MEDLINE]Related articles PMCID: PMC2796901 ; Wills J, Jones J.Haggerty T, Duka V, Joyce JN, Sidhu A (2010). Elevated taupathy and alpha-synuclein pathology in postmortem Parkinson's disease brains with and without dementia. Exp Neurol. 2010 Sep;225(1):210-8. Epub 2010. PMCID: PMC2922478].
The blockade of GSK-3P in vivo with therapeutically effective levels of lithium (e.g., at ~ 0.8 mEq/L) reverses synucleinopathy and tauopathy in a PD mouse model (see Examples). Thus, 8-10 month old mice
overexpressing a-Syn under the PDGF promoter, and who show
synucleinopathy and tauopathy, were treated for one month with therapeutic levels of lithium in chow. Lithium reversed motor impairments, reduced a- Syn levels to that of control WT mice, while abolishing the toxic increases in p-Tau levels. Moreover, aggregates of both a-Syn and p-Tau were abolished.
GSK-3P can be targeted with lithium, providing for a novel therapeutic approach in the treatment of PD. Importantly, lithium is FDA-approved and has been long used in the treatment of bipolar disorders. Moreover, its use is associated with very few side effects, with no psychotropic effects in humans; it is not a sedative, depressant, or euphoriant [Marcus WL. Lithium: a review of its pharmacokinetics, health effects, and toxicology. J Environ Pathol Toxicol Oncol. 1994 -13(2):73-9]. Inhibitors of GSK-3 , such as lithium, are a novel group of drugs for the treatment of PD. Moreover, evidence shows that the treatment of individuals carrying SNCA multiplications with low levels of lithium will prevent the onset of PD. The ability of lithium, a FDA-approved drug widely used in the treatment of bipolar disease, and which is a known inhibitor of GSK-3p, as well as an activator of autophagy, has been tested in PDGF-a- Syn overexpressing mice, a widely used model of sporadic PD, the most common form of PD.
The term tauopathies is used to refer to a class of neurodegenerative diseases associated with the pathological aggregation of tau protein in the human brain. The term synucleinopathies is used to name a group of neurodegenerative disorders characterized by fibrillary aggregates of alpha- synuclein protein in the cytoplasm of selective populations of neurons and glia. These disorders include Parkinson's disease (PD), dementia with Lewy bodies (DLB), pure autonomic failure (PAF), and multiple system atrophy (MSA). Clinically, they are characterized by a chronic and progressive decline in motor, cognitive, behavioural, and autonomic functions, depending on the distribution of the lesions. Because of clinical overlap, differential diagnosis is sometimes very difficult. Parkinsonism is the predominant symptom of PD, but it can be indistinguishable from the parkinsonism of DLB and MSA. Autonomic dysfunction, which is an isolated finding in PAF, may be present in PD and DLB, but is usually more prominent and appears earlier in MSA. DLB could be the same disease as PD but with widespread cortical pathological states, leading to dementia, fluctuating cognition, and the characteristic visual hallucinations. The deposition of aggregates of synuclein in neurons and glia suggests that a common pathogenic mechanism may exist for these disorders.
In one embodiment, a method of treating a mammal suffering from a malady associated with synucleinopathy and/or tauopathy involves administering to said mammal a therapeutically effective amount of lithium and/or a lithium salt. In another embodiment, the malady is Parkinson's Disease (PD), In still another embodiment, the method involves the administration to a mammal suffering from a malady associated with aggregates of both a-Syn and p-Tau, and involves the administration of an aggregate-reducing or - eliminating amount of lithium. And in still another embodiment, the method involves the prevention and/or treatment of a mammal suffering from overproduction of GSK-3P, or from the activation of p-GSK-3p.
The lithium may be co-administered, complexed or covalently linked with other carriers or active agents. As used herein, and unless stated otherwise, the term "lithium" includes elemental lithium, lithium ion, lithium salts, and lithium complexed with, or covalently bonded to, other carriers or active agents. In one embodiment, lithium is co-administered (or complexed or covalently linked) with TDZD-8 or with methylmaleimide.
In various embodiments, the mammal is a human, murine, porcine, bovine, ovine, equine, canine, feline, or gibbon. In another embodiment, the mammal is human, the malady is Parkinson's Disease, and the lithium is administered as a lithium salt (e.g., lithium carbonate, lithium citrate, lithium sulfonates, lithium orotate). Other lithium salts are known, and may also be used in these methods.
The lithium-containing active agent may be administered orally, parenterally (e.g., intravenous, intramuscular, etc.), subcutaneously, transdermally, or by sublingual, intranasal, or suppository, or by any means commonly known and accepted in the art for the administration of lithium or lithium salts. In one embodiment, the lithium agent is administered orally.
The therapeutically effective amount for treating, reversing, or preventing these maladies can be readily determined by those of ordinary skill in the art, and will vary based upon the patient and the seventy of his or her condition. The severity of the patient's condition will be affected by a constellation of factors including genetic predisposition, environmental exposure, and other factors. One skilled in the art will be able to assess the therapeutic efficacy of a particular dosage regimen based upon routine screening and diagnostic testing.
In these methods, the lithium agent can be administered orally, with blood levels reaching therapeutic blood levels in the patient of about 0.01 - 3 mEq/L. Alternatively, lithium is administered so as to achieve therapeutically effective blood levels of about 0.05 - 2.0 mEq/L. In other embodiments, lithium is administered to achieve patient blood levels of about 0.1-1.5 mEq/L. And in still other embodiments, the method is employed to administer lithium to patient blood levels of about 0.6 to about 0.8 mEq/L. Again, and as depending on the severity of the patient's condition, and the response of the patient to the lithium therapy, one skilled in the art will appreciate that therapeutically effective blood levels of lithium may vary, and perhaps decline over time.
Patient treatment according to these methods can be achieved by repeated and routine administration of lithium. For example, the lithium may be administered daily for periods of six months or more. Treatment regimens will generally be about one month or more.
When administering lithium orally according to these methods, lithium can be combined with food for ease of administration. Dosage regimens of about 1-20 g lithium per kg food can be used. In other embodiments, about
1- 10 g lithium per kg food can be used. And in still other embodiments, about 3-6 g lithium per kg food can be used.
EXAMPLES
These Examples show the utility of lithium administration in a-Syn overexpressing Tg mice. The Examples are illustrative, and should not be interpreted as limiting the scope of the invention.
Mice overexpressing human a-Syn were obtained [Masliah E, Rockenstein E, Veinbergs I, Mallory M, Hashimoto M, Takeda A, Sagara Y, Sisk A, Mucke L. Dopaminergic loss and inclusion body formation in alpha- synuclein mice: implications for neurodegenerative disorders. Science. 2000 Feb 18;287(5456): 1265-9] and bred. These mice have been used in several studies and both synucleinopathy and tauopathy are well- characterized in these animals [Haggerty T, Credle J, Rodriguez O, Wills J, Duka V, Oaks AW, Masliah E, Sidhu A. Hyperphosphorylated Tau in an a- synuclein overexpressing transgenic model of Parkinson's disease. 33(9) Eur. J. Neurosci. 1598-610 (2011 May); Kaul T, Credle J, Haggerty T, Oaks A, Masliah E, Sidhu A (20 1 ) Region-specific tauopathic changes in brain of the alpha-synuclein overexpressing mouse model of Parkinson's disease. 12 BMC Neurosci. 79 (201 1 Aug 3)]. Accumulation of a-Syn and p-Tau develops in these mice at >8 months of age; younger mice do not show such pathology. From IHC, it is shown that these mice have large intraneuronal aggregates of a-Syn and p-Tau in striatum, which resemble Lewy bodies, with proteins co-localizing with one another (Figure 1) and with p-GSK-3 [Haggerty T, Credle J, Rodriguez O, Wills J, Duka V, Oaks AW, Masliah E, Sidhu A. Hyperphosphorylated Tau in an a-synuclein overexpressing transgenic model of Parkinson's disease. 33(9) Eur. J. Neurosci. 1598-610 (201 1 May)]. Moreover, it has been shown that activation of p-GSK-3 (phosphorylation at Tyr216) occurs in these mice, resulting in
hyperphosphorylation of Tau [Haggerty T, Credle J, Rodriguez O, Wills J, Duka V, Oaks AW, Masliah E, Sidhu A. Hyperphosphorylated Tau in an a- synuclein overexpressing transgenic model of Parkinson's disease. 33(9) Eur. J. Neurosci. 1598-610 (201 1 May)]. a-Syn overexpressing mice also display abnormal motor behavior, when compared to WT mice, as indexed by reduced latency to fall and latency to passive rotation (see Figure 3).
A vast majority of studies using lithium are based on daily injections of lithium (i.v. 30 μΙ of a 0.6 M solution for 4 weeks). This is extremely toxic to mice, resulting in -35% mortality, even in WT animals. Accordingly, administering lithium orally, where lithium carbonate is mixed in with chow (4.4 gm/kg of chow), is used. There is no mortality or loss of weight, indicating that this treatment paradigm is well tolerated. As described herein, 8-10 month old PDGF-a-Syn mice and age- matched litter mate WT mice were used. There were 4 treatment
paradigms: WT mice fed normal chow; Tg mice fed normal chow; WT mice fed lithium chow and Tg mice fed lithium chow. There were 4-6 animals per treatment group. Treatments were conducted for 1 month. Mice were euthanized, brains removed and blood plasma collected. Levels of lithium in blood plasma were analyzed by RP-HPLC and lithium was found to be present at 0.6-0.8 mEq/L (Figure 2), which is mid-level of the therapeutic range of lithium for bipolar disorders, 0.2-1.5 mEq/L [Marcus WL. Lithium: a review of its pharmacokinetics, health effects, and toxicology. J Environ Pathol Toxicol Oncol. 1994;13(2):73-9],
Groups of WT or Tg mice (N=8-9) were fed normal chow or lithium chow (4.4 gm/kg of chow) for 4 weeks; mice were 10-12 months of age. Motor behavior was measured using an accelerating rotarod as described previously [Graham DR, Sidhu (2010) Mice expressing the A53T mutant form of human alpha-synuclein exhibit hyperactivity and reduced anxiety-like behavior. J Neurosci Res. 2010 Jun;88(8):1777-83. [Epub ahead of print] PMCID: PMC2861296J and results of the days 2-3 are summarized in
Figure 3. Motor behavior in Tg mice fed normal chow was significantly severely impaired compared to WT mice, as indexed by both latency to fall and latency to passive rotation. However, upon treatment with lithium for 4 weeks, motor behavior of Tg mice were restored to that of WT mice fed normal chow. Lithium did not cause any changes in motor behavior in WT mice. These studies show that lithium reverses the impairment in motor behavior seen in these Tg mice, improving the parkinsonism-like motor dysfunction.
In control Tg mice, a-Syn and p-GSK-3p (Tyr216) levels were significantly (P<0.01 ) elevated by -700 and 65%, respectively (Figure 4). Moreover, total GSK-3P were also significantly (P<0.01) increased by ~50%. Treatment with lithium for 1 month significantly (P<0.01 ) reduced levels of both a-Syn and total GSK-3 to levels seen in WT mice fed normal chow, p- ΘβΚ-3β levels were reduced to -60% below levels seen in control WT mice fed normal chow (Figure 4). These data show that lithium blocks activation of p-GSK-3p, while simultaneously reducing the very high levels of a-Syn seen in these mice.
In WT mice fed lithium, levels of a-Syn were slightly increased, while p-GSK-3 levels were slightly decreased compared to WT animals fed normal chow (Figure 5). Lower levels of lithium in chow can be used to obtain plasma levels that are at the low end of the therapeutic range.
As p-GSK-3p is sufficient and necessary for the development of tauopathy in PD, blockade of p-GSK-3 will lead to a reduction in p-Tau levels. In Tg mice fed normal chow, levels of p-Tau hyperphosphorylated at pathogenic epitopes were significantly (P<0.01 ) increased. Thus, pSer202 levels were increased by -220%, while pSer262 and pSer396/404 levels were increased by 280 and 240%, respectively, compared to control WT mice fed normal chow (Figure 5).
In Tg mice fed lithium, the levels of all 3 epitopes of p-Tau were significantly reduced to control levels (Figure 5). These data show that lithium reverses tauopathy in a-Syn Tg mice. In WT mice, lithium did not significantly increase levels of pSer202, although a non-significant increase of -120% was seen in pSer262 levels.
Thus, lithium may act to not only inhibit GSK-3P activation but also to remove existing aggregates of a-Syn and p-Tau through increase in proteasomal activity. Moreover, lithium is also a known activator of autophagy [Sarkar S, Rubinsztein DC. Inositol and IP3 levels regulate autophagy: biology and therapeutic speculations. Autophagy. 2006 Apr- Jun;2(2):132-4. Epub 2006 Apr 6. Review; Pasquali L, Longone P, Isidoro C, Ruggieri S, Paparelli A, Fornai F. Autophagy, lithium, and amyotrophic lateral sclerosis. Muscle Nerve. 2009 Aug;40(2): 173-94. Review] and clearance of aggregates of a-Syn and p-Tau may also occur through induction of autophagy. This provides strong support for using lithium in treatment of PD, since lithium reverses motor impairment and PD-associated pathology in striatum in vivo in a well-characterized mouse model of PD.
There are several objectives to be studied in pre-clinical trials, in order to assess whether a specific treatment will be successful. These are: (1) to establish the optimal dosage, using various doses of the drug; and (2) to determine if treatment of target population with a specific drug can reverse the observed pathology.
Example 1 : To test whether long-term treatment of aged Tg mice, who have both synucleinopathy and tauopathy, with lithium will reverse PD- like pathology. PD is most commonly seen in the elderly population. In aged mice (18 months) PDGF-a-Syn is administered at different doses of lithium in chow for 2 months. Improvement in biochemical,
neuropathological and behavioral correlates is assessed. These studies enable the estimation of the lowest dose of lithium that can reverse PD-like pathology.
It has been demonstrated that GSK-3 is central to the tauopathic process seen in PD, and that its inhibition in neurons and transfected neuronal cells with lithium, TDZD-8 or with methylmaleimide inhibitors of GSK-3 , rescues cells from MPP+-mediated cell death, with abolition of synucleinopathy and tauopathy [Duka T, Sidhu A. (2006) The neurotoxin, MPP+, induces hyperphosphorylation of Tau, in the presence of alpha- Synuclein, in SH-SY5Y neuroblastoma cells. Neurotox Res. 10: 1-10; Alan P. Kozikowski, Irina N. Gaisina, Pavel A. Petukhov, Jayalakshmi Sridhar, LaShaunda T. King, Sylvie Y. Blond, Tetyana Duka, Milan Rusnak, Anita Sidhu (2006). Highly Potent and Specific GSK-3beta Inhibitors That Block Tau Phosphorylation. ChemMedChem. 1 :256-66; Duka T, Duka V, Joyce JN, Sidhu (2009) Alpha-Synuclein contributes to GSK-3beta-catalyzed Tau phosphorylation in Parkinson's disease models. A FASEB J;23(9):2820-30. PMID: 19369384 [PubMed - indexed for MEDLINE]Related articles PMCID: PMC2796901 ; Wills J, Jones J.Haggerty T, Duka V, Joyce JN, Sidhu A (2010). Elevated taupathy and alpha-synuclein pathology in postmortem Parkinson's disease brains with and without dementia. Exp Neurol. 2010 Sep;225(1 ):210-8. Epub 2010. PMCID: PMC2922478]. Described herein, in 8-10 month old Tg mice overexpressing a-Syn, it is shown that lithium can reverse both synucleinopathy and tauopathy.
Lithium is a specific inhibitor of ΘβΚ-3β (Ki = 1 mM, 17), and is also an activator of the autophagy pathway [Sarkar S, Rubinsztein DC. Inositol and IP3 levels regulate autophagy: biology and therapeutic speculations. Autophagy. 2006 Apr-Jun;2(2):132-4. Epub 2006 Apr 6. Review; Pasquali L, Longone P, Isidoro C, Ruggieri S, Paparelli A, Fornai F. Autophagy, lithium, and amyotrophic lateral sclerosis. Muscle Nerve. 2009 Aug;40(2):173-94. Review], and is therefore an ideal drug to prevent ongoing toxicity and for clearing aggregates of a-Syn and p-Tau that have already formed. In cells, misfolded aggregated proteins, such as a-Syn and Tau accumulate, and their clearance is enhanced when autophagy is stimulated. Moreover, lithium can be administered orally, with blood levels reaching therapeutic levels (0.2-1 .5 mEq/L). Other ΘΘΚ-3β inhibitors are not as advantageous for several reasons: (1 ) there are very few GSK-3 inhibitors which cross the blood brain barrier; (2) all of the available GSK-3P inhibitors which do cross the blood brain barrier have to be injected daily [Kalinichev M, Dawson LA. Evidence for antimanic efficacy of glycogen synthase kinase-3 (GSK3) inhibitors in a strain-specific model of acute mania. Int J
Neuropsychopharmacol. 201 1 Jan 6:1 -17. [Epub ahead of print]], which is not suitable for long-term treatment in either mice or humans; and (3) none of the specific GSK-3P inhibitors stimulate autophagy. Lithium has been used for the long-term treatment of bipolar disease, and its side-effects and toxicity are well established [Marcus WL. Lithium: a review of its
pharmacokinetics, health effects, and toxicology. J Environ Pathol Toxicol Oncol. 1994;13(2):73-9].
The PDGF-a-Syn mouse is an ideal model for our studies, as it mimics the vast majority of sporadic PD, which is caused by multiplications of the SNCA [Hofer A, Berg D, Asmus F, Niwar M, Ransmayr G, et al (2005). The role of alpha-synuclein in gene multiplications in early-onset Parkinson's disease and dementia with Lewy bodies. J Neural Transm.
1 12(9): 1249-1254; Ross OA, Braithwaite AT, Skipper LM, Kachergus J, Hulihan MM, Middleton FA, Nishioka K, Fuchs J, Gasser T, Maraganore DM, Adler CH, Larvor L, Chartier-Harlin MC, Nilsson C, Langston JW, Gwinn K, Hattori N, Farrer MJ. Genomic investigation of alpha-synuclein multiplication and parkinsonism. Ann Neurol. 2008 Jun;63(6):743-50; Ahn TB, Kim SY, Kim JY, Park SS, Lee DS, Min HJ, Kim YK, Kim SE, Kim JM, Kim HJ, Cho J, Jeon BS. alpha-Synuclein gene duplication is present in sporadic Parkinson disease. Neurology. 2008 Jan 1 ;70(1 ):43-9. Epub 2007 Jul 11 ]. Importantly, it has key features of PD pathology seen in humans: (1 ) elevation of a-Syn to levels similar to those seen in PD; (2) hyperphosphorylation of Tau at sites identical to those seen in PD striatum, namely, at Ser202, Ser262 and Ser396/404; (3) increases in p-Tau to levels similar to those seen in PD; (4) increases in p-GSK-3 to levels similar to those seen in PD [Wills J, Jones J,Haggerty T, Duka V, Joyce JN, Sidhu A (2010). Elevated taupathy and alpha-synuclein pathology in postmortem Parkinson's disease brains with and without dementia. Exp Neurol. 2010 Sep;225(1 ):210-8. Epub 2010. PMCID: PMC2922478; Haggerty T, Credle J, Rodriguez O, Wills J, Duka V, Oaks AW, Masliah E, Sidhu A. Hyperphosphorylated Tau in an a-synuclein overexpressing transgenic model of Parkinson's disease. 33(9) Eur. J.
Neurosci. 1598-610 (201 1 May)]; (5) accumulation and aggregation of a-Syn and p-Tau into aggresomes that resemble Lewy bodies in PD patients
[Haggerty T, Credle J, Rodriguez O, Wills J, Duka V, Oaks AW, Masliah E, Sidhu A. Hyperphosphorylated Tau in an a-synuclein overexpressing transgenic model of Parkinson's disease. 33(9) Eur. J. Neurosci. 1598-610 (201 1 May)]; (6) an age-dependent development of synucleinopathy and tauopathy similar to PD; and (7) reduced motor behavior upon aging (>12 months). The results herein were conducted in adult (8-10 month), but not aged (>16 month), mice, however, the evidence suggests that lithium will reverse synucleinopathy and tauopathy in aged mice. In this Example, 16 month old mice are treated with varying doses of lithium (reaching plasma levels of -0.2, 0.5 and 0.8 mEq/L). After 2 months, mice are euthanized and tested.
Study to correlate levels of lithium in chow to levels of lithium in serum. The normal therapeutic range of serum lithium levels for treatment of bipolar disorder is very narrow, with serum levels of 0.2-1 .5 mEq/L
[Marcus WL. Lithium: a review of its pharmacokinetics, health effects, and toxicology. J Environ Pathol Toxicol Oncol. 1994;13(2):73-9]; 4.4 gm/kg of lithium in chow results in serum plasma levels of ~0.6-0.8 mEq/L, which is at the mid-level range of the therapeutic scale for bipolar disorder. In this Example, the amount of lithium in chow is correlated to the lithium in serum. 16 month old WT mice (4/group) will receive lithium in their feed for 1 month; the levels of lithium in feed are: 0, 0.25, 0.5, 1.0, 2.0 and 4.0 gm/kg. After 1 month, mice are sacrificed and serum levels of lithium will be measured by HPLC.
Treatment of mice. WT and a-Syn Tg mice at age 16 months are used, since this is the age which most closely models the onset of PD in humans. Mice are treated with low, medium and high levels of lithium in chow in order to obtain serum levels of ~0.2, 0.5 and 0.8 mEq/L. The treatment groups are: WT + Tg mice fed normal chow; WT + Tg mice fed low levels of lithium chow; WT + Tg mice fed medium levels of lithium; WT + Tg mice fed high levels of lithium. There are 12 mice per group. After 2 months of treatments, all mice are sacrificed. Results from lithium-fed mice are compared to Tg and WT mice fed normal chow.
Assessment of the efficacy of treatments by analyzing behavior. PDGF-a-Syn mice develop abnormal motor behavior [Masliah E,
Rockenstein E, Veinbergs I, Mallory M, Hashimoto M, Takeda A, Sagara Y, Sisk A, Mucke L. Dopaminergic loss and inclusion body formation in alpha- synuclein mice: implications for neurodegenerative disorders. Science. 2000 Feb 18;287(5456): 1265-9]. Motor function is analyzed by rotor rod and by the wire hang test [Perez-Lloret S, Rascol O. Dopamine receptor agonists for the treatment of early or advanced Parkinson's disease. CNS Drugs. 2010 Nov 1 ;24(1 1 ):941-68. Review; Bronstein JM, Tagliati M, Alterman RL, Lozano AM, Volkmann J, Stefani A, Horak FB, Okun MS, Foote KD, Krack P, Pahwa R, Henderson JM, Hariz Ml, Bakay RA, Rezai A, Marks WJ Jr, Mora E, Vitek JL, Weaver FM, Gross RE, DeLong MR. Deep brain stimulation for Parkinson disease: an expert consensus and review of key issues. Arch Neurol. 201 1 Feb;68(2):165. Epub 2010 Oct 1 1. Review;
Masliah E, Rockenstein E, Veinbergs I, Mallory M, Hashimoto M, Takeda A, Sagara Y, Sisk A, Mucke L. Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neurodegenerative disorders.
Science. 2000 Feb 18;287(5456):1265-9; Graham DR, Sidhu (2010) Mice expressing the A53T mutant form of human alpha-synuclein exhibit hyperactivity and reduced anxiety-like behavior. J Neurosci Res. 2010
Jun;88(8): 1777-83. [Epub ahead of print] PMCID: PMC2861296; Sarkar S, Rubinsztein DC. Inositol and IP3 levels regulate autophagy: biology and therapeutic speculations. Autophagy. 2006 Apr-Jun;2(2):132-4. Epub 2006 Apr 6. Review; Pasquali L, Longone P, Isidoro C, Ruggieri S, Paparelli A, Fornai F. Autophagy, lithium, and amyotrophic lateral sclerosis. Muscle Nerve. 2009 Aug;40(2):173-94. Review; Kalinichev M, Dawson LA.
Evidence for antimanic efficacy of glycogen synthase kinase-3 (GSK3) inhibitors in a strain-specific model of acute mania. Int J
Neuropsychopharmacol. 201 1 Jan 6:1-17. [Epub ahead of print]; Hofer A, Berg D, Asmus F, Niwar M, Ransmayr G, et al (2005). The role of alpha- synuclein in gene multiplications in early-onset Parkinson's disease and dementia with Lewy bodies. J Neural Transm. 1 12(9): 1249-1254; Ross OA, Braithwaite AT, Skipper LM, Kachergus J, Hulihan MM, Middleton FA, Nishioka K, Fuchs J, Gasser T, Maraganore DM, Adler CH, Larvor L, Chartier-Harlin MC, Nilsson C, Langston JW, Gwinn K, Hattori N, Farrer MJ. Genomic investigation of alpha-synuclein multiplication and parkinsonism. Ann Neurol. 2008 Jun;63(6):743-50; Ahn TB, Kim SY, Kim JY, Park SS, Lee DS, Min HJ, Kim YK, Kim SE, Kim JM, Kim HJ, Cho J, Jeon BS. alpha- Synuclein gene duplication is present in sporadic Parkinson disease.
Neurology. 2008 Jan 1 ;70(1 ):43-9. Epub 2007 Jul 1 1 ; Martins-Silva, C, X. De Jaeger, M.S. Guzman, R.D. Lima, M.S. Santos, C. Kushmerick, M.V. Gomez, M.G. Caron, M.A. Prado, and V.F. Prado, Novel strains of mice deficient for the vesicular acetylcholine transporter: insights on
transcriptional regulation and control of locomotor behavior. PLoS One, 201 . 6(3): p. e17611 ; Yao, I., K. Takao, T. Miyakawa, S. Ito, and M. Setou, Synaptic E3 Ligase SCRAPPER in Contextual Fear Conditioning: Extensive Behavioral Phenotyping of Scrapper Heterozygote and Overexpressing Mutant Mice. PLoS One, 201 1. 6(2): p. e17317; West MJ, Gundersen HJ (1990) Unbiased stereological estimation of the number of neurons in the human hippocampus. Journal of Comparative Neurology 296:1-22]. The rotor rod test is conducted as described [Graham DR, Sidhu (2010) Mice expressing the A53T mutant form of human alpha-synuclein exhibit hyperactivity and reduced anxiety-like behavior. J Neurosci Res. 2010 Jun;88(8):1777-83. [Epub ahead of print] PMCID: PMC2861296], where latency to fall from an accelerating rod will be measured after training of animals. For the wire hang test [Yao, I., K. Takao, T. Miyakawa, S. Ito, and M. Setou, Synaptic E3 Ligase SCRAPPER in Contextual Fear Conditioning: Extensive Behavioral Phenotyping of Scrapper Heterozygote and
Overexpressing Mutant Mice. PLoS One, 2011 . 6(2): p. e17317; West MJ, Gundersen HJ (1990) Unbiased stereological estimation of the number of neurons in the human hippocampus. Journal of Comparative Neurology 296:1-22], which measures subtle motor behavior, mice are induced to grip the wire screen cover from a rodent housing cage and the screen will be inverted and suspended 45 cm above an empty housing cage [Mata IF, Yearout D, Alvarez V, Coto E, de Mena L, Ribacoba R, Lorenzo-Betancor O, Samaranch L, Pastor P, Cervantes S, Infante J, Garcia-Gorostiaga I, Sierra M, Combarros O, Snapinn KW, Edwards KL, Zabetian CP. Replication of MAPT and SNCA, but not PARK16-18, as susceptibility genes for
Parkinson's disease. 26(5) Mov Disord. 819-23. Epub 201 1 Mar 21 ;
International Parkinson Disease Genomics Consortium, Nails MA, Plagnol V, Hernandez DG, Sharma M, Sheerin UM, Saad M, Simon-Sanchez J, Schulte C, Lesage S, Sveinbjornsdottir S, Stefansson K, Martinez M, Hardy J, Heutink P, Brice A, Gasser T, Singleton AB, Wood NW. Imputation of sequence variants for identification of genetic risks for Parkinson's disease: a meta-analysis of genome-wide association studies. Lancet. 201 1 Feb 19;377(9766):641-9. Epub 201 1 Feb 1 ; Edwards TL, Scott WK, Almonte C, Burt A, Powell EH, Beecham GW, Wang L, Zuchner S, Konidari I, Wang G, Singer C, Nahab F, Scott B, Stajich JM, Pericak-Vance M, Haines J, Vance JM, Martin ER. Genome-wide association study confirms SNPs in SNCA and the MAPT region as common risk factors for Parkinson disease. Ann Hum Genet. 2010 Mar;74(2):97-109. Epub 2010 Jan 8]. Latency to fall from the screen is recorded as the average of six trials, conducted twice per day over three days, with a five minute cut-off time per trial [Martins-Silva, C, X. De Jaeger, M.S. Guzman, R.D. Lima, M.S. Santos, C. Kushmerick, M.V. Gomez, M.G. Caron, M.A. Prado, and V.F. Prado, Novel strains of mice deficient for the vesicular acetylcholine transporter: insights on
transcriptional regulation and control of locomotor behavior. PLoS One,
201 . 6(3): p. e1761 1 ; Yao, I., K. Takao, T. Miyakawa, S. Ito, and M. Setou, Synaptic E3 Ligase SCRAPPER in Contextual Fear Conditioning: Extensive Behavioral Phenotyping of Scrapper Heterozygote and Overexpressing Mutant Mice. PLoS One, 201 1. 6(2): p. e17317]. Improvements in scores compared to transgenic (Tg) mice fed a normal feed indicate improvement in parkinsonism motor symptoms.
Measurement of dopamine metabolites and loss of dopaminergic neurons. Dopamine metabolites (DOPAC and HVA) are measured by HPLC, as previously described [Duka T, Sidhu A. (2006) The neurotoxin, MPP+, induces hyperphosphorylation of Tau, in the presence of alpha-
Synuclein, in SH-SY5Y neuroblastoma cells. Neurotox Res. 10:1-10], using striatum. Brain slices (at least 4 animals/per group) are stained with tyrosine hydroxylase (TH) antibody and non-biased stereological counting of TH- positive neurons of the Substania nigra is conducted to assess number of dopaminergic neurons of S. nigra [West MJ, Gundersen HJ (1990) Unbiased stereological estimation of the number of neurons in the human
hippocampus. Journal of Comparative Neurology 296:1-22] as follows:
brains are sectioned in a 1 :2 series at 40 pm and IHC performed for TH and counterstained with a Nissl stain. Counts are made at regular predetermined intervals (x, 140 pm, y 140 pm). Systematic samples of the area occupied by the nuclei are made from a random starting point. An unbiased counting frame of known area (45 pm x 35 pm) is superimposed on the image of the tissue sections, using a 63 times oil objective NA 1 .36.
Immunohistochemical assessment of the efficacy of treatments. Immunohistochemical studies followed by confocal microscopy, are conducted, as described [Wills J, Credle J, Haggerty T, Lee J-H, Oaks AW, Sidhu A. Tauopathic changes in the striatum of A53T a-synuclein mutant mouse model of Parkinson's disease. 6(3) Plos One, e17953 (201 1 Mar 21); Smolinsky C, Wills J, Duka V, Oaks A, Sidhu A. (201 1) Taupathic changes in brain of alpha-synuclein A53T mutant mouse model of Parkinson's disease is restricted to the straitum and is dependent on oxidative stress. In Press; Kaul T, Credle J, Haggerty T, Oaks A, Masliah E, Sidhu A (201 1 ) Region- specific tauopathic changes in brain of the alpha-synuclein overexpressing mouse model of Parkinson's disease. 12 BMC Neurosci. 79 (201 1 Aug 3)]. Co-immunostaining of a-Syn and PHF-1 Tau (Tau hyperphosphorylated at Ser396/Ser404), a-Syn and p-GSK-3 (phosphorylation at Tyr216) and PHF- 1 Tau and p-GSK-3p, along with DAPI is conducted. These studies demonstrate that lithium abolished the large aggregates of a-Syn and Tau previously observed in these mice [Wills J, Credle J, Haggerty T, Lee J-H, Oaks AW, Sidhu A. Tauopathic changes in the striatum of A53T a-synuclein mutant mouse model of Parkinson's disease. 6(3) Plos One, e17953 (201 1 Mar 21)]. Assessment of the efficacy of treatments by analyzing GSK-3p.
Active p-GSK-3 is phosphorylated at Tyr216, while inactive GSK-3p is phosphorylated at Ser9. Using specific antibodies against the various epitopes, as well as total GSK-3p, levels of p-GSK-3p and GSK-3p-Ser9 are determined by conducting Western blots. Total GSK-3 levels are
examined. Striatal lysates are used. Comparison of results obtained with the lithium treated Tg and WT groups will be made to controls fed normal chow.
Assessment of the efficacy of treatments by examining
synucleinopathy and tauopathy. a-Syn levels are measured in striata of mice after lithium treatments, by Western blots. p-a-Syn levels are also measured. p-Tau levels are measured by Western blots. The epitopes examined are: Ser202, Ser262 and Ser396/404. Assessment of these sites is sufficient to determine if tauopathy is reduced. Comparison of results obtained with the lithium treated Tg and WT groups is made to controls fed normal chow.
Example 2: To test the ability of lithium to prevent onset of PD-like pathology and behavioral deficits in pre-symptomatic PDGF-a-Syn
overexpressing mice. Gene multiplications of a-Syn is causal in sporadic
PD, the most common form of PD. Pre-symptomatic adult mice (6 months of age) are continuously fed very low doses of lithium for 4 months to test the ability of lithium to prevent onset of PD-like pathology. Improvement in biochemical, neuropathological and behavioral correlates is assessed.
Treatment of pre-symptomatic mice with low levels of lithium prevents the onset of PD pathology through partial inhibition of GSK-3 and
stimulation of autophagy.
Gene multiplications of a-Syn are causal in sporadic PD, the most common form of PD. Genotyping can identify individuals carrying SNCA multiplications; as a preventive measure, administration of low levels of lithium is useful in preventing onset of PD. Moreover, other individuals identified at high risk for developing PD, particularly through work-related exposure to environmental toxins and agrichemicals, are administered low levels of lithium as a precautionary measure. The a-Syn overexpressing Tg mice develop synucleinopathy and tauopathy at 8 months of age. Treatment of 6 month old pre-symptomatic mice with low levels of lithium prevents synucleinopathy and tauopathy at a later age in mice. This approach is very useful in prevention strategies aimed at individuals who carry multiplications of the a-Syn gene, and who are at the highest risk of developing PD.
Through genotyping, such individuals are easily identified and preventive measures are conducted.
Treatment of mice with lithium for 4 months. Mice 6 months of age (12/group) are treated with lithium in their chow as described above, and treatment will be continued for 4 months. As a control, a group of Tg mice will be fed normal chow and allowed to age to 10 months. Another group of WT mice will be fed normal chow and a group of WT mice will be fed lithium chow. The purpose of this study is to determine if long-term treatment of mice will prevent onset of PD-like pathology.
Assessment of the efficacy of treatments by analyzing behavior, measurement of dopamine metabolites and loss of dopaminergic neurons, assessment of the efficacy of treatments by analyzing GSK-3P, and assessment of the efficacy of treatments by examining protein correlates of synucleinopathy and tauopathy is performed. The assessment of long-term treatment with sub-therapeutic levels of lithium is conducted as described in Example 1 . Results in lithium-treated mice are compared to mice that were treated with normal chow.
Although the present invention has been described using specific implementations, it is understood that several variations and modifications may be grafted to said implementations, and the present invention aims to cover such modifications, uses, or adaptations of the present invention that in general follow the principles of the invention and that include any variation of the present description which will become known or conventional in the field of activity in which the present invention lies. All publications cited herein are expressly incorporated herein by reference in their entireties for all purposes.

Claims

WE CLAIM:
1. A method of treating a mammal suffering from a malady associated with synucleinopathy and/or tauopathy comprising administering to said mammal a therapeutically effective amount of lithium and/or lithium salt.
2. The method of claim 1 , wherein the malady is Parkinson's Disease.
3. The method of claim 1 , wherein the lithium salt is lithium carbonate, lithium citrate, lithium sulfonate, or lithium orotate.
4. The method of claim 1 , wherein the therapeutically effective amount of lithium and/or lithium salt reduces or eliminates aggregates of both a-Syn and p-Tau.
5. The method of claim 1 , wherein the lithium and/or lithium salt is coadministered, complexed or covalently linked with other carriers or active agents.
6. The method of claim 5, wherein the lithium and/or lithium salt is coadministered with TDZD-8 or methylmaleimide.
7. The method of claim 1 , wherein the lithium and/or lithium salt is administered orally.
8. The method of claim 1 , wherein the therapeutically effective amount of lithium and/or lithium salt is that producing in said mammal a blood level of lithium of about 0.05 - 2.0 mEq/L.
9. The method of claim 1 , wherein the therapeutically effective amount of lithium and/or lithium salt is that producing in said mammal a blood level of lithium of about 0.1 - 1.5 mEq/L.
10. The method of claim 1 , wherein the therapeutically effective amount of lithium and/or lithium salt is that producing in said mammal a blood level of lithium of about 0.6 - 0.8 mEq/L.
1 1 . A method of treating a mammal suffering from an overproduction of GSK-3P and/or from the activation of p-GSK- p comprising administering to said mammal a therapeutically effective amount of lithium and/or lithium salt.
12. The method of claim 11 , wherein the lithium and/or lithium salt is coadministered, complexed or covalently linked with other carriers or active agents.
13. The method of claim 1 1 , wherein the lithium and/or lithium salt is coadministered with TDZD-8 or methylmaleimide.
14. The method of claim 1 1 , wherein the lithium salt is lithium carbonate, lithium citrate, lithium sulfonate, or lithium orotate.
15. The method of claim 1 1 , wherein the lithium and/or lithium salt is administered orally.
16. The method of claim 1 1 , wherein the therapeutically effective amount of lithium and/or lithium salt is that producing in said mammal a blood level of lithium of about 0.05 - 2.0 mEq/L.
17. The method of claim 11 , wherein the therapeutically effective amount of lithium and/or lithium salt is that producing in said mammal a blood level of lithium of about 0.1 - 1.5 mEq/L.
18. The method of claim 1 1 , wherein the therapeutically effective amount of lithium and/or lithium salt is that producing in said mammal a blood level of lithium of about 0.6 - 0.8 mEq/L.
19. A method of treating a human patient suffering from Parkinson's Disease comprising orally administering to said human patient a lithium salt at a dose sufficient to achieve a blood level of lithium in said human patient of about 0.1 - 1.5 mEq/L.
20. The method of claim 19, wherein the lithium salt is lithium carbonate, and wherein the dose is selected to achieve a blood level of lithium in said human patient of about 0.6 - 0.8 mEq/L.
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