Sirtuin 2 inhibitors rescue alpha-synuclein-mediated toxicity in models of Parkinson's disease.

The sirtuins are members of the histone deacetylase family of proteins that participate in a variety of cellular functions and play a role in aging. We identified a potent inhibitor of sirtuin 2 (SIRT2) and found that inhibition of SIRT2 rescued alpha-synuclein toxicity and modified inclusion morphology in a cellular model of Parkinson's disease. Genetic inhibition of SIRT2 via small interfering RNA similarly rescued alpha-synuclein toxicity. Furthermore, the inhibitors protected against dopaminergic cell death both in vitro and in a Drosophila model of Parkinson's disease. The results suggest a link between neurodegeneration and aging.

Myopathy as a first symptom of Huntington's disease in a Marathon runner.

A semi professional marathon runner at risk for Huntington's disease (HD) (43 CAG repeats) developed signs of a slowly progressive myopathy with exercise-induced muscle fatigue, pain, elevated creatine kinase level, and worsening of his running performance many years before first signs of chorea were detected. Muscle biopsy displayed a mild myopathy with mitochondrial pathology including a complex IV deficiency and analysis of the patient's fibroblast culture demonstrated deficits in mitochondrial function. Challenging skeletal muscle by excessive training might have disclosed myopathy in HD even years before the appearance of other neurological symptoms.

Discovery of a novel small-molecule targeting selective clearance of mutant huntingtin fragments.

CAG-triplet repeat extension, translated into polyglutamines within the coding frame of otherwise unrelated gene products, causes 9 incurable neurodegenerative disorders, including Huntington's disease. Although an expansion in the CAG repeat length is the autosomal dominant mutation that causes the fully penetrant neurological phenotypes, the repeat length is inversely correlated with the age of onset. The precise molecular mechanism(s) of neurodegeneration remains elusive, but compelling evidence implicates the protein or its proteolytic fragments as the cause for the gain of novel pathological function(s). The authors sought to identify small molecules that target the selective clearance of polypeptides containing pathological polyglutamine extension. In a high-throughput chemical screen, they identified compounds that facilitate the clearance of a small huntingtin fragment with extended polyglutamines fused to green fluorescent protein reporter. Identified hits were validated in dose-response and toxicity tests. Compounds have been further tested in an assay for clearance of a larger huntingtin fragment, containing either pathological or normal polyglutamine repeats. In this assay, the authors identified compounds selectively targeting the clearance of mutant but not normal huntingtin fragments. These compounds were subjected to a functional assay, which yielded a lead compound that rescues cells from induced mutant polyglutamine toxicity.

Discovery of bioactive small-molecule inhibitor of poly adp-ribose polymerase: implications for energy-deficient cells.

Poly (ADP-ribose) polymerase (PARP1) is a nuclear protein that, when overactivated by oxidative stress-induced DNA damage, ADP ribosylates target proteins leading to dramatic cellular ATP depletion. We have discovered a biologically active small-molecule inhibitor of PARP1. The discovered compound inhibited PARP1 enzymatic activity in vitro and prevented ATP loss and cell death in a surrogate model of oxidative stress in vivo. We also investigated a new use for PARP1 inhibitors in energy-deficient cells by using Huntington's disease as a model. Our results showed that insult with the oxidant hydrogen peroxide depleted cellular ATP in mutant cells below the threshold of viability. The protective role of PARP1 inhibitors against oxidative stress has been shown in this model system.

Interactions between metabotropic glutamate 5 and adenosine A2A receptors in normal and parkinsonian mice.

Evidence for heteromeric receptor complexes comprising adenosine A2A and metabotropic glutamate 5 (mGlu5) receptors in striatum has raised the possibility of synergistic interactions between striatal A2A and mGlu5 receptors. We investigated the role of striatal A2A receptors in the locomotor stimulant and antiparkinsonian properties of mGlu5 antagonists using complementary pharmacologic and genetic approaches. Locomotion acutely stimulated by the mGlu5 antagonist [2-methyl-6-(phenylethynyl)-pyridine (MPEP)] was absent in mGlu5 knock-out (KO) mice and was potentiated by an A2A antagonist KW-6002 [(E)-1,3-diethyl-8-(3,4-dimethoxystyryl)-7-methylxanthine], both in normal and in dopamine-depleted (reserpinized) mice. Conversely, the MPEP-induced motor response was markedly attenuated in single and double A2A and D2 receptor KO mice. In contrast, motor stimulation by a D1 dopamine agonist was not attenuated in the KO mice. The A2A receptor dependence of MPEP-induced motor stimulation was investigated further using a postnatal forebrain-specific conditional (Cre/loxP system) KO of the A2A receptor. MPEP loses the ability to stimulate locomotion in conditional KO mice, suggesting that this mGlu5 antagonist effect requires the postdevelopmental action of striatal A2A receptors. The potentiation of mGlu5 antagonist-induced motor stimulation by an A2A antagonist and its dependence on both D2 and forebrain A2A receptors highlight the functional interdependence of these receptors. These data also strengthen a rationale for pursuing a combinational drug strategy for enhancing the antiparkinsonian effects of A2A and mGlu5 antagonists.

A potent small molecule inhibits polyglutamine aggregation in Huntington's disease neurons and suppresses neurodegeneration in vivo.

Polyglutamine (polyQ) disorders, including Huntington's disease (HD), are caused by expansion of polyQ-encoding repeats within otherwise unrelated gene products. In polyQ diseases, the pathology and death of affected neurons are associated with the accumulation of mutant proteins in insoluble aggregates. Several studies implicate polyQ-dependent aggregation as a cause of neurodegeneration in HD, suggesting that inhibition of neuronal polyQ aggregation may be therapeutic in HD patients. We have used a yeast-based high-throughput screening assay to identify small-molecule inhibitors of polyQ aggregation. We validated the effects of four hit compounds in mammalian cell-based models of HD, optimized compound structures for potency, and then tested them in vitro in cultured brain slices from HD transgenic mice. These efforts identified a potent compound (IC50=10 nM) with long-term inhibitory effects on polyQ aggregation in HD neurons. Testing of this compound in a Drosophila HD model showed that it suppresses neurodegeneration in vivo, strongly suggesting an essential role for polyQ aggregation in HD pathology. The aggregation inhibitors identified in this screen represent four primary chemical scaffolds and are strong lead compounds for the development of therapeutics for human polyQ diseases.

Analysis of cellular, transgenic and human models of Huntington's disease reveals tyrosine hydroxylase alterations and substantia nigra neuropathology.

Huntington's disease (HD) is a progressive, autosomal dominant neurodegenerative disorder that is pathologically characterized by a striatal-specific degeneration. Aberrant dopamine neurotransmission has been proposed as a mechanism underlying the movement disorder of HD. We report that the enzymatic activity of tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine biosynthesis, is decreased in a transgenic mouse model of HD. In addition, mutant huntingtin was found to disrupt transcription of TH and dopamine beta-hydroxylase (DbetaH) promoter reporter constructs. In situ hybridization revealed extensive loss of TH mRNA and decreased dopaminergic cell size in human HD substantia nigra. TH-immunoreactive protein was reduced in human grade 4 HD substantia nigra by 32% compared to age-matched controls. These findings implicate abnormalities in dopamine neurotransmission in HD and may provide new insights into targets for pharmacotherapy.

Differential D1 and D2 receptor-mediated effects on immediate early gene induction in a transgenic mouse model of Huntington's disease.

The diminished expression of D1 and D2 dopamine receptors is a well-documented hallmark of Huntington's disease (HD), but relatively little is known about how these changes in receptor populations affect the dopaminergic responses of striatal neurons. Using transgenic mice expressing an N-terminal portion of mutant huntingtin (R6/2 mice), we have examined immediate early gene (IEG) expression as an index of dopaminergic signal transduction. c-fos, jun B, zif268, and N10 mRNA levels and expression patterns were analyzed using quantitative in situ hybridization histochemistry following intraperitoneal administration of selective D1 and D2 family pharmacological agents (SKF-82958 and eticlopride). Basal IEG levels were generally lower in the dorsal subregion of R6/2 striata relative to wild-type control striata at 10-11 weeks of age, a finding in accord with previously reported decreases in D1 and adenosine A2A receptors. D2-antagonist-stimulated IEG expression was significantly reduced in the striata of transgenic animals. In contrast, D1-agonist-induced striatal R6/2 IEG mRNA levels were either equivalent or significantly enhanced relative to control levels, an unexpected result given the reduced level of D1 receptors in R6/2 animals. Understanding the functional bases for these effects may further elucidate the complex pathophysiology of Huntington's disease.

Dysregulation of gene expression in the R6/2 model of polyglutamine disease: parallel changes in muscle and brain.

Previous analyses of gene expression in a mouse model of Huntington's disease (R6/2) indicated that an N-terminal fragment of mutant huntingtin causes downregulation of striatal signaling genes and particularly those normally induced by cAMP and retinoic acid. The present study expands the regional and temporal scope of this previous work by assessing whether similar changes occur in other brain regions affected in Huntington's disease and other polyglutamine diseases and by discerning whether gene expression changes precede the appearance of disease signs. Oligonucleotide microarrays were employed to survey the expression of approximately 11,000 mRNAs in the cerebral cortex, cerebellum and striatum of symptomatic R6/2 mice. The number and nature of gene expression changes were similar among these three regions, influenced as expected by regional differences in baseline gene expression. Time-course studies revealed that mRNA changes could only reliably be detected after 4 weeks of age, coincident with development of early pathologic and behavioral changes in these animals. In addition, we discovered that skeletal muscle is also a target of polyglutamine-related perturbations in gene expression, showing changes in mRNAs that are dysregulated in brain and also muscle-specific mRNAs. The complete dataset is available at www.neumetrix.info.

Polyglutamine and transcription: gene expression changes shared by DRPLA and Huntington's disease mouse models reveal context-independent effects.

Recent evidence indicates that transcriptional abnormalities may play an important role in the pathophysiology of polyglutamine diseases. In the present study, we have explored the extent to which polyglutamine-related changes in gene expression may be independent of protein context by comparing mouse models of dentatorubral-pallidoluysian atrophy (DRPLA) and Huntington's disease (HD). Microarray gene expression profiling was conducted in mice of the same background strain in which the same promoter was employed to direct the expression of full-length atrophin-1 or partial huntingtin transproteins (At-65Q or N171-82Q mice). A large number of overlapping gene expression changes were observed in the cerebella of At-65Q and N171-82Q mice. Six of the gene expression changes common to both huntingtin and atrophin-1 transgenic mice were also observed in the cerebella of mouse models expressing full-length mutant ataxin-7 or the androgen receptor. These results demonstrate that some of the gene expression effects of expanded polyglutamine proteins occur independently of protein context.

Increased huntingtin protein length reduces the number of polyglutamine-induced gene expression changes in mouse models of Huntington's disease.

Both transcriptional dysregulation and proteolysis of mutant huntingtin (htt) are postulated to be important components of Huntington's disease (HD) pathogenesis. In previous studies, we demonstrated that transgenic mice that express short mutant htt fragments containing 171 or fewer N-terminal residues (R6/2 and N171-82Q mice) recapitulate many of the mRNA changes observed in human HD brain. To examine whether htt protein length influences the ability of its expanded polyglutamine domain to alter gene expression, we conducted mRNA profiling analyses of mice that express an extended N-terminal fragment (HD46, HD100; 964 amino acids) or full-length (YAC72; 3144 amino acids) mutant htt transprotein. Oligonucleotide microarray analyses of HD46 and YAC72 mice identified fewer differentially expressed mRNAs than were seen in transgenic mice expressing short N-terminal mutant htt fragments. Histologic analyses also detected limited changes in these mice (small decreases in adenosine A2a receptor mRNA and dopamine D2 receptor binding in HD100 animals; small increases in dopamine D1 receptor binding in HD46 and HD100 mice). Neither HD46 nor YAC72 mice exhibited altered mRNA levels similar to those observed previously in R6/2 mice, N171-82Q mice or human HD patients. These findings suggest that htt protein length influences the ability of an expanded polyglutamine domain to alter gene expression. Furthermore, our findings suggest that short N-terminal fragments of mutant htt might be responsible for the gene expression alterations observed in human HD brain.

Sp1 and TAFII130 transcriptional activity disrupted in early Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine tract in the huntingtin protein. Transcriptional dysregulation has been implicated in HD pathogenesis. Here, we report that huntingtin interacts with the transcriptional activator Sp1 and coactivator TAFII130. Coexpression of Sp1 and TAFII130 in cultured striatal cells from wild-type and HD transgenic mice reverses the transcriptional inhibition of the dopamine D2 receptor gene caused by mutant huntingtin, as well as protects neurons from huntingtin-induced cellular toxicity. Furthermore, soluble mutant huntingtin inhibits Sp1 binding to DNA in postmortem brain tissues of both presymptomatic and affected HD patients. Understanding these early molecular events in HD may provide an opportunity to interfere with the effects of mutant huntingtin before the development of disease symptoms.