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Pyrazolone derivatives have previously been found to be inhibitors of Cu/Zn superoxide dismutase 1 (SOD1)-dependent protein aggregation, which extended survival of an amyotrophic lateral sclerosis (ALS) mouse model. On the basis of ADME analysis, we describe herein a new series of tertiary amine-containing pyrazolones and their structure-activity relationships. Further conversion to the conjugate salts greatly improved their solubility. Phosphate compound 17 exhibited numerous benefits both to cellular activity and to CNS-related drug-like properties in vitro and in vivo, including microsomal stability, tolerated toxicity, and blood-brain barrier permeation. These results indicate that tertiary amine pyrazolones comprise a valuable class of ALS drug candidates.

The Wnt receptor Ryk is an evolutionary-conserved protein important during neuronal differentiation through several mechanisms, including γ-secretase cleavage and nuclear translocation of its intracellular domain (Ryk-ICD). Although the Wnt pathway may be neuroprotective, the role of Ryk in neurodegenerative disease remains unknown. We found that Ryk is up-regulated in neurons expressing mutant huntingtin (HTT) in several models of Huntington's disease (HD). Further investigation in Caenorhabditis elegans and mouse striatal cell models of HD provided a model in which the early-stage increase of Ryk promotes neuronal dysfunction by repressing the neuroprotective activity of the longevity-promoting factor FOXO through a noncanonical mechanism that implicates the Ryk-ICD fragment and its binding to the FOXO co-factor β-catenin. The Ryk-ICD fragment suppressed neuroprotection by lin-18/Ryk loss-of-function in expanded-polyQ nematodes, repressed FOXO transcriptional activity, and abolished β-catenin protection of mutant htt striatal cells against cell death vulnerability. Additionally, Ryk-ICD was increased in the nucleus of mutant htt cells, and reducing γ-secretase PS1 levels compensated for the cytotoxicity of full-length Ryk in these cells. These findings reveal that the Ryk-ICD pathway may impair FOXO protective activity in mutant polyglutamine neurons, suggesting that neurons are unable to efficiently maintain function and resist disease from the earliest phases of the pathogenic process in HD., Competing Interests: The authors have declared that no competing interests exist. Dr. Robert Ferrante is listed as an author of our paper, but at the time of acceptance was not reachable or able to confirm details of his author contributions to the manuscript. The corresponding author, Christian Neri, has therefore supplied the information regarding his contribution to the manuscript and his competing interests and it is correct to the best of Christian Neri's knowledge.

Inhibition of sirtuin 2 (SIRT2) deacetylase mediates protective effects in cell and invertebrate models of Parkinson's disease and Huntington's disease (HD). Here we report the in vivo efficacy of a brain-permeable SIRT2 inhibitor in two genetic mouse models of HD. Compound treatment resulted in improved motor function, extended survival, and reduced brain atrophy and is associated with marked reduction of aggregated mutant huntingtin, a hallmark of HD pathology. Our results provide preclinical validation of SIRT2 inhibition as a potential therapeutic target for HD and support the further development of SIRT2 inhibitors for testing in humans., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disease. Approximately 20 percent of familial ALS cases are caused by mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Rodents expressing mutant SOD1 transgenes develop progressive, fatal motor neuron disease and disease onset and progression is dependent on the level of SOD1. We investigated the possibility that a reduction in SOD1 protein may be of therapeutic benefit in ALS and screened 30,000 compounds for inhibition of SOD1 transcription. The most effective inhibitor identified was N-{4-[4-(4-methylbenzoyl)-1-piperazinyl]phenyl}-2-thiophenecarboxamide (Compound ID 7687685), which in PC12 cells showed an EC50 of 10.6 microM for inhibition of SOD1 expression and an LD50 more than 30 microM. This compound was subsequently shown to reduce endogenous SOD1 levels in HeLa cells and to exhibit a modest reduction of SOD1 protein levels in mouse spinal cord tissue. These data suggest that the efficacy of compound 7687685 as an inhibitor of SOD1 gene expression is not likely to be clinically useful, although the strategy reported could be applied broadly to screening for small molecule inhibitors of gene expression.

Cyclohexane 1,3-diones were identified as a class of molecules exhibiting a protective effect against mutant SOD1 induced toxicity in PC-12 cells, but an optimized analogue had little or no effect on life extension in the G93A SOD1 mouse model for amyotrophic lateral sclerosis (ALS). Additional testing showed that these compounds were inactive in neurons and further analogue synthesis was carried out to identify compounds with neuronal activity. Starting from two racemic derivatives that were active in cortical neurons, two potent analogues (1b and 2b) were resolved, which were protective against mutant SOD1 induced toxicity in PC-12 cells. Both compounds were found to be active in cortical neurons and presented good ADME profiles in vitro. On the basis of these results, an ALS mouse trial with 1b was carried out, which showed slightly greater life extension than the FDA-approved ALS drug riluzole, thereby validating cyclohexane 1,3-diones as a novel therapeutic class for the treatment of ALS.

Amyotrophic lateral sclerosis (ALS) is an orphan neurodegenerative disease currently without a cure. The arylsulfanyl pyrazolone (ASP) scaffold was one of the active scaffolds identified in a cell-based high throughput screening assay targeting mutant Cu/Zn superoxide dismutase 1 (SOD1) induced toxicity and aggregation as a marker for ALS. The initial ASP hit compounds were potent and had favorable ADME properties but had poor microsomal and plasma stability. Here, we identify the microsomal metabolite and describe synthesized analogues of these ASP compounds to address the rapid metabolism. Both in vitro potency and pharmacological properties of the ASP scaffold have been dramatically improved via chemical modification to the corresponding sulfone and ether derivatives. One of the ether analogues (13), with superior potency and in vitro pharmacokinetic properties, was tested in vivo for its pharmacokinetic profile, brain penetration, and efficacy in an ALS mouse model. The analogue showed sustained blood and brain levels in vivo and significant activity in the mouse model of ALS, thus validating the new aryloxanyl pyrazolone scaffold as an important novel therapeutic lead for the treatment of this neurodegenerative disorder.

Huntington disease (HD) is a progressive neurodegenerative disease that affects 30,000 individuals in North America. Treatments that slow its relentless course are not yet available, and biomarkers that can reliably measure disease activity and therapeutic response are urgently needed to facilitate their development. Here, we interrogated 119 human blood samples for transcripts associated with HD. We found that the dynamic regulator of chromatin plasticity H2A histone family, member Y (H2AFY) is specifically overexpressed in the blood and frontal cortex of patients with HD compared with controls. This association precedes the onset of clinical symptoms, was confirmed in two mouse models, and was independently replicated in cross-sectional and longitudinal clinical studies comprising 142 participants. A histone deacetylase inhibitor that suppresses neurodegeneration in animal models reduces H2AFY levels in a randomized phase II clinical trial. This study identifies the chromatin regulator H2AFY as a potential biomarker associated with disease activity and pharmacodynamic response that may become useful for enabling disease-modifying therapeutics for HD.

Huntington's disease (HD) is an autosomal dominant, progressive, and fatal neurodegenerative disorder caused by an expanded polyglutamine cytosine-adenine-guanine repeat in the gene coding for the protein huntingtin. Despite great progress over the past two decades since the identification of the gene mutation, a direct causative pathway from the HD gene mutation to neuronal dysfunction and death has not yet been established. One important advance in understanding the pathogenic mechanisms of this disease has been the development of experimental mouse models that replicate many of the clinical, neuropathological, and molecular events in HD patients. These murine models have played a critical role in providing accurate and experimentally accessible systems to study multiple features of disease pathogenesis and to test potential therapeutic strategies. A better understanding of the pathophysiological mechanisms of disease and how they interrelate has become important in identifying a treatment for HD and in the design of human clinical trials. In this chapter, we review the current state of HD mouse models and their successes in elucidating disease pathogenesis and in developing pharmacotherapies. There is no clinically proven treatment for HD that can halt or ameliorate the inexorable disease progression. As such, a guide to assessing studies in mouse models and salient issues related to translation from mice to humans are included., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Although a direct causative pathway from the gene mutation to the selective neostriatal neurodegeneration remains unclear in Huntington's disease (HD), one putative pathological mechanism reported to play a prominent role in the pathogenesis of this neurological disorder is mitochondrial dysfunction. We examined mitochondria in preferentially vulnerable striatal calbindin-positive neurons in moderate-to-severe grade HD patients, using antisera against mitochondrial markers of COX2, SOD2 and cytochrome c. Combined calbindin and mitochondrial marker immunofluorescence showed a significant and progressive grade-dependent reduction in the number of mitochondria in spiny striatal neurons, with marked alteration in size. Consistent with mitochondrial loss, there was a reduction in COX2 protein levels using western analysis that corresponded with disease severity. In addition, both mitochondrial transcription factor A, a regulator of mtDNA, and peroxisome proliferator-activated receptor-co-activator gamma-1 alpha, a key transcriptional regulator of energy metabolism and mitochondrial biogenesis, were also significantly reduced with increasing disease severity. Abnormalities in mitochondrial dynamics were observed, showing a significant increase in the fission protein Drp1 and a reduction in the expression of the fusion protein mitofusin 1. Lastly, mitochondrial PCR array profiling in HD caudate nucleus specimens showed increased mRNA expression of proteins involved in mitochondrial localization, membrane translocation and polarization and transport that paralleled mitochondrial derangement. These findings reveal that there are both mitochondrial loss and altered mitochondrial morphogenesis with increased mitochondrial fission and reduced fusion in HD. These findings provide further evidence that mitochondrial dysfunction plays a critical role in the pathogenesis of HD.

Coenzyme Q10 (CoQ(10)), a potential neuroprotective compound, was previously investigated at a dosage of 600 mg/day in Huntington's disease (HD) patients and demonstrated a trend toward slowing disease progression. Higher CoQ(10) dosages may prove beneficial. We investigated the tolerability and blood levels associated with 1,200, 2,400, and 3,600 mg/day of CoQ(10) in HD and healthy subjects. Twenty-eight subjects (20 HD, 8 healthy) enrolled in a 20-week open-label trial. Subjects started on 1,200 mg/day of CoQ(10), increasing every 4 weeks by 1,200 mg to a maximum dosage of 3,600 mg/day. Monthly evaluations included review of adverse events and CoQ(10) blood levels. Twenty-three subjects (82%) achieved the target dosage of 3,600 mg/day. Six subjects (2 healthy, 4 HD) withdrew prematurely (gastrointestinal (GI) symptoms in 3, worsening HD in 2, and 1 because of a fall). All three serious adverse events occurred in a single subject, and were deemed unrelated to CoQ(10). The most common adverse events seen were GI symptoms. Mean (± SD) CoQ10 blood levels achieved over the course of the trial were as follows: 1.26 ± 1.27 μg/mL (baseline, n = 28), 5.59 ± 2.24 μg/mL (1,200 mg/day, week 4, n = 26), 6.38 ± 3.25 μg/mL (2,400 mg/day, week 8, n = 25), 7.49 ± 4.09 μg/mL (3,600 mg/day, week 12, n = 23), and 6.78 ± 3.36 μg/mL (3,600 mg/day, week 20, n = 20). CoQ(10) was well tolerated with over 80% of subjects achieving the target dosage. Dosages of 2,400 mg/day may provide the best balance between tolerability and blood level achieved. Further studies examining the efficacy of 2,400 mg/day are planned., (© 2010 Movement Disorder Society.)

Huntington's disease (HD) is a neurodegenerative disorder previously thought to be of primary neuronal origin, despite ubiquitous expression of mutant huntingtin (mHtt). We tested the hypothesis that mHtt expressed in astrocytes may contribute to the pathogenesis of HD. To better understand the contribution of astrocytes in HD in vivo, we developed a novel mouse model using lentiviral vectors that results in selective expression of mHtt into striatal astrocytes. Astrocytes expressing mHtt developed a progressive phenotype of reactive astrocytes that was characterized by a marked decreased expression of both glutamate transporters, GLAST and GLT-1, and of glutamate uptake. These effects were associated with neuronal dysfunction, as observed by a reduction in DARPP-32 and NR2B expression. Parallel studies in brain samples from HD subjects revealed early glial fibrillary acidic protein expression in striatal astrocytes from Grade 0 HD cases. Astrogliosis was associated with morphological changes that increased with severity of disease, from Grades 0 through 4 and was more prominent in the putamen. Combined immunofluorescence showed co-localization of mHtt in astrocytes in all striatal HD specimens, inclusive of Grade 0 HD. Consistent with the findings from experimental mice, there was a significant grade-dependent decrease in striatal GLT-1 expression from HD subjects. These findings suggest that the presence of mHtt in astrocytes alters glial glutamate transport capacity early in the disease process and may contribute to HD pathogenesis.

Huntington's disease (HD) is an autosomal dominant, progressive, and fatal neurodegenerative disorder caused by an expanded polyglutamine cytosine-adenine-guanine repeat in the gene coding for the protein huntingtin. Despite great progress, a direct causative pathway from the HD gene mutation to neuronal dysfunction and death has not yet been established. One important advance in understanding the pathogenic mechanisms of this disease has been the development of multiple murine models that replicate many of the clinical, neuropathological, and molecular events in HD patients. These models have played an important role in providing accurate and experimentally accessible systems to study multiple aspects of disease pathogenesis and to test potential therapeutic treatment strategies. Understanding how disease processes interrelate has become important in identifying a pharmacotherapy in HD and in the design of clinical trials. A review of the current state of HD mouse models and their successes in elucidating disease pathogenesis are discussed. There is no clinically proven treatment for HD that can halt or ameliorate the inexorable disease progression. As such, a guide to assessing studies in mouse models and salient issues related to translation from mice to humans are included.

Coenzyme Q(10) (CoQ(10)) and creatine are promising agents for neuroprotection in neurodegenerative diseases via their effects on improving mitochondrial function and cellular bioenergetics and their properties as antioxidants. We examined whether a combination of CoQ(10) with creatine can exert additive neuroprotective effects in a MPTP mouse model of Parkinson's disease, a 3-NP rat model of Huntington's disease (HD) and the R6/2 transgenic mouse model of HD. The combination of the two agents produced additive neuroprotective effects against dopamine depletion in the striatum and loss of tyrosine hydroxylase neurons in the substantia nigra pars compacta (SNpc) following chronic subcutaneous administration of MPTP. The combination treatment resulted in significant reduction in lipid peroxidation and pathologic alpha-synuclein accumulation in the SNpc neurons of the MPTP-treated mice. We also observed additive neuroprotective effects in reducing striatal lesion volumes produced by chronic subcutaneous administration of 3-NP to rats. The combination treatment showed significant effects on blocking 3-NP-induced impairment of glutathione homeostasis and reducing lipid peroxidation and DNA oxidative damage in the striatum. Lastly, the combination of CoQ(10) and creatine produced additive neuroprotective effects on improving motor performance and extending survival in the transgenic R6/2 HD mice. These findings suggest that combination therapy using CoQ(10) and creatine may be useful in the treatment of neurodegenerative diseases such as Parkinson's disease and HD.

Accumulation of expanded polyglutamine proteins is considered to be a major pathogenic biomarker of Huntington disease. We isolated SCAMP5 as a novel regulator of cellular accumulation of expanded polyglutamine track protein using cell-based aggregation assays. Ectopic expression of SCAMP5 augments the formation of ubiquitin-positive and detergent-resistant aggregates of mutant huntingtin (mtHTT). Expression of SCAMP5 is markedly increased in the striatum of Huntington disease patients and is induced in cultured striatal neurons by endoplasmic reticulum (ER) stress or by mtHTT. The increase of SCAMP5 impairs endocytosis, which in turn enhances mtHTT aggregation. On the contrary, down-regulation of SCAMP5 alleviates ER stress-induced mtHTT aggregation and endocytosis inhibition. Moreover, stereotactic injection into the striatum and intraperitoneal injection of tunicamycin significantly increase mtHTT aggregation in the striatum of R6/2 mice and in the cortex of N171-82Q mice, respectively. Taken together, these results suggest that exposure to ER stress increases SCAMP5 in the striatum, which positively regulates mtHTT aggregation via the endocytosis pathway.

Release of mitochondrial cytochrome c resulting in downstream activation of cell death pathways has been suggested to play a role in neurologic diseases featuring cell death. However, the specific biologic importance of cytochrome c release has not been demonstrated in Huntington's disease (HD). To evaluate the role of cytochrome c release, we screened a drug library to identify new inhibitors of cytochrome c release from mitochondria. Drugs effective at the level of purified mitochondria were evaluated in a cellular model of HD. As proof of principle, one drug was chosen for in depth evaluation in vitro and a transgenic mouse model of HD. Our findings demonstrate the utility of mitochondrial screening to identify inhibitors of cell death and provide further support for the important functional role of cytochrome c release in HD. Given that many of these compounds have been approved by the Food and Drug Administration for clinical usage and cross the blood-brain barrier, these drugs may lead to trials in patients.

Chromatin remodeling is tightly controlled under physiological conditions. Alterations in chromatin structure are involved in the pathogenesis of neuronal systems. We found that the monoallelic deletion of CREB binding protein (CBP) results in the induction of ERG-associated protein with SET domain (ESET) and increases trimethylation of histone H3 (K9) and condensation of pericentromeric heterochromatin structure in neurons. Nested deletion and mutational analysis of the ESET promoter further demonstrated that the Ets-2 transcription factor regulates transcriptional activity of the ESET gene. In CBP+/- mice, Ets-2 occupancy in the ESET promoter DNA was markedly elevated. Our results suggest that CBP is a transcriptional repressor of ESET gene expression by limiting Ets-2 transcriptional activity, while CBP siRNA enhances basal and Ets-2-dependent ESET transcriptional activity. Altered expression of the ESET gene and hypertrimethylation of H3 (K9) correlate with striatal neuron atrophy and dysfunction in CBP+/- mice. These results establish an alternative pathway that loss of CBP leads to the pericentric heterochromatin condensation through ESET expression and trimethylation of H3 (K9).

Mitochondria are power organelles generating biochemical energy, ATP, in the cell. Mitochondria play a variety of roles, including integrating extracellular signals and executing critical intracellular events, such as neuronal cell survival and death. Increasing evidence suggests that a cross-talk mechanism between mitochondria and the nucleus is closely related to neuronal function and activity. Nuclear receptors (estrogen receptors, thyroid (T3) hormone receptor, peroxisome proliferators-activated receptor gamma2) and transcription factors (cAMP response binding protein, p53) have been found to target mitochondria and exert prosurvival and prodeath pathways. In this context, the regulation of mitochondrial function via the translocation of nuclear receptors and transcription factors may underlie some of the mechanisms involved in neuronal survival and death. Understanding the function of nuclear receptors and transcription factors in the mitochondria may provide important pharmacological utility in the treatment of neurodegenerative conditions. Thus, the modulation of signaling pathways via mitochondria-targeting nuclear receptors and transcription factors is rapidly emerging as a novel therapeutic target.

To test the hypotheses that mutant huntingtin protein length and wild-type huntingtin dosage have important effects on disease-related transcriptional dysfunction, we compared the changes in mRNA in seven genetic mouse models of Huntington's disease (HD) and postmortem human HD caudate. Transgenic models expressing short N-terminal fragments of mutant huntingtin (R6/1 and R6/2 mice) exhibited the most rapid effects on gene expression, consistent with previous studies. Although changes in the brains of knock-in and full-length transgenic models of HD took longer to appear, 15- and 22-month CHL2(Q150/Q150), 18-month Hdh(Q92/Q92) and 2-year-old YAC128 animals also exhibited significant HD-like mRNA signatures. Whereas it was expected that the expression of full-length huntingtin transprotein might result in unique gene expression changes compared with those caused by the expression of an N-terminal huntingtin fragment, no discernable differences between full-length and fragment models were detected. In addition, very high correlations between the signatures of mice expressing normal levels of wild-type huntingtin and mice in which the wild-type protein is absent suggest a limited effect of the wild-type protein to change basal gene expression or to influence the qualitative disease-related effect of mutant huntingtin. The combined analysis of mouse and human HD transcriptomes provides important temporal and mechanistic insights into the process by which mutant huntingtin kills striatal neurons. In addition, the discovery that several available lines of HD mice faithfully recapitulate the gene expression signature of the human disorder provides a novel aspect of validation with respect to their use in preclinical therapeutic trials.

Transcriptional dysregulation and aberrant chromatin remodeling are central features in the pathology of Huntington's disease (HD). In order to more fully characterize these pathogenic events, an assessment of histone profiles and associated gene changes were performed in transgenic N171-82Q (82Q) and R6/2 HD mice. Analyses revealed significant chromatin modification, resulting in reduced histone acetylation with concomitant increased histone methylation, consistent with findings observed in HD patients. While there are no known interventions that ameliorate or arrest HD progression, DNA/RNA-binding anthracyclines may provide significant therapeutic potential by correcting pathological nucleosome changes and realigning transcription. Two such anthracyclines, chromomycin and mithramycin, improved altered nucleosome homeostasis in HD mice, normalizing the chromatin pattern. There was a significant shift in the balance between methylation and acetylation in treated HD mice to that found in wild-type mice, resulting in greater acetylation of histone H3 at lysine 9 and promoting gene transcription. Gene expression profiling in anthracycline-treated HD mice showed molecular changes that correlate with disease correction, such that a subset of downregulated genes were upregulated with anthracycline treatment. Improved nucleosomal dynamics were concurrent with a significant improvement in the behavioral and neuropathological phenotype observed in HD mice. These data show the ability of anthracycline compounds to rebalance epigenetic histone modification and, as such, may provide the rationale for the design of human clinical trials in HD patients.

Amyotrophic lateral sclerosis (ALS) is a clinically severe and fatal neurodegenerative disease characterized by a loss of both upper and lower motor neurons, resulting in progressive muscle loss and paralysis. While the exact cause of neuronal death in ALS remains unknown, it is proposed that multiple molecular defects trigger motor neuron cell death. These pathophysiological mechanisms include oxidative stress, mitochondrial impairment, protein aggregation, glutamate cytotoxicity, transcription dysfunction, inflammation, and apoptotic cell death. An understanding of how these potential therapeutic targets interrelate will provide direction both in the development of a pharmacotherapy and in the design of clinical trials in ALS. Important issues related to therapeutic development are the principals that should be followed in designing and conducting experiments using genetic animal models and what body of evidence is desirable to fully inform clinical decision making. In the context of ALS, we review some of the salient issues related to the use of genetic models in providing a guide to assessing studies in translating therapeutic strategies to patients with ALS and discuss therapeutic targets and pharmacological approaches to slowing disease progression. As in other neurodegenerative diseases, the most effective neuroprotection may result from combined treatment strategies.

Significant progress has been made in identifying neuroprotective agents and their translation to patients with neurological disorders. While the direct causative pathways of neurodegeneration remain unclear, they are under great clinical and experimental investigation. There are a number of interrelated pathogenic mechanisms triggering molecular events that lead to neuronal death. One putative mechanism reported to play a prominent role in the pathogenesis of neurological diseases is impaired energy metabolism. If reduced energy stores play a role in neuronal loss, then therapeutic strategies that buffer intracellular energy levels may prevent or impede the neurodegenerative process. Recent studies suggest that impaired energy production promotes neurological disease onset and progression. Sustained ATP levels are critical to cellular homeostasis and may have both direct and indirect influence on pathogenic mechanisms associated with neurological disorders. Creatine is a critical component in maintaining cellular energy homeostasis, and its administration has been reported to be neuroprotective in a wide number of both acute and chronic experimental models of neurological disease. In the context of this chapter, we will review the experimental evidence for creatine supplementation as a neurotherapeutic strategy in patients with neurological disorders, including Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Alzheimer's disease, as well as in ischemic stroke, brain and spinal cord trauma, and epilepsy.

Interactions between mutant huntingtin (Htt) and a variety of transcription factors including specificity proteins (Sp) have been suggested as a central mechanism in Huntington disease (HD). However, the transcriptional activity induced by Htt in neurons that triggers neuronal death has yet to be fully elucidated. In the current study, we characterized the relationship of Sp1 to Htt protein aggregation and neuronal cell death. We found increased levels of Sp1 in neuronal-like PC12 cells expressing mutant Htt, primary striatal neurons, and brain tissue of HD transgenic mice. Sp1 levels were also elevated when 3-nitropropionate (3-NP) was used to induce cell death in PC12 cells. To assess the effects of knocking down Sp1 in HD pathology, we used Sp1 siRNA, a heterozygous Sp1 knock-out mouse, and mithramycin A, a DNA-intercalating agent that inhibits Sp1 function. The three approaches consistently yielded reduced levels of Sp1 which ameliorated toxicity caused by either mutant Htt or 3-NP. In addition, when HD mice were crossed with Sp1 heterozygous knock-out mice, the resulting offspring did not experience the loss of dopamine D2 receptor mRNA characteristic of HD mice, and survived longer than their HD counterparts. Our data suggest that enhancement of transcription factor Sp1 contributes to the pathology of HD and demonstrates that its suppression is beneficial.

Cyclic AMP response element-binding protein (CREB) is a widely expressed transcription factor whose role in neuronal protection is now well established. Here we report that CREB is present in the mitochondrial matrix of neurons and that it binds directly to cyclic AMP response elements (CREs) found within the mitochondrial genome. Disruption of CREB activity in the mitochondria decreases the expression of a subset of mitochondrial genes, including the ND5 subunit of complex I, down-regulates complex I-dependent mitochondrial respiration, and increases susceptibility to 3-nitropropionic acid, a mitochondrial toxin that induces a clinical and pathological phenotype similar to Huntington disease. These results demonstrate that regulation of mitochondrial gene expression by mitochondrial CREB, in part, underlies the protective effects of CREB and raise the possibility that decreased mitochondrial CREB activity contributes to the mitochondrial dysfunction and neuronal loss associated with neurodegenerative disorders.

Genetic murine models play an important role in the study of human neurological disorders by providing accurate and experimentally accessible systems to study pathogenesis and to test potential therapeutic treatments. One of the most widely employed models of Huntington's disease (HD) is the R6/2 transgenic mouse. To characterize this model further, we have performed behavioral and neuropathological analyses that provide a foundation for the use of R6/2 mice in preclinical therapeutic trials. Behavioral analyses of the R6/2 mouse reveal age-related impairments in dystonic movements, motor performance, grip strength, and body weight that progressively worsen until death. Significant neuropathological sequela, identified as increasing marked reductions in brain weight, are present from 30 days, whereas decreased brain volume is present from 60 days and decreased neostriatal volume and striatal neuron area, with a concomitant reduction in striatal neuron number, are present at 90 days of age. Huntingtin-positive aggregates are present at postnatal day 1 and increase in number and size with age. Our findings suggest that the R6/2 HD model exhibits a progressive HD-like behavioral and neuropathological phenotype that more closely corresponds to human HD than previously believed, providing further assurance that the R6/2 mouse is an appropriate model for testing potential therapies for HD., (Copyright 2005 Wiley-Liss, Inc.)

Genetic animal models of inherited neurological diseases provide an opportunity to test potential treatments and explore their promise for translation to humans experiencing these diseases. Therapeutic trials conducted in mouse models of Huntington's disease have identified a growing number of potential therapies that are candidates for clinical trials. Although it is very exciting to have these candidates, there has been increasing concern about the feasibility and desirability of taking all of the compounds that may work in mice and testing them in patients with HD. There is a need to begin to prioritize leads emerging from transgenic mouse studies; however, it is difficult to compare results between compounds and laboratories, and there are also many additional factors that can affect translation to humans. Among the important issues are what constitutes an informative genetic model, what principals should be followed in designing and conducting experiments using genetic animal models, how can results from different laboratories and in different models be compared, what body of evidence is desirable to fully inform clinical decision making, and what factors contribute to the equipoise in determining whether preclinical information about a therapy makes clinical study warranted. In the context of Huntington's disease, we will review the current state of genetic models and their successes in putting forward therapeutic leads, provide a guide to assessing studies in mouse models, and discuss some of the salient issues related to translation from mice to humans.

Creatine mediates remarkable neuroprotection in experimental models of amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, and traumatic brain injury. Because caspase-mediated pathways are shared functional mechanistic components in these diseases, as well as in ischemia, we evaluated the effect of creatine supplementation on an experimental stroke model. Oral creatine administration resulted in a remarkable reduction in ischemic brain infarction and neuroprotection after cerebral ischemia in mice. Postischemic caspase-3 activation and cytochrome c release were significantly reduced in creatine-treated mice. Creatine administration buffered ischemia-mediated cerebral ATP depletion. These data provide the first direct correlation between the preservation of bioenergetic cellular status and the inhibition of activation of caspase cell-death pathways in vivo. An alternative explanation to our findings is that creatine is neuroprotective through other mechanisms that are independent of mitochondrial cell-death pathways, and therefore postischemic ATP preservation is the result of tissue sparing. Given its safety record, creatine might be considered as a novel therapeutic agent for inhibition of ischemic brain injury in humans. Prophylactic creatine supplementation, similar to what is recommended for an agent such as aspirin, may be considered for patients in high stroke-risk categories.

Adenosine A2A receptor (A2AR) antagonism attenuates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration and quinolinic acid-induced excitotoxicity in the neostriatum. As A2ARs are enriched in striatum, we investigated the effect of genetic and pharmacological A2A inactivation on striatal damage produced by the mitochondrial complex II inhibitor 3-nitropriopionic acid (3-NP). 3-NP was administered to A2AR knockout (KO) and wild-type (WT) littermate mice over 5 days. Bilateral striatal lesions were analyzed from serial brain tissue sections. Whereas all of the 3-NP-treated WT mice (C57BL/6 genetic background) had bilateral striatal lesions, only one of eight of the 3-NP-treated A2AR KO mice had detectable striatal lesions. Similar attenuation of 3-NP-induced striatal damage was observed in A2AR KO mice in a 129-Steel background. In addition, the effect of pharmacological antagonism on 3-NP-induced striatal neurotoxicity was tested by pre-treatment of C57Bl/6 mice with the A2AR antagonist 8-(3-chlorostyryl) caffeine (CSC). Although bilateral striatal lesions were observed in all mice treated either with 3-NP alone or 3-NP plus vehicle, there were no demonstrable striatal lesions in mice treated with CSC (5 mg/kg) plus 3-NP and in five of six mice treated with CSC (20 mg/kg) plus 3-NP. We conclude that both genetic and pharmacological inactivation of the A2AR attenuates striatal neurotoxicity produced by 3-NP. Since the clinical and neuropathological features of 3-NP-induced striatal damage resemble those observed in Huntington's disease, the results suggest that A2AR antagonism may be a potential therapeutic strategy in Huntington's disease patients.

Huntington's disease (HD) is caused by a mutation in the gene encoding for huntingtin resulting in selective neuronal degeneration. Because HD is an autosomal dominant disorder, affected individuals have one copy of the mutant and one copy of the wild-type allele. Huntingtin has antiapoptotic properties and is critical for cell survival. However, the important role of wild-type huntingtin in both HD and other neurological diseases has not been fully recognized. We demonstrate disease-associated decreased levels of full-length huntingtin in brains of transgenic mouse models of HD, ischemia, trauma, and in spinal cord after injury. In addition, overexpression of wild-type huntingtin confers in vivo protection of neurodegeneration after ischemia. We propose that in HD, in addition to a toxic gain-of-function of mutant huntingtin, a parallel depletion of wild-type huntingtin results in a detrimental loss-of-function, playing an important role in disease progression.

Minocycline is broadly protective in neurologic disease models featuring cell death and is being evaluated in clinical trials. We previously demonstrated that minocycline-mediated protection against caspase-dependent cell death related to its ability to prevent mitochondrial cytochrome c release. These results do not explain whether or how minocycline protects against caspase-independent cell death. Furthermore, there is no information on whether Smac/Diablo or apoptosis-inducing factor might play a role in chronic neurodegeneration. In a striatal cell model of Huntington's disease and in R6/2 mice, we demonstrate the association of cell death/disease progression with the recruitment of mitochondrial caspase-independent (apoptosis-inducing factor) and caspase-dependent (Smac/Diablo and cytochrome c) triggers. We show that minocycline is a drug that directly inhibits both caspase-independent and -dependent mitochondrial cell death pathways. Furthermore, this report demonstrates recruitment of Smac/Diablo and apoptosis-inducing factor in chronic neurodegeneration. Our results further delineate the mechanism by which minocycline mediates its remarkably broad neuroprotective effects.

While there have been enormous strides in the understanding of Huntington's disease (HD) pathogenesis, treatment to slow or prevent disease progression remains elusive. We previously reported that dietary creatine supplementation significantly improves the clinical and neuropathological phenotype in transgenic HD mice lines starting at weaning, before clinical symptoms appear. We now report that creatine administration started after onset of clinical symptoms significantly extends survival in the R6/2 transgenic mouse model of HD. Creatine treatment started at 6, 8, and 10 weeks of age, analogous to early, middle, and late stages of human HD, significantly extended survival at both the 6- and 8-week starting points. Significantly improved motor performance was present in both the 6- and 8-week treatment paradigms, while reduced body weight loss was only observed in creatine-supplemented R6/2 mice started at 6 weeks. Neuropathological sequelae of gross brain and neuronal atrophy and huntingtin aggregates were delayed in creatine-treated R6/2 mice started at 6 weeks. We show significantly reduced brain levels of both creatine and ATP in R6/2 mice, consistent with a bioenergetic defect. Oral creatine supplementation significantly increased brain concentrations of creatine and ATP to wild-type control levels, exerting a neuroprotective effect. These findings have important therapeutic implications, suggesting that creatine therapy initiated after diagnosis may provide significant clinical benefits to HD patients.

L-Arginine is the only endogenous nitrogen-containing substrate of NO synthase (NOS), and it thus governs the production of NO during nervous system development as well as in disease states such as stroke, multiple sclerosis, Parkinson's disease, and HIV dementia. The "arginine paradox" refers to the dependence of cellular NO production on exogenous L-arginine concentration despite the theoretical saturation of NOS enzymes with intracellular L-arginine. Herein, we report that decreased availability of L-arginine blocked induction of NO production in cytokine-stimulated astrocytes, owing to inhibition of inducible NOS (iNOS) protein expression. However, activity of the promoter of the iNOS gene, induction of iNOS mRNA, and stability of iNOS protein were not inhibited under these conditions. Our results indicate that inhibition of iNOS activity by arginine depletion in stimulated astrocyte cultures occurs via inhibition of translation of iNOS mRNA. After stimulation by cytokines, uptake of L-arginine negatively regulates the phosphorylation status of the eukaryotic initiation factor (eIF2 alpha), which, in turn, regulates translation of iNOS mRNA. eIF2 alpha phosphorylation correlates with phosphorylation of the mammalian homolog of yeast GCN2 eIF2 alpha kinase. As the kinase activity of GCN2 is activated by phosphorylation, these findings suggest that GCN2 activity represents a proximal step in the iNOS translational regulation by availability of l-arginine. These results provide an explanation for the arginine paradox for iNOS and define a distinct mechanism by which a substrate can regulate the activity of its associated enzyme.

Oxidative stress is believed to be an important mediator of neurodegeneration. However, the transcriptional pathways induced in neurons by oxidative stress that activate protective gene responses have yet to be fully delineated. We report that the transcription factor Sp1 is acetylated in response to oxidative stress in neurons. Histone deacetylase (HDAC) inhibitors augment Sp1 acetylation, Sp1 DNA binding, and Sp1-dependent gene expression and confer resistance to oxidative stress-induced death in vitro and in vivo. Sp1 activation is necessary for the protective effects of HDAC inhibitors. Together, these results demonstrate that HDAC inhibitors inhibit oxidative death independent of polyglutamine expansions by activating an Sp1-dependent adaptive response.

Minocycline mediates neuroprotection in experimental models of neurodegeneration. It inhibits the activity of caspase-1, caspase-3, inducible form of nitric oxide synthetase (iNOS) and p38 mitogen-activated protein kinase (MAPK). Although minocycline does not directly inhibit these enzymes, the effects may result from interference with upstream mechanisms resulting in their secondary activation. Because the above-mentioned factors are important in amyotrophic lateral sclerosis (ALS), we tested minocycline in mice with ALS. Here we report that minocycline delays disease onset and extends survival in ALS mice. Given the broad efficacy of minocycline, understanding its mechanisms of action is of great importance. We find that minocycline inhibits mitochondrial permeability-transition-mediated cytochrome c release. Minocycline-mediated inhibition of cytochrome c release is demonstrated in vivo, in cells, and in isolated mitochondria. Understanding the mechanism of action of minocycline will assist in the development and testing of more powerful and effective analogues. Because of the safety record of minocycline, and its ability to penetrate the blood-brain barrier, this drug may be a novel therapy for ALS.

There is substantial evidence that bioenergetic defects and excitotoxicity may play a role in the pathogenesis of Huntington's disease (HD). Potential therapeutic strategies for neurodegenerative diseases in which there is reduced energy metabolism and NMDA-mediated excitotoxicity are the administration of the mitochondrial cofactor coenzyme Q10 and the NMDA antagonist remacemide. We found that oral administration of either coenzyme Q10 or remacemide significantly extended survival and delayed the development of motor deficits, weight loss, cerebral atrophy, and neuronal intranuclear inclusions in the R6/2 transgenic mouse model of HD. The combined treatment, using coenzyme Q10 and remacemide together, was more efficacious than either compound alone, resulting in an approximately 32 and 17% increase in survival in the R6/2 and N171-82Q mice, respectively. Magnetic resonance imaging showed that combined treatment significantly attenuated ventricular enlargement in vivo. These studies further implicate defective energy metabolism and excitotoxicity in the R6/2 and N171-82Q transgenic mouse models of HD and are of interest in comparison with the outcome of a recent clinical trial examining coenzyme Q10 and remacemide in HD patients.

There is increasing evidence implicating apoptosis-mediated cell death in the pathogenesis of neurodegenerative diseases. One important event in the apoptotic cascade is the release of cytochrome c by mitochondria into the cytoplasm, activating caspase-9, leading to the subsequent activation of downstream executioner caspases. In the present study, we examined the distribution of cytochrome c and caspase-9 in Huntington's disease (HD) patients and in a transgenic model of HD (R6/2 line). Neuronal cytochrome c immunoreactivity increased with neuropathological severity in HD patients. Concomitant with this finding, Western-blot analysis showed a shift in the distribution of cytochrome c from the mitochondrial to the cytosolic fraction with incremental cytosolic expression associated with greater striatal degeneration. Active caspase-9 immunoreactivity was present in both HD striatal neurons and in Western blots of severe-grade specimens. Similar findings were observed in the R6/2 mice. There was a temporal increase in expression and shift of cytochrome c from the mitochondrial to the cytosolic fraction from 4-13 wk of age. Activated caspase-9 and caspase 3 activities were present only at endstage disease. Although the present results provide evidence that key components of the intrinsic mitochondrial apoptotic pathway are activated in both HD patients and a transgene murine model of HD, these phenomena are prominent in only severe neuropathological grades in HD patients and HD mice, suggesting that apoptosis may play a greater role in neuronal death at endstage disease.