Existing protocols for the neural differentiation of mouse embryonic stem (ES) cells require extended in vitro culture, yield variable differentiation results or are limited to the generation of selected neural subtypes. Here we provide a set of coculture conditions that allows rapid and efficient derivation of most central nervous system phenotypes. The fate of both fertilization- and nuclear transfer-derived ES (ntES) cells was directed selectively into neural stem cells, astrocytes, oligodendrocytes or neurons. Specific differentiation into gamma-aminobutyric acid (GABA), dopamine, serotonin or motor neurons was achieved by defining conditions to induce forebrain, midbrain, hindbrain and spinal cord identity. Neuronal function of ES cell-derived dopaminergic neurons was shown in vitro by electron microscopy, measurement of neurotransmitter release and intracellular recording. Furthermore, transplantation of ES and ntES cell-derived dopaminergic neurons corrected the phenotype of a mouse model of Parkinson disease, demonstrating an in vivo application of therapeutic cloning in neural disease.

A growing body of evidence suggests a relationship between oxidative stress and b-amyloid (Ab) peptide accumulation, a hallmark in the pathogenesis of Alzheimer's disease (AD). However, a direct causal relationship between oxidative stress and Ab pathology has not been established in vivo. Therefore, we crossed mice with a knockout of one allele of manganese superoxide dismutase (MnSOD), a critical antioxidant enzyme, with Tg19959 mice, which overexpress a doubly mutated human b-amyloid precursor protein (APP). Partial deficiency of MnSOD, which is well established to cause elevated oxidative stress, significantly increased brain Ab levels and Ab plaque burden in Tg19959 mice. These results indicate that oxidative stress can promote the pathogenesis of AD and further support the feasibility of antioxidant approaches for AD therapy.

We investigated the role of PPAR gamma coactivator 1alpha (PGC-1alpha) in muscle dysfunction in Huntington's disease (HD). We observed reduced PGC-1alpha and target genes expression in muscle of HD transgenic mice. We produced chronic energy deprivation in HD mice by administering the catabolic stressor beta-guanidinopropionic acid (GPA), a creatine analogue that reduces ATP levels, activates AMP-activated protein kinase (AMPK), which in turn activates PGC-1alpha. Treatment with GPA resulted in increased expression of AMPK, PGC-1alpha target genes, genes for oxidative phosphorylation, electron transport chain and mitochondrial biogenesis, increased oxidative muscle fibers, numbers of mitochondria and motor performance in wild-type, but not in HD mice. In muscle biopsies from HD patients, there was decreased PGC-1alpha, PGC-1beta and oxidative fibers. Oxygen consumption, PGC-1alpha, NRF1 and response to GPA were significantly reduced in myoblasts from HD patients. Knockdown of mutant huntingtin resulted in increased PGC-1alpha expression in HD myoblast. Lastly, adenoviral-mediated delivery of PGC-1alpha resulted increased expression of PGC-1alpha and markers for oxidative muscle fibers and reversal of blunted response for GPA in HD mice. These findings show that impaired function of PGC-1alpha plays a critical role in muscle dysfunction in HD, and that treatment with agents to enhance PGC-1alpha function could exert therapeutic benefits. Furthermore, muscle may provide a readily accessible tissue in which to monitor therapeutic interventions.

In Alzheimer's disease (AD), oxidative stress is present early and contributes to disease pathogenesis. We previously reported that in Tg19959 transgenic AD mice, partial deficiency of the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) exacerbated amyloid pathology. We therefore asked whether MnSOD overexpression would prove beneficial against AD pathogenesis, by studying the offspring of Tg19959 mice crossed with MnSOD-overexpressing mice. At 4 mo of age, there was a 2- to 3-fold increase in MnSOD protein levels in Tg19959-MnSOD mice compared to Tg19959 littermates. Tg19959-MnSOD mice also had a 50% increase in catalase protein levels, a 50% decrease in levels of oxidized protein, and a 33% reduction in cortical plaque burden compared to Tg19959 littermates. Spatial memory was impaired and synaptophysin levels were decreased in Tg19959 mice compared to wild-type littermates, but memory and synaptophysin levels were restored to wild-type levels in Tg19959-MnSOD littermates. These benefits occurred without changes in sodium dodecyl sulfate-soluble or formic acid-soluble Abeta pools or Abeta oligomers in Tg19959-MnSOD mice compared to Tg19959 littermates. These data demonstrate that facilitation of the mitochondrial antioxidant response improves resistance to Abeta, slows plaque formation or increases plaque degradation, and markedly attenuates the phenotype in a transgenic AD mouse model.

A promising approach to neurotherapeutics involves activating the nuclear-factor-E2-related factor 2 (Nrf2)/antioxidant response element signaling, which regulates expression of antioxidant, anti-inflammatory, and cytoprotective genes. Tecfidera, a putative Nrf2 activator, is an oral formulation of dimethylfumarate (DMF) used to treat multiple sclerosis. We compared the effects of DMF and its bioactive metabolite monomethylfumarate (MMF) on Nrf2 signaling and their ability to block 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced experimental Parkinson's disease (PD). We show that in vitro DMF and MMF activate the Nrf2 pathway via S-alkylation of the Nrf2 inhibitor Keap1 and by causing nuclear exit of the Nrf2 repressor Bach1. Nrf2 activation by DMF but not MMF was associated with depletion of glutathione, decreased cell viability, and inhibition of mitochondrial oxygen consumption and glycolysis rates in a dose-dependent manner, whereas MMF increased these activities in vitro. However, both DMF and MMF upregulated mitochondrial biogenesis in vitro in an Nrf2-dependent manner. Despite the in vitro differences, both DMF and MMF exerted similar neuroprotective effects and blocked MPTP neurotoxicity in wild-type but not in Nrf2 null mice. Our data suggest that DMF and MMF exhibit neuroprotective effects against MPTP neurotoxicity because of their distinct Nrf2-mediated antioxidant, anti-inflammatory, and mitochondrial functional/biogenetic effects, but MMF does so without depleting glutathione and inhibiting mitochondrial and glycolytic functions. Given that oxidative damage, neuroinflammation, and mitochondrial dysfunction are all implicated in PD pathogenesis, our results provide preclinical evidence for the development of MMF rather than DMF as a novel PD therapeutic.Key words: fumarates; inflammation; mitochondria; MPTP; Nrf2; oxidative stress Significance StatementAlmost two centuries since its first description by James Parkinson, Parkinson's disease (PD) remains an incurable disease with limited symptomatic treatment. The current study provides preclinical evidence that a Food and Drug Administration-approved drug, dimethylfumarate (DMF), and its metabolite monomethylfumarate (MMF) can block nigrostriatal dopaminergic neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of PD. We elucidated mechanisms by which DMF and its active metabolite MMF activates the redox-sensitive transcription factor nuclear-factor-E2-related factor 2 (Nrf2) to upregulate antioxidant, anti-inflammatory, mitochondrial biosynthetic and cytoprotective genes to render neuroprotection via distinct S-alkylating properties and depletion of glutathione. Our data suggest that targeting Nrf2-mediated gene transcription using MMF rather than DMF is a promising approach to block oxidative stress, neuroinflammation, and mitochondrial dysfunction for therapeutic intervention in PD while minimizing side effects.