Nicotinamide improves motor deficits and upregulates PGC-1α and BDNF gene expression in a mouse model of Huntington's disease
Research Highlights
► Nicotinamide (NAM) treatment improves motor dysfunction in HD mice. ► Nicotinamide also improves BDNF and PGC-1α mRNA and protein levels. ► This drug should therefore be strongly considered as a symptomatic HD therapeutic.
Introduction
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor dysfunction, emotional disturbances, cognitive dysfunction, weight loss, and premature death. HD is caused by an expansion of a trinucleotide (CAG)n repeat in the coding sequence of the huntingtin (HTT) gene on chromosome-4, resulting in an expanded polyglutamine (polyQ) tract in huntingtin (htt), with the age of onset inversely correlated with repeat number (Duyao et al., 1993). HD, along with Alzheimer's disease, Parkinson's disease (PD), spinal cerebellar ataxia, and prion-based dementia, is classified as a misfolded protein disorder, based on the characteristic intranuclear and cytoplasmic pathogenic aggregations of truncated and full-length htt protein observed in human patients and in mouse models of HD (Davies et al., 1997, DiFiglia et al., 1997). The HDR6/1 transgenic mouse model contains human huntingtin gene exon-1 under the control of the endogenous promoter, with approximately 115 CAG repeats. In this model, overt neurological symptoms similar to human HD become evident at 15–21 weeks of age, with premature death occurring at 32–40 weeks (Mangiarini et al., 1996). Behavioral abnormalities have been documented as early as 4 weeks of age (Bolivar et al., 2004).
Altered transcription of genes involved in cellular processes vital to neuronal function and survival has been demonstrated in mouse and human HD (Luthi-Carter et al., 2000, Zuccato et al., 2001, Zuccato et al., 2003) suggesting a direction for therapies. Acetylation and deacetylation of histones play an important role in regulation of chromatin condensation and gene transcription (Roth et al., 2001); therefore, increasing histone acetyltransferase activity, through inhibition of the histone deacetylases (HDACs), has been used as a strategy to slow the progression of many neurological disorders. The efficacy of HDAC inhibition in HD was first observed in a Drosophila model (Steffan et al., 2001), with suberoylanilide hydroxamic acid blocking photoreceptor neurodegeneration and increasing survival. In addition, HDAC inhibitors have provided phenotypic improvement in HD transgenic mouse models (Ferrante et al., 2003, Hockly et al., 2003).
The superfamily of HDACs consists of five main subtypes (Butler and Bates, 2006), including the structurally distinct class III which contains the family of sirtuins. Sirtuins comprise a unique class of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases (Imai et al., 2000) that are involved in diverse biological functions such as metabolism, cell division and aging (Taylor et al., 2008). To date, seven mammalian homologues have been identified, with mammalian SIRT1 closest evolutionarily to yeast Sir2 (silent information regulator 2), the founding member of the sirtuin family (Rine et al., 1979).
SIRT1 is a multifunctional protein that regulates diverse cellular functions through deacetylation of important transcription factors, such as p53, forkhead subgroup O (FOXO) proteins and the DNA repair factor KU (Guarente, 2006). The observation that sirtuin activity requires NAD+ suggests a mechanistic link between sirtuin activity and intracellular energetics (Imai et al., 2000). The peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) is another well-known target of SIRT1 and its activation requires SIRT1 deacetylation (Nemoto et al., 2005). PGC-1α is a key regulator of mitochondrial biogenesis and respiration, modulating expression of several transcription factors (McGill and Beal, 2006).
Although the actions of sirtuins in the nervous system are only beginning to be explored, it has been reported that pharmacological modulation of SIRT1 can have significance in managing neurodegenerative diseases (Green et al., 2008, Lagouge et al., 2006). An early in vivo study using nicotinamide (NAM), also known as vitamin B3 or niacin, identified it as an effective suppressor of polyglutamine toxicity in a model of spinocerebellar ataxia (Ghosh and Feany, 2004). In addition to being a precursor for ATP, NAM is classified as a class III HDAC inhibitor, because of its role as a SIRT1 inhibitor (Bitterman et al., 2002). More recently, researchers have shown that high doses of NAM improved cognition in a mouse model of Alzheimer's disease (Green et al., 2008), reduced motor deficits in a rodent PD model (Jia et al., 2008) and reduced both neuronal loss and motor impairment in the Drosophila model of HD (Pallos et al., 2008). Thus, we hypothesized that NAM will be effective in alleviating HD pathology in the B6.HDR6/1 mouse model.
NAM has been evaluated for its potential as a therapeutic for a range of disorders, from heart disease to juvenile diabetes; with doses as high as 6 g/day with no major signs of toxicity (Knip et al., 2000). Most important to this study, NAM injected sub-cutaneously into rats has been shown to increase NAM levels in plasma, CSF, and brain (Erb and Klein, 1998). NAM is therefore pharmacologically feasible as an HD therapeutic.
Section snippets
Animals
B6.HD6/1 mice were generated and maintained by breeding transgenic males to C57BL/6J females (Jax # 000664). Offspring were genotyped by polymerase chain reaction (PCR) from tail biopsy. HDR6/1 mice, a moderate fragment model of HD, have human HTT Exon 1 with 1kB of the endogenous promoter (Mangiarini et al., 1996). This model recapitulates several phenotypes found in human HD patients, including neuropathology with nuclear and cytoplasmic aberrations in the corpus striatum and hippocampus,
NAM improves transcriptional dysregulation of BDNF and PGC-1α, restores BDNF protein levels and increases acetylated PGC-1α
Eight week old B6.HDR6/1 mice were treated with 250 mg/kg of NAM for 10 weeks via two consecutive minipumps, or for 12 weeks in drinking water. At the end of the treatment period, the animals were assessed for gene expression changes that we hypothesized would improve after NAM treatment. Importantly, neither mode of administration nor sex affected outcomes.
Brain-derived neurotrophic factor (BDNF) is critical to neuronal survival in the CNS, and has been shown to be down-regulated in HD patients
Discussion
The use of HDAC inhibitors to improve HD-mediated gene dysregulation is an appealing strategy, although it may result in non-specific targeting effects (Shao and Diamond, 2007). In addition, the long-term use of HDAC inhibitors in humans may be limited by high toxicity. In this study we use a class III HDAC (sirtuin) inhibitor that is in long-term human clinical use for other disorders, and that produces no apparent toxic effects here in the HD mice. Our results compare favorably with those
Conclusions
This study demonstrates that NAM can increase gene expression of both BDNF and PGC-1α, indicating a beneficial effect. We hypothesized that this correction would alleviate the motor phenotype seen in HD mice. Indeed, we found that this small molecule can alleviate motor deficits in B6.HDR6/1 mice. NAM administration via either of two modes was able to increase exploratory behavior in B6.HDR6/1 mice. More extensive investigation of behavioral changes demonstrated improvements in rotarod
Acknowledgments
We thank Kevin Manley for expert assistance with the statistical analyses, Lizbeth Day for HD mouse assistance, Drs. David Butler for valuable consults on the SDS-AGE and RT-PCR, Valerie Bolivar for discussions of the behavioral testing, and Erik Kvam for valuable discussions and suggestions. This work was supported by NIH/NINDS to A. M. (NS053912), and a graduate student diversity supplement of NS053912 to T.H.
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