The inherited motor neuron disease spinal muscular atrophy (SMA) is caused by mutation of the telomeric survival motor neuron 1 (SMN1) gene with retention of the centromeric SMN2 gene. We sought to establish whether the potent and specific hydroxamic acid class of histone deacetylase (HDAC) inhibitors activates SMN2 gene expression in vivo and modulates the SMA disease phenotype when delivered after disease onset. Single intraperitoneal doses of 10 mg/kg trichostatin A (TSA) in nontransgenic and SMA model mice resulted in increased levels of acetylated H3 and H4 histones and modest increases in SMN gene expression. Repeated daily doses of TSA caused increases in both SMN2-derived transcript and SMN protein levels in neural tissues and muscle, which were associated with an improvement in small nuclear ribonucleoprotein (snRNP) assembly. When TSA was delivered daily beginning on P5, after the onset of weight loss and motor deficit, there was improved survival, attenuated weight loss, and enhanced motor behavior. Pathological analysis showed increased myofiber size and number and increased anterior horn cell size. These results indicate that the hydroxamic acid class of HDAC inhibitors activates SMN2 gene expression in vivo and has an ameliorating effect on the SMA disease phenotype when administered after disease onset.

Increasing survival motor neuron 2 (SMN2) gene expression may be an effective strategy for the treatment of spinal muscular atrophy (SMA). Histone deacetylase (HDAC) inhibitors have been shown to increase SMN transcript and protein levels, but the specific role of histone acetylation in regulating SMN gene expression has not been explored. Using chromatin immunopreciptation, we investigated the levels of acetylated H3 and H4 histones and HDACs associated with different regions of the human and mouse SMN genes in both cultured cells and tissues. We show that the SMN gene has a reproducible pattern of histone acetylation that is largely conserved among different tissues and species. A limited region of the promoter surrounding the transcriptional start site has relatively high levels of histone acetylation, whereas regions further upstream or downstream have lower levels. After HDAC inhibitor treatment, acetylated histone levels increased, particularly at upstream regions, correlating with a 2-fold increase in promoter activity. During development in mouse tissues, histone acetylation levels decreased and associated HDAC2 levels increased at the region closest to the transcriptional start site, correlating with a 40-60% decrease in SMN transcript and protein levels. These data indicate that histone acetylation modulates SMN gene expression and that pharmacological manipulation of this epigenetic determinant is feasible. HDAC2, in particular, may be a future therapeutic target for SMA.

ABSTRACT3 H]epibatidine were found: one that was increased about 4-fold in cells grown for 2 to 4 days in the presence of nicotine and one that was increased 5-fold in cells grown for 2 to 4 days in the presence of nerve growth factor (NGF). The actions of the two treatments were superadditive, resulting in approximately a 13-fold increase in binding sites in cells grown in the combination of the two treatments. The pharmacology of the binding sites in the nicotine-and NGF-treated cells was compared with the pharmacology of defined ␣3␤2 and ␣3␤4 nicotinic acetylcholine receptor (nAChR) subtypes heterologously expressed in human embryonic kidney 293 cells. Nicotine treatment predominantly increased a receptor with characteristics of an ␣3␤2 subtype, whereas the NGF treatment exclusively increased a receptor with characteristics of an ␣3␤4 subtype. Nicotinic receptormediated function measured with the 86 Rb ϩ efflux assay was evident only in the NGF-treated cells, and it had a pharmacological profile that was, again, nearly identical to that of the heterologously expressed ␣3␤4 receptor subtype. Receptor function measured with the [ 3 H]norepinephrine release assay was measurable in both nicotine-treated and NGF-treated cells; however, cytisine-stimulated [ 3 H]norepinephrine release indicated that nicotine treatment increased an nAChR containing ␤2 subunits, whereas NGF increased a receptor containing ␤4 subunits. NGF treatment increased mRNA only for ␤4 subunits in these cells, whereas nicotine treatment did not affect mRNA for any of the subunits measured. After withdrawal of the treatments, the receptors increased by nicotine were much less stable than those increased by NGF.Neuronal nicotinic acetylcholine receptors (nAChRs) are found throughout the central nervous system as well as in autonomic ganglia and the adrenal gland, where they mediate cholinergic neurotransmission critical to the functions of the autonomic nervous system. These receptors are composed of two types of subunits, ␣ and ␤. To date, nine neuronal ␣ subunit genes (␣2-␣10) and three ␤ subunit genes (␤2-␤4) have been found in vertebrate tissues. Different combinations of subunits compose subtypes of nicotinic receptors, all of which are ligand-gated cation channels that conduct Na ϩ , K ϩ , and Ca 2ϩ when activated by the endogenous neurotransmitter acetylcholine or by nicotine or other nicotinic agonists. However, the different receptor subtypes have distinguishing biophysical and/or pharmacological properties, including channel conductances, rates of desensitization and recovery, and sensitivity to drugs (for reviews, see Sargent, 1993;Colquhoun and Patrick, 1997;Lukas, 1998).Certain subtypes of nAChRs are strongly regulated by nicotine. Previous studies have shown that chronic administration of nicotine increases nicotinic receptor binding sites in rat and mouse brain (Schwartz and Kellar, 1983;Marks et al., 1983; for review, see Gentry and Lukas, 2002), and a similar increase is found in human brains from people who smoked tobacco (Benwe...