To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine stretch in the protein huntingtin (Htt). HD neurons are dysfunctional at multiple levels and have increased susceptibility to stress and apoptotic stimuli. We have discovered that synthesis of the ganglioside GM1 is reduced in fibroblasts from HD patients and in cell and animal models of HD, and that decreased GM1 levels contribute to heighten HD cell susceptibility to apoptosis. The apoptotic susceptibility is recapitulated through inhibition of ganglioside synthesis in wild-type striatal cells, suggesting that decreased GM1 levels might be one of the key events leading to HD pathogenesis and progression. Administration of GM1 restores ganglioside levels in HD cells and promotes activation of AKT and phosphorylation of mutant Htt, leading to decreased mutant Htt toxicity and increased survival of HD cells. Our data identify GM1 as a potential treatment for HD.

Huntington disease (HD) is a progressive neurodegenerative monogenic disorder caused by expansion of a polyglutamine stretch in the huntingtin (Htt) protein. Mutant huntingtin triggers neural dysfunction and death, mainly in the corpus striatum and cerebral cortex, resulting in pathognomonic motor symptoms, as well as cognitive and psychiatric decline. Currently, there is no effective treatment for HD. We report that intraventricular infusion of ganglioside GM1 induces phosphorylation of mutant huntingtin at specific serine amino acid residues that attenuate huntingtin toxicity, and restores normal motor function in already symptomatic HD mice. Thus, our studies have identified a potential therapy for HD that targets a posttranslational modification of mutant huntingtin with critical effects on disease pathogenesis.H untington disease (HD) is an inherited neurodegenerative monogenic disorder caused by the expansion of a polyglutamine stretch beyond 36 residues in the amino-terminal domain of huntingtin (Htt), a protein expressed in most tissues and cells. The mutation causes huntingtin to acquire toxic conformation/s and to affect neuronal function and viability. Medium-sized spiny neurons in the corpus striatum are most affected, but neurodegeneration also occurs in the cerebral cortex and, to a minor extent, in other brain areas, resulting in motor and psychiatric symptoms, as well as cognitive decline.The cellular and molecular mechanisms underlying HD pathogenesis are complex. Both loss and gain of function of mutant huntingtin contribute to cause a wide array of neuronal dysfunctions affecting cell signaling, gene transcription, axonal transport, cell and mitochondrial metabolism as well as neurotransmission (1).In recent years, a breakthrough in HD research has been the discovery that posttranslational modifications of mutant Htt are crucial modulators of mutant Htt toxicity (2-4). Phosphorylation at various serine residues prevents cleavage of mutant huntingtin into more toxic fragments, decreases neural cell death in vitro (5-10), and/or restores Htt functions that are compromised by the mutation (8, 11). The most dramatic effects have been described for huntingtin phosphorylation at serine 13 and serine 16. These two amino acid residues are part of the highly conserved amino-terminal "N17" domain of huntingtin, a domain that regulates huntingtin intracellular localization and association to cellular membranes (12, 13), as well as kinetics of mutant huntingtin aggregation (14,15). Phosphomimetic mutations of serine 13 and serine 16 by aspartic or glutamic acid substitution (S13D and S16D or S13E and S16E) decrease the toxicity of mutant huntingtin fragments in vitro (10, 16). In line with these studies, expression of a phosphomimetic (S13D and S16D) mutant form of expanded full-length huntingtin in a BACHD transgenic mouse model was shown to result in a normal phenotype, with no detectable signs of HD pathology by 12 mo (17).These findings suggest that pharmacological interventions that modulate cell sig...

Huntington disease (HD) is a genetic neurodegenerative disorder for which there is currently no cure and no way to stop or even slow the brain changes it causes. In the present study, we aimed to investigate whether FTY720, the first approved oral therapy for multiple sclerosis, may be effective in HD models and eventually constitute an alternative therapeutic approach for the treatment of the disease. Here, we utilized preclinical target validation paradigms and examined the in vivo efficacy of chronic administration of FTY720 in R6/2 HD mouse model. Our findings indicate that FTY720 improved motor function, prolonged survival and reduced brain atrophy in R6/2 mice. The beneficial effect of FTY720 administration was associated with a significant strengthening of neuronal activity and connectivity and, with reduction of mutant huntingtin aggregates, and it was also paralleled by increased phosphorylation of mutant huntingtin at serine 13/16 residues that are predicted to attenuate protein toxicity.

Huntington’s disease is characterized by a complex and heterogeneous pathogenic profile. Studies have shown that disturbance in lipid homeostasis may represent a critical determinant in the progression of several neurodegenerative disorders. The recognition of perturbed lipid metabolism is only recently becoming evident in HD. In order to provide more insight into the nature of such a perturbation and into the effect its modulation may have in HD pathology, we investigated the metabolism of Sphingosine-1-phosphate (S1P), one of the most important bioactive lipids, in both animal models and patient samples. Here, we demonstrated that S1P metabolism is significantly disrupted in HD even at early stage of the disease and importantly, we revealed that such a dysfunction represents a common denominator among multiple disease models ranging from cells to humans through mouse models. Interestingly, the in vitro anti-apoptotic and the pro-survival actions seen after modulation of S1P-metabolizing enzymes allows this axis to emerge as a new druggable target and unfolds its promising therapeutic potential for the development of more effective and targeted interventions against this incurable condition.

Huntington disease (HD) is a neurodegenerative disorder for which new treatments are urgently needed. Pridopidine is a new dopaminergic stabilizer, recently developed for the treatment of motor symptoms associated with HD. The therapeutic effect of pridopidine in patients with HD has been determined in two double-blind randomized clinical trials, however, whether pridopidine exerts neuroprotection remains to be addressed. The main goal of this study was to define the potential neuroprotective effect of pridopidine, in HD in vivo and in vitro models, thus providing evidence that might support a potential disease-modifying action of the drug and possibly clarifying other aspects of pridopidine mode-of-action. Our data corroborated the hypothesis of neuroprotective action of pridopidine in HD experimental models. Administration of pridopidine protected cells from apoptosis, and resulted in highly improved motor performance in R6/2 mice. The anti-apoptotic effect observed in the in vitro system highlighted neuroprotective properties of the drug, and advanced the idea of sigma-1-receptor as an additional molecular target implicated in the mechanism of action of pridopidine. Coherent with protective effects, pridopidine-mediated beneficial effects in R6/2 mice were associated with an increased expression of pro-survival and neurostimulatory molecules, such as brain derived neurotrophic factor and DARPP32, and with a reduction in the size of mHtt aggregates in striatal tissues. Taken together, these findings support the theory of pridopidine as molecule with disease-modifying properties in HD and advance the idea of a valuable therapeutic strategy for effectively treating the disease.

Carboxylesterase 3/triacylglycerol hydrolase (Ces3/TGH) participates in hepatic very lowdensity lipoprotein (VLDL) assembly and in adipose tissue basal lipolysis. Global ablation of Ces3/Tgh expression decreases serum triacylglycerol (TG) and nonesterified fatty acid levels and improves insulin sensitivity. To understand the tissue-specific role of Ces3/TGH in lipid and glucose homeostasis, we generated mice with a liver-specific deletion of Ces3/ Tgh expression (L-TGH knockout [KO]). Elimination of hepatic Ces3/Tgh expression dramatically decreased plasma VLDL TG and VLDL cholesterol concentrations but only moderately increased liver TG levels in mice fed a standard chow diet. Significantly reduced plasma TG and cholesterol without hepatic steatosis were also observed in L-TGH KO mice challenged with a high-fat, high-cholesterol diet. L-TGH KO mice presented with increased plasma ketone bodies and hepatic fatty acid oxidation. Intrahepatic TG in L-TGH KO mice was stored in significantly smaller lipid droplets. Augmented hepatic TG levels in chow-fed L-TGH KO mice did not affect glucose tolerance or glucose production from hepatocytes, but impaired insulin tolerance was observed in female mice. Conclusion: Our data suggest that ablation of hepatic Ces3/Tgh expression decreases plasma lipid levels without causing severe hepatic steatosis. (HEPATOLOGY 2012;56:2154-2162 I ncreased plasma triacylglycerol (TG) concentration represents an independent risk factor for cardiovascular disease.1,2 Elevated levels of circulating TGrich, apolipoprotein B (apoB)-containing lipoproteins (very low-density lipoprotein [VLDL] and chylomicrons) accompany insulin resistance and visceral obesity.1,3-5 Secretion of VLDL depends on the availability of lipids for apoB lipidation, and therefore, intracellular VLDL assembly presents a potential pharmacological target for the treatment of hypertriglyceridemia and associated cardiovascular and metabolic diseases. 6,7 Accumulating evidence suggests that the formation of apoB-containing lipoproteins is accomplished by a two-step process, where a primordial lipoprotein with low amounts of TG is initially synthesized, followed by the addition of TG from preexisting lipid storage pools. [8][9][10] Preformed intrahepatic TG was shown to be the source of 60%-80% of VLDL-TG; however, this TG is not transferred to the primordial lipoprotein en bloc but is delivered via a process involving lipolysis and re-esterification.11-15 Triacylglycerol hydrolase (TGH), also termed carboxylesterase 3 (Ces3) in mice and carboxylesterase 1 in humans, has been suggested to play an important role in the provision of TG for the assembly of apoB-containing lipoproteins.

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