Stimulation of S1PR5 with A-971432, a selective agonist, preserves blood-brain barrier integrity and exerts therapeutic effect in an animal model of Huntington's disease

Huntington's disease (HD) is themost common neurodegenerative disorder for which no effective cure is yet available. Although several agents have been identified to provide benefits so far, the number of therapeutic options remains limited with only symptomatic treatment available. Over the past few years, we have demonstrated that sphingolipid-based approachesmay open the door to newandmore targeted treatments for the disease. In this study, we investigated the therapeutic potential of stimulating sphingosine-1-phosphate (S1P) receptor 5 by the new selective agonist A-971432 (provided by AbbVie) in R6/2mice, a widely used HD animalmodel. Chronic administration of low-dose (0.1mg/kg) A-971432 slowed down the progression of the disease and significantly prolonged lifespan in symptomatic R6/2mice. Such beneficial effects were associated with activation of pro-survival pathways (BDNF, AKT and ERK) and with reduction of mutant huntingtin aggregation. A-971432 also protected blood-brain barrier (BBB) homeostasis in the same mice. Interestingly, when administered early in the disease, before any overt symptoms, A-971432 completely protected HDmice fromthe classic progressivemotor deficit and preserved BBB integrity. Beside representing a promising strategy to take into consideration for the development of alternative therapeutic options for HD, selective stimulation of S1P receptor 5may be also seen as an effective approach to target brain vasculature defects in the disease

Lipid dysfunction in Huntington Disease -"Molecular Mechanisms and Therapy"

Huntington disease (HD) is a neurodegenerative disorder characterized by motor and cognitive symptoms. In HD patients, the protein huntingtin contains an abnormal expansion of a polyglutamine tract, which leads to the selective dysfunction and death of striatal and cortical neurons. Among other cellular dysfunctions, cholesterol and ganglioside GM1 synthesis are affected in HD neurons. In this thesis I demonstrated that impaired cholesterol metabolism in HD cells results from aberrant interaction of mutant huntingtin with the transcription factor Sterol Regulatory Element-Binding Protein 2 (SREBP2). I also showed that administration of GM1 restores normal motor behavior in HD mice.My studies have led to a better understanding of the causes of cholesterol metabolism dysregulation in HD, and have identified GM1 as a potential therapy for the diseas

Sphingolipids and impaired hypoxic stress responses in Huntington disease.

Huntington disease (HD) is a debilitating, currently incurable disease. Protein aggregation and metabolic deficits are pathological hallmarks but their link to neurodegeneration and symptoms remains debated. Here, we summarize alterations in the levels of different sphingolipids in an attempt to characterize sphingolipid patterns specific to HD, an additional molecular hallmark of the disease. Based on the crucial role of sphingolipids in maintaining cellular homeostasis, the dynamic regulation of sphingolipids upon insults and their involvement in cellular stress responses, we hypothesize that maladaptations or blunted adaptations, especially following cellular stress due to reduced oxygen supply (hypoxia) contribute to the development of pathology in HD. We review how sphingolipids shape cellular energy metabolism and control proteostasis and suggest how these functions may fail in HD and in combination with additional insults. Finally, we evaluate the potential of improving cellular resilience in HD by conditioning approaches (improving the efficiency of cellular stress responses) and the role of sphingolipids therein. Sphingolipid metabolism is crucial for cellular homeostasis and for adaptations following cellular stress, including hypoxia. Inadequate cellular management of hypoxic stress likely contributes to HD progression, and sphingolipids are potential mediators. Targeting sphingolipids and the hypoxic stress response are novel treatment strategies for HD

Population stratification may bias analysis of PGC-1α as a modifier of age at Huntington disease motor onset

Huntington’s disease (HD) is an inherited neurodegenerative disorder characterized by motor, cognitive and behavioral disturbances, caused by the expansion of a CAG trinucleotide repeat in the HD gene. The CAG allele size is the major determinant of age at onset (AO) of motor symptoms, although the remaining variance in AO is highly heritable. The rs7665116 SNP in PPARGC1A, encoding the mitochondrial regulator PGC-1α, has been reported to be a significant modifier of AO in three European HD cohorts, perhaps due to affected cases from Italy. We attempted to replicate these findings in a large collection of (1,727) HD patient DNA samples of European origin. In the entire cohort, rs7665116 showed a significant effect in the dominant model (p value = 0.008) and the additive model (p value = 0.009). However, when examined by origin, cases of Southern European origin had an increased rs7665116 minor allele frequency (MAF), consistent with this being an ancestry-tagging SNP. The Southern European cases, despite similar mean CAG allele size, had a significantly older mean AO (p < 0.001), suggesting population-dependent phenotype stratification. When the generalized estimating equations models were adjusted for ancestry, the effect of the rs7665116 genotype on AO decreased dramatically. Our results do not support rs7665116 as a modifier of AO of motor symptoms, as we found evidence for a dramatic effect of phenotypic (AO) and genotypic (MAF) stratification among European cohorts that was not considered in previously reported association studies. A significantly older AO in Southern Europe may reflect population differences in genetic or environmental factors that warrant further investigation

Candidate glutamatergic and dopaminergic pathway gene variants do not influence Huntington’s disease motor onset

Huntington’s disease (HD) is a neurodegenerative disorder characterized by motor, cognitive, and behavioral disturbances. It is caused by the expansion of the HTT CAG repeat, which is the major determinant of age at onset (AO) of motor symptoms. Aberrant function of N-methyl-D-aspartate receptors and/or overexposure to dopamine has been suggested to cause significant neurotoxicity, contributing to HD pathogenesis. We used genetic association analysis in 1,628 HD patients to evaluate candidate polymorphisms in N-methyl-D-aspartate receptor subtype genes (GRIN2A rs4998386 and rs2650427, and GRIN2B rs1806201) and functional polymorphisms in genes in the dopamine pathway (DAT1 3′ UTR 40-bp variable number tandem repeat (VNTR), DRD4 exon 3 48-bp VNTR, DRD2 rs1800497, and COMT rs4608) as potential modifiers of the disease process. None of the seven polymorphisms tested was found to be associated with significant modification of motor AO, either in a dominant or additive model, after adjusting for ancestry. The results of this candidate-genetic study therefore do not provide strong evidence to support a modulatory role for these variations within glutamatergic and dopaminergic genes in the AO of HD motor manifestations

Differential expression of sphingolipid metabolizing enzymes in spontaneously hypertensive rats: a possible substrate for susceptibility to brain and kidney damage

Alterations in the metabolism of sphingolipids, a class of biologically active molecules in cell membranes with direct effect on vascular homeostasis, are increasingly recognized as important determinant in different vascular disorders. However, it is not clear whether sphingolipids are implicated in the pathogenesis of hypertension-related cerebrovascular and renal damage. In this study, we evaluated the existence of possible abnormalities related to the sphingolipid metabolism in the brain and kidneys of two well validated spontaneously hypertensive rat strains, the stroke-prone (SHRSP) and the stroke-resistant (SHRSR) models, as compared to the normotensive Wistar Kyoto (WKY) rat strain. Our results showed a global alteration in the metabolism of sphingolipids in both cerebral and renal tissues of both hypertensive strains as compared to the normotensive rat. However, few defects, such as reduced expression of enzymes involved in the metabolism/catabolism of sphingosine-1-phosphate and in the de novo biosynthetic pathways, were exclusively detected in the SHRSP. Although further studies are necessary to fully understand the significance of these findings, they suggest that defects in specific lipid molecules and/or their related metabolic pathways may likely contribute to the pathogenesis of hypertensive target organ damage and may eventually serve as future therapeutic targets to reduce the vascular consequences of hypertension

DNA Damage, Homology-Directed Repair, and DNA Methylation

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

Decipher non-canonical SPAST splicing mutations with the help of functional assays in patients affected by spastic paraplegia 4 (SPG4)

Flowchart showing the molecular approach used to decipher the non-canonical splicing mutations