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Down syndrome (DS), a complex genetic disorder caused by chromosome 21 trisomy, is associated with mitochondrial dysfunction leading to the accumulation of damaged mitochondria. Here we report that mitophagy, a form of selective autophagy activated to clear damaged mitochondria is deficient in primary human fibroblasts derived from individuals with DS leading to accumulation of damaged mitochondria with consequent increases in oxidative stress. We identified two molecular bases for this mitophagy deficiency: PINK1/PARKIN impairment and abnormal suppression of macroautophagy. First, strongly downregulated PARKIN and the mitophagic adaptor protein SQSTM1/p62 delays PINK1 activation to impair mitophagy induction after mitochondrial depolarization by CCCP or antimycin A plus oligomycin. Secondly, mTOR is strongly hyper-activated, which globally suppresses macroautophagy induction and the transcriptional expression of proteins critical for autophagosome formation such as ATG7, ATG3 and FOXO1. Notably, inhibition of mTOR complex 1 (mTORC1) and complex 2 (mTORC2) using AZD8055 (AZD) restores autophagy flux, PARKIN/PINK initiation of mitophagy, and the clearance of damaged mitochondria by mitophagy. These results recommend mTORC1-mTORC2 inhibition as a promising candidate therapeutic strategy for Down Syndrome. post-print 3199 KB

Although mechanisms underlying early steps in cerebellar development are known, evidence is lacking on genetic and epigenetic changes during the establishment of the synaptic circuitry. Using metagene analysis, we report pivotal changes in multiple reactomes of epigenetic pathway genes in cerebellar granule cells (GCs) during circuit formation. During this stage, Tet genes are up-regulated and vitamin C activation of Tet enzymes increases the levels of 5-hydroxymethylcytosine (5hmC) at exon start sites of up-regulated genes, notably axon guidance genes and ion channel genes. Knockdown of Tet1 and Tet3 by RNA interference in ex vivo cerebellar slice cultures inhibits dendritic arborization of developing GCs, a critical step in circuit formation. These findings demonstrate a role for Tet genes and chromatin remodeling genes in the formation of cerebellar circuitry.

5-hydroxymethylcytosine (5hmC) occurs at maximal levels in postmitotic neurons, where its accumulation is cell-specific and correlated with gene expression. Here we demonstrate that the distribution of 5hmC in CG and non-CG dinucleotides is distinct and that it reflects the binding specificity and genome occupancy of methylcytosine binding protein 2 (MeCP2). In expressed gene bodies, accumulation of 5hmCG acts in opposition to 5mCG, resulting in "functional" demethylation and diminished MeCP2 binding, thus facilitating transcription. Non-CG hydroxymethylation occurs predominantly in CA dinucleotides (5hmCA) and it accumulates in regions flanking active enhancers. In these domains, oxidation of 5mCA to 5hmCA does not alter MeCP2 binding or expression of adjacent genes. We conclude that the role of 5-hydroxymethylcytosine in postmitotic neurons is to functionally demethylate expressed gene bodies while retaining the role of MeCP2 in chromatin organization.

12 páginas, 8 figuras -- PAGS nros. 1279-1290, Autophagy is a homoeostatic process necessary for the clearance of damaged or superfluous proteins and organelles. The recycling of intracellular constituents also provides energy during periods of metabolic stress, thereby contributing to cell viability. In addition, disruption of autophagic machinery interferes with embryonic development in several species, although the underlying cellular processes affected remain unclear. Here, we investigate the role of autophagy during the early stages of chick retina development, when the retinal neuroepithelium proliferates and starts to generate the first neurons, the retinal ganglion cells. These two developmental processes are accompanied by programmed cell death. Upon treatment with the autophagic inhibitor 3-methyladenine, retinas accumulated numerous TdT-mediated dUTP nick-end labelling-positive cells that correlated with a lack of the ‘eat-me’ signal phosphatidylserine (PS). In consequence, neighbouring cells did not engulf apoptotic bodies and they persisted as individual cell corpses, a phenotype that was also observed after blockade of phagocytosis with phospho-L-Serine. Supplying the retinas with methylpyruvate, a cell-permeable substrate for ATP production, restored ATP levels and the presentation of PS at the cell surface. Hence, engulfment and lysosomal degradation of apoptotic bodies were also re-established. Together, these data point to a novel role for the autophagic machinery during the development of the central nervous system, This research was supported by grants from the Spanish Ministerio de Educación y Ciencia (BFU2006-00508 to PB and SAF2007-66175 to EJdlR) and Comunidad de Madrid (CCG06-CSIC/SAL-0821 to PB). MAM is a FPU Fellow and PB is a Ramón y Cajal Fellow (both Ministerio de Educación y Ciencia programs)

The high level of 5-hydroxymethylcytosine (5hmC) present in neuronal genomes suggests that mechanisms interpreting 5hmC in the central nervous system (CNS) may differ from those present in embryonic stem cells. Here we present quantitative, genome-wide analysis of 5hmC, 5-methylcytosine (5mC) and gene expression in differentiated CNS cell types in vivo. We report that 5hmC is enriched in active genes, and that surprisingly strong depletion of 5mC is observed over these regions. The contribution of these epigenetic marks to gene expression depends critically on cell type. We identify methyl-CpG binding protein 2 (MeCP2) as the major 5hmC binding protein in the brain, and demonstrate that MeCP2 binds 5hmC and 5mC containing DNA with similar high affinities. The Rett Syndromecausing mutation R133C preferentially inhibits 5hmC binding. These findings support a model in which 5hmC and MeCP2 constitute a cell specific epigenetic mechanism for regulation of chromatin structure and gene expression.