The accumulation of beta-sheet-rich amyloid fibrils or aggregates is a complex, multistep process that is associated with cellular toxicity in a number of human protein misfolding disorders, including Parkinson's and Alzheimer's diseases. It involves the formation of various transient and intransient, on- and off-pathway aggregate species, whose structure, size and cellular toxicity are largely unclear. Here we demonstrate redirection of amyloid fibril formation through the action of a small molecule, resulting in off-pathway, highly stable oligomers. The polyphenol (-)-epigallocatechin gallate efficiently inhibits the fibrillogenesis of both alpha-synuclein and amyloid-beta by directly binding to the natively unfolded polypeptides and preventing their conversion into toxic, on-pathway aggregation intermediates. Instead of beta-sheet-rich amyloid, the formation of unstructured, nontoxic alpha-synuclein and amyloid-beta oligomers of a new type is promoted, suggesting a generic effect on aggregation pathways in neurodegenerative diseases.

DNA methylation is essential for the control of a number of biological mechanisms in mammals [1]. Mammalian development is accompanied by two major waves of genome-wide demethylation and remethylation: one during germ-cell development and the other after fertilisation [2] [3] [4] [5] [6] [7]. Most previous studies have suggested that the genome-wide demethylation observed after fertilisation occurs passively, that is, by the lack of maintenance methylation following DNA replication and cell division [6] [7], although one other study has reported that replication-independent demethylation may also occur during early embryogenesis [8]. Here, we report that genes that are highly methylated in sperm are rapidly demethylated in the zygote only hours after fertilisation, before the first round of DNA replication commences. By contrast, the oocyte-derived maternal alleles are unaffected by this reprogramming. They either remain methylated after fertilisation or become further methylated de novo. These results provide the first direct evidence for active demethylation of single-copy genes in the mammalian zygote and, moreover, reveal a striking asymmetry in epigenetic methylation reprogramming. Whereas paternally (sperm)-derived sequences are exposed to putative active demethylases in the oocyte cytoplasm, maternally (oocyte)-derived sequences are protected from this reaction. These results, whose generality is supported by findings of Mayer et al. [9], have important implications for the establishment of biparental genetic totipotency after fertilisation, the establishment and maintenance of genomic imprinting, and the reprogramming of somatic cells during cloning.

Huntington's disease (HD) is a progressive neurodegenerative disorder for which only symptomatic treatments of limited effectiveness are available. Preventing early misfolding steps and thereby aggregation of the polyglutamine (polyQ)-containing protein huntingtin (htt) in neurons of patients may represent an attractive therapeutic strategy to postpone the onset and progression of HD. Here, we demonstrate that the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) potently inhibits the aggregation of mutant htt exon 1 protein in a dose-dependent manner. Dot-blot assays and atomic force microscopy studies revealed that EGCG modulates misfolding and oligomerization of mutant htt exon 1 protein in vitro, indicating that it interferes with very early events in the aggregation process. Also, EGCG significantly reduced polyQ-mediated htt protein aggregation and cytotoxicity in an yeast model of HD. When EGCG was fed to transgenic HD flies overexpressing a pathogenic htt exon 1 protein, photoreceptor degeneration and motor function improved. These results indicate that modulators of htt exon 1 misfolding and oligomerization like EGCG are likely to reduce polyQ-mediated toxicity in vivo. Our studies may provide the basis for the development of a novel pharmacotherapy for HD and related polyQ disorders.

Polyglutamine (polyQ) disorders, including Huntington's disease (HD), are caused by expansion of polyQ-encoding repeats within otherwise unrelated gene products. In polyQ diseases, the pathology and death of affected neurons are associated with the accumulation of mutant proteins in insoluble aggregates. Several studies implicate polyQ-dependent aggregation as a cause of neurodegeneration in HD, suggesting that inhibition of neuronal polyQ aggregation may be therapeutic in HD patients. We have used a yeast-based high-throughput screening assay to identify small-molecule inhibitors of polyQ aggregation. We validated the effects of four hit compounds in mammalian cell-based models of HD, optimized compound structures for potency, and then tested them in vitro in cultured brain slices from HD transgenic mice. These efforts identified a potent compound (IC 50 ‫؍‬ 10 nM) with long-term inhibitory effects on polyQ aggregation in HD neurons. Testing of this compound in a Drosophila HD model showed that it suppresses neurodegeneration in vivo, strongly suggesting an essential role for polyQ aggregation in HD pathology. The aggregation inhibitors identified in this screen represent four primary chemical scaffolds and are strong lead compounds for the development of therapeutics for human polyQ diseases.high-throughput screen ͉ small-molecule therapeutics ͉ Drosophila ͉ R6͞2 brain slices ͉ genetic disease A t least nine inherited neurodegenerative diseases, including Huntington's disease (HD), are caused by expansion of polyglutamine (polyQ)-encoding repeats within otherwise unrelated proteins (1, 2). In HD, expansion of polyQ repeats within the huntingtin (Htt) protein causes an adult-onset neurodegenerative disease characterized by movement disorder, psychiatric symptoms, and cognitive dysfunction (3-5). As in several major neurological disorders, including Alzheimer's and Parkinson's diseases, the pathology and death of affected neurons in polyQ diseases are associated with accumulation of mutant polypeptides in insoluble aggregates (6-9). These polyQ-containing aggregates, or inclusions, have been found in the nuclei of affected neurons in postmortem patient tissues and brains from HD transgenic mice (10-12) and have emerged as a hallmark of HD pathology.Mutant polypeptides with extended polyQ tracts aggregate in vitro and in vivo in a polyQ length-dependent manner, which closely correlates with the age of onset in HD and other polyQ-expansion diseases (2,(13)(14)(15). Although the precise role of neuronal aggregates in disease pathogenesis is not clear, therapeutic strategies aimed at inhibiting polyQ aggregation have shown some efficacy in vivo in both Drosophila and mouse models of HD (16,17). These and other studies (18, 19) implicate polyQ-dependent aggregation as a cause of neurodegeneration in HD and suggest that inhibition of neuronal polyQ aggregation may be therapeutic in HD patients (8).Chemical compounds that directly target polyQ aggregation have been identified in high-throughput screens using cell-free ...

Preventing the formation of insoluble polyglutamine containing protein aggregates in neurons may represent an attractive therapeutic strategy to ameliorate Huntington's disease (HD). Therefore, the ability to screen for small molecules that suppress the self-assembly of huntingtin would have potential clinical and significant research applications. We have developed an automated filter retardation assay for the rapid identification of chemical compounds that prevent HD exon 1 protein aggregation in vitro. Using this method, a total of 25 benzothiazole derivatives that inhibit huntingtin fibrillogenesis in a dose-dependent manner were discovered from a library of Ϸ184,000 small molecules. The results obtained by the filter assay were confirmed by immunoblotting, electron microscopy, and mass spectrometry. Furthermore, cell culture studies revealed that 2-amino-4,7-dimethylbenzothiazol-6-ol, a chemical compound similar to riluzole, significantly inhibits HD exon 1 aggregation in vivo. These findings may provide the basis for a new therapeutic approach to prevent the accumulation of insoluble protein aggregates in Huntington's disease and related glutamine repeat disorders.

The clustered organization of most imprinted genes in mammals suggests coordinated genetic and epigenetic control mechanisms. Comparisons between human and mouse will help in elucidating these mechanisms by identifying structural and functional similarities. Previously we reported on such a comparison in the central part of the mouse imprinting cluster on distal chromosome 7 with the homologous Beckwith-Wiedemann syndrome (BWS) gene cluster on human chromosome 11p15.5. Here we focus on the adjacent sequences of 0.5 Mb including the KCNQ1/Kcnq1 and CDKN1C/Cdkn1c genes, which are implicated in BWS, and on one of the proposed boundary regions of the imprinting cluster. As in the previously analysed central region, this part of the cluster exhibits a highly conserved arrangement and structure of genes. The most striking similarity is found in the 3' part of the KCNQ1/Kcnq1 genes in large stretches of mostly non-coding sequences. The conserved region includes the recently identified KCNQ1OT1/Kcnq1ot1 antisense transcripts, flanked by a strikingly conserved cluster of LINE/Line elements and a CpG island which we show to carry a maternal germline methylation imprint. This region is likely to be the proposed second imprinting centre (IC2) in the BWS cluster. We also identified several novel genes inside and outside the previously proposed boundaries of the imprinting cluster. One of the genes outside the cluster, Obph1, is imprinted in mouse placenta indicating that at least in extra-embryonic tissues the imprinting cluster extends into a larger domain.

In human and mouse most imprinted genes are arranged in chromosomal clusters. This linked organization suggests coordinated mechanisms controlling imprinted expression. We have sequenced 250 kb in the centre of the mouse imprinting cluster on distal chromosome 7 and compared it with the orthologous Beckwith-Wiedemann gene cluster on human chromosome 11p15.5. This first comparative imprinting cluster analysis revealed a high structural and functional conservation of the six orthologous genes identified. However, several striking differences were also discovered. First, compared with the mouse the human sequence is approximately 40% longer, mostly due to insertions of two large repetitive clusters. One of these clusters encompasses an additional gene coding for a homologue of the ribosomal protein L26. Second, pronounced blocks of unique direct repeats characteristic of imprinted genes were only found in the human sequence. Third, two of the orthologous gene pairs Tssc4/TSSC4 and Ltrpc5/LTRPC5 showed apparent differences in imprinting between human and mouse, whereas others like Tssc6/TSSC6 were not imprinted in either organism. Together these results suggest a significant functional and structural variability in the centre of the imprinting cluster. Some genes escape imprinting in both organisms whereas others exhibit tissue- and species-specific imprinting. Hence the control of imprinting in the cluster appears to be a highly dynamic process under fast evolutionary adaptation. Intriguingly, whereas imprinted genes within the cluster contain CpG islands the non-imprinted Ltrpc5 and Tssc6/TSSC6 do not. This and additional comparisons with other imprinted and non-imprinted regions suggest that CpG islands are key features of imprinted domains.

No abstract