The C-terminus of α-synuclein regulates its dynamic cellular internalization by neurexin 1β

The aggregation of the disordered neuronal protein, α-Synuclein (αS), is the primary pathological feature of Parkinson's disease. Current hypotheses favor cell-to-cell spread of αS species as underlying disease progression, driving interest in identifying the molecular species and cellular processes involved in cellular internalization of αS. Prior work from our lab identified the chemically specific interaction between αS and the pre-synaptic adhesion protein neurexin 1β (N1β) to be capable of driving cellular internalization of both monomer and aggregated forms of αS. Here we explore the physical basis of N1β-driven internalization of αS. Specifically, we show that spontaneous internalization of αS by SH-SY5Y and HEK293 cells expressing N1β requires essentially all of the membrane-binding domain of αS; αS constructs truncated beyond residue 90 bind to N1β in the plasma membrane of HEK cells, but are not internalized. Interestingly, prior to internalization, αS and N1β co-diffuse rapidly in the plasma membrane. αS constructs that are not internalized show very slow mobility themselves, as well as slow N1β diffusion. Finally, we find that truncated αS is capable of blocking internalization of full-length αS. Our results draw attention to the potential therapeutic value of blocking αS-N1β interactions

Novel Tools for Conservation Genomics: Comparing Two High-Throughput Approaches for SNP Discovery in the Transcriptome of the European Hake

The growing accessibility to genomic resources using next-generation sequencing (NGS) technologies has revolutionized the application of molecular genetic tools to ecology and evolutionary studies in non-model organisms. Here we present the case study of the European hake (Merluccius merluccius), one of the most important demersal resources of European fisheries. Two sequencing platforms, the Roche 454 FLX (454) and the Illumina Genome Analyzer (GAII), were used for Single Nucleotide Polymorphisms (SNPs) discovery in the hake muscle transcriptome. De novo transcriptome assembly into unique contigs, annotation, and in silico SNP detection were carried out in parallel for 454 and GAII sequence data. High-throughput genotyping using the Illumina GoldenGate assay was performed for validating 1,536 putative SNPs. Validation results were analysed to compare the performances of 454 and GAII methods and to evaluate the role of several variables (e.g. sequencing depth, intron-exon structure, sequence quality and annotation). Despite well-known differences in sequence length and throughput, the two approaches showed similar assay conversion rates (approximately 43%) and percentages of polymorphic loci (67.5% and 63.3% for GAII and 454, respectively). Both NGS platforms therefore demonstrated to be suitable for large scale identification of SNPs in transcribed regions of non-model species, although the lack of a reference genome profoundly affects the genotyping success rate. The overall efficiency, however, can be improved using strict quality and filtering criteria for SNP selection (sequence quality, intron-exon structure, target region score)