Parkinson's disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites: neuronal inclusions immunopositive for the protein α-synuclein. In-depth ultrastructural analysis of Lewy pathology is crucial to understanding pathogenesis of this disease. Using correlative light and electron microscopy/tomography on post-mortem human brain tissue from Parkinson's disease brain donors, we identified α-synuclein immunopositive Lewy pathology and show a crowded environment of membranes therein, including vesicular structures and dysmorphic organelles. Filaments interspersed between the membranes and organelles were identifiable in many, but not all aSyn inclusions. Crowding of organellar components was confirmed by STED-based superresolution microscopy, and high lipid content within α-synuclein immunopositive inclusions was corroborated by confocal imaging, CARS/FTIRimaging and lipidomics. Applying such correlative high-resolution imaging and biophysical approaches, we discovered an aggregated protein-lipid compartmentalization not previously described in the PD brain.

Paraspeckles are mammalian subnuclear bodies built on a long noncoding RNA and are enriched in RNA binding proteins with prion-like domains; two of these proteins, RBM14 and FUS, use these domains to hold paraspeckles together.

Accumulation of abnormally phosphorylated TDP-43 (pTDP-43) is the main pathology in affected neurons of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Morphological diversity and neuroanatomical distribution of pTDP-43 accumulations allowed classification of FTD cases into at least four different subtypes, which correlate with clinical presentations and genetic causes. To understand the molecular basis of this heterogeneity, we developed SarkoSpin, a new method for extremely pure biochemical isolation of pathological TDP-43. Combining SarkoSpin with mass spectrometry, we revealed proteins beyond TDP-43, which become abnormally insoluble in a disease subtype-specific manner. We show that pTDP-43 extracted from disease brain forms large and stable assemblies of distinct densities and morphologies that correlate with disease subtypes. Importantly, biochemically extracted pTDP-43 assemblies displayed differential neurotoxicity and seeding that correlated with disease duration of FTLD patients. Our data indicate that disease heterogeneity may originate from alternate pathological TDP-43 conformations, reminiscent of prion strains. 5 developed SarkoSpin, a novel and simple extraction method for physical separation of pathological TDP-43 from more than 99% of total protein mass including the extreme bulk of physiological, monomeric and oligomeric 11 TDP-43. Using SarkoSpin on brain cortical samples from over 80 patients, we found that TDP-43 forms large and buoyant assemblies of distinct densities, polyubiquitination levels and morphologies that correlate with specific neuropathological classifications. Importantly, coupling SarkoSpin with mass spectrometry, we illustrate that a specific subset of proteins, beyond TDP-43, become insoluble in each disease subtype. These proteins are rarely co-aggregated with pTDP-43 and most likely represent a downstream effect of TDP-43 pathology. One of these proteins depicts a distinct astrocytic reaction discriminating FTLD-TDP-A from FTLD-TDP-C patients, illustrating divergent pathogenic mechanisms within these two disease subtypes. Most importantly, we show evidence that SarkoSpin extracted pTDP-43 assemblies exhibit cytotoxicity and protein seeding ability. Remarkably, pathological aggregates extracted from FTLD-TDP-A were significantly more cyto-and neurotoxic than those extracted from FTLD-TDP-C, thereby correlating with the significant difference in disease duration between these two subtypes. Collectively, our data demonstrate that ALS and FTLD heterogeneity is consistently reflected in the biochemical, neurotoxic and seeding properties of TDP-43 and the associated insoluble proteome. We propose that alternative TDP-43 pathological conformations may underlie the diversity of TDP-43 proteinopathies, reminiscent of prion strains 33,34. Results Summary of patient cohort and characterization of FTLD-TDP-A and FTLD-TDP-C cases Brain cortical samples from over 80 patients, including control patients with no apparent CNS pathology or with non-TDP...

The field of structural biology has the unique advantage of being able to provide a comprehensive picture of biological mechanisms at the molecular and atomic level. Long noncoding RNAs (lncRNAs) represent the new frontier in the molecular biology of complex organisms yet remain the least characterised of all the classes of RNA. Thousands of new lncRNAs are being reported each year yet very little structural data exists for this rapidly expanding field. The length of lncRNAs ranges from 200 nt to over 100 kb in length and they generally exhibit low cellular abundance. Therefore, obtaining sufficient quantities of lncRNA to use for structural analysis is challenging. However, as technologies develop structures of lncRNAs are starting to emerge providing important information regarding their mechanism of action. Here we review the current methods used to determine the structure of lncRNA and lncRNA:protein complexes and describe the significant contribution structural biology has and will make to the field of lncRNA research. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa.

Proteins of the pentatricopeptide repeat (PPR) superfamily are characterized by tandem arrays of a degenerate 35-amino-acid α-hairpin motif. PPR proteins are typically single-stranded RNA-binding proteins with essential roles in organelle biogenesis, RNA editing and mRNA maturation. A modular, predictable code for sequence-specific binding of RNA by PPR proteins has recently been revealed, which opens the door to the de novo design of bespoke proteins with specific RNA targets, with widespread biotechnological potential. Here, the design and production of a synthetic PPR protein based on a consensus sequence and the determination of its crystal structure to 2.2 Å resolution are described. The crystal structure displays helical disorder, resulting in electron density representing an infinite superhelical PPR protein. A structural comparison with related tetratricopeptide repeat (TPR) proteins, and with native PPR proteins, reveals key roles for conserved residues in directing the structure and function of PPR proteins. The designed proteins have high solubility and thermal stability, and can form long tracts of PPR repeats. Thus, consensus-sequence synthetic PPR proteins could provide a suitable backbone for the design of bespoke RNA-binding proteins with the potential for high specificity.