SMN regulates GEMIN5 expression and acts as a modifier of GEMIN5-mediated neurodegeneration.

GEMIN5 is essential for core assembly of small nuclear Ribonucleoproteins (snRNPs), the building blocks of spliceosome formation. Loss-of-function mutations in GEMIN5 lead to a neurodevelopmental syndrome among patients presenting with developmental delay, motor dysfunction, and cerebellar atrophy by perturbing SMN complex protein expression and assembly. Currently, molecular determinants of GEMIN5-mediated disease have yet to be explored. Here, we identified SMN as a genetic suppressor of GEMIN5-mediated neurodegeneration in vivo. We discovered that an increase in SMN expression by either SMN gene therapy replacement or the antisense oligonucleotide (ASO), Nusinersen, significantly upregulated the endogenous levels of GEMIN5 in mammalian cells and mutant GEMIN5-derived iPSC neurons. Further, we identified a strong functional association between the expression patterns of SMN and GEMIN5 in patient Spinal Muscular Atrophy (SMA)-derived motor neurons harboring loss-of-function mutations in the SMN gene. Interestingly, SMN binds to the C-terminus of GEMIN5 and requires the Tudor domain for GEMIN5 binding and expression regulation. Finally, we show that SMN upregulation ameliorates defective snRNP biogenesis and alternative splicing defects caused by loss of GEMIN5 in iPSC neurons and in vivo. Collectively, these studies indicate that SMN acts as a regulator of GEMIN5 expression and neuropathologies.

CAPRIN1P512L causes aberrant protein aggregation and associates with early-onset ataxia.

CAPRIN1 is a ubiquitously expressed protein, abundant in the brain, where it regulates the transport and translation of mRNAs of genes involved in synaptic plasticity. Here we describe two unrelated children, who developed early-onset ataxia, dysarthria, cognitive decline and muscle weakness. Trio exome sequencing unraveled the identical de novo c.1535C > T (p.Pro512Leu) missense variant in CAPRIN1, affecting a highly conserved residue. In silico analyses predict an increased aggregation propensity of the mutated protein. Indeed, overexpressed CAPRIN1P512L forms insoluble ubiquitinated aggregates, sequestrating proteins associated with neurodegenerative disorders (ATXN2, GEMIN5, SNRNP200 and SNCA). Moreover, the CAPRIN1P512L mutation in isogenic iPSC-derived cortical neurons causes reduced neuronal activity and altered stress granule dynamics. Furthermore, nano-differential scanning fluorimetry reveals that CAPRIN1P512L aggregation is strongly enhanced by RNA in vitro. These findings associate the gain-of-function Pro512Leu mutation to early-onset ataxia and neurodegeneration, unveiling a critical residue of CAPRIN1 and a key role of RNA-protein interactions.

Long-Term SMN- and Ncald-ASO Combinatorial Therapy in SMA Mice and NCALD-ASO Treatment in hiPSC-Derived Motor Neurons Show Protective Effects.

For SMA patients with only two SMN2 copies, available therapies might be insufficient to counteract lifelong motor neuron (MN) dysfunction. Therefore, additional SMN-independent compounds, supporting SMN-dependent therapies, might be beneficial. Neurocalcin delta (NCALD) reduction, an SMA protective genetic modifier, ameliorates SMA across species. In a low-dose SMN-ASO-treated severe SMA mouse model, presymptomatic intracerebroventricular (i.c.v.) injection of Ncald-ASO at postnatal day 2 (PND2) significantly ameliorates histological and electrophysiological SMA hallmarks at PND21. However, contrary to SMN-ASOs, Ncald-ASOs show a shorter duration of action limiting a long-term benefit. Here, we investigated the longer-term effect of Ncald-ASOs by additional i.c.v. bolus injection at PND28. Two weeks after injection of 500 µg Ncald-ASO in wild-type mice, NCALD was significantly reduced in the brain and spinal cord and well tolerated. Next, we performed a double-blinded preclinical study combining low-dose SMN-ASO (PND1) with 2× i.c.v. Ncald-ASO or CTRL-ASO (100 µg at PND2, 500 µg at PND28). Ncald-ASO re-injection significantly ameliorated electrophysiological defects and NMJ denervation at 2 months. Moreover, we developed and identified a non-toxic and highly efficient human NCALD-ASO that significantly reduced NCALD in hiPSC-derived MNs. This improved both neuronal activity and growth cone maturation of SMA MNs, emphasizing the additional protective effect of NCALD-ASO treatment.

Evaluating Protocols for Porcine Faecal Microbiome Recollection, Storage and DNA Extraction: from the Farm to the Lab.

There is a growing interest in understanding the role of the gut microbiome on productive and meat quality-related traits in livestock species in order to develop new useful tools for improving pig production systems and industry. Faecal samples are analysed as a proxy of gut microbiota and here the selection of suitable protocols for faecal sampling and DNA isolation is a critical first step in order to obtain reliable results, even more to compare results obtained from different studies. The aim of the current study was to establish in a cost-effective way, using automated ribosomal intergenic spacer analysis technique, a protocol for porcine faecal sampling and storage at farm and slaughterhouse and to determine the most efficient microbiota DNA isolation kit among those most widely used. Operational Taxonomic Unit profiles were compared from Iberian pig faecal samples collected from rectum or ground, stored with liquid N2, room temperature or RNAlater, and processed with QIAamp DNA Stool (Qiagen), PowerFecal DNA Isolation (Mobio) or SpeedTools Tissue DNA extraction (Biotools) commercial kits. The results, focused on prokaryote sampling, based on DNA yield and quality, OTU number and Sørensen similarity Indexes, indicate that the recommended protocol for porcine faecal microbiome sampling at farm should include: the collection from porcine rectum to avoid contamination; the storage in liquid N2 or even at room temperature, but not in RNAlater; and the isolation of microbiota DNA using PowerFecal DNA Isolation kit. These conditions provide more reliable DNA samples for further microbiome analysis.

Deciphering the regulation of porcine genes influencing growth, fatness and yield-related traits through genetical genomics.

Genetical genomics approaches aim at identifying quantitative trait loci for molecular traits, also known as intermediate phenotypes, such as gene expression, that could link variation in genetic information to physiological traits. In the current study, an expression GWAS has been carried out on an experimental Iberian × Landrace backcross in order to identify the genomic regions regulating the gene expression of those genes whose expression is correlated with growth, fat deposition, and premium cut yield measures in pig. The analyses were conducted exploiting Porcine 60K SNP BeadChip genotypes and Porcine Expression Microarray data hybridized on mRNA from Longissimus dorsi muscle. In order to focus the analysis on productive traits and reduce the number of analyses, only those probesets whose expression showed significant correlation with at least one of the seven phenotypes of interest were selected for the eGWAS. A total of 63 eQTL regions were identified with effects on 36 different transcripts. Those eQTLs overlapping with phenotypic QTLs on SSC4, SSC9, SSC13, and SSC17 chromosomes previously detected in the same animal material were further analyzed. Moreover, candidate genes and SNPs were analyzed. Among the most promising results, a long non-coding RNA, ALDBSSCG0000001928, was identified, whose expression is correlated with premium cut yield. Association analysis and in silico sequence domain annotation support TXNRD3 polymorphisms as candidate to regulate ALDBSSCG0000001928 expression, which can be involved in the transcriptional regulation of surrounding genes, affecting productive and meat quality traits.