Investigating the role of nidogens, a family of basement membrane proteins, at the neuromuscular junction in health and disease

The basement membrane is a specialised form of the extracellular matrix that surrounds cells and tissues to provide structural support, regulate tissue development and modulate homeostatic signalling. Despite the ubiquitous presence of this matrix throughout the body, dysregulation of individual components can result in tissue-specific human diseases. Recently nidogens, a family of basement membrane glycoproteins, have been identified as candidate genes in a family with hydrocephalus and muscle weakness. Increasing evidence suggests that nidogens are involved in signalling processes underlying neurological function and development, and certain nidogen isoforms appear to be specific to the neuromuscular junction (NMJ). My aim was to investigate the active signalling role of nidogens at the NMJ using a combination of biochemical and imaging techniques. As the basement membrane can play an important role in tissue remodelling, I investigated the changes in expression profile of nidogens in conditions of NMJ plasticity. Expression was measured during postnatal muscle maturation and disease progression in a model of amyotrophic lateral sclerosis (ALS) - the SOD1G93A mouse. As neurotrophins are crucial to the development and maintenance of motor neurons and NMJs, I also investigated the interaction of nidogens with several trophic molecules. My work shows that nidogens are internalised by motor neurons and may be co-transported with neurotrophin receptors inside signalling endosomes. While expression of nidogens appears unaffected by disease progression in the SOD1G93A mouse, there may be increased levels of nidogens and altered isoform expression during early postnatal muscle maturation. I also introduce a novel nidogen-1 knock-out mouse model, which displays neurological phenotypes and compensatory upregulation of nidogen-2 in certain tissues. Defining the nidogen signalling complex and its downstream pathways will increase our understanding of the homeostatic mechanisms governing the NMJ and provide new targets for therapeutic strategies to maintain neuromuscular function in pathological conditions

Widespread FUS mislocalization is a molecular hallmark of amyotrophic lateral sclerosis

Mutations causing amyotrophic lateral sclerosis (ALS) clearly implicate ubiquitously expressed and predominantly nuclear RNA binding proteins, which form pathological cytoplasmic inclusions in this context. However, the possibility that wild-type RNA binding proteins mislocalize without necessarily becoming constituents of cytoplasmic inclusions themselves remains relatively unexplored. We hypothesized that nuclear-to-cytoplasmic mislocalization of the RNA binding protein fused in sarcoma (FUS), in an unaggregated state, may occur more widely in ALS than previously recognized. To address this hypothesis, we analysed motor neurons from a human ALS induced-pluripotent stem cell model caused by the VCP mutation. Additionally, we examined mouse transgenic models and post-mortem tissue from human sporadic ALS cases. We report nuclear-to-cytoplasmic mislocalization of FUS in both VCP-mutation related ALS and, crucially, in sporadic ALS spinal cord tissue from multiple cases. Furthermore, we provide evidence that FUS protein binds to an aberrantly retained intron within the SFPQ transcript, which is exported from the nucleus into the cytoplasm. Collectively, these data support a model for ALS pathogenesis whereby aberrant intron retention in SFPQ transcripts contributes to FUS mislocalization through their direct interaction and nuclear export. In summary, we report widespread mislocalization of the FUS protein in ALS and propose a putative underlying mechanism for this process

Intron retention and nuclear loss of SFPQ are molecular hallmarks of ALS

Mutations causing amyotrophic lateral sclerosis (ALS) strongly implicate ubiquitously expressed regulators of RNA processing. To understand the molecular impact of ALS-causing mutations on neuronal development and disease, we analysed transcriptomes during in vitro differentiation of motor neurons (MNs) from human control and patient-specific VCP mutant induced-pluripotent stem cells (iPSCs). We identify increased intron retention (IR) as a dominant feature of the splicing programme during early neural differentiation. Importantly, IR occurs prematurely in VCP mutant cultures compared with control counterparts. These aberrant IR events are also seen in independent RNAseq data sets from SOD1- and FUS-mutant MNs. The most significant IR is seen in the SFPQ transcript. The SFPQ protein binds extensively to its retained intron, exhibits lower nuclear abundance in VCP mutant cultures and is lost from nuclei of MNs in mouse models and human sporadic ALS. Collectively, we demonstrate SFPQ IR and nuclear loss as molecular hallmarks of familial and sporadic ALS

Incidência de suicídios e uso de agrotóxicos por trabalhadores rurais em Luz (MG), Brasil

Este artigo apresenta um estudo sobre a incidência de suicídios em Luz (MG), verificando as eventuais relações com agrotóxicos. Para tanto, adotou-se estudo descritivo com 50 moradores de uma micro-região, entrevistados mediante questionário. Realizaram-se dosagens de acetilcolinesterase, gama-glutamil-transferase (GGT), transaminase glutâmico-oxalacética (TGO) e transaminase glutâmico-pirúvica (TGP) no soro e pesquisas nos prontuários hospitalares e no cartório do registro civil, estudando-se os casos de suicídios entre os anos de 2000 e 2004. A pesquisa nos prontuários e no cartório permitiu verificar a ocorrência de 8,1 atendimentos/mês de casos de intoxicação por agrotóxicos, sendo 19 suicídios (22,6/100.000 hab./ano) no período. Desses, 18 eram trabalhadores rurais do sexo masculino. O mecanismo de suicídio foi, em 57,9% dos casos, envenenamento com agrotóxicos. Dos entrevistados, 98% relataram usar regularmente agrotóxicos, 72% não utilizaram nenhum equipamento de proteção, 56% nunca leram as bulas e 40% afirmaram ter tido intoxicação. Encontrou-se elevação da TGO e da TGP em 33,33% dos homens e da GGT em 21,42% das mulheres e 13,88% dos homens. Não foi detectada redução da atividade da acetilcolinesterase. Constatou-se que a incidência de suicídios foi mais que o dobro da maior média estadual brasileira e que o número de atendimentos de intoxicações foi alto se comparado aos do Sinitox