Leonardo Almeida-Souza

Endophilin marks and controls a clathrin-independent endocytic pathway

Endocytosis is required for internalization of micronutrients and turnover of membrane components. Endophilin has been assigned as a component of clathrin-mediated endocytosis. Here we show in mammalian cells that endophilin marks and controls a fast-acting tubulovesicular endocytic pathway that is independent of AP2 and clathrin, activated upon ligand binding to cargo receptors, inhibited by inhibitors of dynamin, Rac, phosphatidylinositol-3-OH kinase, PAK1 and actin polymerization, and activated upon Cdc42 inhibition. This pathway is prominent at the leading edges of cells where phosphatidylinositol-3,4-bisphosphate—produced by the dephosphorylation of phosphatidylinositol-3,4,5-triphosphate by SHIP1 and SHIP2—recruits lamellipodin, which in turn engages endophilin. This pathway mediates the ligand-triggered uptake of several G-protein-coupled receptors such as α2a- and β1-adrenergic, dopaminergic D3 and D4 receptors and muscarinic acetylcholine receptor 4, the receptor tyrosine kinases EGFR, HGFR, VEGFR, PDGFR, NGFR and IGF1R, as well as interleukin-2 receptor. We call this new endocytic route fast endophilin-mediated endocytosis (FEME).

Missense Mutations in the Copper Transporter Gene ATP7A Cause X-Linked Distal Hereditary Motor Neuropathy

Distal hereditary motor neuropathies comprise a clinically and genetically heterogeneous group of disorders. We recently mapped an X-linked form of this condition to chromosome Xq13.1-q21 in two large unrelated families. The region of genetic linkage included ATP7A, which encodes a copper-transporting P-type ATPase mutated in patients with Menkes disease, a severe infantile-onset neurodegenerative condition. We identified two unique ATP7A missense mutations (p.P1386S and p.T994I) in males with distal motor neuropathy in two families. These molecular alterations impact highly conserved amino acids in the carboxyl half of ATP7A and do not directly involve the copper transporters known critical functional domains. Studies of p.P1386S revealed normal ATP7A mRNA and protein levels, a defect in ATP7A trafficking, and partial rescue of a S. cerevisiae copper transport knockout. Although ATP7A mutations are typically associated with severe Menkes disease or its milder allelic variant, occipital horn syndrome, we demonstrate here that certain missense mutations at this locus can cause a syndrome restricted to progressive distal motor neuropathy without overt signs of systemic copper deficiency. This previously unrecognized genotype-phenotype correlation suggests an important role of the ATP7A copper transporter in motor-neuron maintenance and function.

Mutations in the SPTLC2 Subunit of Serine Palmitoyltransferase Cause Hereditary Sensory and Autonomic Neuropathy Type I

Hereditary sensory and autonomic neuropathy type I (HSAN-I) is an axonal peripheral neuropathy associated with progressive distal sensory loss and severe ulcerations. Mutations in the first subunit of the enzyme serine palmitoyltransferase (SPT) have been associated with HSAN-I. The SPT enzyme catalyzes the first and rate-limiting step in the de novo sphingolipid synthesis pathway. However, different studies suggest the implication of other genes in the pathology of HSAN-I. Therefore, we screened the two other known subunits of SPT, SPTLC2 and SPTLC3, in a cohort of 78 HSAN patients. No mutations were found in SPTLC3, but we identified three heterozygous missense mutations in the SPTLC2 subunit of SPT in four families presenting with a typical HSAN-I phenotype. We demonstrate that these mutations result in a partial to complete loss of SPT activity in vitro and in vivo. Moreover, they cause the accumulation of the atypical and neurotoxic sphingoid metabolite 1-deoxy-sphinganine. Our findings extend the genetic heterogeneity in HSAN-I and enlarge the group of HSAN neuropathies associated with SPT defects. We further show that HSAN-I is consistently associated with an increased formation of the neurotoxic 1-deoxysphinganine, suggesting a common pathomechanism for HSAN-I.

Acute injury in the peripheral nervous system triggers an alternative macrophage response

BackgroundThe activation of the immune system in neurodegeneration has detrimental as well as beneficial effects. Which aspects of this immune response aggravate the neurodegenerative breakdown and which stimulate regeneration remains an open question. To unravel the neuroprotective aspects of the immune system we focused on a model of acute peripheral nerve injury, in which the immune system was shown to be protective.MethodsTo determine the type of immune response triggered after axotomy of the sciatic nerve, a model for Wallerian degeneration in the peripheral nervous system, we evaluated markers representing the two extremes of a type I and type II immune response (classical vs. alternative) using real-time quantitative polymerase chain reaction (RT-qPCR), western blot, and immunohistochemistry.ResultsOur results showed that acute peripheral nerve injury triggers an anti-inflammatory and immunosuppressive response, rather than a pro-inflammatory response. This was reflected by the complete absence of classical macrophage markers (iNOS, IFNγ, and IL12p40), and the strong up-regulation of tissue repair markers (arginase-1, Ym1, and Trem2). The signal favoring the alternative macrophage environment was induced immediately after nerve damage and appeared to be established within the nerve, well before the infiltration of macrophages. In addition, negative regulators of the innate immune response, as well as the anti-inflammatory cytokine IL-10 were induced. The strict regulation of the immune system dampens the potential tissue damaging effects of an over-activated response.ConclusionsWe here demonstrate that acute peripheral nerve injury triggers an inherent protective environment by inducing the M2 phenotype of macrophages and the expression of arginase-1. We believe that the M2 phenotype, associated with a sterile inflammatory response and tissue repair, might explain their neuroprotective capacity. As such, shifting the neurodegeneration-induced immune responses towards an M2/Th2 response could be an important therapeutic strategy.

Small Heat-Shock Protein HSPB1 Mutants Stabilize Microtubules in Charcot-Marie-Tooth Neuropathy

Mutations in the small heat shock protein HSPB1 (HSP27) are causative for Charcot-Marie-Tooth (CMT) neuropathy. We previously showed that a subset of these mutations displays higher chaperone activity and enhanced affinity to client proteins. We hypothesized that this excessive binding property might cause the HSPB1 mutant proteins to disturb the function of proteins essential for the maintenance or survival of peripheral neurons. In the present work, we explored this hypothesis further and compared the protein complexes formed by wild-type and mutant HSPB1. Tubulin came out as the most striking differential interacting protein, with hyperactive mutants binding more strongly to both tubulin and microtubules. This anomalous binding leads to a stabilization of the microtubule network in a microtubule-associated protein-like manner as reflected by resistance to cold depolymerization, faster network recovery after nocodazole treatment, and decreased rescue and catastrophe rates of individual microtubules. In a transgenic mouse model for mutant HSPB1 that recapitulates all features of CMT, we could confirm the enhanced interaction of mutant HSPB1 with tubulin. Increased stability of the microtubule network was also clear in neurons isolated from these mice. Since neuronal cells are particularly vulnerable to disturbances in microtubule dynamics, this mechanism might explain the neuron-specific CMT phenotype caused by HSPB1 mutations.

Loss-of-function mutations in HINT1 cause axonal neuropathy with neuromyotonia.

Inherited peripheral neuropathies are frequent neuromuscular disorders known for their clinical and genetic heterogeneity. In 33 families, we identified 8 mutations in HINT1 (encoding histidine triad nucleotide–binding protein 1) by combining linkage analyses with next-generation sequencing and subsequent cohort screening of affected individuals. Our study provides evidence that loss of functional HINT1 protein results in a distinct phenotype of autosomal recessive axonal neuropathy with neuromyotonia.

Increased Monomerization of Mutant HSPB1 Leads to Protein Hyperactivity in Charcot-Marie-Tooth Neuropathy

Small heat shock proteins are molecular chaperones capable of maintaining denatured proteins in a folding-competent state. We have previously shown that missense mutations in the small heat shock protein HSPB1 (HSP27) cause distal hereditary motor neuropathy and axonal Charcot-Marie-Tooth disease. Here we investigated the biochemical consequences of HSPB1 mutations that are known to cause peripheral neuropathy. In contrast to other chaperonopathies, our results revealed that particular HSPB1 mutations presented higher chaperone activity compared with wild type. Hyperactivation of HSPB1 was accompanied by a change from its wild-type dimeric state to a monomer without dissociation of the 24-meric state. Purification of protein complexes from wild-type and HSPB1 mutants showed that the hyperactive isoforms also presented enhanced binding to client proteins. Furthermore, we show that the wild-type HSPB1 protein undergoes monomerization during heat-shock activation, strongly suggesting that the monomer is the active form of the HSPB1 protein.

Mutant HSPB8 causes motor neuron-specific neurite degeneration.

Missense mutations (K141N and K141E) in the α-crystallin domain of the small heat shock protein HSPB8 (HSP22) cause distal hereditary motor neuropathy (distal HMN) or Charcot-Marie-Tooth neuropathy type 2L (CMT2L). The mechanism through which mutant HSPB8 leads to a specific motor neuron disease phenotype is currently unknown. To address this question, we compared the effect of mutant HSPB8 in primary neuronal and glial cell cultures. In motor neurons, expression of both HSPB8 K141N and K141E mutations clearly resulted in neurite degeneration, as manifested by a reduction in number of neurites per cell, as well as in a reduction in average length of the neurites. Furthermore, expression of the K141E (and to a lesser extent, K141N) mutation also induced spheroids in the neurites. We did not detect any signs of apoptosis in motor neurons, showing that mutant HSPB8 resulted in neurite degeneration without inducing neuronal death. While overt in motor neurons, these phenotypes were only very mildly present in sensory neurons and completely absent in cortical neurons. Also glial cells did not show an altered phenotype upon expression of mutant HSPB8. These findings show that despite the ubiquitous presence of HSPB8, only motor neurons appear to be affected by the K141N and K141E mutations which explain the predominant motor neuron phenotype in distal HMN and CMT2L.

MIR137 variants identified in psychiatric patients affect synaptogenesis and neuronal transmission gene sets

Sequence analysis of 13 microRNA (miRNA) genes expressed in the human brain and located in genomic regions associated with schizophrenia and/or bipolar disorder, in a northern Swedish patient/control population, resulted in the discovery of two functional variants in the MIR137 gene. On the basis of their location and the allele frequency differences between patients and controls, we explored the hypothesis that the discovered variants impact the expression of the mature miRNA and consequently influence global mRNA expression affecting normal brain functioning. Using neuronal-like SH-SY5Y cells, we demonstrated significantly reduced mature miR-137 levels in the cells expressing the variant miRNA gene. Subsequent transcriptome analysis showed that the reduction in miR-137 expression led to the deregulation of gene sets involved in synaptogenesis and neuronal transmission, all implicated in psychiatric disorders. Our functional findings add to the growing data, which implicate that miR-137 has an important role in the etiology of psychiatric disorders and emphasizes its involvement in nervous system development and proper synaptic function.

Distal myopathy with upper limb predominance caused by filamin C haploinsufficiency.

Objective: In this study, we investigated the detailed clinical findings and underlying genetic defect in 3 presumably related Bulgarian families displaying dominantly transmitted adult onset distal myopathy with upper limb predominance. Methods: We performed neurologic, electrophysiologic, radiologic, and histopathologic analyses of 13 patients and 13 at-risk but asymptomatic individuals from 3 generations. Genome-wide parametric linkage analysis was followed by bidirectional sequencing of the filamin C (FLNC) gene. We characterized the identified nonsense mutation at cDNA and protein level. Results: Based on clinical findings, no known myopathy subtype was implicated in our distal myopathy patients. Light microscopic analysis of affected muscle tissue showed no specific hallmarks; however, the electron microscopy revealed changes compatible with myofibrillar myopathy. Linkage studies delineated a 9.76 Mb region on chromosome 7q22.1-q35 containing filamin C (FLNC), a gene previously associated with myofibrillar myopathy. Mutation analysis revealed a novel c.5160delC frameshift deletion in all patients of the 3 families. The mutation results in a premature stop codon (p.Phe1720LeufsX63) that triggers nonsense-mediated mRNA decay. FLNC transcript levels were reduced in muscle and lymphoblast cells from affected subjects and partial loss of FLNC in muscle tissue was confirmed by protein analysis. Conclusions: The FLNC mutation that we identified is distinct in terms of the associated phenotype, muscle morphology, and underlying molecular mechanism, thus extending the currently recognized clinical and genetic spectrum of filaminopathies. We conclude that filamin C is a dosage-sensitive gene and that FLNC haploinsufficiency can cause a specific type of myopathy in humans.