RESUMO
Charcot-Marie-Tooth type 4B1 (CMT4B1) is a severe autosomal recessive demyelinating neuropathy with childhood onset, caused by loss-of-function mutations in the myotubularin-related 2 (MTMR2) gene. MTMR2 is a ubiquitously expressed catalytically active 3-phosphatase, which in vitro dephosphorylates the 3-phosphoinositides PtdIns3P and PtdIns(3,5)P2, with a preference for PtdIns(3,5)P2 A hallmark of CMT4B1 neuropathy are redundant loops of myelin in the nerve termed myelin outfoldings, which can be considered the consequence of altered growth of myelinated fibers during postnatal development. How MTMR2 loss and the resulting imbalance of 3'-phosphoinositides cause CMT4B1 is unknown. Here we show that MTMR2 by regulating PtdIns(3,5)P2 levels coordinates mTORC1-dependent myelin synthesis and RhoA/myosin II-dependent cytoskeletal dynamics to promote myelin membrane expansion and longitudinal myelin growth. Consistent with this, pharmacological inhibition of PtdIns(3,5)P2 synthesis or mTORC1/RhoA signaling ameliorates CMT4B1 phenotypes. Our data reveal a crucial role for MTMR2-regulated lipid turnover to titrate mTORC1 and RhoA signaling thereby controlling myelin growth.
Assuntos
Doença de Charcot-Marie-Tooth/metabolismo , Bainha de Mielina/metabolismo , Fosfatos de Fosfatidilinositol/biossíntese , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Transdução de Sinais , Animais , Doença de Charcot-Marie-Tooth/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Knockout , Bainha de Mielina/genética , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Fosfatos de Fosfatidilinositol/genética , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
BACKGROUND AND PURPOSE: Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of disorders caused by mutations in at least 100 genes. However, approximately 60% of cases with axonal neuropathies (CMT2) still remain without a genetic diagnosis. We aimed at identifying novel disease genes responsible for CMT2. METHODS: We performed whole exome sequencing and targeted next generation sequencing panel analyses on a cohort of CMT2 families with evidence for autosomal recessive inheritance. We also performed functional studies to explore the pathogenetic role of selected variants. RESULTS: We identified rare, recessive variants in the MYO9B (myosin IX) gene in two families with CMT2. MYO9B has not yet been associated with a human disease. MYO9B is an unconventional single-headed processive myosin motor protein with signaling properties, and, consistent with this, our results indicate that a variant occurring in the MYO9B motor domain impairs protein expression level and motor activity. Interestingly, a Myo9b-null mouse has degenerating axons in sciatic nerves and optic nerves, indicating that MYO9B plays an essential role in both peripheral nervous system and central nervous system axons, respectively. The degeneration observed in the optic nerve prompted us to screen for MYO9B mutations in a cohort of patients with optic atrophy (OA). Consistent with this, we found compound heterozygous variants in one case with isolated OA. CONCLUSIONS: Novel or very rare variants in MYO9B are associated with CMT2 and isolated OA.
Assuntos
Doença de Charcot-Marie-Tooth , Miosinas , Animais , Humanos , Camundongos , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Mutação/genética , Linhagem , Fenótipo , Proteínas , Nervo Isquiático/patologia , Miosinas/genéticaRESUMO
Ageing is associated with notorious alterations in neurons, i.e., in gene expression, mitochondrial function, membrane degradation or intercellular communication. However, neurons live for the entire lifespan of the individual. One of the reasons why neurons remain functional in elderly people is survival mechanisms prevail over death mechanisms. While many signals are either pro-survival or pro-death, others can play both roles. Extracellular vesicles (EVs) can signal both pro-toxicity and survival. We used young and old animals, primary neuronal and oligodendrocyte cultures and neuroblastoma and oligodendrocytic lines. We analysed our samples using a combination of proteomics and artificial neural networks, biochemistry and immunofluorescence approaches. We found an age-dependent increase in ceramide synthase 2 (CerS2) in cortical EVs, expressed by oligodendrocytes. In addition, we show that CerS2 is present in neurons via the uptake of oligodendrocyte-derived EVs. Finally, we show that age-associated inflammation and metabolic stress favour CerS2 expression and that oligodendrocyte-derived EVs loaded with CerS2 lead to the expression of the antiapoptotic factor Bcl2 in inflammatory conditions. Our study shows that intercellular communication is altered in the ageing brain, which favours neuronal survival through the transfer of oligodendrocyte-derived EVs containing CerS2.
Assuntos
Vesículas Extracelulares , Neurônios , Animais , Vesículas Extracelulares/metabolismo , Encéfalo/metabolismo , Inflamação/metabolismoRESUMO
Microtubule-based kinesin motors have many cellular functions, including the transport of a variety of cargos. However, unconventional roles have recently emerged, and kinesins have also been reported to act as scaffolding proteins and signaling molecules. In this work, we further extend the notion of unconventional functions for kinesin motor proteins, and we propose that Kif13b kinesin acts as a signaling molecule regulating peripheral nervous system (PNS) and central nervous system (CNS) myelination. In this process, positive and negative signals must be tightly coordinated in time and space to orchestrate myelin biogenesis. Here, we report that in Schwann cells Kif13b positively regulates myelination by promoting p38γ mitogen-activated protein kinase (MAPK)-mediated phosphorylation and ubiquitination of Discs large 1 (Dlg1), a known brake on myelination, which downregulates the phosphatidylinositol 3-kinase (PI3K)/v-AKT murine thymoma viral oncogene homolog (AKT) pathway. Interestingly, Kif13b also negatively regulates Dlg1 stability in oligodendrocytes, in which Dlg1, in contrast to Schwann cells, enhances AKT activation and promotes myelination. Thus, our data indicate that Kif13b is a negative regulator of CNS myelination. In summary, we propose a novel function for the Kif13b kinesin in glial cells as a key component of the PI3K/AKT signaling pathway, which controls myelination in both PNS and CNS.
Assuntos
Sistema Nervoso Central/fisiologia , Cinesinas/fisiologia , Proteínas de Membrana/fisiologia , Bainha de Mielina/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Sistema Nervoso Periférico/fisiologia , Animais , Proteína 1 Homóloga a Discs-Large , Camundongos , Camundongos Knockout , Oligodendroglia/metabolismo , Proteínas Associadas SAP90-PSD95 , Células de Schwann/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Proteins containing C2 domains are the sensors for Ca(2+) and PI(4,5)P2 in a myriad of secretory pathways. Here, the use of a free-mounting system has enabled us to capture an intermediate state of Ca(2+) binding to the C2A domain of rabphilin 3A that suggests a different mechanism of ion interaction. We have also determined the structure of this domain in complex with PI(4,5)P2 and IP3 at resolutions of 1.75 and 1.9 Å, respectively, unveiling that the polybasic cluster formed by strands ß3-ß4 is involved in the interaction with the phosphoinositides. A comparative study demonstrates that the C2A domain is highly specific for PI(4,5)P2/PI(3,4,5)P3, whereas the C2B domain cannot discriminate among any of the diphosphorylated forms. Structural comparisons between C2A domains of rabphilin 3A and synaptotagmin 1 indicated the presence of a key glutamic residue in the polybasic cluster of synaptotagmin 1 that abolishes the interaction with PI(4,5)P2. Together, these results provide a structural explanation for the ability of different C2 domains to pull plasma and vesicle membranes close together in a Ca(2+)-dependent manner and reveal how this family of proteins can use subtle structural changes to modulate their sensitivity and specificity to various cellular signals.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Cálcio/química , Proteínas do Tecido Nervoso/química , Fosfatidilinositol 4,5-Difosfato/química , Sinaptotagmina I/química , Proteínas de Transporte Vesicular/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cálcio/metabolismo , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Cristalografia por Raios X , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Rabfilina-3ARESUMO
C2 domains are widely-spread protein signaling motifs that in classical PKCs act as Ca(2+)-binding modules. However, the molecular mechanisms of their targeting process at the plasma membrane remain poorly understood. Here, the crystal structure of PKCalpha-C2 domain in complex with Ca(2+), 1,2-dihexanoyl-sn-glycero-3-[phospho-L-serine] (PtdSer), and 1,2-diayl-sn-glycero-3-[phosphoinositol-4,5-bisphosphate] [PtdIns(4,5)P(2)] shows that PtdSer binds specifically to the calcium-binding region, whereas PtdIns(4,5)P(2) occupies the concave surface of strands beta3 and beta4. Strikingly, the structure reveals a PtdIns(4,5)P(2)-C2 domain-binding mode in which the aromatic residues Tyr-195 and Trp-245 establish direct interactions with the phosphate moieties of the inositol ring. Mutations that abrogate Tyr-195 and Trp-245 recognition of PtdIns(4,5)P(2) severely impaired the ability of PKCalpha to localize to the plasma membrane. Notably, these residues are highly conserved among C2 domains of topology I, and a general mechanism of C2 domain-membrane docking mediated by PtdIns(4,5)P(2) is presented.
Assuntos
Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteína Quinase C-alfa/química , Proteína Quinase C-alfa/metabolismo , Aminoácidos/metabolismo , Animais , Cátions/metabolismo , Membrana Celular/enzimologia , Sequência Conservada , Genes Dominantes , Modelos Moleculares , Proteínas Mutantes/metabolismo , Células PC12 , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Relação Estrutura-AtividadeRESUMO
PKCα is a key mediator of the neuronal differentiation controlled by NGF and ATP. However, its downstream signaling pathways remain to be elucidated. To identify the signaling partners of PKCα, we analyzed proteins coimmunoprecipitated with this enzyme in PC12 cells differentiated with NGF and ATP and compared them with those obtained with NGF alone or growing media. Mass spectrometry analysis (LC-MS/MS) identified plectin, peripherin, filamin A, fascin, and ß-actin as potential interacting proteins. The colocalization of PKCα and its interacting proteins increased when PC12 cells were differentiated with NGF and ATP. Peripherin and plectin organization and the cortical remodeling of ß-actin were dramatically affected when PKCα was down-regulated, suggesting that all three proteins might be functional targets of ATP-dependent PKCα signaling. Taken together, these data demonstrate that PKCα is essential for controlling the neuronal development induced by NGF and ATP and interacts with the cytoskeletal components at two levels: assembly of the intermediate filament peripherin and organization of cortical actin.
Assuntos
Trifosfato de Adenosina/metabolismo , Diferenciação Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Neurônios/metabolismo , Proteína Quinase C-alfa/metabolismo , Animais , Western Blotting , Regulação para Baixo , Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/metabolismo , Espaço Intracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Fator de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Neurônios/citologia , Neurônios/enzimologia , Células PC12 , Periferinas , Plectina/metabolismo , Mapeamento de Interação de Proteínas , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Ratos , Reprodutibilidade dos Testes , Transdução de Sinais/fisiologia , Espectrometria de Massas em TandemRESUMO
C2 domains are conserved protein modules in many eukaryotic signaling proteins, including the protein kinase (PKCs). The C2 domains of classical PKCs bind to membranes in a Ca(2+)-dependent manner and thereby act as cellular Ca(2+) effectors. Recent findings suggest that the C2 domain of PKCalpha interacts specifically with phosphatidylinositols 4,5-bisphosphate (PtdIns(4,5)P(2)) through its lysine rich cluster, for which it shows higher affinity than for POPS. In this work, we compared the three C2 domains of classical PKCs. Isothermal titration calorimetry revealed that the C2 domains of PKCalpha and beta display a greater capacity to bind to PtdIns(4,5)P(2)-containing vesicles than the C2 domain of PKCgamma. Comparative studies using lipid vesicles containing both POPS and PtdIns(4,5)P(2) as ligands revealed that the domains behave as PtdIns(4,5)P(2)-binding modules rather than as POPS-binding modules, suggesting that the presence of the phosphoinositide in membranes increases the affinity of each domain. When the magnitude of PtdIns(4,5)P(2) binding was compared with that of other polyphosphate phosphatidylinositols, it was seen to be greater in both PKCbeta- and PKCgamma-C2 domains. The concentration of Ca(2+) required to bind to membranes was seen to be lower in the presence of PtdIns(4,5)P(2) for all C2 domains, especially PKCalpha. In vivo experiments using differentiated PC12 cells transfected with each C2 domain fused to ECFP and stimulated with ATP demonstrated that, at limiting intracellular concentration of Ca(2+), the three C2 domains translocate to the plasma membrane at very similar rates. However, the plasma membrane dissociation event differed in each case, PKCalpha persisting for the longest time in the plasma membrane, followed by PKCgamma and, finally, PKCbeta, which probably reflects the different levels of Ca(2+) needed by each domain and their different affinities for PtdIns(4,5)P(2).
Assuntos
Membrana Celular/enzimologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Animais , Cálcio/farmacologia , Membrana Celular/efeitos dos fármacos , Cinética , Modelos Biológicos , Modelos Moleculares , Células PC12 , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Ratos , Receptores de Superfície Celular/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , TermodinâmicaRESUMO
Charcot-Marie-Tooth (CMT) neuropathies are highly heterogeneous disorders caused by mutations in more than 70 genes, with no available treatment. Thus, it is difficult to envisage a single suitable treatment for all pathogenetic mechanisms. Axonal Neuregulin 1 (Nrg1) type III drives Schwann cell myelination and determines myelin thickness by ErbB2/B3-PI3K-Akt signaling pathway activation. Nrg1 type III is inhibited by the α-secretase Tace, which negatively regulates PNS myelination. We hypothesized that modulation of Nrg1 levels and/or secretase activity may constitute a unifying treatment strategy for CMT neuropathies with focal hypermyelination as it could restore normal levels of myelination. Here we show that in vivo delivery of Niaspan, a FDA-approved drug known to enhance TACE activity, efficiently rescues myelination in the Mtmr2-/- mouse, a model of CMT4B1 with myelin outfoldings, and in the Pmp22+/- mouse, which reproduces HNPP (hereditary neuropathy with liability to pressure palsies) with tomacula. Importantly, we also found that Niaspan reduces hypermyelination of Vim (vimentin)-/- mice, characterized by increased Nrg1 type III and Akt activation, thus corroborating the hypothesis that Niaspan treatment downregulates Nrg1 type III signaling.