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1.
J Biol Chem ; 299(2): 102839, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36581210

RESUMO

Data from gnomAD indicate that a missense mutation encoding the T118M variation in human peripheral myelin protein 22 (PMP22) is found in roughly one of every 75 genomes of western European lineage (1:120 in the overall human population). It is unusual among PMP22 variants that cause Charcot-Marie-Tooth (CMT) disease in that it is not 100% penetrant. Here, we conducted cellular and biophysical studies to determine why T118M PMP22 predisposes humans to CMT, but with only incomplete penetrance. We found that T118M PMP22 is prone to mistraffic but differs even from the WT protein in that increased expression levels do not result in a reduction in trafficking efficiency. Moreover, the T118M mutant exhibits a reduced tendency to form large intracellular aggregates relative to other disease mutants and even WT PMP22. NMR spectroscopy revealed that the structure and dynamics of T118M PMP22 resembled those of WT. These results show that the main consequence of T118M PMP22 in WT/T118M heterozygous individuals is a reduction in surface-trafficked PMP22, unaccompanied by formation of toxic intracellular aggregates. This explains the incomplete disease penetrance and the mild neuropathy observed for WT/T118M CMT cases. We also analyzed BioVU, a biobank linked to deidentified electronic medical records, and found a statistically robust association of the T118M mutation with the occurrence of long and/or repeated episodes of carpal tunnel syndrome. Collectively, our results illuminate the cellular effects of the T118M PMP22 variation leading to CMT disease and indicate a second disorder for which it is a risk factor.


Assuntos
Doença de Charcot-Marie-Tooth , Proteínas da Mielina , Humanos , Doença de Charcot-Marie-Tooth/genética , Mutação de Sentido Incorreto , Proteínas da Mielina/genética , Predisposição Genética para Doença
2.
J Pathol ; 258(1): 69-82, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35686747

RESUMO

The development of neural structures within tumors is now considered vital for carcinogenesis. However, the time course of this development in human pre-invasive neoplasia has been incompletely described. Therefore, we performed a detailed analysis of nerves across the neoplastic spectrum in resected intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. Histology and multiplexed immunochemistry demonstrated that nerve density increased from low-grade (LG) to high-grade dysplasia (HG) but did not further increase once invasive IPMN (INV IPMN) was present. Higher nerve density correlated with increasing expression of nerve growth factor (NGF) by the tumor cells. Intra-tumoral nerves were immature and lacked markers of sympathetic, parasympathetic, and sensory lineages. Here, we show for the first time the presence of neural precursor cells (NPCs) within the stroma of pancreatic tumors. The density of these doublecortin (DCX)-positive NPCs increased from LG to HG, but not from HG to INV IPMN. We conclude that peak neural density of tumors is reached in high-grade dysplasia (often termed carcinoma in situ) rather than after invasion. These findings suggest that nerve-tumor interactions are important in IPMN progression and may serve as the basis for future mechanistic studies and novel therapeutic modalities. © 2022 The Pathological Society of Great Britain and Ireland.


Assuntos
Adenocarcinoma Mucinoso , Carcinoma Ductal Pancreático , Células-Tronco Neurais , Neoplasias Intraductais Pancreáticas , Neoplasias Pancreáticas , Adenocarcinoma Mucinoso/patologia , Carcinoma Ductal Pancreático/patologia , Humanos , Hiperplasia/patologia , Células-Tronco Neurais/metabolismo , Neurônios/patologia , Pâncreas/patologia , Neoplasias Intraductais Pancreáticas/patologia , Neoplasias Pancreáticas/patologia
3.
J Neurosci ; 41(42): 8710-8724, 2021 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-34507952

RESUMO

We report that the neurotrophin receptor p75 contributes to sensory neuron survival through the regulation of cholesterol metabolism in Schwann cells. Selective deletion of p75 in mouse Schwann cells of either sex resulted in a 30% loss of dorsal root ganglia (DRG) neurons and diminished thermal sensitivity. P75 regulates Schwann cell cholesterol biosynthesis in response to BDNF, forming a co-receptor complex with ErbB2 and activating ErbB2-mediated stimulation of sterol regulatory element binding protein 2 (SREBP2), a master regulator of cholesterol synthesis. Schwann cells lacking p75 exhibited decreased activation of SREBP2 and a reduction in 7-dehydrocholesterol (7-DHC) reductase (DHCR7) expression, resulting in accumulation of the neurotoxic intermediate, 7-dehyrocholesterol in the sciatic nerve. Restoration of DHCR7 in p75 null Schwann cells in mice significantly attenuated DRG neuron loss. Together, these results reveal a mechanism by which the disruption of lipid metabolism in glial cells negatively influences sensory neuron survival, which has implications for a wide range of peripheral neuropathies.SIGNIFICANCE STATEMENT Although expressed in Schwann cells, the role of p75 in myelination has remained unresolved in part because of its dual expression in sensory neurons that Schwann cells myelinate. When p75 was deleted selectively among Schwann cells, myelination was minimally affected, while sensory neuron survival was reduced by 30%. The phenotype is mainly due to dysregulation of cholesterol biosynthesis in p75-deficient Schwann cells, leading to an accumulation of neurotoxic cholesterol precursor, 7-dehydrocholesterol (7-DHC). Mechanism-wise, we discovered that in response to BDNF, p75 recruits and activates ErbB2 independently of ErbB3, thereby stimulating the master regulator, sterol regulatory element binding protein 2 (SREBP2). These results together highlight a novel role of p75 in Schwann cells in regulating DRG neuron survival by orchestrating proper cholesterol metabolism.


Assuntos
Receptores de Fator de Crescimento Neural/deficiência , Receptores de Fator de Crescimento Neural/genética , Células de Schwann/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Células de Schwann/ultraestrutura , Células Receptoras Sensoriais/ultraestrutura
4.
PLoS Pathog ; 16(2): e1008380, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32109948

RESUMO

Several barriers protect the central nervous system (CNS) from pathogen invasion. Yet viral infections of the CNS are common and often debilitating. Understanding how neurotropic viruses co-opt host machinery to overcome challenges to neuronal entry and transmission is important to combat these infections. Neurotropic reovirus disseminates through neural routes and invades the CNS to cause lethal encephalitis in newborn animals. To define mechanisms of reovirus neuronal entry and directional transport, we used primary neuron cultures, which reproduce in vivo infection patterns displayed by different reovirus serotypes. Treatment of neurons with small-molecule inhibitors of different endocytic uptake pathways allowed us to discover that the cellular machinery mediating macropinocytosis is required for reovirus neuronal entry. This mechanism of reovirus entry differs from clathrin-mediated endocytosis, which is used by reovirus to invade non-neuronal cells. Analysis of reovirus transport and release from isolated soma or axonal termini of neurons cultivated in microfluidic devices indicates that reovirus is capable of retrograde but only limited anterograde neuronal transmission. The dynamics of retrograde reovirus movement are consistent with fast axonal transport coordinated by dynein along microtubules. Further analysis of viral transport revealed that multiple virions are transported together in axons within non-acidified vesicles. Reovirus-containing vesicles acidify after reaching the soma, where disassembly of virions and release of the viral core into the cytoplasm initiates replication. These results define mechanisms of reovirus neuronal entry and transport and establish a foundation to identify common host factors used by neuroinvasive viruses. Furthermore, our findings emphasize consideration of cell type-specific entry mechanisms in the tailored design of neurotropic viruses as tracers, oncolytic agents, and delivery vectors.


Assuntos
Transporte Axonal/fisiologia , Infecções por Reoviridae/metabolismo , Reoviridae/metabolismo , Animais , Axônios/virologia , Linhagem Celular , Sistema Nervoso Central , Citoplasma/metabolismo , Endocitose , Masculino , Camundongos , Microtúbulos/metabolismo , Neurônios/metabolismo , Neurônios/virologia , Pinocitose/fisiologia , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Reoviridae/genética , Vírion/metabolismo , Internalização do Vírus
5.
J Biol Chem ; 295(34): 11963-11970, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32647009

RESUMO

Charcot-Marie-Tooth disease (CMT) is a neuropathy of the peripheral nervous system that afflicts ∼1:2500 people. The most common form of this disease (CMT1A, 1:4000) is associated with duplication of chromosome fragment 17p11.2-12, which results in a third WT PMP22 allele. In rodent models overexpressing the PMP22 (peripheral myelin protein 22) protein and in dermal fibroblasts from patients with CMT1A, PMP22 aggregates have been observed. This suggests that overexpression of PMP22 under CMT1A conditions overwhelms the endoplasmic reticulum quality control system, leading to formation of cytotoxic aggregates. In this work, we used a single-cell flow-cytometry trafficking assay to quantitatively examine the relationship between PMP22 expression and trafficking efficiency in individual cells. We observed that as expression of WT or disease variants of PMP22 is increased, the amount of intracellular PMP22 increases to a greater extent than the amount of surface-trafficked protein. This was true for both transiently transfected cells and PMP22 stable expressing cells. Our results support the notion that overexpression of PMP22 in CMT1A leads to a disproportionate increase in misfolding and mistrafficking of PMP22, which is likely a contributor to disease pathology and progression.


Assuntos
Doença de Charcot-Marie-Tooth/metabolismo , Regulação da Expressão Gênica , Proteínas da Mielina/biossíntese , Agregação Patológica de Proteínas/metabolismo , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Células HEK293 , Humanos , Proteínas da Mielina/genética , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia , Transporte Proteico
6.
J Biol Chem ; 294(32): 12054-12065, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31213528

RESUMO

Charcot-Marie-Tooth (CMT) disease is a peripheral neuropathy associated with gene duplication and point mutations in the peripheral myelin protein 22 (PMP22) gene. However, the role of PMP22 in Schwann cell physiology and the mechanisms by which PMP22 mutations cause CMT are not well-understood. On the basis of homology between PMP22 and proteins associated with modulation of ion channels, we hypothesized that PMP22 alters ion channel activity. Using whole-cell electrophysiology, we show here that heterologous PMP22 expression increases the amplitude of currents similar to those ascribed to store-operated calcium (SOC) channels, particularly those involving transient receptor canonical channel 1 (TrpC1). These channels help replenish Ca2+ in the endoplasmic reticulum (ER) following stimulus-induced depletion. Currents with similar properties were recorded in WT but not pmp22-/- mouse Schwann cells. Heterologous expression of the CMT-associated PMP22_L16P variant, which fails to reach the plasma membrane and localizes to the ER, led to larger currents than WT PMP22. Similarly, Schwann cells isolated from Trembler J (TrJ; PMP22_L16P) mice had larger currents than WT littermates. Calcium imaging in live nerves and cultured Schwann cells revealed elevated intracellular Ca2+ in TrJ mice compared with WT. Moreover, we found that PMP22 co-immunoprecipitated with stromal interaction molecule 1 (STIM1), the Ca2+ sensor SOC channel subunit in the ER. These results suggest that in the ER, PMP22 interacts with STIM1 and increases Ca2+ influx through SOC channels. Excess or mutant PMP22 in the ER may elevate intracellular Ca2+ levels, which could contribute to CMT pathology.


Assuntos
Canais de Cálcio/metabolismo , Proteínas da Mielina/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/química , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Retículo Endoplasmático/metabolismo , Potenciais Evocados/efeitos dos fármacos , Gadolínio/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Proteínas da Mielina/deficiência , Proteínas da Mielina/genética , Células de Schwann/citologia , Células de Schwann/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Canais de Cátion TRPC/metabolismo
7.
J Neurosci ; 38(24): 5606-5619, 2018 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-29789375

RESUMO

The p75 neurotrophin receptor (p75NTR) plays an integral role in patterning the sympathetic nervous system during development. Initially, p75NTR is expressed at low levels as sympathetic axons project toward their targets, which enables neurotrophin-3 (NT3) to activate TrkA receptors and promote growth. Upon reaching nerve growth factor (NGF) producing tissues, p75NTR is upregulated, resulting in formation of TrkA-p75 complexes, which are high-affinity binding sites selective for NGF, thereby blunting NT3 signaling. The level of p75NTR expressed on the neuron surface is instrumental in regulating trophic factor response; however, the mechanisms by which p75NTR expression is regulated are poorly understood. Here, we demonstrate a rapid, translation independent increase in surface expression of p75NTR in response to NGF in rat sympathetic neurons. p75NTR was mobilized to the neuron surface from GGA3-postitive vesicles through activation of the GTPase Arf6, which was stimulated by NGF, but not NT3 binding to TrkA. Arf6 activation required PI3 kinase activity and was prevented by an inhibitor of the cytohesin family of Arf6 guanine nucleotide exchange factors. Overexpression of a constitutively active Arf6 mutant (Q67L) was sufficient to significantly increase surface expression of p75NTR even in the absence of NGF. Functionally, expression of active Arf6 markedly attenuated the ability of NT3 to promote neuronal survival and neurite outgrowth, whereas the NGF response was unaltered. These data suggest that NGF activation of Arf6 through TrkA is critical for the increase in p75NTR surface expression that enables the switch in neurotrophin responsiveness during development in the sympathetic nervous system.SIGNIFICANCE STATEMENT p75NTR is instrumental in the regulation of neuronal survival and apoptosis during development and is also implicated as a contributor to aberrant neurodegeneration in numerous conditions. Therefore, a better understanding of the mechanisms that mediate p75NTR surface availability may provide insight into how and why neurodegenerative processes manifest and reveal new therapeutic targets. Results from this study indicate a novel mechanism by which p75NTR can be rapidly shuttled to the cell surface from existing intracellular pools and explores a unique pathway by which NGF regulates the sympathetic innervation of target tissues, which has profound consequences for the function of these organs.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Neurônios/metabolismo , Neurotrofina 3/metabolismo , Receptor trkA/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Fator 6 de Ribosilação do ADP , Animais , Proteínas do Tecido Nervoso , Neurogênese/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de Fatores de Crescimento , Sistema Nervoso Simpático/crescimento & desenvolvimento , Sistema Nervoso Simpático/metabolismo
9.
J Neurosci ; 37(24): 5885-5899, 2017 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-28522736

RESUMO

Remyelination of CNS axons by Schwann cells (SCs) is not efficient, in part due to the poor migration of SCs into the adult CNS. Although it is known that migrating SCs avoid white matter tracts, the molecular mechanisms underlying this exclusion have never been elucidated. We now demonstrate that myelin-associated glycoprotein (MAG), a well known inhibitor of neurite outgrowth, inhibits rat SC migration and induces their death via γ-secretase-dependent regulated intramembrane proteolysis of the p75 neurotrophin receptor (also known as p75 cleavage). Blocking p75 cleavage using inhibitor X (Inh X), a compound that inhibits γ-secretase activity before exposing to MAG or CNS myelin improves SC migration and survival in vitro Furthermore, mouse SCs pretreated with Inh X migrate extensively in the demyelinated mouse spinal cord and remyelinate axons. These results suggest a novel role for MAG/myelin in poor SC-myelin interaction and identify p75 cleavage as a mechanism that can be therapeutically targeted to enhance SC-mediated axon remyelination in the adult CNS.SIGNIFICANCE STATEMENT Numerous studies have used Schwann cells, the myelin-making cells of the peripheral nervous system to remyelinate adult CNS axons. Indeed, these transplanted cells successfully remyelinate axons, but unfortunately they do not migrate far and so remyelinate only a few axons in the vicinity of the transplant site. It is believed that if Schwann cells could be induced to migrate further and survive better, they may represent a valid therapy for remyelination. We show that myelin-associated glycoprotein or CNS myelin, in general, inhibit rodent Schwann cell migration and induce their death via cleavage of the neurotrophin receptor p75. Blockade of p75 cleavage using a specific inhibitor significantly improves migration and survival of the transplanted Schwann cells in vivo.


Assuntos
Apoptose/fisiologia , Movimento Celular/fisiologia , Glicoproteína Associada a Mielina/metabolismo , Crescimento Neuronal/fisiologia , Células de Schwann/citologia , Células de Schwann/fisiologia , Animais , Células Cultivadas , Feminino , Camundongos , Camundongos Nus , Bainha de Mielina/metabolismo
10.
J Cell Sci ; 127(Pt 9): 1966-79, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24569882

RESUMO

The p75 neurotrophin receptor (p75, also known as NGFR) is a multifaceted signalling receptor that regulates neuronal physiology, including neurite outgrowth, and survival and death decisions. A key cellular aspect regulating neurotrophin signalling is the intracellular trafficking of their receptors; however, the post-endocytic trafficking of p75 is poorly defined. We used sympathetic neurons and rat PC12 cells to study the mechanism of internalisation and post-endocytic trafficking of p75. We found that p75 internalisation depended on the clathrin adaptor protein AP2 and on dynamin. More surprisingly, p75 evaded the lysosomal route at the level of the early endosome, instead accumulating in two different types of endosomes, Rab11-positive endosomes and multivesicular bodies (MVBs) positive for CD63, a marker of the exosomal pathway. Consistently, depolarisation by KCl induced the liberation of previously endocytosed full-length p75 into the extracellular medium in exosomes. Thus, p75 defines a subpopulation of MVBs that does not mature to lysosomes and is available for exosomal release by neuronal cells.


Assuntos
Endossomos/metabolismo , Exossomos/metabolismo , Lisossomos/metabolismo , Corpos Multivesiculares/metabolismo , Neurônios/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Microscopia de Fluorescência , Proteínas do Tecido Nervoso , Células PC12 , Interferência de RNA , Ratos , Receptores de Fatores de Crescimento , Receptores de Fator de Crescimento Neural/genética
11.
J Biol Chem ; 289(31): 21205-16, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24939843

RESUMO

The p75 neurotrophin receptor (p75(NTR)) mediates the death of specific populations of neurons during the development of the nervous system or after cellular injury. The receptor has also been implicated as a contributor to neurodegeneration caused by numerous pathological conditions. Because many of these conditions are associated with increases in reactive oxygen species, we investigated whether p75(NTR) has a role in neurodegeneration in response to oxidative stress. Here we demonstrate that p75(NTR) signaling is activated by 4-hydroxynonenal (HNE), a lipid peroxidation product generated naturally during oxidative stress. Exposure of sympathetic neurons to HNE resulted in neurite degeneration and apoptosis. However, these effects were reduced markedly in neurons from p75(NTR-/-) mice. The neurodegenerative effects of HNE were not associated with production of neurotrophins and were unaffected by pretreatment with a receptor-blocking antibody, suggesting that oxidative stress activates p75(NTR) via a ligand-independent mechanism. Previous studies have established that proteolysis of p75(NTR) by the metalloprotease TNFα-converting enzyme and γ-secretase is necessary for p75(NTR)-mediated apoptotic signaling. Exposure of sympathetic neurons to HNE resulted in metalloprotease- and γ-secretase-dependent cleavage of p75(NTR). Pharmacological blockade of p75(NTR) proteolysis protected sympathetic neurons from HNE-induced neurite degeneration and apoptosis, suggesting that cleavage of p75(NTR) is necessary for oxidant-induced neurodegeneration. In vivo, p75(NTR-/-) mice exhibited resistance to axonal degeneration associated with oxidative injury following administration of the neurotoxin 6-hydroxydopamine. Together, these data suggest a novel mechanism linking oxidative stress to ligand-independent cleavage of p75(NTR), resulting in axonal fragmentation and neuronal death.


Assuntos
Apoptose/fisiologia , Axônios , Estresse Oxidativo , Receptores de Fator de Crescimento Neural/fisiologia , Animais , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso , Ratos , Ratos Sprague-Dawley , Receptores de Fatores de Crescimento , Testes de Campo Visual
12.
J Biol Chem ; 289(12): 8067-85, 2014 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-24519935

RESUMO

Malignant gliomas are highly invasive, proliferative, and resistant to treatment. Previously, we have shown that p75 neurotrophin receptor (p75NTR) is a novel mediator of invasion of human glioma cells. However, the role of p75NTR in glioma proliferation is unknown. Here we used brain tumor-initiating cells (BTICs) and show that BTICs express neurotrophin receptors (p75NTR, TrkA, TrkB, and TrkC) and their ligands (NGF, brain-derived neurotrophic factor, and neurotrophin 3) and secrete NGF. Down-regulation of p75NTR significantly decreased proliferation of BTICs. Conversely, exogenouous NGF stimulated BTIC proliferation through α- and γ-secretase-mediated p75NTR cleavage and release of its intracellular domain (ICD). In contrast, overexpression of the p75NTR ICD induced proliferation. Interestingly, inhibition of Trk signaling blocked NGF-stimulated BTIC proliferation and p75NTR cleavage, indicating a role of Trk in p75NTR signaling. Further, blocking p75NTR cleavage attenuated Akt activation in BTICs, suggesting role of Akt in p75NTR-mediated proliferation. We also found that p75NTR, α-secretases, and the four subunits of the γ-secretase enzyme were elevated in glioblastoma multiformes patients. Importantly, the ICD of p75NTR was commonly found in malignant glioma patient specimens, suggesting that the receptor is activated and cleaved in patient tumors. These results suggest that p75NTR proteolysis is required for BTIC proliferation and is a novel potential clinical target.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Neoplasias Encefálicas/metabolismo , Encéfalo/patologia , Glioma/metabolismo , Células-Tronco Neoplásicas/patologia , Fatores de Crescimento Neural/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Encéfalo/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Silenciamento de Genes , Glioma/genética , Glioma/patologia , Humanos , Mutação , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Receptor de Fator de Crescimento Neural/genética
13.
J Am Chem Soc ; 137(27): 8758-68, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-26102530

RESUMO

Despite broad biochemical relevance, our understanding of the physiochemical reactions that limit the assembly and cellular trafficking of integral membrane proteins remains superficial. In this work, we report the first experimental assessment of the relationship between the conformational stability of a eukaryotic membrane protein and the degree to which it is retained by cellular quality control in the secretory pathway. We quantitatively assessed both the conformational equilibrium and cellular trafficking of 12 variants of the α-helical membrane protein peripheral myelin protein 22 (PMP22), the intracellular misfolding of which is known to cause peripheral neuropathies associated with Charcot-Marie-Tooth disease (CMT). We show that the extent to which these mutations influence the energetics of Zn(II)-mediated PMP22 folding is proportional to the observed reduction in cellular trafficking efficiency. Strikingly, quantitative analyses also reveal that the reduction of motor nerve conduction velocities in affected patients is proportional to the extent of the mutagenic destabilization. This finding provides compelling evidence that the effects of these mutations on the energetics of PMP22 folding lie at the heart of the molecular basis of CMT. These findings highlight conformational stability as a key factor governing membrane protein biogenesis and suggest novel therapeutic strategies for CMT.


Assuntos
Doença de Charcot-Marie-Tooth/genética , Mutação de Sentido Incorreto , Proteínas da Mielina/química , Proteínas da Mielina/genética , Dobramento de Proteína , Sequência de Aminoácidos , Animais , Doença de Charcot-Marie-Tooth/metabolismo , Cães , Humanos , Células Madin Darby de Rim Canino , Metais/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas da Mielina/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/metabolismo , Conformação Proteica , Estabilidade Proteica , Transporte Proteico , Termodinâmica
14.
Ann Neurol ; 75(2): 255-65, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24339129

RESUMO

OBJECTIVE: The peripheral myelin protein-22 (PMP22) gene is associated with the most common types of inherited neuropathies, including hereditary neuropathy with liability to pressure palsies (HNPP) caused by PMP22 deficiency. However, the function of PMP22 has yet to be defined. Our previous study has shown that PMP22 deficiency causes an impaired propagation of nerve action potentials in the absence of demyelination. In the present study, we tested an alternative mechanism relating to myelin permeability. METHODS: Utilizing Pmp22(+) (/) (-) mice as a model of HNPP, we evaluated myelin junctions and their permeability using morphological, electrophysiological, and biochemical approaches. RESULTS: We show disruption of multiple types of cell junction complexes in peripheral nerve, resulting in increased permeability of myelin and impaired action potential propagation. We further demonstrate that PMP22 interacts with immunoglobulin domain-containing proteins known to regulate tight/adherens junctions and/or transmembrane adhesions, including junctional adhesion molecule-C (JAM-C) and myelin-associated glycoprotein (MAG). Deletion of Jam-c or Mag in mice recapitulates pathology in HNPP. INTERPRETATION: Our study reveals a novel mechanism by which PMP22 deficiency affects nerve conduction not through removal of myelin, but through disruption of myelin junctions.


Assuntos
Artrogripose/genética , Artrogripose/metabolismo , Neuropatia Hereditária Motora e Sensorial/genética , Neuropatia Hereditária Motora e Sensorial/metabolismo , Proteínas da Mielina/deficiência , Bainha de Mielina/metabolismo , Junções Íntimas/patologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Fatores Etários , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Axônios/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Moléculas de Adesão Juncional/metabolismo , Camundongos , Camundongos Transgênicos , Mutação/genética , Condução Nervosa/efeitos dos fármacos , Condução Nervosa/genética , Nervos Periféricos/metabolismo , Nervos Periféricos/patologia , Potássio/farmacologia , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/genética , Junções Íntimas/metabolismo
15.
J Neurosci ; 33(6): 2388-97, 2013 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-23392668

RESUMO

In the developing peripheral nervous system, axon-derived signals stimulate Schwann cells to undergo a global genetic reprogramming involving the cessation of cellular division and the upregulation of myelin genes. How such a comprehensive change in gene transcription is regulated is poorly understood. Here we report that BRG1/SMARCA4, the central helicase of the mammalian SWI/SNF-related chromatin remodeling complex, is required for Schwann cells to differentiate and form myelin, both in vitro and in vivo, in the mouse. BRG1 was highly activated in Schwann cells at early stages of myelination, and loss of the enzyme inhibited their differentiation and completely prevented myelin formation. Furthermore, we identify NF-κB as a key transcription factor that associates with the BRG1 complex in response to neuregulin 1 type III. During myelination, BRG1 was activated through the formation of a complex with NF-κB, and both proteins bound to the promoter region of Sox10, an inducer of myelination. These findings delineate a novel mechanism whereby axonal signals promote myelination through the remodeling of chromatin structure.


Assuntos
Diferenciação Celular/fisiologia , Cromatina/metabolismo , DNA Helicases/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Células de Schwann/fisiologia , Fatores de Transcrição/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Cromatina/fisiologia , Técnicas de Cocultura , DNA Helicases/fisiologia , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Camundongos Transgênicos , NF-kappa B/fisiologia , Proteínas Nucleares/fisiologia , Ratos , Células de Schwann/citologia , Fatores de Transcrição/fisiologia
16.
J Neurosci ; 32(38): 13022-31, 2012 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-22993420

RESUMO

During the development of the peripheral nervous system there is extensive apoptosis, and these neuronal corpses need to be cleared to prevent an inflammatory response. Recently, Jedi-1 and MEGF10, both expressed in glial precursor cells, were identified in mouse as having an essential role in this phagocytosis (Wu et al., 2009); however, the mechanisms by which they promote engulfment remained unknown. Both Jedi-1 and MEGF10 are homologous to the Drosophila melanogaster receptor Draper, which mediates engulfment through activation of the tyrosine kinase Shark. Here, we identify Syk, the mammalian homolog of Shark, as a signal transducer for both Jedi-1 and MEGF10. Syk interacted with each receptor independently through the immunoreceptor tyrosine-based activation motifs (ITAMs) in their intracellular domains. The interaction was enhanced by phosphorylation of the tyrosines in the ITAMs by Src family kinases (SFKs). Jedi association with Syk and activation of the kinase was also induced by exposure to dead cells. Expression of either Jedi-1 or MEGF10 in HeLa cells facilitated engulfment of carboxylated microspheres to a similar extent, and there was no additive effect when they were coexpressed. Mutation of the ITAM tyrosines of Jedi-1 and MEGF10 prevented engulfment. The SFK inhibitor PP2 or a selective Syk inhibitor (BAY 61-3606) also blocked engulfment. Similarly, in cocultures of glial precursors and dying sensory neurons from embryonic mice, addition of PP2 or knock down of endogenous Syk decreased the phagocytosis of apoptotic neurons. These results indicate that both Jedi-1 and MEGF10 can mediate phagocytosis independently through the recruitment of Syk.


Assuntos
Apoptose/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/genética , Motivos de Aminoácidos , Animais , Proteínas de Arabidopsis/metabolismo , Contagem de Células , Células Cultivadas , Técnicas de Cocultura , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Feminino , Gânglios Espinais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Humanos , Imunoprecipitação , Motivo de Ativação do Imunorreceptor Baseado em Tirosina/efeitos dos fármacos , Motivo de Ativação do Imunorreceptor Baseado em Tirosina/genética , Motivo de Ativação do Imunorreceptor Baseado em Tirosina/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Transferases Intramoleculares/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Microglia , Mutagênese Sítio-Dirigida , Mutação/genética , Neurônios , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Fagocitose/efeitos dos fármacos , Fagocitose/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteínas Tirosina Quinases/genética , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estaurosporina/farmacologia , Quinase Syk , Transfecção
17.
Biochemistry ; 52(19): 3229-41, 2013 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-23639031

RESUMO

Misfolding of the α-helical membrane protein peripheral myelin protein 22 (PMP22) has been implicated in the pathogenesis of the common neurodegenerative disease known as Charcot-Marie-Tooth disease (CMTD) and also several other related peripheral neuropathies. Emerging evidence suggests that the propensity of PMP22 to misfold in the cell may be due to an intrinsic lack of conformational stability. Therefore, quantitative studies of the conformational equilibrium of PMP22 are needed to gain insight into the molecular basis of CMTD. In this work, we have investigated the folding and unfolding of wild type (WT) human PMP22 in mixed micelles. Both kinetic and thermodynamic measurements demonstrate that the denaturation of PMP22 by n-lauroyl sarcosine (LS) in dodecylphosphocholine (DPC) micelles is reversible. Assessment of the conformational equilibrium indicates that a significant fraction of unfolded PMP22 persists even in the absence of the denaturing detergent. However, we find the stability of PMP22 is increased by glycerol, which facilitates quantitation of thermodynamic parameters. To our knowledge, this work represents the first report of reversible unfolding of a eukaryotic multispan membrane protein. The results indicate that WT PMP22 possesses minimal conformational stability in micelles, which parallels its poor folding efficiency in the endoplasmic reticulum. Folding equilibrium measurements for PMP22 in micelles may provide an approach to assess the effects of cellular metabolites or potential therapeutic agents on its stability. Furthermore, these results pave the way for future investigation of the effects of pathogenic mutations on the conformational equilibrium of PMP22.


Assuntos
Proteínas da Mielina/química , Dicroísmo Circular , Reagentes de Ligações Cruzadas , Glicerol , Humanos , Cinética , Micelas , Modelos Moleculares , Proteínas da Mielina/metabolismo , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Estrutura Quaternária de Proteína , Termodinâmica , Resposta a Proteínas não Dobradas
18.
J Biol Chem ; 287(2): 1600-8, 2012 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-22128191

RESUMO

Brain-derived neurotrophic factor (BDNF) was shown to play a role in Schwann cell myelination by recruiting Par3 to the axon-glial interface, but the underlying mechanism has remained unclear. Here we report that Par3 regulates Rac1 activation by BDNF but not by NRG1-Type III in Schwann cells, although both ligands activate Rac1 in vivo. During development, active Rac1 signaling is localized to the axon-glial interface in Schwann cells by a Par3-dependent polarization mechanism. Knockdown of p75 and Par3 individually inhibits Rac1 activation, whereas constitutive activation of Rac1 disturbs the polarized activation of Rac1 in vivo. Polarized Rac1 activation is necessary for myelination as Par3 knockdown attenuates myelination in mouse sciatic nerves as well as in zebrafish. Specifically, Par3 knockdown in zebrafish disrupts proper alignment between the axon and Schwann cells without perturbing Schwann cell migration, suggesting that localized Rac1 activation at the axon-glial interface helps identify the initial wrapping sites. We therefore conclude that polarization of Rac1 activation is critical for myelination.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular/metabolismo , Bainha de Mielina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/metabolismo , Células de Schwann/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Axônios/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Proteínas de Transporte/genética , Moléculas de Adesão Celular/genética , Proteínas de Ciclo Celular , Movimento Celular/fisiologia , Células Cultivadas , Camundongos , Proteínas do Tecido Nervoso/genética , Neuroglia/citologia , Neuroglia/metabolismo , Neuropeptídeos/genética , Ratos , Células de Schwann/citologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genética
19.
Nat Chem Biol ; 7(11): 803-9, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22053353

RESUMO

Cyclooxygenase-2 (COX-2) catalyzes the oxygenation of arachidonic acid and the endocannabinoids 2-arachidonoylglycerol and arachidonoylethanolamide. Evaluation of a series of COX-2 inhibitors revealed that many weak competitive inhibitors of arachidonic acid oxygenation are potent inhibitors of endocannabinoid oxygenation. (R) enantiomers of ibuprofen, naproxen and flurbiprofen, which are considered to be inactive as COX-2 inhibitors, are potent 'substrate-selective inhibitors' of endocannabinoid oxygenation. Crystal structures of the COX-2­(R)-naproxen and COX-2­(R)-flurbiprofen complexes verified this unexpected binding and defined the orientation of the (R) enantiomers relative to (S) enantiomers. (R)-Profens selectively inhibited endocannabinoid oxygenation by lipopolysaccharide-stimulated dorsal root ganglion (DRG) cells. Substrate-selective inhibition provides new tools for investigating the role of COX-2 in endocannabinoid oxygenation and a possible explanation for the ability of (R)-profens to maintain endocannabinoid tone in models of neuropathic pain.


Assuntos
Moduladores de Receptores de Canabinoides/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Ciclo-Oxigenase 2/metabolismo , Endocanabinoides , Ácido Araquidônico/metabolismo , Ácidos Araquidônicos/metabolismo , Domínio Catalítico , Química Farmacêutica , Inibidores de Ciclo-Oxigenase 2/química , Glicerídeos/metabolismo , Modelos Moleculares , Oxirredução , Ligação Proteica , Conformação Proteica , Especificidade por Substrato
20.
Cancer Cell ; 8(2): 155-67, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16098468

RESUMO

Germline NF1, c-RET, SDH, and VHL mutations cause familial pheochromocytoma. Pheochromocytomas derive from sympathetic neuronal precursor cells. Many of these cells undergo c-Jun-dependent apoptosis during normal development as NGF becomes limiting. NF1 encodes a GAP for the NGF receptor TrkA, and NF1 mutations promote survival after NGF withdrawal. We found that pheochromocytoma-associated c-RET and VHL mutations lead to increased JunB, which blunts neuronal apoptosis after NGF withdrawal. We also found that the prolyl hydroxylase EglN3 acts downstream of c-Jun and is specifically required among the three EglN family members for apoptosis in this setting. Moreover, EglN3 proapoptotic activity requires SDH activity because EglN3 is feedback inhibited by succinate. These studies suggest that failure of developmental apoptosis plays a role in pheochromocytoma pathogenesis.


Assuntos
Neoplasias das Glândulas Suprarrenais/enzimologia , Neoplasias das Glândulas Suprarrenais/genética , Apoptose , Feocromocitoma/enzimologia , Feocromocitoma/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Regulação Neoplásica da Expressão Gênica , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia , Proteínas Imediatamente Precoces/metabolismo , Mutação , Fator de Crescimento Neural/metabolismo , Neurônios/enzimologia , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais , Succinato Desidrogenase/metabolismo , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau
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