Note:

Mitochondria are dynamic organelles that constantly undergo opposing fission and fusion events which impact many aspects of mitochondrial and cellular homeostasis including bioenergetic activity, calcium buffering and organelle transport. The large GTPase dynamin-related protein 1 (Drp1) acts as a mechanoenzyme to catalyze fission of mitochondria. Drp1 activity is regulated through a series of reversible posttranslational modifications. Phosphorylation of the conserved serine residue, S656, by cAMP dependent protein kinase A (PKA) acts as a master regulator of Drp1 activity. Two phosphatases oppose PKA by dephosporylating Drp1 S656, a mitochondrial isoform of protein phosphatase 2A and the calcium-calmodulin dependent phosphatase calcineurin (CaN). Here I report the characterization of a conserved CaN docking site on Drp1, an LxVP motif, just upstream of the Drp1 S656 site. Mutational modification of the Drp1 LxVP motif resulted in selective bidirectional modulation of formation of the CaN:Drp1 complex. Stability of the CaN:Drp1 LxVP motif mutant complexes was qualitatively described by affinity purification and quantitatively described by isothermal titration calorimetry. Stability of the CaN:Drp1 complex was found to directly correlate with Drp1 S656 dephosphorylation kinetics as demonstrated by studies conducted in vitro and in intact cells. Further, the CaN:Drp1 signaling axis was shown to shape basal mitochondrial morphology in a heterologous cell line system and in primary hippocampal neurons. Finally, disruption of the CaN:Drp1 signaling axis was found to protect neurons from oxygen-glucose deprivation, an in vitro model of ischemic injury. While these results suggest that the CaN:Drp1 signaling axis may be a potential target for neuroprotective therapeutic exploitation, the mechanism by which disruption of the CaN:Drp1 signaling axis specifically and mitochondrial elongation generally results in resistance to ischemic injury remains unknown. Additional studies reported here demonstrate that mitochondrial fragmentation remains a prominent feature of injured neurons regardless of the fidelity of the CaN:Drp1 signaling axis. Mitochondrial fragmentation at the time of injury was found to occur in a Drp1-independent manner. Chronic mitochondrial elongation was also found to leave unaltered the ability of neurons to detoxify reactive oxygen species, buffer intracellular calcium and supply ATP for homeostatic function.

Leukocyte-specific protein 1 (LSP1), an F-actin binding protein, is involved in neutrophil recruitment into peritoneum. Because mechanisms of excessive migration of activated neutrophils into inflamed lungs, credited with tissue damage, are not fully understood, we explored the hitherto unknown expression and role of LSP1 in neutrophil migration in a mouse model of acute lung inflammation. We induced acute lung inflammation through intranasal E. coli lipopolysacharide (LPS) (80μg) in wild-type 129/SvJ (WT) and LSP1 deficient (LSP1-/-) mice. WT (n=10) and LSP1-/- (n=11) mice showed significant neutrophilia and more neutrophils in bronchoalveolar lavage (BAL) at 9 hour post-LPS challenge compared to respective saline-treated controls (WT=7; LSP1-/-=10). LPS treatment induced more BAL neutrophils (P<0.001), myeloperoxidase concentrations and Gr-1+ neutrophils in lung tissues in WT mice compared to LSP1-/- mice. Lung myeloperoxidase and Gr-1+ (P<0.05) were higher in LPS-treated WT compared to the LSP1-/- mice. Lung tissue and BAL fluid KC, MCP-1, MIP-1α and MIP-1β concentration and vascular permeability were not different between LPS-treated WT and LSP1-/- mice but TNF-α concentration was higher in LPS-treated WT mice. Hematoxylin and eosin staining showed more septal congestion in LPS-treated WT mice compared to LSP1-/- mice. LSP1 expression was increased in lungs from LPS-treated mice compared to saline control. The autopsied lungs from septic humans, compared to their respective controls, showed increased expression of LSP1. These data show that LSP1 expression is modulated in acute lung inflammation and that LSP1 deficiency reduces neutrophil migration into acute lung inflammation.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 76-102).

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2017. / Submitted by Albânia Cézar de Melo (albania@bce.unb.br) on 2017-06-23T16:23:53Z No. of bitstreams: 1 2017_SamuelCoelhoMandacaru.pdf: 9292757 bytes, checksum: 1a53ba8cc877a93480f8a2d77b63ff6d (MD5) / Approved for entry into archive by Raquel Viana (raquelviana@bce.unb.br) on 2017-07-27T20:55:36Z (GMT) No. of bitstreams: 1 2017_SamuelCoelhoMandacaru.pdf: 9292757 bytes, checksum: 1a53ba8cc877a93480f8a2d77b63ff6d (MD5) / Made available in DSpace on 2017-07-27T20:55:36Z (GMT). No. of bitstreams: 1 2017_SamuelCoelhoMandacaru.pdf: 9292757 bytes, checksum: 1a53ba8cc877a93480f8a2d77b63ff6d (MD5) Previous issue date: 2017-07-27 / Geleia Real (GR) dispara o desenvolvimento de larvas de abelhas fêmeas até rainhas. Este efeito tem sido atribuído à presença de Major Royal Jelly Protein 1 (MRJP1) presente na geleia real. MRJP1 isolada de GR está intimamente associada a apisimina, um peptídeo helicoidal com 54 resíduos de aminoácidos que promove uma associação não covalente de MRJP1 em oligômeros de diferentes tamanhos. Não existem dados de alta resolução disponíveis para essas estruturas e até mesmo sua estequiometria ainda não é clara. Nesta tese, examinamos a relação MRJP1/apisimina usando um arsenal de técnicas biofísicas. Também, investigamos o comportamento de MRJP1/apisimina em amostras após remoção de seus carboidratos e de apisimina associada. Nossos dados de espectrometria de massas (MS) nativos demonstraram que os complexos existem predominantemente numa estequiometria de MRJP14/apisimina4. Blue native PAGE demonstrou a prevalência de estruturas tetraméricas e monoméricas. Microscopia de força atômica demonstrou a presença de populações que puderam ser agrupadas em dois grandes grupos. Troca do hidrogênio por deutério (HDX) seguida de análises por espectrometria de massas revelaram que MRJP1, nesses complexos, é desordenada na extensão dos resíduos 20-265. Estruturas secundárias (provavelmente folhas beta antiparalelas) estáveis são encontradas marginalmente ao redor dos resíduos 266-432. Estas são regiões fracamente estruturadas com conformações que variam entre estruturada e desestruturada, gerando uma distribuição isotópica bimodal (EX1). Nós propomos que os complexos nativos (tetrâmeros) têm uma estrutura quaternária formada por “dímero de dímero”, onde as cadeias de MRJP1 são ligadas por apisimina. Especificamente, nossos dados sugerem que apisimina age como um ligante que forma contatos hidrofóbicos envolvendo o segmento 316VLFFGLV322 de MRJP1. Esta proteína tem dois sítios de glicosilação localizados nos resíduos de aminoácidos 144 e 177. Por 2DE podemos ver 9 proteoformas de MRJP1, mesmo após a remoção dos carboidratos. Deglicosilação produz grandes agregados solúveis, enfatizando o papel dos glicanos como inibidores de agregação. Amostras com apisimina parcialmente removida formam complexos diméricos com estequiometria (MRJP12/apisimina1). As informações produzidas neste trabalho podem contribuir para uma melhor compreensão da relação estrutura/função de MRJP1, que possui papéis únicos na biologia da abelha. / Royal jelly (RJ) triggers the development of female honeybee larvae into queens. This effect has been attributed to the presence of major royal jelly protein 1 (MRJP1) in RJ. MRJP1 isolated from royal jelly is tightly associated with apisimin, a 54-residue -helical peptide that promotes the noncovalent assembly of MRJP1 into multimers. No high resolution structural data are available for these complexes, and their binding stoichiometry remains uncertain. We examined MRJP1/apisimin using a range of biophysical techniques. In addition, we investigated the behavior of deglycosylated samples, as well as samples with reduced apisimin content. Our mass spectrometry (MS) data demonstrated that the native complexes predominantly exist in a (MRJP14 apisimin4) stoichiometry. Blue native and showed the prevalence of tetrameric and monomeric structures in native conditions. Atomic force microscopy also showed two populations. Hydrogen/deuterium exchange (HDX) MS revealed that MRJP1 within these complexes is extensively disordered in the range of the residues 20-265. Marginally stable secondary structure (likely antiparallel -sheet) exists around residues 266-432. These weakly structured regions interchange with conformations that are extensively unfolded, giving rise to bimodal (EX1) isotope distributions. We propose that the native complexes have a “dimer of dimers” quaternary structure in which MRJP1 chains are bridged by apisimin. Specifically, our data suggest that apisimin acts as a linker that forms hydrophobic contacts involving the MRJP1 segment 316VLFFGLV322. MRJP1 has 2 glycosites located at amino acids 144 and 177. By using 2-DE, we observed 9 MRJP1 proteoforms, even after carbohydrate removal. Deglycosylation produces large soluble aggregates, highlighting the role of glycans as aggregation inhibitors. Samples with reduced apisimin content form dimeric complexes with a (MRJP12 apisimin1) stoichiometry. Therefore, the information uncovered in this work should help pave the way towards a better understanding of the structure/function relationship for MRJP1, which possesses unique roles in the honey bee biology.

Le virus d’Epstein-Barr (EBV) est un herpèsvirus humain qui infecte plus de 90% de la population généralement de façon bénigne et asymptomatique. Cependant, de nombreuses données démontrent que ce virus peut également contribuer à certains processus de cancérisation. En effet, l’EBV est associé à de nombreuses pathologies malignes telles que le lymphome de Burkitt, le lymphome hodgkinien et le carcinome du nasopharynx. Dans la grande majorité de ces cancers associées à ce virus, l’EBV exprime un programme de latence de type II durant lequel la protéine LMP1 est exprimée. Elle est décrite comme l’oncogène majeur de l’EBV car son expression est nécessaire à la survie et à la prolifération des lignées transformées in vitro. Cette protéine membranaire est fonctionnellement apparentée aux membres de la famille des récepteurs du TNF. LMP1 est constitutivement active et son expression conduit à l’activation de voies de signalisation telles que les voies NF-&#954;B, PI3K et des MAPK. L’activation de ces voies de signalisation cellulaire confère à LMP1 des propriétés oncogéniques, cependant, des effets toxiques liés à son expression ont également été décrits. Effectivement, LMP1 est capable d’induire l’apoptose dans différents types cellulaires. Dans ce contexte, nous avons d’abord développé et caractérisé, des variants dérivés de LMP1 constitués de sa partie C-terminale signalisatrice, complète ou partielle, fusionnée à la protéine GFP. Nous montrons que ces variants sont capables de séquestrer les protéines adaptatrices se fixant à LMP1 ou au récepteur TNFR1, et d’inhiber le signal et les phénotypes induits par ces derniers. Ces protéines à effet dominant négatif peuvent ainsi contrecarrer les effets transformants de LMP1 dans des modèles de latence II et III. Ces dominants négatifs peuvent aussi inhiber l’activation du TNFR1 et les phénotypes qui en découlent. Puis, nous avons étudié les propriétés de LMP1 en dehors d’un contexte infectieux et son rôle dans la transformation épithéliale. Nous démontrons que LMP1 induit la mort des cellules épithéliales MDCK mais certaines cellules outrepassent ses effets cytotoxiques générant des lignées qui expriment stablement LMP1 et dans lesquelles cet oncogène viral favorise la survie et exacerbe les phénotypes induits par le facteur de croissance HGF. Le caractère ambivalent de LMP1 pourrait limiter le pouvoir oncogène de l’EBV mais en contrepartie favoriser l’émergence de cellules résistantes à l’apoptose et capables de répondre de façon accrue à des facteurs de croissance. Nos travaux ont permis de mieux comprendre la dualité fonctionnelle de LMP1, d’une part ses effets oncogènes favorisant la survie cellulaire et d’autre part ses propriétés pro-apoptotiques, induites directement ou révélées suite à son inhibition, limitant la tumorigenèse. La caractérisation des mécanismes moléculaires impliquant LMP1 pourrait ainsi participer à la définition de potentielles stratégies thérapeutiques pour le traitement de cancers associés à l’EBV et où LMP1 est exprimée. / Epstein-Barr virus (EBV) is a human herpesvirus that infects more than 90% of worldwide population, generally asymptomatically. However, numerous studies show that EBV promotes tumorigenesis. Indeed, EBV infection is associated with many human malignancies including Burkitt’s lymphoma, Hodgkin’s lymphoma and nasopharyngeal carcinoma. In most of these cancers associated with EBV, it expresses latency II program in which the latent membrane protein 1 (LMP1) is expressed. LMP1 is described as the major EBV oncogene because its expression is necessary in vitro for survival and proliferation of transformed cell lines. This membrane protein is functionally related to members of the TNF receptors superfamily. LMP1 is constitutively active and its expression leads to activation of NF-&#954;B, PI3K and MAPK signaling pathways. These activation confers oncogenic properties to LMP1, however, toxic effects associated with its expression are also described. Indeed, LMP1 can induce cell death in different cell types. In this context, we first developed and characterized LMP1 derivative variants consisting of its C-terminal signal, complete or partial, fused to GFP. We show that these variants are able to sequester adaptors binding to LMP1 and TNFR1, and inhibit signal and phenotypes induced by them. These proteins have dominant negative effect and may counteract LMP1 transformant properties in latency II cellular models. In addition, these dominant negatives impair TNFR1 signaling and associated phenotypes. Then, we studied LMP1 properties outside infectious context and its involvement in epithelial transformation. We show that LMP1 induces cell death in MDCK epithelial cells, but some go beyond its cytotoxic effects generating lines stably expressing LMP1 and in which this viral oncogene promotes survival and exacerbates HGF-induced phenotypes. Ambivalent character of LMP1 could limit the oncogenic potential of EBV but in return support the emergence of cells resistant to apoptosis and able to enhance growth factor responses. Our work allowed us to better understand the functional duality of LMP1 on the one hand its oncogenic effects favoring cell survival and other pro-apoptotic properties, induced directly or reveal by its inhibition, limiting tumorigenesis. Thus, characterization of molecular mechanisms involving LMP1 could participate in the definition of potential therapeutic strategies for treating cancers associated with EBV and where LMP1 is expressed.

Indiana University-Purdue University Indianapolis (IUPUI) / Chronic inflammation is a major driver of tumor progression in over fifty percent of breast cancers. Tumors activate inflammatory processes by secreting factors that recruit and trigger inflammatory cells to release cytokines such as Interleukin 6 (IL-6). IL-6 stimulates the activity of signal transducers and activators of transcription 3 (STAT3), a transcription factor that has been extensively studied for its role in promoting breast cancer. Recently, downregulated HIPPO signaling was shown to drive the pro-growth effects of IL 6. Reduced HIPPO signaling allows for the nuclear translocation of transcriptional co-activator yes associated protein (YAP), implicating IL-6 in the co-activation of several transcription factors such as the TEADs that trigger pro growth programs. While IL-6/STAT3 stimulation has been shown to increase YAP activity, the mechanism driving this remains undocumented. The Angiomotins (Amots) are adapters of the HIPPO pathway that directly bind and regulate YAP activity. Molecular characterization of Amot transcriptional regulation unexpectedly revealed a single promoter controlling the expression of its two major isoforms: Amot 130 and Amot 80. Through immunofluorescent analysis, this study found that total Amot levels were elevated across multiple breast tumor subtypes and highest in samples with increased presence of stromal inflammatory cells. Further, the induction of total Amot expression by IL 6 was found to be essential for YAP dependent growth of breast cancer cells. The activation of Amot transcription by IL-6 was found to be through Specificity Protein 1 (Sp1), a transcription factor that is activated by STAT3. This work connects the activation of YAP1 by IL-6/STAT3 through the elevation of Amot expression by Sp1. Taken together, this explains a new avenue whereby breast cancer cells acquire enhanced oncogenic properties in response to inflammatory signaling.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20068号 / 農博第2197号 / 新制||農||1046(附属図書館) / 学位論文||H28||N5024(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 河田 照雄, 教授 金本 龍平, 教授 谷 史人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Introdução: Dentre as neuropatias hereditárias, as neuropatias hereditárias motoras e sensitivas (HMSN), também conhecidas como Doença de Charcot-MarieTooth (CMT) são as mais comuns, podendo acometer 1:2500 pessoas. Elas podem ser classificadas com base nas características clínicas, eletrofisiológicas, padrão de herança e mutação em localização gênica/mutação. Atualmente, mais de 80 genes estão relacionados à CMT, dentre eles o GDAP1 que é responsável pelas formas CMT4A, AR-CMT2, CMTRIA e CMT2K. O gene codifica a proteína GDAP1 que é expressa pelos neurônios do sistema nervoso periférico e central e também pelas células de Schwann. Mutações no GDAP1 geralmente estão relacionados com CMT de herança recessiva, mas autossômica dominante também pode ocorrer. Geralmente, as neuropatias recessivas são de diagnóstico molecular mais difíceis, são mais graves e tem rápida progressão. É necessário que haja um maior número de casos de pacientes com CMT envolvendo mutações no GDAP1, juntamente com os dados clínicos, patológicos e de eletrofisiologia detalhados, para estabelecer uma relação de confiança entre o genótipo e o fenótipo das diferentes formas da doença. O objetivo do trabalho foi investigar mutações no gene GDAP1 em uma amostra da população brasileira com quadro clínico de CMT tanto axonal quanto desmielinizantes. Métodos: Screening mutacional do gene GDAP1 por sequenciamento direto em 100 pacientes com diagnóstico clínico sugestivo de CMT4, CMT2, CMTi e CMT esporádico, onde mutações nos genes MPZ, MFN2 e GJB1 foram previamente excluídos. Resultados: Foram encontradas alterações no GDAP1 em 6 pacientes índices não relacionados, sendo que 1 deles foi homozigoto para a alteração Q163*, 1 heterozigoto composto para as alterações N64S e R125*, dois pacientes não relacionados foram heterozigotos compostos para as alterações P119T e Q163*, 1 paciente em heterozigose para a alteração P119T e 1 paciente em heterozigose para alteração K207T. Dentre essas alterações, as variantes N64S, P119T, K207T ainda não foram descritas na literatura. Conclusão: Os resultados obtidos mostraram que mutações no gene GDAP1 estão presentes em pacientes da população brasileira com fenótipo de CMT2, AR-CMT2 e CMT esporádico. A frequência de ocorrência pode ser considerada alta (3,88%). Ademais, foram encontradas na população de estudo, duas mutações conhecidamente patogênicas (Gln163Ter e Arg125Ter) e três novas variantes (Asn64Ser, Pro119Thr, Lys207Thr) que ainda não haviam sido relacionadas ao fenótipo de CMT. / Introduction: Among the inherited neuropathies, the hereditary motor and sensory neuropathies (HMSN), also known as Charcot-Marie-Tooth disease, are the most common ones and may affect 1:2500 people. They can be classified based on their clinical features, electrophysiology, inheritance pattern and mutation on a gene location/mutation. Today, more than 80 genes have been related to the CMT disease and among them the GDAP1 gene, responsible for the CMT4A, AR-CMT2, CMTRIA and CMT2K forms. This gene codes the GDAP1 protein, which is expressed by neurons from the peripheral and central nervous system and also by Schwann cells. GDAP1 mutations are usually related to recessive inheritance, although autosomal dominant cases can also occur. Most of the times, recessive neuropathies tend to have a more difficult molecular diagnosis, besides being more severe and presenting fast progression. A larger number of patients with CMT related to GDAP1 mutations, along with detailed clinical, pathological and electrophysiological data, is necessary in order to establish a trustful relation between genotype and phenotype in the different forms of this disease. The objective of this research was to investigate mutations in the GDAP1 gene in a brazilian sample of patients with clinical picture of both axonal and demyelinating CMT. Methods: Mutational screening of the GDAP1 gene by direct sequencing of 100 patients presenting suggestive clinical diagnosis of CMT4, CMT2, CMTi and sporadic CMT, where mutations in the MPZ, MFN2 and GJB1 genes have been previously excluded. Results: Alterations in the GDAP1 were found in 6 unrelated index patients, including 1 homozygous for the Q163* alteration, 1 compound heterozygous for the N64S and R125* alterations, 2 compound heterozygous for the P119T and Q163* alterations, 1 heterozygous for the P119T alteration alone and 1 heterozygous for the K207T alteration. Among these alterations, the variants N64S, P119T, K207T haven\'t been described in the literature yet. Conclusion: The results obtained showed that mutations in GDAP1 gene are present in patients of brazilian population with phenotype of CMT2, AR-CMT2 and sporadic CMT. The occurrence frequency can be considered high (3,88%). Furthermore, were found in the studied population two mutations known as pathogenic (Gln163Ter and Arg125Ter) and three news variants (Asn64Ser, Pro119Thr, Lys207Thr), that had not been related to the CMT phenotype.

Introdução: Dentre as neuropatias hereditárias, as neuropatias hereditárias motoras e sensitivas (HMSN), também conhecidas como Doença de Charcot-MarieTooth (CMT) são as mais comuns, podendo acometer 1:2500 pessoas. Elas podem ser classificadas com base nas características clínicas, eletrofisiológicas, padrão de herança e mutação em localização gênica/mutação. Atualmente, mais de 80 genes estão relacionados à CMT, dentre eles o GDAP1 que é responsável pelas formas CMT4A, AR-CMT2, CMTRIA e CMT2K. O gene codifica a proteína GDAP1 que é expressa pelos neurônios do sistema nervoso periférico e central e também pelas células de Schwann. Mutações no GDAP1 geralmente estão relacionados com CMT de herança recessiva, mas autossômica dominante também pode ocorrer. Geralmente, as neuropatias recessivas são de diagnóstico molecular mais difíceis, são mais graves e tem rápida progressão. É necessário que haja um maior número de casos de pacientes com CMT envolvendo mutações no GDAP1, juntamente com os dados clínicos, patológicos e de eletrofisiologia detalhados, para estabelecer uma relação de confiança entre o genótipo e o fenótipo das diferentes formas da doença. O objetivo do trabalho foi investigar mutações no gene GDAP1 em uma amostra da população brasileira com quadro clínico de CMT tanto axonal quanto desmielinizantes. Métodos: Screening mutacional do gene GDAP1 por sequenciamento direto em 100 pacientes com diagnóstico clínico sugestivo de CMT4, CMT2, CMTi e CMT esporádico, onde mutações nos genes MPZ, MFN2 e GJB1 foram previamente excluídos. Resultados: Foram encontradas alterações no GDAP1 em 6 pacientes índices não relacionados, sendo que 1 deles foi homozigoto para a alteração Q163*, 1 heterozigoto composto para as alterações N64S e R125*, dois pacientes não relacionados foram heterozigotos compostos para as alterações P119T e Q163*, 1 paciente em heterozigose para a alteração P119T e 1 paciente em heterozigose para alteração K207T. Dentre essas alterações, as variantes N64S, P119T, K207T ainda não foram descritas na literatura. Conclusão: Os resultados obtidos mostraram que mutações no gene GDAP1 estão presentes em pacientes da população brasileira com fenótipo de CMT2, AR-CMT2 e CMT esporádico. A frequência de ocorrência pode ser considerada alta (3,88%). Ademais, foram encontradas na população de estudo, duas mutações conhecidamente patogênicas (Gln163Ter e Arg125Ter) e três novas variantes (Asn64Ser, Pro119Thr, Lys207Thr) que ainda não haviam sido relacionadas ao fenótipo de CMT. / Introduction: Among the inherited neuropathies, the hereditary motor and sensory neuropathies (HMSN), also known as Charcot-Marie-Tooth disease, are the most common ones and may affect 1:2500 people. They can be classified based on their clinical features, electrophysiology, inheritance pattern and mutation on a gene location/mutation. Today, more than 80 genes have been related to the CMT disease and among them the GDAP1 gene, responsible for the CMT4A, AR-CMT2, CMTRIA and CMT2K forms. This gene codes the GDAP1 protein, which is expressed by neurons from the peripheral and central nervous system and also by Schwann cells. GDAP1 mutations are usually related to recessive inheritance, although autosomal dominant cases can also occur. Most of the times, recessive neuropathies tend to have a more difficult molecular diagnosis, besides being more severe and presenting fast progression. A larger number of patients with CMT related to GDAP1 mutations, along with detailed clinical, pathological and electrophysiological data, is necessary in order to establish a trustful relation between genotype and phenotype in the different forms of this disease. The objective of this research was to investigate mutations in the GDAP1 gene in a brazilian sample of patients with clinical picture of both axonal and demyelinating CMT. Methods: Mutational screening of the GDAP1 gene by direct sequencing of 100 patients presenting suggestive clinical diagnosis of CMT4, CMT2, CMTi and sporadic CMT, where mutations in the MPZ, MFN2 and GJB1 genes have been previously excluded. Results: Alterations in the GDAP1 were found in 6 unrelated index patients, including 1 homozygous for the Q163* alteration, 1 compound heterozygous for the N64S and R125* alterations, 2 compound heterozygous for the P119T and Q163* alterations, 1 heterozygous for the P119T alteration alone and 1 heterozygous for the K207T alteration. Among these alterations, the variants N64S, P119T, K207T haven\'t been described in the literature yet. Conclusion: The results obtained showed that mutations in GDAP1 gene are present in patients of brazilian population with phenotype of CMT2, AR-CMT2 and sporadic CMT. The occurrence frequency can be considered high (3,88%). Furthermore, were found in the studied population two mutations known as pathogenic (Gln163Ter and Arg125Ter) and three news variants (Asn64Ser, Pro119Thr, Lys207Thr), that had not been related to the CMT phenotype.