Microglial depletion has no impact on disease progression in a mouse model of machado–joseph disease

Machado–Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is an autosomal dominant neurodegenerative disorder (ND). While most research in NDs has been following a neuron-centric point of view, microglia are now recognized as crucial in the brain. Previous work revealed alterations that point to an increased activation state of microglia in the brain of CMVMJD135 mice, a MJD mouse model that replicates the motor symptoms and neuropathology of the human condition. Here, we investigated the extent to which microglia are actively contributing to MJD pathogenesis and symptom progression. For this, we used PLX3397 to reduce the number of microglia in the brain of CMVMJD135 mice. In addition, a set of statistical and machine learning models were further implemented to analyze the impact of PLX3397 on the morphology of the surviving microglia. Then, a battery of behavioral tests was used to evaluate the impact of microglial depletion on the motor phenotype of CMVMJD135 mice. Although PLX3397 treatment substantially reduced microglia density in the affected brain regions, it did not affect the motor deficits seen in CMVMJD135 mice. In addition to reducing the number of microglia, the treatment with PLX3397 induced morphological changes suggestive of activation in the surviving microglia, the microglia of wild-type animals becoming similar to those of CMVMJD135 animals. These results suggest that microglial cells are not key contributors for MJD progression. Furthermore, the impact of PLX3397 on microglial activation should be taken into account in the interpretation of findings of ND modification seen upon treatment with this CSF1R inhibitor.Fundação para a Ciência e a Tecnologia (FCT) (PTDC/NEUNMC/3648/2014) and COMPETE-FEDER (POCI-01-0145-FEDER-016818). It was also supported by Portuguese funds through FCT in the framework of the Project POCI-01-0145-FEDER-031987 (PTDC/MED-OUT/31987/2017). A.B.C. was supported by a doctoral fellowship from FCT (PD/BD/127828/2016). B.C. was also supported by FCT (2020.03898.CEECIND). This work was funded by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI (Portuguese Platform of Bioimaging) (PPBIPOCI-01-0145-FEDER-022122), and by National funds, through FCT—project UIDB/50026/2020 and UIDP/50026/2020

Profiling microglia in a mouse model of Machado-Joseph disease

Microglia have been increasingly implicated in neurodegenerative diseases (NDs), and specific disease associated microglia (DAM) profiles have been defined for several of these NDs. Yet, the microglial profile in Machado–Joseph disease (MJD) remains unexplored. Here, we characterized the profile of microglia in the CMVMJD135 mouse model of MJD. This characterization was performed using primary microglial cultures and microglial cells obtained from disease-relevant brain regions of neonatal and adult CMVMJD135 mice, respectively. Machine learning models were implemented to identify potential clusters of microglia based on their morphological features, and an RNA-sequencing analysis was performed to identify molecular perturbations and potential therapeutic targets. Our findings reveal morphological alterations that point to an increased activation state of microglia in CMVMJD135 mice and a disease-specific transcriptional profile of MJD microglia, encompassing a total of 101 differentially expressed genes, with enrichment in molecular pathways related to oxidative stress, immune response, cell proliferation, cell death, and lipid metabolism. Overall, these results allowed us to define the cellular and molecular profile of MJD-associated microglia and to identify genes and pathways that might represent potential therapeutic targets for this disorder.This work was supported by Fundação para a Ciência e a Tecnologia (FCT) (PTDC/NEUNMC/3648/2014) and COMPETE-FEDER (POCI-01-0145-FEDER-016818). It was also supported by Portuguese funds through FCT in the framework of the Project POCI-01-0145-FEDER-031987 (PTDC/MED-OUT/31987/2017). A.B.C. was supported by a doctoral fellowship from FCT (PD/BD/ 127828/2016). S.P.N. was also supported by FCT (PD/BD/114120/2015). Work in the JBR laboratory was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operational Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT in the framework of the Project POCI-01-0145- FEDER030647 (PTDC/MED-NEU/31318/2017). This work was funded by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI (Portuguese Platform of Bioimaging) (PPBIPOCI-01-0145-FEDER-022122), and by National funds, through FCT—project UIDB/50026/2020 and UIDP/50026/2020

Microglial Depletion Has No Impact on Disease Progression in a Mouse Model of Machado–Joseph Disease

Machado–Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is an autosomal dominant neurodegenerative disorder (ND). While most research in NDs has been following a neuron-centric point of view, microglia are now recognized as crucial in the brain. Previous work revealed alterations that point to an increased activation state of microglia in the brain of CMVMJD135 mice, a MJD mouse model that replicates the motor symptoms and neuropathology of the human condition. Here, we investigated the extent to which microglia are actively contributing to MJD pathogenesis and symptom progression. For this, we used PLX3397 to reduce the number of microglia in the brain of CMVMJD135 mice. In addition, a set of statistical and machine learning models were further implemented to analyze the impact of PLX3397 on the morphology of the surviving microglia. Then, a battery of behavioral tests was used to evaluate the impact of microglial depletion on the motor phenotype of CMVMJD135 mice. Although PLX3397 treatment substantially reduced microglia density in the affected brain regions, it did not affect the motor deficits seen in CMVMJD135 mice. In addition to reducing the number of microglia, the treatment with PLX3397 induced morphological changes suggestive of activation in the surviving microglia, the microglia of wild-type animals becoming similar to those of CMVMJD135 animals. These results suggest that microglial cells are not key contributors for MJD progression. Furthermore, the impact of PLX3397 on microglial activation should be taken into account in the interpretation of findings of ND modification seen upon treatment with this CSF1R inhibitor

Profiling Microglia in a Mouse Model of Machado–Joseph Disease

Microglia have been increasingly implicated in neurodegenerative diseases (NDs), and specific disease associated microglia (DAM) profiles have been defined for several of these NDs. Yet, the microglial profile in Machado–Joseph disease (MJD) remains unexplored. Here, we characterized the profile of microglia in the CMVMJD135 mouse model of MJD. This characterization was performed using primary microglial cultures and microglial cells obtained from disease-relevant brain regions of neonatal and adult CMVMJD135 mice, respectively. Machine learning models were implemented to identify potential clusters of microglia based on their morphological features, and an RNA-sequencing analysis was performed to identify molecular perturbations and potential therapeutic targets. Our findings reveal morphological alterations that point to an increased activation state of microglia in CMVMJD135 mice and a disease-specific transcriptional profile of MJD microglia, encompassing a total of 101 differentially expressed genes, with enrichment in molecular pathways related to oxidative stress, immune response, cell proliferation, cell death, and lipid metabolism. Overall, these results allowed us to define the cellular and molecular profile of MJD-associated microglia and to identify genes and pathways that might represent potential therapeutic targets for this disorder

O papel da disfunção microglial associada ao envelhecimento nos processos neurodegenerativos da doença de Machado-Joseph

Programa doutoral em Aging and Chronic DiseasesCélulas microgliais têm sido, cada vez mais, associadas às doenças neurodegenerativas, apresentando diferentes perfis para várias destas doenças. Contudo, o perfil da microglia na doença de Machado-Joseph (DMJ) permanece desconhecido, bem como a contribuição destas células para a doença. Deste modo, este trabalho começou pela caracterização do perfil das células da microglia no modelo de ratinho que mimetiza a DMJ, o modelo CMVMJD135, através do uso das células da microglia obtidas das regiões do cérebro mais afetadas por esta doença. Diversos modelos foram usados para identificar perturbações celulares e moleculares, e potenciais alvos terapêuticos. Mais tarde, perguntamos se a microglia contribuía, ou não, para a patogénese da DMJ. A resposta surgiu pela depleção da microglia nos cérebros dos ratinhos CMVMJD135 usando o fármaco PLX3397. Um conjunto de testes comportamentais foi depois aplicado para avaliar o impacto da depleção destas células no fenótipo motor dos ratinhos CMVMJD135. Os resultados obtidos revelam alterações morfológicas que apontam para um aumento do estado de ativação da microglia nos ratinhos CMVMJD135 e um perfil transcricional específico da microglia associada à DMJ, abrangendo um total de 101 genes diferencialmente expressos, com enriquecimento em vias relacionadas com o stress oxidativo, resposta imune e metabolismo lipídico. Apesar do tratamento com o fármaco ter promovido uma redução substancial do número de células microgliais, isto não alterou os défices motores presentes neste modelo de ratinho. O fármaco induziu também alterações morfológicas na microglia sobrevivente dos ratinhos de controlo, o que poderá trazer implicações para outros estudos que utilizam este fármaco para reduzir o número de células microgliais. No geral, estes resultados permitiram-nos definir o perfil celular e molecular da microglia associada à DMJ e identificar genes e vias que podem representar potenciais alvos terapêuticos para combater esta doença. No entanto, estes resultados também sugerem que, apesar das alterações morfológicas, fenotípicas e transcriptómicas observadas na microglia dos ratinhos da DMJ, estas células poderão não ser contribuidores chave para a progressão desta doença.Microglia have been increasingly implicated in neurodegenerative diseases (NDs), with non-homeostatic or pathological microglial profiles being defined for several of these NDs. Yet, the microglial profile in Machado- Joseph disease (MJD) remains unexplored as well as their contribution to the disease. Hence, in this study we first characterized the microglial profile in the CMVMJD135 mouse model of MJD, using microglial cells obtained from disease-relevant brain regions. Machine learning models and an RNA-sequencing analysis were used to identify cellular and molecular perturbations and potential therapeutic targets. Afterwards, we asked whether microglia are, or not, actively contributing for MJD pathogenesis. This was addressed by depleting microglia in the brains of CMVMJD135 mice, through the pharmacological inhibition of colony stimulating factor 1 receptor signaling, using PLX3397. A battery of behavioral tests was then applied to evaluate the impact of microglial depletion on the motor phenotype of CMVMJD135 mice. Our findings reveal morphological alterations that point to an increased activation state of microglia in CMVMJD135 mice and a disease-specific transcriptional profile of MJD microglia, encompassing a total of 101 differentially expressed genes, with enrichment in molecular pathways related to oxidative stress, immune response, cell proliferation, cell death, and lipid metabolism. Although PLX3397 treatment substantially reduced microglia numbers in the affected brain regions, it did not affect the motor deficits seen in this mouse model of MJD. Our results also show that, in addition to reducing the number of microglial cells, the treatment with PLX3397 induces morphological changes in the surviving microglia of wild-type mice that are a finding that has implications for other studies using this drug as a microglia depletion tool. Overall, these results allowed us to define the cellular and molecular profile of MJD-associated microglia and to identify genes and pathways that might represent potential therapeutic targets for this disorder. However, these results also suggest that despite the morphological, phenotypic, and transcriptomic changes seen in microglia in MJD mice, these cells may not be significant key contributors for MJD progression

Vesicles from different Trypanosoma cruzi strains trigger differential innate and chronic immune responses

Submitted by Nuzia Santos ([email protected]) on 2016-09-08T12:35:46Z No. of bitstreams: 1 ve_Paula_Nogueira_Vesicles_CPqRR_2015.pdf: 7053221 bytes, checksum: 38b07d08c52b86bcd4b207e402020ad9 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2016-09-08T18:36:59Z (GMT) No. of bitstreams: 1 ve_Paula_Nogueira_Vesicles_CPqRR_2015.pdf: 7053221 bytes, checksum: 38b07d08c52b86bcd4b207e402020ad9 (MD5)Made available in DSpace on 2016-09-08T18:36:59Z (GMT). No. of bitstreams: 1 ve_Paula_Nogueira_Vesicles_CPqRR_2015.pdf: 7053221 bytes, checksum: 38b07d08c52b86bcd4b207e402020ad9 (MD5) Previous issue date: 2015Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte,MG, Brasil/Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilUniversidade Federal de São Paulo. Departamento de Ciências Biológicas. Laboratório de Imunologia Celular e Bioquímica de Fungos e Protozoários. São Paulo, SP, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilUniversidade Federal de São Paulo. Departamento de Ciências Biológicas. Laboratório de Imunologia Celular e Bioquímica de Fungos e Protozoários. São Paulo, SP, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilUniversidade de São Paulo. Instituto de Química. Departamento de Bioquímica. São Paulo, SP, BrasilUniversidade de São Paulo. Instituto de Química. Departamento de Bioquímica. São Paulo, SP, BrasilUniversidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte,MG, Brasil/Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilUniversidade Federal de São Paulo. Departamento de Ciências Biológicas. Laboratório de Imunologia Celular e Bioquímica de Fungos e Protozoários. São Paulo, SP, BrasilTrypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas Disease, shed extracellular vesicles (EVs) enriched with glycoproteins of the gp85/trans-sialidase (TS) superfamily and other α-galactosyl (α-Gal)-containing glycoconjugates, such as mucins. Here, purified vesicles from T. cruzi strains (Y, Colombiana, CL-14 and YuYu) were quantified according to size, intensity and concentration. Qualitative analysis revealed differences in their protein and α-galactosyl contents. Later, those polymorphisms were evaluated in the modulation of immune responses (innate and in the chronic phase) in C57BL/6 mice. EVs isolated from YuYu and CL-14 strains induced in macrophages higher levels of proinflammatory cytokines (TNF-α and IL-6) and nitric oxide via TLR2. In general, no differences were observed in MAPKs activation (p38, JNK and ERK 1/2) after EVs stimulation. In splenic cells derived from chronically infected mice, a different modulation pattern was observed, where Colombiana (followed by Y strain) EVs were more proinflammatory. This modulation was independent of the T. cruzi strain used in the mice infection. To test the functional importance of this modulation, the expression of intracellular cytokines after in vitro exposure was evaluated using EVs from YuYu and Colombiana strains. Both EVs induced cytokine production with the appearance of IL-10 in the chronically infected mice. A high frequency of IL-10 in CD4+ and CD8+ T lymphocytes was observed. A mixed profile of cytokine induction was observed in B cells with the production of TNF-α and IL-10. Finally, dendritic cells produced TNF-α after stimulation with EVs. Polymorphisms in the vesicles surface may be determinant in the immunopathologic events not only in the early steps of infection but also in the chronic phase

Platinum(II) ring-fused chlorins as efficient theranostic agents: Dyes for tumor-imaging and photodynamic therapy of cancer

The discovery of Pt-chlorin-type theranostic agents is described. Luminescent Pt(II) 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused chlorins, with different degrees of hydrophilicity, have been synthesized and their in vitro photocytotoxicity against human melanoma, oesophageal and bladder carcinomas was studied. A di(hydroxymethyl)-substituted chlorin was identified as a privileged molecule to explore imaging-guided photodynamic therapy. In addition to the high activity as PDT agent and absence of cytotoxicity per se, this molecule showed the ideal photophysical and photochemical properties. In vivo studies using a A375 melanoma mouse model, proved the extraordinary properties of this chlorin as a luminescent probe and the ability to impair tumor growth, making image guided treatment and follow up a possibility