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.

Genotypic resistance of cytomegalovirus to antivirals in hematopoietic stem cell transplant recipients from Portugal: A retrospective study.

The aim of this study was to characterize Human Cytomegalovirus (HCMV) drug resistance mutations in UL97 and UL54 genes in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients in Portugal. We have performed a retrospective study with 22 patients from a cohort of patients with different haematological malignancies submitted to allo-HSCT between 2010 and 2014. Patients were selected according to clinical and laboratory data of HCMV infection and management. HCMV resistance mutations were characterized by sequencing of UL97 and UL54 genes. Sequence data were compared with: 1) HCMV genome reference strain AD169; and also 2) UL97 from Merlin strain (GenBank: AY446894.2), and UL54 from TB40/E strain (GenBank: ABV71585.1). Resistance mutations were identified in seven patients (32%): five harboured resistance mutations in UL97: A594V (n = 2), C592G (n = 1), L595W (n = 1), and C603W (n = 1); and two harboured resistance mutations in UL54: P522S and L957F, one in each patient. Several natural polymorphisms and unknown mutations were found in both UL97 and UL54, with the majority of the patients harbouring more than one unknown mutation in UL97 but only one in UL54. No simultaneous mutations were found. This is the first study in Portugal to characterize HCMV UL97 and UL54 sequences and to identify HCMV drug-resistance mutations in allo-HSCT patients. The UL97 resistance mutations found were amongst the most frequent resistant mutations, while UL54 L957F mutation was here reported for the first time in a clinical specimen. This information provides important information regarding HCMV strains and antiviral resistance in our population.

Human cytomegalovirus antiviral drug resistance in hematopoietic stem cell transplantation: current state of the art.

Human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant recipients. The significant clinical impact of HCMV infection and progression to HCMV disease among allogeneic hematopoietic stem cell transplant recipients has been reduced by prophylactic, preemptive, and curative treatments using ganciclovir, valganciclovir, foscarnet, and cidofovir. Resistance to (val)ganciclovir results from mutations localized in HCMV UL97 gene (encoding the pUL97 phosphotransferase), UL54 gene (encoding the pUL54 DNA polymerase), or both genes, whereas foscarnet and cidofovir resistance results from mutations localized within UL54 gene only. This review is focused on HCMV antiviral drug resistance, including the functions of target genes of antivirals, the mechanisms of antiviral resistance, the different mutations in pUL97 and pUL54 that have been identified in either clinical isolates or laboratory strains, and their impact on HCMV susceptibility to antiviral drugs. It emphasizes the importance of proving that observed genetic changes confer resistance so they can be distinguished from polymorphisms. Because of the emergence of HCMV resistance to currently available drugs, novel drugs are urgently needed for the therapeutic management of HCMV-resistant infections in hematopoietic stem cell transplant patients.