G2019S selective LRRK2 kinase inhibitor abrogates mitochondrial DNA damage.

Pathogenic mutations in LRRK2 cause Parkinson's disease (PD). The G2019S variant is the most common, which results in abnormally high kinase activity. Compounds that target LRRK2 kinase activity are currently being developed and tested in clinical trials. We recently found that G2019S LRRK2 causes mitochondrial DNA (mtDNA) damage and treatment with multiple classes of LRRK2 kinase inhibitors at concentrations associated with dephosphorylation of LRRK2 reversed mtDNA damage to healthy control levels. Because maintaining the normal function of LRRK2 in heterozygous G2019S LRRK2 carriers while specifically targeting the G2019S LRRK2 activity could have an advantageous safety profile, we explored the efficacy of a G2019S mutant selective LRRK2 inhibitor to reverse mtDNA damage in G2019S LRRK2 models and patient cells relative to non-selective LRRK2 inhibitors. Potency of LRRK2 kinase inhibition by EB-42168, a G2019S mutant LRRK2 kinase inhibitor, and MLi-2, a non-selective inhibitor, was determined by measuring phosphorylation of LRRK2 at Ser935 and/or Ser1292 using quantitative western immunoblot analysis. The Mito DNADX assay, which allows for the accurate real-time quantification of mtDNA damage in a 96-well platform, was performed in parallel. We confirmed that EB-42168 selectively inhibits LRRK2 phosphorylation on G2019S LRRK2 relative to wild-type LRRK2. On the other hand, MLi-2 was equipotent for wild-type and G2019S LRRK2. Acute treatment with EB-42168 inhibited LRRK2 phosphorylation and also restored mtDNA damage to healthy control levels. We further investigated the relationship between LRRK2 kinase activity, mtDNA damage and mitophagy. Levels of mtDNA damage caused by G2019S LRRK2 were fully re-established within 2 h of a LRRK2 inhibitor wash out and recovery experiment, indicating the mtDNA damage phenotype is highly dynamic. G2019S LRRK2 mitophagy defects were not alleviated with LRRK2 kinase inhibition, suggesting that mitophagy is not mechanistically regulating LRRK2 kinase-mediated reversal of mtDNA damage in this acute timeframe. Abrogation of mtDNA damage with the mutant selective tool inhibitor EB-42168 demonstrates the potential of a precision medicine approach for LRRK2 G2019S PD. Levels of mtDNA damage may serve as a potential pharmacodynamic biomarker of altered kinase activity that could be useful for small molecule development and clinical trials.

A blood-based marker of mitochondrial DNA damage in Parkinson's disease.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, and neuroprotective or disease-modifying interventions remain elusive. High-throughput markers aimed at stratifying patients on the basis of shared etiology are required to ensure the success of disease-modifying therapies in clinical trials. Mitochondrial dysfunction plays a prominent role in the pathogenesis of PD. Previously, we found brain region-specific accumulation of mitochondrial DNA (mtDNA) damage in PD neuronal culture and animal models, as well as in human PD postmortem brain tissue. To investigate mtDNA damage as a potential blood-based marker for PD, we describe herein a PCR-based assay (Mito DNADX) that allows for the accurate real-time quantification of mtDNA damage in a scalable platform. We found that mtDNA damage was increased in peripheral blood mononuclear cells derived from patients with idiopathic PD and those harboring the PD-associated leucine-rich repeat kinase 2 (LRRK2) G2019S mutation in comparison with age-matched controls. In addition, mtDNA damage was elevated in non-disease-manifesting LRRK2 mutation carriers, demonstrating that mtDNA damage can occur irrespective of a PD diagnosis. We further established that Lrrk2 G2019S knock-in mice displayed increased mtDNA damage, whereas Lrrk2 knockout mice showed fewer mtDNA lesions in the ventral midbrain, compared with wild-type control mice. Furthermore, a small-molecule kinase inhibitor of LRRK2 mitigated mtDNA damage in a rotenone PD rat midbrain neuron model and in idiopathic PD patient-derived lymphoblastoid cell lines. Quantifying mtDNA damage using the Mito DNADX assay may have utility as a candidate marker of PD and for measuring the pharmacodynamic response to LRRK2 kinase inhibitors.

Anal cancer screening in a high-risk behavior group: A local picture / Rastreamento de câncer anal em um grupo de comportamento de alto risco: uma imagem local

ABSTRACT Study objectives To perform anal lesion and anal cancer screening in men living with HIV/AIDS. Methods This is a descriptive, observational, cross-sectional study. Data were obtained from the Specialized Assistance Service (SAE) in Divinópolis, Minas Gerais. A sociodemographic, epidemiological, and sexual behavior questionnaire was applied; material was collected for cytology, high-resolution anoscopy (AAR) was performed, and an acceptability questionnaire applied. Main results Of the 50 men living with HIV/AIDS invited to participate in this study, 6% were excluded because they were illiterate, 40% refused to participate, and 54% participated in the survey. Among these, all answered the self-administered questionnaire. However, ten (37.0%) underwent proctological examination and anal cytology. Of these, two did not respond to the acceptability questionnaire. No anal lesions were identified during AAR and no biopsy was required. A 10% change in anal cytology was found. Conclusions Through the study it was possible to construct a flow of referrals from the SAE to the UFSJ Coloproctology outpatient clinic. Moreover, the existence of internal stigmas on the part of the participants regarding the proctological examination and the lack of information about anal cancer screening are challenges to be overcome.

RESUMO Objetivos do estudo Realizar o rastreamento de lesões anais e câncer anal em homens vivendo com HIV/AIDS. Métodos Trata-se de estudo descritivo observacional transversal, cujos dados foram obtidos no Serviço de Assistência Especializada (SAE) em Divinópolis, Minas Gerais. Foi aplicado questionário sociodemográfico, epidemiológico e de comportamento sexual; realizada coleta de material para citologia, Anuscopia de Alta Resolução (AAR) e aplicado questionário de aceitabilidade do exame. Principais resultados Dos 50 homens vivendo com HIV/AIDS convidados a participar do presente estudo, 6% foram excluídos por serem analfabetos, 40% se recusaram a participar e 54% participaram da pesquisa. Entre estes, todos responderam o questionário autoaplicado. Entretanto, 10 (37.0%) realizaram o exame proctológico e a citologia anal. Desses, dois não responderam ao questionário de aceitabilidade. Não foram identificadas lesões anais durante a AAR, não sendo necessária a realização de biópsia. Foi encontrado 10% de alteração à citologia anal. Conclusões Por meio do estudo foi possível construir um fluxo de encaminhamentos do SAE para o ambulatório de Coloproctologia da UFSJ. Ademais, a existência de estigmas internos por parte dos participantes no que concerne à realização do exame proctológico e a falta de informação a respeito do rastreamento do câncer anal são desafios a serem vencidos.