Biology subjects

Santos, A. C.

Publications and source records attributed to Santos, A. C..

3 recordsLinked to original sources

bioRxiv2026

PARG activity is required for cell death by parthanatos

Cell death by a non-apoptotic pathway termed parthanatos is induced by hyperactivation of the DNA damage sensor PARP1, which uses NAD+ as a substrate to catalyse the poly-ADP-ribosylation (PARylation) of proteins. Parthanatos has been implicated in several pathological conditions such as ischaemia-reperfusion injury and neurodegenerative processes, including Alzheime[r]s and Parkinso[n]s diseases, as well as rare genetic disorders characterized by PARP1 hyperactivation. However, the precise sequence of molecular events by which excessive PARP1 activity causes cell death is currently unclear. Here we show that, in addition to PARP1-dependent PARylation, the execution of parthanatos also requires the hydrolysis of poly-ADP-ribose (PAR) chains by the glycohydrolase PARG. While complete inhibition of PARG activity prevents parthanatos, low levels of residual PARG activity are sufficient to support cell death by this pathway. Due to a phenotypic discrepancy between CRISPR/Cas9-generated PARG KO cells and PARG inhibitor-treated cells, we uncovered a new PARG splice variant predicted to encode an isoform of 53 kDa, termed PARG53, and provide evidence for the incorrect annotation of the previously reported isoforms PARG55 and PARG60. PARP1 hyperactivation during parthanatos leads to rapid and profound depletion of NAD+ and ATP pools, but full PARG inhibition only prevents the depletion of ATP, indicating that the depletion of these core metabolites can be uncoupled, and that ATP depletion is more closely correlated with cell death. This work sheds light on the initial steps of parthanatos induction, demonstrating that PAR formation and PAR hydrolysis are both required for this type of cell death.

biochemistry↗

bioRxiv2023

Essential role of the CCL2-CCR2 axis in Mayaro virus-induced disease

Mayaro virus (MAYV) is an emerging arbovirus member of the Togaviridae family and Alphavirus genus. MAYV infection causes an acute febrile illness accompanied by persistent polyarthralgia and myalgia. Understanding the mechanisms involved in arthritis caused by alphaviruses is necessary to develop specific therapies. In this work, we investigated the role of the CCL2/CCR2 axis in the pathogenesis of MAYV-induced disease. For this, WT C57BL/6J and CCR2-/- mice were infected with MAYV subcutaneously and evaluated for disease development. MAYV infection induced an acute inflammatory disease in WT mice. The immune response profile was characterized by an increase in the production of inflammatory mediators, such as IL-6, TNF and CCL2. Higher levels of CCL2 at the local and systemic levels, was followed by significant recruitment of CCR2+ macrophages and a cellular response orchestrated by these cells. CCR2-/- mice showed an increase in CXCL-1 levels, followed by a replacement of the macrophage inflammatory infiltrate by neutrophils. Additionally, absence of the CCR2 receptor protected mice from bone loss induced by MAYV. Accordingly, the silencing of CCL2 chemokine expression in vivo and the pharmacological blockade of CCR2 promoted a partial improvement in disease. Cell culture data support the mechanism underlying MAYVs bone pathology in which: i) MAYV infection promoted a pro-osteoclastogenic microenvironment mediated by IL-6, TNF and CCL2 and ii) migration of osteoclast precursors was dependent on the CCR2/CCL2 axis. Overall, these data contribute to the understanding of the pathophysiology of MAYV infection and to the identification future of specific therapeutic targets in MAYV-induced disease. ImportanceThis work demonstrates the role of the CCL2/CCR2 axis in MAYV-induced disease. Infection of WT C57BL/6J and CCR2-/- mice was associated with high levels of CCL2, an important chemoattractant involved in the recruitment of macrophages, the main precursor of osteoclasts. In the absence of the CCR2 receptor there is a mitigation of macrophage migration to the target organs of infection and protection of these mice against bone loss induced by MAYV infection. Much evidence has shown that host immune response factors contribute significantly to the tissue damage associated to alfavirus infections. Thus, this work highlights molecular and cellular targets involved in the pathogenesis of arthritis triggered by MAYV, and identifies novel therapeutic possibilities directed to the host inflammatory response unleashed by MAYV.

cell biology↗

bioRxiv2021

Secreted reporter assay enables quantitative and longitudinal monitoring of neuronal activity

The ability to measure changes in neuronal activity in a quantifiable and precise manner is of fundamental importance to understand neuron development and function. Repeated monitoring of neuronal activity of the same population of neurons over several days is challenging and, typically, low-throughput. Here, we describe a new biochemical reporter assay that allows for repeated measurements of neuronal activity in a cell type-specific manner. We coupled activity-dependent elements from the Arc/Arg3.1 gene with a secreted reporter, Gaussia luciferase, to quantify neuronal activity without sacrificing the neurons. The reporter predominantly senses calcium and NMDA receptor-dependent activity. By repeatedly measuring the accumulation of the reporter in cell media, we can profile the developmental dynamics of neuronal activity in cultured neurons from male and female mice. The assay also allows for longitudinal analysis of pharmacological treatments, thus distinguishing acute from delayed responses. Moreover, conditional expression of the reporter allows for monitoring cell type-specific changes. This simple, quantitative, cost-effective, automatable, and cell type-specific activity reporter is a valuable tool to study the development of neuronal activity in normal and disease-model conditions, and to identify small molecules or protein factors that selectively modulate the activity of a specific population of neurons. SignificanceNeurological and neurodevelopmental disorders are prevalent worldwide. Despite significant advances in our understanding of synapse formation and function, developing effective therapeutics remains challenging, in part due to the lack of simple and robust high-throughput screening assays of neuronal activity. Here, we describe a simple biochemical assay that allows for repeated measurements of neuronal activity in a cell type-specific manner. Thus filling the need for assays amenable to longitudinal studies, such as those related to neural development. Other advantages include its simple and quantitative nature, logitudinal profiling, cell type-specificity, and being multiplexed with other invasive techniques.

neuroscience↗