Biology subjects

Benitez, C.

Publications and source records attributed to Benitez, C..

5 recordsLinked to original sources

bioRxiv2024

Minimally invasive serial collection of cerebrospinal fluid reveals sex-dependent differences in neuroinflammation in a rat model of mild traumatic brain injury

Traumatic brain injuries (TBI) are the seventh leading cause of disability globally with 48.99 million prevalent cases and 7.08 million years lived with diability. Approximately 80% of TBI patients are diagnosed with mild TBI (mTBI), or concussion, caused by nonpenetrating mechanical trauma to the head or body along with sudden rotational motion of the head. Studies investigating the temporal dynamics of neuroinflammation after mTBI are greatly needed. Without longitudinal studies, translating preclinical studies to clinical studies remains challenging as the difference in timing remains poorly understood. In this study, we describe a method of minimally invasive serial cerebrospinal fluid (CSF) collection that enables longitudinal investigation of CSF inflammation. The method described in this study can easily be adapted by any laboratory prepared for animal studies. Multiplex immunoassay of serially collected and singly collected CSF samples show collection frequency does not alter protein expression in the CSF. Further, sex-dependent differences in TBI have been reported, but remain poorly understood. This study establishes a framework for assessing sex difference in neuroinflammation after a concussion. We showed that results vary based on the framing of the statistical test. However, it is evident that males experience a more robust inflammatory response to a single concussion than females.

neuroscience↗

bioRxiv2023

CRISPRi-based screens in iAssembloids to elucidate neuron-glia interactions

The sheer complexity of the brain has complicated our ability to understand the cellular and molecular mechanisms underlying its function in health and disease. Genome-wide association studies have uncovered genetic variants associated with specific neurological phenotypes and diseases. In addition, single-cell transcriptomics have provided molecular descriptions of specific brain cell types and the changes they undergo during disease. Although these approaches provide a giant leap forward towards understanding how genetic variation can lead to functional changes in the brain, they do not establish molecular mechanisms. To address this need, we developed a 3D co-culture system termed iAssembloids (induced multi-lineage assembloids) that enables the rapid generation of homogenous neuron-glia spheroids. We characterize these iAssembloids with immunohistochemistry and single-cell transcriptomics and combine them with large-scale CRISPRi-based screens. In our first application, we ask how glial and neuronal cells interact to control neuronal death and survival. Our CRISPRi-based screens identified that GSK3{beta} inhibits the protective NRF2-mediated oxidative stress response in the presence of reactive oxygen species elicited by high neuronal activity, which was not previously found in 2D monoculture neuron screens. We also apply the platform to investigate the role of APOE- 4, a risk variant for Alzheimers Disease, in its effect on neuronal survival. We find that APOE- 4-expressing astrocytes may promote more neuronal activity as compared to APOE- 3- expressing astrocytes. This platform expands the toolbox for the unbiased identification of mechanisms of cell-cell interactions in brain health and disease.

neuroscience↗

bioRxiv2022

Astrocytic engagement of the corticostriatal synaptic cleft is disrupted in a mouse model of Huntington disease

Astroglial dysfunction contributes to the pathogenesis of Huntingtons disease (HD), and glial replacement can ameliorate disease course. To establish the topographic relationship of diseased astrocytes to medium spiny neuron (MSN) synapses in HD, we used 2-photon imaging to map the relationship of tRFP-tagged striatal astrocytes and rabies-traced, EGFP-tagged coupled neuronal pairs, in R6/2 HD and wild-type (WT) mice. The tagged, prospectively-identified corticostriatal synapses were then studied by correlated light electron microscopy followed by serial block-face scanning EM, allowing nm scale assessment of synaptic structure in 3D. By this means, we compared the astrocytic engagement of single striatal synapses in HD and WT brains. R6/2 HD astrocytes exhibited constricted domains, with significantly less coverage of mature dendritic spines than WT astrocytes, despite enhanced engagement of immature, thin spines. These data suggest that disease-dependent changes in astroglial engagement and sequestration of MSN synapses enable the high synaptic and extrasynaptic levels of glutamate and K+ that underlie the striatal hyperexcitability of HD. As such, these data suggest that astrocytic structural pathology may causally contribute to the synaptic dysfunction and disease phenotype of those neurodegenerative disorders characterized by network overexcitation. Significance StatementAstrocytic physiological dysfunction contributes to development of the neurodegenerative phenotype in Huntingtons disease (HD), but the structural correlates to this dysfunction are unclear. Here, we used a combination of viral tracing, phenotype-specific tagging, and ultrastructural modalities to reconstruct and study HD synapses at nm scale, in the neostriata of HD mice. We discovered significant impairment in the glial engagement of mature striatal synapses. In light of the known deficiencies in glutamate and potassium uptake by HD astrocytes, these findings suggest the potential for leakage of excitatory synaptic contents during neurotransmission, and hance a structural basis for neuronal hyperexcitability in HD. More broadly, our data suggest that astrocytic structural pathology may causally contribute to those neurodegenerative disorders associated with central hyperexcitability.

neuroscience↗

bioRxiv2022

Endocytic trafficking determines cellular tolerance of presynaptic opioid signaling

Opioid tolerance is well described physiologically but its mechanistic basis remains incompletely understood. An important site of opioid action in vivo is the presynaptic terminal, where opioids inhibit transmitter release. This response characteristically resists desensitization over minutes yet becomes gradually tolerant over hours, and how this is possible remains unknown. Here we delineate a cellular mechanism underlying this longer-term form of opioid tolerance. Our results support a model in which presynaptic tolerance is mediated by a gradual depletion of cognate receptors from the axon surface through iterative rounds of receptor endocytosis and recycling. For the -opioid receptor (MOR), we show that the agonist-induced endocytic process which initiates iterative receptor cycling requires GRK2/3-mediated phosphorylation of the receptors cytoplasmic tail, and that partial or biased agonist drugs which have reduced ability to drive phosphorylation-dependent endocytosis in terminals produce correspondingly less presynaptic tolerance. We then show that the {delta}-opioid receptor (DOR) conforms to the same general paradigm except that endocytosis of DOR from the presynapse, in marked contrast to MOR, does not require phosphorylation of the receptors cytoplasmic tail. Further, we show that DOR recycles less efficiently than MOR in axons and, consistent with this, that DOR tolerance develops more strongly. Together, these results delineate a cellular basis for the development of presynaptic tolerance to opioids and describe a methodology useful for investigating presynaptic neuromodulation more broadly.

neuroscience↗

bioRxiv2020

Riluzole suppresses growth and enhances response to endocrine therapy in ER+ breast cancer

BackgroundResistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer remains a significant clinical problem. Riluzole is FDA-approved for the treatment of amyotrophic lateral sclerosis. A benzothiazole-based glutamate release inhibitor with several context-dependent mechanism(s) of action, Riluzole has shown anti-tumor activity in multiple malignancies, including melanoma, glioblastoma, and breast cancer. We previously reported that the acquisition of Tamoxifen resistance in a cellular model of invasive lobular breast cancer is accompanied by the upregulation of GRM mRNA expression and growth inhibition by Riluzole. MethodsWe tested the ability of Riluzole to reduce cell growth, alone and in combination with endocrine therapy, in a diverse set of ER+ invasive ductal and lobular breast cancer-derived cell lines, primary breast tumor explant cultures, and the estrogen-independent, ESR1-mutated invasive lobular breast cancer patient-derived xenograft model HCI-013EI. ResultsSingle-agent Riluzole suppressed the growth of ER+ invasive ductal and lobular breast cancer cell lines in vitro, inducing a histologic subtype-associated cell cycle arrest (G0-G1 for ductal, G2-M for lobular). Riluzole induced apoptosis and ferroptosis and reduced phosphorylation of multiple pro-survival signaling molecules, including Akt/mTOR, CREB, and Src/Fak family kinases. Riluzole, in combination with either Fulvestrant or 4-hydroxytamoxifen, additively suppressed ER+ breast cancer cell growth in vitro. Single-agent Riluzole significantly inhibited HCI-013EI patient-derived xenograft growth in vivo, and the combination of Riluzole plus Fulvestrant significantly reduced proliferation in primary breast tumor explant cultures. ConclusionsRiluzole, alone or combined with endocrine therapy, may offer therapeutic benefits in diverse ER+ breast cancers, including lobular breast cancer.

cancer biology↗