Biology subjects

Chen, H.-R.

Publications and source records attributed to Chen, H.-R..

6 recordsLinked to original sources

bioRxiv2026

Optimizing CRISPR/Cas9 genome editing in primary human hematopoietic cells to advance studies into HIV biology

Defining how human host factors shape HIV-1 infection in vivo remains essential for the development of genetically engineered cell therapies. Here, we established a non-viral CRISPR/Cas9 ribonucleoprotein-based platform for efficient single and multiplex gene editing in primary human CD34+ hematopoietic stem and progenitor cells (HSPCs). Edited HSPCs retained viability, proliferative capacity, primitive immunophenotypes, and multilineage differentiation potential and supported targeted reporter knock-in independently of the cell source, cord blood (CB), bone marrow (BM), and mobilized from peripheral blood (MPB). Following differentiation, SAMHD1 knockout increased HIV-1 susceptibility of HSPC-derived macrophages, MX2 knockout also increased HIV infection in cells pre-stimulated with IFN-2a, and CXCR4 knockout blocked X4-tropic HIV-1 infection of HSPC-derived megakaryocytes. We then transplanted CCR5-, SAMHD1-, or non-targeting control-edited HSPCs into immunodeficient mice and challenged reconstituted animals with R5-tropic HIV-1. CCR5 knockout prevented detectable viral spread, validating the model using a clinically relevant dependency factor. In contrast, SAMHD1 knockout accelerated viral dissemination, with earlier plasma viremia and 3.6-fold higher cumulative viremia, although endpoint viral burden was not significantly different from controls. These findings establish transplantation of CRISPR/Cas9-edited human HSPCs as a modular platform for dissecting HIV-1 host-factor function across hematopoietic lineages ex vivo and in vivo and for evaluating engineered cell-based strategies.

bioengineering↗

bioRxiv2026

Meningeal neutrophil infiltration drives inflammation-exacerbated pediatric stroke through IL-36γ signaling

Infection and systemic inflammation have been identified as major risk factors for pediatric stroke; however, the immune mechanisms underlying these clinical observations remain poorly understood. Here, we utilized an LPS-sensitized photothrombotic stroke model (LPS/PT) to investigate the contribution of immune cell infiltration to pediatric stroke pathogenesis and identified a previously unrecognized route of neutrophil entry into the injured brain. Compared with photothrombotic stroke alone (PT), LPS/PT mice exhibited markedly increased neutrophil infiltration accompanied by a hyperactivated phenotype. Depletion of neutrophils, but not monocytes, significantly reduced infarct size in LPS/PT animals, indicating a central role for neutrophils in inflammation-exacerbated pediatric stroke. Notably, we observed that neutrophils first accumulated within the leptomeningeal space before entering the brain parenchyma during the early phase of stroke, suggesting that the meninges may serve as an initial staging site for neutrophil recruitment. Using KikGR photoconvertible reporter mice combined with two-photon imaging, we further demonstrated that neutrophils infiltrate the ischemic brain through a compromised meningeal barrier following stroke. Transcriptomic analysis of infiltrating neutrophils revealed distinct gene expression signatures between meningeal neutrophils and circulating blood-derived neutrophils. Among these, IL-36{gamma} was highly enriched in meninges-associated neutrophils during pediatric stroke. Consistently, single-cell RNA sequencing of meningeal immune cells confirmed elevated IL-36{gamma} expression within the neutrophil cluster in LPS/PT animals. Importantly, intracisternal administration of IL-36 receptor antagonist (IL-36Ra) or anti-ICAM-1 antibody significantly reduced infarct volume in this pediatric stroke model. Together, our findings identify the meningeal barrier as a critical gateway for neutrophil infiltration and reveal IL-36{gamma} as a key inflammatory mediator regulating neuroinflammation in pediatric stroke, highlighting a potential therapeutic target for limiting immune-mediated brain injury.

neuroscience↗

bioRxiv2025

Intrahippocampal delivery of hyperphosphorylated human tau oligomers induces neurodegeneration in non-transgenic wildtype mice

Hyperphosphorylated tau (p-tau) forms neurofibrillary tangles, a key biomarker for Alzheimers disease and additional neurodegenerative tauopathies. However, neurofibrillary tangles are not sufficient to cause neuronal dysfunction or death. Intrahippocampal injection of tau isolated from AD patients has limited effects on the cognitive functions of non-transgenic mice, despite the recapitulation of pathological tau deposits in the mouse brain. It therefore remains uncertain as to whether all hyperphosphorylated tau is directly responsible for AD neurodegeneration. We examined this issue by injecting recombinant p-tau oligomers to the hippocampus of non-transgenic, wildtype mice and found progressive cognitive deficits that correlate with neuron death spreading from the ipsilateral hippocampus to the cortex. Apomorphine, which retards p-tau aggregation and cytotoxicity in vitro, antagonized p-tau-induced cognitive deficits and neuron death. These results suggest the pathogenic role of p-tau oligomers and a novel AD model facilitating drug development.

neuroscience↗

bioRxiv2023

P-tau Inflicts Intracellular Stress Responses That Are Mitigated by Apomorphine

BackgroundAbnormal phosphorylation of the microtubule-binding protein tau in the brain is a key pathological marker for Alzheimers disease and additional neurodegenerative tauopathies. However, how hyperphosphorylated tau causes cellular dysfunction or death that underlie neurodegeneration remains an unsolved question critical for the understanding of disease mechanism and the design of efficacious drugs. MethodsUsing a recombinant hyperphosphorylated tau protein (p-tau) synthesized by the PIMAX approach, we examined how cells responded to the cytotoxic tau and explored means to enhance cellular resistance to tau attack. ResultsUpon p-tau uptake, the intracellular calcium levels rose promptly. Gene expression analyses revealed that p-tau potently triggered endoplasmic reticulum (ER) stress, Unfolded Protein Response (UPR), ER stress-associated apoptosis, and pro-inflammation in cells. Proteomics studies showed that p-tau diminished heme oxygenase-1 (HO-1), an ER stress associated anti-inflammation and anti-oxidative stress regulator, while stimulated the accumulation of MIOS and other proteins. P-tau-induced ER stress-associated apoptosis and pro-inflammation are ameliorated by apomorphine, a brain-permeable prescription drug widely used to treat Parkinsons disease symptoms, and by overexpression of HO-1. ConclusionOur results reveal probable cellular functions targeted by hyperphosphorylated tau. Some of these dysfunctions and stress responses have been linked to neurodegeneration in Alzheimers disease. The observations that the ill effects of p-tau can be mitigated by a small compound and by overexpressing HO-1 that is otherwise diminished in the treated cells inform new directions of Alzheimers disease drug discovery.

biochemistry↗

bioRxiv2021

Hypothermia Suppresses Uncoupling of Oxidative-Phosphorylation after Neonatal Cerebral Hypoxia-Ischemia

Hypoxia-ischemia (HI), which disrupts the oxygen supply-demand balance in the brain by impairing blood oxygen supply and the cerebral metabolic rate of oxygen (CMRO2), is a leading cause of neonatal brain injury. However, it is unclear how post-HI hypothermia helps to restore the balance, as cooling reduces CMRO2. Also, how transient HI leads to secondary energy failure (SEF) in neonatal brains remains elusive. Using photoacoustic microscopy, we examined the effects of HI on CMRO2 in awake 10-day-old mice, supplemented by bioenergetic analysis of purified cortical mitochondria. Our results show that while HI suppresses ipsilateral CMRO2, it sparks a prolonged CMRO2-surge post-HI, associated with increased mitochondrial oxygen consumption, superoxide emission, and reduced mitochondrial membrane potential necessary for ATP synthesis--indicating oxidative phosphorylation (OXPHOS) uncoupling. Post-HI hypothermia prevents the CMRO2-surge by constraining oxygen extraction fraction, reduces mitochondrial oxidative stress, and maintains ATP and N-acetylaspartate levels, resulting in attenuated infarction at 24 hours post-HI. Our findings suggest that OXPHOS-uncoupling induced by the post-HI CMRO2-surge underlies SEF and blocking the surge is a key mechanism of hypothermia protection. Also, our study highlights the potential of optical CMRO2-measurements for detecting neonatal HI brain injury and guiding the titration of therapeutic hypothermia at the bedside.

neuroscience↗

bioRxiv2021

Capillary-associated microglia regulate vascular structure and function through PANX1-P2RY12 coupling

Microglia are brain-resident immune cells with a repertoire of functions in the developing, mature and pathological brain. Their wide-ranging roles in physiology include the clearance of cellular debris, elimination of excess synapses, regulation of neuronal activity and contributions to blood vessel development. Despite these known roles for microglia, the extent of their interactions with the vasculature and potential regulation of vascular physiology has been insufficiently explored. Here, using in vivo acute and longitudinal two-photon imaging in transgenic mice combined with electron microscopy, fixed tissue immunohistochemistry, pharmacological treatments and laser speckle imaging, we document the steady-state interactions between ramified CX3CR1+ myeloid cell somata and capillaries in the brain. We first confirm that these myeloid cells are bona fide microglia by molecular, morphological and ultrastructural approaches. Then we give a detailed spatio-temporal characterization of these capillary-associated microglia (CAMs) comparing and contrasting them with parenchymal microglia (PCMs) in their static, dynamic and chronic morphological activities including during microglial depletion and repopulation. Molecularly, we identify microglial-specific purinergic P2RY12 receptors as a receptor regulating CAM interactions under the control of released purines from pannexin 1 (PANX1) channels. Furthermore, to elucidate roles for microglia in vascular structure and function, we eliminated microglia and showed that this triggered capillary dilation, blood flow increase, and impaired vasodilative responses. We find that P2RY12-/- and PANX1-/- mice recapitulate these vascular impairments suggesting purines released through PANX1 channels play important roles in activating microglial P2RY12 receptors to regulate neurovascular structure and function.

neuroscience↗