Tapatee Das

Deterioration of the blood–brain barrier (BBB), including impaired neurovascular uncoupling, contributes to cognitive decline in aging. The BBB is formed principally by brain microvascular endothelial cells (ECs), and ECs throughout the body are enriched for the transcription factor Krüppel-like factor 4 (KLF4). Because KLF4 levels in ECs decrease with age, we tested whether that decline contributes to aging-related BBB deterioration, neurovascular dysfunction, and cognitive impairment. Using EC-specific Klf4 knockout mice (EC-K4KO), we show that loss of EC KLF4 accelerates multiple age-related brain pathologies. Indeed, middle-aged EC-K4KO mice display pathological features that are not normally observed until advanced age, including marked BBB leakage, impaired neurovascular coupling, loss of microvessels, increased oxidative damage, neuroinflammation, neurodegeneration, anxiety-like behavior, and cognitive deficits. Single-cell RNA sequencing of brain vasculature reveals dysregulation of immune response and barrier-related genes in ECs lacking KLF4, indicating that KLF4 maintains brain endothelial homeostasis by constraining proinflammatory and senescence programs at the chromatin level. Together, these results identify loss of EC KLF4 as a key driver of neurovascular decline and age-associated cognitive dysfunction.

Understanding the molecular basis of sex differences in neural response to acute hypoxic insult has profound implications for the effective prevention and treatment of ischemic stroke. Global hypoxic-ischemic induced neural damage has been studied recently under the well-controlled, non-invasive, reproducible conditions using zebrafish model. Our earlier report on sex difference in global acute hypoxia induced neural damage and recovery in zebrafish prompted us for comprehensive study on the mechanisms underlying the recovery. An omics approach for studying quantitative changes in brain proteome upon hypoxia insult following recovery was undertaken using iTRAQ-based LC-MS/MS approach. The results shed light on altered expression of many regulatory proteins in zebrafish brain upon acute hypoxia following recovery. The sex difference in differentially expressed proteins along with the proteins expressed in uniform direction in both the sexes was studied. Core expression analysis by Ingenuity Pathway analysis (IPA) showed a distinct sex difference in the disease function heatmap. Most of the upstream regulators obtained through IPA were validated at the transcriptional level. Translational upregulation of H3K9me3 in male led us to elucidate the mechanism of recovery by confirming transcriptional targets through ChIP-qPCR. The upregulation of H3K9me3 level in male at 4 hr post-hypoxia appears to affect the early neurogenic markers nestin, klf4 and sox2, which might explain the late recovery in male, compared to female. Acute hypoxia-induced sex-specific comparison of brain proteome led us to reveal many differentially expressed proteins, which can be further studied for the development of novel targets for better therapeutic strategy.

Hypoxia, the major cause of ischemia, leads to debilitating disease in infants via birth asphyxia and cerebral palsy, whereas in adults via heart attack and stroke. A widespread, natural protective phenomenon termed ‘Hypoxic Preconditioning’ occurs when prior exposures to hypoxia eventually results in robust hypoxia resistance. Accordingly, we have developed a novel model of sex-specific hypoxic preconditioning in adult zebrafish to mimic the tolerance of mini stroke(s) in human, which appears to protect against the severe damage inflicted by a major stroke event. Remarkable difference in the progression pattern of neuroprotection between preconditioning hypoxia followed by acute hypoxia (PH) group, and acute hypoxia (AH) group were observed with noticeable sex difference. Since gender difference has been reported in stroke, it was pertinent to investigate whether any such sex difference also exists in PH’s protective mechanism against acute ischemic stroke. In order to elucidate the neural molecular mechanisms behind sex difference in neuroprotection induced by PH, a high throughput proteomics approach utilizing iTRAQ was performed, followed by protein enrichment analysis using Ingenuity Pathway Analysis. Out of thousands of altered proteins in zebrafish brain, the ones having critical role either in neuroglial proliferation/differentiation or neurotrophic functions, were validated by analyzing their expression levels in PH, AH and normoxia groups. Results indicate that female zebrafish brains are more protected against the severity of AH. The study also sheds light on the involvement of many signaling pathways underlying sex difference in pre-conditioning induced neuroprotective mechanism, which can be further validated for the therapeutic approach.