Development of CRID3-Based Anti-inflammatory Agents to Ameliorate Chronic Hypoxia-Induced Memory Impairment in Zebrafish Models.

Chronic hypoxic exposure triggers the onset and progression of cognitive dysfunction; however, the mechanisms underlying chronic hypoxia-induced neuroinflammation and its contribution to cognitive dysfunction remain poorly understood. Although inflammation and hypoxia are interdependent, numerous recent studies have linked the development of various human diseases to hypoxia-induced inflammation. In this study, we focused on the NLRP3 inflammasome with novel analogues of cytokine release inhibitory drug 3 (CRID3), a class of small molecule inhibitors for the NLRP3 inflammasome, to investigate their potential contribution to alleviating chronic hypoxia-induced neuroinflammation using the zebrafish model. The designed CRID3 analogues 6a-q were prepared from 2-methyl furan-3-carboxylate, following a four-step reaction sequence and fully characterized by NMR and mass spectral analysis. The administration of CRID3 analogues 6a-q led to a notable reduction in neuroinflammation and an increase in glial proliferation markers in both sexes. In addition, we investigated the potential effects of CRID3 analogues 6a-q through various behavioral tasks to assess their role in ameliorating post-hypoxic behavioral deficits and cognitive impairment. Notably, the study revealed that post-chronic hypoxia, male zebrafish exhibited significantly higher levels of inflammatory marker expression than females. Furthermore, we observed that the neurogenic response to treatment with CRID3 derivative 6o varied depending on the sex, with females showing a sex-specific differential increase in neurogenesis compared to males. This work emphasizes the significance of considering sex differences into account in developing therapeutic strategies for neurological disorders, as shown by the sex-specific molecular and behavioral changes in zebrafish cognitive impairment and neuroinflammation.

KLF15 controls brown adipose tissue transcriptional flexibility and metabolism in response to various energetic demands.

Brown adipose tissue (BAT) is a specialized metabolic organ responsible for non-shivering thermogenesis. Recently, its activity has been shown to be critical in systemic metabolic health through its utilization and consumption of macronutrients. In the face of energetically demanding states, metabolic flexibility and systemic coordination of nutrient partitioning is requisite for health and survival. In this study, we elucidate BAT's differential transcriptional adaptations in response to multiple nutrient challenges and demonstrate its context-dependent prioritization of lipid, glucose, and amino acid metabolism. We show that the transcription factor Krüppel-like factor 15 (KLF15) plays a critical role in BAT metabolic flexibility. BAT-specific loss of KLF15 results in widespread changes in circulating metabolites and severely compromised thermogenesis in response to high energy demands, indicative of impaired nutrient utilization and metabolic flexibility. Together, our data demonstrate KLF15 in BAT plays an indispensable role in partitioning resources to maintain homeostasis and ensure survival.

Proteome profile of telencephalon associates attenuated neurogenesis with chronic stress induced mood disorder phenotypes in zebrafish model.

Debilitating mental illness like depression and related mood disorders is due to the disruption in circuitry that controls emotion, motivation, and reward, characterized by disparate phenotypes like decrease in socialization, motivation, threshold for threat apprehension, etc. Chronic stress is a major factor in the etiology of these disorders. Here, using a chronic unpredictable stress (CUS) paradigm the characterization of an array of mood disorder phenotypes in adult zebrafish, in comparison to normal control unstressed fish, was achieved using a battery of behavioral assays including novel ones comprising social interaction test, feed approach test, threat response test and novel tank test. For the predictive validity of the model for mood disorders, the mitigative role of a slow (imipramine) and fast (ketamine) acting antidepressant was assessed. The molecular changes associated with CUS-induced mood disorder phenotype was investigated utilizing a high throughput method called isobaric tag for relative and absolute quantification (iTRAQ) in telencephalon, the region critically associated with the processing of emotional information in the fish brain. Out of 222 proteins identified to be significantly altered, 58 were differentially expressed across the stress and antidepressant-treatment groups at more than one fold (in log2) change. Of these proteins, some were implicated in earlier studies on mood disorders such as CABP1, PER2, mTOR, etc. The enrichment of altered proteins by Ingenuity Pathway Analysis (IPA) led us to mTOR and opioid signaling pathways, the top canonical pathways affected in the fish telencephalon. Interestingly, most of the pathways affected converge at the one controlling cell proliferation thus indicating altered neurogenesis, which was validated using immunohistochemistry for cell proliferation markers BrdU, SOX2, and BLBP. The study concludes that molecules that regulate telencephalon neural progenitor cell proliferation or neurogenesis are crucially involved in chronic stress-induced mood disorders by affecting the circuitry that controls emotion and reward.

Hypoxia Induced Sex-Difference in Zebrafish Brain Proteome Profile Reveals the Crucial Role of H3K9me3 in Recovery From Acute Hypoxia.

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 well-controlled, non-invasive, reproducible conditions using a zebrafish model. Our earlier report on sex difference in global acute hypoxia-induced neural damage and recovery in zebrafish prompted us to conduct a 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 the altered expression of many regulatory proteins in the zebrafish brain upon acute hypoxia following recovery. The sex difference in differentially expressed proteins along with the proteins expressed in a 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 males led us to elucidate the mechanism of recovery by confirming transcriptional targets through ChIP-qPCR. The upregulation of H3K9me3 level in males at 4 h post-hypoxia appears to affect the early neurogenic markers nestin, klf4, and sox2, which might explain the late recovery in males, compared to females. 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.

Differential modulation of GR signaling and HDACs in the development of resilient/vulnerable phenotype and antidepressant-like response of vorinostat.

The present study explored the antidepressant potential of vorinostat (VOR) against chronic social defeat stress (CSDS) in mice. Since this model has the remarkable capacity to delineate the resilient and the defeated mice, we also looked for their molecular deviations. Defeated mice showed classical phenotypic alterations such as anhedonia, social avoidance, anxiety and despair. Whereas, resilient mice were immune to the development of those. Both defeated and resilient mice demonstrated marked CORT elevation in blood. Development of resilience vs. defeat to CSDS was found to be associated with the differential nuclear levels of GR, HDAC3 and HDAC6 in the hippocampus. Activation of a stress responsive adaptive mechanism involving these mediators at the nuclear level might be offering resilience while maladaptive mechanisms leading to defeat. Interestingly, an elevated hippocampal HDAC6 level in defeated mice was also observed, which was restored by VOR treatment. Further studies will be necessary to delineate the HDAC6 associated antidepressant mechanisms. As HDAC3 and HDAC6 are crucial mediators of GR signaling, further molecular studies may aid in understanding the basis of development of resilience to target MDD with new prospective.

Molecular Basis of Sex Difference in Neuroprotection induced by Hypoxia Preconditioning in Zebrafish.

Hypoxia, the major cause of ischemic injury, 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' (PH) occurs when prior exposures to hypoxia eventually result in robust hypoxia resistance. Accordingly, we have developed and optimized a novel model of 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. Here, we observed a remarkable difference in the progression pattern of neuroprotection between preconditioning hypoxia followed by acute hypoxia (PH) group, and acute hypoxia (AH) only group, with noticeable sex difference when compared with normoxia behaviour upon recovery. Since gender difference has been reported in stroke risk factors and disease history, 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 (IPA) tool. Out of thousands of significantly 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 preconditioned (PH), acute hypoxia (AH), and normoxia groups. The data indicate that female zebrafish brains are more protected against the severity of AH when exposed to the hypoxic preconditioning. The study also sheds light on the involvement of many signalling pathways underlying sex difference in preconditioning-induced neuroprotective mechanism, which can be further validated for the therapeutic approach.

Potential Therapeutic Application of Zinc Oxide Nanoflowers in the Cerebral Ischemia Rat Model through Neuritogenic and Neuroprotective Properties.

Neuritogenesis, a complex process of the sprouting of neurites, plays a vital role in the structural and functional restoration of cerebral ischemia-injured neuronal tissue. Practically, there is no effective long-term treatment strategy for cerebral ischemia in clinical practice to date due to several limitations of conventional therapies, facilitating the urgency to develop new alternative therapeutic approaches. Herein, for the first time we report that pro-angiogenic nanomaterials, zinc oxide nanoflowers (ZONF), exhibit neuritogenic activity by elevating mRNA expression of different neurotrophins, following PI3K/Akt-MAPK/ERK signaling pathways. Further, ZONF administration to global cerebral ischemia-induced Fischer rats shows improved neurobehavior and enhanced synaptic plasticity of neurons via upregulation of Neurabin-2 and NT-3, revealing their neuroprotective activity. Altogether, this study offers the basis for exploitation of angio-neural cross talk of other pro-angiogenic nano/biomaterials for future advancement of alternative treatment strategies for cerebral ischemia, where neuritogenesis and neural repair are highly critical.

Revealing sex-specific molecular changes in hypoxia-ischemia induced neural damage and subsequent recovery using zebrafish model.

Functional recovery from hypoxia-ischemia depends on an individual's response to the ischemic damage and recovery. Many of the neurological disorders, including cerebral stroke have sex-specific characteristics. Deciphering the differential molecular mechanisms of sex-specific recovery from hypoxic-ischemic insult can improve medical practice in the treatment of cerebral stroke. In the present study, we describe the establishment of a sex-specific global hypoxia-ischemia neural damage and repair model in zebrafish. During hypoxic exposure a delayed behavioural response was observed in female fish that resumed normal swimming pattern earlier than their male counterparts. Moreover, female appeared more affected as they showed restricted locomotor and exploratory behaviour in novel tank test, reduced mitochondrial enzyme activity, enhanced DNA damage, and cell death after hypoxia insult. However, they showed a faster recovery as compared to male. Analysis of mRNA and protein expression levels of some characteristic hypoxic-ischemic markers showed notable sex-specific differences. Using zebrafish model, we have uncovered cellular and molecular differences in sex-specific systemic responses during the post-hypoxia recovery. This insight might help in devising better therapeutic strategy for stroke in female patients.

A 2-oxa-spiro[5.4]decane scaffold displays neurotrophic, neurogenic and anti-neuroinflammatory activities with high potential for development as a versatile CNS therapeutic.

Following our recent discovery of a new scaffold exhibiting significant neurotrophic and neurogenic activities, a structurally tweaked analogue, embodying a 2-oxa-spiro [5.4]decane framework, has been conceptualised and found to be more potent and versatile. It exhibits enhanced neurotrophic and neurogenic action in in vitro, ex vivo and in vivo models and also shows robust neuroprotection in mouse acute cerebral stroke model. The observed attributes are traceable to the predominant activation of the TrkB-PI3K-AKT-CREB pathway. In addition, it also exhibits remarkable anti-neuroinflammatory activity by concurrently down-regulating pro-inflammatory cytokines IL-1α and IL-6, thereby providing a unique molecule with a trinity of neuroactivities, i.e. neurotrophic, neurogenic and anti-inflammatory. The new chemical entity disclosed here has the potential to be advanced as a versatile therapeutic molecule to treat stroke, depression, and possibly other neuropsychiatric disorders associated with attenuated neurotrophic/ neurogenic activity, together with heightened neuroinflammation.

A novel natural product inspired scaffold with robust neurotrophic, neurogenic and neuroprotective action.

In search for drugs to treat neuropsychiatric disorders wherein neurotrophic and neurogenic properties are affected, two neurotrophically active small molecules specially crafted following natural product leads based on 2-oxa-spiro[5.5]-undecane scaffold, have been thoroughly evaluated for their neurotrophic, neurogenic and neuroprotective potential in ex vivo primary culture and in vivo zebrafish and mouse models. The outcome of in vivo investigations suggest that one of these molecules is more neurotrophic than neurogenic while the other one is more neurogenic than neurotrophic and the former exhibits remarkable neuroprotection in a mouse acute ischemic stroke model. The molecular mechanisms of action of these compounds appear to be through the TrkB-MEK-ERK-CREB-BDNF pathway as pre-treatment with neurotrophin receptor TrkB inhibitor ANA-12 and MEK inhibitor PD98059 attenuates the neurotrophic action of compounds.

Chronic unpredictable stress (CUS)-induced anxiety and related mood disorders in a zebrafish model: altered brain proteome profile implicates mitochondrial dysfunction.

Anxiety and depression are major chronic mood disorders, and the etiopathology for each appears to be repeated exposure to diverse unpredictable stress factors. Most of the studies on anxiety and related mood disorders are performed in rodents, and a good model is chronic unpredictable stress (CUS). In this study, we have attempted to understand the molecular basis of the neuroglial and behavioral changes underlying CUS-induced mood disorders in the simplest vertebrate model, the zebrafish, Danio rerio. Zebrafish were subjected to a CUS paradigm in which two different stressors were used daily for 15 days, and thorough behavioral analyses were performed to assess anxiety and related mood disorder phenotypes using the novel tank test, shoal cohesion and scototaxis. Fifteen days of exposure to chronic stressors appears to induce an anxiety and related mood disorder phenotype. Decreased neurogenesis, another hallmark of anxiety and related disorders in rodents, was also observed in this zebrafish model. The common molecular markers of rodent anxiety and related disorders, corticotropin-releasing factor (CRF), calcineurin (ppp3r1a) and phospho cyclic AMP response element binding protein (pCREB), were also replicated in the fish model. Finally, using 2DE FTMS/ITMSMS proteomics analyses, 18 proteins were found to be deregulated in zebrafish anxiety and related disorders. The most affected process was mitochondrial function, 4 of the 18 differentially regulated proteins were mitochondrial proteins: PHB2, SLC25A5, VDAC3 and IDH2, as reported in rodent and clinical samples. Thus, the zebrafish CUS model and proteomics can facilitate not only uncovering new molecular targets of anxiety and related mood disorders but also the routine screening of compounds for drug development.