Arg4810Lys mutation in RNF213 among Eastern Indian non-MMD ischemic stroke patients: a genotype-phenotype correlation.

INTRODUCTION: RNF213 mutations have been reported mostly in moyamoya disease (MMD) with varying frequencies across different ethnicities. However, its prevalence in non-MMD adult-onset ischemic stroke is still not well explored. AIMS AND OBJECTIVES: This present study thus aims to screen the most common RNF213 variant (Arg4810Lys, among East Asians) in the Eastern Indian non-MMD ischemic stroke patients and correlate it with long-term progression and prognosis of the patients. The subjects were analyzed for this variant using PCR-RFLP and confirmed using Sanger sequencing method. RESULT AND CONCLUSION: We have identified Arg4810Lys variant among eleven young-onset familial ischemic stroke patients in heterozygous manner. A positive correlation of the variant with positive family history (P = 0.001), earlier age at onset (P = 0.002), and history of recurrent stroke (P = 0.015) was observed. However, the carriers showed better cognitive performances in memory (P = 0.042) and executive function (P = 0.004). Therefore, we can conclude that Arg4810Lys/RNF213 - a pathogenic variant for young-onset familial ischemic stroke with higher incidence of recurrent events unlike in MMD cases, have no additional impact on cognition among Eastern Indians.

Decoding the proregenerative competence of regulatory T cells through complex tissue regeneration in zebrafish.

Regulatory T cells (Tregs ) are specific subtype of T cells that play a central role in sustaining self-antigen tolerance and restricting inflammatory tissue damage. More recently, additional direct functions of Tregs in mammalian tissue repair have emerged, but the regenerative potential of Tregs in non-mammalian vertebrates has not been explored despite the latter possessing a highly developed adaptive immune system. Why complex organs such as the caudal fin, heart, brain, spinal cord and retina regenerate in certain non-mammalian vertebrates, but not in mammals, is an interesting but unresolved question in the field of regenerative biology. Inflammation has traditionally been thought to be an impediment to regeneration due to the formation of scars. Regenerative decline in higher organisms has been speculated to be the evolutionary advent of adaptive immunity. Recent studies, however, have shown that the innate inflammatory response in non-mammalian organisms is required for organ regeneration. It has also been found that highly advanced adaptive immunity is no longer incompatible with regeneration and for that, Tregs are important. Zebrafish regulatory T cells (zTregs ) migrate rapidly to the injury site in damaged organs, where they facilitate the proliferation of regeneration precursor cells by generating tissue-specific regenerative factors by a process distinct from the canonical anti-inflammatory pathway. We review both reparative and proregenerative roles of Tregs in mammals and zebrafish, respectively, and also give an overview of the forkhead box protein 3 (FoxP3) -dependent immunosuppressive function of Tregs in zebrafish, which makes it a useful model organism for future Treg biology and research.

The loss of regeneration competency in the animal kingdom at the expense of immunity: A journey in retrospect.

Regeneration refers to the structural growth of damaged organs or tissues and their functional integration into the existing system. Injury induced regenerative response is extremely variable across the animal kingdom. On one hand the early acoelomates can reform the entire animal even from dissociated cells, on the other; the capacity in humans is mostly restricted to wound healing. A general trend of regenerative ability is the existence of an inverse relationship between the robustness of immune system and the degree of regeneration throughout the animal kingdom. This review summarizes the evolutionary advancement of immune system in different groups and gives an account of their respective regenerative competency.

Regeneration of Zebrafish CNS: Adult Neurogenesis.

Regeneration in the animal kingdom is one of the most fascinating problems that have allowed scientists to address many issues of fundamental importance in basic biology. However, we came to know that the regenerative capability may vary across different species. Among vertebrates, fish and amphibians are capable of regenerating a variety of complex organs through epimorphosis. Zebrafish is an excellent animal model, which can repair several organs like damaged retina, severed spinal cord, injured brain and heart, and amputated fins. The focus of the present paper is on spinal cord regeneration in adult zebrafish. We intend to discuss our current understanding of the cellular and molecular mechanism(s) that allows formation of proliferating progenitors and controls neurogenesis, which involve changes in epigenetic and transcription programs. Unlike mammals, zebrafish retains radial glia, a nonneuronal cell type in their adult central nervous system. Injury induced proliferation involves radial glia which proliferate, transcribe embryonic genes, and can give rise to new neurons. Recent technological development of exquisite molecular tools in zebrafish, such as cell ablation, lineage analysis, and novel and substantial microarray, together with advancement in stem cell biology, allowed us to investigate how progenitor cells contribute to the generation of appropriate structures and various underlying mechanisms like reprogramming.

Expression pattern of Nogo-A, MAG, and NgR in regenerating urodele spinal cord.

BACKGROUND: The mammalian central nervous system is incapable of substantial axon regeneration after injury partially due to the presence of myelin-associated inhibitory molecules including Nogo-A and myelin associated glycoprotein (MAG). In contrast, axolotl salamanders are capable of considerable axon regrowth during spinal cord regeneration. RESULTS: Here, we show that Nogo-A and MAG, and their receptor, Nogo receptor (NgR), are present in the axolotl genome and are broadly expressed in the central nervous system (CNS) during development, adulthood, and importantly, during regeneration. Furthermore, we show that Nogo-A and NgR are co-expressed in Sox2 positive neural progenitor cells. CONCLUSIONS: These expression patterns suggest myelin-associated proteins are permissive for neural development and regeneration in axolotls.

Epigenetic Cross-Talk Between Sirt1 and Dnmt1 Promotes Axonal Regeneration After Spinal Cord Injury in Zebrafish.

Though spinal cord injury (SCI) causes irreversible sensory and motor impairments in human, adult zebrafish retain the potent regenerative capacity by injury-induced proliferation of central nervous system (CNS)-resident progenitor cells to develop new functional neurons at the lesion site. The hallmark of SCI in zebrafish lies in a series of changes in the epigenetic landscape, specifically DNA methylation and histone modifications. Decoding the post-SCI epigenetic modifications is therefore critical for the development of therapeutic remedies that boost SCI recovery process. Here, we have studied on Sirtuin1 (Sirt1), a non-classical histone deacetylase that potentially plays a critical role in neural progenitor cells (NPC) proliferation and axonal regrowth following SCI in zebrafish. We investigated the role of Sirt1 in NPC proliferation and axonal regrowth in response to injury in the regenerating spinal cord and found that Sirt1 is involved in the induction of NPC proliferation along with glial bridging during spinal cord regeneration. We also demonstrate that Sirt1 plays a pivotal role in regulating the HIPPO pathway through deacetylation-mediated inactivation of Dnmt1 and subsequent hypomethylation of yap1 promoter, leading to the induction of ctgfa expression, which drives the NPC proliferation and axonal regrowth to complete the regenerative process. In conclusion, our study reveals a novel cross-talk between two important epigenetic effectors, Sirt1 and Dnmt1, in the context of spinal cord regeneration, establishing a previously undisclosed relation between Sirt1 and Yap1 which provides a deeper understanding of the underlying mechanisms governing injury-induced NPC proliferation and axonal regrowth. Therefore, we have identified Sirt1 as a novel, major epigenetic regulator of spinal cord regeneration by modulating the HIPPO pathway in zebrafish.

Fruit ripening retardant Daminozide induces cognitive impairment, cell specific neurotoxicity, and genotoxicity in Drosophila melanogaster.

BACKGROUND: We explored neurotoxic and genotoxic effects of Daminozide, a fruit ripening retardant, on the brain of Drosophila melanogaster, based on our previous finding of DNA fragmentation in larval brain cell in the flies experimentally exposed to this chemicals. METHODS: Adult flies were subjected to two distinct concentrations of daminozide (200 mg/L and 400 mg/L) mixed in culture medium, followed by an examination of specific behaviors such as courtship conditioning and aversive phototaxis, which serve as indicators of cognitive functions. We investigated brain histology and histochemistry to assess the overall toxicity of daminozide, focusing on neuron type-specific effects. Additionally, we conducted studies on gene expression specific to neuronal function. Statistical comparisons were then made between the exposed and control flies across all tested attributes. RESULTS: The outcome of behavioral assays suggested deleterious effects of Daminozide on learning, short term and long term memory function. Histological examination of brain sections revealed cellular degeneration, within Kenyon cell neuropiles in Daminozide-exposed flies. Neurone specific Immuno-histochemistry study revealed significant reduction of dopaminergic and glutaminergic neurones with discernible reduction in cellular counts, alteration in cell and nuclear morphology among daminozide exposed flies. Gene expression analyses demonstrated upregulation of rutabaga (rut), hb9 and down regulation of PKa- C1, CrebB, Ace and nAchRbeta-1 in exposed flies which suggest dysregulation of gene functions involved in motor neuron activity, learning, and memory. CONCLUSION: Taken together, our findings suggests that Daminozide induces multifaceted harmful impacts on the neural terrain of Drosophila melanogaster, posing a threat to its cognitive abilities.

Clinical Implication of Time of Ischaemic Stroke Among Post-Stroke Survivors from Eastern India: A Circadian Perspective.

The circadian variation in stroke occurrence is a well-documented phenomenon. However, the circadian effect on stroke outcome, particularly on post-stroke cognition, has not yet been fully elucidated. We aim to evaluate the influence of diurnal variation of stroke onset upon post-stroke cognition and development of post-stroke depression. Based on 4-hourly time period of stroke occurrence, 249 recruited cohorts were categorized into 6 groups. Several clinical and cognitive parameters were compared among the groups. Then, the mRNA expression of core clock genes in Peripheral Blood Mononuclear Cells were quantified and correlated with post-stroke outcomes among 24 acute phase cases with day-time or night-time stroke occurrence. Furthermore, the genetic susceptibility towards a higher number of cases in the morning was examined by genotyping CLOCK (rs1801260T/C, rs4580704G/C) and CRY2 (rs2292912C/G) genes variants in cases and 292 controls. In our study, the peak for highest incidence although observed during the early morning from 4 to 8 am, the nocturnal-onset stroke cases showed more severity (12.2 ± 5.67) at the time of admission irrespective of arterial territory involved. The night onset cases were also found to be more susceptible to develop language impairment and post-stroke depression in due course of time. Upon transcript analysis, circadian genes (BMAL1 and CRY1) were found to be downregulated in night-time cases than day-time ones during the acute phase of onset. In addition, those mRNA levels also showed a correlation with raw scores for language and depression. However, the difference in incidence frequency along a day did not reveal any genetic correlation. Therefore, we suggest night-time stroke to be positively associated with higher immediate severity and poor cognitive outcome than day-time injury and propose downregulation of circadian genes during the acute phase could be the underlying molecular mechanism for this.

Regenerative Potential of Injured Spinal Cord in the Light of Epigenetic Regulation and Modulation.

A spinal cord injury is a form of physical harm imposed on the spinal cord that causes disability and, in many cases, leads to permanent mammalian paralysis, which causes a disastrous global issue. Because of its non-regenerative aspect, restoring the spinal cord's role remains one of the most daunting tasks. By comparison, the remarkable regenerative ability of some regeneration-competent species, such as some Urodeles (Axolotl), Xenopus, and some teleost fishes, enables maximum functional recovery, even after complete spinal cord transection. During the last two decades of intensive research, significant progress has been made in understanding both regenerative cells' origins and the molecular signaling mechanisms underlying the regeneration and reconstruction of damaged spinal cords in regenerating organisms and mammals, respectively. Epigenetic control has gradually moved into the center stage of this research field, which has been helped by comprehensive work demonstrating that DNA methylation, histone modifications, and microRNAs are important for the regeneration of the spinal cord. In this review, we concentrate primarily on providing a comparison of the epigenetic mechanisms in spinal cord injuries between non-regenerating and regenerating species. In addition, we further discuss the epigenetic mediators that underlie the development of a regeneration-permissive environment following injury in regeneration-competent animals and how such mediators may be implicated in optimizing treatment outcomes for spinal cord injurie in higher-order mammals. Finally, we briefly discuss the role of extracellular vesicles (EVs) in the context of spinal cord injury and their potential as targets for therapeutic intervention.

Genetic Variations and Altered Blood mRNA Level of Circadian Genes and BDNF as Risk Factors of Post-Stroke Cognitive Impairment Among Eastern Indians.

Post-stroke cognitive impairment (PSCI) is a clinical outcome in around 30% of post-stroke survivors. BDNF is a major gene in this regard. It is regulated by circadian rhythm. The circadian genes are correlated with stroke timings at molecular level. However, studies suggesting the role of these on susceptibility to PSCI are limited. We aim here to determine: (a) genetic risk variants in circadian clock genes, BDNF and (b) dysregulation in expression level of CLOCK, BMAL1, and BDNF that may be associated with PSCI. BDNF (rs6265G/A, rs56164415C/T), CLOCK (rs1801260T/C, rs4580704G/C), and CRY2 (rs2292912C/G) genes variants were genotyped among 119 post-stroke survivors and 292 controls from Eastern part of India. In addition, we analyzed their gene expression in Peripheral blood Mononuclear cells (PBMC) from 15 PSCI cases and 12 controls. The mRNA data for BDNF was further validated by its plasma level through ELISA (n = 38). Among the studied variants, only rs4580704/CLOCK showed an overall association with PSCI (P = 0.001) and lower Bengali Mini-Mental State Examination (BMSE) score. Its 'C' allele showed a correlation with attention deficiency. The language and memory impairments showed association with rs6265/BDNF, while the 'CC' genotype of rs2292912/CRY2 negatively influenced language and executive function. A significant decrease in gene expression for CLOCK and BDNF in PBMC (influenced by specific genotypes) of PSCI patients was observed than controls. Unlike Pro-BDNF, plasma-level mBDNF was also lower in them. Our results suggest the genetic variants in CLOCK, CRY2, and BDNF as risk factors for PSCI among eastern Indians. At the same time, a lowering expression of CLOCK and BDNF genes in PSCI patients than controls describes their transcriptional dysregulation as underlying mechanism for post-stroke cognitive decline.

Celsr family genes are dynamically expressed in embryonic and juvenile zebrafish.

The Cadherin EGF LAG seven-pass G-type receptor (Celsr) family belongs to the adhesion G-protein coupled receptor superfamily. In most vertebrates, the Celsr family has three members (CELSR1-3), whereas zebrafish display four paralogues (celsr1a, 1b, 2, 3). Although studies have shown the importance of the Celsr family in planar cell polarity, axonal guidance, and dendritic growth, the molecular mechanisms of the Celsr family regulating these cellular processes in vertebrates remain elusive. Zebrafish is an experimentally more amenable model to study vertebrate development, as zebrafish embryos develop externally, optically transparent, remain alive with malformed organs, and zebrafish is genetically similar to humans. Understanding the detailed expression pattern is the first step of exploring the functional mechanisms of the genes involved in development. Thus, we report the spatiotemporal expression pattern of Celsr family members in zebrafish nervous tissues. Our analysis shows that celsr1b and celsr2 are expressed maternally. In embryos, celsr1a, celsr1b, and celsr2 are expressed in the neural progenitors, and celsr3 is expressed in all five primary neural clusters of the brain and mantle layer of the spinal cord. In juvenile zebrafish, celsr1a, celsr1b, and celsr2 are presumably expressed in the neural progenitor enriched regions of the CNS. Therefore, the expression pattern of zebrafish Celsr family members is reminiscent of patterns described in other vertebrates or mammalian speciate. This indicates the conserved role of Celsr family genes in nervous system development and suggests zebrafish as an excellent model to explore the cellular and molecular mechanisms of Celsr family genes in vertebrate neurogenesis.

Cellular response after crush injury in adult zebrafish spinal cord.

Zebrafish proves to be an excellent model system to study spinal cord regeneration because it can repair its disengaged axons and replace lost cells after injury, allowing the animal to make functional recovery. We have characterized injury response following crush injury, which is comparable to the mammalian mode of injury. Infiltrations of blood cells during early phases involve macrophages that are important in debris clearance and probably in suppression of inflammatory response. Unlike mammals where secondary injury mechanisms lead to apoptotic death of both neurons and glia, here we observe a beneficial role of apoptotic cell death. Injury-induced proliferation, presence of radial glia cells, and their role as progenitor all contribute to cellular replacement and successful neurogenesis after injury in adult zebrafish. Together with cell replacement phenomenon, there is creation of a permissive environment that includes the absence or clearance of myelin debris, presence of Schwann cells, and absence of inflammatory response.

An insight on established retinal injury mechanisms and prevalent retinal stem cell activation pathways in vertebrate models.

Implementing different tools and injury mechanisms in multiple animal models of retina regeneration, researchers have discovered the existence of retinal stem/progenitor cells. Although they appear to be distributed uniformly across the vertebrate lineage, the reparative potential of the retina is mainly restricted to lower vertebrates. Regenerative repair post-injury requires the creation of a proliferative niche, vital for proper stem cell activation, propagation, and lineage differentiation. This seems to be lacking in mammals. Hence, in this review, we first discuss the many forms of retinal injuries that have been generated using animal models. Next, we discuss how they are utilized to stimulate regeneration and mimic eye disease pathologies. The key to driving stem cell activation in mammals relies on the information we can gather from these models. Lastly, we present a brief update about the genes, growth factors, and signaling pathways that have been brought to light using these models.

Neural cells and their progenitors in regenerating zebrafish spinal cord.

The zebrafish (Danio rerio), among all amniotes is emerging as a powerful model to study vertebrate organogenesis and regeneration. In contrast to mammals, the adult zebrafish is capable of regenerating damaged axonal tracts; it can replace neurons and glia lost after spinal cord injury (SCI) and functionally recover. In the present paper, we report ultrastructural and cell biological analyses of regeneration processes after SCI. We have focused on event specific analyses of spinal cord regeneration involving different neuronal and glial cell progenitors, such as radial glia, oligodendrocyte progenitors (OPC), and Schwann cells. While comparing the different events, we frequently refer to previous ultrastructural analyses of central nervous system (CNS) injury in higher vertebrates. Our data show (a) the cellular events following injury, such as cell death and proliferation; (b) demyelination and remyelination followed by target innervation and regeneration of synaptic junctions and c) the existence of different progenitors and their roles during regeneration. The present ultrastructural analysis corroborates the cellular basis of regeneration in the zebrafish spinal cord and confirms the presence of both neuronal and different glial progenitors.

Axonal regeneration in zebrafish spinal cord.

In the present review we discuss two interrelated events-axonal damage and repair-known to occur after spinal cord injury (SCI) in the zebrafish. Adult zebrafish are capable of regenerating axonal tracts and can restore full functionality after SCI. Unlike fish, axon regeneration in the adult mammalian central nervous system is extremely limited. As a consequence of an injury there is very little repair of disengaged axons and therefore functional deficit persists after SCI in adult mammals. In contrast, peripheral nervous system axons readily regenerate following injury and hence allow functional recovery both in mammals and fish. A better mechanistic understanding of these three scenarios could provide a more comprehensive insight into the success or failure of axonal regeneration after SCI. This review summarizes the present understanding of the cellular and molecular basis of axonal regeneration, in both the peripheral nervous system and the central nervous system, and large scale gene expression analysis is used to focus on different events during regeneration. The discovery and identification of genes involved in zebrafish spinal cord regeneration and subsequent functional experimentation will provide more insight into the endogenous mechanism of myelination and remyelination. Furthermore, precise knowledge of the mechanism underlying the extraordinary axonal regeneration process in zebrafish will also allow us to unravel the potential therapeutic strategies to be implemented for enhancing regrowth and remyelination of axons in mammals.

Characterization of Proliferating Neural Progenitors after Spinal Cord Injury in Adult Zebrafish.

Zebrafish can repair their injured brain and spinal cord after injury unlike adult mammalian central nervous system. Any injury to zebrafish spinal cord would lead to increased proliferation and neurogenesis. There are presences of proliferating progenitors from which both neuronal and glial loss can be reversed by appropriately generating new neurons and glia. We have demonstrated the presence of multiple progenitors, which are different types of proliferating populations like Sox2+ neural progenitor, A2B5+ astrocyte/ glial progenitor, NG2+ oligodendrocyte progenitor, radial glia and Schwann cell like progenitor. We analyzed the expression levels of two common markers of dedifferentiation like msx-b and vimentin during regeneration along with some of the pluripotency associated factors to explore the possible role of these two processes. Among the several key factors related to pluripotency, pou5f1 and sox2 are upregulated during regeneration and associated with activation of neural progenitor cells. Uncovering the molecular mechanism for endogenous regeneration of adult zebrafish spinal cord would give us more clues on important targets for future therapeutic approach in mammalian spinal cord repair and regeneration.

Genome wide expression profiling during spinal cord regeneration identifies comprehensive cellular responses in zebrafish.

BACKGROUND: Among the vertebrates, teleost and urodele amphibians are capable of regenerating their central nervous system. We have used zebrafish as a model to study spinal cord injury and regeneration. Relatively little is known about the molecular mechanisms underlying spinal cord regeneration and information based on high density oligonucleotide microarray was not available. We have used a high density microarray to profile the temporal transcriptome dynamics during the entire phenomenon. RESULTS: A total of 3842 genes expressed differentially with significant fold changes during spinal cord regeneration. Cluster analysis revealed event specific dynamic expression of genes related to inflammation, cell death, cell migration, cell proliferation, neurogenesis, neural patterning and axonal regrowth. Spatio-temporal analysis of stat3 expression suggested its possible function in controlling inflammation and cell proliferation. Genes involved in neurogenesis and their dorso-ventral patterning (sox2 and dbx2) are differentially expressed. Injury induced cell proliferation is controlled by many cell cycle regulators and some are commonly expressed in regenerating fin, heart and retina. Expression pattern of certain pathway genes are identified for the first time during regeneration of spinal cord. Several genes involved in PNS regeneration in mammals like stat3, socs3, atf3, mmp9 and sox11 are upregulated in zebrafish SCI thus creating PNS like environment after injury. CONCLUSION: Our study provides a comprehensive genetic blue print of diverse cellular response(s) during regeneration of zebrafish spinal cord. The data highlights the importance of different event specific gene expression that could be better understood and manipulated further to induce successful regeneration in mammals.