Proteins from the Bcl-2 family play an essential role in the regulation of apoptosis. However, they also possess cell death-unrelated activities that are less well understood. This prompted us to study apoptosis-unrelated activities of the Bax and Bak, pro-apoptotic members of the Bcl-2 family. We prepared Bax/Bak-deficient human cancer cells of different origin and found that while respiration in the glioblastoma U87 Bax/Bak-deficient cells was greatly enhanced, respiration of Bax/Bak-deficient B lymphoma HBL-2 cells was slightly suppressed. Bax/Bak-deficient U87 cells also proliferated faster in culture, formed tumours more rapidly in mice, and showed modulation of metabolism with a considerably increased NAD+/NADH ratio. Follow-up analyses documented increased/decreased expression of mitochondria-encoded subunits of respiratory complexes and stabilization/destabilization of the mitochondrial transcription elongation factor TEFM in Bax/Bak-deficient U87 and HBL-2 cells, respectively. TEFM downregulation using shRNAs attenuated mitochondrial respiration in Bax/Bak-deficient U87 as well as in parental HBL-2 cells. We propose that (post)translational regulation of TEFM levels in Bax/Bak-deficient cells modulates levels of subunits of mitochondrial respiratory complexes that, in turn, contribute to respiration and the accompanying changes in metabolism and proliferation in these cells.
- MeSH
- apoptóza * genetika MeSH
- dýchání MeSH
- lidé MeSH
- mitochondrie genetika metabolismus MeSH
- myši MeSH
- protein Bak * genetika metabolismus MeSH
- protein X asociovaný s bcl-2 genetika metabolismus MeSH
- protoonkogenní proteiny c-bcl-2 genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Astrocytes are crucial for the functioning of the nervous system as they maintain the ion homeostasis via volume regulation. Pathological states, such as amyotrophic lateral sclerosis (ALS), affect astrocytes and might even cause a loss of such functions. In this study, we examined astrocytic swelling/volume recovery in both the brain and spinal cord of the SOD1 animal model to determine the level of their impairment caused by the ALS-like pathology. Astrocyte volume changes were measured in acute brain or spinal cord slices during and after exposure to hyperkalemia. We then compared the results with alterations of extracellular space (ECS) diffusion parameters, morphological changes, expression of the Kir4.1 channel and the potassium concentration measured in the cerebrospinal fluid, to further disclose the link between potassium and astrocytes in the ALS-like pathology. Morphological analysis revealed astrogliosis in both the motor cortex and the ventral horns of the SOD1 spinal cord. The activated morphology of SOD1 spinal astrocytes was associated with the results from volume measurements, which showed decreased swelling of these cells during hyperkalemia. Furthermore, we observed lower shrinkage of ECS in the SOD1 spinal ventral horns. Immunohistochemical analysis then confirmed decreased expression of the Kir4.1 channel in the SOD1 spinal cord, which corresponded with the diminished volume regulation. Despite astrogliosis, cortical astrocytes in SOD1 mice did not show alterations in swelling nor changes in Kir4.1 expression, and we did not identify significant changes in ECS parameters. Moreover, the potassium level in the cerebrospinal fluid did not deviate from the physiological concentration. The results we obtained thus suggest that ALS-like pathology causes impaired potassium uptake associated with Kir4.1 downregulation in the spinal astrocytes, but based on our data from the cortex, the functional impairment seems to be independent of the morphological state.
- Publikační typ
- časopisecké články MeSH
Glial cells expressing neuron-glial antigen 2 (NG2), also known as oligodendrocyte progenitor cells (OPCs), play a critical role in maintaining brain health. However, their ability to differentiate after ischemic injury is poorly understood. The aim of this study was to investigate the properties and functions of NG2 glia in the ischemic brain. Using transgenic mice, we selectively labeled NG2-expressing cells and their progeny in both healthy brain and after focal cerebral ischemia (FCI). Using single-cell RNA sequencing, we classified the labeled glial cells into five distinct subpopulations based on their gene expression patterns. Additionally, we examined the membrane properties of these cells using the patch-clamp technique. Of the identified subpopulations, three were identified as OPCs, whereas the fourth subpopulation had characteristics indicative of cells likely to develop into oligodendrocytes. The fifth subpopulation of NG2 glia showed astrocytic markers and had similarities to neural progenitor cells. Interestingly, this subpopulation was present in both healthy and post-ischemic tissue; however, its gene expression profile changed after ischemia, with increased numbers of genes related to neurogenesis. Immunohistochemical analysis confirmed the temporal expression of neurogenic genes and showed an increased presence of NG2 cells positive for Purkinje cell protein-4 at the periphery of the ischemic lesion 12 days after FCI, as well as NeuN-positive NG2 cells 28 and 60 days after injury. These results suggest the potential development of neuron-like cells arising from NG2 glia in the ischemic tissue. Our study provides insights into the plasticity of NG2 glia and their capacity for neurogenesis after stroke.
- MeSH
- antigeny metabolismus MeSH
- astrocyty metabolismus MeSH
- ischemie mozku * metabolismus MeSH
- mozek metabolismus MeSH
- myši transgenní MeSH
- myši MeSH
- nervové kmenové buňky * metabolismus MeSH
- neuroglie metabolismus MeSH
- oligodendroglie metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
INTRODUCTION: Astrocytic Transient receptor potential vanilloid 4 (TRPV4) channels, together with Aquaporin 4 (AQP4), are suspected to be the key players in cellular volume regulation, and therefore may affect the development and severity of cerebral edema during ischemia. In this study, we examined astrocytic swelling/volume recovery in mice with TRPV4 and/or AQP4 deletion in response to in vitro ischemic conditions, to determine how the deletion of these channels can affect the development of cerebral edema. METHODS: We used three models of ischemia-related pathological conditions: hypoosmotic stress, hyperkalemia, and oxygenglucose deprivation (OGD), and observed their effect on astrocyte volume changes in acute brain slices of Aqp4-/-, Trpv4-/- and double knockouts. In addition, we employed single-cell RT-qPCR to assess the effect of TRPV4 and AQP4 deletion on the expression of other ion channels and transporters involved in the homeostatic functioning of astrocytes. RESULTS: Quantification of astrocyte volume changes during OGD revealed that the deletion of AQP4 reduces astrocyte swelling, while simultaneous deletion of both AQP4 and TRPV4 leads to a disruption of astrocyte volume recovery during the subsequent washout. Of note, astrocyte exposure to hypoosmotic stress or hyperkalemia revealed no differences in astrocyte swelling in the absence of AQP4, TRPV4, or both channels. Moreover, under ischemia-mimicking conditions, we identified two distinct subpopulations of astrocytes with low and high volumetric responses (LRA and HRA), and their analyses revealed that mainly HRA are affected by the deletion of AQP4, TRPV4, or both channels. Furthermore, gene expression analysis revealed reduced expression of the ion transporters KCC1 and ClC2 as well as the receptors GABAB and NMDA in Trpv4-/- mice. The deletion of AQP4 instead caused reduced expression of the serine/cysteine peptidase inhibitor Serpina3n. DISCUSSION: Thus, we showed that in AQP4 or TRPV4 knockouts, not only the specific function of these channels is affected, but also the expression of other proteins, which may modulate the ischemic cascade and thus influence the final impact of ischemia.
- Publikační typ
- časopisecké články MeSH
Traumatic brain injury (TBI) is one of the most common pathological conditions impacting the central nervous system (CNS). A neurological deficit associated with TBI results from a complex of pathogenetic mechanisms including glutamate excitotoxicity, inflammation, demyelination, programmed cell death, or the development of edema. The critical components contributing to CNS response, damage control, and regeneration after TBI are glial cells-in reaction to tissue damage, their activation, hypertrophy, and proliferation occur, followed by the formation of a glial scar. The glial scar creates a barrier in damaged tissue and helps protect the CNS in the acute phase post-injury. However, this process prevents complete tissue recovery in the late/chronic phase by producing permanent scarring, which significantly impacts brain function. Various glial cell types participate in the scar formation, but this process is mostly attributed to reactive astrocytes and microglia, which play important roles in several brain pathologies. Novel technologies including whole-genome transcriptomic and epigenomic analyses, and unbiased proteomics, show that both astrocytes and microglia represent groups of heterogenic cell subpopulations with different genomic and functional characteristics, that are responsible for their role in neurodegeneration, neuroprotection and regeneration. Depending on the representation of distinct glia subpopulations, the tissue damage as well as the regenerative processes or delayed neurodegeneration after TBI may thus differ in nearby or remote areas or in different brain structures. This review summarizes TBI as a complex process, where the resultant effect is severity-, region- and time-dependent and determined by the model of the CNS injury and the distance of the explored area from the lesion site. Here, we also discuss findings concerning intercellular signaling, long-term impacts of TBI and the possibilities of novel therapeutical approaches. We believe that a comprehensive study with an emphasis on glial cells, involved in tissue post-injury processes, may be helpful for further research of TBI and be the decisive factor when choosing a TBI model.
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
INTRODUCTION: Modification of the extracellular matrix (ECM) is one of the major processes in the pathology of brain damage following an ischemic stroke. However, our understanding of how age-related ECM alterations may affect stroke pathophysiology and its outcome is still very limited. METHODS: We conducted an ECM-targeted re-analysis of our previously obtained RNA-Seq dataset of aging, ischemic stroke and their interactions in young adult (3-month-old) and aged (18-month-old) mice. The permanent middle cerebral artery occlusion (pMCAo) in rodents was used as a model of ischemic stroke. Altogether 56 genes of interest were chosen for this study. RESULTS: We identified an increased activation of the genes encoding proteins related to ECM degradation, such as matrix metalloproteinases (MMPs), proteases of a disintegrin and metalloproteinase with the thrombospondin motifs (ADAMTS) family and molecules that regulate their activity, tissue inhibitors of metalloproteinases (TIMPs). Moreover, significant upregulation was also detected in the mRNA of other ECM molecules, such as proteoglycans, syndecans and link proteins. Notably, we identified 8 genes where this upregulation was enhanced in aged mice in comparison with the young ones. Ischemia evoked a significant downregulation in only 6 of our genes of interest, including those encoding proteins associated with the protective function of ECM molecules (e.g., brevican, Hapln4, Sparcl1); downregulation in brevican was more prominent in aged mice. The study was expanded by proteome analysis, where we observed an ischemia-induced overexpression in three proteins, which are associated with neuroinflammation (fibronectin and vitronectin) and neurodegeneration (link protein Hapln2). In fibronectin and Hapln2, this overexpression was more pronounced in aged post-ischemic animals. CONCLUSION: Based on these results, we can conclude that the ratio between the protecting and degrading mechanisms in the aged brain is shifted toward degradation and contributes to the aged tissues' increased sensitivity to ischemic insults. Altogether, our data provide fresh perspectives on the processes underlying ischemic injury in the aging brain and serve as a freely accessible resource for upcoming research.
- Publikační typ
- časopisecké články MeSH
The role of glia in amyotrophic lateral sclerosis (ALS) is undeniable. Their disease-related activity has been extensively studied in the spinal cord, but only partly in the brain. We present herein a comprehensive study of glia in the cortex of SOD1(G93A) mice-a widely used model of ALS. Using single-cell RNA sequencing (scRNA-seq) and immunohistochemistry, we inspected astrocytes, microglia, and oligodendrocytes, in four stages of the disease, respecting the factor of sex. We report minimal changes of glia throughout the disease progression and regardless of sex. Pseudobulk and single-cell analyses revealed subtle disease-related transcriptional alterations at the end-stage in microglia and oligodendrocytes, which were supported by immunohistochemistry. Therefore, our data support the hypothesis that the SOD1(G93A) mouse cortex does not recapitulate the disease in patients, and we recommend the use of a different model for future studies of the cortical ALS pathology.
- MeSH
- amyotrofická laterální skleróza * genetika patologie MeSH
- mícha patologie MeSH
- modely nemocí na zvířatech MeSH
- motorické neurony patologie MeSH
- myši transgenní MeSH
- myši MeSH
- neuroglie * patologie MeSH
- superoxid dismutáza 1 * genetika MeSH
- superoxiddismutasa genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Transient receptor potential cation channels subfamily V member 4 (TRPV4) are non-selective cation channels expressed in different cell types of the central nervous system. These channels can be activated by diverse physical and chemical stimuli, including heat and mechanical stress. In astrocytes, they are involved in the modulation of neuronal excitability, control of blood flow, and brain edema formation. All these processes are significantly impaired in cerebral ischemia due to insufficient blood supply to the tissue, resulting in energy depletion, ionic disbalance, and excitotoxicity. The polymodal cation channel TRPV4, which mediates Ca2+ influx into the cell because of activation by various stimuli, is one of the potential therapeutic targets in the treatment of cerebral ischemia. However, its expression and function vary significantly between brain cell types, and therefore, the effect of its modulation in healthy tissue and pathology needs to be carefully studied and evaluated. In this review, we provide a summary of available information on TRPV4 channels and their expression in healthy and injured neural cells, with a particular focus on their role in ischemic brain injury.
INTRODUCTION: Astrocytic Aquaporin 4 (AQP4) and Transient receptor potential vanilloid 4 (TRPV4) channels form a functional complex that likely influences cell volume regulation, the development of brain edema, and the severity of the ischemic injury. However, it remains to be fully elucidated whether blocking these channels can serve as a therapeutic approach to alleviate the consequences of having a stroke. METHODS AND RESULTS: In this study, we used in vivo magnetic resonance imaging (MRI) to quantify the extent of brain lesions one day (D1) and seven days (D7) after permanent middle cerebral artery occlusion (pMCAO) in AQP4 or TRPV4 knockouts and mice with simultaneous deletion of both channels. Our results showed that deletion of AQP4 or TRPV4 channels alone leads to a significant worsening of ischemic brain injury at both time points, whereas their simultaneous deletion results in a smaller brain lesion at D1 but equal tissue damage at D7 when compared with controls. Immunohistochemical analysis 7 days after pMCAO confirmed the MRI data, as the brain lesion was significantly greater in AQP4 or TRPV4 knockouts than in controls and double knockouts. For a closer inspection of the TRPV4 and AQP4 channel complex in the development of brain edema, we applied a real-time iontophoretic method in situ to determine ECS diffusion parameters, namely volume fraction (α) and tortuosity (λ). Changes in these parameters reflect alterations in cell volume, and tissue structure during exposure of acute brain slices to models of ischemic conditions in situ, such as oxygen-glucose deprivation (OGD), hypoosmotic stress, or hyperkalemia. The decrease in α was comparable in double knockouts and controls when exposed to hypoosmotic stress or hyperkalemia. However, during OGD, there was no decrease in α in the double knockouts as observed in the controls, which suggests less swelling of the cellular components of the brain. CONCLUSION: Although simultaneous deletion of AQP4 and TRPV4 did not improve the overall outcome of ischemic brain injury, our data indicate that the interplay between AQP4 and TRPV4 channels plays a critical role during neuronal and non-neuronal swelling in the acute phase of ischemic injury.
- Publikační typ
- časopisecké články MeSH
Maternal immune activation has been identified as a significant risk factor for schizophrenia. Using rodent models, past work has demonstrated various behavioral and brain impairments in offspring after immune-activating events. We applied 5 mg/kg of poly(I:C) on gestation day 9 to pregnant mouse dams, whose offspring were then stressed during puberty. We show impairments in attentional set-shifting in a T-maze, and a decreased number of parvalbumin-positive interneurons in the hippocampus as a result of peripubertal stress specifically in females.
- MeSH
- chování zvířat fyziologie MeSH
- exekutivní funkce fyziologie MeSH
- hipokampus cytologie MeSH
- infekční komplikace v těhotenství * chemicky indukované imunologie MeSH
- interneurony cytologie MeSH
- kognitivní dysfunkce etiologie patologie patofyziologie MeSH
- modely nemocí na zvířatech MeSH
- myši inbrední C57BL MeSH
- poly I-C aplikace a dávkování MeSH
- pozornost fyziologie MeSH
- psychický stres komplikace patologie patofyziologie MeSH
- schizofrenie etiologie imunologie patologie patofyziologie MeSH
- těhotenství MeSH
- zpožděný efekt prenatální expozice chemicky indukované patologie patofyziologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
