Socrates: A Novel N-Ethyl-N-nitrosourea-Induced Mouse Mutant with Audiogenic Epilepsy.

Epilepsy is one of the common neurological diseases that affects not only adults but also infants and children. Because epilepsy has been studied for a long time, there are several pharmacologically effective anticonvulsants, which, however, are not suitable as therapy for all patients. The genesis of epilepsy has been extensively investigated in terms of its occurrence after injury and as a concomitant disease with various brain diseases, such as tumors, ischemic events, etc. However, in the last decades, there are multiple reports that both genetic and epigenetic factors play an important role in epileptogenesis. Therefore, there is a need for further identification of genes and loci that can be associated with higher susceptibility to epileptic seizures. Use of mouse knockout models of epileptogenesis is very informative, but it has its limitations. One of them is due to the fact that complete deletion of a gene is not, in many cases, similar to human epilepsy-associated syndromes. Another approach to generating mouse models of epilepsy is N-Ethyl-N-nitrosourea (ENU)-directed mutagenesis. Recently, using this approach, we generated a novel mouse strain, soc (socrates, formerly s8-3), with epileptiform activity. Using molecular biology methods, calcium neuroimaging, and immunocytochemistry, we were able to characterize the strain. Neurons isolated from soc mutant brains retain the ability to differentiate in vitro and form a network. However, soc mutant neurons are characterized by increased spontaneous excitation activity. They also demonstrate a high degree of Ca2+ activity compared to WT neurons. Additionally, they show increased expression of NMDA receptors, decreased expression of the Ca2+-conducting GluA2 subunit of AMPA receptors, suppressed expression of phosphoinositol 3-kinase, and BK channels of the cytoplasmic membrane involved in protection against epileptogenesis. During embryonic and postnatal development, the expression of several genes encoding ion channels is downregulated in vivo, as well. Our data indicate that soc mutation causes a disruption of the excitation-inhibition balance in the brain, and it can serve as a mouse model of epilepsy.

Overexpression of Brain- and Glial Cell Line-Derived Neurotrophic Factors Is Neuroprotective in an Animal Model of Acute Hypobaric Hypoxia.

Currently, the role of the neurotrophic factors BDNF and GDNF in maintaining the brain's resistance to the damaging effects of hypoxia and functional recovery of neural networks after exposure to damaging factors are actively studied. The assessment of the effect of an increase in the level of these neurotrophic factors in brain tissues using genetic engineering methods on the resistance of laboratory animals to hypoxia may pave the way for the future clinical use of neurotrophic factors BDNF and GDNF in the treatment of hypoxic damage. This study aimed to evaluate the antihypoxic and neuroprotective properties of BDNF and GDNF expression level increase using adeno-associated viral vectors in modeling hypoxia in vivo. To achieve overexpression of neurotrophic factors in the central nervous system's cells, viral constructs were injected into the brain ventricles of newborn male C57Bl6 (P0) mice. Acute hypobaric hypoxia was modeled on the 30th day after the injection of viral vectors. Survival, cognitive, and mnestic functions in the late post-hypoxic period were tested. Evaluation of growth and weight characteristics and the neurological status of animals showed that the overexpression of neurotrophic factors does not affect the development of mice. It was found that the use of adeno-associated viral vectors increased the survival rate of male mice under hypoxic conditions. The present study indicates that the neurotrophic factors' overexpression, induced by the specially developed viral constructs carrying the BDNF and GDNF genes, is a prospective neuroprotection method, increasing the survival rate of animals after hypoxic injury.

The Influence of Neurotrophic Factors BDNF and GDNF Overexpression on the Functional State of Mice and Their Adaptation to Audiogenic Seizures.

The high prevalence of diagnosed cases of severe neurological disorders, a significant proportion of which are epilepsy, contributes to a high level of mortality and disability in the population. Neurotrophic factors BDNF and GNDF are considered promising agents aimed at increasing the central nervous system's adaptive potential for the development of the epileptiform activity. Despite the pronounced neuroprotective and anticonvulsant potential, an appropriate way to stimulate these endogenous signaling molecules with minimal risk of side effects remains an open question. Herein, we assessed the safety of gene therapy using original adeno-associated viral constructs carrying the genes of neurotrophic factors BDNF and GDNF in the early postnatal period of development of experimental animals. The intraventricular injection of AAV-Syn-BDNF-eGFP and AAV-Syn-GDNF-eGFP viral constructs into newborn mice was found to provide persistent overexpression of target genes in the hippocampus and cerebral cortex in vivo for four weeks after injection. The application of viral constructs has a multidirectional effect on the weight and body length characteristics of mice in the early postnatal period; however, it ensures the animals' resistance to the development of seizure activity under audiogenic stimulation in the late postnatal period and preserves basic behavioral reactions, emotional status, as well as the mnestic and cognitive abilities of mice after simulated stress. Our results demonstrated the safety of using the AAV-Syn-BDNF-eGFP and AAV-Syn-GDNF-eGFP viral constructs in vivo, which indicates the expediency of further testing the constructs as therapeutic anticonvulsants.

Role of Satb1 and Satb2 Transcription Factors in the Glutamate Receptors Expression and Ca2+ Signaling in the Cortical Neurons In Vitro.

Transcription factors Satb1 and Satb2 are involved in the processes of cortex development and maturation of neurons. Alterations in the expression of their target genes can lead to neurodegenerative processes. Molecular and cellular mechanisms of regulation of neurotransmission by these transcription factors remain poorly understood. In this study, we have shown that transcription factors Satb1 and Satb2 participate in the regulation of genes encoding the NMDA-, AMPA-, and KA- receptor subunits and the inhibitory GABA(A) receptor. Deletion of gene for either Satb1 or Satb2 homologous factors induces the expression of genes encoding the NMDA receptor subunits, thereby leading to higher amplitudes of Ca2+-signals in neurons derived from the Satb1-deficient (Satb1fl/+ * NexCre/+) and Satb1-null mice (Satb1fl/fl * NexCre/+) in response to the selective agonist reducing the EC50 for the NMDA receptor. Simultaneously, there is an increase in the expression of the Gria2 gene, encoding the AMPA receptor subunit, thus decreasing the Ca2+-signals of neurons in response to the treatment with a selective agonist (5-Fluorowillardiine (FW)). The Satb1 deletion increases the sensitivity of the KA receptor to the agonist (domoic acid), in the cortical neurons of the Satb1-deficient mice but decreases it in the Satb1-null mice. At the same time, the Satb2 deletion decreases Ca2+-signals and the sensitivity of the KA receptor to the agonist in neurons from the Satb1-null and the Satb1-deficient mice. The Satb1 deletion affects the development of the inhibitory system of neurotransmission resulting in the suppression of the neuron maturation process and switching the GABAergic responses from excitatory to inhibitory, while the Satb2 deletion has a similar effect only in the Satb1-null mice. We show that the Satb1 and Satb2 transcription factors are involved in the regulation of the transmission of excitatory signals and inhibition of the neuronal network in the cortical cell culture.

The Dynamics of ß-Amyloid Proteoforms Accumulation in the Brain of a 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the leading cause of dementia among the elderly. Neuropathologically, AD is characterized by the deposition of a 39- to 42-amino acid long ß-amyloid (Aß) peptide in the form of senile plaques. Several post-translational modifications (PTMs) in the N-terminal domain have been shown to increase the aggregation and cytotoxicity of Aß, and specific Aß proteoforms (e.g., Aß with isomerized D7 (isoD7-Aß)) are abundant in the senile plaques of AD patients. Animal models are indispensable tools for the study of disease pathogenesis, as well as preclinical testing. In the presented work, the accumulation dynamics of Aß proteoforms in the brain of one of the most widely used amyloid-based mouse models (the 5xFAD line) was monitored. Mass spectrometry (MS) approaches, based on ion mobility separation and the characteristic fragment ion formation, were applied. The results indicated a gradual increase in the Aß fraction of isoD7-Aß, starting from approximately 8% at 7 months to approximately 30% by 23 months of age. Other specific PTMs, in particular, pyroglutamylation, deamidation, and oxidation, as well as phosphorylation, were also monitored. The results for mice of different ages demonstrated that the accumulation of Aß proteoforms correlate with the formation of Aß deposits. Although the mouse model cannot be a complete analogue of the processes occurring in the human brain in AD, and several of the observed parameters differ significantly from human values supposedly due to the limited lifespan of the model animals, this dynamic study provides evidence on at least one of the possible mechanisms that can trigger amyloidosis in AD, i.e., the hypothesis on the relationship between the accumulation of isoD7-Aß and the progression of AD-like pathology.

Brain-Derived Neurotrophic Factor (BDNF) Preserves the Functional Integrity of Neural Networks in the ß-Amyloidopathy Model in vitro.

Alzheimer's disease (AD) is a widespread chronic neurodegenerative pathology characterized by synaptic dysfunction, partial neuronal death, cognitive decline and memory impairments. The major hallmarks of AD are extracellular senile amyloid plaques formed by various types of amyloid proteins (Aß) and the formation and accumulation of intracellular neurofibrillary tangles. However, there is a lack of relevant experimental models for studying changes in neural network activity, the features of intercellular signaling or the effects of drugs on the functional activity of nervous cells during AD development. In this work, we examined two experimental models of amyloidopathy using primary hippocampal cultures. The first model involves the embryonic brains of 5xFAD mice; the second uses chronic application of amyloid beta 1-42 (Aß1-42). The model based on primary hippocampal cells obtained from 5xFAD mice demonstrated changes in spontaneous network calcium activity characterized by a decrease in the number of cells exhibiting Ca2+ activity, a decrease in the number of Ca2+ oscillations and an increase in the duration of Ca2+ events from day 21 of culture development in vitro. Chronic application of Aß1-42 resulted in the rapid establishment of significant neurodegenerative changes in primary hippocampal cultures, leading to marked impairments in neural network calcium activity and increased cell death. Using this model and multielectrode arrays, we studied the influence of amyloidopathy on spontaneous bioelectrical neural network activity in primary hippocampal cultures. It was shown that chronic Aß application decreased the number of network bursts and spikes in a burst. The spatial structure of neural networks was also disturbed that characterized by reduction in both the number of key network elements (hubs) and connections between network elements. Moreover, application of brain-derived neurotrophic factor (BDNF) recombinant protein and BDNF hyperexpression by an adeno-associated virus vector partially prevented these amyloidopathy-induced neurodegenerative phenomena. BDNF maintained cell viability and spontaneous bioelectrical and calcium network activity in primary hippocampal cultures.

Intracellular Neuroprotective Mechanisms in Neuron-Glial Networks Mediated by Glial Cell Line-Derived Neurotrophic Factor.

Glial cell line-derived neurotrophic factor (GDNF) has a pronounced neuroprotective effect in various nervous system pathologies, including ischaemic brain damage and neurodegenerative diseases. In this work, we studied the effect of GDNF on the ultrastructure and functional activity of neuron-glial networks during acute hypoxic exposure, a key damaging factor in numerous brain pathologies. We analysed the molecular mechanisms most likely involved in the positive effects of GDNF. Hypoxia modelling was performed on day 14 of culturing primary hippocampal cells obtained from mouse embryos (E18). GDNF (1 ng/ml) was added to the culture medium 20 min before oxygen deprivation. Acute hypoxia-induced irreversible changes in the ultrastructure of neurons and astrocytes led to the loss of functional Сa2+ activity and neural network disruption. Destructive changes in the mitochondrial apparatus and its functional activity characterized by an increase in the basal oxygen consumption rate and respiratory chain complex II activity during decreased stimulated respiration intensity were observed 24 hours after hypoxic injury. At a concentration of 1 ng/ml, GDNF maintained the functional metabolic network activity in primary hippocampal cultures and preserved the structure of the synaptic apparatus and number of mature chemical synapses, confirming its neuroprotective effect. GDNF maintained the normal structure of mitochondria in neuronal outgrowth but not in the soma. Analysis of the possible GDNF mechanism revealed that RET kinase, a component of the receptor complex, and the PI3K/Akt pathway are crucial for the neuroprotective effect of GDNF. The current study also revealed the role of GDNF in the regulation of HIF-1α transcription factor expression under hypoxic conditions.

AAV-Syn-BDNF-EGFP Virus Construct Exerts Neuroprotective Action on the Hippocampal Neural Network during Hypoxia In Vitro.

Brain-derived neurotrophic factor (BDNF) is one of the key signaling molecules that supports the viability of neural cells in various brain pathologies, and can be considered a potential therapeutic agent. However, several methodological difficulties, such as overcoming the blood⁻brain barrier and the short half-life period, challenge the potential use of BDNF in clinical practice. Gene therapy could overcome these limitations. Investigating the influence of viral vectors on the neural network level is of particular interest because viral overexpression affects different aspects of cell metabolism and interactions between neurons. The present work aimed to investigate the influence of the adeno-associated virus (AAV)-Syn-BDNF-EGFP virus construct on neural network activity parameters in an acute hypobaric hypoxia model in vitro. MATERIALS AND METHODS: An adeno-associated virus vector carrying the BDNF gene was constructed using the following plasmids: AAV-Syn-EGFP, pDP5, DJvector, and pHelper. The developed virus vector was then tested on primary hippocampal cultures obtained from C57BL/6 mouse embryos (E18). Acute hypobaric hypoxia was induced on day 21 in vitro. Spontaneous bioelectrical and calcium activity of neural networks in primary cultures and viability tests were analysed during normoxia and during the posthypoxic period. RESULTS: BDNF overexpression by AAV-Syn-BDNF-EGFP does not affect cell viability or the main parameters of spontaneous bioelectrical activity in normoxia. Application of the developed virus construct partially eliminates the negative hypoxic consequences by preserving cell viability and maintaining spontaneous bioelectrical activity in the cultures. Moreover, the internal functional structure, including the activation pattern of network bursts, the number of hubs, and the number of connections within network elements, is also partially preserved. BDNF overexpression prevents a decrease in the number of cells exhibiting calcium activity and maintains the frequency of calcium oscillations. CONCLUSION: This study revealed the pronounced antihypoxic and neuroprotective effects of AAV-Syn-BDNF-EGFP virus transduction in an acute normobaric hypoxia model.

Effect of brain injury on immunophenotype of peripheral blood lymphocytes in rats.

Strong immunosuppression occurs after severe traumatic brain injury (TBI) and most likely contributes substantially to the patient morbidity and mortality. However, the mechanisms of this immunosuppression are unknown. For the lowering of stressful factors, severe TBI was induced in anaesthetized rats. The lymphocyte subsets from 60 rats with severe TBI were analyzed using monoclonal antibody by the indirect immunofluorescence method. The blood of 30 rats without TBI was used as a control. When compared to the control group, the rats with TBI showed a remarkable reduction in the relative number of CD4(+), RT-Ia(+), Thy-1(+) and ICO-111(+) lymphocytes during the first 2 h after injury. Further reduction in the number of CD4(+) cells was determined in the rats during all the period of the experimental observation. The number of lymphocytes expressing membrane Thy-1 and ICO-111 antigens was significantly decreased 7 days after the trauma. The relative number of RT-Ia(+) lymphocytes was significantly reduced in rats with TBI during 14 days following the trauma. A significant decrease in the luminescence intensity of all the analyzed antigen-positive cells was also observed in rats with TBI. Between the 7th and the 14th days after the trauma a positive correlation between the number of Thy-1(+) PBLs and the number of RT-Ia(+) lymphocytes was determined. Similar results on lymphocyte immunophenotype were seen in patients with TBI. Thus, the cellular immune response is identical in patients and in animals with TBI. Severe brain traumatic injury leads to a reduced expression of cell surface antigens and causes a decrease in the number of antigen-positive lymphocytes and in the intensity of their luminescence.