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

peer reviewedAlzheimer'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 (Aβ) 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 (Aβ1-42). 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 Ca(2+) activity, a decrease in the number of Ca(2+) oscillations and an increase in the duration of Ca(2+) events from day 21 of culture development in vitro. Chronic application of Aβ1-42 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 Aβ 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

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

peer reviewedThe 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

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

peer reviewedCurrently, 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

Novel Algorithm of Network Calcium Dynamics Analysis for Studying the Role of Astrocytes in Neuronal Activity in Alzheimer’s Disease Models

Accumulated experimental data strongly suggest that astrocytes play an important role in the pathogenesis of neurodegeneration, including Alzheimer’s disease (AD). The effect of astrocytes on the calcium activity of neuron–astroglia networks in AD modelling was the object of the present study. We have expanded and improved our approach’s capabilities to analyze calcium activity. We have developed a novel algorithm to construct dynamic directed graphs of both astrocytic and neuronal networks. The proposed algorithm allows us not only to identify functional relationships between cells and determine the presence of network activity, but also to characterize the spread of the calcium signal from cell to cell. Our study showed that Alzheimer’s astrocytes can change the functional pattern of the calcium activity of healthy nerve cells. When healthy nerve cells were cocultivated with astrocytes treated with Aβ42, activation of calcium signaling was found. When healthy nerve cells were cocultivated with 5xFAD astrocytes, inhibition of calcium signaling was observed. In this regard, it seems relevant to further study astrocytic–neuronal interactions as an important factor in the regulation of the functional activity of brain cells during neurodegenerative processes. The approach to the analysis of streaming imaging data developed by the authors is a promising tool for studying the collective calcium dynamics of nerve cells

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

The Dynamics of β-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 β-amyloid (Aβ) 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 Aβ, and specific Aβ proteoforms (e.g., Aβ with isomerized D7 (isoD7-Aβ)) 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 Aβ 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 Aβ fraction of isoD7-Aβ, 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 Aβ proteoforms correlate with the formation of Aβ 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-Aβ and the progression of AD-like pathology