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Immunosenescence and chronic inflammation associated with old age accompany brain aging and the loss of complex behaviors. Neuroinflammation in the hippocampus plays a pivotal role in the development of cognitive impairment and anxiety. However, the underlying mechanisms have not been fully explained. In this study, we aimed to investigate the disruption of insulin signaling and the mechanisms underlying metabolic inflammation ("metaflammation") in the brains of wild-type (WT) and NLRP3 knockout (KO) mice of different ages. We found a significant upregulation of the NLRP3 inflammasome in the hippocampus during aging, leading to an increase in the expression of phosphorylated metaflammation proteinases and inflammatory markers, along with an increase in the number of senescent cells. Additionally, metaflammation causes anxiety and impairs social preference behavior in aged mice. On the other hand, deletion of NLRP3 improves some behavioral and biochemical characteristics associated with aging, such as signal memory, neuroinflammation, and metabolic inflammation, but not anxious behavior. These results are associated with reduced IL-18 signaling and the PKR/IKKβ/IRS1 pathway as well as the SASP phenotype. In NLRP3 gene deletion conditions, PKR is down-regulated. Therefore, it is likely that slowing aging through various NLRP3 inhibition mechanisms will lessen the corresponding cognitive decline with aging. Thus, the genetic knockout of the NLRP3 inflammasome can be seen as a new therapeutic strategy for slowing down central nervous system (CNS) aging.

Spinocerebellar ataxia type 1 (SCA1) is an intractable progressive neurodegenerative disease that leads to a range of movement and motor defects and is eventually lethal. Purkinje cells (PC) are typically the first to show signs of degeneration. SCA1 is caused by an expansion of the polyglutamine tract in the ATXN1 gene and the subsequent buildup of mutant Ataxin-1 protein. In addition to its toxicity, mutant Ataxin-1 protein interferes with gene expression and signal transduction in cells. Recently, it is evident that ATXN1 is not only expressed in neurons but also in glia, however, it is unclear the extent to which either contributes to the overall pathology of SCA1. There are various ways to model SCA1 in mice. Here, functional deficits at cerebellar synapses were investigated in two mouse models of SCA1 in which mutant ATXN1 is either nonspecifically expressed in all cell types of the cerebellum (SCA1 knock-in (KI)), or specifically in Bergmann glia with lentiviral vectors expressing mutant ATXN1 under the control of the astrocyte-specific GFAP promoter. We report impairment of motor performance in both SCA1 models. In both cases, prominent signs of astrocytosis were found using immunohistochemistry. Electrophysiological experiments revealed alteration of presynaptic plasticity at synapses between parallel fibers and PCs, and climbing fibers and PCs in SCA1 KI mice, which is not observed in animals expressing mutant ATXN1 solely in Bergmann glia. In contrast, short- and long-term synaptic plasticity was affected in both SCA1 KI mice and glia-targeted SCA1 mice. Thus, non-neuronal mechanisms may underlie some aspects of SCA1 pathology in the cerebellum. By combining the outcomes of our current work with our previous data from the B05 SCA1 model, we further our understanding of the mechanisms of SCA1.

The current prevalence of neurodevelopmental, neurodegenerative diseases, stroke and brain injury stimulates studies aimed to identify new molecular targets, to select the drug candidates, to complete the whole set of preclinical and clinical trials, and to implement new drugs into routine neurological practice. Establishment of protocols based on microfluidics, blood-brain barrier- or neurovascular unit-on-chip, and microphysiological systems allowed improving the barrier characteristics and analyzing the regulation of local microcirculation, angiogenesis, and neurogenesis. Reconstruction of key mechanisms of brain development and even some aspects of experience-driven brain plasticity would be helpful in the establishment of brain in vitro models with the highest degree of reliability. Activity, metabolic status and expression pattern of cells within the models can be effectively assessed with the protocols of system biology, cell imaging, and functional cell analysis. The next generation of in vitro models should demonstrate high scalability, 3D or 4D complexity, possibility to be combined with other tissues or cell types within the microphysiological systems, compatibility with bio-inks or extracellular matrix-like materials, achievement of adequate vascularization, patient-specific characteristics, and opportunity to provide high-content screening. In this review, we will focus on currently available and prospective brain tissue in vitro models suitable for experimental and preclinical studies with the special focus on models enabling 4D reconstruction of brain tissue for the assessment of brain development, brain plasticity, and drug kinetics., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

Background: Alzheimer's disease (AD) is a devastating neurodegenerative disorder. In recent years, attention of researchers has increasingly been focused on studying the role of brain insulin resistance (BIR) in the AD pathogenesis. Neuroinflammation makes a significant contribution to the BIR due to the activation of NLRP3 inflammasome. This study was devoted to the understanding of the potential therapeutic roles of the NLRP3 inflammasome in neurodegeneration occurring concomitant with BIR and its contribution to the progression of emotional disorders., Methods: To test the impact of innate immune signaling on the changes induced by Aβ1-42 injection, we analyzed animals carrying a genetic deletion of the Nlrp3 gene. Thus, we studied the role of NLRP3 inflammasomes in health and neurodegeneration in maintaining brain insulin signaling using behavioral, electrophysiological approaches, immunohistochemistry, ELISA and real-time PCR., Results: We revealed that NLRP3 inflammasomes are required for insulin-dependent glucose transport in the brain and memory consolidation. Conclusions NLRP3 knockout protects mice against the development of BIR: Taken together, our data reveal the protective role of Nlrp3 deletion in the regulation of fear memory and the development of Aβ-induced insulin resistance, providing a novel target for the clinical treatment of this disorder.

Spinocerebellar ataxias are a family of fatal inherited diseases affecting the brain. Although specific mutated proteins are different, they may have a common pathogenetic mechanism, such as insufficient glutamate clearance. This function fails in reactive glia, leading to excitotoxicity and overactivation of NMDA receptors. Therefore, NMDA receptor blockers could be considered for the management of excitotoxicity. One such drug, memantine, currently used for the treatment of Alzheimer's disease, could potentially be used for the treatment of other forms of neurodegeneration, for example, spinocerebellar ataxias (SCA). We previously demonstrated close parallels between optogenetically induced cerebellar degeneration and SCA1. Here we induced reactive transformation of cerebellar Bergmann glia (BG) using this novel optogenetic approach and tested whether memantine could counteract changes in BG and Purkinje cell (PC) morphology and expression of the main glial glutamate transporter-excitatory amino acid transporter 1 (EAAT1). Reactive BG induced by chronic optogenetic stimulation presented increased GFAP immunoreactivity, increased thickness and decreased length of its processes. Oral memantine (~90 mg/kg/day for 4 days) prevented thickening of the processes (1.57 to 1.81 vs. 1.62 μm) and strongly antagonized light-induced reduction in their average length (186.0 to 150.8 vs. 171.9 μm). Memantine also prevented the loss of the key glial glutamate transporter EAAT1 on BG. Finally, memantine reduced the loss of PC (4.2 ± 0.2 to 3.2 ± 0.2 vs. 4.1 ± 0.3 cells per 100 μm of the PC layer). These results identify memantine as potential neuroprotective therapeutics for cerebellar ataxias.

Blood-brain barrier (BBB) is a structural and functional element of the neurovascular unit (NVU), which includes cells of neuronal, glial, and endothelial nature. The main functions of NVU include maintenance of the control of metabolism and chemical homeostasis in the brain tissue, ensuring adequate blood flow in active regions, regulation of neuroplasticity processes, which is realized through intercellular interactions under normal conditions, under stress, in neurodegeneration, neuroinfection, and neurodevelopmental diseases. Current versions of the BBB and NVU models, static and dynamic, have significantly expanded research capabilities, but a number of issues remain unresolved, in particular, personification of the models for a patient. In addition, application of both static and dynamic models has an important problem associated with the difficulty in reproducing pathophysiological mechanisms responsible for the damage of the structural and functional integrity of the barrier in the diseases of the central nervous system. More knowledge on the cellular and molecular mechanisms of BBB and NVU damage in pathology is required to solve this problem. This review discusses current state of the cellular and molecular mechanisms that control BBB permeability, pathobiochemical mechanisms and manifestations of BBB breakdown in stress and neurodegenerative diseases, as well as the problems and prospects of creating in vitro BBB and NVU models for translational studies in neurology and neuropharmacology. Deciphering BBB (patho)physiology will open up new opportunities for further development in the related areas of medicine such as regenerative medicine, neuropharmacology, and neurorehabilitation.

Pathophysiology of chronic neurodegeneration is mainly based on complex mechanisms related to aberrant signal transduction, excitation/inhibition imbalance, excitotoxicity, synaptic dysfunction, oxidative stress, proteotoxicity and protein misfolding, local insulin resistance and metabolic dysfunction, excessive cell death, development of glia-supported neuroinflammation, and failure of neurogenesis. These mechanisms tightly associate with dramatic alterations in the structure and activity of the neurovascular unit (NVU) and the blood-brain barrier (BBB). NVU is an ensemble of brain cells (brain microvessel endothelial cells (BMECs), astrocytes, pericytes, neurons, and microglia) serving for the adjustment of cell-to-cell interactions, metabolic coupling, local microcirculation, and neuronal excitability to the actual needs of the brain. The part of the NVU known as a BBB controls selective access of endogenous and exogenous molecules to the brain tissue and efflux of metabolites to the blood, thereby providing maintenance of brain chemical homeostasis critical for efficient signal transduction and brain plasticity. In Alzheimer's disease, mitochondria are the target organelles for amyloid-induced neurodegeneration and alterations in NVU metabolic coupling or BBB breakdown. In this review we discuss understandings on mitochondria-driven NVU and BBB dysfunction, and how it might be studied in current and prospective NVU/BBB in vitro models for finding new approaches for the efficient pharmacotherapy of Alzheimer's disease.

The dose-dependent effects of plasma exposure to a unipolar nanosecond sliding discharge over the surface of the culture medium in a closed plate on the cells of cerebral endothelium in vitro were studied. Using a 24-well plate, the surface plasma energy density of one pulse was 360 μJ/cm2 at a pulse frequency of 100 Hz. It has been shown that in the creeping discharge plasma there is an active excitation of air molecules, the formation of positive nitrogen and oxygen ions, and the formation of carbon monoxide. In the dose density range of 0-32 J/cm2, the dose-dependent effects were assessed in the 4-12 h post-radiation period. Cell death was analyzed with an assessment of the total number of cells, necrotic cells, cells in apoptosis (phosphatidylserine externalization, internucleosomal DNA fragmentation) and their proliferative activity (Ki67-immunopositive cells). A preliminary assessment of subtle dose-dependent effects indicates the peculiarities of the effect of small doses.

In the central nervous system of mammals, there are specialized areas in which neurogenesis - neurogenic niches - is observed in the postnatal period. It is believed that astrocytes in the composition of neurogenic niches play a significant role in the regulation of neurogenesis, and therefore they are considered as a promising "target" for the possible control of neurogenesis, including the use of optogenetics. In the framework of this work, we formed an in vitro model of a neurogenic niche, consisting of cerebral endothelial cells, astrocytes and neurospheres. Astrocytes in the neurogenic niche model expressed canalorodopsin ChR2 and underwent photoactivation. The effect of photoactivated astrocytes on the expression profile of neurogenic niche cells was evaluated using immunocytochemical analysis methods. It was found that intact astrocytes in the composition of the neurogenic niche contribute to neuronal differentiation of stem cells, as well as the activation of astroglia expressing photosensitive proteins, changes the expression of molecules characterized by intercellular interactions of pools of resting and proliferating cells in the composition of the neurogenic niche with the participation of NAD+ (Cx43, CD38, CD157), lactate (MCT1). In particular, the registered changes reflect a violation of the paracrine intercellular interactions of two subpopulations of cells, one of which acts as a source of NAD+, and the second as a consumer of NAD+ to ensure the processes of intracellular signal transduction; a change in the mechanisms of lactate transport due to aberrant expression of the lactate transporter MCT1 in cells forming a pool of cells developing along the neuronal path of differentiation. In general, with photostimulation of niche astrocytes, the total proliferative activity increases mainly due to neural progenitor cells, but not neural stem cells. Thus, optogenetic activation of astrocytes can become a promising tool for controlling the activity of neurogenesis processes and the formation of a local proneurogenic microenvironment in an in vitro model of a neurogenic niche.

Impairment of hippocampal adult neurogenesis in aging or degenerating brain is a well-known phenomenon caused by the shortage of brain stem cell pool, alterations in the local microenvironment within the neurogenic niches, or deregulation of stem cell development. Environmental enrichment (EE) has been proposed as a potent tool to restore brain functions, to prevent aging-associated neurodegeneration, and to cure neuronal deficits seen in neurodevelopmental and neurodegenerative disorders. Here, we report our data on the effects of environmental enrichment on hippocampal neurogenesis in vivo and neurosphere-forming capacity of hippocampal stem/progenitor cells in vitro. Two models - Alzheimer's type of neurodegeneration and physiological brain aging - were chosen for the comparative analysis of EE effects. We found that environmental enrichment greatly affects the expression of markers specific for stem cells, progenitor cells and differentiated neurons (Pax6, Ngn2, NeuroD1, NeuN) in the hippocampus of young adult rats or rats with Alzheimer's disease (AD) model but less efficiently in aged animals. Application of time-lag mathematical model for the analysis of impedance traces obtained in real-time monitoring of cell proliferation in vitro revealed that EE could restore neurosphere-forming capacity of hippocampal stem/progenitor cells more efficiently in young adult animals (fourfold greater in the control group comparing to the AD model group) but not in the aged rats (no positive effect of environmental enrichment at all). In accordance with the results obtained in vivo , EE was almost ineffective in the recovery of hippocampal neurogenic reserve in vitro in aged, but not in amyloid-treated or young adult, rats. Therefore, EE-based neuroprotective strategies effective in Aβ-affected brain could not be directly extrapolated to aged brain.

Neuroinflammation is a complex inflammatory process in the central nervous system, which is sought to play an important defensive role against various pathogens, toxins or factors that induce neurodegeneration. The onset of neurodegenerative diseases and various microbial infections are counted as stimuli that can challenge the host immune system and trigger the development of neuroinflammation. The homeostatic nature of neuroinflammation is essential to maintain the neuroplasticity. Neuroinflammation is regulated by the activity of neuronal, glial, and endothelial cells within the neurovascular unit, which serves as a "platform" for the coordinated action of pro- and anti-inflammatory mechanisms. Production of inflammatory mediators (cytokines, chemokines, reactive oxygen species) by brain resident cells or cells migrating from the peripheral blood, results in the impairment of blood-brain barrier integrity, thereby further affecting the course of local inflammation. In this review, we analyzed the most recent data on the central nervous system inflammation and focused on major mechanisms of neurovascular unit dysfunction caused by neuroinflammation and infections.

Barriergenesis is the process of maturation of the primary vascular network of the brain responsible for the establishment of the blood-brain barrier. It represents a combination of factors that, on the one hand, contribute to the process of migration and tubulogenesis of endothelial cells (angiogenesis), on the other hand, contribute to the formation of new connections between endothelial cells and other elements of the neurovascular unit. Astrocytes play a key role in barriergenesis, however, mechanisms of their action are still poorly examined. We have studied the effects of HIF-1 modulators acting on the cells of non-endothelial origin (neurons and astrocytes) on the development of the blood-brain barrier in vitro. Application of FM19G11 regulating expression of HIF-1 activity and GSI-1 suppressing gamma-secretase and/or proteasomal activity resulted in the elevated expression of thrombospondins and matrix metalloproteinases in the developing blood-brain barrier. However, it caused the opposite effect on VEGF expression thus promoting barrier maturation in vitro.

The peculiarities in expression of transport proteins and the proteins implicated in the control of glycolysis by the cellular components of neurovascular units were examined in animals of different age under normal conditions and after modeled perinatal stress or hypoxic brain injury. In both cases, the specialties in expression of transport proteins in ontogenesis were revealed. The perinatal hypoxic brain injury resulted in up-regulation of MCT1, MCT4, and GLUT4 expression in endotheliocytes of hippocampal microvessels accompanied by transient elevation of HIF-1α and GSK3 expression.

Metabolic activity of cells within a neurovascular unit is among the factors determining structural and functional integritY of the blood-brain barrier and the an- giogenesis process. in order to verify the hypothesis about the role Of g1YcolYtic activity in the perivascula astroglialcells associated with lactate release in the development of functioning of cerebral microvessel endothelial cells, we have used a three-component model of the brain neurovascular unit in vitro. The cells o f n o n -en d o th elia l o rig in w ere in c u b a te d in th e p rese n ce o f m o d u la to rs o f la c ta te pro d u c n ago ni glu c ose ta a G ly c o s o) , bas t h e oe t a n t a at- blocker of monocarboxylate transporters MCTlprCT and recepltiors of3Ctate0produasan (2-donisyoflactate G e8 breceptor) Iasa estbishe vthat that te suppression of lactate production and transport, prdc o1,adrcpin(C-O-Aa n (2gdoxysgflucoase as a glycolysis inhibitor), transport (phloretin as a sukr of lacaroduto transport , aswellasastimultionof3lactate receptors in astroglial cells, lead to aberrant development of endothelial layer, ther by u g g e tin t h efor atio o f anti ngi gencmi roen ircm ent for cerebral endothelium due to inappropriate lactate-m ediated effects. KeYw.ords:-n-eur-ovascular unit; metabolism; glYcolysis; lactate.

Formation and functional plasticity of the blood-brain barrier is associated with the molecular events that occur in the brain neurovascular unit in the embryonic and early postnatal development. To study the characteristics of barriergenesis under physiological conditions, as well as recovering from perinatal hypoxia and early life stress, we examined the expression of proteins of cerebral endothelial tight junctions (JAM, ZO1, CLDN5) in rats aged 7, 28 and 70 days of postnatal development (P7—P70). Under physiological conditions, we have found that the number of endothelial cells expressing JAM, ZO1, CLDN5 slightly increases in the cortex, hippocampus and amygdala of the brain in the period from P7 to P70. Perinatal hypoxia significantly increased the number of cells expressing proteins of tight junction proteins (JAM, CLDN5) up to the age P28—P70, whereas the number of cells expressing ZO1 was reduced in the same period of time. Early life stress led to an imbalance between the number of cells expressing ZO1 proteins and that expressing tight junctions proteins, but these changes were in opposite direction to that observed in perinatal hypoxia

Glutamine transporter protein SLC1A5 and glutamate transporter protein EAAT2 responsible for cell-cell communication and energetic coupling were studied using in vitro model of multicellular neurovascular unit consisting of astrocytes, neurons, and endotheliocytes under standard conditions and during chemical hypoxia in vitro. Hypoxic damage to the neurovascular unit cells increased the number of SLC1A5-expressing cells and reduced the number of EAAT2-expressing astrocytes. Metabolic uncoupling in the neurovascular unit cells under hypoxic conditions resulted from abnormal expression of glutamine and glutamate transporter proteins, which is indicative of impaired glutamine and glutamate transport.

We studied in vitro development of brain progenitor cells isolated from healthy 7-9-month-old Wistar rats and rats with experimental Alzheimer's disease kept under standard conditions and in enriched (multistimulus) environment in vivo. Progenitor cells from healthy animals more rapidly formed neurospheres. Considerable changes at the early stages of in vitro development of brain progenitor cells were observed in both groups kept in enriched environment.

Suspension of magnetic nanoparticles (0.7 g/liter) obtained from Klebsiella oxytoca culture was injected intraperitoneally (1 ml), intramuscularly (in the hip; 100 μl), and subcutaneously (200 μl) or administered orally instead of drinking water for 2 days. The presence of magnetic nanoparticles was evaluated detected by MRI in 15 min and 2 h after injections and in 1 and 2 days after the beginning of oral consumption of the suspension. Magnetic nanoparticles were eliminated from the site of intramuscular and intraperitoneal injections and after oral consumption. The period of elimination after intramuscular and intraperitoneal injections did not exceed 2 h, while after oral consumption it corresponded to the time of gastrointestinal tract contents evacuation.

There are many ways to model blood brain barrier and neurovascular unit in vitro. All existing models have their disadvantages, advantages and some peculiarities of preparation and usage. We obtained the three-cells neurovascular unit model in vitro using progenitor cells isolated from the rat embryos brain (Wistar, 14-16 d). After withdrawal of the progenitor cells the neurospheres were cultured with subsequent differentiation into astrocytes and neurons. Endothelial cells were isolated from embryonic brain too. During the differentiation of progenitor cells the astrocytes monolayer formation occurs after 7-9 d, neurons monolayer--after 10-14 d, endothelial cells monolayer--after 7 d. Our protocol for simultaneous isolation and cultivation of neurons, astrocytes and endothelial cells reduces the time needed to obtain neurovascular unit model in vitro, consisting of three cells types and reduce the number of animals used. It is also important to note the cerebral origin of all cell types, which is also an advantage of our model in vitro.

Perinatal hypoxic-ischemic brain injury is a relevant medical and social problem. Among many pathological processes in the neonatal period perinatal hypoxic-ischemic injury is a major cause of further hemorrhage, necrotic and atrophic changes in the brain. This review presents recent data on the basic mechanisms of the hypoxic-ischemic brain injury along the concept of neurovascular unit (neurons, astrocytes, endothelial cells, pericytes) with the focus on alterations in cell-to-cell communication. Pathological changes caused by ischemia-hypoxia are considered within two phases of injury (ischemic phase and reperfusion phase). The review highlights changes in each individual component of the neurovascular unit and their interactions. Molecular targets for pharmacological improvement of intercellular communication within neurovascular unit as a therapeutic strategy in perinatal brain injury are discussed.