Adult neural stem cell activation in mice is regulated by the day/night cycle and intracellular calcium dynamics.

Neural stem cells (NSCs) in the adult brain transit from the quiescent state to proliferation to produce new neurons. The mechanisms regulating this transition in freely behaving animals are, however, poorly understood. We customized in vivo imaging protocols to follow NSCs for several days up to months, observing their activation kinetics in freely behaving mice. Strikingly, NSC division is more frequent during daylight and is inhibited by darkness-induced melatonin signaling. The inhibition of melatonin receptors affected intracellular Ca2+ dynamics and promoted NSC activation. We further discovered a Ca2+ signature of quiescent versus activated NSCs and showed that several microenvironmental signals converge on intracellular Ca2+ pathways to regulate NSC quiescence and activation. In vivo NSC-specific optogenetic modulation of Ca2+ fluxes to mimic quiescent-state-like Ca2+ dynamics in freely behaving mice blocked NSC activation and maintained their quiescence, pointing to the regulatory mechanisms mediating NSC activation in freely behaving animals.

A light-inducible protein clustering system for in vivo analysis of α-synuclein aggregation in Parkinson disease.

Neurodegenerative disorders refer to a group of diseases commonly associated with abnormal protein accumulation and aggregation in the central nervous system. However, the exact role of protein aggregation in the pathophysiology of these disorders remains unclear. This gap in knowledge is due to the lack of experimental models that allow for the spatiotemporal control of protein aggregation, and the investigation of early dynamic events associated with inclusion formation. Here, we report on the development of a light-inducible protein aggregation (LIPA) system that enables spatiotemporal control of α-synuclein (α-syn) aggregation into insoluble deposits called Lewy bodies (LBs), the pathological hallmark of Parkinson disease (PD) and other proteinopathies. We demonstrate that LIPA-α-syn inclusions mimic key biochemical, biophysical, and ultrastructural features of authentic LBs observed in PD-diseased brains. In vivo, LIPA-α-syn aggregates compromise nigrostriatal transmission, induce neurodegeneration and PD-like motor impairments. Collectively, our findings provide a new tool for the generation, visualization, and dissection of the role of α-syn aggregation in PD.

In vivo live imaging of postnatal neural stem cells.

Neural stem cells (NSCs) are maintained in specific regions of the postnatal brain and contribute to its structural and functional plasticity. However, the long-term renewal potential of NSCs and their mode of division remain elusive. The use of advanced in vivo live imaging approaches may expand our knowledge of NSC physiology and provide new information for cell replacement therapies. In this Review, we discuss the in vivo imaging methods used to study NSC dynamics and recent live-imaging results with respect to specific intracellular pathways that allow NSCs to integrate and decode different micro-environmental signals. Lastly, we discuss future directions that may provide answers to unresolved questions regarding NSC physiology.

SARS-CoV-2 deregulates the vascular and immune functions of brain pericytes via Spike protein.

Coronavirus disease 19 (COVID-19) is a respiratory illness caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). COVID-19 pathogenesis causes vascular-mediated neurological disorders via elusive mechanisms. SARS-CoV-2 infects host cells via the binding of viral Spike (S) protein to transmembrane receptor, angiotensin-converting enzyme 2 (ACE2). Although brain pericytes were recently shown to abundantly express ACE2 at the neurovascular interface, their response to SARS-CoV-2 S protein is still to be elucidated. Using cell-based assays, we found that ACE2 expression in human brain vascular pericytes was increased upon S protein exposure. Pericytes exposed to S protein underwent profound phenotypic changes associated with an elongated and contracted morphology accompanied with an enhanced expression of contractile and myofibrogenic proteins, such as α-smooth muscle actin (α-SMA), fibronectin, collagen I, and neurogenic locus notch homolog protein-3 (NOTCH3). On the functional level, S protein exposure promoted the acquisition of calcium (Ca2+) signature of contractile ensheathing pericytes characterized by highly regular oscillatory Ca2+ fluctuations. Furthermore, S protein induced lipid peroxidation, oxidative and nitrosative stress in pericytes as well as triggered an immune reaction translated by activation of nuclear factor-kappa-B (NF-κB) signaling pathway, which was potentiated by hypoxia, a condition associated with vascular comorbidities that exacerbate COVID-19 pathogenesis. S protein exposure combined to hypoxia enhanced the production of pro-inflammatory cytokines involved in immune cell activation and trafficking, namely macrophage migration inhibitory factor (MIF). Using transgenic mice expressing the human ACE2 that recognizes S protein, we observed that the intranasal infection with SARS-CoV-2 rapidly induced hypoxic/ischemic-like pericyte reactivity in the brain of transgenic mice, accompanied with an increased vascular expression of ACE2. Moreover, we found that SARS-CoV-2 S protein accumulated in the intranasal cavity reached the brain of mice in which the nasal mucosa is deregulated. Collectively, these findings suggest that SARS-CoV-2 S protein impairs the vascular and immune regulatory functions of brain pericytes, which may account for vascular-mediated brain damage. Our study provides a better understanding for the mechanisms underlying cerebrovascular disorders in COVID-19, paving the way to develop new therapeutic interventions.

Heavy metals pollution levels and children health risk assessment of Yerevan kindergartens soils.

Children, the most vulnerable urban population group, are exceptionally sensitive to polluted environments, particularly urban soils, which can lead to adverse health effects upon exposure. In this study, the total concentrations of Ag, As, Ba, Cd, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Ti, V, and Zn were determined in 111 topsoil samples collected from kindergartens in Yerevan. The objectives of this study were to evaluate heavy metal pollution levels of kindergarten's soils in Yerevan, compare with national legal and international requirements on heavy metal contents in kindergarten soil, and assess related child health risk. Multivariate geostatistical analyses suggested that the concentrations of Ag, As, Ba, Cd, Cu, Hg, Mo, Pb, and Zn observed in the kindergarten's topsoil may have originated from anthropogenic sources, while Co, Cr, Fe, Mn, Ni, Ti, and V mostly come from natural sources. According to the Summary pollution index (Zc), 102 kindergartens belong to the low pollution level, 7 to the moderate and only 2 to the high level of pollution. Summary concentration index (SCI) showed that 109 kindergartens were in the allowable level, while 2 featured in the low level of pollution. The health risk assessment showed that in all kindergartens except for seven, non-carcinogenic risk for children was detected (HI>1), while carcinogenic risk from arsenic belongs to the very low (allowable) level. Cr and multi-element carcinogenic risk (RI) exceeded the safety level (1.0E- 06) in all kindergartens and showed that the potential of developing cancer, albeit small, does exist. Therefore, city's kindergartens require necessary remedial actions to eliminate or reduce soil pollution and heavy metal-induced health risks.

The Role of Adult-Born Neurons in the Constantly Changing Olfactory Bulb Network.

The adult mammalian brain is remarkably plastic and constantly undergoes structurofunctional modifications in response to environmental stimuli. In many regions plasticity is manifested by modifications in the efficacy of existing synaptic connections or synapse formation and elimination. In a few regions, however, plasticity is brought by the addition of new neurons that integrate into established neuronal networks. This type of neuronal plasticity is particularly prominent in the olfactory bulb (OB) where thousands of neuronal progenitors are produced on a daily basis in the subventricular zone (SVZ) and migrate along the rostral migratory stream (RMS) towards the OB. In the OB, these neuronal precursors differentiate into local interneurons, mature, and functionally integrate into the bulbar network by establishing output synapses with principal neurons. Despite continuous progress, it is still not well understood how normal functioning of the OB is preserved in the constantly remodelling bulbar network and what role adult-born neurons play in odor behaviour. In this review we will discuss different levels of morphofunctional plasticity effected by adult-born neurons and their functional role in the adult OB and also highlight the possibility that different subpopulations of adult-born cells may fulfill distinct functions in the OB neuronal network and odor behaviour.

Activity of the principal cells of the olfactory bulb promotes a structural dynamic on the distal dendrites of immature adult-born granule cells via activation of NMDA receptors.

The adult olfactory bulb is continuously supplied with neuronal precursors that differentiate into granule and periglomerular cells. Little is known about the structural dynamic of adult-born granule cells (GCs) at their different maturational stages, the mechanisms controlling the integration of new neurons into the pre-existing neuronal circuitry, or the role of principal cell activity in these processes. We used two-photon time-lapse imaging to reveal a high level of filopodia formation and retraction on the distal dendrites of adult-born GCs at their early maturational stages. This dynamic decreased as the adult-born interneurons matured. Filopodia formation/retraction on the dendrites of adult-born GCs at the early maturational stages depended on the activation of NMDA receptors (NMDARs). The stimulation of mitral cells using a pattern that mimics activity of these principal neurons to odor presentation promotes the NMDAR-dependent filopodia dynamic of adult-born GCs during their early but not late maturational stages. Moreover, NMDA iontophoresis was sufficient to induce the formation of new filopodia on the distal dendrites of immature adult-born GCs. The maturation of adult-born interneurons was accompanied by a progressive hyperpolarization of the membrane potential and an increased Mg(2+) block of NMDARs. Decreasing the extracellular Mg(2+) concentration led to filopodia formation on the dendrites of mature adult-born GCs following NMDA iontophoresis. Our findings reveal an increased structural dynamic of adult-born GCs during the early stages of their integration into the mouse bulbar circuitry and highlight a critical period during which the principal cells' activity influences filopodia formation/retraction on the dendrites of interneurons.

The extracellular matrix glycoprotein tenascin-R affects adult but not developmental neurogenesis in the olfactory bulb.

Neuronal precursors produced in the subventricular zone throughout an animal's life migrate tangentially along the rostral migratory stream and, once in the olfactory bulb (OB), turn to migrate radially to the bulbar layers, where they differentiate into interneurons. Despite extensive investigations, it has remained largely unknown whether the same molecular mechanisms control OB neurogenesis during early postnatal development and in adulthood. In this study, we show that the extracellular matrix glycoprotein tenascin-R (TNR) is produced in the granule cell layer of the OB and that its expression increases during postnatal development. Time-lapse video imaging and morphological analyses revealed that a lack of TNR decreases the radial migration of neuronal precursors in the adult, but not in the developing OB. A lack of TNR also reduces spine development of newborn neurons in adult mice. To understand the functional consequences of a lack of TNR, we performed electrophysiological and behavioral studies on young and adult mice. Electrophysiological recordings showed that mitral cells, the target cells of newly generated interneurons, receive reduced spontaneous and evoked inhibitory activity in adult, but not young, TNR knock-out mice. Moreover, the synchronized activity of mitral cells was decreased in the OB of adult TNR knock-out mice. Behavioral studies revealed that the lower numbers of newborn interneurons in the adult OB induce alterations in short-term odor memory. Our results indicate that TNR modulates adult but not developmental neurogenesis in the OB and also highlight that the regulation of OB neurogenesis can vary during an animal's lifetime.

Distinct forms of structural plasticity of adult-born interneuron spines in the mouse olfactory bulb induced by different odor learning paradigms.

The morpho-functional properties of neural networks constantly adapt in response to environmental stimuli. The olfactory bulb is particularly prone to constant reshaping of neural networks because of ongoing neurogenesis. It remains unclear whether the complexity of distinct odor-induced learning paradigms and sensory stimulation induces different forms of structural plasticity. In the present study, we automatically reconstructed spines in 3D from confocal images and performed unsupervised clustering based on morphometric features. We show that while sensory deprivation decreased the spine density of adult-born neurons without affecting the morphometric properties of these spines, simple and complex odor learning paradigms triggered distinct forms of structural plasticity. A simple odor learning task affected the morphometric properties of the spines, whereas a complex odor learning task induced changes in spine density. Our work reveals distinct forms of structural plasticity in the olfactory bulb tailored to the complexity of odor-learning paradigms and sensory inputs.

Astrocytes control the development of the migration-promoting vasculature scaffold in the postnatal brain via VEGF signaling.

New neurons are constantly being generated in the postnatal subventricular zone. They have to migrate long distances via the rostral migratory stream (RMS) to reach their final destination in the olfactory bulb (OB). In adults, these neuronal precursors migrate in chains, ensheathed by astrocytic processes, and travel toward the OB along blood vessels (BVs) that topographically outline the RMS. The molecular and cellular mechanisms leading to the development of the RMS and the formation of the migration-promoting vasculature scaffold in the adult mice remain unclear. We now reveal that astrocytes orchestrate the formation and structural reorganization of the vasculature scaffold in the RMS and, during early developmental stages, the RMS contains only a few BVs oriented randomly with respect to the migrating neuroblasts. The first parallel BVs appeared at the outer border of the RMS, where vascular endothelial growth factor (VEGF)-expressing astrocytes are located. Gain-of-function and loss-of-function experiments revealed that astrocyte-derived VEGF plays a crucial role in the formation and growth of new BVs. Real-time videoimaging also showed that the migration of neuronal precursors in the developing RMS differs substantially from neuronal displacement in the adult migratory stream partially because of not yet fully developed vasculature scaffold. The downregulation of VEGF in vivo, specifically in the astrocytes of the developing RMS, affected the development of the vasculature scaffold and led to alterations in neuroblast migration. Altogether, our results demonstrate that astrocytes orchestrate the formation and growth of parallel BVs, crucial migration-promoting scaffolds in the adult migratory stream, via VEGF signaling.

Calcium signaling as an integrator and decoder of niche factors to control somatic stem cell quiescence and activation.

Somatic stem cells (SSCs) play a major role in tissue homeostasis and respond to a panoply of micro-environmental cues by adjusting their quiescence and activation profiles. How these cells integrate and decode multiple niche signals remains elusive. In recent years, Ca2+ signaling has emerged as one of the key intracellular pathways that allow stem cells to dynamically adjust their fate and either to remain quiescent for future needs or to become activated to generate new progeny. Interestingly, not only distinct Ca2+ signatures are associated with the quiescence and activation states of stem cells, but also various extracellular cues impinge on Ca2+ pathways to dynamically regulate the responses of stem cells to different niche signals. This Viewpoint article deals with how Ca2+ signaling may be used to decode and integrate different niche factors and how Ca2+ fluctuations of distinct amplitudes, frequencies, and overall intracellular levels may trigger the differential gene transcription program. Knowledge about mechanisms that allow SSCs to translate the complexity of extracellular niche signaling into intrinsic states of cell quiescence and activation is crucial for understanding life-long tissue homeostasis and regeneration.

Metformin rescues migratory deficits of cells derived from patients with periventricular heterotopia.

Periventricular neuronal heterotopia (PH) is one of the most common forms of cortical malformation in the human cortex. We show that human neuronal progenitor cells (hNPCs) derived from PH patients with a DCHS1 or FAT4 mutation as well as isogenic lines had altered migratory dynamics when grafted in the mouse brain. The affected migration was linked to altered autophagy as observed in vivo with an electron microscopic analysis of grafted hNPCs, a Western blot analysis of cortical organoids, and time-lapse imaging of hNPCs in the presence of bafilomycin A1. We further show that deficits in autophagy resulted in the accumulation of paxillin, a focal adhesion protein involved in cell migration. Strikingly, a single-cell RNA-seq analysis of hNPCs revealed similar expression levels of autophagy-related genes. Bolstering AMPK-dependent autophagy by metformin, an FDA-approved drug, promoted migration of PH patients-derived hNPCs. Our data indicate that transcription-independent homeostatic modifications in autophagy contributed to the defective migratory behavior of hNPCs in vivo and suggest that modulating autophagy in hNPCs might rescue neuronal migration deficits in some forms of PH.

Deciphering heterogeneous populations of migrating cells based on the computational assessment of their dynamic properties.

Neuronal migration is a highly dynamic process, and multiple cell movement metrics can be extracted from time-lapse imaging datasets. However, these parameters alone are often insufficient to evaluate the heterogeneity of neuroblast populations. We developed an analytical pipeline based on reducing the dimensions of the dataset by principal component analysis (PCA) and determining sub-populations using k-means, supported by the elbow criterion method and validated by a decision tree algorithm. We showed that neuroblasts derived from the same adult neural stem cell (NSC) lineage as well as across different lineages are heterogeneous and can be sub-divided into different clusters based on their dynamic properties. Interestingly, we also observed overlapping clusters for neuroblasts derived from different NSC lineages. We further showed that genetic perturbations or environmental stimuli affect the migratory properties of neuroblasts in a sub-cluster-specific manner. Our data thus provide a framework for assessing the heterogeneity of migrating neuroblasts.

Role of blood vessels in the neuronal migration.

The proper development and functioning of the vertebrate brain depends on the correct positioning of neuronal precursors which is achieved by the widespread and far-ranging migration of cells from their birthplaces. The vast majority of neuronal precursors use cellular substrates for their migration. Until very recently, it was assumed that these cellular substrates were either glial (glia-mediated or gliophilic migration) or neuronal (neuron-mediated or neurophilic migration) in nature. The widely studied examples of gliophilic and neurophilic migrations in the developing brain are displacement of neuronal precursors along the processes of radial glia in the developing cortex and migration of neurons expressing gonadotropin-releasing hormone (GnRH) along the vomeronasal axons, respectively. Recent data indicate, however, that neuronal precursors might also use blood vessels as a physical substrate for their migration. The vasculature-guided (vasophilic) migration of neuronal precursors has been observed not only under normal conditions, in the healthy brain, but also following strokes. The purpose of this review is to highlight emerging principles and delineate putative mechanisms of vasculature-guided neuronal migration under both normal and pathological conditions.

AMPK-induced autophagy as a key regulator of cell migration.

Cell migration is a highly dynamic and energy-intensive process that ensures the correct targeting of cells during embryonic and postnatal development. In recent work, we highlighted the importance of macroautophagy/autophagy in regulating the dynamics of cell migration under baseline conditions and in response to a diverse set of molecular factors. Genetic suppression of autophagy-related genes induced longer stationary phases in migrating cells and cell stalling at the beginning of the migratory stream. We also showed that autophagy is required for recycling of the focal adhesion molecule PXN (paxillin), and is induced by energy levels of cells via AMPK activation. This recent study revealed the importance of autophagy in the maintenance of cell migration, and showed that the dynamic interplay between autophagy and energy levels is required to sustain neuronal migration and to cope with diverse micro-environmental factors.

Live imaging of adult neural stem cells in freely behaving mice using mini-endoscopes.

During adulthood, the activation of adult neural stem cells (NSCs) has been mostly studied ex vivo in post-mortem tissues or in vivo in anesthetized animals. This protocol presents an approach that allows for the long-term and minimally invasive investigation of adult NSC activation and physiology in freely behaving animals. By combining specific NSC labeling and mini-endoscopic microscopy, live imaging of NSC division and Ca2+ activity can be performed continuously for 2-3 days and even up to several months. For complete details on the use and execution of this protocol, please refer to Gengatharan et al. (2021).

Identification of spatial patterns, geochemical associations and assessment of origin-specific health risk of potentially toxic elements in soils of Armavir region, Armenia.

The study of soil potentially toxic elements (PTE) contents and establishment of the geochemical characterization of areas which have never been studied is of great concern. In 2019, soil survey of the Armavir region (Armenia) was conducted in order to investigate the spatial pattern of PTE, reveal PTE geochemical associations and assess the origin-specific health risks. The application of compositional data analysis and geospatial mapping allowed to identify two clusters of samples. The first cluster was spatially located on volcanic rocks and was represented by Fe, Co, Mn, Ti, Zn, Ba, Pb suggesting a natural origin of PTE in these areas. The second cluster was allocated on the alluvial, deluvial, and proluvial sediments and represented by As, Cu, Cr, Ni. Such combination of elements in the same group indicates the anthropogenic introduction of some quantities of PTE. The latter is confirmed by the presence of outliers and extreme values for As, Cu and Ni, as well as by the spatial colocation of Fe, Mn, Co, Pb, Zn outliers and extreme contents. The health risk assessment showed that for children the multi-elemental non-carcinogenic risk was detected, while for the adults the non-carcinogenic risk and carcinogenic risk were below the allowable level. The detailed study of the risk levels showed that in first cluster comparatively higher risk were observed for Pb, V, Ba, Zn while in the second cluster: Fe, Co, Mn, As, Cr, Cu, Ni. The results indicated the necessity of additional in-depth studies with special focus on bioavailability of PTE.

Yerevan soil radioactivity: Radiological and geochemical assessment.

Spatial pattern of naturally occurring radionuclides (NOR): 226Ra, 232Th, 40K, and artificial 137Cs was studied using soil samples of the multipurpose geochemical survey of the city of Yerevan, capital of Armenia. High purity Ge detector-based gamma spectrometry system was used for the determination of radionuclides activity concentrations in urban soils. A combination of compositional data analysis, geochemical mapping and radiological assessment were applied to reveal potential factors of technologically enhanced natural radioactivity and excess lifetime cancer risk for Yerevan's population due to NOR and artificial 137Cs in the urban environment. Statistical methods with the geochemical mapping revealed the great contribution of soil-forming rocks to NOR distribution in urban soils. The spatial distribution of calculated radiological indices and dose rates levels follows the distribution patterns of NOR. The activity concentration of fallout radionuclide 137Cs was within the range typical for the studied altitudes. Above baseline activity of 137Cs was observed in the north-western and western part of the city that is in typical ranges of 137Cs content in soil derived from global radioactive fallout. Urban soils of Yerevan were found radiologically safe, however, igneous rock derived soils are a sink of NOR and the main environmental source of continuous exposure to the residents. Values of excess lifetime cancer risk were higher than mean global value.

Sensitive period for rescuing parvalbumin interneurons connectivity and social behavior deficits caused by TSC1 loss.

The Mechanistic Target Of Rapamycin Complex 1 (mTORC1) pathway controls several aspects of neuronal development. Mutations in regulators of mTORC1, such as Tsc1 and Tsc2, lead to neurodevelopmental disorders associated with autism, intellectual disabilities and epilepsy. The correct development of inhibitory interneurons is crucial for functional circuits. In particular, the axonal arborisation and synapse density of parvalbumin (PV)-positive GABAergic interneurons change in the postnatal brain. How and whether mTORC1 signaling affects PV cell development is unknown. Here, we show that Tsc1 haploinsufficiency causes a premature increase in terminal axonal branching and bouton density formed by mutant PV cells, followed by a loss of perisomatic innervation in adult mice. PV cell-restricted Tsc1 haploinsufficient and knockout mice show deficits in social behavior. Finally, we identify a sensitive period during the third postnatal week during which treatment with the mTOR inhibitor Rapamycin rescues deficits in both PV cell innervation and social behavior in adult conditional haploinsufficient mice. Our findings reveal a role of mTORC1 signaling in the regulation of the developmental time course and maintenance of cortical PV cell connectivity and support a mechanistic basis for the targeted rescue of autism-related behaviors in disorders associated with deregulated mTORC1 signaling.

Interneurons produced in adulthood are required for the normal functioning of the olfactory bulb network and for the execution of selected olfactory behaviors.

Olfactory bulb (OB) interneurons are continuously renewed throughout an animal's lifespan. Despite extensive investigation of this phenomenon, little is known about bulbar circuitry functioning and olfactory performances under conditions of ablated arrival of new neurons into the adult OB. To address this issue we performed morphological, electrophysiological, and behavioral analysis in mice with suppressed bulbar neurogenesis. Infusion of the antimitotic drug AraC to the lateral ventricle via 28 d osmotic minipumps abolished the arrival of newly born neurons into the adult OB without affecting the total number of granule cells. The number, dendritic arborization, and spine density of interneurons generated in adulthood, before pump installation, were also not affected by AraC treatment. As a result of ablated neurogenesis, mitral cells--the principal output neurons in the OB--receive fewer inhibitory synapses, display reduced frequency of spontaneous IPSCs, experience smaller dendrodendritic inhibition, and exhibit decreased synchronized activity. Consequently, short-term olfactory memory was drastically reduced in AraC-treated mice. In contrast, olfactory performances of AraC-treated animals were undistinguishable from those of control mice in other odor-associated tests, such as spontaneous odor discrimination and long-term odor-associative memory tasks. Altogether, our data highlight the importance of adult neurogenesis for the proper functioning of the OB network and imply that new bulbar interneurons are involved in some, but not all, odor-associated tasks.