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During adult rodent life, newborn neurons are added to the olfactory bulb (OB) in a tightly controlled manner. Upon arrival in the OB, input synapses from the local bulbar network and the higher olfactory cortex precede the formation of functional output synapses, indicating a possible role for these regions in newborn neuron survival. An interplay between the environment and the piriform cortex in the regulation of newborn neuron survival has been suggested. However, the specific network and the neuronal cell types responsible for this effect have not been elucidated. Furthermore, the role of the other olfactory cortical areas in this process is not known. Here we demonstrate that pyramidal neurons in the mouse anterior olfactory nucleus, the first cortical area for odor processing, have a key role in the survival of newborn neurons. Using DREADD (Designer Receptors Exclusively Activated by Designer Drugs) technology, we applied chronic stimulation to the anterior olfactory nucleus and observed a decrease in newborn neurons in the OB through induction of apoptosis. These findings provide further insight into the network regulating neuronal survival in adult neurogenesis and strengthen the importance of the surrounding network for sustained integration of new neurons., (© 2021 International Society for Neurochemistry.)

Pathogenic mutations in the leucine-rich repeat kinase 2 (LRRK2) gene belong to the most common genetic causes of inherited Parkinson's disease (PD) and variations in its locus increase the risk to develop sporadic PD. Extensive research efforts aimed at understanding how changes in the LRRK2 function result in molecular alterations that ultimately lead to PD. Cellular LRRK2-based models revealed several potential pathophysiological mechanisms including apoptotic cell death, LRRK2 protein accumulation and deficits in neurite outgrowth. However, highly variable outcomes between different cellular models have been reported. Here, we have investigated the effect of different experimental conditions, such as the use of different tags and gene transfer methods, in various cellular LRRK2 models. Readouts included cell death, sensitivity to oxidative stress, LRRK2 relocalization, α-synuclein aggregation and neurite outgrowth in cell culture, as well as neurite maintenance in vivo. We show that overexpression levels and/or the tag fused to LRRK2 affect the relocalization of LRRK2 to filamentous and skein-like structures. We found that overexpression of LRRK2 per se is not sufficient to induce cellular toxicity or to affect α-synuclein-induced toxicity and aggregate formation. Finally, neurite outgrowth/retraction experiments in cell lines and in vivo revealed that secondary, yet unknown, factors are required for the pathogenic LRRK2 effects on neurite length. Our findings stress the importance of technical and biological factors in LRRK2-induced cellular phenotypes and hence imply that conclusions based on these types of LRRK2-based assays should be interpreted with caution., (© 2020 The Author(s).)

Aggregation of alpha-synuclein (α-SYN) is the pathological hallmark of several diseases named synucleinopathies, including Parkinson's disease (PD), which is the most common neurodegenerative motor disorder. Alpha-SYN has been linked to synaptic function both in physiological and pathological conditions. However, the exact link between neuronal activity, α-SYN toxicity and disease progression in PD is not clear. In this study, we aimed to investigate the effect of chronic neuromodulation in an α-SYN-based rat model for PD using chemogenetics. To do this, we expressed excitatory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) combined with mutant A53T α-SYN, using two different recombinant adeno-associated viral (rAAV) vectors (serotypes 2/7 and 2/8) in rat substantia nigra (SN) and investigated the effect on motor behavior, synapses and neuropathology. We found that chronic neuromodulation aggravates motor deficits induced by α-SYN, without altering dopaminergic neurodegeneration. In addition, neuronal activation led to changes in post-translational modification and subcellular localization of α-SYN, linking neuronal activity to the pathophysiological role of α-SYN in PD.

Adult hippocampal neurogenesis plays a critical role in a wide spectrum of hippocampus-dependent functions. Brain pathologies that involve the hippocampus like epilepsy, stroke, and traumatic brain injury, are commonly associated with cognitive impairments and mood disorders. These insults can affect neural stem cells and the subsequent neurogenic cascade in the hippocampus, resulting in the induction of aberrant neurogenesis, which is thought to compromise hippocampal network function, thereby hampering hippocampus-dependent behavior. We here summarize recent preclinical literature on hippocampal insult-induced changes in neurogenesis and based on that, we propose that normalizing aberrant neurogenesis post-insult may help to prevent or rescue behavioral deficits which could help develop novel therapeutic strategies., (Copyright © 2019. Published by Elsevier B.V.)

Neurons in macaque inferior temporal (IT) cortex show a decrease in the response with stimulus repetition, known as repetition suppression (RS). Several mechanisms may contribute to RS in IT, such as firing rate-dependent fatigue and transsynaptic mechanisms, like synaptic depression or reduced input from neurons within the same area or from up- or downstream areas. We examined the role of firing rate fatigue and transsynaptic mechanisms by stimulating directly IT neurons using optogenetics and measured the effect of photo-stimulation on their responses using timing parameters that resulted in RS for visual stimuli. Photo-stimulation of IT neurons resulted in a marginally decreased probability of spiking activity to a subsequent photo-stimulation or to a subsequent low-contrast visual stimulus. This response reduction was small relative to that for repeated visual stimuli and was related to post-stimulation inhibition of the activity during the interval between adapter and test stimuli. Presentation of a visual adapter did not change the response to subsequent photo-stimulation. In neurons whose response to the visual adapter was inhibited by simultaneous photo-stimulation, RS to visual stimuli was unaffected. Overall, these data imply that RS in IT has a transsynaptic origin, with little or no contribution of intrinsic firing rate fatigue. In addition, they suggest a limited contribution of both local synaptic depression and reduced input from nearby IT neurons, whose responses were postulated to be decreased by firing rate fatigue, to RS in IT., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Glaucoma is characterized by a progressive loss of retinal ganglion cells (RGCs) in the eye, which ultimately results in visual impairment or even blindness. Because current therapies often fail to halt disease progression, there is an unmet need for novel neuroprotective therapies to support RGC survival. Various research lines suggest that visual target centers in the brain support RGC functioning and survival. Here, we explored whether increasing neuronal activity in one of these projection areas could improve survival of RGCs in a mouse glaucoma model. Prolonged activation of an important murine RGC target area, the superior colliculus (SC), was established via a novel optogenetic stimulation paradigm. By leveraging the unique channel kinetics of the stabilized step function opsin (SSFO), protracted stimulation of the SC was achieved with only a brief light pulse. SSFO-mediated collicular stimulation was confirmed by immunohistochemistry for the immediate-early gene c-Fos and behavioral tracking, which both demonstrated consistent neuronal activity upon repeated stimulation. Finally, the neuroprotective potential of optogenetic collicular stimulation was investigated in mice of either sex subjected to a glaucoma model and a 63% reduction in RGC loss was found. This work describes a new paradigm for optogenetic collicular stimulation and a first demonstration that increasing target neuron activity can increase survival of the projecting neurons. SIGNIFICANCE STATEMENT Despite glaucoma being a leading cause of blindness and visual impairment worldwide, no curative therapies exist. This study describes a novel paradigm to reduce retinal ganglion cell (RGC) degeneration underlying glaucoma. Building on previous observations that RGC survival is supported by the target neurons to which they project and using an innovative optogenetic approach, we increased neuronal activity in the mouse superior colliculus, a main projection target of rodent RGCs. This proved to be efficient in reducing RGC loss in a glaucoma model. Our findings establish a new optogenetic paradigm for target stimulation and encourage further exploration of the molecular signaling pathways mediating retrograde neuroprotective communication., (Copyright © 2019 the authors 0270-6474/19/392313-13$15.00/0.)

The enteric nervous system controls a variety of gastrointestinal functions including intestinal motility. The minimal neuronal circuit necessary to direct peristalsis is well-characterized but several intestinal regions display also other motility patterns for which the underlying circuits and connectivity schemes that coordinate the transition between those patterns are poorly understood. We investigated whether in regions with a richer palette of motility patterns, the underlying nerve circuits reflect this complexity. Using Ca 2+ imaging, we determined the location and response fingerprint of large populations of enteric neurons upon focal network stimulation. Complemented by neuronal tracing and volumetric reconstructions of synaptic contacts, this shows that the multifunctional proximal colon requires specific additional circuit components as compared to the distal colon, where peristalsis is the predominant motility pattern. Our study reveals that motility control is hard-wired in the enteric neural networks and that circuit complexity matches the motor pattern portfolio of specific intestinal regions., Competing Interests: ZL, MH, CV, VB, WB, PV No competing interests declared, (© 2019, Li et al.)

Optogenetic manipulations are widely used for investigating the contribution of genetically identified cell types to behavior. Simultaneous electrophysiological recordings are less common, although they are critical for characterizing the specific impact of optogenetic manipulations on neural circuits in vivo. This is at least in part because combining photostimulation with large-scale electrophysiological recordings remains technically challenging, which also poses a limitation for performing extracellular identification experiments. Currently available interfaces that guide light of the appropriate wavelength into the brain combined with an electrophysiological modality suffer from various drawbacks such as a bulky size, low spatial resolution, heat dissipation, or photovoltaic artifacts. To address these challenges, we have designed and fabricated an integrated ultrathin neural interface with 12 optical outputs and 24 electrodes. We used the device to measure the effect of localized stimulation in the anterior olfactory cortex, a paleocortical structure involved in olfactory processing. Our experiments in adult mice demonstrate that because of its small dimensions, our novel tool causes far less tissue damage than commercially available devices. Moreover, optical stimulation and recording can be performed simultaneously, with no measurable electrical artifact during optical stimulation. Importantly, optical stimulation can be confined to small volumes with approximately single-cortical layer thickness. Finally, we find that even highly localized optical stimulation causes inhibition at more distant sites. NEW & NOTEWORTHY In this study, we establish a novel tool for simultaneous extracellular recording and optogenetic photostimulation. Because the device is built using established microchip technology, it can be fabricated with high reproducibility and reliability. We further show that even very localized stimulation affects neural firing far beyond the stimulation site. This demonstrates the difficulty in predicting circuit-level effects of optogenetic manipulations and highlights the importance of closely monitoring neural activity in optogenetic experiments.

Neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD) impose a pressing burden on our developed and consequently aging society. Misfolded protein aggregates are a critical aspect of several neurodegenerative diseases. Nevertheless, several questions remain unanswered regarding the role of misfolded protein aggregates and the cause of neuronal cell death. Recently, it has been postulated that neuroinflammatory processes might play a crucial role in the pathogenesis of PD. Numerous postmortem, brain imaging, epidemiological, and animal studies have documented the involvement of the innate and adaptive immunity in neurodegeneration. Whether these inflammatory processes are directly involved in the etiology of PD or represent secondary consequences of nigrostriatal pathway injury is the subject of intensive research. Immune alterations in response to extracellular α -synuclein may play a critical role in modulating Parkinson's disease progression. In this review, we address the current concept of neuroinflammation and its involvement in PD-associated neurodegeneration.

Novel neuromodulation techniques in the field of brain research, such as optogenetics, prompt to target specific cell populations. However, not every subpopulation can be distinguished based on brain area or activity of specific promoters, but rather on topology and connectivity. A fascinating tool to detect neuronal circuitry is based on the transsynaptic tracer, wheat germ agglutinin (WGA). When expressed in neurons, it is transported throughout the neuron, secreted, and taken up by synaptically connected neurons. Expression of a WGA and Cre recombinase fusion protein using a viral vector technology in Cre-dependent transgenic animals allows to trace neuronal network connections and to induce topological transgene expression. In this study, we applied and evaluated this technology in specific areas throughout the whole rodent brain, including the hippocampus, striatum, substantia nigra, and the motor cortex. Adeno-associated viral vectors (rAAV) encoding the WGA-Cre fusion protein under control of a CMV promoter were stereotactically injected in Rosa26-STOP-EYFP transgenic mice. After 6 weeks, both the number of transneuronally labeled YFP + /mCherry - cells and the transduced YFP + /mCherry + cells were quantified in the connected regions. We were able to trace several connections using WGA-Cre transneuronal labeling; however, the labeling efficacy was region-dependent. The observed transneuronal labeling mostly occurred in the anterograde direction without the occurrence of multi-synaptic labeling. Furthermore, we were able to visualize a specific subset of newborn neurons derived from the subventricular zone based on their connectivity.

Cell assemblies manipulation by optogenetics is pivotal to advance neuroscience and neuroengineering. In in vivo applications, photostimulation often broadly addresses a population of cells simultaneously, leading to feed-forward and to reverberating responses in recurrent microcircuits. The former arise from direct activation of targets downstream, and are straightforward to interpret. The latter are consequence of feedback connectivity and may reflect a variety of time-scales and complex dynamical properties. We investigated wide-field photostimulation in cortical networks in vitro, employing substrate-integrated microelectrode arrays and long-term cultured neuronal networks. We characterized the effect of brief light pulses, while restricting the expression of channelrhodopsin to principal neurons. We evoked robust reverberating responses, oscillating in the physiological gamma frequency range, and found that such a frequency could be reliably manipulated varying the light pulse duration, not its intensity. By pharmacology, mathematical modelling, and intracellular recordings, we conclude that gamma oscillations likely emerge as in vivo from the excitatory-inhibitory interplay and that, unexpectedly, the light stimuli transiently facilitate excitatory synaptic transmission. Of relevance for in vitro models of (dys)functional cortical microcircuitry and in vivo manipulations of cell assemblies, we give for the first time evidence of network-level consequences of the alteration of synaptic physiology by optogenetics.

Malfunctioning of the protein α-synuclein is critically involved in the demise of dopaminergic neurons relevant to Parkinson's disease. Nonetheless, the precise mechanisms explaining this pathogenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triggers DNA degradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminergic neurons of human Parkinson-diseased patients, while EndoG depletion largely reduces α-synuclein-induced cell death in human neuroblastoma cells. Xenogenic expression of human α-synuclein in yeast cells triggers mitochondria-nuclear translocation of EndoG and EndoG-mediated DNA degradation through a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α-synuclein-driven degeneration of dopaminergic neurons. Moreover, the locomotion and survival of α-synuclein-expressing flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phylogenetically conserved pathway that involves EndoG as a critical downstream executor of α-synuclein cytotoxicity.

Adult hippocampal neurogenesis is to a large degree controlled at the level of cell survival, and a number of potential mediators of this effect have been postulated. Here, we investigated the small heat shock protein Hspb8, which, because of its pleiotropic prosurvival effects in other systems, was considered a particularly promising candidate factor. Hspb8 is, for example, found in plaques of Alzheimer disease but exerts neuroprotective effects. We found that expression of Hspb8 increased during differentiation in vitro and was particularly associated with later stages (48-96 h) of differentiation. Gain-of-function and loss-of-function experiments supported the hypothesis that Hspb8 regulates cell survival of new neurons in vitro. In the dentate gyrus of adult mice in vivo, lentiviral overexpression of Hspb8 doubled the surviving cells and concomitantly promoted differentiation and net neurogenesis without affecting precursor cell proliferation. We also discovered that the truncated form of the crystallin domain of Hspb8 was sufficient to affect cell survival and neuronal differentiation in vitro and in vivo. Precursor cell experiments in vitro revealed that Hspb8 increases the phosphorylation of Akt and suggested that the prosurvival effect can be produced by a cell-autonomous mechanism. Analysis of hippocampal Hspb8 expression in mice of 69 strains of the recombinant inbred set BXD revealed that Hspb8 is a cis-acting gene whose expression was associated with clusters of transcript enriched in genes linked to growth factor signaling and apoptosis. Our results strongly suggest that Hspb8 and its α-crystallin domain might act as pleiotropic prosurvival factor in the adult hippocampus.

Neurogenesis has been the subject of active research in recent years and many authors have explored the phenomenology of the process, its regulation and its purported purpose. Recent developments in bioluminescent imaging (BLI) allow direct in vivo imaging of neurogenesis, and in order to interpret the experimental results, mathematical models are necessary. This study proposes such a mathematical model that describes adult mammalian neurogenesis occurring in the subventricular zone and the subsequent migration of cells through the rostral migratory stream to the olfactory bulb (OB). This model assumes that a single chemoattractant is responsible for cell migration, secreted both by the OB and in an endocrine fashion by the cells involved in neurogenesis. The solutions to the system of partial differential equations are compared with the physiological rodent process, as previously documented in the literature and quantified through the use of BLI, and a parameter space is described, the corresponding solution to which matches that of the rodent model. A sensitivity analysis shows that this parameter space is stable to perturbation and furthermore that the system as a whole is sloppy. A large number of parameter sets are stochastically generated, and it is found that parameter spaces corresponding to physiologically plausible solutions generally obey constraints similar to the conditions reported in vivo. This further corroborates the model and its underlying assumptions based on the current understanding of the investigated phenomenon. Concomitantly, this leaves room for further quantitative predictions pertinent to the design of future proposed experiments.

Endogenous neural stem cells have the potential to facilitate therapy for various neurodegenerative brain disorders. To increase our understanding of neural stem and progenitor cell biology in healthy and diseased brain, methods to label and visualize stem cells and their progeny in vivo are indispensable. Iron oxide particle based cell-labeling approaches enable cell tracking by MRI with high resolution and good soft tissue contrast in the brain. However, in addition to important concerns about unspecific labeling and low labeling efficiency, the dilution effect upon cell division is a major drawback for longitudinal follow-up of highly proliferating neural progenitor cells with MRI. Stable viral vector-mediated marking of endogenous stem cells and their progeny with a reporter gene for MRI could overcome these limitations. We stably and efficiently labeled endogenous neural stem/progenitor cells in the subventricular zone in situ by injecting a lentiviral vector expressing ferritin, a reporter for MRI. We developed an image analysis pipeline to quantify MRI signal changes at the level of the olfactory bulb as a result of migration of ferritin-labeled neuroblasts along the rostral migratory stream. We were able to detect ferritin-labeled endogenous neural stem cell progeny into the olfactory bulb of individual animals with ex vivo MRI at 30 weeks post injection, but could not demonstrate reliable in vivo detection and longitudinal tracking of neuroblast migration to the OB in individual animals. Therefore, although LV-mediated labeling of endogenous neural stem and progenitor cells resulted in efficient and stable ferritin-labeling of stem cell progeny in the OB, even with quantitative image analysis, sensitivity remains a limitation for in vivo applications., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The adult rodent brain contains neural progenitor cells (NPCs), generated in the subventricular zone (SVZ), which migrate along the rostral migratory stream (RMS) towards the olfactory bulb (OB) where they differentiate into neurons. The aim of this study was to visualize endogenous NPC migration along the RMS with magnetic resonance imaging (MRI) in adult healthy mice. We evaluated various in situ (in vivo) labeling approaches using micron-sized iron oxide particles (MPIOs) on their efficiency to label endogenous NPCs. In situ labeling and visualization of migrating NPCs were analyzed by a longitudinal MRI study and validated with histology. Here, we visualized endogenous NPC migration in the mouse brain by in vivo MRI and demonstrated accumulation of MPIO-labeled NPCs in the OB over time with ex vivo MRI. Furthermore, we investigated the influence of in situ injection of MPIOs on adult neurogenesis. Quantitative analysis of bromodeoxyuridine labeled cells revealed altered proliferation in the SVZ and NPC migration after in situ MPIO injection. From the labeling strategies presented in this report, intraventricular injection of a small number of MPIOs combined with the transfection agent poly-l-lysine hydrobromide was the best method as labeling of the NPCs was successful and proliferation in the SVZ was only marginally affected. While MRI visualization of endogenous NPC migration can provide insight into aberrant NPC migration in disease models, this work emphasizes the importance to carefully explore the impact on adult neurogenesis when new in situ labeling strategies are developed., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Leucine-rich repeat kinase 2 (LRRK2) functions as a putative protein kinase of ezrin, radixin, and moesin (ERM) family proteins. A Parkinson's disease-related G2019S substitution in the kinase domain of LRRK2 further enhances the phosphorylation of ERM proteins. The phosphorylated ERM (pERM) proteins are restricted to the filopodia of growing neurites in which they tether filamentous actin (F-actin) to the cytoplasmic membrane and regulate the dynamics of filopodia protrusion. Here, we show that, in cultured neurons derived from LRRK2 G2019S transgenic mice, the number of pERM-positive and F-actin-enriched filopodia was significantly increased, and this correlates with the retardation of neurite outgrowth. Conversely, deletion of LRRK2, which lowered the pERM and F-actin contents in filopodia, promoted neurite outgrowth. Furthermore, inhibition of ERM phosphorylation or actin polymerization rescued the G2019S-dependent neuronal growth defects. These data support a model in which the G2019S mutation of LRRK2 causes a gain-of-function effect that perturbs the homeostasis of pERM and F-actin in sprouting neurites critical for neuronal morphogenesis.

Deposition of the amyloid-beta peptide is a pathological hallmark of Alzheimer's disease. A high-throughput functional genomics screen identified G protein-coupled receptor 3 (GPR3), a constitutively active orphan G protein-coupled receptor, as a modulator of amyloid-beta production. Overexpression of GPR3 stimulated amyloid-beta production, whereas genetic ablation of GPR3 prevented accumulation of the amyloid-beta peptide in vitro and in an Alzheimer's disease mouse model. GPR3 expression led to increased formation and cell-surface localization of the mature gamma-secretase complex in the absence of an effect on Notch processing. GPR3 is highly expressed in areas of the normal human brain implicated in Alzheimer's disease and is elevated in the sporadic Alzheimer's disease brain. Thus, GPR3 represents a potential therapeutic target for the treatment of Alzheimer's disease.

It is now generally accepted that continuous neurogenesis occurs in the adult mammalian brain, including that of humans. Modulation of adult neurogenesis can provide therapeutic benefits for various brain disorders, including stroke and Parkinson's disease. The subventricular zone-olfactory bulb pathway is one of the preferred model systems by which to study neural stem cell proliferation, migration, and differentiation in adult rodent brain. Research on adult neurogenesis would greatly benefit from reliable methods for long-term noninvasive in vivo monitoring. We have used lentiviral vectors encoding firefly luciferase to stably mark endogenous neural stem cells in the mouse subventricular zone. We show that bioluminescence imaging (BLI) allows quantitative follow-up of the migration of adult neural stem cells into the olfactory bulb in time. Moreover, we propose a model to fit the kinetic data that allows estimation of migration and survival times of the neural stem cells using in vivo BLI. Long-term expression of brain-derived neurotrophic factor in the subventricular zone attenuated neurogenesis, as detected by histology and BLI. In vivo monitoring of the impact of drugs or genes on adult neurogenesis is now within reach.

Aggregation of alpha-synuclein (alpha-SYN) plays a key role in Parkinson's disease. We have previously shown that aggregation of alpha-SYN in vitro is accelerated by addition of FK506 binding proteins (FKBP) and that this effect can be counteracted by FK506, a specific inhibitor of these enzymes. In this paper, we investigated in detail the effect of FKBP12 on early aggregation and on fibril formation of wild-type, A53T and A30P alpha-SYN. FKBP12 has a much smaller effect on the fibril formation of these two clinical mutants alpha-SYN. Using an inactive enzyme, we were able to discriminate between catalytic and non-catalytic effects that differentially influence the two processes. A model explaining non-linear concentration dependencies is proposed.

Parkinson disease (PD) is a progressive neurodegenerative disorder affecting millions of people worldwide. To date, treatment strategies are mainly symptomatic and aimed at increasing dopamine levels in the degenerating nigrostriatal system. Hope rests upon the development of effective neurorestorative or neuroregenerative therapies based on gene and stem cell therapy or a combination of both. The results of experimental therapies based on transplanting exogenous dopamine-rich fetal cells or glial cell line-derived neurotrophic factor overexpression into the brain of Parkinson disease patients encourage future cell- and gene-based strategies. The endogenous neural stem cells of the adult brain provide an alternative and attractive cell source for neuroregeneration. Prior to designing endogenous stem cell therapies, the possible impact of PD on adult neuronal stem cell pools and their neurogenic potential must be investigated. We review the experimental data obtained in animal models or based on analysis of patients' brains prior to describing different treatment strategies. Strategies aimed at enhancing neuronal stem cell proliferation and/or differentiation in the striatum or the substantia nigra will have to be compared in animal models and selected prior to clinical studies.