Neuronal Machinery Research Articles

Endo-IP and lyso-IP toolkit for endolysosomal profiling of human-induced neurons

Plasma membrane protein degradation and recycling are regulated by the endolysosomal system, wherein endocytic vesicles bud from the plasma membrane into the cytoplasm and mature into endosomes and then degradative lysosomes. As such, the endolysosomal system plays a critical role in determining the abundance of proteins on the cell surface and influencing cellular identity and function. Highly polarized cells, like neurons, rely on the endolysosomal system for axonal and dendritic specialization and synaptic compartmentalization. The importance of this system to neuronal function is reflected by the prevalence of risk variants in components of the system in several neurodegenerative diseases, ranging from Parkinson's to Alzheimer's disease. Nevertheless, our understanding of endocytic cargo and core endolysosomal machinery in neurons is limited, in part due to technical limitations. Here, we develop a toolkit for capturing EEA1-positive endosomes (termed Endo-IP) and TMEM192-positive lysosomes (termed Lyso-IP) in stem cell-derived induced neurons (iNeurons). We demonstrate its utility by revealing the endolysosomal protein landscapes for stem cells and cortical-like iNeurons, and profiling endosomes in response to potassium-mediated neuronal depolarization. Through global profiling of endocytic cargo, we identify hundreds of transmembrane proteins, including neurogenesis and synaptic proteins, as well as endocytic cargo with predicted SNX17 or SNX27 recognition motifs. By contrast, parallel lysosome profiling reveals a simpler protein repertoire, reflecting in part temporally controlled recycling or degradation for many endocytic targets. This system will facilitate mechanistic interrogation of endolysosomal components found as risk factors in neurodegenerative disease.

Regulation of leptin signaling and diet-induced obesity by SEL1L-HRD1 ER-associated degradation in POMC expressing neurons

Endoplasmic reticulum (ER) homeostasis in the hypothalamus has been implicated in the pathogenesis of diet-induced obesity (DIO) and type 2 diabetes; however, the underlying molecular mechanism remain vague and debatable. Here we report that SEL1L-HRD1 protein complex of the highly conserved ER-associated protein degradation (ERAD) machinery in POMC-expressing neurons ameliorates diet-induced obesity and its associated complications, partly by regulating the turnover of the long isoform of Leptin receptors (LepRb). Loss of SEL1L in POMC-expressing neurons attenuates leptin signaling and predisposes mice to HFD-associated pathologies including fatty liver, glucose intolerance, insulin and leptin resistance. Mechanistically, nascent LepRb, both wildtype and disease-associated Cys604Ser variant, are misfolding prone and bona fide substrates of SEL1L-HRD1 ERAD. In the absence of SEL1L-HRD1 ERAD, LepRb are largely retained in the ER, in an ER stress-independent manner. This study uncovers an important role of SEL1L-HRD1 ERAD in the pathogenesis of central leptin resistance and leptin signaling.

Endo-IP and Lyso-IP Toolkit for Endolysosomal Profiling of Human Induced Neurons.

Plasma membrane protein degradation and recycling is regulated by the endolysosomal system, wherein endosomes bud from the plasma membrane into the cytosol and mature into degradative lysosomes. As such, the endolysosomal system plays a critical role in determining the abundance of proteins on the cell surface, influencing cellular identity and function. Highly polarized cells, like neurons, rely on the endolysosomal system for axonal and dendritic specialization and synaptic compartmentalization. The importance of this system to neuronal function is reflected by the prevalence of risk variants in components of the system in several neurodegenerative diseases, ranging from Parkinson's to Alzheimer's disease. Nevertheless, our understanding of endocytic cargo and core endolysosomal machinery in neurons is limited, in part due to technical limitations. Here, we developed a toolkit for capturing EEA1-postive endosomes (Endo-IP) and TMEM192-positive lysosomes (Lyso-IP) in stem cell-derived induced neurons (iNeurons). We demonstrated its utility by revealing the endolysosomal protein landscapes for cortical-like iNeurons and stem cells. This allowed us to globally profile endocytic cargo, identifying hundreds of transmembrane proteins, including neurogenesis and synaptic proteins, as well as endocytic cargo with predicted SNX17 or SNX27 recognition motifs. By contrast, parallel lysosome profiling reveals a simpler protein repertoire, reflecting in part temporally controlled recycling or degradation for many endocytic targets. This system will facilitate mechanistic interrogation of endolysosomal components found as risk factors in neurodegenerative disease.

SIRT-1/RHOT-1/PGC-1α loop modulates mitochondrial biogenesis and transfer to offer resilience following endovascular stem cell therapy in ischemic stroke

Current clinical interventions for stroke majorly involve thrombolysis or thrombectomy, however, cessation of the progressive deleterious cellular cascades post-stroke and long-term neuroprotection are yet to be explored. Mitochondria are highly vulnerable organelles and their dysfunction is one of the detrimental consequences following stroke. Mitochondria dysregulation activate unfavourable cellular events over a period of time that leads to the collapse of neuronal machinery in the brain. Hence, strategies to protect and replenish mitochondria in injured neurons may be useful and needs to be explored. Stem cell therapy in ischemic stroke holds a great promise. Past studies have shown beneficial outcomes of endovascularly delivered stem cells in both pre-clinical and clinical settings. Intra-arterial (IA) administration can provide more cells to the stroke foci and affected brain regions than intravenous administration. Supplying new mitochondria to the stroke-compromised neurons either in the core or penumbra by infused stem cells can help increase their survival and longevity. Previously, our lab has demonstrated that IA 1∗105 mesenchymal stem cells (MSCs) in rats were safe, efficacious and rendered neuroprotection by regulating neuronal calcineurin, modulating sirtuin1(SIRT-1) mediated inflammasome signaling, ameliorating endoplasmic reticulum-stress, alleviation of post-stroke edema and reducing cellular apoptosis. To explore further, our present study aims to investigate the potential of IA MSCs in protecting and replenishing mitochondria in the injured neurons post-stroke and the involvement of SIRT-1/RHOT-1/PGC-1α loop towards mitochondria transfer, biogenesis, and neuroprotection. This study will open new avenues for using stem cells for ischemic stroke in clinics as one of the future adjunctive therapies.

A miR-383-5p Signaling Hub Coordinates the Axon Regeneration Response to Inflammation.

Neuroinflammation can positively influence axon regeneration following injury in the central nervous system. Inflammation promotes the release of neurotrophic molecules and stimulates intrinsic proregenerative molecular machinery in neurons, but the detailed mechanisms driving this effect are not fully understood. We evaluated how microRNAs are regulated in retinal neurons in response to intraocular inflammation to identify their potential role in axon regeneration. We found that miR-383-5p is downregulated in retinal ganglion cells in response to zymosan-induced intraocular inflammation. MiR-383-5p downregulation in neurons is sufficient to promote axon growth in vitro, and the intravitreal injection of a miR-383-5p inhibitor into the eye promotes axon regeneration following optic nerve crush. MiR-383-5p directly targets ciliary neurotrophic factor (CNTF) receptor components, and miR-383-5p inhibition sensitizes adult retinal neurons to the outgrowth-promoting effects of CNTF. Interestingly, we also demonstrate that CNTF treatment is sufficient to reduce miR-383-5p levels in neurons, constituting a positive-feedback module, whereby initial CNTF treatment reduces miR-383-5p levels, which then disinhibits CNTF receptor components to sensitize neurons to the ligand. Additionally, miR-383-5p inhibition derepresses the mitochondrial antioxidant protein peroxiredoxin-3 (PRDX3) which was required for the proregenerative effects associated with miR-383-5p loss-of-function in vitro. We have thus identified a positive-feedback mechanism that facilitates neuronal CNTF sensitivity in neurons and a new molecular signaling module that promotes inflammation-induced axon regeneration.

Topographic Organization of Glutamatergic and GABAergic Parvalbumin-Positive Neurons in the Lateral Habenula.

Parvalbumin-expressing (PV) neurons, classified by their expression of the calcium-binding protein parvalbumin, play crucial roles in the function and plasticity of the lateral habenular nucleus (LHb). This study aimed to deepen our understanding of the LHb by collecting information about the heterogeneity of LHb PV neurons in mice. To achieve this, we investigated the proportions of the transmitter machinery in LHb PV neurons, including GABAergic, glutamatergic, serotonergic, cholinergic, and dopaminergic neurotransmitter markers, using transcriptome analysis, mRNA in situ hybridization chain reaction, and immunohistochemistry. LHb PV neurons comprise three subsets: glutamatergic, GABAergic, and double-positive for glutamatergic and GABAergic machinery. By comparing the percentages of the subsets, we found that the LHb was topographically organized anteroposteriorly; the GABAergic and glutamatergic PV neurons were preferentially distributed in the anterior and posterior LHb, respectively, uncovering the anteroposterior topography of the LHb. In addition, we confirmed the mediolateral topography of lateral GABAergic PV neurons. These findings suggest that PV neurons play distinct roles in different parts of the LHb along the anteroposterior and mediolateral axes, facilitating the topographic function of the LHb. It would be interesting to determine whether their topography is differentially involved in various cognitive and motivational processes associated with the LHb, particularly the involvement of posterior glutamatergic PV neurons.

A roadmap to human hippocampal neurogenesis in adulthood, aging and AD.

In the rodent, hippocampal neurogenesis plays critical roles in learning and memory1,2, is tightly regulated by inhibitory neurons3-7 and contributes to memory dysfunction in Alzheimer's disease (AD) mouse models8-10. In contrast, the mechanisms regulating neurogenesis in the adult human hippocampus, the dynamic shifts in the transcriptomic and epigenomic profiles in aging and AD and putative niche interactions within the cellular environment, remain largely unknown. Using single nuclei multi-omics of postmortem human hippocampi we map the molecular mechanisms of hippocampal neurogenesis across aging, cognitive decline, and AD neuropathology. Transcriptomic and epigenetic profiling of neural stem cells (NSCs), neuroblasts and immature neurons suggests that the earliest shift in the characteristics of neurogenesis takes place in NSCs in aging. Cognitive impairment was associated with changes in neuroblast profile. In AD, there was a widespread cessation of the transcription machinery in immature neurons, with robust downregulation of genes regulating ribosomal and mitochondrial function. Further, there was substantial loss of parvalbumin+ inhibitory neurons in the hippocampus in aging. The number of the rest of inhibitory neurons were reduced as a function of age and diagnosis. Notably, a similar system-level effect was observed between immature and inhibitory neurons in the transition from aging to AD, manifested by common molecular pathways that were ultimately lost in AD. The numbers of neuroblasts, immature and GABAergic neurons inversely correlated with extent of neuropathology. Using CellChat and NeuronChat, we inferred the ligands and receptors by which neurogenic cells communicate with their cellular environment. Loss of synaptic adhesion molecules and neurotransmitters, either sent or received by neurogenic cells, was observed in AD. Together, this study delineates the molecular mechanisms and dynamics of human neurogenesis, functional association with inhibitory neurons and a mechanism of hippocampal hyperexcitability in AD.

Metabolic Function of Leucine-Rich Repeat Transmembrane Neuronal 4 Expressed by Hypothalamic AgRP Neurons

It is well known that the agouti-related peptide (AgRP)-expressing neurons of the arcuate nucleus of the hypothalamus regulate energy balance and glucose homeostasis. While it was previously shown that excitatory and inhibitory synaptic input onto the AgRP neurons influence the activity and metabolic function of AgRP neurons, little is known about the role of synaptic adhesion molecules therein. In this study, we focused on a synaptic adhesion molecule, leucine-rich repeat transmembrane neuronal 4 (LRRTM4), which is expressed by post-synaptic part of excitatory synapses to be involved in synapse formation and synaptic transmission. We generated conditional knockout mice which lacks LRRTM4 specifically in the AgRP neurons, and assessed the electrophysiological and metabolic phenotypes. Our results should provide insight how synaptic machinery of hypothalamic AgRP neurons can shape in vivo metabolic function. This work was supported by the National Research Foundation of Korea (NRF-2022R1A2C3005613 to Jong-Woo Sohn) funded by the Korean Ministry of Science and ICT. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

New Views of the DNA Repair Protein Ataxia-Telangiectasia Mutated in Central Neurons: Contribution in Synaptic Dysfunctions of Neurodevelopmental and Neurodegenerative Diseases.

Ataxia-Telangiectasia Mutated (ATM) is a serine/threonine protein kinase principally known to orchestrate DNA repair processes upon DNA double-strand breaks (DSBs). Mutations in the Atm gene lead to Ataxia-Telangiectasia (AT), a recessive disorder characterized by ataxic movements consequent to cerebellar atrophy or dysfunction, along with immune alterations, genomic instability, and predisposition to cancer. AT patients show variable phenotypes ranging from neurologic abnormalities and cognitive impairments to more recently described neuropsychiatric features pointing to symptoms hardly ascribable to the canonical functions of ATM in DNA damage response (DDR). Indeed, evidence suggests that cognitive abilities rely on the proper functioning of DSB machinery and specific synaptic changes in central neurons of ATM-deficient mice unveiled unexpected roles of ATM at the synapse. Thus, in the present review, upon a brief recall of DNA damage responses, we focus our attention on the role of ATM in neuronal physiology and pathology and we discuss recent findings showing structural and functional changes in hippocampal and cortical synapses of AT mouse models. Collectively, a deeper knowledge of ATM-dependent mechanisms in neurons is necessary not only for a better comprehension of AT neurological phenotypes, but also for a higher understanding of the pathological mechanisms in neurodevelopmental and degenerative disorders involving ATM dysfunctions.

IFNγ protects motor neurons from oxidative stress via enhanced global protein synthesis in FUS-associated amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis type 6 (ALS6) is a familial subtype of ALS linked to Fused in Sarcoma (FUS) gene mutation. FUS mutations lead to decreased global protein synthesis, but the mechanism that drives this has not been established. Here, we used ALS6 patient-derived induced pluripotent stem cells (hIPSCs) to study the effect of the ALS6 FUSR521H mutation on the translation machinery in motor neurons (MNs). We find, in agreement with findings of others, that protein synthesis is decreased in FUSR521H MNs. Furthermore, FUSR521H MNs are more sensitive to oxidative stress and display reduced expression of TGF-β and mTORC gene pathways when stressed. Finally, we show that IFNγ treatment reduces apoptosis of FUSR521H MNs exposed to oxidative stress and partially restores the translation rates in FUSR521H MNs. Overall, these findings suggest that a functional IFNγ response is important for FUS-mediated protein synthesis, possibly by FUS nuclear translocation in ALS6.

Anesthesia and the Merry-go-round of Information in the Brain.

Anesthesia and the Merry-go-round of Information in the Brain.

Social Network Plasticity of Mice Parental Behavior.

Neural plasticity occurs during developmental stages and is essential for sexual differentiation of the brain and the ensuing sex-dependent behavioral changes in adults. Maternal behavior is primarily affected by sex-related differences in the brain; however, chronic social isolation even in mature male mice can induce maternal retrieving and crouching behavior when they are first exposed to pups. Social milieus influence the inherent behavior of adults and alter the molecular architecture in the brain, thereby allowing higher levels of associated gene expression and molecular activity. This review explores the possibility that although the development of neural circuits is closely associated with maternal behavior, the brain can still retain its neuroplasticity in adults from a neuromolecular perspective. In addition, neuronal machinery such as neurotransmitters and neuropeptides might influence sociobehavioral changes. This review also discusses that the neural circuits regulating behaviors such as parenting and infanticide (including neglect behavior), might be controlled by neural relay on melanin concentrating hormone (MCH)–oxytocin in the hypothalamus during the positive and negative mode of action in maternal behavior. Furthermore, MCH–oxytocin neural relay might contribute to the anxiolytic effect on maternal behavior, which is involved with reward circuits.

Neuropeptide repertoire and 3D anatomy of the ctenophore nervous system

Ctenophores are gelatinous marine animals famous for locomotion by ciliary combs. Due to the uncertainties of the phylogenetic placement of ctenophores and the absence of some key bilaterian neuronal genes, it has been hypothesized that their neurons evolved independently. Additionally, recent whole-body, single-cell RNA sequencing (scRNA-seq) analysis failed to identify ctenophore neurons using any of the known neuronal molecular markers. To reveal the molecular machinery of ctenophore neurons, we have characterized the neuropeptide repertoire of the ctenophore Mnemiopsis leidyi. Using the machine learning NeuroPID tool, we predicted 129 new putative neuropeptide precursors. Sixteen of them were localized to the subepithelial nerve net (SNN), sensory aboral organ (AO), and epithelial sensory cells (ESCs), providing evidence that they are neuropeptide precursors. Four of these putative neuropeptides had a behavioral effect and increased the animals' swimming speed. Intriguingly, these putative neuropeptides finally allowed us to identify neuronal cell types in single-cell transcriptomic data and reveal the molecular identity of ctenophore neurons. High-resolution electron microscopy and 3D reconstructions of the nerve net underlying the comb plates confirmed a more than 100-year-old hypothesis of anastomoses between neurites of the same cell in ctenophores and revealed that they occur through a continuous membrane. Our work demonstrates the unique ultrastructure of the peptidergic nerve net and a rich neuropeptide repertoire of ctenophores, supporting the hypothesis that the first nervous system(s) evolved as nets of peptidergic cells.

Screening of a Focused Ubiquitin-Proteasome Pathway Inhibitor Library Identifies Small Molecules as Novel Modulators of Botulinum Neurotoxin Type A Toxicity.

Botulinum neurotoxins (BoNTs) are known as the most potent bacterial toxins, which can cause potentially deadly disease botulism. BoNT Serotype A (BoNT/A) is the most studied serotype as it is responsible for most human botulism cases, and its formulations are extensively utilized in clinics for therapeutic and cosmetic applications. BoNT/A has the longest-lasting effect in neurons compared to other serotypes, and there has been high interest in understanding how BoNT/A manages to escape protein degradation machinery in neurons for months. Recent work demonstrated that an E3 ligase, HECTD2, leads to efficient ubiquitination of the BoNT/A Light Chain (A/LC); however, the dominant activity of a deubiquitinase (DUB), VCIP135, inhibits the degradation of the enzymatic component. Another DUB, USP9X, was also identified as a potential indirect contributor to A/LC degradation. In this study, we screened a focused ubiquitin-proteasome pathway inhibitor library, including VCIP135 and USP9X inhibitors, and identified ten potential lead compounds affecting BoNT/A mediated SNAP-25 cleavage in neurons in pre-intoxication conditions. We then tested the dose-dependent effects of the compounds and their potential toxic effects in cells. A subset of the lead compounds demonstrated efficacy on the stability and ubiquitination of A/LC in cells. Three of the compounds, WP1130 (degrasyn), PR-619, and Celastrol, further demonstrated efficacy against BoNT/A holotoxin in an in vitro post-intoxication model. Excitingly, PR-619 and WP1130 are known inhibitors of VCIP135 and USP9X, respectively. Modulation of BoNT turnover in cells by small molecules can potentially lead to the development of effective countermeasures against botulism.

Galantamine prevents and reverses neuroimmune induction and loss of adult hippocampal neurogenesis following adolescent alcohol exposure

BackgroundBinge ethanol exposure during adolescence reduces hippocampal neurogenesis, a reduction which persists throughout adulthood despite abstinence. This loss of neurogenesis, indicated by reduced doublecortin+ immunoreactivity (DCX+IR), is paralleled by an increase in hippocampal proinflammatory signaling cascades. As galantamine, a cholinesterase inhibitor, has anti-inflammatory actions, we tested the hypothesis that galantamine would prevent (study 1) or restore (study 2) AIE induction of proinflammatory signals within the hippocampus as well as AIE-induced loss of hippocampal neurogenesis.MethodsGalantamine (4 mg/kg) or vehicle (saline) was administered to Wistar rats during adolescent intermittent ethanol (AIE; 5.0 g/kg ethanol, 2 days on/2 days off, postnatal day [P] 25-54) (study 1, prevention) or after AIE during abstinent maturation to adulthood (study 2, restoration).ResultsResults indicate AIE reduced DCX+IR and induced cleaved caspase3 (Casp3) in DCX-expressing immature neurons. Excitingly, AIE induction of activated Casp3 in DCX-expressing neurons is both prevented and reversed by galantamine treatment, which also resulted in prevention and restoration of neurogenesis (DCX+IR). Similarly, galantamine prevented and/or reversed AIE induction of proinflammatory markers, including the chemokine (C-C motif) ligand 2 (CCL2), cyclooxygenase-2 (COX-2), and high mobility group box 1 (HMGB1) protein, suggesting that AIE induction of proinflammatory signaling mediates both cell death cascades and hippocampal neurogenesis. Interestingly, galantamine treatment increased Ki67+IR generally as well as increased pan-Trk expression specifically in AIE-treated rats but failed to reverse AIE induction of NADPH-oxidase (gp91phox).ConclusionsCollectively, our studies suggest that (1) loss of neurogenesis after AIE is mediated by persistent induction of proinflammatory cascades which drive activation of cell death machinery in immature neurons, and (2) galantamine can prevent and restore AIE disruptions in the hippocampal environmental milieu to then prevent and restore AIE-mediated loss of neurogenesis.

VGLUT2 Is a Determinant of Dopamine Neuron Resilience in a Rotenone Model of Dopamine Neurodegeneration.

Parkinson's disease (PD) is characterized by progressive dopamine (DA) neuron loss in the SNc. In contrast, DA neurons in the VTA are relatively protected from neurodegeneration, but the underlying mechanisms for this resilience remain poorly understood. Recent work suggests that expression of the vesicular glutamate transporter 2 (VGLUT2) selectively impacts midbrain DA neuron vulnerability. We investigated whether altered DA neuron VGLUT2 expression determines neuronal resilience in rats exposed to rotenone, a mitochondrial complex I inhibitor and toxicant model of PD. We discovered that VTA/SNc DA neurons that expressed VGLUT2 are more resilient to rotenone-induced DA neurodegeneration. Surprisingly, the density of neurons with detectable VGLUT2 expression in the VTA and SNc increases in response to rotenone. Furthermore, dopaminergic terminals within the NAc, where the majority of VGLUT2-expressing DA neurons project, exhibit greater resilience compared with DA terminals in the caudate/putamen. More broadly, VGLUT2-expressing terminals are protected throughout the striatum from rotenone-induced degeneration. Together, our data demonstrate that a distinct subpopulation of VGLUT2-expressing DA neurons are relatively protected from rotenone neurotoxicity. Rotenone-induced upregulation of the glutamatergic machinery in VTA and SNc neurons and their projections may be part of a broader neuroprotective mechanism. These findings offer a putative new target for neuronal resilience that can be manipulated to prevent toxicant-induced DA neurodegeneration in PD.SIGNIFICANCE STATEMENT Environmental exposures to pesticides contribute significantly to pathologic processes that culminate in Parkinson's disease (PD). The pesticide rotenone has been used to generate a PD model that replicates key features of the illness, including dopamine neurodegeneration. To date, longstanding questions remain: are there dopamine neuron subpopulations resilient to rotenone; and if so, what are the molecular determinants of this resilience? Here we show that the subpopulation of midbrain dopaminergic neurons that express the vesicular glutamate transporter 2 (VGLUT2) are more resilient to rotenone-induced neurodegeneration. Rotenone also upregulates VGLUT2 more broadly in the midbrain, suggesting that VGLUT2 expression generally confers increased resilience to rotenone. VGLUT2 may therefore be a new target for boosting neuronal resilience to prevent toxicant-induced DA neurodegeneration in PD.

Involvement of Bcl-xL in Neuronal Function and Development

The B-cell lymphoma (Bcl-2) family of proteins are mainly known for their role in the regulation of apoptosis by preventing pore formation at the mitochondrial outer membrane and subsequent caspase activation. However, Bcl-2 proteins also have non-canonical functions, independent of apoptosis. Indeed, the cell death machinery, including Bcl-2 homologs, was reported to be essential for the central nervous system (CNS), notably with respect to synaptic transmission and axon pruning. Here we focused on Bcl-xL, a close Bcl-2 homolog, which plays a major role in neuronal development, as bclx knock out mice prematurely die at embryonic day 13.5, showing massive apoptosis in the CNS. In addition, we present evidence that Bcl-xL fosters ATP generation by the mitochondria to fuel high energy needs by neurons, and its contribution to synaptic transmission. We discuss how Bcl-xL might control local and transient activation of caspases in neurons without causing cell death. Consistently, Bcl-xL may contribute to morphological changes, such as sprouting and retractation of axon branches, in the context of CNS plasticity. Regarding degenerative diseases and aging, a better understanding of the numerous roles of the cell death machinery in neurons may have future clinical implications.

An Improved CRISPR/dCas9 Interference Tool for Neuronal Gene Suppression.

The expression of genetic material governs brain development, differentiation, and function, and targeted manipulation of gene expression is required to understand contributions of gene function to health and disease states. Although recent improvements in CRISPR/dCas9 interference (CRISPRi) technology have enabled targeted transcriptional repression at selected genomic sites, integrating these techniques for use in non-dividing neuronal systems remains challenging. Previously, we optimized a dual lentivirus expression system to express CRISPR-based activation machinery in post-mitotic neurons. Here we used a similar strategy to adapt an improved dCas9-KRAB-MeCP2 repression system for robust transcriptional inhibition in neurons. We find that lentiviral delivery of a dCas9-KRAB-MeCP2 construct driven by the neuron-selective human synapsin promoter enabled transgene expression in primary rat neurons. Next, we demonstrate transcriptional repression using CRISPR sgRNAs targeting diverse gene promoters, and show superiority of this system in neurons compared to existing RNA interference methods for robust transcript specific manipulation at the complex Brain-derived neurotrophic factor (Bdnf) gene. Our findings advance this improved CRISPRi technology for use in neuronal systems for the first time, potentially enabling improved ability to manipulate gene expression states in the nervous system.

Spontaneous Activity of Neuronal Ensembles in Mouse Motor Cortex: Changes after GABAergic Blockade

The mouse motor cortex exhibits spontaneous activity in the form of temporal sequences of neuronal ensembles in vitro without the need of tissue stimulation. These neuronal ensembles are defined as groups of neurons with a strong correlation between its firing patterns, generating what appears to be a predetermined neural conduction mode that needs study. Each ensemble is commonly accompanied by one or more parvalbumin expressing neurons (PV+) or fast spiking interneurons. Many of these interneurons have functional connections between them, helping to form a circuit configuration similar to a small-world network. However, rich club metrics show that most connected neurons are neurons not expressing parvalbumin, mainly pyramidal neurons (PV−) suggesting feed-forward propagation through pyramidal cells. Ensembles with PV+ neurons are connected to these hubs. When ligand-gated fast GABAergic transmission is blocked, temporal sequences of ensembles collapse into a unique synchronous and recurrent ensemble, showing the need of inhibition for coding cortical spontaneous activity. This new ensemble has a duration and electrophysiological characteristics of brief recurrent interictal epileptiform discharges (IEDs) composed by the coactivity of both PV− and PV+ neurons, demonstrating that GABA transmission impedes its occurrence. Synchronous ensembles are clearly divided into two clusters one of them lasting longer and mainly composed by PV+ neurons. Because an ictal-like event was not recorded after several minutes of IEDs recording, it is inferred that an external stimulus and/or fast GABA transmission are necessary for its appearance, making this preparation ideal to study both the neuronal machinery to encode cortical spontaneous activity and its transformation into brief non-ictal epileptiform discharges.

Core cell cycle machinery is crucially involved in both life and death of post-mitotic neurons.

A persistent dogma in neuroscience supported the idea that terminally differentiated neurons permanently withdraw from the cell cycle. However, since the late 1990s, several studies have shown that cell cycle proteins are expressed in post-mitotic neurons under physiological conditions, indicating that the cell cycle machinery is not restricted to proliferating cells. Moreover, many studies have highlighted a clear link between cell cycle-related proteins and neurological disorders, particularly relating to apoptosis-induced neuronal death. Indeed, cell cycle-related proteins can be upregulated or overactivated in post-mitotic neurons in case of acute or degenerative central nervous system disease. Given the considerable lack of effective treatments for age-related neurological disorders, new therapeutic approaches targeting the cell cycle machinery might thus be considered. This review aims at summarizing current knowledge about the role of the cell cycle machinery in post-mitotic neurons in healthy and pathological conditions.