The Alzheimer's disease risk gene BIN1 regulates activity-dependent gene expression in human-induced glutamatergic neurons.

Bridging Integrator 1 (BIN1) is the second most important Alzheimer's disease (AD) risk gene, but its physiological roles in neurons and its contribution to brain pathology remain largely elusive. In this work, we show that BIN1 plays a critical role in the regulation of calcium homeostasis, electrical activity, and gene expression of glutamatergic neurons. Using single-cell RNA-sequencing on cerebral organoids generated from isogenic BIN1 wild type (WT), heterozygous (HET) and homozygous knockout (KO) human-induced pluripotent stem cells (hiPSCs), we show that BIN1 is mainly expressed by oligodendrocytes and glutamatergic neurons, like in the human brain. Both BIN1 HET and KO cerebral organoids show specific transcriptional alterations, mainly associated with ion transport and synapses in glutamatergic neurons. We then demonstrate that BIN1 cell-autonomously regulates gene expression in glutamatergic neurons by using a novel protocol to generate pure culture of hiPSC-derived induced neurons (hiNs). Using this system, we also show that BIN1 plays a key role in the regulation of neuronal calcium transients and electrical activity via its interaction with the L-type voltage-gated calcium channel Cav1.2. BIN1 KO hiNs show reduced activity-dependent internalization and higher Cav1.2 expression compared to WT hiNs. Pharmacological blocking of this channel with clinically relevant doses of nifedipine, a calcium channel blocker, partly rescues electrical and gene expression alterations in BIN1 KO glutamatergic neurons. Further, we show that transcriptional alterations in BIN1 KO hiNs that affect biological processes related to calcium homeostasis are also present in glutamatergic neurons of the human brain at late stages of AD pathology. Together, these findings suggest that BIN1-dependent alterations in neuronal properties could contribute to AD pathophysiology and that treatment with low doses of clinically approved calcium blockers should be considered as an option to slow disease-onset and progression.

Switch of innate to adaptative immune responses in the brain of patients with Alzheimer's disease correlates with tauopathy progression.

Neuroinflammation is a key feature of Alzheimer's disease (AD). In this work, analysis of single- cell RNA-sequencing (scRNA-seq) data obtained from the brain of patients with AD provides evidence supporting a switch from an innate to an adaptative immune response during tauopathy progression, with both disease-associated microglia (DAM) and CD8+ T cells becoming more frequent at advanced Braak stages.

Amyloid-Beta Peptides Trigger Premature Functional and Gene Expression Alterations in Human-Induced Neurons.

Alzheimer's disease (AD) is the most prevalent cause of dementia in the elderly, characterized by the presence of amyloid-beta (Aß) plaques, neurofibrillary tangles, neuroinflammation, synapse loss and neurodegeneration in the brain. The amyloid cascade hypothesis postulates that deposition of Aß peptides is the causative agent of AD pathology, but we still lack comprehensive understanding of the molecular mechanisms connecting Aß peptides to neuronal dysfunctions in AD. In this work, we investigate the early effects of Aß peptide accumulation on the functional properties and gene expression profiles of human-induced neurons (hiNs). We show that hiNs acutely exposed to low concentrations of both cell-secreted Aß peptides or synthetic Aß1-42 exhibit alterations in the frequency of calcium transients suggestive of increased neuronal excitability. Using single-cell RNA sequencing, we also show that cell-secreted Aß up-regulates the expression of several synapse-related genes and down-regulates the expression of genes associated with metabolic stress mainly in glutamatergic neurons and, to a lesser degree, in GABAergic neurons and astrocytes. These neuronal alterations correlate with activation of the SEMA5, EPHA and NECTIN signaling pathways, which are important regulators of synaptic plasticity. Altogether, our findings indicate that slight elevations in Aß concentrations are sufficient to elicit transcriptional changes in human neurons, which can contribute to early alterations in neural network activity.

Analysis of modular gene co-expression networks reveals molecular pathways underlying Alzheimer's disease and progressive supranuclear palsy.

A comprehensive understanding of the pathological mechanisms involved at different stages of neurodegenerative diseases is key for the advance of preventive and disease-modifying treatments. Gene expression alterations in the diseased brain is a potential source of information about biological processes affected by pathology. In this work, we performed a systematic comparison of gene expression alterations in the brains of human patients diagnosed with Alzheimer's disease (AD) or Progressive Supranuclear Palsy (PSP) and animal models of amyloidopathy and tauopathy. Using a systems biology approach to uncover biological processes associated with gene expression alterations, we could pinpoint processes more strongly associated with tauopathy/PSP and amyloidopathy/AD. We show that gene expression alterations related to immune-inflammatory responses preponderate in younger, whereas those associated to synaptic transmission are mainly observed in older AD patients. In PSP, however, changes associated with immune-inflammatory responses and synaptic transmission overlap. These two different patterns observed in AD and PSP brains are fairly recapitulated in animal models of amyloidopathy and tauopathy, respectively. Moreover, in AD, but not PSP or animal models, gene expression alterations related to RNA splicing are highly prevalent, whereas those associated with myelination are enriched both in AD and PSP, but not in animal models. Finally, we identify 12 AD and 4 PSP genetic risk factors in cell-type specific co-expression modules, thus contributing to unveil the possible role of these genes to pathogenesis. Altogether, this work contributes to unravel the potential biological processes affected by amyloid versus tau pathology and how they could contribute to the pathogenesis of AD and PSP.

New insights into the genetic etiology of Alzheimer's disease and related dementias.

Characterization of the genetic landscape of Alzheimer's disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/'proxy' AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele.

The Alzheimer susceptibility gene BIN1 induces isoform-dependent neurotoxicity through early endosome defects.

The Bridging Integrator 1 (BIN1) gene is a major susceptibility gene for Alzheimer's disease (AD). Deciphering its pathophysiological role is challenging due to its numerous isoforms. Here we observed in Drosophila that human BIN1 isoform1 (BIN1iso1) overexpression, contrary to human BIN1 isoform8 (BIN1iso8) and human BIN1 isoform9 (BIN1iso9), induced an accumulation of endosomal vesicles and neurodegeneration. Systematic search for endosome regulators able to prevent BIN1iso1-induced neurodegeneration indicated that a defect at the early endosome level is responsible for the neurodegeneration. In human induced neurons (hiNs) and cerebral organoids, BIN1 knock-out resulted in the narrowing of early endosomes. This phenotype was rescued by BIN1iso1 but not BIN1iso9 expression. Finally, BIN1iso1 overexpression also led to an increase in the size of early endosomes and neurodegeneration in hiNs. Altogether, our data demonstrate that the AD susceptibility gene BIN1, and especially BIN1iso1, contributes to early-endosome size deregulation, which is an early pathophysiological hallmark of AD pathology.

Differential Expression Levels of Sox9 in Early Neocortical Radial Glial Cells Regulate the Decision between Stem Cell Maintenance and Differentiation.

Radial glial progenitor cells (RGCs) in the dorsal telencephalon directly or indirectly produce excitatory projection neurons and macroglia of the neocortex. Recent evidence shows that the pool of RGCs is more heterogeneous than originally thought and that progenitor subpopulations can generate particular neuronal cell types. Using single-cell RNA sequencing, we have studied gene expression patterns of RGCs with different neurogenic behavior at early stages of cortical development. At this early age, some RGCs rapidly produce postmitotic neurons, whereas others self-renew and undergo neurogenic divisions at a later age. We have identified candidate genes that are differentially expressed among these early RGC subpopulations, including the transcription factor Sox9. Using in utero electroporation in embryonic mice of either sex, we demonstrate that elevated Sox9 expression in progenitors affects RGC cell cycle duration and leads to the generation of upper layer cortical neurons. Our data thus reveal molecular differences between progenitor cells with different neurogenic behavior at early stages of corticogenesis and indicates that Sox9 is critical for the maintenance of RGCs to regulate the generation of upper layer neurons.SIGNIFICANCE STATEMENT The existence of heterogeneity in the pool of RGCs and its relationship with the generation of cellular diversity in the cerebral cortex has been an interesting topic of debate for many years. Here we describe the existence of RGCs with reduced neurogenic behavior at early embryonic ages presenting a particular molecular signature. This molecular signature consists of differential expression of some genes including the transcription factor Sox9, which has been found to be a specific regulator of this subpopulation of progenitor cells. Functional experiments perturbing expression levels of Sox9 reveal its instructive role in the regulation of the neurogenic behavior of RGCs and its relationship with the generation of upper layer projection neurons at later ages.

Disentangling chemical and electrical effects of status epilepticus-induced dentate gyrus abnormalities.

In rodents, status epilepticus (SE) triggered by chemoconvulsants can differently affect the proliferation and fate of adult-born dentate granule cells (DGCs). It is unknown whether abnormal neurogenesis results from intracellular signaling associated with drug-receptor interaction, paroxysmal activity, or both. To test the contribution of these factors, we systematically compared the effects of kainic acid (KA)- and pilocarpine (PL)-induced SE on the morphology and localization of DGCs generated before or after SE in the ipsi- and contralateral hippocampi of mice. Hippocampal insult was induced by unilateral intrahippocampal (ihpc) administration of KA or PL. We employed conditional doublecortin-dependent expression of the green fluorescent protein (GFP) to label adult-born cells committed to neuronal lineage either one month before (mature DGCs) or seven days after (immature DGCs) SE. Unilateral ihpc administration of KA and PL led to bilateral epileptiform discharges and focal and generalized behavioral seizures. However, drastic granule cell layer (GCL) dispersion occurred only in the ipsilateral side of KA injection, but not in PL-treated animals. Granule cell layer dispersion was accompanied by a significant reduction in neurogenesis after SE in the ipsilateral side of KA-treated animals, while neurogenesis increased in the contralateral side of KA-treated animals and both hippocampi of PL-treated animals. The ratio of ectopic neurons in the ipsilateral hippocampus was higher among immature as compared to mature neurons in the KA model (32.8% vs. 10.0%, respectively), while the occurrence of ectopic neurons in PL-treated animals was lower than 3% among both mature and immature DGCs. Collectively, our results suggest that KA- and PL-induced SE leads to distinct cellular alterations in mature and immature DGCs. We also show different local and secondary effects of KA or PL in the histological organization of the adult DG, suggesting that these unique epilepsy models may be complementary to our understanding of the disease. NEWroscience 2018.

Evidence of Progenitor Cell Lineage Rerouting in the Adult Mouse Hippocampus After Status Epilepticus.

Cell lineage in the adult hippocampus comprises multipotent and neuron-committed progenitors. In the present work, we fate-mapped neuronal progenitors using Dcx-CreERT2 and CAG-CAT-EGFP double-transgenic mice (cDCX/EGFP). We show that 3 days after tamoxifen-mediated recombination in cDCX/EGFP adult mice, GFP+ cells in the dentate gyrus (DG) co-expresses DCX and about 6% of these cells are proliferative neuronal progenitors. After 30 days, 20% of GFP+ generated from these progenitors differentiate into GFAP+ astrocytes. Unilateral intrahippocampal administration of the chemoconvulsants kainic acid (KA) or pilocarpine (PL) triggered epileptiform discharges and led to a significant increase in the number of GFP+ cells in both ipsi and contralateral DG. However, while PL favored the differentiation of neurons in both ipsi- and contralateral sides, KA stimulated neurogenesis only in the contralateral side. In the ipsilateral side, KA injection led to an unexpected increase of astrogliogenesis in the Dcx-lineage. We also observed a small number of GFP+/GFAP+ cells displaying radial-glia morphology ipsilaterally 3 days after KA administration, suggesting that some Dcx-progenitors could regress to a multipotent stage. The boosted neurogenesis and astrogliogenesis observed in the Dcx-lineage following chemoconvulsants administration correlated, respectively, with preservation or degeneration of the parvalbuminergic plexus in the DG. Increased inflammatory response, by contrast, was observed both in the DG showing increased neurogenesis or astrogliogenesis. Altogether, our data support the view that cell lineage progression in the adult hippocampus is not unidirectional and could be modulated by local network activity and GABA-mediated signaling.

Ablation of sensory nerves favours melanoma progression.

The tumour mass is composed not only of heterogeneous neoplastic cells, but also a variety of other components that may affect cancer cells behaviour. The lack of detailed knowledge about all the constituents of the tumour microenvironment restricts the design of effective treatments. Nerves have been reported to contribute to the growth and maintenance of numerous tissues. The effects of sensory innervations on tumour growth remain unclear. Here, by using state-of-the-art techniques, including Cre/loxP technologies, confocal microscopy, in vivo-tracing and chemical denervation, we revealed the presence of sensory nerves infiltrating within the melanoma microenvironment, and affecting cancer progression. Strikingly, melanoma growth in vivo was accelerated following genetic ablation or chemical denervation of sensory nerves. In humans, a retrospective analysis of melanoma patients revealed that increased expression of genes related to sensory nerves in tumours was associated with better clinical outcomes. These findings suggest that sensory innervations counteract melanoma progression. The emerging knowledge from this research provides a novel target in the tumour microenvironment for therapeutic benefit in cancer patients.

Pyk2 overexpression in postsynaptic neurons blocks amyloid ß1-42-induced synaptotoxicity in microfluidic co-cultures.

Recent meta-analyses of genome-wide association studies identified a number of genetic risk factors of Alzheimer's disease; however, little is known about the mechanisms by which they contribute to the pathological process. As synapse loss is observed at the earliest stage of Alzheimer's disease, deciphering the impact of Alzheimer's risk genes on synapse formation and maintenance is of great interest. In this article, we report a microfluidic co-culture device that physically isolates synapses from pre- and postsynaptic neurons and chronically exposes them to toxic amyloid ß peptides secreted by model cell lines overexpressing wild-type or mutated (V717I) amyloid precursor protein. Co-culture with cells overexpressing mutated amyloid precursor protein exposed the synapses of primary hippocampal neurons to amyloid ß1-42 molecules at nanomolar concentrations and induced a significant decrease in synaptic connectivity, as evidenced by distance-based assignment of postsynaptic puncta to presynaptic puncta. Treating the cells with antibodies that target different forms of amyloid ß suggested that low molecular weight oligomers are the likely culprit. As proof of concept, we demonstrate that overexpression of protein tyrosine kinase 2 beta-an Alzheimer's disease genetic risk factor involved in synaptic plasticity and shown to decrease in Alzheimer's disease brains at gene expression and protein levels-selectively in postsynaptic neurons is protective against amyloid ß1-42-induced synaptotoxicity. In summary, our lab-on-a-chip device provides a physiologically relevant model of Alzheimer's disease-related synaptotoxicity, optimal for assessing the impact of risk genes in pre- and postsynaptic compartments.

Differential immunoglobulin and complement levels in leprosy prior to development of reversal reaction and erythema nodosum leprosum.

BACKGROUND: Leprosy is a treatable infectious disease caused by Mycobacterium leprae. However, there is additional morbidity from leprosy-associated pathologic immune reactions, reversal reaction (RR) and erythema nodosum leprosum (ENL), which occur in 1 in 3 people with leprosy, even with effective treatment of M. leprae. There is currently no predictive marker in use to indicate which people with leprosy will develop these debilitating immune reactions. Our peripheral blood mononuclear cell (PBMC) transcriptome analysis revealed that activation of the classical complement pathway is common to both RR and ENL. Additionally, differential expression of immunoglobulin receptors and B cell receptors during RR and ENL support a role for the antibody-mediated immune response during both RR and ENL. In this study, we investigated B-cell immunophenotypes, total and M. leprae-specific antibodies, and complement levels in leprosy patients with and without RR or ENL. The objective was to determine the role of these immune mediators in pathogenesis and assess their potential as biomarkers of risk for immune reactions in people with leprosy. METHODOLOGY/FINDINGS: We followed newly diagnosed leprosy cases (n = 96) for two years for development of RR or ENL. They were compared with active RR (n = 35), active ENL (n = 29), and healthy household contacts (n = 14). People with leprosy who subsequently developed ENL had increased IgM, IgG1, and C3d-associated immune complexes with decreased complement 4 (C4) at leprosy diagnosis. People who developed RR also had decreased C4 at leprosy diagnosis. Additionally, elevated anti-M. leprae antibody levels were associated with subsequent RR or ENL. CONCLUSIONS: Differential co-receptor expression and immunoglobulin levels before and during immune reactions intimate a central role for humoral immunity in RR and ENL. Decreased C4 and elevated anti-M. leprae antibodies in people with new diagnosis of leprosy may be risk factors for subsequent development of leprosy immune reactions.

Instructing neuronal identity during CNS development and astroglial-lineage reprogramming: Roles of NEUROG2 and ASCL1.

The adult mammalian brain contains an enormous variety of neuronal types, which are generally categorized in large groups, based on their neurochemical identity, hodological properties and molecular markers. This broad classification has allowed the correlation between individual neural progenitor populations and their neuronal progeny, thus contributing to probe the cellular and molecular mechanisms involved in neuronal identity determination during central nervous system (CNS) development. In this review, we discuss the contribution of the proneural genes Neurogenin2 (Neurog2) and Achaete-scute homolog 1 (Ascl1) for the specification of neuronal phenotypes in the developing neocortex, cerebellum and retina. Then, we revise recent data on astroglia cell lineage reprogramming into induced neurons using the same proneural proteins to compare the neuronal phenotypes obtained from astroglial cells originated in those CNS regions. We conclude that Ascl1 and Neurog2 have different contributions to determine neuronal fates, depending on the neural progenitor or astroglial population expressing those proneural factors. Finally, we discuss some possible explanations for these seemingly conflicting effects of Ascl1 and Neurog2 and propose future approaches to further dissect the molecular mechanisms of neuronal identity specification.

Evidence of Müller Glia Conversion Into Retina Ganglion Cells Using Neurogenin2.

Degenerative retinopathies are the leading causes of irreversible visual impairment in the elderly, affecting hundreds of millions of patients. Müller glia cells (MGC), the main type of glia found in the vertebrate retina, can resume proliferation in the rodent adult injured retina but contribute weakly to tissue repair when compared to zebrafish retina. However, postnatal and adult mouse MGC can be genetically reprogrammed through the expression of the transcription factor (TF) Achaete-scute homolog 1 (ASCL1) into induced neurons (iNs), displaying key hallmarks of photoreceptors, bipolar and amacrine cells, which may contribute to regenerate the damaged retina. Here, we show that the TF neurogenin 2 (NEUROG2) is also sufficient to lineage-reprogram postnatal mouse MGC into iNs. The efficiency of MGC lineage conversion by NEUROG2 is similar to that observed after expression of ASCL1 and both TFs induce the generation of functionally active iNs. Treatment of MGC cultures with EGF and FGF2 prior to Neurog2 or Ascl1 expression enhances reprogramming efficiencies, what can be at least partially explained by an increase in the frequency of MGCs expressing sex determining region Y (SRY)-box 2 (SOX2). Transduction of either Neurog2 or Ascl1 led to the upregulation of key retina neuronal genes in MGC-derived iNs, but only NEUROG2 induced a consistent increase in the expression of putative retinal ganglion cell (RGC) genes. Moreover, in vivo electroporation of Neurog2 in late progenitors from the neonatal rat retina, which are transcriptionally similar to MGCs, also induced a shift in the generation of retinal cell subtypes, favoring neuronal differentiation at the expense of MGCs and resuming the generation of RGCs. Altogether, our data indicate that NEUROG2 induces lineage conversion of postnatal rodent MGCs into RGC-like iNs in vitro and resumes the generation of this neuronal type from late progenitors of the retina in vivo.

Direct Reprogramming of Adult Human Somatic Stem Cells Into Functional Neurons Using Sox2, Ascl1, and Neurog2.

Reprogramming of somatic cells into induced pluripotent stem cells (iPS) or directly into cells from a different lineage, including neurons, has revolutionized research in regenerative medicine in recent years. Mesenchymal stem cells are good candidates for lineage reprogramming and autologous transplantation, since they can be easily isolated from accessible sources in adult humans, such as bone marrow and dental tissues. Here, we demonstrate that expression of the transcription factors (TFs) SRY (sex determining region Y)-box 2 (Sox2), Mammalian achaete-scute homolog 1 (Ascl1), or Neurogenin 2 (Neurog2) is sufficient for reprogramming human umbilical cord mesenchymal stem cells (hUCMSC) into induced neurons (iNs). Furthermore, the combination of Sox2/Ascl1 or Sox2/Neurog2 is sufficient to reprogram up to 50% of transfected hUCMSCs into iNs showing electrical properties of mature neurons and establishing synaptic contacts with co-culture primary neurons. Finally, we show evidence supporting the notion that different combinations of TFs (Sox2/Ascl1 and Sox2/Neurog2) may induce multiple and overlapping neuronal phenotypes in lineage-reprogrammed iNs, suggesting that neuronal fate is determined by a combination of signals involving the TFs used for reprogramming but also the internal state of the converted cell. Altogether, the data presented here contribute to the advancement of techniques aiming at obtaining specific neuronal phenotypes from lineage-converted human somatic cells to treat neurological disorders.

Subtle Alterations in Spatial Memory Induced by Amyloid Peptides Infusion in Rats.

The cause of Alzheimer's disease (AD) remains uncertain. The accumulation of amyloid peptides (Aß) is the main pathophysiological hallmark of the disease. Spatial deficit is an important initial sign of AD, while other types of memory impairments that appear in later stages. The Barnes maze allows the detection of subtle alterations in spatial search by the analysis of use of different strategies. Previous findings showed a general performance deficit in this task following long-term (35 days) infusion of Aß, which corresponds to the moderate or severe impairments of the disease. In the present study, we evaluated the effects of a low-dose 15-day long treatment with Aß peptides on spatial and non-spatial strategies of rats tested in the Barnes maze. Aß peptides (0.5 µL/site/day; 30 pmoL solution of Aß1-40:Aß1-42 10:1) or saline were bilaterally infused into the CA1 (on the first treatment day) and intraventricularly (on the following 15 days) in 6-month-old Wistar male rats. Aß infusion induced a deficit in the performance (increased latency and distance traveled to reach the target compared to saline group). In addition, a significant association between treatment and search strategy in the retrieval trial was found: Aß group preferred the non-spatial search strategy, while saline group preferred the spatial search. In conclusion, the protocol of Aß infusion used here induced a subtle cognitive deficit that was specific to spatial aspects. Indeed, animals under Aß treatment still showed retrieval, but using non-spatial strategies. We suggest that this approach is potentially useful to the study of the initial memory deficits in early AD.

Activity-Independent Effects of CREB on Neuronal Survival and Differentiation during Mouse Cerebral Cortex Development.

Neuronal survival and morphological maturation depends on the action of the transcription factor calcium responsive element binding protein (CREB), which regulates expression of several target genes in an activity-dependent manner. However, it remains largely unknown whether CREB-mediated transcription could play a role at early stages of neuronal differentiation, prior to the establishment of functional synaptic contacts. Here, we show that CREB is phosphorylated at very early stages of neuronal differentiation in vivo and in vitro, even in the absence of depolarizing agents. Using genetic tools, we also show that inhibition of CREB-signaling affects neuronal growth and survival in vitro without affecting cell proliferation and neurogenesis. Expression of A-CREB or M-CREB, 2 dominant-negative inhibitors of CREB, decreases cell survival and the complexity of neuronal arborization. Similar changes are observed in neurons treated with protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitors, which also show decreased levels of pCREBSer133. Notably, expression of CREB-FY, a Tyr134Phe CREB mutant with a lower Km for phosphorylation, partly rescues the effects of PKA and CaMKII inhibition. Our data indicate that CREB-mediated signaling play important roles at early stages of cortical neuron differentiation, prior to the establishment of fully functional synaptic contacts.

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations.

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

Gene Manipulation Strategies to Identify Molecular Regulators of Axon Regeneration in the Central Nervous System.

Limited axon regeneration in the injured adult mammalian central nervous system (CNS) usually results in irreversible functional deficits. Both the presence of extrinsic inhibitory molecules at the injury site and the intrinsically low capacity of adult neurons to grow axons are responsible for the diminished capacity of regeneration in the adult CNS. Conversely, in the embryonic CNS, neurons show a high regenerative capacity, mostly due to the expression of genes that positively control axon growth and downregulation of genes that inhibit axon growth. A better understanding of the role of these key genes controlling pro-regenerative mechanisms is pivotal to develop strategies to promote robust axon regeneration following adult CNS injury. Genetic manipulation techniques have been widely used to investigate the role of specific genes or a combination of different genes in axon regrowth. This review summarizes a myriad of studies that used genetic manipulations to promote axon growth in the injured CNS. We also review the roles of some of these genes during CNS development and suggest possible approaches to identify new candidate genes. Finally, we critically address the main advantages and pitfalls of gene-manipulation techniques, and discuss new strategies to promote robust axon regeneration in the mature CNS.

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System.

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.