A new age in understanding adult hippocampal neurogenesis in Alzheimer's disease.

Several lines of evidence have established that proliferation and differentiation of neural stem cells into neurons within the sub-granular zone of the dentate gyrus, a process named adult hippocampal neurogenesis, contribute to maintaining healthy cognitive functions throughout life. The rate of adult hippocampal neurogenesis decreases with aging and a premature impairment of adult hippocampal neurogenesis has been observed both in animal models of Alzheimer's disease and human post-mortem tissues. The causal relationship between adult hippocampal neurogenesis and the development of Alzheimer's disease pathology has, however, not been established. This is partly due to the limitation of recapitulating the development of Alzheimer's disease pathology in rodent models and the lack of translatable biomarkers to identify tractable targets in humans. While it is tempting to postulate that adult hippocampal neurogenesis could be leveraged to improve cognitive deficits in Alzheimer's disease, consensual results have yet to be reached to fully explore this hypothesis. In this review, we discuss how the recent progress in identifying molecular pathways in adult hippocampal neurogenesis provides a good framework to initiate strategies for drug-based intervention in neurodegenerative diseases, especially in Alzheimer's disease. We outline how discrepancies in pre-clinical disease models and experimental methodology have resulted in contradictory findings and propose a shift towards using more translatable approaches to model neurogenesis in Alzheimer's disease. In particular, we review how exploring novel experimental paradigms including the use of human induced pluripotent stem cells and more complex cell culture systems, as well as standardizing protocols used to investigate evidence of neurogenesis in human tissues, could deliver deeper mechanistic insights that would kick-start innovative drug discovery efforts to promote healthy aging and cellular rejuvenation.

Fluorescence-Activated Nuclei Negative Sorting of Neurons Combined with Single Nuclei RNA Sequencing to Study the Hippocampal Neurogenic Niche.

Adult Hippocampal Neurogenesis (AHN), which consists of a lifelong maintenance of proliferative and quiescent neural stem cells (NSCs) within the sub-granular zone (SGZ) of the dentate gyrus (DG) and their differentiation from newly born neurons into granule cells in the granule cell layer, is well validated across numerous studies. Using genetically modified animals, particularly rodents, is a valuable tool to investigate signaling pathways regulating AHN and to study the role of each cell type that compose the hippocampal neurogenic niche. To address the latter, methods combining single nuclei isolation with next generation sequencing have had a significant impact in the field of AHN to identify gene signatures for each cell population. Further refinement of these techniques is however needed to phenotypically profile rarer cell populations within the DG. Here, we present a method that utilizes Fluorescence Activated Nuclei Sorting (FANS) to exclude most neuronal populations from a single nuclei suspension isolated from freshly dissected DG, by selecting unstained nuclei for the NeuN antigen, in order to perform single nuclei RNA sequencing (snRNA-seq). This method is a potential steppingstone to further investigate intercellular regulation of the AHN and to uncover novel cellular markers and mechanisms across species.

Targeting impaired adult hippocampal neurogenesis in ageing by leveraging intrinsic mechanisms regulating Neural Stem Cell activity.

Deficits in adult neurogenesis may contribute to the aetiology of many neurodevelopmental, psychiatric and neurodegenerative diseases. Genetic ablation of neurogenesis provides proof of concept that adult neurogenesis is required to sustain complex and dynamic cognitive functions, such as learning and memory, mostly by providing a high degree of plasticity to neuronal circuits. In addition, adult neurogenesis is reactive to external stimuli and the environment making it particularly susceptible to impairment and consequently contributing to comorbidity. In the human brain, the dentate gyrus of the hippocampus is the main active source of neural stem cells that generate granule neurons throughout life. The regulation and preservation of the pool of neural stem cells is central to ensure continuous and healthy adult hippocampal neurogenesis (AHN). Recent advances in genetic and metabolic profiling alongside development of more predictive animal models have contributed to the development of new concepts and the emergence of molecular mechanisms that could pave the way to the implementation of new therapeutic strategies to treat neurological diseases. In this review, we discuss emerging molecular mechanisms underlying AHN that could be embraced in drug discovery to generate novel concepts and targets to treat diseases of ageing including neurodegeneration. To support this, we review cellular and molecular mechanisms that have recently been identified to assess how AHN is sustained throughout life and how AHN is associated with diseases. We also provide an outlook on strategies for developing correlated biomarkers that may accelerate the translation of pre-clinical and clinical data and review clinical trials for which modulation of AHN is part of the therapeutic strategy.

Isolation of Chromatin-bound Proteins from Subcellular Fractions for Biochemical Analysis.

Shuttling of proteins between different cellular compartments controls their proteostasis and can contribute in some cases to regulate their activity. Biochemical analysis of chromatin-bound proteins, such as transcription factors, is often difficult because of their low yield and due to the interference from nucleic acids. This protocol describes a method to efficiently fractionate cells combined with a mechanical (i.e., sonication) or an enzymatic treatment (i.e., benzonase) that facilitates analysis of chromatin-bound protein extracts by Western blot analysis or by protein pull-down assays. This approach can be valuable to enrich a particular protein within a particular subcellular fraction either to study specific post-translational modification patterns or to identify specific protein-protein interactions.

Subcellular localisation modulates ubiquitylation and degradation of Ascl1.

The proneural transcription factor Ascl1 is a master regulator of neurogenesis, coordinating proliferation and differentiation in the central nervous system. While its expression is well characterised, post-translational regulation is much less well understood. Here we demonstrate that a population of chromatin-bound Ascl1 can be found associated with short chains of ubiquitin while cytoplasmic Ascl1 harbours much longer ubiquitin chains. Only cytoplasmic ubiquitylation targets Ascl1 for destruction, which occurs by conjugation of ubiquitin to lysines in the basic helix-loop-helix domain of Ascl1 and requires the E3 ligase Huwe1. In contrast, chromatin-bound Ascl1 associated with short ubiquitin-chains, which can occur on lysines within the N-terminal region or the bHLH domain and is not mediated by Huwe1, is not targeted for ubiquitin-mediated destruction. We therefore offer further insights into post-translational regulation of Ascl1, highlighting complex regulation of ubiquitylation and degradation in the cytoplasm and on chromatin.

Neurogenin3 phosphorylation controls reprogramming efficiency of pancreatic ductal organoids into endocrine cells.

ß-cell replacement has been proposed as an effective treatment for some forms of diabetes, and in vitro methods for ß-cell generation are being extensively explored. A potential source of ß-cells comes from fate conversion of exocrine pancreatic cells into the endocrine lineage, by overexpression of three regulators of pancreatic endocrine formation and ß-cell identity, Ngn3, Pdx1 and MafA. Pancreatic ductal organoid cultures have recently been developed that can be expanded indefinitely, while maintaining the potential to differentiate into the endocrine lineage. Here, using mouse pancreatic ductal organoids, we see that co-expression of Ngn3, Pdx1 and MafA are required and sufficient to generate cells that express insulin and resemble ß-cells transcriptome-wide. Efficiency of ß-like cell generation can be significantly enhanced by preventing phosphorylation of Ngn3 protein and further augmented by conditions promoting differentiation. Taken together, our new findings underline the potential of ductal organoid cultures as a source material for generation of ß-like cells and demonstrate that post-translational regulation of reprogramming factors can be exploited to enhance ß-cell generation.

Micro-chromatin Immunoprecipation (µChIP) Protocol for Real-time PCR Analysis of a Limited Amount of Cells.

Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) is an important strategy to study gene regulation. When availability of cells is limited, however, it can be useful to focus on specific genes to investigate in depth the role of transcription factors or histone marks. Unfortunately, performing ChIP experiments to study transcription factors' binding to DNA can be difficult when biological material is restricted. This protocol describes a robust method to perform µChIP for over-expressed or endogenous transcription factors using ~100,000 cells per ChIP experiment (Masserdotti et al., 2015). We also describe optimization steps, which we think are critical for this protocol to work and which can be used to further reduce the number of cells.

The N-terminus of a novel isoform of human iASPP is required for its cytoplasmic localization.

ASPP1 and ASPP2 are both proteins that interact with p53 and enhance its ability to induce apoptosis by selectively elevating the expression of proapoptotic p53-responsive genes. iASPP(RAI) is a third member of the family that is the most conserved inhibitor of p53-mediated apoptosis. Here, we have described iASPP, a longer form of iASPP(RAI), which at 828 amino acids is more than twice the size of iASPP(RAI). Using two antibodies that recognize both iASPP and iASPP(RAI), we report that this longer form of iASPP is the predominant form of the molecule expressed in cells. Like iASPP(RAI), iASPP also binds to p53 and inhibits apoptosis induced by p53 overexpression. However, whereas iASPP(RAI) is predominantly nuclear, the N-terminus of iASPP is entirely cytoplasmic, and the longer iASPP is located in both the cytoplasm and the nucleus. The effect upon subcellular localization of the longer N-terminus of iASPP means that this new, longer form of the molecule may be subject to greater regulation and provides another layer in the control of p53-induced apoptosis.

Transcriptional Mechanisms of Proneural Factors and REST in Regulating Neuronal Reprogramming of Astrocytes.

Direct lineage reprogramming induces dramatic shifts in cellular identity, employing poorly understood mechanisms. Recently, we demonstrated that expression of Neurog2 or Ascl1 in postnatal mouse astrocytes generates glutamatergic or GABAergic neurons. Here, we take advantage of this model to study dynamics of neuronal cell fate acquisition at the transcriptional level. We found that Neurog2 and Ascl1 rapidly elicited distinct neurogenic programs with only a small subset of shared target genes. Within this subset, only NeuroD4 could by itself induce neuronal reprogramming in both mouse and human astrocytes, while co-expression with Insm1 was required for glutamatergic maturation. Cultured astrocytes gradually became refractory to reprogramming, in part by the repressor REST preventing Neurog2 from binding to the NeuroD4 promoter. Notably, in astrocytes refractory to Neurog2 activation, the underlying neurogenic program remained amenable to reprogramming by exogenous NeuroD4. Our findings support a model of temporal hierarchy for cell fate change during neuronal reprogramming.

Ascl1 Coordinately Regulates Gene Expression and the Chromatin Landscape during Neurogenesis.

The proneural transcription factor Ascl1 coordinates gene expression in both proliferating and differentiating progenitors along the neuronal lineage. Here, we used a cellular model of neurogenesis to investigate how Ascl1 interacts with the chromatin landscape to regulate gene expression when promoting neuronal differentiation. We find that Ascl1 binding occurs mostly at distal enhancers and is associated with activation of gene transcription. Surprisingly, the accessibility of Ascl1 to its binding sites in neural stem/progenitor cells remains largely unchanged throughout their differentiation, as Ascl1 targets regions of both readily accessible and closed chromatin in proliferating cells. Moreover, binding of Ascl1 often precedes an increase in chromatin accessibility and the appearance of new regions of open chromatin, associated with de novo gene expression during differentiation. Our results reveal a function of Ascl1 in promoting chromatin accessibility during neurogenesis, linking the chromatin landscape at Ascl1 target regions with the temporal progression of its transcriptional program.

The ASPP proteins complex and cooperate with p300 to modulate the transcriptional activity of p53.

Understanding how p53 is able to specifically respond to particular stress signals and regulate many different signalling pathways remains a challenge. Several studies have demonstrated that p53's interactions with different protein partners are essential for it to be able to coordinate specific responses. In particular, the apoptotic pathway is regulated by p53 in cooperation with the Apoptosis Stimulating Proteins of p53 (ASPP) proteins. In this study, we showed that the ASPP proteins are able to bind and cooperate with p300, a well defined co-factor of p53, to selectively regulate p53's transcriptional activity on promoters such as p53-inducible gene 3 but not on p21waf1. This is the first demonstration that the ASPPs can function together with p300 in regulating the transcriptional activity of p53.

Mutation at Ser392 specifically sensitizes mutant p53H175 to mdm2-mediated degradation.

Mdm2 is one of the main E3 ubiquitin ligases, which targets both wild type and mutant p53 for degradation. The ability of post-translational modifications, such as phosphorylation, to modulate the function and stability of wild type p53 has been extensively studied. However, their ability to modulate the functions and stability of mutant forms of p53 remains poorly documented. Here we show, for the first time, that the stability of mutant p53 can be regulated by phosphorylation. Mutation of serine 392 to alanine shortens the half life of p53H175, and renders p53H175A392 more sensitive to mdm2-mediated degradation than p53H175. This effect of Ser392 phosphorylation specifically affects p53H175, a misfolded mutant, and does not affect p53W248 which maintains a native conformation. Detailed analysis subsequently showed that the reduced stability of p53H175A392 is not due to an increase in mdm2/p300 binding or polyubiquitin chain formation, uncoupling the extent of polyubiquitin chain formation and the stability of mutant p53. This is supported by the observation that Ser392 mutation enhances polyubiquitin chain formation on p53W248, without reducing its stability. These results suggest that the inhibition of phosphorylation at Ser392 of p53, together with the use of an mdm2-enhancing agent such as nutlin, could present a new therapeutic strategy with which to treat tumors expressing mutant p53H175.