Postmitotic Neurons Research Articles

Alternative Functions of Cell Cycle-Related and DNA Repair Proteins in Post-mitotic Neurons.

Proper regulation of neuronal morphological changes is essential for neuronal migration, maturation, synapse formation, and high-order function. Many cytoplasmic proteins involved in the regulation of neuronal microtubules and the actin cytoskeleton have been identified. In addition, some nuclear proteins have alternative functions in neurons. While cell cycle-related proteins basically control the progression of the cell cycle in the nucleus, some of them have an extra-cell cycle-regulatory function (EXCERF), such as regulating cytoskeletal organization, after exit from the cell cycle. Our expression analyses showed that not only cell cycle regulators, including cyclin A1, cyclin D2, Cdk4/6, p21cip1, p27kip1, Ink4 family, and RAD21, but also DNA repair proteins, including BRCA2, p53, ATM, ATR, RAD17, MRE11, RAD9, and Hus1, were expressed after neurogenesis, suggesting that these proteins have alternative functions in post-mitotic neurons. In this perspective paper, we discuss the alternative functions of the nuclear proteins in neuronal development, focusing on possible cytoplasmic roles.

Progenitor‐derived ribosomal RNA supports protein synthesis inDrosophilaneurons

AbstractGlobal mRNA translation may differ dramatically between progenitor cells and their differentiated progeny. One way cell type‐specific translation is established is through ribosome concentration. In addition to addressing unique metabolic needs, changes in ribosome concentration may influence cell fate. The mechanisms that determine ribosome abundance in progenitors versus differentiated progeny are not fully understood. Here, we investigated this process by focusing on ribosomal RNA (rRNA) synthesis inDrosophilaneural progenitors and neurons. We found that rRNA synthesis is robust in neural progenitors but is limited in postmitotic neurons. Newly born neurons inherit rRNA from their progenitor parent and this inherited rRNA is sufficient for protein synthesis in neurons. Our findings support a model in which neuron‐specific translation programs are established by rRNA inheritance.Key PointsThis work links changes in ribosomal RNA (rRNA) synthesis, often assumed to be an invariant feature of cells, to neural differentiation.We describe a process of rRNA inheritance, where rRNA made in neural progenitors is passed to their progeny.We show that rRNA inheritance is sufficient for neural development and protein synthesis in neurons.

Persistent DNA damage alters the neuronal transcriptome suggesting cell cycle dysregulation and altered mitochondrial function.

Oxidative DNA damage induces changes in the neuronal cell cycle and activates a DNA damage response (DDR) to promote repair, but these processes may be altered under a chronic oxidative environment, leading to the accumulation of unrepaired DNA damage and continued activation of a DDR. Failure to repair DNA damage can lead to apoptosis or senescence, which is characterized by a permanent cell cycle arrest. Increased oxidative stress and accumulation of oxidative DNA damage are features of brain ageing and neurodegeneration, but the effects of persistent DNA damage in neurons are not well characterized. We developed a model of persistent oxidative DNA damage in immortalized post-mitotic neurons in vitro by exposing them to a sublethal concentration of hydrogen peroxide following a 'double stress' protocol and performed a detailed characterization of the neuronal transcriptome using microarray analysis. Persistent DNA damage significantly altered the expression of genes involved in cell cycle regulation, DDR and repair mechanisms, and mitochondrial function, suggesting an active DDR response to replication stress and alterations in mitochondrial electron transport chain. Quantitative polymerase chain reaction (qPCR) and functional validation experiments confirmed hyperactivation of mitochondrial Complex I in response to persistent DNA damage. These changes in response to persistent oxidative DNA damage may lead to further oxidative stress, contributing to neuronal dysfunction and ultimately neurodegeneration.

Genetic dissection of the glutamatergic neuron system in cerebral cortex

Diverse types of glutamatergic pyramidal neurons mediate the myriad processing streams and output channels of the cerebral cortex1,2, yet all derive from neural progenitors of the embryonic dorsal telencephalon3,4. Here we establish genetic strategies and tools for dissecting and fate-mapping subpopulations of pyramidal neurons on the basis of their developmental and molecular programs. We leverage key transcription factors and effector genes to systematically target temporal patterning programs in progenitors and differentiation programs in postmitotic neurons. We generated over a dozen temporally inducible mouse Cre and Flp knock-in driver lines to enable the combinatorial targeting of major progenitor types and projection classes. Combinatorial strategies confer viral access to subsets of pyramidal neurons defined by developmental origin, marker expression, anatomical location and projection targets. These strategies establish an experimental framework for understanding the hierarchical organization and developmental trajectory of subpopulations of pyramidal neurons that assemble cortical processing networks and output channels.

BAF45b Is Required for Efficient Zika Virus Infection of HAP1 Cells.

The 2016 Zika virus (ZIKV) epidemic illustrates the impact of flaviviruses as emerging human pathogens. For unknown reasons, ZIKV replicates more efficiently in neural progenitor cells (NPCs) than in postmitotic neurons. Here, we identified host factors used by ZIKV using the NCI-60 library of cell lines and COMPARE analysis, and cross-analyzed this library with two other libraries of host factors with importance for ZIKV infection. We identified BAF45b, a subunit of the BAF (Brg1/Brm-associated factors) protein complexes that regulate differentiation of NPCs to post-mitotic neurons. ZIKV (and other flaviviruses) infected HAP1 cells deficient in expression of BAF45b and other BAF subunits less efficiently than wildtype (WT) HAP1 cells. We concluded that subunits of the BAF complex are important for infection of ZIKV and other flavivirus. Given their function in cell and tissue differentiation, such regulators may be important determinants of tropism and pathogenesis of arthropod-borne flaviviruses.

Protocols for Characterization of Cdk5 Kinase Activity.

Cyclin-dependent kinases (Cdks) are generally known to be involved in controlling the cell cycle, but Cdk5 is a unique member of this protein family for being most active in post-mitotic neurons. Cdk5 is developmentally important in regulating neuronal migration, neurite outgrowth, and axon guidance. Cdk5 is enriched in synaptic membranes and is known to modulate synaptic activity. Postnatally, Cdk5 can also affect neuronal processes such as dopaminergic signaling and pain sensitivity. Dysregulated Cdk5, in contrast, has been linked to neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Despite primarily being implicated in neuronal development and activity, Cdk5 has lately been linked to non-neuronal functions including cancer cell growth, immune responses, and diabetes. Since Cdk5 activity is tightly regulated, a method for measuring its kinase activity is needed to fully understand the precise role of Cdk5 in developmental and disease processes. This article includes methods for detecting Cdk5 kinase activity in cultured cells or tissues, identifying new substrates, and screening for new kinase inhibitors. Furthermore, since Cdk5 shares homology and substrate specificity with Cdk1 and Cdk2, the Cdk5 kinase assay can be used, with modification, to measure the activity of other Cdks as well. © 2021 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Measuring Cdk5 activity from protein lysates Support Protocol 1: Immunoprecipitation of Cdk5 using Dynabeads Alternate Protocol: Non-radioactive protocols to measure Cdk5 kinase activity Support Protocol 2: Western blot analysis for the detection of Cdk5, p35, and p39 Support Protocol 3: Immunodetection analysis for Cdk5, p35, and p39 Support Protocol 4: Genetically engineered mice (+ and - controls) Basic Protocol 2: Identifying new Cdk5 substrates and kinase inhibitors.

Phospho-Tau and Chromatin Landscapes in Early and Late Alzheimer's Disease.

Cellular identity is determined through complex patterns of gene expression. Chromatin, the dynamic structure containing genetic information, is regulated through epigenetic modulators, mainly by the histone code. One of the main challenges for the cell is maintaining functionality and identity, despite the accumulation of DNA damage throughout the aging process. Replicative cells can remain in a senescent state or develop a malign cancer phenotype. In contrast, post-mitotic cells such as pyramidal neurons maintain extraordinary functionality despite advanced age, but they lose their identity. This review focuses on tau, a protein that protects DNA, organizes chromatin, and plays a crucial role in genomic stability. In contrast, tau cytosolic aggregates are considered hallmarks of Alzheimer´s disease (AD) and other neurodegenerative disorders called tauopathies. Here, we explain AD as a phenomenon of chromatin dysregulation directly involving the epigenetic histone code and a progressive destabilization of the tau–chromatin interaction, leading to the consequent dysregulation of gene expression. Although this destabilization could be lethal for post-mitotic neurons, tau protein mediates profound cellular transformations that allow for their temporal survival.

Notch-mediated re-specification of neuronal identity during central nervous system development

Neuronal identity has long been thought of as immutable, so that once a cell acquires a specific fate, it is maintained for life.1 Studies using the overexpression of potent transcription factors to experimentally reprogram neuronal fate in the mouse neocortex2,3 and retina4,5 have challenged this notion by revealing that post-mitotic neurons can switch their identity. Whether fate reprogramming is part of normal development in the central nervous system (CNS) is unclear. While there are some reports of physiological cell fate reprogramming in invertebrates,6,7 and in the vertebrate peripheral nervous system,8 endogenous fate reprogramming in the vertebrate CNS has not been documented. Here, we demonstrate spontaneous fate re-specification in an interneuron lineage in the zebrafish retina. We show that the visual system homeobox 1 (vsx1)-expressing lineage, which has been associated exclusively with excitatory bipolar cell (BC) interneurons,9-12 also generates inhibitory amacrine cells (ACs). We identify a role for Notch signaling in conferring plasticity to nascent vsx1 BCs, allowing suitable transcription factor programs to re-specify them to an AC fate. Overstimulating Notch signaling enhances this physiological phenotype so that both daughters of a vsx1 progenitor differentiate into ACs and partially differentiated vsx1 BCs can be converted into ACs. Furthermore, this physiological re-specification can be mimicked to allow experimental induction of an entirely distinct fate, that of retinal projection neurons, from the vsx1 lineage. Our observations reveal unanticipated plasticity of cell fate during retinal development.

An XRCC4 mutant mouse, a model for human X4 syndrome, reveals interplays with Xlf, PAXX, and ATM in lymphoid development.

We developed an Xrcc4M61R separation of function mouse line to overcome the embryonic lethality of Xrcc4-deficient mice. XRCC4M61R protein does not interact with Xlf, thus obliterating XRCC4-Xlf filament formation while preserving the ability to stabilize DNA ligase IV. X4M61R mice, which are DNA repair deficient, phenocopy the Nhej1-/- (known as Xlf -/-) setting with a minor impact on the development of the adaptive immune system. The core non-homologous end-joining (NHEJ) DNA repair factor XRCC4 is therefore not mandatory for V(D)J recombination aside from its role in stabilizing DNA ligase IV. In contrast, Xrcc4M61R mice crossed on Paxx-/-, Nhej1-/-, or Atm-/- backgrounds are severely immunocompromised, owing to aborted V(D)J recombination as in Xlf-Paxx and Xlf-Atm double Knock Out (DKO) settings. Furthermore, massive apoptosis of post-mitotic neurons causes embryonic lethality of Xrcc4M61R -Nhej1-/- double mutants. These in vivo results reveal new functional interplays between XRCC4 and PAXX, ATM and Xlf in mouse development and provide new insights into the understanding of the clinical manifestations of human XRCC4-deficient condition, in particular its absence of immune deficiency.

Molecular targets and approaches to restore autophagy and lysosomal capacity in neurodegenerative disorders

Autophagy is a catabolic process that promotes cellular fitness by clearing aggregated protein species, pathogens and damaged organelles through lysosomal degradation. The autophagic process is particularly important in the nervous system where post-mitotic neurons rely heavily on protein and organelle quality control in order to maintain cellular health throughout the lifetime of the organism. Alterations of autophagy and lysosomal function are hallmarks of various neurodegenerative disorders. In this review, we conceptualize some of the mechanistic and genetic evidence pointing towards autophagy and lysosomal dysfunction as a causal driver of neurodegeneration. Furthermore, we discuss rate-limiting pathway nodes and potential approaches to restore pathway activity, from autophagy initiation, cargo sequestration to lysosomal capacity.

Discrete cis-acting element regulates developmentally timed gene-lamina relocation and neural progenitor competence invivo.

SUMMARYThe nuclear lamina is typically associated with transcriptional silencing, and peripheral relocation of genes highly correlates with repression. However, the DNA sequences and proteins regulating gene-lamina interactions are largely unknown. Exploiting the developmentally timed hunchback gene movement to the lamina in Drosophila neuroblasts, we identified a 250 bp intronic element (IE) both necessary and sufficient for relocation. The IE can target a reporter transgene to the lamina and silence it. Endogenously, however, hunchback is already repressed prior to relocation. Instead, IE-mediated relocation confers a heritably silenced gene state refractory to activation in descendent neurons, which terminates neuroblast competence to specify early-born identity. Surprisingly, we found that the Polycomb group chromatin factors bind the IE and are required for lamina relocation, revealing a nuclear architectural role distinct from their well-known function in transcriptional repression. Together, our results uncover in vivo mechanisms underlying neuroblast competence and lamina association in heritable gene silencing.

Identification of neural progenitor cells and their progeny reveals long distance migration in the developing octopus brain.

Cephalopods have evolved nervous systems that parallel the complexity of mammalian brains in terms of neuronal numbers and richness in behavioral output. How the cephalopod brain develops has only been described at the morphological level, and it remains unclear where the progenitor cells are located and what molecular factors drive neurogenesis. Using histological techniques, we located dividing cells, neural progenitors and postmitotic neurons in Octopus vulgaris embryos. Our results indicate that an important pool of progenitors, expressing the conserved bHLH transcription factors achaete-scute or neurogenin, is located outside the central brain cords in the lateral lips adjacent to the eyes, suggesting that newly formed neurons migrate into the cords. Lineage-tracing experiments then showed that progenitors, depending on their location in the lateral lips, generate neurons for the different lobes, similar to the squid Doryteuthis pealeii. The finding that octopus newborn neurons migrate over long distances is reminiscent of vertebrate neurogenesis and suggests it might be a fundamental strategy for large brain development.

Sustained expression of unc-4 homeobox gene and unc-37/Groucho in postmitotic neurons specifies the spatial organization of the cholinergic synapses in C. elegans.

Neuronal cell fate determinants establish the identities of neurons by controlling gene expression to regulate neuronal morphology and synaptic connectivity. However, it is not understood if neuronal cell fate determinants have postmitotic functions in synapse pattern formation. Here we identify a novel role for UNC-4 homeobox protein and its corepressor UNC-37/Groucho, in tiled synaptic patterning of the cholinergic motor neurons in Caenorhabditis elegans. We show that unc-4 is not required during neurogenesis but is required in the postmitotic neurons for proper synapse patterning. In contrast, unc-37 is required in both developing and postmitotic neurons. The synaptic tiling defects of unc-4 mutants are suppressed by bar-1/β-catenin mutation, which positively regulates the expression of ceh-12/HB9. Ectopic ceh-12 expression partly underlies the synaptic tiling defects of unc-4 and unc-37 mutants. Our results reveal a novel postmitotic role of neuronal cell fate determinants in synapse pattern formation through inhibiting the canonical Wnt signaling pathway.

Role of Hydrazine-Related Chemicals in Cancer and Neurodegenerative Disease.

Hydrazine-related chemicals (HRCs) with carcinogenic and neurotoxic potential are found in certain mushrooms and plants used for food and in products employed in various industries, including aerospace. Their propensity to induce DNA damage (mostly O6-, N7- and 8-oxo-guanine lesions) resulting in multiple downstream effects is linked with both cancer and neurological disease. For cycling cells, unrepaired DNA damage leads to mutation and uncontrolled mitosis. By contrast, postmitotic neurons attempt to re-enter the cell cycle but undergo apoptosis or nonapoptotic cell death. Biomarkers of exposure to HRCs can be used to explore whether these substances are risk factors for sporadic amyotrophic laterals sclerosis and other noninherited neurodegenerative diseases, which is the focus of this paper.

GABAB Receptor-Mediated Impairment of Intermediate Progenitor Maturation During Postnatal Hippocampal Neurogenesis of Newborn Rats.

The neurotransmitter GABA and its receptors assume essential functions during fetal and postnatal brain development. The last trimester of a human pregnancy and early postnatal life involves a vulnerable period of brain development. In the second half of gestation, there is a developmental shift from depolarizing to hyperpolarizing in the GABAergic system, which might be disturbed by preterm birth. Alterations of the postnatal GABA shift are associated with several neurodevelopmental disorders. In this in vivo study, we investigated neurogenesis in the dentate gyrus (DG) in response to daily administration of pharmacological GABAA (DMCM) and GABAB (CGP 35348) receptor inhibitors to newborn rats. Six-day-old Wistar rats (P6) were daily injected (i.p.) to postnatal day 11 (P11) with DMCM, CGP 35348, or vehicle to determine the effects of both antagonists on postnatal neurogenesis. Due to GABAB receptor blockade by CGP 35348, immunohistochemistry revealed a decrease in the number of NeuroD1 positive intermediate progenitor cells and a reduction of proliferative Nestin-positive neuronal stem cells at the DG. The impairment of hippocampal neurogenesis at this stage of differentiation is in line with a significantly decreased RNA expression of the transcription factors Pax6, Ascl1, and NeuroD1. Interestingly, the number of NeuN-positive postmitotic neurons was not affected by GABAB receptor blockade, although strictly associated transcription factors for postmitotic neurons, Tbr1, Prox1, and NeuroD2, displayed reduced expression levels, suggesting impairment by GABAB receptor antagonization at this stage of neurogenesis. Antagonization of GABAB receptors decreased the expression of neurotrophins (BDNF, NT-3, and NGF). In contrast to the GABAB receptor blockade, the GABAA receptor antagonization revealed no significant changes in cell counts, but an increased transcriptional expression of Tbr1 and Tbr2. We conclude that GABAergic signaling via the metabotropic GABAB receptor is crucial for hippocampal neurogenesis at the time of rapid brain growth and of the postnatal GABA shift. Differentiation and proliferation of intermediate progenitor cells are dependent on GABA. These insights become more pertinent in preterm infants whose developing brains are prematurely exposed to spostnatal stress and predisposed to poor neurodevelopmental disorders, possibly as sequelae of early disruption in GABAergic signaling.

Mid-pregnancy maternal immune activation increases Pax6-positive and Tbr2-positive neural progenitor cells and causes integrated stress response in the fetal brain in a mouse model of maternal viral infection.

Maternal immune activation (MIA) in midpregnancy is a risk factor for neurodevelopmental disorders. Improper brain development may cause malformations of the brain; maldevelopment induced by MIA may lead to a pathology-related phenotype. In this study, a single intraperitoneal injection of 20 mg/kg polyriboinosinic–polyribocytidylic acid [poly(I:C)] was administered to C57BL/6J mice on embryonic day (E) 12.5 to mimic maternal viral infection. Histopathological analysis of neurogenesis was performed using markers for Pax6, Tbr2, and Tbr1. In these fetuses, significant increases were observed in the proportion of Pax6-positive neural progenitor cells and Pax6/Tbr2 double-positive cells 24 h after poly(I:C) injection. There were no differences in the proportion of Tbr1-positive postmitotic neurons 48 h after poly(I:C) injection. At E18.5, there were more Pax6-positive and Tbr2-positive neural progenitor cells in the poly(I:C)-injected group than in the saline-injected group. Gene ontology enrichment analysis of poly(I:C)-induced differentially expressed genes in the fetal brain at E12.5 demonstrated that these genes were enriched in terms including response to cytokine, response to decreased oxygen levels in the category of biological process. At E13.5, activating transcription factor 4 (Atf4), which is an effector of integrated stress response, was significantly upregulated in the fetal brain. Our results show that poly(I:C)-induced MIA at E12.5 leads to dysregulated neurogenesis and upregulates Atf4 in the fetal brain. These findings provide a new insight in the mechanism of MIA causing improper brain development and subsequent neurodevelopmental disorders.

Necdin: A purposive integrator of molecular interaction networks for mammalian neuron vitality

Necdin was originally found in 1991 as a hypothetical protein encoded by a neural differentiation‐specific gene transcript in murine embryonal carcinoma cells. Virtually all postmitotic neurons and their precursor cells express the necdin gene (Ndn) during neuronal development. Necdin mRNA is expressed only from the paternal allele through genomic imprinting, a placental mammal‐specific epigenetic mechanism. Necdin and its homologous MAGE (melanoma antigen) family, which have evolved presumedly from a subcomplex component of the SMC5/6 complex, are expressed exclusively in placental mammals. Paternal Ndn‐mutated mice totally lack necdin expression and exhibit various types of neuronal abnormalities throughout the nervous system. Ndn‐null neurons are vulnerable to detrimental stresses such as DNA damage. Necdin also suppresses both proliferation and apoptosis of neural stem/progenitor cells. Functional analyses using Ndn‐manipulated cells reveal that necdin consistently exerts antimitotic, anti‐apoptotic and prosurvival effects. Necdin interacts directly with a number of regulatory proteins including E2F1, p53, neurotrophin receptors, Sirt1 and PGC‐1α, which serve as major hubs of protein–protein interaction networks for mitosis, apoptosis, differentiation, neuroprotection and energy homeostasis. This review focuses on necdin as a pleiotropic protein that integrates molecular interaction networks to promote neuronal vitality in modern placental mammals.

Three-dimensional chromatin organization in brain function and dysfunction.

The three-dimensional (3D) organization of chromatin within the nucleus is now recognized as a bona fide epigenetic property influencing genome function, replication, and maintenance. In the recent years, several studies have revealed how 3D chromatin organization is associated with brain function and its emerging role in disorders of the brain. 3D chromatin organization plays a crucial role in the development of different cell types of the nervous system and some neuronal cell types have adapted unique modifications to this organization that deviates from all other cell types. In post-mitotic neurons, dynamic changes in chromatin interactions in response to neuronal activity underlie learning and memory formation. Finally, new evidence directly links 3D chromatin organization to several disorders of the brain. These recent findings position 3D chromatin organization as a fundamental regulatory mechanism poised to reveal the etiology of brain function and dysfunctions.

Gadd45α is involved in regulating activity-dependent and exon-specific BDNF expression in postmitotic cortical neurons.

This study aimed to explore the epigenetic regulation of activity-dependent and exon-specific brain-derived neurotrophic factor (BDNF) expression under KCl depolarization in primary cortical neurons. We investigated BDNF exon I, exon IV and the growth arrest and DNA damage-inducible protein 45 alpha (Gadd45α) transcription levels under KCl-induced neuronal activation in postmitotic neurons. Gadd45α occupancy at BDNF I and IV promoter was measured by chromatin immunoprecipitation (ChIP) followed by quantitative PCR; DNA methylation level was checked by methylated DNA immunoprecipitation (MeDIP) followed by qPCR. In addition, lentiviral shRNA targeting Gadd45α was used to knockdown Gadd45α expression. BDNF exon I and IV mRNA expressions were both highly induced by KCl depolarization. However, ChIP analysis demonstrated a significantly increased Gadd45α occupancy only at the BDNF P1 promotor, but not P4, which is associated with reducing DNA methylation within BDNF P1 promoter. Furthermore, after the lentiviral-mediated knockdown of Gadd45α, the increased Gadd45α occupancy at the BDNF P1 was inhibited, which was accompanying the complete blocking of the demethylation effect at P1. Nonetheless, the induction of BDNF exon I mRNA by KCl was only partially prevented by Gadd45α shRNA, indicting other mechanisms involved in regulating BDNF exon I expression. DNA demethylation mediated by Gadd45α protein involves promoting the regulation of activity-dependent BDNF exon I expression in neurons.

RGCC balances self-renewal and neuronal differentiation of neural stem cells in the developing mammalian neocortex.

During neocortical development, neural stem cells (NSCs) divide symmetrically to self-renew at the early stage and then divide asymmetrically to generate post-mitotic neurons. The molecular mechanisms regulating the balance between NSC self-renewal and neurogenesis are not fully understood. Using mouse in utero electroporation (IUE) technique and invitro human NSC differentiation models including cerebral organoids (hCOs), we show here that regulator of cell cycle (RGCC) modulates NSC self-renewal and neuronal differentiation by affecting cell cycle regulation and spindle orientation. RGCC deficiency hampers normal cell cycle process and dysregulates the mitotic spindle, thus driving more cells to divide asymmetrically. These modulations diminish the NSC population and cause NSC pre-differentiation that eventually leads to brain developmental malformation in hCOs. We further show that RGCC might regulate NSC spindle orientation by affecting the organization of centrosome and microtubules. Our results demonstrate that RGCC is essential to maintain the NSC pool during cortical development and suggest that RGCC defects could have etiological roles in human brain malformations.