Neural Cell Cultures Research Articles

Chitosan-alginate porous scaffold incorporated with hydroalcoholic Zingiber officinale Roscoe extract for neural tissue engineering

Context: Preparing a suitable substrate for the culture of neural stem cells and their proliferation in neural tissue engineering is of paramount importance. Aims: To evaluate the effect of the hydroalcoholic Zingiber officinale extract incorporated in the chitosan-alginate scaffold (Chi-Alg-Zo) on nerve tissue. Methods: The porous scaffolds developed in the present study were investigated in terms of their surface properties, chemical interaction, crystallinity, thermal stability, porosity percentage, pore sizes, degradability, and water absorption properties. To this end, the following tests were performed: Field emission scanning electron microscope (FE-SEM), Fourier-transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), thermal gravimetric analysis (TGA), porosity based on liquid replacement, Image-J analysis, and water degradation and absorption test. Mouse neural stem/progenitor cells (NS/PCs) were harvested from the embryonic mouse brain. NSCs were seeded on scaffolds incorporated with hydroalcoholic Z. officinale extract. The MTT assay was done for the survival and the proliferation of neural stem/progenitor cells (NS/PCs) on scaffolds. Results: Results indicated the good capacity of Chi-Alg-Zo for proliferation and differentiation into glial (astrocytes and oligodendrocytes) lineages. A suitable surface, which was provided for cellular interaction, led to the advancement of cell survival, connectivity, proliferation, and separation of NSCs. Conclusions: The present study evaluated the separation of stem cells on the scaffold, finding that the expression of the glial fibrillary acidic protein (GFAP) and Oligo4 markers was higher in Chi-Alg scaffolds containing hydroalcoholic Z. officinale extract. Chi-Alg-Zo scaffolds could be suitable candidates for neural tissue engineering.

Characterisation of mesenchymal stem cells conditioned media obtained at different conditioning times: their effect on glial cells in in vitro scratch model

In this study, the bone marrow mesenchymal stem cells conditioned media (BMMSC-CM) obtained by conditioning for 24(CM24), 48(CM48) and 72(CM72) hours was characterised. In vitro, the impact of BMMSC-CM on the astrocyte migratory response and oligodendrocyte density was evaluated using the scratch model. The proteomic profiles of individual secretomes were analysed by mass spectrometry and the concentrations of four selected neurotrophins (BDNF, NGF, GDNF and VEGF) were determined by ELISA. Our results revealed an increased number of proteins at CM72, many of which are involved in neuroregenerative processes. ELISA documented a gradual increase in the concentration of two neurotrophins (NGF, VEGF), peaking at CM72. In vitro, the different effect of individual BMMSC-CM on astrocyte migration response and oligodendrocyte density was observed, most pronounced with CM72. The outcomes demonstrate that the prolonged conditioning results in increased release of detectable proteins, neurotrophic factors concentration and stronger effect on reparative processes in neural cell cultures.

Development of Brain-Derived Bioscaffolds for Neural Progenitor Cell Culture.

Biomaterials derived from brain extracellular matrix (ECM) have the potential to promote neural tissue regeneration by providing instructive cues that can direct cell survival, proliferation, and differentiation. This study focused on the development and characterization of microcarriers derived from decellularized brain tissue (DBT) as a platform for neural progenitor cell culture. First, a novel detergent-free decellularization protocol was established that effectively reduced the cellular content of porcine and rat brains, with a >97% decrease in the dsDNA content, while preserving collagens (COLs) and glycosaminoglycans (GAGs). Next, electrospraying methods were applied to generate ECM-derived microcarriers incorporating the porcine DBT that were stable without chemical cross-linking, along with control microcarriers fabricated from commercially sourced bovine tendon COL. The DBT microcarriers were structurally and biomechanically similar to the COL microcarriers, but compositionally distinct, containing a broader range of COL types and higher sulfated GAG content. Finally, we compared the growth, phenotype, and neurotrophic factor gene expression levels of rat brain-derived progenitor cells (BDPCs) cultured on the DBT or COL microcarriers within spinner flask bioreactors over 2 weeks. Both microcarrier types supported BDPC attachment and expansion, with immunofluorescence staining results suggesting that the culture conditions promoted BDPC differentiation toward the oligodendrocyte lineage, which may be favorable for cell therapies targeting remyelination. Overall, our findings support the further investigation of the ECM-derived microcarriers as a platform for neural cell derivation for applications in regenerative medicine.

Mechanical dissection and culture of mouse cranial neural crest cells.

Owing to the contribution of cranial neural crest cells (CNCCs) to the majority of craniofacial structures, they have been studied extensively for the pathogenesis of craniofacial diseases. To investigate and summarize how to isolate and culture the CNCCs from wild-type mice, a literature search was performed in online databases (PubMed and Web of Science) using optimized keywords "mouse," "cranial neural crest cell" and "culture." The literature was checked by two investigators according to the screening and exclusion criteria. Initially, 197 studies were retrieved from PubMed and 169 from Web of Science, and after excluding replicate studies, 293 articles were considered. Finally, 17 studies met all the criteria and were included in this review. The results showed that obtaining purified stem cells and balancing the need to promote cell growth and prevent unwanted early cell differentiation were the two key points in the isolation and culture of CNCCs. However, no standard criteria are available for answering these questions. Thus, it is important to emphasize the necessity for standardization of CNCC isolation, culture, and identification in research on craniofacial diseases.

High aspect ratio arrays of Si nano-pillars using displacement Talbot lithography and gas-MacEtch

Structuring Si in arrays of vertical high aspect ratio pillars, ranging from nanoscale to macroscale feature dimensions, is essential for producing functional interfaces for many applications. Arrays of silicon 3D nanostructures are needed to realize photonic and phononic crystals, waveguides, metalenses, X-ray wavefront sensors, detectors. In particular, arrays of Si nanopillars are used as bio-interfaces in neural activity recording, cell culture, microfluidics, sensing and on-chip manipulation. Here, we demonstrate a strategy for realizing arrays of protruding sharp Si nanopillars, using displacement Talbot lithography combined with metal-assisted chemical etching (MacEtch) in gas phase. Such combination enables reliable and low cost pathway for fabrication of ordered nanopillars arrays on large scale. With the double exposure of a linear grating mask in orthogonal orientations and the lift-off technique, we realized a catalyst pattern of holes in a Pt thin film with a period of 1 μm and hole diameter in the range of 100–250 nm. MacEtch in gas phase by using vapor HF and oxygen from air allows to etch arrays of protruding Si nanopillars 200 nm-thick and aspect ratio in the range of 200 (pillar height/width) with an etching rate up to 1 μm/min. Gas-MacEtch has the advantage of no capillary stiction, no ion beam damage of the Si substrate, nanometric resolution and high fidelity of pattern transfer. In combination with controlled uniformity of feature size on large area, spatial frequency doubling and high resolution of Talbot lithography, the proposed method is an easy-to-scale-up processing that can support the fabrication of Si pillars arrays for many valuable applications both at micro and nano-scale.

Ex vivo analysis platforms for monitoring amyloid precursor protein cleavage.

Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder and the most common cause of dementia in the elderly. The presence of large numbers of senile plaques, neurofibrillary tangles, and cerebral atrophy is the characteristic feature of AD. Amyloid β peptide (Aβ), derived from the amyloid precursor protein (APP), is the main component of senile plaques. AD has been extensively studied using methods involving cell lines, primary cultures of neural cells, and animal models; however, discrepancies have been observed between these methods. Dissociated cultures lose the brain's tissue architecture, including neural circuits, glial cells, and extracellular matrix. Experiments with animal models are lengthy and require laborious monitoring of multiple parameters. Therefore, it is necessary to combine these experimental models to understand the pathology of AD. An experimental platform amenable to continuous observation and experimental manipulation is required to analyze long-term neuronal development, plasticity, and progressive neurodegenerative diseases. In the current study, we provide a practical method to slice and cultivate rodent hippocampus to investigate the cleavage of APP and secretion of Aβ in an ex vivo model. Furthermore, we provide basic information on Aβ secretion using slice cultures. Using our optimized method, dozens to hundreds of long-term stable slice cultures can be coordinated simultaneously. Our findings are valuable for analyses of AD mouse models and senile plaque formation culture models.

Mass spectrometry-based quantitation combined with time-dependent metabolomics to discover metabolic features in human neurogenesis using neural constructs generated from neural progenitor cells.

Direct studies focusing on the human brain are difficult to plan and conduct due to ethical and practical reasons. The advent of human pluripotent stem cell (hPSC)-derived neurons has revolutionized the research of the human brain and central nervous system, but relevant analytical techniques have been much less explored. Herein, we have designed a novel bioanalytical strategy to discover the characteristics of human neurogenesis using liquid chromatography-mass spectrometry-based quantitation and time-dependent metabolomics in combination with hPSC-derived neural constructs. To examine the growth of neurons in vitro, a quantitative method for the simultaneous measurement of N-acetylaspartic acid (NAA) and N-acetylglutamic acid (NAG) in a culture medium was established. The analysis of endogenous NAA and NAG concentrations over 28 days of neural cell culture not only illustrated the growth and maturation process of neural progenitors, but also confirmed the successful achievement of human neural constructs. Depending on the quantitative results, day 0, 10, 18, and 28 samples representing different growth phases were selected for further investigation of the global metabolic changes in developing human neurons. A versatile non-targeted, time-dependent metabolomics study identified 17 significantly changed metabolites and revealed the altered metabolic pathways including amino acid metabolism (tryptophan, phenylalanine, aspartate and beta-alanine metabolisms), pantothenate and coenzyme A biosynthesis, fatty acid metabolism, and purine and pyrimidine metabolism. The new metabolite profiles and overall metabolic pathways advance our understanding of human neurodevelopment. Additionally, the bioanalytical approach proposed in this study opens an interesting window for the capture and evaluation of the complex metabolic states of human neural cells, which would potentially be utilized in other in vitro models relevant to pathophysiology and treatment of neurological disorders, benefiting biomarker discovery and metabolic mechanism interpretation.

Characteristic of migration in culture of newborn and fetal rat neural cells

Characteristic of migration in culture of newborn and fetal rat neural cells

Message in a Scaffold: Natural Biomaterials for Three-Dimensional (3D) Bioprinting of Human Brain Organoids

Brain organoids are invaluable tools for pathophysiological studies or drug screening, but there are still challenges to overcome in making them more reproducible and relevant. Recent advances in three-dimensional (3D) bioprinting of human neural organoids is an emerging approach that may overcome the limitations of self-organized organoids. It requires the development of optimal hydrogels, and a wealth of research has improved our knowledge about biomaterials both in terms of their intrinsic properties and their relevance on 3D culture of brain cells and tissue. Although biomaterials are rarely biologically neutral, few articles have reviewed their roles on neural cells. We here review the current knowledge on unmodified biomaterials amenable to support 3D bioprinting of neural organoids with a particular interest in their impact on cell homeostasis. Alginate is a particularly suitable bioink base for cell encapsulation. Gelatine is a valuable helper agent for 3D bioprinting due to its viscosity. Collagen, fibrin, hyaluronic acid and laminin provide biological support to adhesion, motility, differentiation or synaptogenesis and optimize the 3D culture of neural cells. Optimization of specialized hydrogels to direct differentiation of stem cells together with an increased resolution in phenotype analysis will further extend the spectrum of possible bioprinted brain disease models.

Pectin-based bioinks for 3D models of neural tissue produced by a pH-controlled kinetics.

Introduction: In the view of 3D-bioprinting with cell models representative of neural cells, we produced inks to mimic the basic viscoelastic properties of brain tissue. Moving from the concept that rheology provides useful information to predict ink printability, this study improves and expands the potential of the previously published 3D-reactive printing approach by introducing pH as a key parameter to be controlled, together with printing time. Methods: The viscoelastic properties, printability, and microstructure of pectin gels crosslinked with CaCO3 were investigated and their composition was optimized (i.e., by including cell culture medium, HEPES buffer, and collagen). Different cell models representative of the major brain cell populations (i.e., neurons, astrocytes, microglial cells, and oligodendrocytes) were considered. Results and Discussion: The outcomes of this study propose a highly controllable method to optimize the printability of internally crosslinked polysaccharides, without the need for additives or post-printing treatments. By introducing pH as a further parameter to be controlled, it is possible to have multiple (pH-dependent) crosslinking kinetics, without varying hydrogel composition. In addition, the results indicate that not only cells survive and proliferate following 3D-bioprinting, but they can also interact and reorganize hydrogel microstructure. Taken together, the results suggest that pectin-based hydrogels could be successfully applied for neural cell culture.

CBP and p300 Jointly Maintain Neural Progenitor Viability but Play Unique Roles in the Differentiation of Neural Lineages.

The paralogous lysine acetyltransferases 3 (KAT3), CBP and P300, play critical roles during neurodevelopment, but their specific roles in neural precursors maintenance and differentiation remain obscure. In fact, it is still unclear whether these proteins are individually or jointly essential in processes such as proliferation of neural precursors, differentiation to specific neural cell types, or both. Here, we use subventricular zone-derived neurospheres as a potential ex vivo developmental model to analyze the proliferation and differentiation of neural stem cells (NSCs) lacking CBP, p300, or both proteins. The results showed that CBP and p300 are not individually essential for maintenance and proliferation of NSCs, although their combined ablation seriously compromised cell division. In turn, the absence of either of the two proteins compromised the differentiation of NSC into the neuronal and astrocytic lineages. Single-nucleus RNA sequencing analysis of neural cell cultures derived from CBP or p300 mutant neurospheres revealed divergent trajectories of neural differentiation upon CBP or p300 ablation, confirming unique functions and nonredundant roles in neural development. These findings contribute to a better understanding of the shared and individual roles of KAT3 proteins in neural differentiation and the etiology of neurodevelopmental disorders caused by their deficiency.

Potential Involvement of Varicella Zoster Virus in Alzheimer’s Disease/Dementia via Reactivation of Herpes Simplex Virus Type 1

AbstractBackgroundInfections are associated with cognitive decline and development of AD/dementia. Epidemiological studies have revealed an increased AD/dementia risk after shingles, caused by varicella zoster virus (VZV), and/or a decreased risk after vaccination against shingles. This suggests that VZV might reside latently in brain, and on reactivation might cause direct damage in brain, as is thought to be the case with herpes simplex virus type 1 (HSV1), a virus strongly implicated in AD. Alternatively, shingles‐induced inflammation could lead to neuroinflammation and reactivation of HSV1 in brain, i.e., to an indirect consequence – a suggestion previously made to account for the risk of AD/dementia conferred by infections (Itzhaki and Dobson 2002), and for the reduction in risk after certain vaccinations ‐ by preventing occurrence of infection and consequent reactivation. Our aim was to investigate these possibilities by infecting cell cultures with VZV.MethodExperiments were carried out infecting human‐induced neural stem cells (hiNSC) cultures, and cultures quiescently infected with HSV1 with VZV. Cultures were observed for the presence of AD‐related phenotypes such as beta amyloid and P‐tau accumulation, gliosis, and neuroinflammation.ResultCells infected with VZV do not show the main AD characteristics ‐ beta amyloid and P‐tau accumulation – which HSV1 does cause ‐ but do show gliosis and increased levels of several cytokines, suggesting that VZV’s action is indirect. However, very intriguingly, we found that VZV infection of cells quiescently infected with HSV1 leads to reactivation of HSV1 and consequent AD‐like changes, including beta amyloid and P‐tau accumulation, supporting the above infection‐reactivation concept.ConclusionOur results support a role for VZV in AD/dementia via reactivation of HSV1 in brain and in combination with some current preliminary results, indicate that the risk of cognitive decline and of dementia conferred by some infectious diseases might indeed result from their causing HSV1 reactivation in brain.

Establishment of neural stem cell culture from the central nervous system of the Iberian ribbed newt Pleurodeles waltl.

Urodele amphibians have exceptional regeneration ability in various organs. Among these, the Iberian ribbed newt (Pleurodeles waltl) has emerged as a useful model organism for investigating the mechanisms underlying regeneration. Neural stem cells (NSCs) are an important source of regeneration in the central nervous system (CNS) and their culture method in vitro has been well established. NSCs form spherical cell aggregates called neurospheres and their formation has been demonstrated in various vertebrates, including some urodele species, but not in P. waltl. In this study, we reported neurosphere formation in brain- and spinal cord-derived cells of post-metamorphic P. waltl. These neurospheres showed proliferative activity and similar expression of marker proteins. However, the surface morphology was found to vary according to their origin, implying that the characteristics of the neurospheres generated from the brain and spinal cord could be similar but not identical. Subsequent in vitro differentiation analysis demonstrated that spinal cord-derived neurospheres gave rise to neurons and glial cells. We also found that cells in neurospheres from P. waltl differentiated to oligodendrocytes, whereas those from axolotls were reported not to differentiate to this cell type under standard culture conditions. Based on our findings, implantation of genetically modified neurospheres together with associated technical advantages in P. waltl could reveal pivotal gene(s) and/or signaling pathway(s) essential for the complete spinal cord regeneration ability in the future.

Soft surfaces induce neural differentiation via the neuron restrictive silencer factor

The physical and mechanical environment influence the differentiation and culture of stem cells. Early research in this field showed that mesenchymal stem cells (MSCs) adopt preferential lineages when cultured on surfaces of varying stiffness. Culture on very soft surfaces promoted neural differentiation. This effect has been observed in mesenchymal and embryonic stem cells and was shown to increase development of neural stem cells. However, providing further information on the molecular characterization of this phenomenon could help to guide the development of materials that enhance neural differentiation. Previously, our laboratory showed that chemical cues, forskolin and IBMX, induced neural differentiation of MSCs by downregulating the Neuron Restrictive Silencer Factor, leading to de-repression of neural gene expression. We sought to determine if the mechanism whereby soft surfaces induce neural differentiation could also involve NRSF function. We show that MSCs cultured on soft polydimethylsiloxane (PDMS) surfaces have reduced expression of NRSF as well as altered localization of NRSF. This suggests the modulation of transcription factors by surface substrate could be capitalized upon in designing materials for neural cell culture.

Effect of Degree of Deacetylation of Chitosan/Chitin on Human Neural Stem Cell Culture.

Stem cell therapy and research for neural diseases depends on reliable reproduction of neural stem cells. Chitosan-based materials have been proposed as a substrate for culturing human neural stem cells (hNSCs) in the pursuit of clinically compatible culture conditions that are chemically defined and compliant with good manufacturing practices. The physical and biochemical properties of chitosan and chitin are strongly regulated by the degree of deacetylation (DD). However, the effect of DD on hNSC behavior has not been systematically investigated. In this study, films with DD ranging from 93% to 14% are fabricated with chitosan and chitin. Under xeno-free conditions, hNSCs proliferate preferentially on films with a higher DD, exhibiting adherent morphology and retaining multipotency. Lowering the DD leads to formation of neural stem cell spheroids due to unsteady adhesion. The neural spheroids present NSC multipotency protein expression reduction and cytoplasmic translocation. This study provides an insight into the influence of the DD on hNSCs behavior and may serve as a guideline for hNSC research using chitosan-based biomaterials. It demonstrates the capability of controlling hNSC fate by simply tailoring the DD of chitosan.

β-N-Methyl-Amino-L-Alanine cyanotoxin promotes modification of undifferentiated cells population and disrupts the inflammatory status in primary cultures of neural stem cells

β-N-Methyl-Amino-L-Alanine (BMAA) produced by 95% of cyanobacteria is in constant augmentation with cyanobacteria worldwide proliferation due to global warming and eutrophication. Previously, it has been shown that this contaminant induced neurological disorders, notably by acting as a developmental toxin. However, very few studies focus on the impact of BMAA on neuroglial cells, like astrocytes and microglial cells, in a developmental context. In the present study, we investigated whether BMAA disturbs neurogenesis from mice subventricular zone (SVZ) cells and whether this neurotoxin induces neuroinflammation. We show that BMAA at 100 µM disturbs the population of undifferentiated cells (B1 and C cells) and promotes their proliferation. Further, BMAA affects the organization of neuroblasts, indicating that SVZ function could be impaired. BMAA affects neuroinflammatory processes by increasing the release of proinflammatory cytokines IL-1β, IL-6 and TNFα. Our study adds to evidence that BMAA may disturb the central nervous system homeostasis by targeting glial cells. We highlighted that BMAA may impair SVZ niches and drives astrocytes and microglial cells into a proinflammatory status, with an ameboid shape for microglia.

Neuronal lack of PDE7a disrupted working memory, spatial learning, and memory but facilitated cued fear memory in mice

BackgroundPDEs regulate cAMP levels which is critical for PKA activity-dependent activation of CREB-mediated transcription in learning and memory. Inhibitors of PDEs like PDE4 and Pde7 improve learning and memory in rodents. However, the role of PDE7 in cognition or learning and memory has not been reported yet. MethodsTherefore, we aimed to explore the cognitive effects of a PDE7 subtype, PDE7a, using combined pharmacological and genetic approaches. ResultsPDE7a-nko mice showed deficient working memory, impaired novel object recognition, deficient spatial learning & memory, and contextual fear memory, contrary to enhanced cued fear memory, highlighting the potential opposite role of PDE7a in the hippocampal neurons. Further, pharmacological inhibition of PDE7 by AGF2.20 selectively strengthens cued fear memory in C57BL/6 J mice, decreasing its extinction but did not affect cognitive processes assessed in other behavioral tests. The further biochemical analysis detected deficient cAMP in neural cell culture with genetic excision of the PDE7a gene, as well as in the hippocampus of PDE7a-nko mice in vivo. Importantly, we found overexpression of PKA-R and the reduced level of pPKA-C in the hippocampus of PDE7a-nko mice, suggesting a novel mechanism of the cAMP regulation by PDE7a. Consequently, the decreased phosphorylation of CREB, CAMKII, eif2a, ERK, and AMPK, and reduced total level of NR2A have been found in the brain of PDE7a-nko animals. Notably, genetic excision of PDE7a in neurons was not able to change the expression of NR2B, BDNF, synapsin1, synaptophysin, or snap25. ConclusionAltogether, our current findings demonstrated, for the first time, the role of PDE7a in cognitive processes. Future studies will untangle PDE7a-dependent neurobiological and molecular-cellular mechanisms related to cAMP-associated disorders.

Ultrasonication effects on graphene composites in neural cell cultures.

Spinal cord injuries and neurodegenerative diseases, including Parkinson’s, Alzheimer’s, and traumatic brain injuries, remain challenging to treat. Nowadays, neural stem cell therapies excite high expectations within academia. The increasing demand for innovative solutions in regenerative medicine has drawn considerable attention to graphene materials. Due to unique properties, carbon materials are increasingly used as cellular scaffolds. They provide a biological microenvironment supporting cell adhesion and proliferation. The topography and mechanical properties of the graphene culture surface influence the forces exerted by the cells on their extracellular matrix. Which consequently affects the cell proliferation and differentiation. As a result, material properties such as stiffness, elasticity and mechanical strength play an important role in stem cells’ growth and life. The ink unification process is crucial while the layer homogeneity is essential for obtaining suitable surface for specific cell growth. Different ink unification processes were tested to achieve appropriate layer homogeneity and resistivity to successfully applied the GNPs layers in neural cell electrostimulation. The GNP coatings were then used to electrostimulate mouse NE-4C neural stem cells. In this study, the authors investigated how the stimulation voltage amplitude’s value affects cell behaviour, particularly the number of cells. Sinusoidal alternating current was used for stimulation. Three different values of stimulation voltage amplitude were investigated: 5, 10, and 15 V. It was noticed that a lower stimulation voltage amplitude had the most favourable effect on the stem cell count.

Potential Involvement of Varicella Zoster Virus in Alzheimer's Disease via Reactivation of Quiescent Herpes Simplex Virus Type 1.

Varicella zoster virus (VZV) has been implicated in Alzheimer's disease (AD), and vaccination against shingles, caused by VZV, has been found to decrease the risk of AD/dementia. VZV might reside latently in brain, and on reactivation might cause direct damage leading to AD, as proposed for herpes simplex virus type 1 (HSV-1), a virus strongly implicated in AD. Alternatively, shingles could induce neuroinflammation and thence, reactivation of HSV-1 in brain. To investigate these possibilities by comparing the effects of VZV and HSV-1 infection of cultured cells, and the action of VZV infection on cells quiescently infected with HSV-1. We infected human-induced neural stem cell (hiNSC) cultures with HSV-1 and/or VZV and sought the presence of AD-related phenotypes such as amyloid-β (Aβ) and P-tau accumulation, gliosis, and neuroinflammation. Cells infected with VZV did not show the main AD characteristics, Aβ and P-tau accumulation, which HSV-1 does cause, but did show gliosis and increased levels of pro-inflammatory cytokines, suggesting that VZV's action relating to AD/dementia is indirect. Strikingly, we found that VZV infection of cells quiescently infected with HSV-1 causes reactivation of HSV-1 and consequent AD-like changes, including Aβ and P-tau accumulation. Our results are consistent with the suggestion that shingles causes reactivation of HSV1 in brain and with the protective effects against AD of various vaccines, as well as the decrease in herpes labialis reported after certain types of vaccination. They support an indirect role for VZV in AD/dementia via reactivation of HSV-1 in brain.

The transcription factor LEF1 interacts with NFIX and switches isoforms during adult hippocampal neural stem cell quiescence.

Stem cells in adult mammalian tissues are held in a reversible resting state, known as quiescence, for prolonged periods of time. Recent studies have greatly increased our understanding of the epigenetic and transcriptional landscapes that underlie stem cell quiescence. However, the transcription factor code that actively maintains the quiescence program remains poorly defined. Similarly, alternative splicing events affecting transcription factors in stem cell quiescence have been overlooked. Here we show that the transcription factor T-cell factor/lymphoid enhancer factor LEF1, a central player in canonical β-catenin-dependent Wnt signalling, undergoes alternative splicing and switches isoforms in quiescent neural stem cells. We found that active β-catenin and its partner LEF1 accumulated in quiescent hippocampal neural stem and progenitor cell (Q-NSPC) cultures. Accordingly, Q-NSPCs showed enhanced TCF/LEF1-driven transcription and a basal Wnt activity that conferred a functional advantage to the cultured cells in a Wnt-dependent assay. At a mechanistic level, we found a fine regulation of Lef1 gene expression. The coordinate upregulation of Lef1 transcription and retention of alternative spliced exon 6 (E6) led to the accumulation of a full-length protein isoform (LEF1-FL) that displayed increased stability in the quiescent state. Prospectively isolated GLAST + cells from the postnatal hippocampus also underwent E6 retention at the time quiescence is established in vivo. Interestingly, LEF1 motif was enriched in quiescence-associated enhancers of genes upregulated in Q-NSPCs and quiescence-related NFIX transcription factor motifs flanked the LEF1 binding sites. We further show that LEF1 interacts with NFIX and identify putative LEF1/NFIX targets. Together, our results uncover an unexpected role for LEF1 in gene regulation in quiescent NSPCs, and highlight alternative splicing as a post-transcriptional regulatory mechanism in the transition from stem cell activation to quiescence.