Low Forces Push the Maturation of Neural Precursors into Neurons.

Mechanical stimulation modulates neural development and neuronal activity. In a previous study, magnetic "nano-pulling" is proposed as a tool to generate active forces. By loading neural cells with magnetic nanoparticles (MNPs), a precise force vector is remotely generated through static magnetic fields. In the present study, human neural stem cells (NSCs) are subjected to a standard differentiation protocol, in the presence or absence of nano-pulling. Under mechanical stimulation, an increase in the length of the neural processes which showed an enrichment in microtubules, endoplasmic reticulum, and mitochondria is found. A stimulation lasting up to 82 days induces a strong remodeling at the level of synapse density and a re-organization of the neuronal network, halving the time required for the maturation of neural precursors into neurons. The MNP-loaded NSCs are then transplanted into mouse spinal cord organotypic slices, demonstrating that nano-pulling stimulates the elongation of the NSC processes and modulates their orientation even in an ex vivo model. Thus, it is shown that active mechanical stimuli can guide the outgrowth of NSCs transplanted into the spinal cord tissue. The findings suggest that mechanical forces play an important role in neuronal maturation which could be applied in regenerative medicine.

Dynamic and Cell-Specific DACH1 Expression in Human Neocortical and Striatal Development.

DACH1 is the human homolog of the Drosophila dachshund gene, which is involved in the development of the eye, nervous system, and limbs in the fly. Here, we systematically investigate DACH1 expression patterns during human neurodevelopment, from 5 to 21 postconceptional weeks. By immunodetection analysis, we found that DACH1 is highly expressed in the proliferating neuroprogenitors of the developing cortical ventricular and subventricular regions, while it is absent in the more differentiated cortical plate. Single-cell global transcriptional analysis revealed that DACH1 is specifically enriched in neuroepithelial and ventricular radial glia cells of the developing human neocortex. Moreover, we describe a previously unreported DACH1 expression in the human striatum, in particular in the striatal medium spiny neurons. This finding qualifies DACH1 as a new striatal projection neuron marker, together with PPP1R1B, BCL11B, and EBF1. We finally compared DACH1 expression profile in human and mouse forebrain, where we observed spatio-temporal similarities in its expression pattern thus providing a precise developmental description of DACH1 in the 2 mammalian species.

Hmga2 is required for neural crest cell specification in Xenopus laevis.

HMGA proteins are small nuclear proteins that bind DNA by conserved AT-hook motifs, modify chromatin architecture and assist in gene expression. Two HMGAs (HMGA1 and HMGA2), encoded by distinct genes, exist in mammals and are highly expressed during embryogenesis or reactivated in tumour progression. We here addressed the in vivo role of Xenopus hmga2 in the neural crest cells (NCCs). We show that hmga2 is required for normal NCC specification and development. hmga2 knockdown leads to severe disruption of major skeletal derivatives of anterior NCCs. We show that, within the NCC genetic network, hmga2 acts downstream of msx1, and is required for msx1, pax3 and snail2 activities, thus participating at different levels of the network. Because of hmga2 early effects in NCC specification, the subsequent epithelial-mesenchymal transition (EMT) and migration of NCCs towards the branchial pouches are also compromised. Strictly paralleling results on embryos, interfering with Hmga2 in a breast cancer cell model for EMT leads to molecular effects largely consistent with those observed on NCCs. These data indicate that Hmga2 is recruited in key molecular events that are shared by both NCCs and tumour cells.

Developmentally coordinated extrinsic signals drive human pluripotent stem cell differentiation toward authentic DARPP-32+ medium-sized spiny neurons.

Medium-sized spiny neurons (MSNs) are the only neostriatum projection neurons, and their degeneration underlies some of the clinical features of Huntington's disease. Using knowledge of human developmental biology and exposure to key neurodevelopmental molecules, human pluripotent stem (hPS) cells were induced to differentiate into MSNs. In a feeder-free adherent culture, ventral telencephalic specification is induced by BMP/TGFß inhibition and subsequent SHH/DKK1 treatment. The emerging FOXG1(+)/GSX2(+) telencephalic progenitors are then terminally differentiated, resulting in the systematic line-independent generation of FOXP1(+)/FOXP2(+)/CTIP2(+)/calbindin(+)/DARPP-32(+) MSNs. Similar to mature MSNs, these neurons carry dopamine and A2a receptors, elicit a typical firing pattern and show inhibitory postsynaptic currents, as well as dopamine neuromodulation and synaptic integration ability in vivo. When transplanted into the striatum of quinolinic acid-lesioned rats, hPS-derived neurons survive and differentiate into DARPP-32(+) neurons, leading to a restoration of apomorphine-induced rotation behavior. In summary, hPS cells can be efficiently driven to acquire a functional striatal fate using an ontogeny-recapitulating stepwise method that represents a platform for in vitro human developmental neurobiology studies and drug screening approaches.

Long-Term Mouse Spinal Cord Organotypic Slice Culture as a Platform for Validating Cell Transplantation in Spinal Cord Injury.

Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches is based on stem cell transplantation to replace lost tissue and promote functional recovery. This approach should be further explored better in vitro and ex vivo for safety and efficacy before proceeding with more expensive and time-consuming animal testing. In this work, we show the establishment of a long-term platform based on mouse spinal cord (SC) organotypic slices transplanted with human neural stem cells to test cellular replacement therapies for SCIs. Standard SC organotypic cultures are maintained for around 2 or 3 weeks in vitro. Here, we describe an optimized protocol for long-term maintenance (≥30 days) for up to 90 days. The medium used for long-term culturing of SC slices was also optimized for transplanting neural stem cells into the organotypic model. Human SC-derived neuroepithelial stem (h-SC-NES) cells carrying a green fluorescent protein (GFP) reporter were transplanted into mouse SC slices. Thirty days after the transplant, cells still show GFP expression and a low apoptotic rate, suggesting that the optimized environment sustained their survival and integration inside the tissue. This protocol represents a robust reference for efficiently testing cell replacement therapies in the SC tissue. This platform will allow researchers to perform an ex vivo pre-screening of different cell transplantation therapies, helping them to choose the most appropriate strategy before proceeding with in vivo experiments.

Human neural progenitor cell models to study the antiviral effects and neuroprotective potential of approved and investigational human cytomegalovirus inhibitors.

Human cytomegalovirus (HCMV) is the viral leading cause of congenital defects in newborns worldwide. Many aspects of congenital CMV (cCMV) infection, which currently lacks a specific treatment, as well as the main determinants of neuropathogenesis in the developing brain during HCMV infection are unclear. In this study, we modeled HCMV infection at different stages of neural development. Moreover, we evaluated the effects of both approved and investigational anti-HCMV drugs on viral replication and gene expression in two different neural progenitor cell lines, i.e., human embryonic stem cells-derived neural stem cells (NSCs) and fetus-derived neuroepithelial stem (NES) cells. Ganciclovir, letermovir, nitazoxanide, and the ozonide OZ418 reduced viral DNA synthesis and the production of infectious virus in both lines of neural progenitors. HCMV infection dysregulated the expression of genes that either are markers of neural progenitors, such as SOX2, NESTIN, PAX-6, or play a role in neurogenesis, such as Doublecortin. Treatment with antiviral drugs had different effects on HCMV-induced dysregulation of the genes under investigation. This study contributes to the understanding of the molecular mechanisms of cCMV neuropathogenesis and paves the way for further consideration of anti-HCMV drugs as candidate therapeutic agents for the amelioration of cCMV-associated neurological manifestations.

Cortico-thalamic development and disease: From cells, to circuits, to schizophrenia.

The human brain is the most complex structure generated during development. Unveiling the ontogenesis and the intrinsic organization of specific neural networks may represent a key to understanding the physio-pathological aspects of different brain areas. The cortico-thalamic and thalamo-cortical (CT-TC) circuits process and modulate essential tasks such as wakefulness, sleep and memory, and their alterations may result in neurodevelopmental and psychiatric disorders. These pathologies are reported to affect specific neural populations but may also broadly alter physiological connections and thus dysregulate brain network generation, communication, and function. More specifically, the CT-TC system is reported to be severely affected in disorders impacting superior brain functions, such as schizophrenia (SCZ), bipolar disorder, autism spectrum disorders or epilepsy. In this review, the focus will be on CT development, and the models exploited to uncover and comprehend its molecular and cellular mechanisms. In parallel to animal models, still fundamental to unveil human neural network establishment, advanced in vitro platforms, such as brain organoids derived from human pluripotent stem cells, will be discussed. Indeed, organoids and assembloids represent unique tools to study and accelerate fundamental research in CT development and its dysfunctions. We will then discuss recent cutting-edge contributions, including in silico approaches, concerning ontogenesis, specification, and function of the CT-TC circuitry that generates connectivity maps in physiological and pathological conditions.

Microcephaly-associated protein WDR62 shuttles from the Golgi apparatus to the spindle poles in human neural progenitors.

WDR62 is a spindle pole-associated scaffold protein with pleiotropic functions. Recessive mutations in WDR62 cause structural brain abnormalities and account for the second most common cause of autosomal recessive primary microcephaly (MCPH), indicating WDR62 as a critical hub for human brain development. Here, we investigated WDR62 function in corticogenesis through the analysis of a C-terminal truncating mutation (D955AfsX112). Using induced Pluripotent Stem Cells (iPSCs) obtained from a patient and his unaffected parent, as well as isogenic corrected lines, we generated 2D and 3D models of human neurodevelopment, including neuroepithelial stem cells, cerebro-cortical progenitors, terminally differentiated neurons, and cerebral organoids. We report that WDR62 localizes to the Golgi apparatus during interphase in cultured cells and human fetal brain tissue, and translocates to the mitotic spindle poles in a microtubule-dependent manner. Moreover, we demonstrate that WDR62 dysfunction impairs mitotic progression and results in alterations of the neurogenic trajectories of iPSC neuroderivatives. In summary, impairment of WDR62 localization and function results in severe neurodevelopmental abnormalities, thus delineating new mechanisms in the etiology of MCPH.

Inhibiting immunoregulatory amidase NAAA blocks ZIKV maturation in Human Neural Stem Cells.

Recent evidence suggests that lipids play a crucial role in viral infections beyond their traditional functions of supplying envelope and energy, and creating protected niches for viral replication. In the case of Zika virus (ZIKV), it alters host lipids by enhancing lipogenesis and suppressing ß-oxidation to generate viral factories at the endoplasmic reticulum (ER) interface. This discovery prompted us to hypothesize that interference with lipogenesis could serve as a dual antiviral and anti-inflammatory strategy to combat the replication of positive sense single-stranded RNA (ssRNA+) viruses. To test this hypothesis, we examined the impact of inhibiting N-Acylethanolamine acid amidase (NAAA) on ZIKV-infected human Neural Stem Cells. NAAA is responsible for the hydrolysis of palmitoylethanolamide (PEA) in lysosomes and endolysosomes. Inhibition of NAAA results in PEA accumulation, which activates peroxisome proliferator-activated receptor-α (PPAR-α), directing ß-oxidation and preventing inflammation. Our findings indicate that inhibiting NAAA through gene-editing or drugs moderately reduces ZIKV replication by approximately one log10 in Human Neural Stem Cells, while also releasing immature virions that have lost their infectivity. This inhibition impairs furin-mediated prM cleavage, ultimately blocking ZIKV maturation. In summary, our study highlights NAAA as a host target for ZIKV infection.

Induced pluripotent stem cell lines from Huntington's disease mice undergo neuronal differentiation while showing alterations in the lysosomal pathway.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an excessive expansion of a CAG trinucleotide repeat in the gene encoding the protein huntingtin, resulting in an elongated stretch of glutamines near the N-terminus of the protein. Here we report the derivation of a collection of 11 induced pluripotent stem (iPS) cell lines generated through somatic reprogramming of fibroblasts obtained from the R6/2 transgenic HD mouse line. We show that CAG expansion has no effect on reprogramming efficiency, cell proliferation rate, brain-derived neurotrophic factor level, or neurogenic potential. However, genes involved in the cholesterol biosynthesis pathway, which is altered in HD, are also affected in HD-iPS cell lines. Furthermore, we found a lysosomal gene upregulation and an increase in lysosome number in HD-iPS cell lines. These observations suggest that iPS cells from HD mice replicate some but not all of the molecular phenotypes typically observed in the disease; additionally, they do not manifest increased cell death propensity either under self-renewal or differentiated conditions. More studies will be necessary to transform a revolutionary technology into a powerful platform for drug screening approaches.