Direct Neuronal Reprogramming Reveals Unknown Functions for Known Transcription Factors

In recent years, the need to derive sources of specialized cell types to be employed for cell replacement therapies and modeling studies has triggered a fast acceleration of novel cell reprogramming methods. In particular, in neuroscience, a number of protocols for the efficient differentiation of somatic or pluripotent stem cells have been established to obtain a renewable source of different neuronal cell types. Alternatively, several neuronal populations have been generated through direct reprogramming/transdifferentiation, which concerns the conversion of fully differentiated somatic cells into induced neurons. This is achieved through the forced expression of selected transcription factors (TFs) in the donor cell population. The reprogramming cocktail is chosen after an accurate screening process involving lists of TFs enriched into desired cell lineages. In some instances, this type of studies has revealed the crucial role of TFs whose function in the differentiation of a given specific cell type had been neglected or underestimated. Herein, we will speculate on how the in vitro studies have served to better understand physiological mechanisms of neuronal development in vivo

Tbr2-positive intermediate (basal) neuronal progenitors safeguard cerebral cortex expansion by controlling amplification of pallial glutamatergic neurons and attraction of subpallial GABAergic interneurons

Little is known about how, during its formidable expansion in development and evolution, the cerebral cortex is able to maintain the correct balance between excitatory and inhibitory neurons. In fact, while the former are born within the cortical primordium, the latter originate outward in the ventral pallium. Therefore, it remains to be addressed how these two neuronal populations might coordinate their relative amounts in order to build a functional cortical network. Here, we show that Tbr2-positive cortical intermediate (basal) neuronal progenitors (INPs) dictate the migratory route and control the amount of subpallial GABAergic interneurons in the subventricular zone (SVZ) through a non-cell-autonomous mechanism. In fact, Tbr2 interneuron attractive activity is moderated by Cxcl12 chemokine signaling, whose forced expression in the Tbr2 mutants can rescue, to some extent, SVZ cell migration. We thus propose that INPs are able to control simultaneously the increase of glutamatergic and GABAergic neuronal pools, thereby creating a simple way to intrinsically balance their relative accumulation

Neurobiology of Disease Inactivation of Arx, the Murine Ortholog of the X-Linked Lissencephaly with Ambiguous Genitalia Gene, Leads to Severe Disorganization of the Ventral Telencephalon with Impaired Neuronal Migration and Differentiation

ARX loss-of-function mutations cause X-linked lissencephaly with ambiguous genitalia (XLAG), a severe neurological condition that results in profound brain malformations, including microcephaly, absence of corpus callosum, and impairment of the basal ganglia. Despite such dramatic defects, their nature and origin remain largely unknown. Here, we used Arx mutant mice as a model to characterize the cellular and molecular mechanisms underlying the basal ganglia alterations. In these animals, the early differentiation of this tissue appeared normal, whereas subsequent differentiation was impaired, leading to the periventricular accumulation of immature neurons in both the lateral ganglionic eminence and medial ganglionic eminence (MGE). Both tangential migration toward the cortex and striatum and radial migration to the globus pallidus and striatum were greatly reduced in the mutants, causing a periventricular accumulation of NPY � or calretinin � neurons in the MGE. Arx mutant neurons retained their differentiation potential in vitro but exhibited deficits in morphology and migration ability. These findings imply that cell-autonomous defects in migration underlie the neuronal localization defects. Furthermore, Arx mutants lacked a large fraction of cholinergic neurons and displayed a strong impairment of thalamocortical projections, in which major axon fiber tracts failed to traverse the basal ganglia. Altogether, these results highlight the critical functions of Arx in promoting neural migration and regulating basal ganglia differentiation in mice, consistent with the phenotype of XLAG patients

ARX Regulates Cortical Intermediate Progenitor Cell Expansion and Upper Layer Neuron Formation Through Repression of Cdkn1c

Mutations in the Aristaless-related homeobox (ARX) gene are found in a spectrum of epilepsy and X-linked intellectual disability disorders. During development Arx is expressed in pallial ventricular zone (VZ) progenitor cells where the excitatory projection neurons of the cortex are born. Arx(-/Y) mice were shown to have decreased proliferation in the cortical VZ resulting in smaller brains; however, the basis for this reduced proliferation was not established. To determine the role of ARX on cell cycle dynamics in cortical progenitor cells, we generated cerebral cortex-specific Arx mouse mutants (cKO). The loss of pallial Arx resulted in the reduction of cortical progenitor cells, particularly the proliferation of intermediate progenitor cells (IPCs) was affected. Later in development and postnatally cKO brains showed a reduction of upper layer but not deeper layer neurons consistent with the IPC defect. Transcriptional profile analysis of E14.5 Arx-ablated cortices compared with control revealed that CDKN1C, an inhibitor of cell cycle progression, is overexpressed in the cortical VZ and SVZ of Arx KOs throughout corticogenesis. We also identified ARX as a direct regulator of Cdkn1c transcription. Together these data support a model where ARX regulates the expansion of cortical progenitor cells through repression of Cdkn1c

GAS2 and GAS4, a Pair of Developmentally Regulated Genes Required for Spore Wall Assembly in Saccharomyces cerevisiae

The GAS multigene family of Saccharomyces cerevisiae is composed of five paralogs (GAS1 to GAS5). GAS1 is the only one of these genes that has been characterized to date. It encodes a glycosylphosphatidylinositol-anchored protein functioning as a β(1,3)-glucan elongase and required for proper cell wall assembly during vegetative growth. In this study, we characterize the roles of the GAS2 and GAS4 genes. These genes are expressed exclusively during sporulation. Their mRNA levels showed a peak at 7 h from induction of sporulation and then decreased. Gas2 and Gas4 proteins were detected and reached maximum levels between 8 and 10 h from induction of sporulation, a time roughly coincident with spore wall assembly. The double null gas2 gas4 diploid mutant showed a severe reduction in the efficiency of sporulation, an increased permeability of the spores to exogenous substances, and production of inviable spores, whereas the single gas2 and gas4 null diploids were similar to the parental strain. An analysis of spore ultrastructure indicated that the loss of Gas2 and Gas4 proteins affected the proper attachment of the glucan to the chitosan layer, probably as a consequence of the lack of coherence of the glucan layer. The ectopic expression of GAS2 and GAS4 genes in a gas1 null mutant revealed that these proteins are redundant versions of Gas1p specialized to function in a compartment at a pH value close to neutral

Reconstitution of the Human Nigro-striatal Pathway on-a-Chip Reveals OPA1-Dependent Mitochondrial Defects and Loss of Dopaminergic Synapses

Stem cell-derived neurons are generally obtained in mass cultures that lack both spatial organization and any meaningful connectivity. We implement a microfluidic system for long-term culture of human neurons with patterned projections and synaptic ter- minals. Co-culture of human midbrain dopaminergic and striatal medium spiny neurons on the microchip establishes an orchestrated nigro-striatal circuitry with functional dopaminergic synapses. We use this platform to dissect the mitochondrial dysfunc- tions associated with a genetic form of Parkinson’s disease (PD) with OPA1 mutations. Remarkably, we find that axons of OPA1 mutant dopaminergic neu- rons exhibit a significant reduction of mitochondrial mass. This defect causes a significant loss of dopa- minergic synapses, which worsens in long-term cultures. Therefore, PD-associated depletion of mitochondria at synapses might precede loss of neuronal connectivity and neurodegeneration. In vitro reconstitution of human circuitries by micro- fluidic technology offers a powerful system to study brain networks by establishing ordered neuronal compartments and correct synapse identity

Direct Conversion of Fibroblasts into Functional Astrocytes by Defined Transcription Factors

Summary: Direct cell reprogramming enables direct conversion of fibroblasts into functional neurons and oligodendrocytes using a minimal set of cell-lineage-specific transcription factors. This approach is rapid and simple, generating the cell types of interest in one step. However, it remains unknown whether this technology can be applied to convert fibroblasts into astrocytes, the third neural lineage. Astrocytes play crucial roles in neuronal homeostasis, and their dysfunctions contribute to the origin and progression of multiple human diseases. Herein, we carried out a screening using several transcription factors involved in defining the astroglial cell fate and identified NFIA, NFIB, and SOX9 to be sufficient to convert with high efficiency embryonic and postnatal mouse fibroblasts into astrocytes (iAstrocytes). We proved both by gene-expression profiling and functional tests that iAstrocytes are comparable to native brain astrocytes. This protocol can be then employed to generate functional iAstrocytes for a wide range of experimental applications. : In this article, Broccoli, Caiazzo, and colleagues developed a direct reprogramming approach to convert fibroblasts into induced astrocytes (iAstrocytes) by forcing the expression of the three astroglial transcription factors NFIA, NFIB, and SOX9. iAstrocytes are functionally comparable to native primary astrocytes as assessed by in vitro analyses and can be transplanted in the mouse brain. This study discloses the possibility to generate also human iAstrocytes for potential translational applications

Remote control of induced dopaminergic neurons in parkinsonian rats

Direct lineage reprogramming through genetic-based strategies enables the conversion of differentiated somatic cells into functional neurons and distinct neuronal subtypes. Induced dopaminergic (iDA) neurons can be generated by direct conversion of skin fibroblasts; however, their in vivo phenotypic and functional properties remain incompletely understood, leaving their impact on Parkinson's disease (PD) cell therapy and modeling uncertain. Here, we determined that iDA neurons retain a transgene-independent stable phenotype in culture and in animal models. Furthermore, transplanted iDA neurons functionally integrated into host neuronal tissue, exhibiting electrically excitable membranes, synaptic currents, dopamine release, and substantial reduction of motor symptoms in a PD animal model. Neuronal cell replacement approaches will benefit from a system that allows the activity of transplanted neurons to be controlled remotely and enables modulation depending on the physiological needs of the recipient; therefore, we adapted a DREADD (designer receptor exclusively activated by designer drug) technology for remote and real-time control of grafted iDA neuronal activity in living animals. Remote DREADD-dependent iDA neuron activation markedly enhanced the beneficial effects in transplanted PD animals. These data suggest that iDA neurons have therapeutic potential as a cell replacement approach for PD and highlight the applicability of pharmacogenetics for enhancing cellular signaling in reprogrammed cell-based approaches