PTRH2 is Necessary for Purkinje Cell Differentiation and Survival and its Loss Recapitulates Progressive Cerebellar Atrophy and Ataxia Seen in IMNEPD Patients.

Hom ozygous variants in the peptidyl-tRNA hydrolase 2 gene (PTRH2) cause infantile-onset multisystem neurologic, endocrine, and pancreatic disease. The objective is to delineate the mechanisms underlying the core cerebellar phenotype in this disease. For this, we generated constitutive (Ptrh2LoxPxhCMVCre, Ptrh2-/- mice) and Purkinje cell (PC) specific (Ptrh2LoxPxPcp2Cre, Ptrh2ΔPCmice) Ptrh2 mutant mouse models and investigated the effect of the loss of Ptrh2 on cerebellar development. We show that Ptrh2-/- knockout mice had severe postnatal runting and lethality by postnatal day 14. Ptrh2ΔPC PC specific knockout mice survived until adult age; however, they showed progressive cerebellar atrophy and functional cerebellar deficits with abnormal gait and ataxia. PCs of Ptrh2ΔPC mice had reduced cell size and density, stunted dendrites, and lower levels of ribosomal protein S6, a readout of the mammalian target of rapamycin pathway. By adulthood, there was a marked loss of PCs. Thus, we identify a cell autonomous requirement for PTRH2 in PC maturation and survival. Loss of PTRH2 in PCs leads to downregulation of the mTOR pathway and PC atrophy. This suggests a molecular mechanism underlying the ataxia and cerebellar atrophy seen in patients with PTRH2 mutations leading to infantile-onset multisystem neurologic, endocrine, and pancreatic disease.

Transcription factors regulating the specification of brainstem respiratory neurons.

Breathing (or respiration) is an unconscious and complex motor behavior which neuronal drive emerges from the brainstem. In simplistic terms, respiratory motor activity comprises two phases, inspiration (uptake of oxygen, O2) and expiration (release of carbon dioxide, CO2). Breathing is not rigid, but instead highly adaptable to external and internal physiological demands of the organism. The neurons that generate, monitor, and adjust breathing patterns locate to two major brainstem structures, the pons and medulla oblongata. Extensive research over the last three decades has begun to identify the developmental origins of most brainstem neurons that control different aspects of breathing. This research has also elucidated the transcriptional control that secures the specification of brainstem respiratory neurons. In this review, we aim to summarize our current knowledge on the transcriptional regulation that operates during the specification of respiratory neurons, and we will highlight the cell lineages that contribute to the central respiratory circuit. Lastly, we will discuss on genetic disturbances altering transcription factor regulation and their impact in hypoventilation disorders in humans.

Early development of the breathing network.

Breathing (or respiration) is a complex motor behavior that originates in the brainstem. In minimalistic terms, breathing can be divided into two phases: inspiration (uptake of oxygen, O2) and expiration (release of carbon dioxide, CO2). The neurons that discharge in synchrony with these phases are arranged in three major groups along the brainstem: (i) pontine, (ii) dorsal medullary, and (iii) ventral medullary. These groups are formed by diverse neuron types that coalesce into heterogeneous nuclei or complexes, among which the preBötzinger complex in the ventral medullary group contains cells that generate the respiratory rhythm (Chapter 1). The respiratory rhythm is not rigid, but instead highly adaptable to the physic demands of the organism. In order to generate the appropriate respiratory rhythm, the preBötzinger complex receives direct and indirect chemosensory information from other brainstem respiratory nuclei (Chapter 2) and peripheral organs (Chapter 3). Even though breathing is a hard-wired unconscious behavior, it can be temporarily altered at will by other higher-order brain structures (Chapter 6), and by emotional states (Chapter 7). In this chapter, we focus on the development of brainstem respiratory groups and highlight the cell lineages that contribute to central and peripheral chemoreflexes.

The Role of Neurod Genes in Brain Development, Function, and Disease.

Transcriptional regulation is essential for the correct functioning of cells during development and in postnatal life. The basic Helix-loop-Helix (bHLH) superfamily of transcription factors is well conserved throughout evolution and plays critical roles in tissue development and tissue maintenance. A subgroup of this family, called neural lineage bHLH factors, is critical in the development and function of the central nervous system. In this review, we will focus on the function of one subgroup of neural lineage bHLH factors, the Neurod family. The Neurod family has four members: Neurod1, Neurod2, Neurod4, and Neurod6. Available evidence shows that these four factors are key during the development of the cerebral cortex but also in other regions of the central nervous system, such as the cerebellum, the brainstem, and the spinal cord. We will also discuss recent reports that link the dysfunction of these transcription factors to neurological disorders in humans.

Olig3 regulates early cerebellar development.

The mature cerebellum controls motor skill precision and participates in other sophisticated brain functions that include learning, cognition, and speech. Different types of GABAergic and glutamatergic cerebellar neurons originate in temporal order from two progenitor niches, the ventricular zone and rhombic lip, which express the transcription factors Ptf1a and Atoh1, respectively. However, the molecular machinery required to specify the distinct neuronal types emanating from these progenitor zones is still unclear. Here, we uncover the transcription factor Olig3 as a major determinant in generating the earliest neuronal derivatives emanating from both progenitor zones in mice. In the rhombic lip, Olig3 regulates progenitor cell proliferation. In the ventricular zone, Olig3 safeguards Purkinje cell specification by curtailing the expression of Pax2, a transcription factor that suppresses the Purkinje cell differentiation program. Our work thus defines Olig3 as a key factor in early cerebellar development.

The SNAG Domain of Insm1 Regulates Pancreatic Endocrine Cell Differentiation and Represses ß- to δ-Cell Transdifferentiation.

The allocation and specification of pancreatic endocrine lineages are tightly regulated by transcription factors. Disturbances in differentiation of these lineages contribute to the development of various metabolic diseases, including diabetes. The insulinoma-associated protein 1 (Insm1), which encodes a protein containing one SNAG domain and five zinc fingers, plays essential roles in pancreatic endocrine cell differentiation and in mature ß-cell function. In the current study, we compared the differentiation of pancreatic endocrine cells between Insm1 null and Insm1 SNAG domain mutants (Insm1delSNAG) to explore the specific function of the SNAG domain of Insm1. We show that the δ-cell number is increased in Insm1delSNAG but not in Insm1 null mutants as compared with the control mice. We also show a less severe reduction of the ß-cell number in Insm1delSNAG as that in Insm1 null mutants. In addition, similar deficits are observed in α-, PP, and ε-cells in Insm1delSNAG and Insm1 null mutants. We further identified that the increased δ-cell number is due to ß- to δ-cell transdifferentiation. Mechanistically, the SNAG domain of Insm1 interacts with Lsd1, the demethylase of H3K4me1/2. Mutation in the SNAG domain of Insm1 results in impaired recruitment of Lsd1 and increased H3K4me1/2 levels at hematopoietically expressed homeobox (Hhex) loci that are bound by Insm1, thereby promoting the transcriptional activity of the δ-cell-specific gene Hhex Our study has identified a novel function of the SNAG domain of Insm1 in the regulation of pancreatic endocrine cell differentiation, particularly in the repression of ß- to δ-cell transdifferentiation.

Context-specific regulation of cell survival by a miRNA-controlled BIM rheostat.

Knockout of the ubiquitously expressed miRNA-17∼92 cluster in mice produces a lethal developmental lung defect, skeletal abnormalities, and blocked B lymphopoiesis. A shared target of miR-17∼92 miRNAs is the pro-apoptotic protein BIM, central to life-death decisions in mammalian cells. To clarify the contribution of miR-17∼92:Bim interactions to the complex miR-17∼92 knockout phenotype, we used a system of conditional mutagenesis of the nine Bim 3' UTR miR-17∼92 seed matches. Blocking miR-17∼92:Bim interactions early in development phenocopied the lethal lung phenotype of miR-17∼92 ablation and generated a skeletal kinky tail. In the hematopoietic system, instead of causing the predicted B cell developmental block, it produced a selective inability of B cells to resist cellular stress; and prevented B and T cell hyperplasia caused by Bim haploinsufficiency. Thus, the interaction of miR-17∼92 with a single target is essential for life, and BIM regulation by miRNAs serves as a rheostat controlling cell survival in specific physiological contexts.

Mutations in Disordered Regions Can Cause Disease by Creating Dileucine Motifs.

Many disease-causing missense mutations affect intrinsically disordered regions (IDRs) of proteins, but the molecular mechanism of their pathogenicity is enigmatic. Here, we employ a peptide-based proteomic screen to investigate the impact of mutations in IDRs on protein-protein interactions. We find that mutations in disordered cytosolic regions of three transmembrane proteins (GLUT1, ITPR1, and CACNA1H) lead to an increased clathrin binding. All three mutations create dileucine motifs known to mediate clathrin-dependent trafficking. Follow-up experiments on GLUT1 (SLC2A1), the glucose transporter causative of GLUT1 deficiency syndrome, revealed that the mutated protein mislocalizes to intracellular compartments. Mutant GLUT1 interacts with adaptor proteins (APs) in vitro, and knocking down AP-2 reverts the cellular mislocalization and restores glucose transport. A systematic analysis of other known disease-causing variants revealed a significant and specific overrepresentation of gained dileucine motifs in structurally disordered cytosolic domains of transmembrane proteins. Thus, several mutations in disordered regions appear to cause "dileucineopathies."

Mutations in MYO1H cause a recessive form of central hypoventilation with autonomic dysfunction.

BACKGROUND: Congenital central hypoventilation syndrome (CCHS) is a rare life-threatening disorder of respiratory and autonomic regulation. It is classically caused by dominant mutations in the transcription factor PHOX2B. The objective of the present study was to identify the molecular cause of a recessive form of central hypoventilation with autonomic dysfunction. METHODS: Here, we used homozygosity mapping and whole-genome sequencing in a consanguineous family with CCHS in combination with functional analyses in CRISPR/Cas9 engineered mice. RESULTS: We report on a consanguineous family with three affected children, all tested PHOX2B mutation negative, presenting with alveolar hypoventilation and symptoms of autonomic dysregulation. Whole-genome sequencing revealed a homozygous frameshift mutation in exon 25 of the MYO1H gene (c.2524_2524delA) segregating with the phenotype in the family. MYO1H encodes for the unconventional myosin IH, which is thought to function as a motor protein in intracellular transport and vesicle trafficking. We show that Myo1h is broadly expressed in the mouse lower medulla, including the CO2-sensitive Phox2b+ retrotrapezoid neurons. To test the pathogenicity of the variant, we engineered two Myo1h mutant mouse strains: the first strain (Myo1h*) resembling the human mutation and the second being a full knock-out (Myo1hFS ). Whole-body plethysmography studies in Myo1h* newborns with the re-engineered human mutation revealed hypoventilation and a blunted response to CO2, recapitulating the breathing phenotype observed in the kindred. CONCLUSIONS: Our results identify MYO1H as an important gene in CO2 sensitivity and respiratory control and as the cause of a rare recessive form of congenital central hypoventilation.

Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function.

Hundreds of circular RNAs (circRNAs) are highly abundant in the mammalian brain, often with conserved expression. Here we show that the circRNA Cdr1as is massively bound by the microRNAs (miRNAs) miR-7 and miR-671 in human and mouse brains. When the Cdr1as locus was removed from the mouse genome, knockout animals displayed impaired sensorimotor gating-a deficit in the ability to filter out unnecessary information-which is associated with neuropsychiatric disorders. Electrophysiological recordings revealed dysfunctional synaptic transmission. Expression of miR-7 and miR-671 was specifically and posttranscriptionally misregulated in all brain regions analyzed. Expression of immediate early genes such as Fos, a direct miR-7 target, was enhanced in Cdr1as-deficient brains, providing a possible molecular link to the behavioral phenotype. Our data indicate an in vivo loss-of-function circRNA phenotype and suggest that interactions between Cdr1as and miRNAs are important for normal brain function.

Homozygous ARHGEF2 mutation causes intellectual disability and midbrain-hindbrain malformation.

Mid-hindbrain malformations can occur during embryogenesis through a disturbance of transient and localized gene expression patterns within these distinct brain structures. Rho guanine nucleotide exchange factor (ARHGEF) family members are key for controlling the spatiotemporal activation of Rho GTPase, to modulate cytoskeleton dynamics, cell division, and cell migration. We identified, by means of whole exome sequencing, a homozygous frameshift mutation in the ARHGEF2 as a cause of intellectual disability, a midbrain-hindbrain malformation, and mild microcephaly in a consanguineous pedigree of Kurdish-Turkish descent. We show that loss of ARHGEF2 perturbs progenitor cell differentiation and that this is associated with a shift of mitotic spindle plane orientation, putatively favoring more symmetric divisions. The ARHGEF2 mutation leads to reduction in the activation of the RhoA/ROCK/MLC pathway crucial for cell migration. We demonstrate that the human brain malformation is recapitulated in Arhgef2 mutant mice and identify an aberrant migration of distinct components of the precerebellar system as a pathomechanism underlying the midbrain-hindbrain phenotype. Our results highlight the crucial function of ARHGEF2 in human brain development and identify a mutation in ARHGEF2 as novel cause of a neurodevelopmental disorder.

The dorsal spinal cord and hindbrain: From developmental mechanisms to functional circuits.

Neurons of the dorsal hindbrain and spinal cord are central in receiving, processing and relaying sensory perception and participate in the coordination of sensory-motor output. Numerous cellular and molecular mechanisms that underlie neuronal development in both regions of the nervous system are shared. We discuss here the mechanisms that generate neuronal diversity in the dorsal spinal cord and hindbrain, and emphasize similarities in patterning and neuronal specification. Insight into the developmental mechanisms has provided tools that can help to assign functions to small subpopulations of neurons. Hence, novel information on how mechanosensory or pain sensation is encoded under normal and neuropathic conditions has already emerged. Such studies show that the complex neuronal circuits that control perception of somatosensory and viscerosensory stimuli are becoming amenable to investigations.

Neuregulin-1 controls an endogenous repair mechanism after spinal cord injury.

Following traumatic spinal cord injury, acute demyelination of spinal axons is followed by a period of spontaneous remyelination. However, this endogenous repair response is suboptimal and may account for the persistently compromised function of surviving axons. Spontaneous remyelination is largely mediated by Schwann cells, where demyelinated central axons, particularly in the dorsal columns, become associated with peripheral myelin. The molecular control, functional role and origin of these central remyelinating Schwann cells is currently unknown. The growth factor neuregulin-1 (Nrg1, encoded by NRG1) is a key signalling factor controlling myelination in the peripheral nervous system, via signalling through ErbB tyrosine kinase receptors. Here we examined whether Nrg1 is required for Schwann cell-mediated remyelination of central dorsal column axons and whether Nrg1 ablation influences the degree of spontaneous remyelination and functional recovery following spinal cord injury. In contused adult mice with conditional ablation of Nrg1, we found an absence of Schwann cells within the spinal cord and profound demyelination of dorsal column axons. There was no compensatory increase in oligodendrocyte remyelination. Removal of peripheral input to the spinal cord and proliferation studies demonstrated that the majority of remyelinating Schwann cells originated within the injured spinal cord. We also examined the role of specific Nrg1 isoforms, using mutant mice in which only the immunoglobulin-containing isoforms of Nrg1 (types I and II) were conditionally ablated, leaving the type III Nrg1 intact. We found that the immunoglobulin Nrg1 isoforms were dispensable for Schwann cell-mediated remyelination of central axons after spinal cord injury. When functional effects were examined, both global Nrg1 and immunoglobulin-specific Nrg1 mutants demonstrated reduced spontaneous locomotor recovery compared to injured controls, although global Nrg1 mutants were more impaired in tests requiring co-ordination, balance and proprioception. Furthermore, electrophysiological assessments revealed severely impaired axonal conduction in the dorsal columns of global Nrg1 mutants (where Schwann cell-mediated remyelination is prevented), but not immunoglobulin-specific mutants (where Schwann cell-mediated remyelination remains intact), providing robust evidence that the profound demyelinating phenotype observed in the dorsal columns of Nrg1 mutant mice is related to conduction failure. Our data provide novel mechanistic insight into endogenous regenerative processes after spinal cord injury, demonstrating that Nrg1 signalling regulates central axon remyelination and functional repair and drives the trans-differentiation of central precursor cells into peripheral nervous system-like Schwann cells that remyelinate spinal axons after injury. Manipulation of the Nrg1 system could therefore be exploited to enhance spontaneous repair after spinal cord injury and other central nervous system disorders with a demyelinating pathology.media-1vid110.1093/brain/aww039_video_abstractaww039_video_abstract.

Insm1 controls development of pituitary endocrine cells and requires a SNAG domain for function and for recruitment of histone-modifying factors.

The Insm1 gene encodes a zinc finger factor expressed in many endocrine organs. We show here that Insm1 is required for differentiation of all endocrine cells in the pituitary. Thus, in Insm1 mutant mice, hormones characteristic of the different pituitary cell types (thyroid-stimulating hormone, follicle-stimulating hormone, melanocyte-stimulating hormone, adrenocorticotrope hormone, growth hormone and prolactin) are absent or produced at markedly reduced levels. This differentiation deficit is accompanied by upregulated expression of components of the Notch signaling pathway, and by prolonged expression of progenitor markers, such as Sox2. Furthermore, skeletal muscle-specific genes are ectopically expressed in endocrine cells, indicating that Insm1 participates in the repression of an inappropriate gene expression program. Because Insm1 is also essential for differentiation of endocrine cells in the pancreas, intestine and adrenal gland, it is emerging as a transcription factor that acts in a pan-endocrine manner. The Insm1 factor contains a SNAG domain at its N-terminus, and we show here that the SNAG domain recruits histone-modifying factors (Kdm1a, Hdac1/2 and Rcor1-3) and other proteins implicated in transcriptional regulation (Hmg20a/b and Gse1). Deletion of sequences encoding the SNAG domain in mice disrupted differentiation of pituitary endocrine cells, and resulted in an upregulated expression of components of the Notch signaling pathway and ectopic expression of skeletal muscle-specific genes. Our work demonstrates that Insm1 acts in the epigenetic and transcriptional network that controls differentiation of endocrine cells in the anterior pituitary gland, and that it requires the SNAG domain to exert this function in vivo.

Robo1 regulates semaphorin signaling to guide the migration of cortical interneurons through the ventral forebrain.

Cortical interneurons, generated predominantly in the medial ganglionic eminence, migrate around and avoid the developing striatum in the subpallium en route to the cortex. This is attributable to the chemorepulsive cues of class 3 semaphorins expressed in the striatal mantle and acting through neuropilin (Nrp1 and Nrp2) receptors expressed in these cells. Cortical interneurons also express Robo receptors, and we show here that in mice lacking Robo1, but not Robo2, these cells migrate aberrantly through the striatum. In vitro experiments demonstrated that interneurons lacking Robo1 function are significantly less responsive to the effects of semaphorins. Failure to respond to semaphorin appears to be attributable to a reduction in Nrp1 and PlexinA1 receptors within these cells. Biochemical studies further demonstrated that Robo1 binds directly to Nrp1, but not to semaphorins, and this interaction is mediated by a region contained within its first two Ig domains. Thus, we show for the first time that Robo1 interacts with Nrp1 to modulate semaphorin signaling in the developing forebrain and direct the migration of interneurons through the subpallium and into the cortex.

Molecules and mechanisms involved in the generation and migration of cortical interneurons.

The GABA (γ-aminobutyric acid)-containing interneurons of the neocortex are largely derived from the ganglionic eminences in the subpallium. Numerous studies have previously defined the migratory paths travelled by these neurons from their origins to their destinations in the cortex. We review here results of studies that have identified many of the genes expressed in the subpallium that are involved in the specification of the subtypes of cortical interneurons, and the numerous transcription factors, motogenic factors and guidance molecules that are involved in their migration.

The role of Robo3 in the development of cortical interneurons.

A number of studies in recent years have shown that members of the Roundabout (Robo) receptor family, Robo1 and Robo2, play significant roles in the formation of axonal tracks in the developing forebrain and in the migration and morphological differentiation of cortical interneurons. Here, we investigated the expression and function of Robo3 in the developing cortex. We found that this receptor is strongly expressed in the preplate layer and cortical hem of the early cortex where it colocalizes with markers of Cajal-Retzius cells and interneurons. Analysis of Robo3 mutant mice at early (embryonic day [E] 13.5) and late (E18.5) stages of corticogenesis revealed no significant change in the number of interneurons, but a change in their morphology at E13.5. However, preliminary analysis on a small number of mice that lacked all 3 Robo receptors indicated a marked reduction in the number of cortical interneurons, but only a limited effect on their morphology. These observations and the results of other recent studies suggest a complex interplay between the 3 Robo receptors in regulating the number, migration and morphological differentiation of cortical interneurons.