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The Golgi apparatus (GA) is essential for intracellular sorting, trafficking and the targeting of proteins to specific cellular compartments. Anatomically, the GA spreads all over the cell but is also particularly enriched close to the base of the primary cilium. This peculiar organelle protrudes at the surface of almost all cells and fulfills many cellular functions, in particular during development, when a dysfunction of the primary cilium can lead to disorders called ciliopathies. While ciliopathies caused by loss of ciliated proteins have been extensively documented, several studies suggest that alterations of GA and GA-associated proteins can also affect ciliogenesis. Here, we aim to discuss how the loss-of-function of genes coding these proteins induces ciliary defects and results in ciliopathies.

How the brain develops and achieves its final size is a fascinating issue that questions cortical evolution across species and man's place in the animal kingdom. Although animal models have so far been highly valuable in understanding the key steps of cortical development, many human specificities call for appropriate models. In particular, microcephaly, a neurodevelopmental disorder that is characterized by a smaller head circumference has been challenging to model in mice, which often do not fully recapitulate the human phenotype. The relatively recent development of brain organoid technology from induced pluripotent stem cells (iPSCs) now makes it possible to model human microcephaly, both due to genetic and environmental origins, and to generate developing cortical tissue from the patients themselves. These 3D tissues rely on iPSCs differentiation into cortical progenitors that self-organize into neuroepithelial rosettes mimicking the earliest stages of human neurogenesis in vitro. Over the last ten years, numerous protocols have been developed to control the identity of the induced brain areas, the reproducibility of the experiments and the longevity of the cultures, allowing analysis of the later stages. In this review, we describe the different approaches that instruct human iPSCs to form cortical organoids, summarize the different microcephalic conditions that have so far been modeled by organoids, and discuss the relevance of this model to decipher the cellular and molecular mechanisms of primary and secondary microcephalies.

Aim: To characterize the cortical structure, developmental, and cognitive profiles of patients with WD repeat domain 62 (WDR62)-related primary microcephaly., Method: In this observational study, we describe the developmental, neurological, cognitive, and brain imaging characteristics of 17 patients (six males, 11 females; mean age 12y 3mo standard deviation [SD] 5y 8mo, range 5y-24y 6mo) and identify 14 new variants of WDR62. We similarly analyse the phenotypes and genotypes of the 59 previously reported families., Results: Brain malformations, including pachygyria, neuronal heterotopia, schizencephaly, and microlissencephaly, were present in 11 out of 15 patients. The mean full-scale IQ of the 11 assessed patients was 51.8 (standard deviation [SD] 12.6, range 40-70). Intellectual disability was severe in four patients, moderate in four, and mild in three. Scores on the Vineland Adaptive Behavior Scales obtained from 10 patients were low for communication and motor skills (mean 38.29, SD 7.74, and 37.71, SD 5.74 respectively). The socialization score was higher (mean 47.14, SD 12.39). We found a significant difference between scores for communication and daily living skills (mean 54.43, SD 11.6; p=0.001, one-way analysis of variance). One patient displayed progressive ataxia., Interpretation: WDR62-related cognitive consequences may be less severe than expected because 3 out of 11 of the assessed patients had only mild intellectual disability and relatively preserved abilities of autonomy in daily life. We identified progressive ataxia in the second decade of life in one patient, which should encourage clinicians to follow up patients in the long term., (© 2021 Mac Keith Press.)

Background: Primary hereditary microcephaly (MCPH) comprises a large group of autosomal recessive disorders mainly affecting cortical development and resulting in a congenital impairment of brain growth. Despite the identification of >25 causal genes so far, it remains a challenge to distinguish between different MCPH forms at the clinical level., Methods: 7 patients with newly identified mutations in CDK5RAP2 (MCPH3) were investigated by performing prospective, extensive and systematic clinical, MRI, psychomotor, neurosensory and cognitive examinations under similar conditions., Results: All patients displayed neurosensory defects in addition to microcephaly. Small cochlea with incomplete partition type II was found in all cases and was associated with progressive deafness in 4 of them. Furthermore, the CDK5RAP2 protein was specifically identified in the developing cochlea from human fetal tissues. Microphthalmia was also present in all patients along with retinal pigmentation changes and lipofuscin deposits. Finally, hypothalamic anomalies consisting of interhypothalamic adhesions, a congenital midline defect usually associated with holoprosencephaly, was detected in 5 cases., Conclusion: This is the first report indicating that CDK5RAP2 not only governs brain size but also plays a role in ocular and cochlear development and is necessary for hypothalamic nuclear separation at the midline. Our data indicate that CDK5RAP2 should be considered as a potential gene associated with deafness and forme fruste of holoprosencephaly. These children should be given neurosensory follow-up to prevent additional comorbidities and allow them reaching their full educational potential., Trial Registration Number: NCT01565005., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Microcephaly is a neurodevelopmental condition characterized by a small brain size associated with intellectual deficiency in most cases and is one of the most frequent clinical sign encountered in neurodevelopmental disorders. It can result from a wide range of environmental insults occurring during pregnancy or postnatally, as well as from various genetic causes and represents a highly heterogeneous condition. However, several lines of evidence highlight a compromised mode of division of the cortical precursor cells during neurogenesis, affecting neural commitment or survival as one of the common mechanisms leading to a limited production of neurons and associated with the most severe forms of congenital microcephaly. In this context, the emergence of the endoplasmic reticulum (ER) and the Golgi apparatus as key guardians of cellular homeostasis, especially through the regulation of proteostasis, has raised the hypothesis that pathological ER and/or Golgi stress could contribute significantly to cortical impairments eliciting microcephaly. In this review, we discuss recent findings implicating ER and Golgi stress responses in early brain development and provide an overview of microcephaly-associated genes involved in these pathways., Competing Interests: Conflict of interest: The authors declare no conflict of interest., (Copyright: © 2019 Passemard et al.)

Whole exome sequencing undertaken in two siblings with delayed psychomotor development, absent speech, severe intellectual disability and postnatal microcephaly, with brain malformations consisting of cerebellar atrophy in the eldest affected and hypoplastic corpus callosum in the younger sister; revealed a homozygous intragenic deletion in VPS51, which encodes the vacuolar protein sorting-associated protein, one the four subunits of the Golgi-associated retrograde protein (GARP) and endosome-associated recycling protein (EARP) complexes that promotes the fusion of endosome-derived vesicles with the trans-Golgi network (GARP) and recycling endosomes (EARP). This observation supports a pathogenic effect of VPS51 variants, which has only been reported previously once, in a single child with microcephaly. It confirms the key role of membrane trafficking in normal brain development and homeostasis., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Autosomal recessive microcephaly or microcephaly primary hereditary (MCPH) is a genetically heterogeneous neurodevelopmental disorder characterized by a reduction in brain volume, indirectly measured by an occipitofrontal circumference (OFC) 2 standard deviations or more below the age- and sex-matched mean (-2SD) at birth and -3SD after 6 months, and leading to intellectual disability of variable severity. The abnormal spindle-like microcephaly gene (ASPM), the human ortholog of the Drosophila melanogaster "abnormal spindle" gene (asp), encodes ASPM, a protein localized at the centrosome of apical neuroprogenitor cells and involved in spindle pole positioning during neurogenesis. Loss-of-function mutations in ASPM cause MCPH5, which affects the majority of all MCPH patients worldwide. Here, we report 47 unpublished patients from 39 families carrying 28 new ASPM mutations, and conduct an exhaustive review of the molecular, clinical, neuroradiological, and neuropsychological features of the 282 families previously reported (with 161 distinct ASPM mutations). Furthermore, we show that ASPM-related microcephaly is not systematically associated with intellectual deficiency and discuss the association between the structural brain defects (strong reduction in cortical volume and surface area) that modify the cortical map of these patients and their cognitive abilities., (© 2017 Wiley Periodicals, Inc.)

Cell division and differentiation are two fundamental physiological processes that need to be tightly balanced to achieve harmonious development of an organ or a tissue without jeopardizing its homeostasis. The role played by the centriolar protein STIL is highly illustrative of this balance at different stages of life as deregulation of the human STIL gene expression has been associated with either insufficient brain development (primary microcephaly) or cancer, two conditions resulting from perturbations in cell cycle and chromosomal segregation. This review describes the recent advances on STIL functions in the control of centriole duplication and mitotic spindle integrity, and discusses how pathological perturbations of its finely tuned expression result in chromosomal instability in both embryonic and postnatal situations, highlighting the concept that common key factors are involved in developmental steps and tissue homeostasis.

The Golgi apparatus (GA) is involved in a whole spectrum of activities, from lipid biosynthesis and membrane secretion to the posttranslational processing and trafficking of most proteins, the control of mitosis, cell polarity, migration and morphogenesis, and diverse processes such as apoptosis, autophagy, and the stress response. In keeping with its versatility, mutations in GA proteins lead to a number of different disorders, including syndromes with multisystem involvement. Intriguingly, however, > 40% of the GA-related genes known to be associated with disease affect the central or peripheral nervous system, highlighting the critical importance of the GA for neural function. We have previously proposed the term "Golgipathies" in relation to a group of disorders in which mutations in GA proteins or their molecular partners lead to consequences for brain development, in particular postnatal-onset microcephaly (POM), white-matter defects, and intellectual disability (ID). Here, taking into account the broader role of the GA in the nervous system, we refine and enlarge this emerging concept to include other disorders whose symptoms may be indicative of altered neurodevelopmental processes, from neurogenesis to neuronal migration and the secretory function critical for the maturation of postmitotic neurons and myelination., (© 2019 S. Karger AG, Basel.)

The Golgi apparatus plays a central role in cell homeostasis, not only in processing and maturing newly synthesized proteins and lipids but also in orchestrating their sorting, packing, routing and recycling on the way to their final destination. These multiple secretory pathways require a complex ballet of vesicular and tubular carriers that continuously bud off from donor membranes and fuse to acceptor membranes. Membrane trafficking is particularly prominent in axons, where cargo molecules have a long way to travel before they reach the synapse, and in oligodendrocytes, which require an immense increase in membrane surface in order to sheathe axons in myelin. Interestingly, in recent years, genes encoding Golgi-associated proteins with a role in membrane trafficking have been found to be defective in an increasing number of inherited disorders whose clinical manifestations include postnatal-onset microcephaly (POM), white matter defects and intellectual disability. Several of these genes encode RAB GTPases, RAB-effectors or RAB-regulating proteins, linking POM and intellectual disability to RAB-dependent Golgi trafficking pathways and suggesting that their regulation is critical to postnatal brain maturation and function. Here, we review the key roles of the Golgi apparatus in post-mitotic neurons and the oligodendrocytes that myelinate them, and provide an overview of these Golgi-associated POM-causing genes, their function in Golgi organization and trafficking and the likely mechanisms that may link dysfunctions in RAB-dependent regulatory pathways with POM., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Cellular homeostasis is maintained by the highly organized cooperation of intracellular trafficking systems, including COPI, COPII, and clathrin complexes. COPI is a coatomer protein complex responsible for intracellular protein transport between the endoplasmic reticulum and the Golgi apparatus. The importance of such intracellular transport mechanisms is underscored by the various disorders, including skeletal disorders such as cranio-lenticulo-sutural dysplasia and osteogenesis imperfect, caused by mutations in the COPII coatomer complex. In this article, we report a clinically recognizable craniofacial disorder characterized by facial dysmorphisms, severe micrognathia, rhizomelic shortening, microcephalic dwarfism, and mild developmental delay due to loss-of-function heterozygous mutations in ARCN1, which encodes the coatomer subunit delta of COPI. ARCN1 mutant cell lines were revealed to have endoplasmic reticulum stress, suggesting the involvement of ER stress response in the pathogenesis of this disorder. Given that ARCN1 deficiency causes defective type I collagen transport, reduction of collagen secretion represents the likely mechanism underlying the skeletal phenotype that characterizes this condition. Our findings demonstrate the importance of COPI-mediated transport in human development, including skeletogenesis and brain growth., (Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Primary microcephaly is a neurodevelopmental disorder that is caused by a reduction in brain size as a result of defects in the proliferation of neural progenitor cells during development. Mutations in genes encoding proteins that localize to the mitotic spindle and centrosomes have been implicated in the pathogenicity of primary microcephaly. In contrast, the contractile ring and midbody required for cytokinesis, the final stage of mitosis, have not previously been implicated by human genetics in the molecular mechanisms of this phenotype. Citron kinase (CIT) is a multi-domain protein that localizes to the cleavage furrow and midbody of mitotic cells, where it is required for the completion of cytokinesis. Rodent models of Cit deficiency highlighted the role of this gene in neurogenesis and microcephaly over a decade ago. Here, we identify recessively inherited pathogenic variants in CIT as the genetic basis of severe microcephaly and neonatal death. We present postmortem data showing that CIT is critical to building a normally sized human brain. Consistent with cytokinesis defects attributed to CIT, multinucleated neurons were observed throughout the cerebral cortex and cerebellum of an affected proband, expanding our understanding of mechanisms attributed to primary microcephaly., (Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Autosomal recessive primary microcephaly results from abnormal brain development linked to proliferation defects in neural progenitors. The most frequent form, caused by ASPM mutations, is usually defined by a reduced brain volume and is associated with intellectual disability. Although many ASPM cases have now been reported, structural brain abnormalities and their link with cognitive disabilities have rarely been investigated. In this study, we used high resolution T1-weighted magnetic resonance imaging in seven patients with ASPM mutations and 39 healthy age-matched controls to quantify regional volumes, thickness, surface area, gyrification index and white matter volumes of 30 cortical regions. We observed a consistent reduction of 50% or more in the volume and surface area of all cortical regions except for the hippocampus and surrounding medial temporal structures, which were significantly less reduced. Neuropsychologic assessment indicated significant impairments of cognitive abilities. However, these impairments were associated with normal mnesic abilities, in keeping with the relative preservation of the hippocampus and medial temporal structures. These results show that, contrary to current opinion, the cortical volume and surface area of patients with ASPM mutations is reduced depending on a regionally specific fashion and their cognitive profile reflects this heterogeneity. The precise characterization of the cortical map and cognitive abilities of patients with ASPM mutations should allow developing more focused reeducative interventions well-suited to their real abilities., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Dymeclin is a Golgi-associated protein whose deficiency causes Dyggve-Melchior-Clausen syndrome (DMC, MIM #223800), a rare recessively inherited spondyloepimetaphyseal dysplasia consistently associated with postnatal microcephaly and intellectual disability. While the skeletal phenotype of DMC patients has been extensively described, very little is known about their cerebral anomalies, which result in brain growth defects and cognitive dysfunction. We used Dymeclin-deficient mice to determine the cause of microcephaly and to identify defective mechanisms at the cellular level. Brain weight and volume were reduced in all mutant mice from postnatal day 5 onward. Mutant mice displayed a narrowing of the frontal cortex, although cortical layers were normally organized. Interestingly, the corpus callosum was markedly thinner, a characteristic we also identified in DMC patients. Consistent with this, the myelin sheath was thinner, less compact and not properly rolled, while the number of mature oligodendrocytes and their ability to produce myelin basic protein were significantly decreased. Finally, cortical neurons from mutant mice and primary fibroblasts from DMC patients displayed substantially delayed endoplasmic reticulum to Golgi trafficking, which could be fully rescued upon Dymeclin re-expression. These findings indicate that Dymeclin is crucial for proper myelination and anterograde neuronal trafficking, two processes that are highly active during postnatal brain maturation., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: Transplanting exogenous neuronal progenitors to replace damaged neurons in the adult brain following injury or neurodegenerative disorders and achieve functional amelioration is a realistic goal. However, studies so far have rarely taken into consideration the preexisting inflammation triggered by the disease process that could hamper the effectiveness of transplanted cells. Here, we examined the fate and long-term consequences of human cerebellar granule neuron precursors (GNP) transplanted into the cerebellum of Harlequin mice, an adult model of progressive cerebellar degeneration with early-onset microgliosis., Methods: Human embryonic stem cell-derived progenitors expressing Atoh1, a transcription factor key to GNP specification, were generated in vitro and stereotaxically transplanted into the cerebellum of preataxic Harlequin mice. The histological and functional impact of these transplants was followed using immunolabeling and Rotarod analysis., Results: Although transplanted GNPs did not survive beyond a few weeks, they triggered the proliferation of endogenous nestin-positive precursors in the leptomeninges that crossed the molecular layer and differentiated into mature neurons. These phenomena were accompanied by the preservation of the granule and Purkinje cell layers and delayed ataxic changes. In vitro neurosphere generation confirmed the enhanced neurogenic potential of the cerebellar leptomeninges of Harlequin mice transplanted with exogenous GNPs., Interpretation: The cerebellar leptomeninges of adult mice contain an endogenous neurogenic niche that can be stimulated to yield mature neurons from an as-yet unidentified population of progenitors. The transplantation of human GNPs not only stimulates this neurogenesis, but, despite the potentially hostile environment, leads to neuroprotection and functional amelioration.

Objectives: Excitotoxicity plays a significant role in the pathogenesis of perinatal brain injuries. Among the consequences of excessive activation of the N-methyl-d-aspartate (NMDA)-type glutamate are oxidative stress caused by free radical release from damaged mitochondria, neuronal death and subsequent loss of connectivity. Drugs that could protect nervous tissue and support regeneration are attractive therapeutic options. The hepatocarcinoma intestine pancreas protein/pancreatitis-associated protein I (HIP/PAP) or Reg3α, which is approved for clinical testing for the protection and regeneration of the liver, is upregulated in the central nervous system following injury or disease. Here, we examined the neuroprotective/neuroregenerative potential of HIP/PAP following excitotoxic brain injury., Methods: We studied the expression of HIP/PAP and two of its putative effectors, cAMP-regulated phosphoprotein 19 (ARPP19) and growth-associated protein 43 (GAP-43), in the neonatal brain, and the protective/regenerative properties of HIP/PAP in three paradigms of perinatal excitotoxicity: intracerebral injection of the NMDA agonist ibotenate in newborn pups, a pediatric model of traumatic brain injury, and cultured primary cortical neurons., Results: HIP/PAP, ARPP19, and GAP-43 were expressed in the neonatal mouse brain. HIP/PAP prevented the formation of cortical and white matter lesions and reduced neuronal death and glial activation following excitotoxic insults in vivo. In vitro, HIP/PAP promoted neuronal survival, preserved neurite complexity and fasciculation, and protected cell contents from reactive oxygen species (ROS)-induced damage., Interpretation: HIP/PAP has strong neuroprotective/neuroregenerative potential following excitotoxic injury to the developing brain, and could represent an interesting therapeutic strategy in perinatal brain injury.

Directing differentiation of embryonic stem cells (ESCs) to specific neuronal subtype is critical for modeling disease pathology in vitro. An attractive means of action would be to combine regulatory differentiation factors and extrinsic inductive signals added to the culture medium. In this study, we have generated mature cerebellar granule neurons by combining a temporally controlled transient expression of Math1, a master gene in granule neuron differentiation, with inductive extrinsic factors involved in cerebellar development. Using a Tetracyclin-On transactivation system, we overexpressed Math1 at various stages of ESCs differentiation and found that the yield of progenitors was considerably increased when Math1 was induced during embryonic body stage. Math1 triggered expression of Mbh1 and Mbh2, two target genes directly involved in granule neuron precursor formation and strong expression of early cerebellar territory markers En1 and NeuroD1. Three weeks after induction, we observed a decrease in the number of glial cells and an increase in that of neurons albeit still immature. Combining Math1 induction with extrinsic factors specifically increased the number of neurons that expressed Pde1c, Zic1, and GABAα6R characteristic of mature granule neurons, formed "T-shaped" axons typical of granule neurons, and generated synaptic contacts and action potentials in vitro. Finally, in vivo implantation of Math1-induced progenitors into young adult mice resulted in cell migration and settling of newly generated neurons in the cerebellum. These results show that conditional induction of Math1 drives ESCs toward the cerebellar fate and indicate that acting on both intrinsic and extrinsic factors is a powerful means to modulate ESCs differentiation and maturation into a specific neuronal lineage., (Copyright © 2012 AlphaMed Press.)

Smith-McCort dysplasia (SMC) is a rare autosomal recessive spondylo-epi-metaphyseal dysplasia with skeletal features identical to those of Dyggve-Melchior-Clausen syndrome (DMC) but with normal intelligence and no microcephaly. Although both syndromes were shown to result from mutations in the DYM gene, which encodes the Golgi protein DYMECLIN, a few SMC patients remained negative in DYM mutation screening. Recently, autozygosity mapping and exome sequencing in a large SMC family have allowed the identification of a missense mutation in RAB33B, another Golgi protein involved in retrograde transport of Golgi vesicles. Here, we report a novel RAB33B mutation in a second SMC case that leads to a marked reduction of the protein as shown by Western blot and immunofluorescence. These data confirm the genetic heterogeneity of SMC dysplasia and highlight the role of Golgi transport in the pathogenesis of SMC and DMC syndromes., (© 2012 Wiley Periodicals, Inc.)

We report two male sibs, born from unrelated French Caribbean parents, presenting with an unclassifiable storage disorder. Pregnancy and delivery were uneventful. Stunted growth was noted during the first year of life. Both children have short stature (below - 4SD) with short trunk, barrel chest, micromelia with rhizomelic shortening, severe kyphoscoliosis, pectus carinatum, short hands and feet with metatarsus adductus, and excessive joint laxity of the small joints. Learning difficulties with borderline intelligence quotient (IQ) were noted in one of them. They had no hepatomegaly, no splenomegaly, and no dysmorphism. Skeletal X-rays survey demonstrated generalized platyspondyly with tongue-like deformity of the anterior part of the vertebral bodies, hypoplasia of the odontoid process, generalized epiphyseal dysplasia and abnormally shaped metaphyses. The acetabular roofs had a trident aspect. Ophthalmologic and cardiac examinations were normal. Spine deformity required surgical correction in one of the patient at age 4 years. Lysosomal enzymes assays including N-acetylgalactosamine-6-sulfate sulfatase and β-galactosidase were normal, excluding mucopolysaccharidoses type IV A and IV B (Morquio syndrome), respectively. Qualitative analysis found traces of dermatan and chondroitin-sulfates in urine, but quantitative glycosaminoglycan excretion fell within normal limits. They were no vacuolated lymphocytes. Abnormal coarse inclusions were present in eosinophils. Mild Alder anomaly was observed in polymorphonuclears. Granulations were discretely metachromatic with toluidine blue. Those morphological anomalies are in favor of a lysosomal storage disease. No inclusions were found in skin fibroblasts. We hypothesize that these two boys have a distinct autosomal recessive or X-linked lysosomal storage disorder of unknown origin that shares clinical and radiological features with Morquio disease., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Cerebral palsy is a major health problem caused by brain damage during pregnancy, delivery, or the immediate postnatal period. Perinatal stroke, intraventricular hemorrhage, and asphyxia are the most common causes of neonatal brain damage. Periventricular white matter damage (periventricular leukomalacia) is the predominant form in premature infants and the most common antecedent of cerebral palsy. Stem cell treatment has proven effective in restoring injured organs and tissues in animal models. The potential of stem cells for self-renewal and differentiation translates into substantial neuroprotection and neuroregeneration in the animal brain, with minimal risks of rejection and side effects. Stem cell treatments described to date have used neural stem cells, embryonic stem cells, mesenchymal stem cells, umbilical cord stem cells, and induced pluripotent stem cells. Most of these treatments are still experimental. In this review, we focus on the efficacy of stem cell therapy in animal models of cerebral palsy, and discuss potential implications for current and future clinical trials., (Copyright © 2011 American Neurological Association.)

Autosomal recessive primary microcephaly (MCPH) is a genetic disorder that causes a reduction of cortical outgrowth without severe interference with cortical patterning. It is associated with mutations in a number of genes encoding protein involved in mitotic spindle formation and centrosomal activities or cell cycle control. We have shown previously that blocking vasoactive intestinal peptide (VIP) during gestation in mice by using a VIP antagonist (VA) results in microcephaly. Here, we have shown that the cortical abnormalities caused by prenatal VA administration mimic the phenotype described in MCPH patients and that VIP blockade during neurogenesis specifically disrupts Mcph1 signaling. VA administration reduced neuroepithelial progenitor proliferation by increasing cell cycle length and promoting cell cycle exit and premature neuronal differentiation. Quantitative RT-PCR and Western blot showed that VA downregulated Mcph1. Inhibition of Mcph1 expression led to downregulation of Chk1 and reduction of Chk1 kinase activity. The inhibition of Mcph1 and Chk1 affected the expression of a specific subset of cell cycle–controlling genes and turned off neural stem cell proliferation in neurospheres. Furthermore, in vitro silencing of either Mcph1 or Chk1 in neurospheres mimicked VA-induced inhibition of cell proliferation. These results demonstrate that VIP blockade induces microcephaly through Mcph1 signaling and suggest that VIP/Mcph1/Chk1 signaling is key for normal cortical development.

Brain damage through excitotoxic mechanisms is a major cause of cerebral palsy in infants. This phenomenon usually occurs during the fetal period in human, and often leads to lifelong neurological morbidity with cognitive and sensorimotor impairment. However, there is currently no effective therapy. Significant recovery of brain function through neural stem cell implantation has been shown in several animal models of brain damage, but remains to be investigated in detail in neonates. In the present study, we evaluated the effect of cell therapy in a well-established neonatal mouse model of cerebral palsy induced by excitotoxicity (ibotenate treatment on postnatal day 5). Neurosphere-derived precursors or control cells (fibroblasts) were implanted into injured and control brains contralateral to the site of injury, and the fate of implanted cells was monitored by immunohistochemistry. Behavioral tests were performed in animals that received early (4 h after injury) or late (72 h after injury) cell implants. We show that neurosphere-derived precursors implanted into the injured brains of 5-day-old pups migrated to the lesion site, remained undifferentiated at day 10, and differentiated into oligodendrocyte and neurons at day 42. Although grafted cells finally die there few weeks later, this procedure triggered a reduction in lesion size and an improvement in memory performance compared with untreated animals, both 2 and 5 weeks after treatment. Although further studies are warranted, cell therapy could be a future therapeutic strategy for neonates with acute excitotoxic brain injury.

White-matter damage is a leading cause of neurological handicap. Although hypoxia-ischemia and excitotoxicity are major pathogenic factors, a role for genetic influences was suggested recently. Thus, protracted gestational hypoxia was associated with white-matter damage (WMD) in rat pups but not in mouse pups. Indeed, microglial activation and vessel-wall density on postnatal days (P)1 and P10 were found increased in both mouse and rat pups, but cell death, astrogliosis, and myelination were only significantly altered in hypoxic rat pups. We investigated whether this species-related difference was ascribable to effects of antenatal hypoxia on the expression of glutamate receptor subunits by using immunocytochemistry, PCR, and excitotoxic double hit insult. Quantitative PCR in hypoxic mouse pups on P1 showed 2- to 4-fold down-regulation of the AMPA-receptor subunits -1, 2, and -4; of the kainate-receptor subunit GluR7; and of the metabotropic receptor subunits mGluR1, -2, -3, -5, and -7. None of the glutamate-receptor subunits was down-regulated in the hypoxic rat pups. NR2B was the only NMDA-receptor subunit that was down-regulated in hypoxic mice but not in hypoxic rat on P1. Ifenprodil administration to induce functional inhibition of NMDA containing NR2B-subunit receptors prevented hypoxia-induced myelination delay in rat pups. Intracerebral injection of a glutamate agonist produced a larger decrease in ibotenate-induced excitotoxic lesions in hypoxic mouse pups than in normoxic mouse pups. Gestational hypoxia may regulate the expression of specific glutamate-receptor subunits in fetal mice but not in fetal rats. Therefore, genetic factors may influence the susceptibility of rodents to WMD.

Apoptosis-inducing factor (AIF) deficiency compromises oxidative phosphorylation. Harlequin mice, in which AIF is downregulated, develop a severe mitochondrial complex I (CI) deficiency, suggesting that Harlequin mice may represent a natural model of the most common oxidative phosphorylation disorders. However, the brain phenotype specifically involves the cerebellum, whereas human CI deficiencies often manifest as complex multifocal neuropathologies. To evaluate whether this model can be used as to study CI-deficient disorders, the whole brain of Harlequin mice was investigated during the course of the disease. Neurodegeneration was not restricted to the cerebellum but progressively affected thalamic, striatal, and cortical regions as well. Strong astroglial and microglial activation with extensive vascular proliferation was observed by 4 months of age in thalamic, striatal, and cerebellar nuclei associated with somatosensory-motor pathways. At 2 months of age, degenerating mitochondria were observed in most cells in these structures, even in nondegenerating neurons, a finding that indicates mitochondrial injury is a cause rather than an effect of neuronal cell death. Thus, apoptosis-inducing factor deficiency induces early mitochondrial degeneration, followed by progressive multifocal neuropathology (a phenotype broader than previously described), and resembles some histopathologic features of devastating human neurodegenerative mitochondriopathies associated with CI deficiency.

Understanding the trafficking of G-protein-coupled receptors (GPCRs) is of particular importance, especially when modifications of the neurochemic environment occur as in pathological or therapeutic circumstances. In the central nervous system, although some GPCRs were reported to internalize in vivo, little is known about their trafficking downstream of the endocytic event. To address this issue, distribution and expression pattern of the major somatostatin receptor subtype, the somatostatin type 2 (sst2), was monitored in the hippocampus using immunofluorescence, autoradiographic and immunogold experiments from 10 minutes to 7 days after in vivo injection of the receptor agonist octreotide. We then analyzed whether postendocytic trafficking of the receptor was dependent upon integrity of the microtubule network using colchicine-injected animals. Together, our results suggest that upon agonist stimulation, dendritic receptors are retrogradely transported through a microtubule-dependent mechanism to a trans Golgi domain enriched in the t-SNARE syntaxin 6 and trans Golgi network 38 proteins, before recycling. Because we show that the exit rate from the trans Golgi apparatus back to the plasma membrane (hours) is slower than the entry rate (minutes), the neuronal postendocytic trafficking of sst2 receptor is likely to have functional consequences in several neurological diseases in which an increase in somatostatin release occurs.

Crouzon Syndrome (CS), Pfeiffer syndrome (PS) and the phenotypically related Jackson-Weiss (JW) variant are three craniosynostotic conditions caused by heterozygous mutations in Fibroblast Growth Factor Receptor (FGFR) genes. Screening a large cohort of 84 patients with clinical features of CS, PS or JW by direct sequencing of genomic DNA, enabled FGFR1, 2 or 3 mutation detection in 79 cases. Mutations preferentially occurred in exons 8 and 10 of FGFR2 encoding the third Ig loop of the receptor. Among the 74 FGFR2 mutations that we identified, four were novel including three missense substitutions causing CS and a 2 bp deletion creating a premature stop codon and producing JW phenotype. Five FGFR2 mutations were found in one of the two tyrosine kinase subdomains and one in the Ig I loop. Interestingly, two FGFR2 mutations creating cysteine residues (W290C and Y340C) caused severe forms of PS while conversion of the same residues into another amino-acid (W290G/R, Y340H) resulted in Crouzon phenotype exclusively. Our data provide conclusive evidence that the mutational spectrum of FGFR2 mutations in CS and PS is wider than originally thought. Genotype-phenotype analyses based on our cohort and previous studies further indicate that in spite of some overlap, PS and CS are preferentially accounted for by two distinct sets of FGFR2 mutations. A limited number of recurrent amino-acid changes (W290C, Y340C, C342R and S351C) is commonly associated with the most severe Pfeiffer phenotypes of poor prognosis.

Dyggve-Melchior-Clausen syndrome (DMC) (MIM 223800) and Smith-McCort dysplasia (SMC) (MIM 607326) are rare allelic autosomal recessive spondylo-epi-metaphyseal dysplasias (SEMDs) characterized by similar skeletal manifestations. Both phenotypes have been mapped to chromosome 18q21.1 and mutations in the DYM (dymeclin) gene were identified in 13 families with DMC and in two families with SMC. Most mutations identified in DMC predict a loss of function, while those identified in SMC are mainly missense mutations, presumably associated with residual DYM activity and a less severe phenotype. We studied three consanguineous families from Turkey, Lebanon, and Georgia, one with SMC and two with DMC and identified different homozygous DYM mutations (IVS3 194-1G > A, 938_942delTGTCT) in the DMC families. No mutation was identified in the SMC family, possibly suggesting genetic heterogeneity of this disorder.

The genes encoding succinate dehydrogenase (SDH) subunits B, C and D, act as tumour suppressors in neuro-endocrine tissues. Tumour formation has been associated with succinate accumulation. In paraganglioma cells, two forms of SDHA (type I, II) were found which might preclude significant succinate accumulation in the case of a mutation in either form. In fibroblasts only SDHA type I is found. In these cells, SDHA type I mutation leads to SDH deficiency, succinate accumulation and hypoxia-inducible factor 1alpha(HIF1alpha) nuclear translocation. HIF1alpha nuclear translocation was not observed in ATPase-deficient fibroblasts with increased superoxide production and was found to be independent of cellular iron availability in SDHA-mutant cells. This suggests that neither superoxides nor iron were causative of HIF1alpha nuclear translocation. Conversely, alpha-ketoglutarate (alpha-KG) inhibits this nuclear translocation. Therefore, the pseudo-hypoxia pathway in SDH-deficient cells depends on the HIF1alphaprolyl hydroxylase product/substrate (succinate/alpha-KG) equilibrium. In SDH deficiency, organic acids thus appear instrumental in the HIF1alpha-dependent cascade suggesting a direct link between SDH and tumourigenesis.

Mitochondrial succinate dehydrogenase (SDH) consists merely of four nuclearly encoded subunits. It participates in the electron transfer in the respiratory chain and in succinate catabolism in the Krebs cycle. Mutations in the four genes, SDHA, B, C and D, have been reported, resulting in strikingly diverse clinical presentations. So far, SDHA mutations have been reported to cause an encephalomyopathy in childhood, while mutations in the genes encoding the other three subunits have been associated only with tumour formation. Following a brief description of SDH genes and subunits, we examine the properties and roles of SDH in the mitochondria. This allows further discussion of the several hypotheses proposed to account for the different clinical presentations resulting from impaired activity of the enzyme. Finally we stress the importance of SDH as a target and/or marker in a number of diseases and the need to better delineate the consequences of SDH deficiency in humans.

Spondyloepimetaphyseal dysplasias (SEMD) represent a heterogeneous group of conditions composed of at least 15 well-defined entities. The classification is based on clinical, radiological and molecular findings. Among them, several conditions also include a mental retardation (MR) syndrome, namely Wolcott-Rallison syndrome, Dyggve-Melchior-Clausen syndrome (DMC) and lysosomal storage disorders. Here, we report on a novel form of SEMD with MR in two Pakistani sisters born to first-cousin parents. SEMD, MR, microcephaly, ataxia, facial dysmorphism and hirsutism of back and legs were noted in the two children. Skeletal findings included flat vertebral bodies with irregular vertebral plates, irregular and flared metaphyses with vertical striations, small and irregular epiphyses, small carpal bones and narrow iliac wings without lacy pelvis iliac crest. Similarities with DMC prompted us to test and eventually exclude the DMC gene, dymeclin, by direct sequencing. Similarly, we excluded the PAPSS2 gene (3'-alpha phosphoadenosine 5'-phosphosulphate synthase 2) responsible for SEMD Pakistani type. The combination of features observed in the two sisters does not fit with any previously reported SEMD and represents therefore a novel form of autosomal recessive SEMD with MR.

Dyggve-Melchior-Clausen (DMC) is a rare autosomal-recessive disorder characterized by the association of a progressive spondylo-epi-metaphyseal dysplasia and mental retardation ranging from mild to severe. Electron microscopy studies of both DMC chondrocytes and fibroblasts reveal an enlarged endoplasmic reticulum network and a large number of intracytoplasmic membranous vesicles, suggesting that DMC syndrome may be a storage disorder. Indeed, DMC phenotype is often compared to that of type IV mucopolysaccharidosis (Morquio disease), a lysosomal disorder due to either N-acetylgalactosamine-6-sulphatase or beta-galactosidase deficiency. To date, however, the lysosomal pathway appears normal in DMC patients and biochemical analyses failed to reveal any enzymatic deficiency or accumulated substrate. Linkage studies using homozygosity mapping have led to the localization of the disease-causing gene on chromosome 18q21.1. The gene was recently identified as a novel transcript (Dym) encoding a 669-amino acid product (Dymeclin) with no known domains or function. Sixteen different Dym mutations have now been described in 21 unrelated families with at least five founder effects in Morocco, Lebanon, and Guam Island. Smith-MacCort syndrome (SMC), a rare variant of DMC syndrome without mental retardation, was shown to be allelic of DMC syndrome and to result from mutations in Dym that would be less deleterious to the brain. The present review focuses on clinical, radiological, and cellular features and evolution of DMC/SMC syndromes and discusses them with regard to identified Dym mutations and possible roles of the Dym gene product.

Premature fusion of cranial sutures underlies the clinical condition of 'craniosynostosis', a common human disorder that occurs in both nonsyndromic and syndromic forms. The subgroup of syndromic craniosynostoses usually associates limb abnormalities and facial dysmorphism to skull distortion. Over the past decade, some of the genes causing these phenotypes have been identified. Among these, the gene encoding FGFR2, one of four members of the fibroblast growth factor receptor(FGFR) family, has been shown to account for several severe conditions including Apert, Pfeiffer, Crouzon, Beare-Stevenson and Jackson-Weiss syndromes. Two other FGFRs, FGFR1 and FGFR3, also account for craniosynostoses of variable severity [Pfeiffer, Crouzon with acanthosis nigricans (a pre-malignant skin disorder), and Muenke syndromes]. By contrast,Saethre-Chotzen syndrome and craniosynostosis (Boston-type) arise from mutations in the Twist and muscle segment homeobox 2 (MSX2) transcription factors, respectively. Whereas most FGFR mutations are likely to cause ligand independent activation of the receptor, leading to an upregulation of signaling pathways, mutations in the basic helix-loop-helix (bHLH) transcription factor Twist appear to induce loss of protein function. This review will summarise and discuss some of the cellular and molecular mechanisms involved in normal and abnormal craniofacial development, focusing on the possible interactions between the different factors controlling membranous ossification.

Saethre-Chotzen syndrome (SCS) is a human autosomal dominant disorder characterized by premature fusion of cranial sutures caused by mutations of the Twist gene encoding a basic helix-loop-helix (bHLH) transcription factor. We previously showed that Twist haploinsufficiency caused by a Y103X nonsense mutation in SCS alters both proliferation and osteoblast gene expression in human calvarial osteoblasts, indicating that Twist is an important regulator of osteoblast differentiation. Here we show that Twist haploinsufficiency alters osteoblast apoptosis in SCS. Analysis of terminal deoxynucleotidyl transferase-mediated nick-end labelling (TUNEL) demonstrated increased osteoblast and osteocyte apoptosis in coronal sutures from two SCS patients with nonsense mutations (Y103X and Q109X) that result in the synthesis of bHLH-truncated proteins, and one patient with a missense mutation in the basic domain (R118C) that abolishes Twist DNA binding. To assess the mechanisms involved, we studied osteoblast apoptosis in mutant (M-Tw) calvarial cells bearing the Y103X mutation resulting in decreased Twist mRNA and protein levels. M-Tw cells cultured in low serum conditions showed enhanced DNA fragmentation compared to normal (Nl) age-matched calvarial cells. Biochemical analysis showed increased activity of initiator caspases-2 and -8 and downstream effector caspases-3, -6 and -7 in mutant osteoblasts. Caspase-2 was upstream of caspase-8 and effector caspases-3, -6 and -7 because their activities were suppressed by a specific caspase-2 inhibitor. M-Tw osteoblasts also showed increased cytochrome c release from the mitochondria. However, the activity of the downstream effector caspase-9 was not increased due to overexpression of the antagonist protein Hsp70. Detection of differentially expressed genes using cDNA expression array revealed increased Bax and TNFalpha mRNA levels in M-Tw compared to Nl cells, a finding confirmed by RT-PCR and western blot analyses. Neutralization of TNFalpha overexpression using anti-TNFalpha or anti-TNF receptor 1 antibodies abolished the increased activity of caspase-2, caspase-8 and caspases-3, -6 and -7 in M-Tw osteoblasts. These studies provide novel evidence that Twist haploinsufficiency in SCS promotes osteoblast apoptosis by a TNFalpha-caspase-2-caspase-8-caspases-3, -6, -7 cascade, and uncover a molecular mechanism in which Twist plays an anti-apoptotic role in human calvarial osteoblasts.