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Kinesins are motor proteins involved in microtubule (MT)-mediated intracellular transport. They contribute to key cellular processes, including intracellular trafficking, organelle dynamics and cell division. Pathogenic variants in kinesin-encoding genes underlie several human diseases characterized by an extremely variable clinical phenotype, ranging from isolated neurodevelopmental/neurodegenerative disorders to syndromic phenotypes belonging to a family of conditions collectively termed as ‘ciliopathies.’ Among kinesins, kinesin-1 is the most abundant MT motor for transport of cargoes towards the plus end of MTs. Three kinesin-1 heavy chain isoforms exist in mammals. Different from KIF5A and KIF5C, which are specifically expressed in neurons and established to cause neurological diseases when mutated, KIF5B is an ubiquitous protein. Three de novo missense KIF5B variants were recently described in four subjects with a syndromic skeletal disorder characterized by kyphomelic dysplasia, hypotonia and DD/ID. Here, we report three dominantly acting KIF5B variants (p.Asn255del, p.Leu498Pro and p.Leu537Pro) resulting in a clinically wide phenotypic spectrum, ranging from dilated cardiomyopathy with adult-onset ophthalmoplegia and progressive skeletal myopathy to a neurodevelopmental condition characterized by severe hypotonia with or without seizures. In vitro and in vivo analyses provide evidence that the identified disease-associated KIF5B variants disrupt lysosomal, autophagosome and mitochondrial organization, and impact cilium biogenesis. All variants, and one of the previously reported missense changes, were shown to affect multiple developmental processes in zebrafish. These findings document pleiotropic consequences of aberrant KIF5B function on development and cell homeostasis, and expand the phenotypic spectrum resulting from altered kinesin-mediated processes.

Spinal muscular atrophy (SMA) is a motor neuron disease caused by loss of function mutations in the Survival Motor Neuron 1 (SMN1) gene and reduced expression of the SMN protein, leading to spinal motor neuron death, muscle weakness and atrophy. Although humans harbour the highly homologous SMN2 gene, its defective splicing regulation yields a truncated and unstable SMN protein. The first therapy for SMA was recently approved by the Food and Drug Administration and consists of an antisense oligonucleotide (Nusinersen) rendering SMN2 functional and thus improving patients' motor activity and quality of life. Nevertheless, not all patients equally respond to this therapy and the long-term tolerability and safety of Nusinersen are still unknown. Herein, in vivo splicing assays indicated that the HDAC inhibitor LBH589 is particularly efficient in rescuing the SMN2 splicing defect in SMA fibroblasts and SMA type-I mice-derived neural stem cells. Western blot analyses showed that LBH589 also causes a significant increase in SMN protein expression in SMA cells. Moreover chromatin immunoprecipitation analyses revealed that LBH589 treatment induces widespread H4 acetylation of the entire SMN2 locus and selectively favors the inclusion of the disease-linked exon 7 in SMN2 mature mRNA. The combined treatment of SMA cells with sub-optimal doses of LBH589 and of an antisense oligonucleotide that mimic Nusinersen (ASO_ISSN1) elicits additive effects on SMN2 splicing and SMN protein expression. These findings suggest that HDAC inhibitors can potentiate the activity of Nusinersen and support the notion that 'SMN-plus' combinatorial therapeutic approaches might represent an enhanced opportunity in the scenario of SMA therapy.

Riboflavin transporter deficiency (RTD) is a rare genetic disorder characterized by motor, sensory and cranial neuropathy. This childhood-onset neurodegenerative disease is caused by biallelic pathogenic variants in either SLC52A2 or SLC52A3 genes, resulting in insufficient supply of riboflavin (vitamin B2) and consequent impairment of flavoprotein-dependent metabolic pathways. Current therapy, empirically based high-dose riboflavin supplementation, ameliorates the progression of the disease, even though response to treatment is variable and partial. Recent studies have highlighted concurrent pathogenic contribution of cellular energy dysmetabolism and cytoskeletal derangement. In this context, patient specific RTD models, based on induced pluripotent stem cell (iPSC) technology, have provided evidence of redox imbalance, involving mitochondrial and peroxisomal dysfunction. Such oxidative stress condition likely causes cytoskeletal perturbation, associated with impaired differentiation of RTD motor neurons. In this review, we discuss the most recent findings obtained using different RTD models. Relevantly, the integration of data from innovative iPSC-derived in vitro models and invertebrate in vivo models may provide essential information on RTD pathophysiology. Such novel insights are expected to suggest custom therapeutic strategies, especially for those patients unresponsive to high-dose riboflavin treatments.

The cytoskeletal network plays a crucial role in the differentiation, morphogenesis, function and homeostasis of the nervous tissue, so that alterations in any of its components may lead to neurodegenerative diseases. Riboflavin transporter deficiency (RTD), a childhood-onset disorder characterized by degeneration of motor neurons (MNs), is caused by biallelic mutations in genes encoding the human riboflavin (RF) transporters. In a patient-specific induced pluripotent stem cells (iPSCs) model of RTD, we recently demonstrated altered cell-cell contacts, energy dysmetabolism and redox imbalance. The present study focuses on cytoskeletal composition and dynamics associated to RTD, utilizing patients' iPSCs and derived MNs. Abnormal expression and distribution of α- and β-tubulin (α- and β-TUB), as well as imbalanced tyrosination of α-TUB, accompanied by an impaired ability to re-polymerize after nocodazole treatment, were found in RTD patient-derived iPSCs. Following differentiation, MNs showed consistent changes in TUB content, which was associated with abnormal morphofunctional features, such as neurite length and Ca2+ homeostasis, suggesting impaired differentiation. Beneficial effects of RF supplementation, alone or in combination with the antioxidant molecule N-acetyl cystine (NAC), were assessed. RF administration resulted in partially improved cytoskeletal features in patients' iPSCs and MNs, suggesting that redundancy of transporters may rescue cell functionality in the presence of adequate concentrations of the vitamin. Moreover, supplementation with NAC was demonstrated to be effective in restoring all the considered parameters, when used in combination with RF, thus supporting the therapeutic use of both compounds., Summary: This study deals with pathomechanisms underlying riboflavin transporter deficiency, a rare recessively inherited early-onset neurodegenerative condition. Using patient-derived iPSCs, we report on cytoskeletal abnormalities, which are reverted by combined riboflavin/antioxidant treatment.

PCDH19-related epilepsy is a rare genetic disease caused by defective function of PCDH19, a calcium-dependent cell–cell adhesion protein of the cadherin superfamily. This disorder is characterized by a heterogeneous phenotypic spectrum, with partial and generalized febrile convulsions that are gradually increasing in frequency. Developmental regression may occur during disease progression. Patients may present with intellectual disability (ID), behavioral problems, motor and language delay, and a low motor tone. In most cases, seizures are resistant to treatment, but their frequency decreases with age, and some patients may even become seizure-free. ID generally persists after seizure remission, making neurological abnormalities the main clinical issue in affected individuals. An effective treatment is lacking. In vitro studies using patient-derived induced pluripotent stem cells (iPSCs) reported accelerated neural differentiation as a major endophenotype associated with PCDH19 mutations. By using this in vitro model system, we show that accelerated in vitro neurogenesis is associated with a defect in the cell division plane at the neural progenitors stage. We also provide evidence that altered PCDH19 function affects proper mitotic spindle orientation. Our findings identify an altered equilibrium between symmetric versus asymmetric cell division as a previously unrecognized mechanism contributing to the pathogenesis of this rare epileptic encephalopathy.

Leukodystrophies are a heterogeneous group of rare inherited disorders that mostly involve the white matter of the CNS. These conditions are characterized by primary glial cell and myelin sheath pathology of variable aetiology, which causes secondary axonal degeneration, generally emerging with disease progression. Whole exome sequencing performed in five large consanguineous nuclear families allowed us to identify homozygosity for two recurrent missense variants affecting highly conserved residues of RNF220 as the causative event underlying a novel form of leukodystrophy with ataxia and sensorineural deafness. We report these two homozygous missense variants (p.R363Q and p.R365Q) in the ubiquitin E3 ligase RNF220 as the underlying cause of this novel form of leukodystrophy with ataxia and sensorineural deafness that includes fibrotic cardiomyopathy and hepatopathy as associated features in seven consanguineous families. Mass spectrometry analysis identified lamin B1 as the RNF220 binding protein and co-immunoprecipitation experiments demonstrated reduced binding of both RNF220 mutants to lamin B1. We demonstrate that RNF220 silencing in Drosophila melanogaster specifically affects proper localization of lamin Dm0, the fly lamin B1 orthologue, promotes its aggregation and causes a neurodegenerative phenotype, strongly supporting the functional link between RNF220 and lamin B1. Finally, we demonstrate that RNF220 plays a crucial role in the maintenance of nuclear morphology; mutations in primary skin fibroblasts determine nuclear abnormalities such as blebs, herniations and invaginations, which are typically observed in cells of patients affected by laminopathies. Overall, our data identify RNF220 as a gene implicated in leukodystrophy with ataxia and sensorineural deafness and document a critical role of RNF220 in the regulation of nuclear lamina. Our findings provide further evidence on the direct link between nuclear lamina dysfunction and neurodegeneration.

Riboflavin transporter deficiency (RTD) is a childhood-onset neurodegenerative disorder characterized by sensorineural deafness and motor neuron degeneration. Since riboflavin plays key functions in biological oxidation-reduction reactions, energy metabolism pathways involving flavoproteins are affected in RTD. We recently generated iPSC lines from affected individuals as an in vitro model of the disease and documented mitochondrial impairment in these cells dramatically impacting cell redox status. In the present work, we extend our study to motor neurons (MNs), i.e., the cell type mostly affected in patients with RTD. Altered intracellular distribution of mitochondria was detected by confocal microscopic analysis, following immunofluorescence for superoxide dismutase 2 (SOD2), as a dual mitochondrial and antioxidant marker, and βIII Tubulin, as neuronal marker. We demonstrate significantly lower SOD2 levels in RTD MNs, as compared to their healthy counterparts. Mitochondrial ultrastructural abnormalities were also assessed by Focused Ion Beam/Scanning Electron Microscopy. Moreover, we investigated the effects of combination treatment using riboflavin and N-acetylcysteine, which is a widely employed antioxidant. Overall, our findings further support the potential of patient specific RTD models, and provide evidence of mitochondrial alterations in RTD-related iPSC-derived MNs, emphasizing oxidative stress involvement in this rare disease. We also provide new clues for possible therapeutic strategies, aimed at correcting mitochondrial defects, based on the use of antioxidants.

Gnathostome jaw patterning involves focal instructive signals from the embryonic surface cephalic ectoderm (SCE) to a fungible population of cranial neural crest. The spatial refinement of these signals, particularly for those patterning the upper jaws, is not fully understood. We demonstrate thatFoxg1, broadly expressed in the SCE overlying the upper jaw primordia, is required for both neurocranial and viscerocranial development, including the sensory capsules, neurocranial base, middle ear, and upper jaws.Foxg1controls upper jaw molecular identity and morphologic development by actively inhibiting the inappropriate acquisition of lower jaw molecular identity within the upper jaw primordia, and is necessary for the appropriate elaboration of the λ-junction, choanae, palate, vibrissae, rhinarium, upper lip and auxiliary eye. It regulates intra-epithelial cellular organization, gene expression, and the topography of apoptosis within the SCE.Foxg1integrates forebrain and skull development and genetically interacts withDlx5to establish a single, rostral cranial midline.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

Riboflavin transporter deficiency (RTD) is a childhood-onset neurodegenerative disorder characterized by sensorineural deafness and motor neuron degeneration. Since riboflavin plays key functions in biological oxidation-reduction reactions, energy metabolism pathways involving flavoproteins are affected in RTD. We recently generated induced pluripotent stem cell (iPSC) lines from affected individuals as an in vitro model of the disease and documented mitochondrial impairment in these cells, dramatically impacting cell redox status. This work extends our study to motor neurons (MNs), i.e., the cell type most affected in patients with RTD. Altered intracellular distribution of mitochondria was detected by confocal microscopic analysis (following immunofluorescence for superoxide dismutase 2 (SOD2), as a dual mitochondrial and antioxidant marker), and &beta, III-Tubulin, as a neuronal marker. We demonstrate significantly lower SOD2 levels in RTD MNs, as compared to their healthy counterparts. Mitochondrial ultrastructural abnormalities were also assessed by focused ion beam/scanning electron microscopy. Moreover, we investigated the effects of combination treatment using riboflavin and N-acetylcysteine, which is a widely employed antioxidant. Overall, our findings further support the potential of patient-specific RTD models and provide evidence of mitochondrial alterations in RTD-related iPSC-derived MNs&mdash, emphasizing oxidative stress involvement in this rare disease. We also provide new clues for possible therapeutic strategies aimed at correcting mitochondrial defects, based on the use of antioxidants.

The development of the nervous system requires cytoskeleton-mediated processes coordinating self-renewal, migration, and differentiation of neurons. It is not surprising that many neurodevelopmental problems and neurodegenerative disorders are caused by deficiencies in cytoskeleton-related genes. For this reason, we focus on the cytoskeletal dynamics in proliferating iPSCs and in iPSC-derived neurons to better characterize the underpinnings of cytoskeletal organization looking at actin and tubulin repolymerization studies using the cell permeable probes SiR-Actin and SiR-Tubulin. During neurogenesis, each neuron extends an axon in a complex and changing environment to reach its final target. The dynamic behavior of the growth cone and its capacity to respond to multiple spatial information allows it to find its correct target. We decided to characterize various parameters of the actin filaments and microtubules. Our results suggest that a rapid re-organization of the cytoskeleton occurs 45 minutes after treatments with de-polymerizing agents in iPSCs and 60 minutes in iPSC-derived neurons in both actin filaments and microtubules. The quantitative data confirm that the actin filaments have a primary role in the re-organization of the cytoskeleton soon after de-polymerization, while microtubules have a major function following cytoskeletal stabilization. In conclusion, we investigate the possibility that de-polymerization of the actin filaments may have an impact on microtubules organization and that de-polymerization of the microtubules may affect the stability of the actin filaments. Our results suggest that a reciprocal influence of the actin filaments occurs over the microtubules and vice versa in both in iPSCs and iPSC-derived neurons.

Induced pluripotent stem cells (iPSCs) hold great promise for developing personalized regenerative medicine, however characterization of their biological features is still incomplete. Moreover, changes occurring in long-term cultured iPSCs have been reported, suggesting these as a model of cellular aging. For this reason, we addressed the ultrastructural characterization of iPSCs, with a focus on possible time-dependent changes, involving specific cell compartments. To this aim, we comparatively analysed cultures at different timepoints, by an innovative electron microscopic technology (FIB/SEM). We observed progressive loss of cell-to-cell contacts, associated with increased occurrence of exosomes. Mitochondria gradually increased, while acquiring an elongated shape, with well-developed cristae. Such mitochondrial maturation was accompanied by their turnover, as assessed by the presence of autophagomes (undetectable in young iPSCs), some containing recognizable mitochondria. This finding was especially frequent in middle-aged iPSCs, while being occasional in aged cells, suggesting early autophagic activation followed by a decreased efficiency of the process with culturing time. Accordingly, confocal microscopy showed age-dependent alterations to the expression and distribution of autophagic markers. Interestingly, responsivity to rapamycin, highest in young iPSCs, was almost lost in aged cells. Overall, our results strongly support long-term cultured iPSCs as a model for studying relevant aspects of cellular senescence, involving intercellular communication, energy metabolism, and autophagy.

Nuclear integrity and mechanical stability of the nuclear envelope (NE) are conferred by the nuclear lamina, a meshwork of intermediate filaments composed of A- and B-type lamins, supporting the inner nuclear membrane and playing a pivotal role in chromatin organization and epigenetic regulation. During cell senescence, nuclear alterations also involving NE architecture are widely described. In the present study, we utilized induced pluripotent stem cells (iPSCs) upon prolonged in vitro culture as a model to study aging and investigated the organization and expression pattern of NE major constituents. Confocal and four-dimensional imaging combined with molecular analyses, showed that aged iPSCs are characterized by nuclear dysmorphisms, nucleoskeletal components (lamin A/C-prelamin isoforms, lamin B1, emerin, and nesprin-2) imbalance, leading to impaired nucleo-cytoplasmic MKL1 shuttling, actin polymerization defects, mitochondrial dysfunctions, SIRT7 downregulation and NF-kBp65 hyperactivation. The observed age-related NE features of iPSCs closely resemble those reported for premature aging syndromes (e.g., Hutchinson-Gilford progeria syndrome) and for somatic cell senescence. These findings validate the use of aged iPSCs as a suitable cellular model to study senescence and for investigating therapeutic strategies aimed to treat premature aging.

PCDH19 gene mutations have been recently associated with an epileptic syndrome characterized by focal and generalized seizures. The PCDH19 gene (Xq22.1) has an unusual X-linked inheritance with a selective involvement for female subjects. A cellular interference mechanism has been hypothesized and male patients can manifest epilepsy only in the case of a mosaicism. So far about 100 female patients, and only one symptomatic male have been described. Using targeted next generation sequencing (NGS) approach we found a PCDH19 point mutation in two male patients with a clinical picture suggestive of PCDH19-related epilepsy. The system allowed us to verify that the two c.1352 C>T; p.(Pro451Leu) and c.918C>G; p.(Tyr306*) variants occurred in mosaic status. Mutations were confirmed by Sanger sequencing and quantified by real-time polymerase chain reaction (PCR). Up to now, the traditional molecular screening for PCDH19-related epilepsy has been targeted to all females with early onset epilepsy with or without cognitive impairment. Male patients were generally excluded. We describe for the first time two mosaic PCDH19 point mutations in two male patients with a clinical picture suggestive of PCDH19-related epilepsy. This finding opens new opportunities for the molecular diagnoses in patients with a peculiar type of epilepsy that remains undiagnosed in male patients.

During the process of neurogenesis, the stem cell committed to the neuronal cell fate starts a series of molecular and morphological changes. The understanding of the physio-pathology of mechanisms controlling the molecular and morphological changes occurring during neuronal differentiation is fundamental to the development of effective therapies for many neurologic diseases. Unfortunately, our knowledge of the biological events occurring in the cell during neuronal differentiation is still poor. In this study, we focus preliminarily on the relevance of the cytoskeletal rearrangements, which earlier drive the morphology of the neuronal precursors, and later the migrating/mature neurons. In fact, neuritogenesis, neurite branching, outgrowth and retraction are seminal to the development of a fully functional nervous system. With this in mind, we highlight the importance of iPSC technology to study the processes of cytoskeletal-driven morphological changes during neuronal differentiation.

// Claudia Compagnucci 1 , Stefania Petrini 2 , Norimichi Higuraschi 3 , Marina Trivisano 4 , Nicola Specchio 4 , Shinichi Hirose 3 , Enrico Bertini 1 and Alessandra Terracciano 1 1 Unit of Neuromuscular and Neurodegenerative Diseases, Bambino Gesu Children’s Hospital, IRCCS, Rome, Italy 2 Confocal Microscopy Core Facility, Research Laboratories, Bambino Gesu Children’s Hospital, IRCCS, Rome, Italy 3 Central Research Institute for the Pathomechanisms of Epilepsy, Fukuoka University, Fukuoka, Japan 4 Division of Neurology, Bambino Gesu Children’s Hospital, IRCCS, Rome, Italy Correspondence to: Alessandra Terracciano, email: // Keywords : PCDH19, human iPSCs, iPSC-derived neurons, neuronal polarity, neural rosettes, Neruoscience Section Received : July 30, 2015 Accepted : September 05, 2015 Published : September 21, 2015 Abstract PCDH19 (Protocadherin 19), a member of the cadherin superfamily, is involved in the pathogenic mechanism of an X-linked model of neurological disease. The biological function of PCHD19 in human neurons and during neurogenesis is currently unknown. Therefore, we decided to use the model of the induced pluripotent stem cells (iPSCs) to characterize the location and timing of expression of PCDH19 during cortical neuronal differentiation. Our data show that PCDH19 is expressed in pluripotent cells before differentiation in a homogeneous pattern, despite its localization is often limited to one pole of the cell. During neuronal differentiation, positional information on the progenitor cells assumes an important role in acquiring polarization. The proper control of the cell orientation ensures a fine balancing between symmetric (giving rise to two progenitor sister cells) versus asymmetric (giving rise to one progenitor cell and one newborn neuron) division. This process results in the polar organization of the neural tube with a lumen indicating the basal part of the polarized neuronal progenitor cell; in the iPSC model the cells are organized in the ‘neural rosette’ and interestingly, PCDH19 is located at the center of the rosette, with other well-known markers of the lumen (N-cadherin and ZO-1). These data suggest that PCDH19 has a role in instructing the apico-basal polarity of the progenitor cells, thus regulating the development of a properly organized human brain.

Riboflavin is essential in numerous cellular oxidation/reduction reactions but is not synthesized by mammalian cells. Riboflavin absorption occurs through the human riboflavin transporters RFVT1 and RFVT3 in the intestine and RFVT2 in the brain. Mutations in these genes are causative for the Brown–Vialetto–Van Laere (BVVL), childhood-onset syndrome characterized by a variety of cranial nerve palsies as well as by spinal cord motor neuron (MN) degeneration. Why mutations in RFVTs result in a neural cell–selective disorder is unclear. As a novel tool to gain insights into the pathomechanisms underlying the disease, we generated MNs from induced pluripotent stem cells (iPSCs) derived from BVVL patients as an in vitro disease model. BVVL-MNs explained a reduction in axon elongation, partially improved by riboflavin supplementation. RNA sequencing profiles and protein studies of the cytoskeletal structures showed a perturbation in the neurofilament composition in BVVL-MNs. Furthermore, exploring the autophagy–lysosome pathway, we observed a reduced autophagic/mitophagic flux in patient MNs. These features represent emerging pathogenetic mechanisms in BVVL-associated neurodegeneration, partially rescued by riboflavin supplementation. Our data showed that this therapeutic strategy could have some limits in rescuing all of the disease features, suggesting the need to develop complementary novel therapeutic strategies.

Much of the gnathostome (jawed vertebrate) evolutionary radiation was dependent on the ability to sense and interpret the environment and subsequently act upon this information through utilization of a specialized mode of feeding involving the jaws. While the gnathostome skull, reflective of the vertebrate bauplan, typically is bilaterally symmetric with right (dextral) and left (sinistral) halves essentially representing mirror images along the midline, both adaptive and abnormal asymmetries have appeared. Herein we provide a basic primer on studies of the asymmetric development of the gnathostome skull, touching briefly on asymmetry as a field of study, then describing the nature of cranial development and finally underscoring evolutionary and functional aspects of left-right asymmetric cephalic development.

The balance between self-renewal and differentiation of neural progenitor cells (NPCs) dictates neurogenesis and proper brain development. We found that the RNA- binding protein Sam68 (Khdrbs1) is strongly expressed in neurogenic areas of the neocortex and supports the self-renewing potential of mouse NPCs. Knockout of Khdrbs1 constricted the pool of proliferating NPCs by accelerating their cell cycle exit and differentiation into post-mitotic neurons. Sam68 function was linked to regulation of Aldh1a3 pre-mRNA 3'-end processing. Binding of Sam68 to an intronic polyadenylation site prevents its recognition and premature transcript termination, favoring expression of a functional enzyme. The lower ALDH1A3 expression and activity in Khdrbs1-/- NPCs results in reduced glycolysis and clonogenicity, thus depleting the embryonic NPC pool and limiting cortical expansion. Our study identifies Sam68 as a key regulator of NPC self-renewal and establishes a novel link between modulation of ALDH1A3 expression and maintenance of high glycolytic metabolism in the developing cortex. DOI: http://dx.doi.org/10.7554/eLife.20750.001, eLife digest Neurons develop from cells called neural progenitors. These cells can either divide to produce more progenitor cells or develop into specific types of neurons. These two activities – known as self-renewal and differentiation – must be balanced to produce the right number of specialized neurons, without depleting the pool of progenitor cells. The self-renewal and differentiation of progenitor cells is balanced by essentially regulating which genes are active, or expressed, within the cells. In the first step of gene expression, the genetic instructions are copied to form a molecule of pre-messenger RNA (or pre-mRNA for short). Each pre-mRNA molecule is then processed to produce a final product that can be translated into protein. Importantly, two copies of the same pre-mRNA may sometimes be processed in different ways, which allows multiple proteins to be produced from a single gene. RNA-binding proteins control pre-mRNA processing. The expression of one such protein, called Sam68, oscillates during the development of the nervous system, such that its expression peaks when there is intense production of new neurons and then declines. However, it was not known whether Sam68 actually helps neurons to develop. La Rosa et al. have now analysed the role of Sam68 in the developing brain of mice. The experiments confirmed that Sam68 is highly expressed in neural progenitor cells and showed that its levels dictate the cell’s fate: high expression encourages a cell to self-renew, while low expression triggers it to develop into a specialized neuron. Further investigation revealed that Sam68 works by promoting the expression of a metabolic enzyme called Aldehyde Dehydrogenase 1A3 or ALDH1A3. This enzyme promotes the release of energy from molecules of glucose via a process known as anaerobic glycolysis. La Rosa et al. found that cells that lack Sam68 make a truncated version of the pre-mRNA encoding ALDH1A3. This truncated pre-mRNA encodes a shortened version of the enzyme that is inactive. Further experiments confirmed that Sam68 normally prevents this from happening by binding to the pre-mRNA and processing it to produce the full-length, working version of the ALDH1A3 enzyme. Also, La Rosa et al. found that progenitor cells need working ALDH1A3 to keep them dividing, and to stop them from developing into specialized neurons too soon. Finally, because the processing of pre-RNA plays a major role in brain development, problems with this process often lead to intellectual disabilities and neurodegenerative diseases, such as autism spectrum disorder and amyotrophic lateral sclerosis. The next step following on from these new findings will be to investigate whether defects in Sam68 contribute to such conditions and, if so, to look for ways to counteract these defects. DOI: http://dx.doi.org/10.7554/eLife.20750.002

Neurogenesis is the developmental process regulating cell proliferation of neural stem cells, determining their differentiation into glial and neuronal cells, and orchestrating their organization into finely regulated functional networks. Can this complex process be recapitulated in vitro using induced pluripotent stem cell (iPSC) technology? Can neurodevelopmental and neurodegenerative diseases be modeled using iPSCs? What is the potential of iPSC technology in neurobiology? What are the recent advances in the field of neurological diseases? Since the applications of iPSCs in neurobiology are based on the capacity to regulate in vitro differentiation of human iPSCs into different neuronal subtypes and glial cells, and the possibility of obtaining iPSC-derived neurons and glial cells is based on and hindered by our poor understanding of human embryonic development, we reviewed current knowledge on in vitro neural differentiation from a developmental and cellular biology perspective. We highlight the importance to further advance our understanding on the mechanisms controlling in vivo neurogenesis in order to efficiently guide neurogenesis in vitro for cell modeling and therapeutical applications of iPSCs technology.

Evolution of Developmental Control Mechanisms.-- et al., The acquisition of jaws constitutes a landmark event in vertebrate evolution, one that in large part potentiated their success and diversification. Jaw development and patterning involves an intricate spatiotemporal series of reciprocal inductive and responsive interactions between the cephalic epithelia and the cranial neural crest (CNC) and cephalic mesodermal mesenchyme. The coordinated regulation of these interactions is critical for both the ontogenetic registration of the jaws and the evolutionary elaboration of variable jaw morphologies and designs. Current models of jaw development and evolution have been built on molecular and cellular evidence gathered mostly in amniotes such as mice, chicks and humans, and augmented by a much smaller body of work on the zebrafish. These have been partnered by essential work attempting to understand the origins of jaws that has focused on the jawless lamprey. Chondrichthyans (cartilaginous fish) are the most distant group to amniotes within extant gnathostomes, and comprise the crucial clade uniting amniotes and agnathans; yet despite their critical phylogenetic position, evidence of the molecular and cellular underpinnings of jaw development in chondrichthyans is still lacking. Recent advances in genome and molecular developmental biology of the lesser spotted dogfish shark, Scyliorhinus canicula, make it ideal for the molecular study of chondrichthyan jaw development. Here, following the 'Hinge and Caps' model of jaw development, we have investigated evidence of heterotopic (relative changes in position) and heterochronic (relative changes in timing) shifts in gene expression, relative to amniotes, in the jaw primordia of S. canicula embryos. We demonstrate the presence of clear proximo-distal polarity in gene expression patterns in the shark embryo, thus establishing a baseline molecular baüplan for branchial arch-derived jaw development and further validating the utility of the 'Hinge and Caps' model in comparative studies of jaw development and evolution. Moreover, we correlate gene expression patterns with the absence of a lambdoidal junction (formed where the maxillary first arch meets the frontonasal processes) in chondrichthyans, further highlighting the importance of this region for the development and evolution of jaw structure in advanced gnathostomes., This work was supported by funding to M.J.D. from the Royal Society, the Dental Institute of King’s College London, and Friends of Guy’s Hospital. C.C. and J.G. were funded by Marie Curie Early Training Fellowships (MEST-CT-2004-504025). J.L.F. was assisted by a HFSP Long Term Fellowship (LT 01061/2007-L). S.M. was funded by Région Centre, Région Bretagne (EVOVERT grant number 049755), National Research Agency (grant ANR-09-BLAN-026201), CNRS, Université d’Orléans and the Université Pierre et Marie Curie. M.C. benefited from a doctoral fellowship from CNRS.

To elucidate the pathogenesis of axonopathy in Friedreich's Ataxia (FRDA), a neurodegenerative disease characterized by axonal retraction, we analyzed the microtubule (MT) dynamics in an in vitro frataxin-silenced neuronal model (shFxn). A typical feature of MTs is their "dynamic instability", in which they undergo phases of growth (polymerization) and shrinkage (depolymerization). MTs play a fundamental role in the physiology of neurons and every perturbation of their dynamicity is highly detrimental for neuronal functions. The aim of this study is to determine whether MTs are S-glutathionylated in shFxn and if the glutathionylation triggers MT dysfunction. We hypothesize that oxidative stress, determined by high GSSG levels, induces axonal retraction by interfering with MT dynamics. We propose a mechanism of the axonopathy in FRDA where GSSG overload and MT de-polymerization are strictly interconnected. Indeed, using a frataxin-silenced neuronal model we show a significant reduction of neurites extension, a shift of tubulin toward the unpolymerized fraction and a consistent increase of glutathione bound to the cytoskeleton. The live cell imaging approach further reveals a significant decrease in MT growth lifetime due to frataxin silencing, which is consistent with the MT destabilization. The in vitro antioxidant treatments trigger the axonal re-growth and the increase in stable MTs in shFxn, thus contributing to identify new neuronal targets of oxidation in this disease and providing a novel approach for antioxidant therapies.

Patient-derived induced pluripotent stem cells (iPSCs) provide a novel tool to investigate the pathophysiology of poorly known diseases, in particular those affecting the nervous system, which has been difficult to study for its lack of accessibility. In this emerging and promising field, recent iPSCs studies are mostly used as "proof-of-principle" experiments that are confirmatory of previous findings obtained from animal models and postmortem human studies; its promise as a discovery tool is just beginning to be realized. A recent number of studies point to the functional similarities between in vitro neurogenesis and in vivo neuronal development, suggesting that similar morphogenetic and patterning events direct neuronal differentiation. In this context, neuronal adhesion, cytoskeletal organization and cell metabolism emerge as an integrated and unexplored processes of human neurogenesis, mediated by the lack of data due to the difficult accessibility of the human neural tissue. These observations raise the necessity to understand which are the players controlling cytoskeletal reorganization and remodeling. In particular, we investigated human in vitro neurogenesis using iPSCs of healthy subjects to unveil the underpinnings of the cytoskeletal dynamics with the aim to shed light on the physiologic events controlling the development and the functionality of neuronal cells. We validate the iPSCs system to better understand the development of the human nervous system in order to set the bases for the future understanding of pathologies including developmental disorders (i.e. intellectual disability), epilepsy but also neurodegenerative disorders (i.e. Friedreich's Ataxia). We investigate the changes of the cytoskeletal components during the 30days of neuronal differentiation and we demonstrate that human neuronal differentiation requires a (time-dependent) reorganization of actin filaments, intermediate filaments and microtubules; and that immature neurons present a finely regulated localization of Glu-, Tyr- and Acet-TUBULINS. This study advances our understanding on cytoskeletal dynamics with the hope to pave the way for future therapies that could be potentially able to target cytoskeletal based neurodevelopmental and neurodegenerative diseases.

SUMMARY Modularity is a key mechanism bridging development and evolution and is fundamental to evolvability. Herein, we investigate modularity of the Vertebrate jaw with the aim of understanding mechanisms of its morphological evolution. Conservation of the basic structural bauplan of Vertebrate jaws led to a Hinge and Caps model, in which polarity in the patterning system of developing jaws predicts modularity. We have tested the hypothesis that the Satb2+ cell population delineates a developmental module within the mandibular jaw. Satb2 is expressed in the mesenchyme of the jaw primordia that gives rise to distal elements of both the upper and lower jaws. Loss of Satb2 specifically affects structural elements of the distal (incisor) domain, reflecting the integration of these elements as well as their independence from other mandibular domains. Reducing Satb2 dosage leads to an increase in variation in mandibular length, providing insight into the developmental potential to generate variation. Inter-taxa comparisons reveal that the Satb2 domain is conserved within gnathostomes. We complement previous loss of function studies in mice with gene knock-down experiments in Xenopus, providing evidence for functional conservation of Satb2 in regulating size. Finally, we present evidence that the relative size of the amniote mandibular Satb2+ domain varies in relation to epithelial Fgf8 expression, suggesting a mechanism for evolutionary change in this domain. Taken together, our data support the Hinge and Caps model and provide evidence that Satb2 regulates coordinated distal jaw modules that are subject to evolutionary modification by signals emanating from the Hinge.

Rho-GTPases have relevant functions in various aspects of neuronal development, such as differentiation, migration, and synaptogenesis. Loss of function of the oligophrenin-1 gene (OPHN1) causes X-linked intellectual disability with cerebellar hypoplasia and leads to hyperactivation of the rho kinase (ROCK) pathway. ROCK mainly acts through phosphorylation of the myosin phosphatase targeting subunit 1, triggering actin-myosin contractility. We show that during in vitro neurogenesis, ROCK activity decreases from day 10 until terminal differentiation, whereas in OPHN1-deficient human induced pluripotent stem cells (h-iPSCs), the levels of ROCK are elevated throughout differentiation. ROCK inhibition favors neuronal-like appearance of h-iPSCs, in parallel with transcriptional upregulation of nuclear receptor NR4A1, which is known to induce neurite outgrowth. This study analyzed the morphological, biochemical, and functional features of OPHN1-deficient h-iPSCs and their rescue by treatment with the ROCK inhibitor fasudil, shedding light on the relevance of the ROCK pathway during neuronal differentiation and providing a neuronal model for human OPHN1 syndrome and its treatment. Significance The analysis of the levels of rho kinase (ROCK) activity at different stages of in vitro neurogenesis of human induced pluripotent stem cells reveals that ROCK activity decreases progressively in parallel with the appearance of neuronal-like morphology and upregulation of nuclear receptor NR4A1. These results shed light on the role of the ROCK pathway during early stages of human neurogenesis and provide a neuronal stem cell-based model for the treatment of OPHN1 syndrome and other neurological disorders due to ROCK dysfunction.

Rho-kinase (ROCK) has been well documented to play a key role in RhoA-induced actin remodeling. ROCK activation results in myosin light chain (MLC) phosphorylation either by direct action on MLC kinase (MLCK) or by inhibition of MLC phosphatase (MLCP), modulating actin-myosin contraction. We found that inhibition of the ROCK pathway in induced pluripotent stem cells, leads to nuclear export of HDAC7 and transcriptional activation of the orphan nuclear receptor NR4A1 while in cells with constitutive ROCK hyperactivity due to loss of function of the RhoGTPase activating protein Oligophrenin-1 (OPHN1), the orphan nuclear receptor NR4A1 is downregulated. Our study identify a new target of ROCK signaling via myosin phosphatase subunit (MYPT1) and Histone Deacetylase (HDAC7) at the nuclear level and provide new insights in the cellular functions of ROCK.

Much of the gnathostome (jawed vertebrate) evolutionary radiation was dependent on the ability to sense and interpret the environment and subsequently act upon this information through utilization of a specialized mode of feeding involving the jaws. While the gnathostome skull, reflective of the vertebrate baüplan, typically is bilaterally symmetric with right (dextral) and left (sinistral) halves essentially representing mirror images along the midline, both adaptive and abnormal asymmetries have appeared. Herein we provide a basic primer on studies of the asymmetric development of the gnathostome skull, touching briefly on asymmetry as a field of study, then describing the nature of cranial development and finally underscoring evolutionary and functional aspects of left-right asymmetric cephalic development.

Neural stem cells (NSCs) are self-renewing cells that can differentiate into multiple neural lineages and repopulate regions of the brain after injury. We have investigated the role of endocannabinoids (eCBs), endogenous cues that modulate neuronal functions including neurogenesis, and their receptors CB(1) and CB(2) in mouse NSCs. Real-time PCR and Western blot analyses indicated that CB(1) is present at higher levels than CB(2) in NSCs. The eCB anandamide (AEA) or the CB(1)-specific agonist ACEA enhanced NSC differentiation into neurons, but not astrocytes and oligodendrocytes, whereas the CB(2)-specific agonist JWH133 was ineffective. Conversely, the effect of AEA was inhibited by CB(1), but not CB(2), antagonist, corroborating the specificity of the response. CB(1) activation also enhanced maturation of neurons, as indicated by morphometric analysis of neurites. CB(1) stimulation caused long-term inhibition of the ERK1/2 pathway. Consistently, pharmacological inhibition of the ERK1/2 pathway recapitulated the effects exerted by CB(1) activation on neuronal differentiation and maturation. Lastly, gene array profiling showed that CB(1) activation augmented the expression of genes involved in neuronal differentiation while decreasing that of stemness genes. These results highlight the role of CB(1) in the regulation of NSC fate and suggest that its activation may represent a pro-neuronal differentiation signal.

Modularity is a key mechanism bridging development and evolution and is fundamental to evolvability. Herein, we investigate modularity of the Vertebrate jaw with the aim of understanding mechanisms of its morphological evolution. Conservation of the basic structural bauplan of Vertebrate jaws led to a Hinge and Caps model, in which polarity in the patterning system of developing jaws predicts modularity. We have tested the hypothesis that the Satb2+ cell population delineates a developmental module within the mandibular jaw. Satb2 is expressed in the mesenchyme of the jaw primordia that gives rise to distal elements of both the upper and lower jaws. Loss of Satb2 specifically affects structural elements of the distal (incisor) domain, reflecting the integration of these elements as well as their independence from other mandibular domains. Reducing Satb2 dosage leads to an increase in variation in mandibular length, providing insight into the developmental potential to generate variation. Inter-taxa comparisons reveal that the Satb2 domain is conserved within gnathostomes. We complement previous loss of function studies in mice with gene knock-down experiments in Xenopus, providing evidence for functional conservation of Satb2 in regulating size. Finally, we present evidence that the relative size of the amniote mandibular Satb2+ domain varies in relation to epithelial Fgf8 expression, suggesting a mechanism for evolutionary change in this domain. Taken together, our data support the Hinge and Caps model and provide evidence that Satb2 regulates coordinated distal jaw modules that are subject to evolutionary modification by signals emanating from the Hinge.

INTRODUCTIONDue to their large size and long generation times, chondrichthyans have been largely ignored by geneticists. However, their key phylogenetic position makes them ideal subjects to study the molecular bases of the important morphological and physiological innovations that characterize jawed vertebrates. Such analyses are crucial to understanding the origin of the complex genetic mechanisms unraveled in osteichthyans. The small spotted dogfish Scyliorhinus canicula, a representative of the largest order of extant sharks, presents a number of advantages in this context. Due to its relatively small size among sharks, its abundance, and easy maintenance, the dogfish has been an important model in comparative anatomy and physiology for more than a century. Recently, revived interest has occurred with the development of large-scale transcriptomic and genomic resources, together with the establishment of facilities allowing massive egg and embryo production. These new tools open the way to molecular analyses of the elaborate physiological and sensory systems used by sharks. They also make it possible to take advantage of unique characteristics of these species, such as organ zonation, in analyses of cell proliferation and differentiation. Finally, they offer important perspectives to evolutionary developmental biology that will provide a better understanding of the origin and diversifications of jawed vertebrates. The dogfish whole-genome sequence, which may shortly become accessible, should establish this species as an essential shark reference, complementary to other chondrichthyan models. These analyses are likely to reveal an organism of an underestimated complexity, far from the primitive prototypical gnathostome anticipated in gradistic views.

Normal patterning and morphogenesis of the complex skeletal structures of the skull requires an exquisite, reciprocal cross-talk between the embryonic cephalic epithelia and mesenchyme. The mesenchyme associated with the jaws and the optic and olfactory capsules is derived from a Hox-negative cranial neural crest (CNC) population that acts much as an equivalence group in its interactions with specific local cephalic epithelial signals. Craniofacial pattern and morphogenesis is therefore controlled in large part through the regulation of these local cephalic epithelial signals. Here, we demonstrate that Pax6 is essential to the formation and maturation of the complex cephalic ectodermal patterning centers that govern the development and morphogenesis of the upper jaws and associated nasal capsules. Previous examinations of the craniofacial skeletal defects associated with Pax6 mutations have suggested that they arise from an optic-associated blockage in the migration of a specific subpopulation of midbrain CNC to the lateral frontonasal processes. We have addressed an alternative explanation for the craniofacial skeletal defects. We show that in Pax6SeyN/SeyN mutants regional CNC is present by E9.25 while there is already specific disruption in the early ontogenetic elaboration of cephalic ectodermal expression, associated with the nascent lambdoidal junction, of secreted signaling factors (including Fgf8 and Bmp4) and transcription factors (including Six1 and Dlx5) essential for upper jaw and/or nasal capsular development. Pax6 therefore regulates craniofacial form, at stages when CNC has just arrived in the frontonasal region, through its control of surface cephalic ectodermal competence to form an essential craniofacial patterning center. genesis 49:307-325, 2011. © 2011 Wiley-Liss, Inc.