Search

Objectives: Ataxic-rigid gait is a characteristic gait pathology in patients with Rett syndrome (RTT). In the present study, we aimed to quantitatively evaluate gait pathology in patients with RTT using three-dimensional gait analysis (3DGA). Methods: We performed 3DGA in 11 patients with RTT ranging from 5 to 18 years (median age, 9 years) and in 33 age-matched healthy female controls. We compared the results of 3DGA, including spatiotemporal gait parameters and comprehensive indices of gait kinematics, such as the Gait Deviation Index (GDI) and Gait Profile Score (GPS), between the two groups. The GPS consists of nine sub-indices called Gait Variable Scores (GVSs). Decline in GDI or elevation of GPS and GVS indicated greater abnormal gait pathology. Results: The patients demonstrated significantly slower walking speed, lower step length/length of the lower extremities, lower cadence, wider step width, and higher coefficient of variation of step length than the controls. Moreover, the patients had a lower GDI and higher GPS than the controls. The patients also exhibited higher GVSs for eight out of nine gait kinematics, particularly the sagittal plane in the pelvis, hip, knee, and ankle joint; coronal plane in the pelvis and hip joint; and horizontal plane in the pelvis than the controls. Conclusions: Quantitative evaluation of gait pathology in patients with RTT is possible using 3DGA. We found that in addition to ataxic-rigid gait, abnormalities in the coronal plane of the pelvis and hip joint and the horizontal plane of the pelvis were prominent.

Congenital genetic disorders often present with neurological manifestations such as neurodevelopmental disorders, motor developmental retardation, epilepsy, and involuntary movement. Through qualitative morphometric evaluation of neuroimaging studies, remarkable structural abnormalities, such as lissencephaly, polymicrogyria, white matter lesions, and cortical tubers, have been identified in these disorders, while no structural abnormalities were identified in clinical settings in a large population. Recent advances in data analysis programs have led to significant progress in the quantitative analysis of anatomical structural magnetic resonance imaging (MRI) and diffusion-weighted MRI tractography, and these approaches have been used to investigate psychological and congenital genetic disorders. Evaluation of morphometric brain characteristics may contribute to the identification of neuroimaging biomarkers for early diagnosis and response evaluation in patients with congenital genetic diseases. This mini-review focuses on the methodologies and attempts employed to study Rett syndrome using quantitative structural brain MRI analyses, including voxel- and surface-based morphometry and diffusion-weighted MRI tractography. The mini-review aims to deepen our understanding of how neuroimaging studies are used to examine congenital genetic disorders.

To clarify the relationship between structural and functional changes in the brains of patients with Rett syndrome (RTT) using multimodal magnetic resonance imaging (MRI).Nine subjects with typical RTT (RTTs) and an equal number of healthy controls (HCs) underwent structural MRI, diffusion tensor imaging (DTI), and resting-state functional MRI (rs-fMRI). The measurements obtained from each modality were statistically compared between RTTs and HCs and examined for their correlation with the clinical severity of RTTs.Structural MRI imaging revealed volume reductions in most cortical and subcortical regions of the brain. Remarkable volume reductions were observed in the frontal and parietal lobes, cerebellum, and subcortical regions including the putamen, hippocampus, and corpus callosum. DTI analysis revealed decreased white matter integrity in broad regions of the brain. Fractional anisotropy values were greatly decreased in the superior longitudinal fasciculus, corpus callosum, and middle cerebellar peduncle. Rs-fMRI analysis showed decreased functional connectivity in the interhemispheric dorsal attention network, and between the visual and cerebellar networks. The clinical severity of RTTs correlated with the volume reduction of the frontal lobe and cerebellum, and with changes in DTI indices in the fronto-occipital fasciculus, corpus callosum, and cerebellar peduncles.Regional volume and white matter integrity of RTT brains were reduced in broad areas, while most functional connections remained intact. Notably, two functional connectivities, between cerebral hemispheres and between the cerebrum and cerebellum, were decreased in RTT brains, which may reflect the structural changes in these brain regions.

Early interventions for autism spectrum disorder (ASD) are increasingly available, while only 42–50% of ASD children are diagnosed before 3 years old (YO). To identify neuroimaging biomarkers for early ASD diagnosis, we evaluated surface- and voxel-based brain morphometry in participants under 3YO who were later diagnosed with ASD. Magnetic resonance imaging data were retrospectively obtained from patients later diagnosed with ASD at Boston Children’s Hospital. The ASD participants with comorbidities such as congenital disorder, epilepsy, and global developmental delay/intellectual disability were excluded from statistical analyses. Eighty-five structural brain magnetic resonance imaging images were collected from 81 participants under 3YO and compared with 45 images from 45 gender- and age-matched nonautistic controls (non-ASD). Using an Infant FreeSurfer pipeline, 236 regionally distributed measurements were extracted from each scan. By t-tests and linear mixed models, the smaller nucleus accumbens and larger bilateral lateral, third, and fourth ventricles were identified in the ASD group. Vertex-wise t-statistical maps showed decreased thickness in the caudal anterior cingulate cortex and increased thickness in the right medial orbitofrontal cortex in ASD. The smaller bilateral accumbens nuclei and larger cerebral ventricles were independent of age, gender, or gestational age at birth, suggesting that there are MRI-based biomarkers in prospective ASD patients before they receive the diagnosis and that the volume of the nucleus accumbens and cerebral ventricles can be key MRI-based early biomarkers to predict the emergence of ASD.

Dysregulation of epigenetic processes involving histone methylation induces neurodevelopmental impairments and has been implicated in schizophrenia (SCZ) and autism spectrum disorder (ASD). Variants in the gene encoding lysine demethylase 4C (KDM4C) have been suggested to confer a risk for such disorders. However, rare genetic variants in KDM4C have not been fully evaluated, and the functional impact of the variants has not been studied using patient-derived cells. In this study, we conducted copy number variant (CNV) analysis in a Japanese sample set (2605 SCZ and 1141 ASD cases, and 2310 controls). We found evidence for significant associations between CNVs in KDM4C and SCZ (p = 0.003) and ASD (p = 0.04). We also observed a significant association between deletions in KDM4C and SCZ (corrected p = 0.04). Next, to explore the contribution of single nucleotide variants in KDM4C, we sequenced the coding exons in a second sample set (370 SCZ and 192 ASD cases) and detected 18 rare missense variants, including p.D160N within the JmjC domain of KDM4C. We, then, performed association analysis for p.D160N in a third sample set (1751 SCZ and 377 ASD cases, and 2276 controls), but did not find a statistical association with these disorders. Immunoblotting analysis using lymphoblastoid cell lines from a case with KDM4C deletion revealed reduced KDM4C protein expression and altered histone methylation patterns. In conclusion, this study strengthens the evidence for associations between KDM4C CNVs and these two disorders and for their potential functional effect on histone methylation patterns.

Schizophrenia (SCZ) is known to be a heritable disorder; however, its multifactorial nature has significantly hampered attempts to establish its pathogenesis. Therefore, in this study, we performed genome-wide copy-number variation (CNV) analysis of 2940 patients with SCZ and 2402 control subjects and identified a statistically significant association between SCZ and exonic CNVs in the ARHGAP10 gene. ARHGAP10 encodes a member of the RhoGAP superfamily of proteins that is involved in small GTPase signaling. This signaling pathway is one of the SCZ-associated pathways and may contribute to neural development and function. However, the ARHGAP10 gene is often confused with ARHGAP21, thus, the significance of ARHGAP10 in the molecular pathology of SCZ, including the expression profile of the ARHGAP10 protein, remains poorly understood. To address this issue, we focused on one patient identified to have both an exonic deletion and a missense variant (p.S490P) in ARHGAP10. The missense variant was found to be located in the RhoGAP domain and was determined to be relevant to the association between ARHGAP10 and the active form of RhoA. We evaluated ARHGAP10 protein expression in the brains of reporter mice and generated a mouse model to mimic the patient case. The model exhibited abnormal emotional behaviors, along with reduced spine density in the medial prefrontal cortex (mPFC). In addition, primary cultured neurons prepared from the mouse model brain exhibited immature neurites in vitro. Furthermore, we established induced pluripotent stem cells (iPSCs) from this patient, and differentiated them into tyrosine hydroxylase (TH)-positive neurons in order to analyze their morphological phenotypes. TH-positive neurons differentiated from the patient-derived iPSCs exhibited severe defects in both neurite length and branch number; these defects were restored by the addition of the Rho-kinase inhibitor, Y-27632. Collectively, our findings suggest that rare ARHGAP10 variants may be genetically and biologically associated with SCZ and indicate that Rho signaling represents a promising drug discovery target for SCZ treatment.

Summary: Rett syndrome (RTT) is a severe neurological disorder, with impaired brain development caused by mutations in MECP2; however, the underlying mechanism remains elusive. We know from previous work that MeCP2 facilitates the processing of a specific microRNA, miR-199a, by associating with the Drosha complex to regulate neuronal functions. Here, we show that the MeCP2/miR-199a axis regulates neural stem/precursor cell (NS/PC) differentiation. A shift occurs from neuronal to astrocytic differentiation of MeCP2- and miR-199a-deficient NS/PCs due to the upregulation of a miR-199a target, Smad1, a downstream transcription factor of bone morphogenetic protein (BMP) signaling. Moreover, miR-199a expression and treatment with BMP inhibitors rectify the differentiation of RTT patient-derived NS/PCs and development of brain organoids, respectively, suggesting that facilitation of BMP signaling accounts for the impaired RTT brain development. Our study illuminates the molecular pathology of RTT and reveals the MeCP2/miR-199a/Smad1 axis as a potential therapeutic target for RTT.

Proper dendritic elaboration of neurons is critical for the formation of functional circuits during brain development. Defects in dendrite morphogenesis are associated with neuropsychiatric disorders, and microRNAs are emerging as regulators of aspects of neuronal maturation such as axonal and dendritic growth, spine formation, and synaptogenesis. Here, we show that miR-214 plays a pivotal role in the regulation of dendritic development. Overexpression of miR-214 increased dendrite size and complexity, whereas blocking of endogenous miR-214-3p, a mature form of miR-214, inhibited dendritic morphogenesis. We also found that miR-214-3p targets quaking (Qki), which is implicated in psychiatric diseases such as schizophrenia, through conserved target sites located in the 3'-untranslated region of Qki mRNA, thereby down-regulating Qki protein levels. Overexpression and knockdown of Qki impaired and enhanced dendritic formation, respectively. Moreover, overexpression of Qki abolished the dendritic growth induced by miR-214 overexpression. Taken together, our findings reveal a crucial role for the miR-214-Qki pathway in the regulation of neuronal dendritic development.

Summary Prenatal exposure to valproic acid (VPA), an established antiepileptic drug, has been reported to impair postnatal cognitive function in children born to VPA-treated epileptic mothers. However, how these defects arise and how they can be overcome remain unknown. Using mice, we found that comparable postnatal cognitive functional impairment is very likely correlated to the untimely enhancement of embryonic neurogenesis, which led to depletion of the neural precursor cell pool and consequently a decreased level of adult neurogenesis in the hippocampus. Moreover, hippocampal neurons in the offspring of VPA-treated mice showed abnormal morphology and activity. Surprisingly, these impairments could be ameliorated by voluntary running. Our study suggests that although prenatal exposure to antiepileptic drugs such as VPA may have detrimental effects that persist until adulthood, these effects may be offset by a simple physical activity such as running., Graphical Abstract, Highlights • Prenatal VPA treatment caused an untimely enhancement of embryonic neurogenesis • Prenatal VPA treatment has the long-term effect of impairing adult neurogenesis • Reduced level of adult neurogenesis is associated with cognitive functional impairments • Voluntary running can ameliorate the persistent detrimental effects caused by VPA, Nakashima and colleagues show that prenatal VPA exposure in mice produced comparable postnatal cognitive functional impairments in children from epileptic mothers. VPA enhances embryonic neurogenesis, which then leads to depletion of the neural precursor cell pool, resulting in a decreased level of adult hippocampal neurogenesis. They further show that voluntary running could reinstate the neurogenesis and restore cognitive functions.

Functional neuronal connectivity requires proper neuronal morphogenesis and its dysregulation causes neurodevelopmental diseases. Transforming growth factor-β (TGF-β) family cytokines play pivotal roles in development, but little is known about their contribution to morphological development of neurons. Here we show that the Smad-dependent canonical signaling of TGF-β family cytokines negatively regulates neuronal morphogenesis during brain development. Mechanistically, activated Smads form a complex with transcriptional repressor TG-interacting factor (TGIF), and downregulate the expression of a neuronal polarity regulator, collapsin response mediator protein 2. We also demonstrate that TGF-β family signaling inhibits neurite elongation of human induced pluripotent stem cell-derived neurons. Furthermore, the expression of TGF-β receptor 1, Smad4, or TGIF, which have mutations found in patients with neurodevelopmental disorders, disrupted neuronal morphogenesis in both mouse (male and female) and human (female) neurons. Together, these findings suggest that the regulation of neuronal morphogenesis by an evolutionarily conserved function of TGF-β signaling is involved in the pathogenesis of neurodevelopmental diseases.SIGNIFICANCE STATEMENTCanonical transforming growth factor-β (TGF-β) signaling plays a crucial role in multiple organ development, including brain, and mutations in components of the signaling pathway associated with several human developmental disorders. In this study, we found that Smads/TG-interacting factor-dependent canonical TGF-β signaling regulates neuronal morphogenesis through the suppression of collapsin response mediator protein-2 (CRMP2) expression during brain development, and that function of this signaling is evolutionarily conserved in the mammalian brain. Mutations in canonical TGF-β signaling factors identified in patients with neurodevelopmental disorders disrupt the morphological development of neurons. Thus, our results suggest that proper control of TGF-β/Smads/CRMP2 signaling pathways is critical for the precise execution of neuronal morphogenesis, whose impairment eventually results in neurodevelopmental disorders.

Rett syndrome (RTT) is a devastating neurodevelopmental disorder resulting from mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MECP2). MECP2 mutations are also associated with other neurodevelopmental diseases, including autism and schizophrenia. Therefore, elucidating the mechanism of RTT can contribute to understanding the pathogenesis of a wide range of neurodevelopmental diseases. Despite its importance, however, the RTT pathogenesis remains unclear, and effective therapeutic treatment has not been developed. Offering an opportunity to move toward this goal, however, is the recent advance in the stem cell research field of the development of induced pluripotent stem cell (iPSC) technology. This technology can yield important insights into disease pathophysiology and has the potential to provide disease models for screening new drugs. Here, we discuss applications of recent stem cell technology to the field of research on RTT and describe the stem cell biology of RTT pathogenesis.

SummaryRett syndrome (RTT) is a neurodevelopmental disorder caused by MECP2 mutations. Although emerging evidence suggests that MeCP2 deficiency is associated with dysregulation of mechanistic target of rapamycin (mTOR), which functions as a hub for various signaling pathways, the mechanism underlying this association and the molecular pathophysiology of RTT remain elusive. We show here that MeCP2 promotes the posttranscriptional processing of particular microRNAs (miRNAs) as a component of the microprocessor Drosha complex. Among the MeCP2-regulated miRNAs, we found that miR-199a positively controls mTOR signaling by targeting inhibitors for mTOR signaling. miR-199a and its targets have opposite effects on mTOR activity, ameliorating and inducing RTT neuronal phenotypes, respectively. Furthermore, genetic deletion of miR-199a-2 led to a reduction of mTOR activity in the brain and recapitulated numerous RTT phenotypes in mice. Together, these findings establish miR-199a as a critical downstream target of MeCP2 in RTT pathogenesis by linking MeCP2 with mTOR signaling.

Post-transcriptional regulation of gene expression is required for multiple aspects of neuronal development and function in the central nervous system. A sub-class of small non-coding RNA, called microRNAs (miRNAs), is emerging as key modulators of post-transcriptional gene regulation in numerous tissues, including the nervous system. Recent evidence has revealed a widespread role for miRNAs in various aspects of neuronal morphogenesis, including axogenesis, dendritogenesis, and synapse formation. Furthermore, dysregulation or altered expression of miRNAs has been associated with the pathogenesis of neurodevelopmental and psychiatric disorders. Here, we highlight recent advances in the study of miRNA-based regulation of neuronal development and their implications in neurological disorders.

Because of their ability to self-renew, to differentiate into multiple lineages, and to migrate toward a damaged site, neural stem cells (NSCs), which can be derived from various sources such as fetal tissues and embryonic stem cells, are currently considered to be promising components of cell replacement strategies aimed at treating injuries of the central nervous system, including the spinal cord. Despite their efficiency in promoting functional recovery, these NSCs are not homogeneous and possess variable characteristics depending on their derivation protocols. The advent of induced pluripotent stem (iPS) cells has provided new prospects for regenerative medicine. We used a recently developed robust and stable protocol for the generation of long-term, self-renewing, neuroepithelial-like stem cells from human iPS cells (hiPS-lt-NES cells), which can provide a homogeneous and well-defined population of NSCs for standardized analysis. Here, we show that transplanted hiPS-lt-NES cells differentiate into neural lineages in the mouse model of spinal cord injury (SCI) and promote functional recovery of hind limb motor function. Furthermore, using two different neuronal tracers and ablation of the transplanted cells, we revealed that transplanted hiPS-lt-NES cell-derived neurons, together with the surviving endogenous neurons, contributed to restored motor function. Both types of neurons reconstructed the corticospinal tract by forming synaptic connections and integrating neuronal circuits. Our findings indicate that hiPS-lt-NES transplantation represents a promising avenue for effective cell-based treatment of SCI. Disclosure of potential conflicts of interest is found at the end of this article.

Astrocyte differentiation and maintenance is promoted by BMP signaling, which induces REST/NRSF to repress neuronal genes., Once they have differentiated, cells retain their individual character and repress genes that are specifically expressed in other cell lineages, but how alternative fate choice is restricted during and/or after differentiation remains unclear. In the mammalian central nervous system, neurons, astrocytes, and oligodendrocytes are generated throughout life from common tripotent neural progenitor cells (NPCs). Bone morphogenetic proteins (BMPs) are well-known astrocyte-inducing cytokines. We show here that the expression of a transcriptional repressor, RE1 silencer of transcription (REST)/neuron-restrictive silencer factor (NRSF), is up-regulated and sustained by BMP signal activation in the course of astrocytic differentiation of NPCs, and restricts neuronal differentiation. We further show that, in differentiated astrocytes, endogenous REST/NRSF associates with various neuronal genes and that disruption of its function resulted in their derepression, thereby explaining how ectopic neuronal gene expression is prevented in cells with astrocytic traits. Collectively, our results suggest that REST/NRSF functions as a molecular regulator of the nonneuronal phenotype in astrocytes.

Rett syndrome (RTT) is a devastating neurodevelopmental disorder that occurs once in every 10,000–15,000 live female births. Despite intensive research, no effective cure is yet available. Valproic acid (VPA) has been used widely to treat mood disorder, epilepsy, and a growing number of other disorders. In limited clinical studies, VPA has also been used to control seizure in RTT patients with promising albeit somewhat unclear efficacy. In this study we tested the effect of VPA on the neurological symptoms of RTT and discovered that short-term VPA treatment during the symptomatic period could reduce neurological symptoms in RTT mice. We found that VPA restores the expression of a subset of genes in RTT mouse brains, and these genes clustered in neurological disease and developmental disorder networks. Our data suggest that VPA could be used as a drug to alleviate RTT symptoms.

Within the developing mammalian cortex, neural progenitors first generate deep-layer neurons and subsequently more superficial-layer neurons, in an inside-out manner. It has been reported recently that mouse embryonic stem cells (mESCs) can, to some extent, recapitulate cortical development in vitro, with the sequential appearance of neurogenesis markers resembling that in the developing cortex. However, mESCs can only recapitulate early corticogenesis; superficial-layer neurons, which are normally produced in later developmental periods in vivo, are under-represented. This failure of mESCs to reproduce later corticogenesis in vitro implies the existence of crucial factor(s) that are absent or uninduced in existing culture systems. Here we show that mESCs can give rise to superficial-layer neurons efficiently when treated with valproic acid (VPA), a histone deacetylase inhibitor. VPA treatment increased the production of Cux1-positive superficial-layer neurons, and decreased that of Ctip2-positive deep-layer neurons. These results shed new light on the mechanisms of later corticogenesis.

The body's capacity to restore damaged neural networks in the injured CNS is severely limited. Although various treatment regimens can partially alleviate spinal cord injury (SCI), the mechanisms responsible for symptomatic improvement remain elusive. Here, using a mouse model of SCI, we have shown that transplantation of neural stem cells (NSCs) together with administration of valproic acid (VPA), a known antiepileptic and histone deacetylase inhibitor, dramatically enhanced the restoration of hind limb function. VPA treatment promoted the differentiation of transplanted NSCs into neurons rather than glial cells. Transsynaptic anterograde corticospinal tract tracing revealed that transplant-derived neurons reconstructed broken neuronal circuits, and electron microscopic analysis revealed that the transplant-derived neurons both received and sent synaptic connections to endogenous neurons. Ablation of the transplanted cells abolished the recovery of hind limb motor function, confirming that NSC transplantation directly contributed to restored motor function. These findings raise the possibility that epigenetic status in transplanted NSCs can be manipulated to provide effective treatment for SCI.

Methyl-CpG-binding protein 2 (MeCP2), a methyl-CpG-binding domain protein family member which is expressed predominantly in neurons in the nervous system, acts as a transcriptional repressor by binding to methylated genes, and mutations in mecp2 cause the neurological disorder known as Rett syndrome (RTT). Although MeCP2 has been reported to regulate neuronal maturation rather than fate specification of neural precursor cells (NPCs), we have previously shown that it inhibits astrocyte differentiation of NPCs when ectopically expressed. Here, we show that expression of MeCP2 in NPCs not only suppresses astrocytic differentiation but actually promotes neuronal differentiation, even in the presence of well-known astrocyte-inducing cytokines. This dual function of MeCP2 was abolished by the MEK inhibitor U0126. Moreover, we observed that a truncated form of MeCP2 found in RTT patients fails to promote neuronal differentiation. We further demonstrate that transplanted MeCP2-expressing NPCs differentiate in vivo into neurons in two non-neurogenic regions, striatum and spinal cord. These results suggest a possible therapeutic application for MeCP2 in neurodegenerative diseases and injuries to the central nervous system.