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The RPGR (retinitis pigmentosa GTPase regulator) gene has been shown to be mutated in 10-20% of patients with X-linked retinitis pigmentosa (XLRP), a severe form of inherited progressive retinal degeneration. A total of 29 different RPGR mutations have been identified in northern European and United States patients. We have performed mutation analysis of the RPGR gene in a cohort of 49 southern European males affected with XLRP. By multiplex SSCA and automatic direct sequencing of all 19 RPGR exons, seven different and novel mutations were identified in eight of the 49 families; these include three splice site mutations, two microdeletions, and two missense mutations. RNA analysis showed that the three splice site defects resulted in the generation of aberrant RPGR transcripts. Six of these mutations were detected in the conserved amino-terminal region of RPGR protein, containing tandem repeats homologous to the RCC1 protein, a guanine nucleotide-exchange factor for RanGTPase. Several exonic and intronic sequence variations were also detected. None of the RPGR mutations reported in other populations were identified in our series. Our results are consistent with the notions of heterogeneity and minority causation of XLRP by mutations in RPGR in Caucasian populations. [ABSTRACT FROM AUTHOR]

Neuroplasticity is a crucial property of the central nervous system to change its activity in response to intrinsic or extrinsic stimuli. This is mainly achieved through the promotion of changes in the epigenome. One of the epi-drivers priming this process is suberoylanilide hydroxamic acid (SAHA or Vorinostat), a pan-histone deacetylase inhibitor that modulates and promotes neuroplasticity in healthy and disease conditions. Knowledge of the specific molecular changes induced by this epidrug is an important area of neuro-epigenetics for the identification of new compounds to treat cognition impairment and/or epilepsy. In this review, we summarize the findings obtained in cellular and animal models of various brain disorders, highlighting the multiple mechanisms activated by SAHA, such as improvement of memory, learning and behavior, and correction of faulty neuronal functioning. Supporting this evidence, in vitro and in vivo data underline how SAHA positively regulates the expression of neuronal genes and microtubule dynamics, induces neurite outgrowth and spine density, and enhances synaptic transmission and potentiation. In particular, we outline studies regarding neurodevelopmental disorders with pharmaco-resistant seizures and/or severe cognitive impairment that to date lack effective drug treatments in which SAHA could ameliorate defective neuroplasticity. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM) is a fatal brain tumor without effective drug treatment. In this study, we highlight, for the first time, the contribution of chromatin remodeling gene Lysine (K)-specific demethylase 5C (KDM5C) in GBM via an extensive analysis of clinical, expression, and functional data, integrated with publicly available omic datasets. The expression analysis on GBM samples (N = 37) revealed two informative subtypes, namely KDM5CHigh and KDM5CLow, displaying higher/lower KDM5C levels compared to the controls. The former subtype displays a strong downregulation of brain-derived neurotrophic factor (BDNF)—a negative KDM5C target—and a robust overexpression of hypoxia-inducible transcription factor-1A (HIF1A) gene, a KDM5C modulator. Additionally, a significant co-expression among the prognostic markers HIF1A, Survivin, and p75 was observed. These results, corroborated by KDM5C overexpression and hypoxia-related functional assays in T98G cells, suggest a role for the HIF1A-KDM5C axis in the hypoxic response in this tumor. Interestingly, fluorescence-guided surgery on GBM sections further revealed higher KDM5C and HIF1A levels in the tumor rim niche compared to the adjacent tumor margin, indicating a regionally restricted hyperactivity of this regulatory axis. Analyzing the TCGA expression and methylation data, we found methylation changes between the subtypes in the genes, accounting for the hypoxia response, stem cell differentiation, and inflammation. High NANOG and IL6 levels highlight a distinctive stem cell-like and proinflammatory signature in the KDM5CHigh subgroup and GBM niches. Taken together, our results indicate HIF1A-KDM5C as a new, relevant cancer axis in GBM, opening a new, interesting field of investigation based on KDM5C as a potential therapeutic target of the hypoxic microenvironment in GBM. [ABSTRACT FROM AUTHOR]

The X-linked gene encoding aristaless-related homeobox (ARX) is a bi-functional transcription factor capable of activating or repressing gene transcription, whose mutations have been found in a wide spectrum of neurodevelopmental disorders (NDDs); these include cortical malformations, paediatric epilepsy, intellectual disability (ID) and autism. In addition to point mutations, duplications of the ARX locus have been detected in male patients with ID. These rearrangements include telencephalon ultraconserved enhancers, whose structural alterations can interfere with the control of ARX expression in the developing brain. Here, we review the structural features of 15 gain copy-number variants (CNVs) of the ARX locus found in patients presenting wide-ranging phenotypic variations including ID, speech delay, hypotonia and psychiatric abnormalities. We also report on a further novel Xp21.3 duplication detected in a male patient with moderate ID and carrying a fully duplicated copy of the ARX locus and the ultraconserved enhancers. As consequences of this rearrangement, the patient-derived lymphoblastoid cell line shows abnormal activity of the ARX-KDM5C-SYN1 regulatory axis. Moreover, the three-dimensional (3D) structure of the Arx locus, both in mouse embryonic stem cells and cortical neurons, provides new insight for the functional consequences of ARX duplications. Finally, by comparing the clinical features of the 16 CNVs affecting the ARX locus, we conclude that—depending on the involvement of tissue-specific enhancers—the ARX duplications are ID-associated risk CNVs with variable expressivity and penetrance. [ABSTRACT FROM AUTHOR]

Abstract: NEMO/IKKγ gene, which is responsible of two allelic diseases in human, EDA-ID and IP, encodes for a protein with a central regulatory role in the activation of the NF-kB pathway. We here provide insights into the molecular mechanism governing NEMO/IKKγ expression. We mapped 4 distinctive NEMO/IKKγ transcription start sites each corresponding to an alternative first exon, controlled by two conserved promoters. A distal promoter, named promoter A, located 10 kb upstream of the coding region and a proximal promoter, promoter B, with strong bi-directional activity driving also the transcription of G6PD gene in the opposite direction. The promoter B is housekeeping, it is embedded in a CpG island, required for proper expression and it is down-regulated by methylation. The promoter A is active in cells of hepatic origin and it directs transcription of the main NEMO/IKKγ 5′ UTR alternative transcript in liver, which starts at a tissue-specific site. Qualitative and quantitative expression analysis revealed that each NEMO/IKKγ 5′ UTR alternative transcript has different expression profiles indicating that the control of NEMO/IKKγ expression is mediated through tissue-specific transcription initiation sites and multiple regulatory regions. [Copyright &y& Elsevier]

Detecting the ultrastructure of brain tissue in human archaeological remains is a rare event that can offer unique insights into the structure of the ancient central nervous system (CNS). Yet ancient brains reported in the literature show only poor preservation of neuronal structures. Using scanning electron microscopy (SEM) and advanced image processing tools, we describe the direct visualization of neuronal tissue in vitrified brain and spinal cord remains which we discovered in a male victim of the AD 79 eruption in Herculaneum. We show exceptionally well preserved ancient neurons from different regions of the human CNS at unprecedented resolution. This tissue typically consists of organic matter, as detected using energy-dispersive X-ray spectroscopy. By means of a self-developed neural image processing network, we also show specific details of the neuronal nanomorphology, like the typical myelin periodicity evidenced in the brain axons. The perfect state of preservation of these structures is due to the unique process of vitrification which occurred at Herculaneum. The discovery of proteins whose genes are expressed in the different region of the human adult brain further agree with the neuronal origin of the unusual archaeological find. The conversion of human tissue into glass is the result of sudden exposure to scorching volcanic ash and the concomitant rapid drop in temperature. The eruptive-induced process of natural vitrification, locking the cellular structure of the CNS, allowed us to study possibly the best known example in archaeology of extraordinarily well-preserved human neuronal tissue from the brain and spinal cord. [ABSTRACT FROM AUTHOR]

Intellectual disability (ID) and epilepsy often occur together and have a dramatic impact on the development and quality of life of the affected children. Polyalanine (polyA)-expansion-encoding mutations of aristaless-related homeobox (ARX) cause a spectrum of X-linked ID (XLID) diseases and chronic epilepsy, including infantile spasms. We show that lysine-specific demethylase 5C (KDM5C), a gene known to be mutated in XLID-affected children and involved in chromatin remodeling, is directly regulated by ARX through the binding in a conserved noncoding element. We have studied altered ARX carrying various polyA elongations in individuals with XLID and/or epilepsy. The changes in polyA repeats cause hypomorphic ARX alterations, which exhibit a decreased trans-activity and reduced, but not abolished, binding to the KDM5C regulatory region. The altered functioning of the mutants tested is likely to correlate with the severity of XLID and/or epilepsy. By quantitative RT-PCR, we observed a dramatic Kdm5c mRNA downregulation in murine Arx-knockout embryonic and neural stem cells. Such Kdm5c mRNA diminution led to a severe decrease in the KDM5C content during in vitro neuronal differentiation, which inversely correlated with an increase in H3K4me3 signal. We established that ARX polyA alterations damage the regulation of KDM5C expression, and we propose a potential ARX-dependent path acting via chromatin remodeling. [Copyright &y& Elsevier]

BACKGROUND Diminished ovarian reserve (DOR) is a heterogeneous disorder causing infertility, characterized by a decreased number of oocytes, the genetic cause of which is still unknown. METHODS AND RESULTS We describe a family with a new unbalanced X;18 translocation der(X) associated with either fully attenuated or DOR phenotype in the same family. Cytogenetics and array comparative genomic hybridization (aCGH) studies have revealed the same partial Xq monosomy and partial 18q trisomy in both the 32-year-old female with DOR and the unaffected mother. The genetic analysis has defined a subtelomeric deletion spanning 13.3 Mb from Xq27.3 to -Xqter, which covers the premature ovarian failure locus 1 (POF1); and a duplication spanning 13.4 Mb, from 18q22.1 to 18qter. From a parental-origin study, we have inferred that the rearranged X chromosome is maternally derived. The Xq27 and 18q22 breakpoint regions fall in a region extremely rich in long interspersed nuclear element, a class of retrotransposons able to trigger mispairing and unusual crossovers. X-inactivation studies reveal a skewing of der(X) both in the mother and the proband. Therefore, the phenotypic expression of der(X) is fully attenuated in the fertile mother and partially attenuated in the DOR daughter. CONCLUSIONS We report on an unbalanced maternally derived translocation (X;18)(q27;q22) with different intra-familial reproductive performances, ranging from fertility to DOR. Skewed X-inactivation seems to restore the unbalanced genetic make-up, fully silencing the 18q22 trisomy and at least in part the Xq27 monosomy. The chromosomal abnormality observed in this family supports the presence of a DOR susceptibility locus in the distal Xq region and targets the POF1 region for further investigation. [ABSTRACT FROM PUBLISHER]

Membrane-associated guanylate kinase (MAGUK) proteins are cell–cell contact organizing molecules that mediate targeting, clustering and anchoring of proteins at synapses and other cell junctions. MAGUK proteins may contain multiple protein–protein interaction motifs including PDZ, SH3 and guanylate kinase (GuK) domains. In this study, we performed a detailed analysis of the expression pattern of MPP4, a recently described member of the MAGUK protein family. We confirmed that this gene is highly expressed in retina, and demonstrate that it is also present, at lower levels, in brain. We identified a new retina specific isoform encoding a predicted protein lacking 71 amino acids. This protein region contains a newly identified L27 domain, another module playing a role in protein–protein interaction. By RNA in situ hybridization, Mpp4 expression was found to be localized to photoreceptor cells in postnatal retina. The MPP4 gene is localized to chromosome 2, in band 2q31–33, where a locus for autosomal recessive retinitis pigmentosa (RP26) has been mapped. Mutation analysis of the entire open reading frame of the MPP4 gene in a RP26 family revealed no pathologic mutations. In addition, we did not identify mutations in a panel of 300 unrelated patients with retinitis pigmentosa. [Copyright &y& Elsevier]

Dysregulation of transcriptional pathways is observed in multiple forms of neurodevelopmental disorders (NDDs), such as intellectual disability (ID), epilepsy and autism spectrum disorder (ASD). We previously demonstrated that the NDD genes encoding lysine-specific demethylase 5C (KDM5C) and its transcriptional regulators Aristaless related-homeobox (ARX), PHD Finger Protein 8 (PHF8) and Zinc Finger Protein 711 (ZNF711) are functionally connected. Here, we show their relation to each other with respect to the expression levels in human and mouse datasets and in vivo mouse analysis indicating that the coexpression of these syntenic X-chromosomal genes is temporally regulated in brain areas and cellular sub-types. In co-immunoprecipitation assays, we found that the homeotic transcription factor ARX interacts with the histone demethylase PHF8, indicating that this transcriptional axis is highly intersected. Furthermore, the functional impact of pathogenic mutations of ARX, KDM5C, PHF8 and ZNF711 was tested in lymphoblastoid cell lines (LCLs) derived from children with varying levels of syndromic ID establishing the direct correlation between defects in the KDM5C-H3K4me3 pathway and ID severity. These findings reveal novel insights into epigenetic processes underpinning NDD pathogenesis and provide new avenues for assessing developmental timing and critical windows for potential treatments. [ABSTRACT FROM AUTHOR]

X-linked congenital stationary night blindness (CSNBX) is a hereditary non-progressive retinal disorder, which can appear in two different clinical forms, complete and incomplete, associated with CSNB1 and CSNB2 loci on Xp. We describe a Sardinian family with complete CSNBX and define better the limits of the CSNB1 genetic locus on Xp11.4 through linkage analysis. Haplotype analysis showed two key recombinants, which restrict the CSNB1 locus to a region of about 3 cM limited by markers DSX1068 and DSX6810 respectively. The locus that we describe is included in the CSNB1 locus defined by previous reports referring to the same clinical form of the disease. These results, in addition to other recent mapping reports about families from different geographical areas, confirm the genetic homogeneity of X-linked complete CSNB. [ABSTRACT FROM AUTHOR]

• Cryopreserved primary mouse cortical cells can be used for single-cell RNA sequencing. • High reproducible single-cell data can be obtained from E15.5 mouse embryos. • Neurostore-cryoconserved samples can be shipped to other laboratories in dry-ice for further perspective studies. The application of single-cell RNA sequencing (scRNASeq) represents a unique approach to identify hundreds to millions of cells in mammalian cortical multilayers at different stages of embryogenesis. ScRNASeq technology applied to neurological studies requires the use of fresh starting materials because standard cryopreservation methods do not guarantee high viability of cortical primary cells derived from dissected brain areas. Here we set up and validate an innovative strategy to perform scRNASeq studies in cryopreserved primary cortical cells isolated from E15.5 mouse embryo. In order to freeze cortical primary cells, we have employed Neurostore, a medium able to guarantee high viability and cell composition of embryonic cortex after thawing. We showed for the first time the possibility to run scRNASeq experiments on primary cortical cells in an off-line set-up, ensuring cellular integrity and diversity. By trypan blue assay and flow cytometry analysis, we found that Neurostore-cryopreserved cortical cells showed approximately 95 % of viability. Satisfactory RNA recovery and cDNA libraries were achieved. Transcriptome sequencing of 35,763 cryoconserved single cells yielded a robust data-set, identifying 25 cell clusters in three biological samples. Prevalence of peculiar neural populations before and after the cryopreservation-resuscitation procedure was verified by marker gene expression and immunofluorescence analysis. Our findings support the evidence that frozen primary cortical cells can be successfully employed in scRNASeq experiments allowing an unprecedented flexibility in experimental procedures, such as sample preparation and subsequent processing steps performed in different locations. [ABSTRACT FROM AUTHOR]

Unstable repeat disorders comprise a variable group of incurable human neurological and neuromuscular diseases caused by an increase in the copy number of tandem repeats located in various regions of their resident genes. It has become clear that dense DNA methylation in hyperexpanded non-coding repeats induces transcriptional silencing and, subsequently, insufficient protein synthesis. However, the ramifications of this paradigm reveal a far more profound role in disease pathogenesis. This review will summarize the significant progress made in a subset of non-coding repeat diseases demonstrating the role of dense landscapes of 5-methylcytosine (5mC) as a common disease modifier. However, the emerging findings suggest context-dependent models of 5mC-mediated silencing with distinct effects of excessive DNA methylation. An in-depth understanding of the molecular mechanisms underlying this peculiar group of human diseases constitutes a prerequisite that could help to discover novel pathogenic repeat loci, as well as to determine potential therapeutic targets. In this regard, we report on a brief description of advanced strategies in DNA methylation profiling for the identification of unstable Guanine-Cytosine (GC)-rich regions and on promising examples of molecular targeted therapies for Fragile X disease (FXS) and Friedrich ataxia (FRDA) that could pave the way for the application of this technique in other hypermethylated expansion disorders. [ABSTRACT FROM AUTHOR]