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P23H is the most common mutation in the RHODOPSIN (RHO) gene leading to a dominant form of retinitis pigmentosa (RP), a rod photoreceptor degeneration that invariably causes vision loss. Specific disruption of the disease P23H RHO mutant while preserving the wild-type (WT) functional allele would be an invaluable therapy for this disease. However, various technologies tested in the past failed to achieve effective changes and consequently therapeutic benefits. We validated a CRISPR/Cas9 strategy to specifically inactivate the P23H RHO mutant, while preserving the WT allele in vitro. We, then, translated this approach in vivo by delivering the CRISPR/Cas9 components in murine Rho+/P23H mutant retinae. Targeted retinae presented a high rate of cleavage in the P23H but not WT Rho allele. This gene manipulation was sufficient to slow photoreceptor degeneration and improve retinal functions. To improve the translational potential of our approach, we tested intravitreal delivery of this system by means of adeno-associated viruses (AAVs). To this purpose, the employment of the AAV9-PHP.B resulted the most effective in disrupting the P23H Rho mutant. Finally, this approach was translated successfully in human cells engineered with the homozygous P23H RHO gene mutation. Overall, this is a significant proof-of-concept that gene allele specific targeting by CRISPR/Cas9 technology is specific and efficient and represents an unprecedented tool for treating RP and more broadly dominant genetic human disorders affecting the eye, as well as other tissues.

Mutations in PARK2, encoding the E3 ubiquitin protein ligase Parkin, are a common cause of autosomal recessive Parkinson's disease (PD). Loss of PARK2 function compromises mitochondrial quality by affecting mitochondrial biogenesis, bioenergetics, dynamics, transport and turnover. We investigated the impact of PARK2 dysfunction on the endoplasmic reticulum (ER)-mitochondria interface, which mediates calcium (Ca 2+ ) exchange between the two compartments and is essential for Parkin-dependent mitophagy. Confocal and electron microscopy analyses showed the ER and mitochondria to be in closer proximity in primary fibroblasts from PARK2 knockout (KO) mice and PD patients with PARK2 mutations than in controls. Ca 2+ flux to the cytosol was also modified, due to enhanced ER-to-mitochondria Ca 2+ transfers, a change that was also observed in neurons derived from induced pluripotent stem cells of a patient with PARK2 mutations. Subcellular fractionation showed the abundance of the Parkin substrate mitofusin 2 (Mfn2), which is known to modulate the ER-mitochondria interface, to be specifically higher in the mitochondrion-associated ER membrane compartment in PARK2 KO tissue. Mfn2 downregulation or the exogenous expression of normal Parkin restored cytosolic Ca 2+ transients in fibroblasts from patients with PARK2 mutations. In contrast, a catalytically inactive PD-related Parkin variant had no effect. Overall, our data suggest that Parkin is directly involved in regulating ER-mitochondria contacts and provide new insight into the role of the loss of Parkin function in PD development., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Rett syndrome (RTT) is a neurodevelopmental disorder with no efficient treatment that is caused in the majority of cases by mutations in the gene methyl-CpG binding-protein 2 (MECP2). RTT becomes manifest after a period of apparently normal development and causes growth deceleration, severe psychomotor impairment and mental retardation. Effective animal models for RTT are available and show morphofunctional abnormalities of synaptic connectivity. However, the molecular consequences of MeCP2 disruption leading to neuronal and synaptic alterations are not known. Protein synthesis regulation via the mammalian target of the rapamycin (mTOR) pathway is crucial for synaptic organization, and its disruption is involved in a number of neurodevelopmental diseases. We investigated the phosphorylation of the ribosomal protein (rp) S6, whose activation is highly dependent from mTOR activity. Immunohistochemistry showed that rpS6 phosphorylation is severely affected in neurons across the cortical areas of Mecp2 mutants and that this alteration precedes the severe symptomatic phase of the disease. Moreover, we found a severe defect of the initiation of protein synthesis in the brain of presymptomatic Mecp2 mutant that was not restricted to a specific subset of transcripts. Finally, we provide evidence for a general dysfunction of the Akt/mTOR, but not extracellular-regulated kinase, signaling associated with the disease progression in mutant brains. Our results indicate that defects in the AKT/mTOR pathway are responsible for the altered translational control in Mecp2 mutant neurons and disclosed a novel putative biomarker of the pathological process. Importantly, this study provides a novel context of therapeutic interventions that can be designed to successfully restrain or ameliorate the development of RTT.

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been shown to cause severe neurodevelopmental disorders including infantile spasms, encephalopathy, West-syndrome and an early-onset variant of Rett syndrome. CDKL5 is a serine/threonine kinase whose involvement in Rett syndrome can be inferred by its ability to directly bind and mediate phosphorylation of MeCP2. However, it remains to be elucidated how CDKL5 exerts its function. Here, we report that CDKL5 localizes to specific nuclear foci referred to as nuclear speckles in both cell lines and tissues. These sub-nuclear structures are traditionally considered as storage/modification sites of pre-mRNA splicing factors. Interestingly, we provide evidence that CDKL5 regulates the dynamic behaviour of nuclear speckles. Indeed, CDKL5 overexpression leads to nuclear speckle disassembly, and this event is strictly dependent on its kinase activity. Conversely, its down-regulation affects nuclear speckle morphology leading to abnormally large and uneven speckles. Similar results were obtained for primary adult fibroblasts isolated from CDKL5-mutated patients. Altogether, these findings indicate that CDKL5 controls nuclear speckle morphology probably by regulating the phosphorylation state of splicing regulatory proteins. Nuclear speckles are dynamic sites that can continuously supply splicing factors to active transcription sites, where splicing occurs. Notably, we proved that CDKL5 influences alternative splicing, at least as proved in heterologous minigene assays. In conclusion, we provide evidence that CDKL5 is involved indirectly in pre-mRNA processing, by controlling splicing factor dynamics. These findings identify a biological process whose disregulation might affect neuronal maturation and activity in CDKL5-related disorders.

ATP-binding cassette (ABC) transporters facilitate unidirectional translocation of chemically diverse substances, ranging from peptides to lipids, across cell or organelle membranes. In peroxisomes, a subfamily of four ABC transporters (ABCD1 to ABCD4) has been related to fatty acid transport, because patients with mutations in ABCD1 (ALD gene) suffer from X-linked adrenoleukodystrophy (X-ALD), a disease characterized by an accumulation of very-long-chain fatty acids (VLCFAs). Inactivation in the mouse of the abcd1 gene leads to a late-onset neurodegenerative condition, comparable to the late-onset form of X-ALD [Pujol, A., Hindelang, C., Callizot, N., Bartsch, U., Schachner, M. and Mandel, J.L. (2002) Late onset neurological phenotype of the X-ALD gene inactivation in mice: a mouse model for adrenomyeloneuropathy. Hum. Mol. Genet., 11, 499-505.]. In the present work, we have generated and characterized a mouse deficient for abcd2, the closest paralog to abcd1. The main pathological feature in abcd2-/- mice is a late-onset cerebellar and sensory ataxia, with loss of cerebellar Purkinje cells and dorsal root ganglia cell degeneration, correlating with accumulation of VLCFAs in the latter cellular population. Axonal degeneration was present in dorsal and ventral columns in spinal cord. We have identified mitochondrial, Golgi and endoplasmic reticulum damage as the underlying pathological mechanism, thus providing evidence of a disturbed organelle cross-talk, which may be at the origin of the pathological cascade.

Rett syndrome (RTT) is a severe neurodevelopmental disorder almost exclusively affecting females and characterized by a wide spectrum of clinical manifestations. Most patients affected by classic RTT and a smaller percentage of patients with the milder form 'preserved speech variant' have either point mutations or deletions/duplications in the MECP2 gene. Recently, mutations in the CDKL5 gene, coding for a putative kinase, have been found in female patients with a phenotype overlapping with that of RTT. Here, we report two patients with the early seizure variant of RTT, bearing two novel CDKL5 truncating mutations, strengthening the correlation between CDKL5 and RTT. Considering the similar phenotypes caused by mutations in MECP2 and CDKL5, it has been suggested that the two genes play a role in common pathogenic processes. We show here that CDKL5 is a nuclear protein whose expression in the nervous system overlaps with that of MeCP2, during neural maturation and synaptogenesis. Importantly, we demonstrate that MeCP2 and CDKL5 interact both in vivo and in vitro and that CDKL5 is indeed a kinase, which is able to phosphorylate itself and to mediate MeCP2 phosphorylation, suggesting that they belong to the same molecular pathway. Furthermore, this paper contributes to the clarification of the phenotype associated with CDKL5 mutations and indicates that CDKL5 should be analyzed in each patient showing a clinical course similar to RTT but characterized by a lack of an early normal period due to the presence of seizures.

The BarH1 and BarH2 ( Bar ) Drosophila genes are homeobox-containing genes, which are required for the fate determination of external sensory organs in the fly. By means of a bioinformatic approach, we have identified in mouse and human two homeobox genes highly related to the Bar Drosophila genes, Barhl1 and Barhl2. While Barhl1 represents a novel gene, Barhl2 turned out to correspond to the mBH1 cDNA recently described in rat. We isolated and sequenced the full-length mouse Barhl1 and mapped both the human BARHL1 and BARHL2 genes to chromosomes 9q34 and 1p22, respectively. Detailed analysis of the murine Barhl1 expression pattern by in situ hybridization revealed that this transcript is exclusively expressed in restricted domains of the developing CNS, which suggests that this gene, similar to its Drosophila counterparts BarH1 and BarH2, may play a crucial role in cell fate determination of neural structures. In particular, Barhl1 showed specific domains of expression in the diencephalon and in the rhombencephalon where it was found to be expressed in migrating cells giving rise to the cerebellar external granular layer and to specific populations of dorsal sensory interneurons of the spinal cord. Thus, Barhl1 function may be required for the generation of these specific subtypes of neuronal progenitors. Furthermore, the mapping assignment and the expression pattern make BARHL1 an attractive positional candidate gene for a form of Joubert syndrome, a rare developmental anomaly of the cerebellum in humans.

We describe the cloning and characterization of a new human Xq13 gene (XH2), extending over a 220 kb genomic stretch between MNK and DXS56. The gene, which undergoes X-inactivation, contains a 4 kb open reading frame and encodes a putative NTP-binding nuclear protein homologous to several members of the helicase II superfamily. The murine homologue maps to the syntenic genetic interval, between Pgk1 and Xist. In situ hybridization studies in mouse reveal precocious, widespread expression of the murine homologue of XH2 at early stages of embryogenesis, and more restricted expression during late developmental stages and at birth. XH2 is a new member of an expanding family of proven and putative helicases, sharing six conserved, collinear domains. In particular, the XH2 protein shows homology with yeast RAD54. Type II helicases have been implicated in nucleotide excision repair and the initiation of transcription. This new gene, represents a potential candidate for several genetic disorders mapped to human Xq13.