De novo variants in SP9 cause a novel form of interneuronopathy characterized by intellectual disability, autism spectrum disorder, and epilepsy with variable expressivity.

PURPOSE: Interneuronopathies are a group of neurodevelopmental disorders characterized by deficient migration and differentiation of gamma-aminobutyric acidergic interneurons resulting in a broad clinical spectrum, including autism spectrum disorders, early-onset epileptic encephalopathy, intellectual disability, and schizophrenic disorders. SP9 is a transcription factor belonging to the Krüppel-like factor and specificity protein family, the members of which harbor highly conserved DNA-binding domains. SP9 plays a central role in interneuron development and tangential migration, but it has not yet been implicated in a human neurodevelopmental disorder. METHODS: Cases with SP9 variants were collected through international data-sharing networks. To address the specific impact of SP9 variants, in silico and in vitro assays were carried out. RESULTS: De novo heterozygous variants in SP9 cause a novel form of interneuronopathy. SP9 missense variants affecting the glutamate 378 amino acid result in severe epileptic encephalopathy because of hypomorphic and neomorphic DNA-binding effects, whereas SP9 loss-of-function variants result in a milder phenotype with epilepsy, developmental delay, and autism spectrum disorder. CONCLUSION: De novo heterozygous SP9 variants are responsible for a neurodevelopmental disease. Interestingly, variants located in conserved DNA-binding domains of KLF/SP family transcription factors may lead to neomorphic DNA-binding functions resulting in a combination of loss- and gain-of-function effects.

Bypassing Mendel's First Law: Transmission Ratio Distortion in Mammals.

Mendel's law of segregation states that the two alleles at a diploid locus should be transmitted equally to the progeny. A genetic segregation distortion, also referred to as transmission ratio distortion (TRD), is a statistically significant deviation from this rule. TRD has been observed in several mammal species and may be due to different biological mechanisms occurring at diverse time points ranging from gamete formation to lethality at post-natal stages. In this review, we describe examples of TRD and their possible mechanisms in mammals based on current knowledge. We first focus on the differences between TRD in male and female gametogenesis in the house mouse, in which some of the most well studied TRD systems have been characterized. We then describe known TRD in other mammals, with a special focus on the farmed species and in the peculiar common shrew species. Finally, we discuss TRD in human diseases. Thus far, to our knowledge, this is the first time that such description is proposed. This review will help better comprehend the processes involved in TRD. A better understanding of these molecular mechanisms will imply a better comprehension of their impact on fertility and on genome evolution. In turn, this should allow for better genetic counseling and lead to better care for human families.

A homozygous splice variant in ATP5PO, disrupts mitochondrial complex V function and causes Leigh syndrome in two unrelated families.

Mitochondrial complex V plays an important role in oxidative phosphorylation by catalyzing the generation of ATP. Most complex V subunits are nuclear encoded and not yet associated with recognized Mendelian disorders. Using exome sequencing, we identified a rare homozygous splice variant (c.87+3A>G) in ATP5PO, the complex V subunit which encodes the oligomycin sensitivity conferring protein, in three individuals from two unrelated families, with clinical suspicion of a mitochondrial disorder. These individuals had a similar, severe infantile and often lethal multi-systemic disorder that included hypotonia, developmental delay, hypertrophic cardiomyopathy, progressive epileptic encephalopathy, progressive cerebral atrophy, and white matter abnormalities on brain MRI consistent with Leigh syndrome. cDNA studies showed a predominant shortened transcript with skipping of exon 2 and low levels of the normal full-length transcript. Fibroblasts from the affected individuals demonstrated decreased ATP5PO protein, defective assembly of complex V with markedly reduced amounts of peripheral stalk proteins, and complex V hydrolytic activity. Further, expression of human ATP5PO cDNA without exon 2 (hATP5PO-∆ex2) in yeast cells deleted for yATP5 (ATP5PO homolog) was unable to rescue growth on media which requires oxidative phosphorylation when compared to the wild type construct (hATP5PO-WT), indicating that exon 2 deletion leads to a non-functional protein. Collectively, our findings support the pathogenicity of the ATP5PO c.87+3A>G variant, which significantly reduces but does not eliminate complex V activity. These data along with the recent report of an affected individual with ATP5PO variants, add to the evidence that rare biallelic variants in ATP5PO result in defective complex V assembly, function and are associated with Leigh syndrome.

Identification of 8-Hydroxyquinoline Derivatives That Decrease Cystathionine Beta Synthase (CBS) Activity.

CBS encodes a pyridoxal 5'-phosphate-dependent enzyme that catalyses the condensation of homocysteine and serine to form cystathionine. Due to its implication in some cancers and in the cognitive pathophysiology of Down syndrome, the identification of pharmacological inhibitors of this enzyme is urgently required. However, thus far, attempts to identify such molecules have only led to the identification of compounds with low potency and limited selectivity. We consequently developed an original, yeast-based screening method that identified three FDA-approved drugs of the 8-hydroxyquinoline family: clioquinol, chloroxine and nitroxoline. These molecules reduce CBS enzymatic activity in different cellular models, proving that the molecular mechanisms involved in yeast phenotypic rescue are conserved in mammalian cells. A combination of genetic and chemical biology approaches also revealed the importance of copper and zinc intracellular levels in the regulation of CBS enzymatic activity-copper promoting CBS activity and zinc inhibiting its activity. Taken together, these results indicate that our effective screening approach identified three new potent CBS inhibitors and provides new findings for the regulation of CBS activity, which is crucial to develop new therapies for CBS-related human disorders.

Cbs overdosage is necessary and sufficient to induce cognitive phenotypes in mouse models of Down syndrome and interacts genetically with Dyrk1a.

Identifying dosage-sensitive genes is a key to understand the mechanisms underlying intellectual disability in Down syndrome (DS). The Dp(17Abcg1-Cbs)1Yah DS mouse model (Dp1Yah) shows cognitive phenotypes that need to be investigated to identify the main genetic driver. Here, we report that three copies of the cystathionine-beta-synthase gene (Cbs) in the Dp1Yah mice are necessary to observe a deficit in the novel object recognition (NOR) paradigm. Moreover, the overexpression of Cbs alone is sufficient to induce deficits in the NOR test. Accordingly, overexpressing human CBS specifically in Camk2a-expressing neurons leads to impaired objects discrimination. Altogether, this shows that Cbs overdosage is involved in DS learning and memory phenotypes. To go further, we identified compounds that interfere with the phenotypical consequence of CBS overdosage in yeast. Pharmacological intervention in Tg(CBS) mice with one selected compound restored memory in the NOR test. In addition, using a genetic approach, we demonstrated an epistatic interaction between Cbs and Dyrk1a, another human chromosome 21-located gene (which encodes the dual-specificity tyrosine phosphorylation-regulated kinase 1a) and an already known target for DS therapeutic intervention. Further analysis using proteomic approaches highlighted several molecular pathways, including synaptic transmission, cell projection morphogenesis and actin cytoskeleton, that are affected by DYRK1A and CBS overexpression. Overall, we demonstrated that CBS overdosage underpins the DS-related recognition memory deficit and that both CBS and DYRK1A interact to control accurate memory processes in DS. In addition, our study establishes CBS as an intervention point for treating intellectual deficiencies linked to DS.

A new mouse model of ARX dup24 recapitulates the patients' behavioral and fine motor alterations.

The aristaless-related homeobox (ARX) transcription factor is involved in the development of GABAergic and cholinergic neurons in the forebrain. ARX mutations have been associated with a wide spectrum of neurodevelopmental disorders in humans, among which the most frequent, a 24 bp duplication in the polyalanine tract 2 (c.428_451dup24), gives rise to intellectual disability, fine motor defects with or without epilepsy. To understand the functional consequences of this mutation, we generated a partially humanized mouse model carrying the c.428_451dup24 duplication (Arxdup24/0) that we characterized at the behavior, neurological and molecular level. Arxdup24/0 males presented with hyperactivity, enhanced stereotypies and altered contextual fear memory. In addition, Arxdup24/0 males had fine motor defects with alteration of reaching and grasping abilities. Transcriptome analysis of Arxdup24/0 forebrains at E15.5 showed a down-regulation of genes specific to interneurons and an up-regulation of genes normally not expressed in this cell type, suggesting abnormal interneuron development. Accordingly, interneuron migration was altered in the cortex and striatum between E15.5 and P0 with consequences in adults, illustrated by the defect in the inhibitory/excitatory balance in Arxdup24/0 basolateral amygdala. Altogether, we showed that the c.428_451dup24 mutation disrupts Arx function with a direct consequence on interneuron development, leading to hyperactivity and defects in precise motor movement control and associative memory. Interestingly, we highlighted striking similarities between the mouse phenotype and a cohort of 33 male patients with ARX c.428_451dup24, suggesting that this new mutant mouse line is a good model for understanding the pathophysiology and evaluation of treatment.

Embryonic forebrain transcriptome of mice with polyalanine expansion mutations in the ARX homeobox gene.

The Aristaless-related homeobox (ARX) gene encodes a paired-type homeodomain transcription factor with critical roles in embryonic development. Mutations in ARX give rise to intellectual disability (ID), epilepsy and brain malformation syndromes. To capture the genetics and molecular disruptions that underpin the ARX-associated clinical phenotypes, we undertook a transcriptome wide RNASeq approach to analyse developing (12.5 dpc) telencephalon of mice modelling two recurrent polyalanine expansion mutations with different phenotypic severities in the ARX gene. Here we report 238 genes significantly deregulated (Log2FC > +/-1.1, P-value <0.05) when both mutations are compared to wild-type (WT) animals. When each mutation is considered separately, a greater number of genes were deregulated in the severe PA1 mice (825) than in the PA2 animals (78). Analysing genes deregulated in either or both mutant strains, we identified 12% as implicated in ID, epilepsy and autism (99/858), with ∼5% of them as putative or known direct targets of ARX transcriptional regulation. We propose a core pathway of transcription regulators, including Hdac4, involved in chromatin condensation and transcriptional repression, and one of its targets, the transcription factor Twist1, as potential drivers of the ID and infantile spasms in patients with ARX polyalanine expansion mutations. We predict that the subsequent disturbance to this pathway is a consequence of ARX protein reduction with a broader and more significant level of disruption in the PA1 in comparison to the PA2 mice. Identifying early triggers of ARX-associated phenotypes contributes to our understanding of particular clusters/pathways underpinning comorbid phenotypes that are shared by many neurodevelopmental disorders.

Oligodendrocyte lineage is severely affected in human alcohol-exposed foetuses.

Prenatal alcohol exposure is a major cause of neurobehavioral disabilities. MRI studies in humans have shown that alcohol is associated with white matter microstructural anomalies but these studies focused on myelin abnormalities only after birth. Only one of these studies evaluated oligodendrocyte lineage, but only for a short period during human foetal life. As data are lacking in humans and alcohol is known to impair oligodendrocyte differentiation in rodents, the present study aimed to compare by immunohistochemistry the oligodendrocyte precursor cells expressing PDGFR-α and immature premyelinating/mature oligodendrocytes expressing Olig2 in the ganglionic eminences and the frontal cortex of 14 human foetuses exposed to alcohol from 15 to 37 weeks' gestation with age-matched controls. The human brains used in this study were obtained at the time of foetal autopsies for medical termination of pregnancy, in utero or post-natal early death. Before birth, PDGFR-α expression was strongly increased in the ganglionic eminences and the cortex of all foetuses exposed to alcohol except at the earliest stage. No massive generation of Olig2 immunoreactive cells was identified in the ganglionic eminences until the end of pregnancy and the density of Olig2-positive cells within the cortex was consistently lower in foetuses exposed to alcohol than in controls. These antenatal data from humans provides further evidence of major oligodendrocyte lineage impairment at specific and key stages of brain development upon prenatal alcohol exposure including defective or delayed generation and maturation of oligodendrocyte precursors.

New insights into the regulation of Cystathionine beta synthase (CBS), an enzyme involved in intellectual deficiency in Down syndrome.

Down syndrome (DS), the most frequent chromosomic aberration, results from the presence of an extra copy of chromosome 21. The identification of genes which overexpression contributes to intellectual disability (ID) in DS is important to understand the pathophysiological mechanisms involved and develop new pharmacological therapies. In particular, gene dosage of Dual specificity tyrosine phosphorylation Regulated Kinase 1A (DYRK1A) and of Cystathionine beta synthase (CBS) are crucial for cognitive function. As these two enzymes have lately been the main targets for therapeutic research on ID, we sought to decipher the genetic relationship between them. We also used a combination of genetic and drug screenings using a cellular model overexpressing CYS4, the homolog of CBS in Saccharomyces cerevisiae, to get further insights into the molecular mechanisms involved in the regulation of CBS activity. We showed that overexpression of YAK1, the homolog of DYRK1A in yeast, increased CYS4 activity whereas GSK3ß was identified as a genetic suppressor of CBS. In addition, analysis of the signaling pathways targeted by the drugs identified through the yeast-based pharmacological screening, and confirmed using human HepG2 cells, emphasized the importance of Akt/GSK3ß and NF-κB pathways into the regulation of CBS activity and expression. Taken together, these data provide further understanding into the regulation of CBS and in particular into the genetic relationship between DYRK1A and CBS through the Akt/GSK3ß and NF-κB pathways, which should help develop more effective therapies to reduce cognitive deficits in people with DS.

Role of cytoskeletal abnormalities in the neuropathology and pathophysiology of type I lissencephaly.

Type I lissencephaly or agyria-pachygyria is a rare developmental disorder which results from a defect of neuronal migration. It is characterized by the absence of gyri and a thickening of the cerebral cortex and can be associated with other brain and visceral anomalies. Since the discovery of the first genetic cause (deletion of chromosome 17p13.3), six additional genes have been found to be responsible for agyria-pachygyria. In this review, we summarize the current knowledge concerning these genetic disorders including clinical, neuropathological and molecular results. Genetic alterations of LIS1, DCX, ARX, TUBA1A, VLDLR, RELN and more recently WDR62 genes cause migrational abnormalities along with more complex and subtle anomalies affecting cell proliferation and differentiation, i.e., neurite outgrowth, axonal pathfinding, axonal transport, connectivity and even myelination. The number and heterogeneity of clinical, neuropathological and radiological defects suggest that type I lissencephaly now includes several forms of cerebral malformations. In vitro experiments and mutant animal studies, along with neuropathological abnormalities in humans are of invaluable interest for the understanding of pathophysiological mechanisms, highlighting the central role of cytoskeletal dynamics required for a proper achievement of cell proliferation, neuronal migration and differentiation.

p53, A Victim of the Prion Fashion.

Identified in the late 1970s as an oncogene, a driving force leading to tumor development, p53 turned out to be a key tumor suppressor gene. Now p53 is considered a master gene regulating the transcription of over 3000 target genes and controlling a remarkable number of cellular functions. The elevated prevalence of p53 mutations in human cancers has led to a recurring questioning about the roles of mutant p53 proteins and their functional consequences. Both mutants and isoforms of p53 have been attributed dominant-negative and gain of function properties among which is the ability to form amyloid aggregates and behave in a prion-like manner. This report challenges the ongoing "prion p53" hypothesis by reviewing evidence of p53 behavior in light of our current knowledge regarding amyloid proteins, prionoids and prions.

Anti-prion Drugs Targeting the Protein Folding Activity of the Ribosome Reduce PABPN1 Aggregation.

Prion diseases are caused by the propagation of PrPSc, the pathological conformation of the PrPC prion protein. The molecular mechanisms underlying PrPSc propagation are still unsolved and no therapeutic solution is currently available. We thus sought to identify new anti-prion molecules and found that flunarizine inhibited PrPSc propagation in cell culture and significantly prolonged survival of prion-infected mice. Using an in silico therapeutic repositioning approach based on similarities with flunarizine chemical structure, we tested azelastine, duloxetine, ebastine, loperamide and metixene and showed that they all have an anti-prion activity. Like flunarizine, these marketed drugs reduced PrPSc propagation in cell culture and in mouse cerebellum organotypic slice culture, and inhibited the protein folding activity of the ribosome (PFAR). Strikingly, some of these drugs were also able to alleviate phenotypes due to PABPN1 nuclear aggregation in cell and Drosophila models of oculopharyngeal muscular dystrophy (OPMD). These data emphasize the therapeutic potential of anti-PFAR drugs for neurodegenerative and neuromuscular proteinopathies.

Evidence for tangential migration disturbances in human lissencephaly resulting from a defect in LIS1, DCX and ARX genes.

During corticogenesis, neurons adopt different migration pathways to reach their final position. The precursors of pyramidal neurons migrate radially, whereas most of the GABA-containing interneurons are generated in the ventral telencephalon and migrate tangentially into the neocortex. Then, they use a radial migration mode to establish themselves in an inside-out manner in the neocortex, similarly to pyramidal neurons. In humans, the most severe defects in radial migration result in lissencephaly. Lately, a few studies suggested that lissencephaly was also associated with tangential neuronal migration deficits. In the present report, we investigated potential anomalies of this migration mode in three agyric/pachygyric syndromes due to defects in the LIS1, DCX and ARX genes. Immunohistochemistry was performed on paraffin-embedded supra- and infratentorial structures using calretinin, calbindin and parvalbumin antisera. The results were compared with age-matched control brain tissue. In the Miller-Dieker syndrome, GABAergic neurons were found both in upper layers of the cortex and in heterotopic positions in the intermediate zone and in ganglionic eminences. In the DCX mutant brain, few interneurons were dispersed in the cortical plate, with a massive accumulation in the intermediate zone and subventricular zone as well as in the ganglionic eminences. In the ARX-mutated brain, the cortical plate contained almost exclusively pyramidal cells and was devoid of interneurons. The ganglionic eminences and basal ganglia were poorly cellular, suggesting an interneuron production and/or differentiation defect. These data argue for different mechanisms of telencephalic tangential migration impairment in these three agyric/pachygyric syndromes.

Human lissencephaly with cerebellar hypoplasia due to mutations in TUBA1A: expansion of the foetal neuropathological phenotype.

Neuronal migration disorders account for a substantial number of cortical malformations, the most severe forms being represented by lissencephalies. Classical lissencephaly has been shown to result from mutations in LIS1 (PAFAH1B1; MIM#601545), DCX (Doublecortin; MIM#300121), ARX (Aristaless-related homeobox gene; MIM#300382), RELN (Reelin; MIM#600514) and VLDLR (Very low density lipoprotein receptor; MIM#224050). More recently, de novo missense mutations in the alpha-tubulin 1a gene (TUBA1A) located on chromosome 12q13.12, have also been associated with more or less severe defects of cortical development, resulting in complete agyria in the most severe cases of lissencephaly. We report here the cerebral lesions in a 36 weeks' gestation female foetus with a novel de novo missense mutation in the TUBA1A gene, presenting the most severe antenatal phenotype reported so far. Using routine immunohistochemistry and confocal microscopy, we show evidence for defects in axonal transport in addition to defects in neuronal migration and differentiation, giving new insights to the pathophysiology of this form of lissencephaly.

Mechanism of cystathionine-ß-synthase inhibition by disulfiram: The role of bis(N,N-diethyldithiocarbamate)-copper(II).

BACKGROUND: Hydrogen sulfide (H2S) is an endogenous mammalian gasotransmitter. Cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) are the principal enzymes responsible for its biogenesis. A recent yeast screen suggested that disulfiram (a well-known inhibitor of aldehyde dehydrogenase and a clinically used drug in the treatment of alcoholism) may inhibit CBS in a cell-based environment. However, prior studies have not observed any direct inhibition of CBS by disulfiram. We investigated the potential role of bioconversion of disulfiram to bis(N,N-diethyldithiocarbamate)-copper(II) complex (CuDDC) in the inhibitory effect of disulfiram on H2S production and assessed its effect in two human cell types with high CBS expression: HCT116 colon cancer cells and Down syndrome (DS) fibroblasts. METHODS: H2S production from recombinant human CBS, CSE and 3-MST was measured using the fluorescent H2S probe AzMC. Mouse liver homogenate (a rich source of CBS) was also employed to measure H2S biosynthesis. The interaction of copper with accessible protein cysteine residues was evaluated using the DTNB method. Cell proliferation and viability were measured using the BrdU and MTT methods. Cellular bioenergetics was evaluated by Extracellular Flux Analysis. RESULTS: While disulfiram did not exert any significant direct inhibitory effect on any of the H2S-producing enzymes, its metabolite, CuDDC was a potent inhibitor of CBS and CSE. The mode of its action is likely related to the complexed copper molecule. In cell-based systems, the effects of disulfiram were variable. In colon cancer cells, no significant effect of disulfiram was observed on H2S production or proliferation or viability. In contrast, in DS fibroblasts, disulfiram inhibited H2S production and improved proliferation and viability. Copper, on its own, failed to have any effects on either cell type, likely due to its low cell penetration. CuDDC inhibited H2S production in both cell types studied and exerted the functional effects that would be expected from a CBS inhibitor: inhibition of cell proliferation of cancer cells and a bell-shaped effect (stimulation of proliferation at low concentration and inhibition of these responses at higher concentration) in DS cells. Control experiments using a chemical H2S donor showed that, in addition to inhibiting CBS and CSE, part of the biological effects of CuDDC relates to a direct reaction with H2S, which occurs through its complexed copper. CONCLUSIONS: Disulfiram, via its metabolite CuDDC acts as an inhibitor of CBS and a scavenger of H2S, which, in turn, potently suppresses H2S levels in various cell types. Inhibition of H2S biosynthesis may explain some of the previously reported actions of disulfiram and CuDDC in vitro and in vivo. Disulfiram or CuDDC may be considered as potential agents for the experimental therapy of various pathophysiological conditions associated with H2S overproduction.

Prenatal alcohol exposure is a leading cause of interneuronopathy in humans.

Alcohol affects multiple neurotransmitter systems, notably the GABAergic system and has been recognised for a long time as particularly damaging during critical stages of brain development. Nevertheless, data from the literature are most often derived from animal or in vitro models. In order to study the production, migration and cortical density disturbances of GABAergic interneurons upon prenatal alcohol exposure, we performed immunohistochemical studies by means of the proliferation marker Ki67, GABA and calretinin antibodies in the frontal cortical plate of 17 foetal and infant brains antenatally exposed to alcohol, aged 15 weeks' gestation to 22 postnatal months and in the ganglionic eminences and the subventricular zone of the dorsal telencephalon until their regression, i.e., 34 weeks' gestation. Results were compared with those obtained in 17 control brains aged 14 weeks of gestation to 35 postnatal months. We also focused on interneuron vascular migration along the cortical microvessels by confocal microscopy with double immunolabellings using Glut1, GABA and calretinin. Semi-quantitative and quantitative analyses of GABAergic and calretininergic interneuron density allowed us to identify an insufficient and delayed production of GABAergic interneurons in the ganglionic eminences during the two first trimesters of the pregnancy and a delayed incorporation into the laminar structures of the frontal cortex. Moreover, a mispositioning of GABAergic and calretininergic interneurons persisted throughout the foetal life, these cells being located in the deep layers instead of the superficial layers II and III. Moreover, vascular migration of calretininergic interneurons within the cortical plate was impaired, as reflected by low numbers of interneurons observed close to the cortical perforating vessel walls that may in part explain their abnormal intracortical distribution. Our results are globally concordant with those previously obtained in mouse models, in which alcohol has been shown to induce an interneuronopathy by affecting interneuron density and positioning within the cortical plate, and which could account for the neurological disabilities observed in children with foetal alcohol disorder spectrum.

Cell-autonomous roles of ARX in cell proliferation and neuronal migration during corticogenesis.

The aristaless-related homeobox (ARX) gene has been implicated in a wide spectrum of disorders ranging from phenotypes with severe neuronal migration defects, such as lissencephaly, to mild forms of X-linked mental retardation without apparent brain abnormalities. To better understand its role in corticogenesis, we used in utero electroporation to knock down or overexpress ARX. We show here that targeted inhibition of ARX causes cortical progenitor cells to exit the cell cycle prematurely and impairs their migration toward the cortical plate. In contrast, ARX overexpression increases the length of the cell cycle. In addition, we report that RNA interference-mediated inactivation of ARX prevents cells from acquiring multipolar morphology in the subventricular and intermediate zones, resulting in decreased neuronal motility. In contrast, ARX overexpression appears to promote the development of tangentially oriented processes of cells in the subventricular and intermediate zones and affects radial migration of pyramidal neurons. We also demonstrate that the level of ARX expression is important for tangential migration of GABA-containing interneurons, because both inactivation and overexpression of the gene impair their migration from the ganglionic eminence. However, our data suggest that ARX is not directly involved in GABAergic cell fate specification. Overall, these results identify multiple and distinct cell-autonomous roles for ARX in corticogenesis.

A yeast-based screening assay identifies repurposed drugs that suppress mitochondrial fusion and mtDNA maintenance defects.

Mitochondria continually move, fuse and divide, and these dynamics are essential for the proper function of the organelles. Indeed, the dynamic balance of fusion and fission of mitochondria determines their morphology and allows their immediate adaptation to energetic needs as well as preserving their integrity. As a consequence, mitochondrial fusion and fission dynamics and the proteins that control these processes, which are conserved from yeast to human, are essential, and their disturbances are associated with severe human disorders, including neurodegenerative diseases. For example, mutations in OPA1, which encodes a conserved factor essential for mitochondrial fusion, lead to optic atrophy 1, a neurodegeneration that affects the optic nerve, eventually leading to blindness. Here, by screening a collection of ∼1600 repurposed drugs on a fission yeast model, we identified five compounds able to efficiently prevent the lethality associated with the loss of Msp1p, the fission yeast ortholog of OPA1. One compound, hexestrol, was able to rescue both the mitochondrial fragmentation and mitochondrial DNA (mtDNA) depletion induced by the loss of Msp1p, whereas the second, clomifene, only suppressed the mtDNA defect. Yeast has already been successfully used to identify candidate drugs to treat inherited mitochondrial diseases; this work may therefore provide useful leads for the treatment of optic atrophies such as optic atrophy 1 or Leber hereditary optic neuropathy.

Both doublecortin and doublecortin-like kinase play a role in cortical interneuron migration.

Type I lissencephaly, a genetic disease characterized by disorganized cortical layers and gyral abnormalities, is associated with severe cognitive impairment and epilepsy. Two genes, LIS1 and doublecortin (DCX), have been shown to be responsible for a large proportion of cases of type I lissencephaly. Both genes encode microtubule-associated proteins that have been shown to be important for radial migration of cortical pyramidal neurons. To investigate whether DCX also plays a role in cortical interneuron migration, we inactivated DCX in the ganglionic eminence of rat embryonic day 17 brain slices using short hairpin RNA. We found that, when DCX expression was blocked, the migration of interneurons from the ganglionic eminence to the cerebral cortex was slowed but not absent, similar to what had previously been reported for radial neuronal migration. In addition, the processes of DCX-deficient migrating interneurons were more branched than their counterparts in control experiments. These effects were rescued by DCX overexpression, confirming the specificity to DCX inactivation. A similar delay in interneuron migration was observed when Doublecortin-like kinase (DCLK), a microtubule-associated protein related to DCX, was inactivated, although the morphology of the cells was not affected. The importance of these genes in interneuron migration was confirmed by our finding that the cortices of Dcx, Dclk, and Dcx/Dclk mutant mice contained a reduced number of such cells in the cortex and their distribution was different compared with wild-type controls. However, the defect was different for each group of mutant animals, suggesting that DCX and DCLK have distinct roles in cortical interneuron migration.

Annexin V is directly involved in cystic fibrosis transmembrane conductance regulator's chloride channel function.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a cAMP-activated chloride channel, which is regulated by protein-protein interactions. The extent to which CFTR is regulated by these interactions remains unknown. Annexin V is overexpressed in cystic fibrosis (CF), and given the functional properties of annexin V and CFTR we considered whether they are associated and if so whether this has implications for CFTR function. Using co-immunoprecipitation and overlay experiments, we show that annexin V is associated with nucleotide-binding domain 1 (NBD1) of CFTR. Surface plasmon resonance (SPR) indicated different KD values in the absence and presence of both calcium and ATP, suggesting that this interaction is calcium- and ATP-dependent. Using an siRNA approach and overexpression, we showed that CFTR chloride channel function and its localization in the cell membranes were dependent on annexin V expression. We concluded that annexin V is necessary for normal CFTR chloride channel activity. Furthermore, we show that CFTR and annexin V are partially co-distributed in normal epithelial cells in human bronchi. In conclusion, we show for the first time that annexin V is associated with CFTR and is involved in its function.