A case report of two Moroccan patients with hereditary neurological disorders and molecular modeling study on the S72L de novo PMP22 variant.

Hereditary neurological disorders represent a wild group of hereditary illnesses affecting mainly the nervous system, the majority of which have a Mendelian inheritance pattern. Here we present the case of two Moroccan patients each affected by a different hereditary neurological disorder. In the first patient WES analysis revealed the presence of the p.Ser72Leu de novo mutation in the PMP22 gene reported for the first time in Africa, specifically in Morocco. This variant is predicted to be in a mutation "hot-spot" region causing Dejerine-Sottas syndrome called also Charcot-Marie-Tooth type 3. The molecular modeling study suggests an important alteration of hydrogen and hydrophobic interactions between the residue in position 72 of the PMP22 protein and its surrounding amino acids. On the other hand, the p.Ala177Thr mutation on the RNASEH2B gene, responsible of Aicardi-Goutières syndrome 2, was carried in a homozygous state by the second patient descending from a consanguineous family. This mutation is common among the Moroccan population as well as in other North African countries. The present results contributed to a better follow-up of both cases allowing better symptom management with convenient treatments.

STochastic Optical Reconstruction Microscopy (STORM) reveals the nanoscale organization of pathological aggregates in human brain.

AIMS: Histological analysis of brain tissue samples provides valuable information about the pathological processes leading to common neurodegenerative disorders. In this context, the development of novel high-resolution imaging approaches is a current challenge in neuroscience. METHODS: To this end, we used a recent super-resolution imaging technique called STochastic Optical Reconstruction Microscopy (STORM) to analyse human brain sections. We combined STORM cell imaging protocols with neuropathological techniques to image cryopreserved brain samples from control subjects and patients with neurodegenerative diseases. RESULTS: This approach allowed us to perform 2D-, 3D- and two-colour-STORM in neocortex, white matter and brainstem samples. STORM proved to be particularly effective at visualizing the organization of dense protein inclusions and we imaged with a <50 nm resolution pathological aggregates within the central nervous system of patients with Alzheimer's disease, Parkinson's disease, Lewy body dementia and fronto-temporal lobar degeneration. Aggregated Aß branches appeared reticulated and cross-linked in the extracellular matrix, with widths from 60 to 240 nm. Intraneuronal Tau and TDP-43 inclusions were denser, with a honeycomb pattern in the soma and a filamentous organization in the axons. Finally, STORM imaging of α-synuclein pathology revealed the internal organization of Lewy bodies that could not be observed by conventional fluorescence microscopy. CONCLUSIONS: STORM imaging of human brain samples opens further gates to a more comprehensive understanding of common neurological disorders. The convenience of this technique should open a straightforward extension of its application for super-resolution imaging of the human brain, with promising avenues to current challenges in neuroscience.

Lipidomics Reveals Cerebrospinal-Fluid Signatures of ALS.

Amyotrophic lateral sclerosis (ALS), the commonest adult-onset motor neuron disorder, is characterized by a survival span of only 2-5 years after onset. Relevant biomarkers or specific metabolic signatures would provide powerful tools for the management of ALS. The main objective of this study was to investigate the cerebrospinal fluid (CSF) lipidomic signature of ALS patients by mass spectrometry to evaluate the diagnostic and predictive values of the profile. We showed that ALS patients (n = 40) displayed a highly significant specific CSF lipidomic signature compared to controls (n = 45). Phosphatidylcholine PC(36:4), higher in ALS patients (p = 0.0003) was the most discriminant molecule, and ceramides and glucosylceramides were also highly relevant. Analysis of targeted lipids in the brain cortex of ALS model mice confirmed the role of some discriminant lipids such as PC. We also obtained good models for predicting the variation of the ALSFRS-r score from the lipidome baseline, with an accuracy of 71% in an independent set of patients. Significant predictions of clinical evolution were found to be correlated to sphingomyelins and triglycerides with long-chain fatty acids. Our study, which shows extensive lipid remodelling in the CSF of ALS patients, provides a new metabolic signature of the disease and its evolution with good predictive performance.

Loss of function of Ywhah in mice induces deafness and cochlear outer hair cells' degeneration.

In vertebrates, 14-3-3 proteins form a family of seven highly conserved isoforms with chaperone activity, which bind phosphorylated substrates mostly involved in regulatory and checkpoint pathways. 14-3-3 proteins are the most abundant protein in the brain and are abundantly found in the cerebrospinal fluid in neurodegenerative diseases, suggesting a critical role in neuron physiology and death. Here we show that 14-3-3eta-deficient mice displayed auditory impairment accompanied by cochlear hair cells' degeneration. We show that 14-3-3eta is highly expressed in the outer and inner hair cells, spiral ganglion neurons of cochlea and retinal ganglion cells. Screening of YWHAH, the gene encoding the 14-3-3eta isoform, in non-syndromic and syndromic deafness, revealed seven non-synonymous variants never reported before. Among them, two were predicted to be damaging in families with syndromic deafness. In vitro, variants of YWHAH induce mild mitochondrial fragmentation and severe susceptibility to apoptosis, in agreement with a reduced capacity of mutated 14-3-3eta to bind the pro-apoptotic Bad protein. This study demonstrates that YWHAH variants can have a substantial effect on 14-3-3eta function and that 14-3-3eta could be a critical factor in the survival of outer hair cells.

[Hereditary optic neuropathies: from clinical signs to diagnosis]. / Les neuropathies optiques héréditaires: du signe clinique au diagnostic.

Inherited optic atrophy must be considered when working up any optic nerve involvement and any systemic disease with signs of optic atrophy, even with a negative family history. There are two classical forms: dominant optic atrophy, characterized by insidious, bilateral, slowly progressive visual loss and temporal disc pallor, and Leber's optic atrophy, characterized by acute loss of central vision followed by the same event in the fellow eye within a few weeks to months, with disc hyperemia in the acute phase. Family history is critical for diagnosis. In the absence of family history, the clinician must rule out an identifiable acquired cause, i.e. toxic, inflammatory, perinatal injury, traumatic or tumoral, with orbital and brain imaging (MRI). Recessive optic atrophies are more rare and more severe and occur as part of multisystemic disorders, particularly Wolfram syndrome (diabetes mellitus, diabetes insipidus, and hearing loss). Effective treatments are limited; alcohol and smoking should be avoided. A cyclosporine trial (taken immediately upon visual loss in the first eye) is in progress in Leber's optic atrophy to prevent involvement of the fellow eye.

TRPV4 channels mediate the infrared laser-evoked response in sensory neurons.

Infrared laser irradiation has been established as an appropriate stimulus for primary sensory neurons under conditions where sensory receptor cells are impaired or lost. Yet, development of clinical applications has been impeded by lack of information about the molecular mechanisms underlying the laser-induced neural response. Here, we directly address this question through pharmacological characterization of the biological response evoked by midinfrared irradiation of isolated retinal and vestibular ganglion cells from rodents. Whole cell patch-clamp recordings reveal that both voltage-gated calcium and sodium channels contribute to the laser-evoked neuronal voltage variations (LEVV). In addition, selective blockade of the LEVV by micromolar concentrations of ruthenium red and RN 1734 identifies thermosensitive transient receptor potential vanilloid channels as the primary effectors of the chain reaction triggered by midinfrared laser irradiation. These results have the potential to facilitate greatly the design of future prosthetic devices aimed at restoring neurosensory capacities in disabled patients.

Multi-system neurological disease is common in patients with OPA1 mutations.

Additional neurological features have recently been described in seven families transmitting pathogenic mutations in OPA1, the most common cause of autosomal dominant optic atrophy. However, the frequency of these syndromal 'dominant optic atrophy plus' variants and the extent of neurological involvement have not been established. In this large multi-centre study of 104 patients from 45 independent families, including 60 new cases, we show that extra-ocular neurological complications are common in OPA1 disease, and affect up to 20% of all mutational carriers. Bilateral sensorineural deafness beginning in late childhood and early adulthood was a prominent manifestation, followed by a combination of ataxia, myopathy, peripheral neuropathy and progressive external ophthalmoplegia from the third decade of life onwards. We also identified novel clinical presentations with spastic paraparesis mimicking hereditary spastic paraplegia, and a multiple sclerosis-like illness. In contrast to initial reports, multi-system neurological disease was associated with all mutational subtypes, although there was an increased risk with missense mutations [odds ratio = 3.06, 95% confidence interval = 1.44-6.49; P = 0.0027], and mutations located within the guanosine triphosphate-ase region (odds ratio = 2.29, 95% confidence interval = 1.08-4.82; P = 0.0271). Histochemical and molecular characterization of skeletal muscle biopsies revealed the presence of cytochrome c oxidase-deficient fibres and multiple mitochondrial DNA deletions in the majority of patients harbouring OPA1 mutations, even in those with isolated optic nerve involvement. However, the cytochrome c oxidase-deficient load was over four times higher in the dominant optic atrophy + group compared to the pure optic neuropathy group, implicating a causal role for these secondary mitochondrial DNA defects in disease pathophysiology. Individuals with dominant optic atrophy plus phenotypes also had significantly worse visual outcomes, and careful surveillance is therefore mandatory to optimize the detection and management of neurological disability in a group of patients who already have significant visual impairment.

OPA1 alternate splicing uncouples an evolutionary conserved function in mitochondrial fusion from a vertebrate restricted function in apoptosis.

In most eucaryote cells, release of apoptotic proteins from mitochondria involves fission of the mitochondrial network and drastic remodelling of the cristae structures. The intramitochondrial dynamin OPA1, as a potential central actor of these processes, exists as eight isoforms resulting from the alternate splicing combinations of exons (Ex) 4, 4b and 5b, which functions remain undetermined. Here, we show that Ex4 that is conserved throughout evolution confers functions to OPA1 involved in the maintenance of the DeltaPsi(m) and in the fusion of the mitochondrial network. Conversely, Ex4b and Ex5b, which are vertebrate specific, define a function involved in cytochrome c release, an apoptotic process also restricted to vertebrates. The drastic changes of OPA1 variant abundance in different organs suggest that nuclear splicing can control mitochondrial dynamic fate and susceptibility to apoptosis and pathologies.

[A murine model of transitory optic neuropathy based on small interference RNA-induced OPA1 silencing in vivo (gene mutation associated with Kjer's disease)]. / Modèle murin de neuropathie optique transitoire par inhibition in vivo de l'expression d'OPA1 (gène impliqué dans la maladie de Kjer).

PURPOSE: Developing a murine model of OPA1 linked optic neuropathy. METHODS: Intravitreal injections (in adult C57BL/6J mice) of small interference RNA (siRNA) specific to OPA1 were performed in the left eye. The right eye served as control, injected with nonspecific siRNA (siRNA scramble). Visual evoked potentials and flash electroretinograms were performed 5 and 12 days after injection. Three months after injection, microscopy of optic nerve sections was performed. RESULTS: The electrophysiological tests showed a significant reduction in the VEP when the siRNA OPA1-injected eye was stimulated, compared with the control eye injected with siRNA scramble. The electroretinogram was normal in both eyes: no significant difference between the right and the left eye was found. Three months after injection, no measurable axonal degeneration was found in either eye. CONCLUSION: The reduced expression of OPA1 based on RNA silencing in adult mice could induce reversible dysfunction of retinal ganglion cells.

Apisalpha2, Apisalpha7-1 and Apisalpha7-2: three new neuronal nicotinic acetylcholine receptor alpha-subunits in the honeybee brain.

Acetylcholine is the principal excitatory neurotransmitter in the central nervous system of insects. Nicotinic acetylcholine receptors, which belong to the ligand-gated ion channel family, constitute important targets for insecticides. In the honeybee Apis mellifera, pharmacological evidence supports the existence of several nicotinic acetylcholine receptors. In this paper, we report the identification of three new genes that encode nicotinic acetylcholine receptor alpha-subunits in the honeybee. Phylogenetic comparisons with other ligand-gated ion channel subunit sequences support their classification as Apisalpha2, Apisalpha7-1 and Apisalpha7-2 subunits. Based on in situ hybridization experiments, we determined their expression patterns in the different brain regions of pupae and adult honeybees. Our results show that these nicotinic acetylcholine receptor subunits are differently expressed among the brain regions and that they appear at different stages of honeybee development.

A phase I-II, dose-escalating trial of ZD9331 in combination with irinotecan (CPT11) in previously pretreated metastatic colorectal cancer patients.

BACKGROUND: To establish the recommended dose (RD) of the thymidylate-synthase inhibitor ZD9331 administered with irinotecan (CPT-11) in patients with pretreated metastatic colorectal cancer, and to assess toxicity profile, pharmacokinetics (PK), and anti-tumor activity in a phase I/II open, multicenter, intrapatient chemotherapy dose escalating trial. PATIENTS AND METHODS: Twenty-one patients who failed first-line therapy (5-fluorouracil/leucovorin +/- oxaliplatin) received every 2 weeks CPT-11 180 mg/m2 D1, followed by ZD9331 30-minute infusion D2 at three dose levels: 90, 120 and 150 mg/m2. RESULTS: RD of ZD9331 was established at 90 mg/m2 for the first two cycles, with possibility to escalate at 120 mg/m2 according to safety evaluation. Grade 3-4 toxicities were neutropenia (67% of patients), grade 3 vomiting (14%), grade 3 nausea (10%) and grade 3 diarrhea (5%). ZD9331 dose level does not affect the PK of CPT-11 or SN-38. Tumor growth control (PR + SD) was achieved for 14 (66.7%) patients. Median time to progression was 6 months, and median survival was 8.4 months. CONCLUSION: ZD9331 90 mg/m2 combined with CPT-11 180 mg/m2 may be a viable option for treatment of metastatic colorectal cancer, with possible escalation to 120 mg/m2 of ZD9331 according to safety evaluation.

Identification and localization of the nicotinic acetylcholine receptor alpha3 mRNA in the brain of the honeybee, Apis mellifera.

The nicotinic acetylcholine receptors are ligand-gated ion channels responsible for rapid neurotransmission and are target sites for pesticides in insects. In the honeybee Apis mellifera, pharmacological and electrophysiological studies have shown that different nicotinic acetylcholine receptor subtypes may exist in the brain. Here, we have identified a honeybee cDNA that encodes a 537 amino acid protein with features typical of nicotinic acetylcholine receptor alpha subunit, and sequence homology to human alpha3. In situ hybridization on cryosections shows that the Apisalpha3 mRNA is differently expressed in larvae and adult. In larvae, Apisalpha3 mRNA expression is restricted to the suboesophageal ganglia. In adult, it is further expressed in the optic lobes, the dorsal lobes, the antennal lobes and the calyces of mushroom bodies. Together our results suggest that Apisalpha3 shows a controlled expression pattern during development.

Mutation spectrum and splicing variants in the OPA1 gene.

Optic atrophy type 1 (OPA1, MIM 165500) is a dominantly inherited optic neuropathy that features low visual acuity leading in many cases to legal blindness. We have recently shown, with others, that mutations in the OPA1 gene encoding a dynamin-related mitochondrial protein, underlie the dominant form of optic atrophy. Here we report that OPA1 has eight mRNA isoforms as a result of the alternative splicing of exon 4 and two novel exons named 4b and 5b. In addition, we screened a cohort of 19 unrelated patients with dominant optic atrophy by direct sequencing of the 30 OPA1 exons (including exons 4b and 5b) and found mutations in 17 (89%) of them of which 8 were novel. A majority of these mutations were truncative (65%) and located in exons 8 to 28, but a number of them were amino acid changes predominantly found in the GTPase domain (exons 8 to 15). We hypothesize that at least two modifications of OPA1 may lead to dominant optic atrophy, that is alteration in GTPase activity and loss of the last seven C-terminal amino acids that putatively interact with other proteins.

Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy.

Optic atrophy type 1 (OPA1, MIM 165500) is a dominantly inherited optic neuropathy occurring in 1 in 50,000 individuals that features progressive loss in visual acuity leading, in many cases, to legal blindness. Phenotypic variations and loss of retinal ganglion cells, as found in Leber hereditary optic neuropathy (LHON), have suggested possible mitochondrial impairment. The OPA1 gene has been localized to 3q28-q29 (refs 13-19). We describe here a nuclear gene, OPA1, that maps within the candidate region and encodes a dynamin-related protein localized to mitochondria. We found four different OPA1 mutations, including frameshift and missense mutations, to segregate with the disease, demonstrating a role for mitochondria in retinal ganglion cell pathophysiology.

Identification of rpaP1-5 and rpaP2-6 genes encoding two additional variants of the 60S acidic ribosomal proteins of Schizosaccharomyces pombe.

In the fission yeast, four genes (rpaP1-1, rpaP1-3, rpaP2-2, and rpaP2-4) encoding two variants of the RpaP1 and RpaP2 ribosomal proteins (rp) have been characterized. We have identified cDNA for additional variants called RpaP1.5 and RpaP2.6. Sequence comparison suggests that RpaP1.5 diverged before RpaP1.1 and RpaP1.3 and that RpaP2.6 is closer to RpaP2.2 than to RpaP2.4. The corresponding genes, rpaP1-5 and rpaP2-6, are transcribed coordinately with other rp genes.

Identification and transcription control of fission yeast genes repressed by an ammonium starvation growth arrest.

In fission yeast Schizosaccharomyces pombe, ammonium starvation induces a growth arrest, a cell cycle exit in G(1) and a further switch to meiosis. This process is regulated by the cAMP-dependent protein kinase and the Wis1-dependent MAP kinase cascade, and downstream transcription factors. In order to understand how cells adapt their genetic programme to the switch from mitotic cycling to starvation, a differential transcript analysis comparing mRNA from exponentially growing and ammonium-starved cells was performed. Genes repressed by this stimulus mainly concern cell growth, i.e. protein synthesis and global metabolism. Comparison of the expression of two of them, the ribosomal proteins Rps6 and TCTP, in many different growing conditions, evidenced a strong correlation, suggesting that their transcriptions are coordinately regulated. Nevertheless, by repeating the ammonium starvation on strains constitutively activated for the PKA pathway (Deltacgs1), or unable to activate the Wis1-dependent MAP kinase pathway (Deltawis1), or with both characteristics (Deltacgs1+Deltawis1), the transcriptional inhibition was found to be governed either by the PKA pathway, or by the Wis1 pathway, or by both. These results suggest that during the switch from exponential growth to ammonium starvation, cell homeostasis is maintained by downregulating the transcription of the most expressed genes by a PKA and a Wis1-dependent process. Accession Nos for the S30 and L14 ribosomal protein cDNA sequences are AJ2731 and AJ2732, respectively.

Cyclin B (p56cdc13) localization in the yeast Schizosaccharomyces pombe: an ultrastructural and immunocytochemical study.

The eucaryote cell cycle is driven by a set of cyclin dependent kinases (CDKs) associated to cyclins, which confer not only the activity but also the substrate specificity and the proper localization of the kinase activity. In the fission yeast Schizosaccharomyces pombe, only one cyclin, the product of the cdc13 gene (p56cdc13), is required to be associated with p34cdc2, to control the complete cell cycle. Earlier studies have localized this complex mainly in the nucleus and its periphery. Using new improved electron microscopy (EM) technologies, based on high pressure freezing fixation, we refined previous studies, evidencing cytoplasmic localization of p56cdc13, in addition to the nuclear localization previously observed. Further immunofluorescence studies, performed on aldehydically fixed cells, confirmed our EM results, emphasizing the major cytoplasmic localization of p56cdc13 in interphase cells and the relocalization towards the nucleus in mitotic cells, suggesting that the S pombe cyclin B localization is cell cycle-regulated.

Pyp3 PTPase acts as a mitotic inducer in fission yeast.

The p34cdc2 M-phase kinase is regulated by inhibitory phosphorylation of Tyr15, largely through the actions of the p107wee1 tyrosine kinase and p80cdc25 protein tyrosine phosphatase (PTPase). In this study we demonstrate that a second PTPase, encoded by pyp3, also contributes to tyrosyl dephosphorylation of p34cdc2. Pyp3 was identified as a high copy suppressor of a cdc25- mutation. The pyp3 gene encodes a 33 kDa PTPase that is more closely related to human PTP1B and fission yeast pyp1 and pyp2 PTPases than to cdc25. Pyp3 does not share an essential overlapping function with pyp1 or pyp2. We demonstrate that disruption of pyp3 causes a mitotic delay that is greatly exacerbated in cells that are partially defective for cdc25 function and that pyp3 function is essential in cdc25-disruption wee1- strains. Pyp3 PTPase effectively dephosphorylates and activates the p34cdc2 kinase in vitro. We conclude that the pyp3 PTPase acts cooperatively with p80cdc25 to dephosphorylate Tyr15 of p34cdc2.