Jorge A. Bevilacqua

Vascular Factors and Risk of Dementia: Design of the Three-City Study and Baseline Characteristics of the Study Population

Objective: To describe the baseline characteristics of the participants in the Three-City (3C) Study, a study aiming to evaluate the risk of dementia and cognitive impairment attributable to vascular factors. Methods: Between 1999 and 2001, 9,693 persons aged 65 years and over, noninstitutionalized, were recruited from the electoral rolls of three French cities, i.e. Bordeaux, Dijon and Montpellier. Health-related data were collected during face-to-face interviews using standardized questionnaires. The baseline examination included cognitive testing and diagnosis of dementia, and assessment of vascular risk factors, including blood pressure measurements, ultrasound examination of the carotid arteries, and measurement of biological parameters (glycemia, total, high-density lipoprotein and low-density lipoprotein cholesterol, triglycerides, creatinemia); 3,442 magnetic resonance imaging (MRI) examinations were performed in subjects aged 65–79. Measurements of ultrasound, blood, and MRI parameters were centralized. Two follow-up examinations (at 2 and 4 years) were planned. Results: After exclusion of the participants who had subsequently refused the medical interview, the 3C Study sample consisted of 3,649 men (39.3%) and 5,645 women, mean age 74.4 years, with a relatively high level of education and income. Forty-two percent of the participants reported to be followed up for hypertension, about one third for hypercholesterolemia, and 8% for diabetes; 65% had elevated blood pressure measures (systolic blood pressure ≧140 or diastolic blood pressure ≧90). The proportion of Mini-Mental State Examination scores below 24 was 7% and dementia was diagnosed in 2.2% of the participants. Conclusion: Distribution of baseline characteristics of the 3C Study participants suggests that this study will provide a unique opportunity to estimate the risk of dementia attributable to vascular factors.

Dynamin 2 Mutations Cause Sporadic Centronuclear Myopathy with Neonatal Onset

We report four heterozygous dynamin 2 (DNM2) mutations in five centronuclear myopathy patients aged 1 to 15 years. They all presented with neonatal hypotonia with weak suckling. Thereafter, their phenotype progressively improved. All patients demonstrated muscle weakness prominent in the lower limbs, and most of them also presented with facial weakness, open mouth, arched palate, ptosis, and ophthalmoparesis. Electrophysiology showed only myopathic changes, and muscle biopsies showed central nuclei and type 1 fiber hypotrophy and predominance. Our results expand the phenotypic spectrum of dynamin 2–related centronuclear myopathy from the classic mild form to the more severe neonatal phenotype. Ann Neurol 2007

Recessive RYR1 mutations cause unusual congenital myopathy with prominent nuclear internalization and large areas of myofibrillar disorganization

J. A. Bevilacqua, N. Monnier, M. Bitoun, B. Eymard, A. Ferreiro, S. Monges, F. Lubieniecki, A. L. Taratuto, A. Laquerrière, K. G. Claeys, I. Marty, M. Fardeau, P. Guicheney, J. Lunardi and N. B. Romero (2011) Neuropathology and Applied Neurobiology37, 271–284
Recessive RYR1 mutations cause unusual congenital myopathy with prominent nuclear internalization and large areas of myofibrillar disorganization

Analysis of the DYSF mutational spectrum in a large cohort of patients

Dysferlinopathies belong to the heterogeneous group of autosomal recessive muscular dystrophies. Mutations in the gene encoding dysferlin (DYSF) lead to distinct phenotypes, mainly Limb Girdle Muscular Dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM). Here, we analysed the mutational data from the largest cohort described to date, a cohort of 134 patients, included based on clinical suspicion of primary dysferlinopathy and/or dysferlin protein deficiency identified on muscle biopsy samples. Data were compiled from 38 patients previously screened for mutations in our laboratory (Nguyen, et al., 2005; Nguyen, et al., 2007), and 96 supplementary patients screened for DYSF mutations using genomic DHPLC analysis, and subsequent sequencing of detected variants, in a routine diagnostic setting. In 89 (66%) out of 134 patients, molecular analysis identified two disease causing mutations, confirming the diagnosis of primary Dysferlinopathy on a genetic basis. Furthermore, one mutation was identified in 30 patients, without identification of a second deleterious allele. We are currently developing complementary analysis for patients in whom only one or no disease‐causing allele could be identified using the genomic screening procedure. Altogether, 64 novel mutations have been identified in this cohort, which corresponds to approximately 25% of all DYSF mutations reported to date. The mutational spectrum of this cohort significantly shows a higher proportion of nonsense mutations, but a lower proportion of deleterious missense changes as compared to previous series.

Dynamin 2 Mutations Associated With Human Diseases Impair Clathrin-Mediated Receptor Endocytosis

Dynamin 2 (DNM2) is a large GTPase involved in the release of nascent vesicles during endocytosis and intracellular membrane trafficking. Distinct DNM2 mutations, affecting the middle domain (MD) and the Pleckstrin homology domain (PH), have been identified in autosomal dominant centronuclear myopathy (CNM) and in the intermediate and axonal forms of the Charcot‐Marie‐Tooth peripheral neuropathy (CMT). We report here the first CNM mutation (c.1948G>A, p.E650 K) in the DNM2 GTPase effector domain (GED), leading to a slowly progressive moderate myopathy. COS7 cells transfected with DNM2 constructs harboring a disease‐associated mutation in MD, PH, or GED show a reduced uptake of transferrin and low‐density lipoprotein (LDL) complex, two markers of clathrin‐mediated receptor endocytosis. A decrease in clathrin‐mediated endocytosis was also identified in skin fibroblasts from one CNM patient. We studied the impact of DNM2 mutant overexpression on epidermal growth factor (EGF)‐induced extracellular signal‐regulated kinase 1 (ERK1) and ERK2 activation, known to be an endocytosis‐ and DNM2‐dependent process. Activation of ERK1/2 was impaired for all the transfected mutants in COS7 cells, but not in CNM fibroblasts. Our results indicate that impairment of clathrin‐mediated endocytosis may play a role in the pathophysiological mechanisms leading to DNM2‐related diseases, but the tissue‐specific impact of DNM2 mutations in both diseases remains unclear. Hum Mutat 30:1–9, 2009.

Muscarinic M1 receptors activate phosphoinositide turnover and Ca2+ mobilisation in rat sympathetic neurones, but this signalling pathway does not mediate M‐current inhibition

1 The relationship between muscarinic receptor activation, phosphoinositide turnover, calcium mobilisation and M‐current inhibition has been studied in rat superior cervical ganglion (SCG) neurones in primary culture. 2 Phosphoinositide‐specific phospholipase C (PLC) stimulation was measured by the accumulation of [3H]‐cytidine monophosphate phosphatidate (CMP‐PA) after incubation with [3H]‐cytidine in the presence of Li+. The muscarinic agonist oxotremorine methiodide (oxo‐M) stimulated PLC in a dose‐dependent manner with an EC50 of approximately 3.5 μm. 3 The concentration‐response curve for oxo‐M was shifted to the right by a factor of about 10 by pirenzepine (100 nm), suggesting a pKB (—log of the apparent dissociation constant) of 7.9 ± 0.4, while himbacine (1 μm) shifted the curve by a factor of about 13 (pKB∼7.1 ± 0.6). This indicates involvement of the M1 muscarinic receptor in this response. 4 The accumulation of CMP‐PA was localised by in situ autoradiography to SCG principal neurones, with no detectable signal in glial cells present in the primary cultures. 5 The ability of oxo‐M to release Ca2+ from inositol(1,4,5)trisphosphate (InsP3)‐sensitive stores was determined by fura‐2 microfluorimetry of SCG neurones voltage clamped in perforated patch mode. Oxo‐M failed to evoke intracellular Ca2+ (Ca2+i) mobilisation in SCG neurones voltage clamped at −60 mV, but produced a significant Ca2+i rise (67 ± 15 nm, n= 9) in cells voltage clamped at −25 mV. 6 Thapsigargin (0.5–1 μm) caused a 70% inhibition of the oxo‐M‐induced Ca2+i increase, indicating its intracellular origin, while oxo‐M‐induced inhibition of M‐current in the same cells was unaffected by thapsigargin. 7 Our results do not support the involvement of InsP3‐sensitive calcium mobilisation in M‐current inhibition.

A NEW CENTRONUCLEAR MYOPATHY PHENOTYPE DUE TO A NOVEL DYNAMIN 2 MUTATION

Autosomal dominant centronuclear myopathy (CNM) is a rare congenital myopathy mostly characterized by delayed motor milestones, slowly progressive muscle weakness, and bilateral ptosis.1 Mutations in the DNM2 gene encoding dynamin 2 (DNM2), a large GTPase involved in membrane trafficking, have been identified in CNM.2,3 Mutations in the middle domain of the protein are mostly associated with the slowly progressive mild late-onset CNM,2 while mutations in the C-terminal part of the Pleckstrin homology (PH) domain cause a more severe neonatal phenotype.3 In addition, mutations in the N-terminal part of PH domain have been reported in intermediate and axonal Charcot-Marie-Tooth disease (CMT).4–6 Here, we report a novel DNM2 CNM mutation in the CMT region. Among the DNM2-related CNM, the phenotype appears intermediate, with an onset at the end of the first decade and a more rapid progression relative to the mild late-onset DNM2-CNM. ### Case report. The patient is a 34-year-old woman from a nonconsanguineous family from central Africa without history of neuromuscular disorders. She had a normal mental and motor postnatal development with independent ambulation acquired at 13 months. Symptoms started at the age of 7 years with difficulty walking and running. At 10 years, she had facial weakness, bilateral ptosis, and a marked weakness in paraspinal, upper, and lower limb muscles. Motor nerve conduction velocities of the left common peroneal (49.4 m/second) …

Dynamin-2 Function and Dysfunction Along the Secretory Pathway

Dynamin-2 is a ubiquitously expressed mechano-GTPase involved in different stages of the secretory pathway. Its most well-known function relates to the scission of nascent vesicles from the plasma membrane during endocytosis; however, it also participates in the formation of new vesicles from the Golgi network, vesicle trafficking, fusion processes and in the regulation of microtubule, and actin cytoskeleton dynamics. Over the last 8 years, more than 20 mutations in the dynamin-2 gene have been associated to two hereditary neuromuscular disorders: Charcot–Marie–Tooth neuropathy and centronuclear myopathy. Most of these mutations are grouped in the pleckstrin homology domain; however, there are no common mutations associated with both disorders, suggesting that they differently impact on dynamin-2 function in diverse tissues. In this review, we discuss the impact of these disease-related mutations on dynamin-2 function during vesicle trafficking and endocytotic processes.

Dynamin-2 in nervous system disorders.

Dynamin‐2 is a pleiotropic GTPase whose best‐known function is related to membrane scission during vesicle budding from the plasma or Golgi membranes. In the nervous system, dynamin‐2 participates in synaptic vesicle recycling, post‐synaptic receptor internalization, neurosecretion, and neuronal process extension. Some of these functions are shared with the other two dynamin isoforms. However, the involvement of dynamin‐2 in neurological illnesses points to a critical function of this isoform in the nervous system. In this regard, mutations in the dynamin‐2 gene results in two congenital neuromuscular disorders. One of them, Charcot‐Marie‐Tooth disease, affects myelination and peripheral nerve conduction, whereas the other, Centronuclear Myopathy, is characterized by a progressive and generalized atrophy of skeletal muscles, yet it is also associated with abnormalities in the nervous system. Furthermore, single nucleotide polymorphisms located in the dynamin‐2 gene have been associated with sporadic Alzheimers disease. In the present review, we discuss the pathogenic mechanisms implicated in these neurological disorders.

Abnormal distribution of inositol 1,4,5-trisphosphate receptors in human muscle can be related to altered calcium signals and gene expression in Duchenne dystrophy-derived cells

Inositol 1,4,5‐trisphosphate (IP3) receptors (IP3Rs) drive calcium signals involved in skeletal muscle excitation‐transcription coupling and plasticity; IP3R subtype distribution and downstream events evoked by their activation have not been studied in human muscle nor has their possible alteration in Duchenne muscular dystrophy (DMD). We studied the expression and localization of IP3R subtypes in normal and DMD human muscle and in normal (RCMH) and dystrophic (RCDMD) human muscle cell lines. In normal muscle, both type 1 IP3Rs (IP3R1) and type 2 IP3Rs (IP3R2) show a higher expression in type II fibers, whereas type 3 IP3Rs (IP3R3) show uniform distribution. In DMD biopsies, all fibers display a homogeneous IP3R2 label, whereas 24 ± 7% of type II fibers have lost the IP3R1 label. RCDMD cells show 5‐fold overexpression of IP3R2 and down‐regulation of IP3R3 compared with RCMH cells. A tetanic stimulus induces IP3‐dependent slow Ca2+ transients significantly larger and faster in RCDMD cells than in RCMH cells as well as significant ERK1/2 phosphorylation in normal but not in dystrophic cells. Excitation‐driven gene expression was different among cell lines; 44 common genes were repressed in RCMH cells and expressed in RCDMD cells or vice versa. IP3‐dependent Ca2+ release may play a significant role in DMD pathophysiology.—Cárdenas, C., Juretić, N., Bevilacqua, J. A., García, I. E., Figueroa, R., Hartley, R., Taratuto, A. L., Gejman, R., Riveros, N., Molgó, J., Jaimovich, E. Abnormal distribution of inositol 1,4,5‐trisphosphate receptors in human muscle can be related to altered calcium signals and gene expression in Duchenne dystrophy‐derived cells. FASEB J. 24, 3210–3221 (2010). www.fasebj.org