The difficult-to-cultivate coxsackieviruses A can productively multiply in primary culture of mouse skeletal muscle.

Coxsackieviruses A (CVA) are associated with several clinical manifestations such as aseptic meningitis and paralytic syndromes in humans. Most CVA are difficult-to-cultivate, which impedes their propagation and isolation from clinical material. Here, we tested the ability of cultivable (CVA-13, CVA-14), and difficult-to-cultivate (CVA-6, CVA-22) strains to infect primary cultures of skeletal muscle cells established from newborn mice. We found that such cultures sustained the multiplication of these CVA, as evidenced by the development of a cytopathic effect, already in the initial preparation or after passaging once. Cultures established for no more than 24h were sensitive to infection whereas older preparations were resistant. Using confocal microscopy after double-immunolabeling of the VP1 capsid protein and the muscle cell marker myosin, we demonstrated that only the myoblasts were infected, resulting in VP1 expression throughout their cytoplasm. Inoculation of infected cultures to suckling mice resulted in paralysis indicating that infection was productive. The nature of candidate receptors for virus entry in such cultures and the influence of cell culture conditions on the expression of these putative receptors are discussed. This work suggests that primary cultures of skeletal muscle cells could be used to propagate and isolate any CVA strain.

Alteration of protein kinase C conformation in red blood cells: a potential marker for Alzheimer's disease but not for Parkinson's disease.

There is a growing evidence of early changes of blood cells in Alzheimer's disease (AD). We have developed an original novel method for quantifying the alteration of protein kinase C (PKC) by its fluorescence spectrum: by using Fim-1, a specific fluorescent probe made for protein kinase C that detects the conformational changes of this. We show that the PKC conformation is altered in red blood cells (RBC) from AD patients as compared to RBC from healthy controls. This alteration is independent of the patient's age and of the stage of the disease. It is not observed in the RBC of non-demented patients suffering from Parkinson's disease (PD). If PKC alteration is proven to be specific to AD as compared with other dementia, this method could be for a simple, low cost screening test among patients suspected of having AD and may have a strong predictive value.

Uptake of Abeta 1-40- and Abeta 1-42-coated yeast by microglial cells: a role for LRP.

Artificial diffuse and amyloid core of neuritic plaques [beta-amyloid peptide (Abeta) deposits] could be prepared using heat-killed yeast particles opsonized with Abeta 1-40 or Abeta 1-42 peptides. Interaction and fate of these artificial deposits with microglial cells could be followed using a method of staining that allows discrimination of adherent and internalized, heat-killed yeast particles. Using this system, it was possible to show that nonfibrillar or fibrillar (f)Abeta peptides, formed in solution upon heating (aggregates), could not impair the internalization of heat-killed yeast particles opsonized with fAbeta 1-40 or fAbeta 1-42. This indicated that depending on their physical state, Abeta peptide(s) do not recognize the same receptors and probably do not follow the same internalization pathway. Using competitive ligands of class A scavenger receptors (SR-A) or low-density lipoprotein-related receptor protein (LRP), it has been shown that SR-A were not involved in the recognition of amyloid peptide deposits, whereas LRP specifically recognized deposits of fAbeta 1-42 (but not fAbeta 1-40) and mediated their phagocytosis.

Senataxin, the ortholog of a yeast RNA helicase, is mutant in ataxia-ocular apraxia 2.

Ataxia-ocular apraxia 2 (AOA2) was recently identified as a new autosomal recessive ataxia. We have now identified causative mutations in 15 families, which allows us to clinically define this entity by onset between 10 and 22 years, cerebellar atrophy, axonal sensorimotor neuropathy, oculomotor apraxia and elevated alpha-fetoprotein (AFP). Ten of the fifteen mutations cause premature termination of a large DEAxQ-box helicase, the human ortholog of yeast Sen1p, involved in RNA maturation and termination.

Functional maturation of nicotinic acetylcholine receptors as an indicator of murine muscular differentiation in a new nerve-muscle co-culture system.

Under normal conditions in situ, muscle fibers and motoneurons, the main partners of motor units, are strongly dependent on each other. This interdependence hinders ex vivo studies of neuromuscular disorders where nervous or muscular components are considered separately. To allow in vitro access to complex nerve-muscle relationships, we developed a novel nerve-muscle co-culture system where mouse muscle innervation is assured by rat spinal cord explants. The degree of muscular maturation during co-culture was evaluated using the distribution of nicotinic acetylcholine receptors (AChRs) and their electrophysiological characteristics before and after innervation. In myotubes from non-innervated cultures, AChRs were diffusely distributed over the entire myotube surface. Their single-channel conductance (33.5+/-0.6 pS) and mean open time (8.1+/-0.7 ms) are characteristic of AChRs described in embryonic or denervated skeletal muscles. In innervated muscle fibers from co-cultures, AChRs appear as discrete aggregates and co-localize with synaptotagmin. In addition to the embryonic type currents, in innervated fibers AChR currents having high conductance (53.3+/-5.9 pS) and short mean open time (2.6+/-0.1 ms), characteristic of AChRs at mature neuromuscular junctions, were observed. Our data support the use of this new nerve-muscle co-culture system as a reliable model for the study of murine muscular differentiation and function.

Mitochondrial respiratory chain function in skeletal muscle of ALS patients.

Evidence implicating mitochondrial dysfunction in the central nervous system of patients with sporadic amyotrophic lateral sclerosis (SALS) has recently been accumulating. In contrast, data on mitochondrial function in skeletal muscle in SALS are scarce and controversial. We investigated the in situ properties of muscle mitochondria in patients with early-stage SALS and sedentary (SED) controls using the skinned fiber technique to determine whether respiration of muscle tissue is altered in early-stage SALS in comparison with SED. Musculus vastus lateralis biopsies were obtained from 7 SED group members and 14 patients with early-stage SALS (mean disease duration, 9 months). Muscle fibers were permeabilized with saponine and then skinned and placed in an oxygraphic chamber to measure basal (V(0)) and maximal (V(max)) adenosine diphosphate-stimulated respiration rates and to assess mitochondrial regulation by adenosine diphosphate. Muscle oxidative capacity, evaluated with V(max), was identical in patients in the SALS and SED groups (V(0): SALS, 1.1 +/- 0.1; SED, 0.8 +/- 0.1, micromol 0(2). min(-1). gm(-1)dw and V(max): SALS, 3.1 +/- 0.3; SED, 2.5 +/- 0.3, micromol 0(2). min(-1). gm(-1)dw). This study shows an absence of large mitochondrial damage in skeletal muscle of patients with early-stage SALS, suggesting that mitochondrial dysfunction in the earlier stages of SALS is almost certainly not systemic.

Construction of a plasmid containing human SMN, the SMA determining gene, coupled to EGFP.

We describe here the construction of plasmid pEGFP-C3/SMN, bearing the human SMN gene coupled to the green fluorescent protein (GFP) sequence. The mutation of the SMN gene is responsible for spinal muscular atrophy (SMA), a frequent human infantile genetic disease. We introduced the SMN cDNA into the multiple cloning site of pEGFP-C3. This plasmid bears the neomycin-resistance sequence and the enhanced green fluorescent protein (EGFP). It results in the expression of a fusion protein bearing SMN coupled to a carboxy-terminal GFP tag, used for fluorescence localization studies. Transfection of primary human myoblasts with pEGFP-C3 or pEGFP-C3/SMN revealed that EGFP is intracellularly localized within the cytosol as well as in the nucleus, while the fusion protein EGFP-SMN localized within the nucleus in prominent dot-like structures termed "gems." These data demonstrate that human primary muscle cells can be efficiently transfected and may have important implications for the development of therapeutic strategies in SMA.

Possible pathogenic role of muscle cell dysfunction in motor neuron death in spinal muscular atrophy.

We have previously shown that myofibers formed by fusion of muscle satellite cells from spinal muscular atrophy (SMA) I or II undergo degeneration 1 to 3 weeks after innervation by rat embryonic spinal cord explants, whereas normal myofibers survive for several months. In the "muscle component" of the coculture, the only cells responsible for the degeneration are the SMA muscle satellite cells. Moreover, SMA muscle satellite cells do not fuse as rapidly as do normal muscle satellite cells. To determine whether death of muscle cells precedes that of motor neurons, we studied the origin and kinetics of release of apoptotic microparticles. In SMA cocultures, motor neuron apoptosis occurred before myofiber degeneration becomes visible, indicating that SMA myofibers were unable to sustain survival of motor neurons. In normal cocultures, motor neuron apoptosis occurred 4 days after innervation. However, it did not continue beyond 2 days. These results strengthen the hypothesis that SMA is due to a defect in neurotrophic muscle cell function.

Homozygous exon 7 deletion of the SMN centromeric gene (SMN2): a potential susceptibility factor for adult-onset lower motor neuron disease.

Mutations in the telomeric copy of the SMN gene (SMN1) are responsible for almost all infantile motor neuron disease (MND). In contrast, the role of the centromeric copy of the SMN gene (SMN2) in MND remains unclear. We searched for deletions of SMN1 and SMN2 in a group of 11 patients with sporadic adult-onset lower motor neuron disease (also referred to as "progressive muscular atrophy") and found an excess of patients carrying homozygous deletions of SMN2 exon 7 (36% versus 5% in the normal population). This result suggests that SMN2 deletions could act as a susceptibility factor for sporadic lower motor neuron disease in adults.