Ruud A. Wolterman

Germline Mutation of INI1/SMARCB1 in Familial Schwannomatosis

Patients with schwannomatosis develop multiple schwannomas but no vestibular schwannomas diagnostic of neurofibromatosis type 2. We report an inactivating germline mutation in exon 1 of the tumor-suppressor gene INI1 in a father and daughter who both had schwannomatosis. Inactivation of the wild-type INI1 allele, by a second mutation in exon 5 or by clear loss, was found in two of four investigated schwannomas from these patients. All four schwannomas displayed complete loss of nuclear INI1 protein expression in part of the cells. Although the exact oncogenetic mechanism in these schwannomas remains to be elucidated, our findings suggest that INI1 is the predisposing gene in familial schwannomatosis.

The Membrane Attack Complex of the Complement System Is Essential for Rapid Wallerian Degeneration

The complement (C) system plays an important role in myelin breakdown during Wallerian degeneration (WD). The pathway and mechanism involved are, however, not clear. In a crush injury model of the sciatic nerve, we show that C6, necessary for the assembly of the membrane attack complex (MAC), is essential for rapid WD. At 3 d after injury, pronounced WD occurred in wild-type animals, whereas the axons and myelin of C6-deficient animals appeared intact. Macrophage recruitment and activation was inhibited in C6-deficient rats. However, 7 d after injury, the distal part of the C6-deficient nerves appeared degraded. As a consequence of a delayed WD, more myelin breakdown products were present than in wild-type nerves. Reconstitution of the C6-deficient animals with C6 restored the wild-type phenotype. Treatment with rhC1INH (recombinant human complement 1 inhibitor) blocked deposition of activated C-cleaved products after injury. These experiments demonstrate that the classical pathway of the complement system is activated after acute nerve trauma and that the entire complement cascade, including MAC deposition, is essential for rapid WD and efficient clearance of myelin after acute peripheral nerve trauma.

Differentiation and proliferation of respiration-deficient human myoblasts

Replication and transcription of mitochondrial DNA were impaired in dividing human myoblasts exposed to ethidium bromide. MtDNA content decreased linearly per cell division and mitochondrial transcript levels declined rapidly, resulting in respiration-deficiency of the myoblasts. Despite the absence of functional mitochondria the cells remained able to proliferate when grown under specific culture conditions. However, the formation of myotubes was severely impaired in respiration-deficient myoblasts. We conclude that differentiation of myoblasts into myotubes is more dependent on mitochondrial function than proliferation of myoblasts.

Genetic localization of Bethlem myopathy

Bethlem myopathy is a rare autosomal dominant myopathy characterized by slowly progressive limb-girdle muscular atrophy and weakness, and contractures of multiple joints. To identify the genetic localization we used highly polymorphic microsatellite markers in a genome-wide search in six Dutch families. After excluding genetic linkage with 52 markers distributed evenly over the autosomes, significant linkage was present with the 21q22.3 locus PFKL (two-point lod score of Zmax = 6.86 at theta = 0.03). There was no indication of genetic heterogeneity. The pattern of recombinations observed with adjacent markers indicated a localization distal to PFKL. Recombination of a marker within the collagen 6a1 gene (COL6A1) excluded this apparent candidate gene in one of the Bethlem myopathy families. The disease gene is most likely located in the region between COL6A1 and the telomere of chromosome 21q.

Soluble Complement Receptor 1 Protects the Peripheral Nerve from Early Axon Loss after Injury

Complement activation is a crucial early event in Wallerian degeneration. In this study we show that treatment of rats with soluble complement receptor 1 (sCR1), an inhibitor of all complement pathways, blocked both systemic and local complement activation after crush injury of the sciatic nerve. Deposition of membrane attack complex (MAC) in the nerve was inhibited, the nerve was protected from axonal and myelin breakdown at 3 days after injury, and macrophage infiltration and activation was strongly reduced. We show that both classical and alternative complement pathways are activated after acute nerve trauma. Inhibition of the classical pathway by C1 inhibitor (Cetor) diminished, but did not completely block, MAC deposition in the injured nerve, blocked myelin breakdown, inhibited macrophage infiltration, and prevented macrophage activation at 3 days after injury. However, in contrast to sCR1 treatment, early signs of axonal degradation were visible in the nerve, linking MAC deposition to axonal damage. We conclude that sCR1 protects the nerve from early axon loss after injury and propose complement inhibition as a potential therapy for the treatment of diseases in which axon loss is the main cause of disabilities.

Myelin and Axon Pathology in a Long-Term Study of PMP22-Overexpressing Mice

We analyzed clinical and pathological disease in 2 peripheral myelin protein-22 (PMP22) overexpressing mouse models for 1.5years. C22 mice have 7 and C3-PMP mice have 3 to 4 copies of the human PMP22 gene. C3-PMP mice showed no overt clinical signs at 3 weeks and developed mild neuromuscular impairment; C22 mice showed signs at 3 weeks that progressed to severe impairment. Adult C3-PMP mice had very similar, stable, low nerve conduction velocities similar to adults with human Charcot-Marie-Tooth disease type 1A (CMT1A); velocities were much lower in C22 mice. Myelination was delayed, and normal myelination was not reached in either model but the degree of dysmyelination in C3-PMP mice was considerably less than that in C22 mice; myelination was stable in the adult mice. Numbers of myelinated, fibers were reduced at 3 weeks inboth models, suggesting that normal numbers of myelinated fibers are not reached during development in the models. In adult C3-PMP and wild-type mice, there was no detectable loss of myelinated fibers,whereas there was clear loss of myelinated fibers in C22 mice.In C3-PMP mice, there is a balance between myelination status and axonal function early in life, whereas in C22 mice, early reduction of axons is more severe and there is major loss of axons in adulthood. We conclude that C3-PMP mice may be an appropriate model for most CMT1A patients, whereas C22 mice may be more relevant to severely affected patients in the CMT1 spectrum.

Complement inhibition accelerates regeneration in a model of peripheral nerve injury.

Complement (C) activation is a crucial event in peripheral nerve degeneration but its effect on the subsequent regeneration is unknown. Here we show that genetic deficiency of the sixth C component, C6, accelerates axonal regeneration and recovery in a rat model of sciatic nerve injury. Foot-flick test and Sciatic Function Index monitored up to 5 weeks post-injury showed a significant improvement of sensory and motor function in the C6 deficient animals compared to wildtypes. Retrograde tracing experiments showed a significantly higher number of regenerated neurons at 1 week post-injury in C6 deficient rats than wildtypes. Pathology showed improved nerve regeneration in tibials of C6 deficient animals compared to wildtypes. Reconstitution with purified human C6 protein re-established the wildtype phenotype whereas pharmacological inhibition of C activation with soluble C receptor 1 (sCR1) facilitated recovery and improved pathology similarly to C6 deficient animals. We suggest that a destructive C-mediated event during nerve degeneration hampers the subsequent regenerative process. These findings provide a rationale for the testing of anti-complement agents in human nerve injury.

Demonstration of a Sandhoff disease-associated autosomal 50-kb deletion by field inversion gel electrophoresis.

SummaryField inversion gel electrophoresis (FIGE) of SfiI-digested chromosomal DNA was used to demonstrate a 50-kb deletion in one allele of the gene encoding the beta subunit of human hexosaminidase (HEXB at 5q13) of two apparently unrelated patients with Sandhoff disease. In conventional electrophoretic restriction analysis, this deletion was masked by hybridization of bands from the other allele.

Myelination competent conditionally immortalized mouse Schwann cells.

Numerous mouse myelin mutants are available to analyze the biology of the peripheral nervous system related to health and disease in vivo. However, robust in vitro biochemical characterizations of players in peripheral nerve processes are still not possible due to the limited growth capacities of Schwann cells. In order to generate cell lines from peripheral nerves that are amenable to experimental manipulation, we have isolated Schwann cells from transgenic mice (H-2Kb-tsA58) carrying the temperature sensitive SV40 large T oncogene under the control of the interferon gamma (IFNgamma) H-2Kb promoter. These cells are immortalized at 33 degrees C when the SV40 large T antigen has a stable conformation. At the non-permissive temperature of 37 degrees C and in the absence of IFNgamma, the growth rate of the cultures reduces and typical Schwann cell markers such as p75(NGFR) become upregulated. The conditionally immortalized Schwann cells allow genetic manipulation as demonstrated here by the generation of a stable eGFP expressing cell line. They regain their characteristic non-immortalized properties at non-permissive temperature and differentiate to myelin-forming cells when seeded on dorsal root ganglia neurons. The Schwann cell lines derived are valuable tools for in vitro studies involving demyelinating diseases.

Expression profiling of sciatic nerve in a Charcot-Marie-Tooth disease type 1a mouse model.

Expression profiling was performed on sciatic nerve of normal mice and of transgenic mice overexpressing the peripheral myelin protein 22 kDa (PMP22). These mice represent a model for the hereditary peripheral neuropathy Charcot‐Marie Tooth type 1A. Comparison of the profiles reveals that the proteasomal degradation pathway and various signaling mechanisms are up‐regulated in the diseased nerve. The down‐regulated processes represent cell shape and adhesion as well as cellular activity and metabolism. In addition, we found that the most significantly up‐regulated differences could not be mapped on known transcripts and thus might represent not identified transcripts. Our data will be helpful to direct future research aimed at deciphering the molecular pathogenesis of the most prevalent hereditary peripheral neuropathy.