Robert L. Wollmann

Gene targeting of BPAG1: Abnormalities in mechanical strength and cell migration in stratified epithelia and neurologic degeneration

BPAG1 is the major antigenic determinant of autoimmune sera of bullous pemphigoid (BP) patients. It is made by stratified squamous epithelia, where it localizes to the inner surface of specialized integrin-mediated adherens junctions (hemidesmosomes). To explore the function of BPAG1 and its relation to BP, we targeted the removal of the BPAG1 gene in mice. Hemidesmosomes are otherwise normal, but they lack the inner plate and have no cytoskeleton attached. Though not affecting cell growth or substratum adhesion, this compromises mechanical integrity and influences migration. Unexpectedly, the mice also develop severe dystonia and sensory nerve degeneration typical of dystonia musculorum (dt/dt) mice. We show that in at least one other strain of dt/dt mice, BPAG1 gene is defective.

Distribution of S-100 protein outside the central nervous system

The distribution of S-100 outside the central nervous system in humans and rats was explored using antiserum to S-100 and the peroxidase anti-peroxidase method of Sternberger. In peripheral nerves the Schwann cells and the outermost part of the myelin sheaths were stained; axons were not. In dorsal root ganglia and ganglia of the autonomic nervous system only satellite cells were stained. In the adrenal medulla a considerable number of cells were stained. In all other organs studied Schwann cells and satellite cells of ganglia were the only elements that were stained. We conclude that S-100 could serve as a marker for Schwann cells in situ.

S-100 protein in granular cell tumors (granular cell myoblastomas).

Outside the central nervous system S‐100 is found only in Schwann cells and satellite cells of ganglia. It has also been demonstrated in Schwannomas and neurofibromas but is absent from soft tissue tumors of non‐neural origin. S‐100 protein was looked for in granular cell myoblastomas using an immuno‐histochemical technique in an attempt to further elucidate the histogenesis of these tumors. All tumor cells in the ten tumors studied were intensely stained with antiserum to S‐100 including one with some malignant features. These results support the idea that granular cell myoblastomas arise from Schwann cells.

Patients with myasthenia gravis and thymoma have in their sera IgG autoantibodies against titin

Patients with myasthenia gravis (MG) and thymoma have in their sera antibodies which react with non‐receptor antigens from striated muscle. The purpose of this investigation was to characterize the antigen(s). Polypeptides in homogenates from rat skeletal muscle were separated by SDS‐PAGE and trans‐blotted to nitrocellulose. Sera from six patients with MG and thymoma stained a large (molecular weight >500 kD) polypeptide. while no staining was observed with sera from 20 non‐thymoma MG patients. Titin is one of the large (> 500 kD) polypeptides of striated muscle and the antibody containing MG sera have antibodies that bind to titin in a preparation of myofibrillary proteins from rabbit skeletal muscle. The staining pattern is identical to that obtained with antiserum to titin, showing that the antigen has the same electrophoretic mobility as titin. Antibodies from the sera of the patients with MG and thymoma, affinity‐purified on the large polypeptide, reacted with skeletal muscle sections in a cross‐striational pattern, near the A/I band junction but within the I band, corresponding to the localization of one of the epitopes of titin. Our findings therefore indicate that the muscle antibodies found in the sera from some MG patients with thymoma are directed against titin.

Late Denervation in Patients with Antecedent Paralytic Poliomyelitis

The development of new weakness, fatigue, and pain decades after acute paralytic poliomyelitis is a recognized syndrome. We conducted a controlled study of this syndrome by analyzing clinical, electromyographic, and muscle-biopsy features in 18 patients with a history of poliomyelitis--13 reporting 1 to 20 years of new weakness and 5 without new symptoms. The patients with new weakness also reported new muscle atrophy (9 of 13) and fatigue (10 of 13), symptoms not reported by the controls. The age at the time of acute poliomyelitis, severity of poliomyelitis, residual disability, number of years since acute poliomyelitis, and age at the time of study were comparable in the weakening and control groups. Evidence of remote denervation consistent with antecedent poliomyelitis was demonstrated in all patients by electromyography or muscle biopsy or both. In addition, active denervation (as evidenced by spontaneous activity on conventional electromyography, increased jitter on single-fiber electromyography, or atrophic myofibers) was found in 12 patients in the weakening group and in all 5 controls. Immunohistochemical detection of myofibers expressing the neural-cell adhesion molecule corroborated ongoing denervation in both patient groups. When muscle data from both groups were pooled, correlations were observed between the extent of past reinnervation and the degree of ongoing motor-unit instability. We conclude that the extensive reinnervation of denervated muscle that occurs in paralytic poliomyelitis may be followed by late denervation of the previously reinnervated muscle fibers. Electromyographic and muscle-biopsy evidence of ongoing denervation does not distinguish between stable patients with prior paralytic poliomyelitis and those with new weakness.

Proprioceptive Sensory Neuropathy in Mice with a Mutation in the Cytoplasmic Dynein Heavy Chain 1 Gene

Mice heterozygous for the radiation-induced Sprawling (Swl) mutation display an early-onset sensory neuropathy with muscle spindle deficiency. The lack of an H reflex despite normal motor nerve function in the hindlimbs of these mutants strongly suggests defective proprioception. Immunohistochemical analyses reveal that proprioceptive sensory neurons are severely compromised in the lumbar dorsal root ganglia of newborn Swl/+ mice, whereas motor neuron numbers remain unaltered even in aged animals. We have used positional cloning to identify a nine base-pair deletion in the cytoplasmic dynein heavy chain 1 gene (Dync1h1) in this mutant. Furthermore, we demonstrate that Loa/+ mice, which have previously been shown to carry a missense point mutation in Dync1h1 that results in late-onset motor neuron loss, also present with a severe, early-onset proprioceptive sensory neuropathy. Interestingly, in contrast to the Loa mutation, the Swl mutation does not delay disease progression in a motor neuron disease mouse model overexpressing a human mutant superoxide dismutase (SOD1G93A) transgene. Together, we provide in vivo evidence that distinct mutations in cytoplasmic dynein can either result in a pure sensory neuropathy or in a sensory neuropathy with motor neuron involvement.

Neurological and behavioral abnormalities, ventricular dilatation, altered cellular functions, inflammation, and neuronal injury in brains of mice due to common, persistent, parasitic infection

BackgroundWorldwide, approximately two billion people are chronically infected with Toxoplasma gondii with largely unknown consequences.MethodsTo better understand long-term effects and pathogenesis of this common, persistent brain infection, mice were infected at a time in human years equivalent to early to mid adulthood and studied 5–12 months later. Appearance, behavior, neurologic function and brain MRIs were studied. Additional analyses of pathogenesis included: correlation of brain weight and neurologic findings; histopathology focusing on brain regions; full genome microarrays; immunohistochemistry characterizing inflammatory cells; determination of presence of tachyzoites and bradyzoites; electron microscopy; and study of markers of inflammation in serum. Histopathology in genetically resistant mice and cytokine and NRAMP knockout mice, effects of inoculation of isolated parasites, and treatment with sulfadiazine or αPD1 ligand were studied.ResultsTwelve months after infection, a time equivalent to middle to early elderly ages, mice had behavioral and neurological deficits, and brain MRIs showed mild to moderate ventricular dilatation. Lower brain weight correlated with greater magnitude of neurologic abnormalities and inflammation. Full genome microarrays of brains reflected inflammation causing neuronal damage (Gfap), effects on host cell protein processing (ubiquitin ligase), synapse remodeling (Complement 1q), and also increased expression of PD-1L (a ligand that allows persistent LCMV brain infection) and CD 36 (a fatty acid translocase and oxidized LDL receptor that mediates innate immune response to beta amyloid which is associated with pro-inflammation in Alzheimers disease). Immunostaining detected no inflammation around intra-neuronal cysts, practically no free tachyzoites, and only rare bradyzoites. Nonetheless, there were perivascular, leptomeningeal inflammatory cells, particularly contiguous to the aqueduct of Sylvius and hippocampus, CD4+ and CD8+ T cells, and activated microglia in perivascular areas and brain parenchyma. Genetically resistant, chronically infected mice had substantially less inflammation.ConclusionIn outbred mice, chronic, adult acquired T. gondii infection causes neurologic and behavioral abnormalities secondary to inflammation and loss of brain parenchyma. Perivascular inflammation is prominent particularly contiguous to the aqueduct of Sylvius and hippocampus. Even resistant mice have perivascular inflammation. This mouse model of chronic T. gondii infection raises questions of whether persistence of this parasite in brain can cause inflammation or neurodegeneration in genetically susceptible hosts.

Mutations in LAMB2 causing a severe form of synaptic congenital myasthenic syndrome

Background: We describe a severe form of congenital myasthenic syndrome (CMS) associated with congenital nephrosis and ocular malformations caused by two truncating mutations in the gene encoding the laminin β2 subunit (LAMB2). Methods and results: Mutational analysis in the affected patient, who has a history of a serious untoward reaction to treatment with acetylcholinesterase inhibition, revealed two frame-shifting heteroallelic mutations, a maternally inherited 1478delG and a paternally inherited 4804delC. An anconeus muscle biopsy demonstrated a profound distortion of the architecture and function of the neuromuscular junction, which was strikingly similar to that seen in mice lacking laminin β2 subunit. The findings included: pronounced reduction of the axon terminal size with encasement of the nerve endings by Schwann cells, severe widening of the primary synaptic cleft and invasion of the synaptic space by the processes of Schwann cells, and moderate simplification of postsynaptic folds and intact expression of the endplate acetylcholinesterase. The endplate potential quantal content was notably reduced, while the frequencies and amplitudes of miniature endplate potentials were only moderately diminished and the decay phases of miniature endplate potentials were normal. Western blot analysis of muscle and kidney tissue and immunohistochemistry of kidney tissue showed no laminin β2 expression. Conclusion: This case, which represents a new type of synaptic CMS, exemplifies the wide variability of phenotypes associated with LAMB2 mutations and underscores the fundamental role that laminin β2 plays in the development of the human neuromuscular junction.

Foamy cells with oligodendroglial phenotype in childhood ataxia with diffuse central nervous system hypomyelination syndrome

Abstract Childhood ataxia with diffuse central nervous system hypomyelination syndrome (CACH) is a recently described leukodystrophy of unknown etiology. To characterize the neuropathological features and gain insight as to the pathogenesis of this disorder, we studied cerebral tissue from six patients with the CACH syndrome. Evaluation of toluidine blue-stained, semithin sections of white matter from CACH patients disclosed unusual cells with “foamy” cytoplasm, small round nuclei and fine chromatin. Electron microscopy (EM) revealed cells in the white matter with abundant cytoplasm containing many mitochondria and loosely clustered, membranous structures, but lacking the lysosomal structures seen in macrophages. Further analysis of tissue sections with antibodies and special stains demonstrated that the abnormal cells with abundant cytoplasm labeled with oligodendroglial markers, but did not react with macrophage or astrocytic markers. Double immunolabeling with macrophage and oligodendroglial markers clearly distinguished macrophages from the “foamy” oligodendroglial cells (FODCs). Proteolipid protein (PLP) mRNA in situ hybridization demonstrated PLP mRNA transcripts in a high proportion of oligodendrocytes in CACH patients compared to control patients, and PLP mRNA transcript signal in cells, morphologically consistent with FODCs. Normal and pathological brain control tissues did not contain FODCs. These neuropathological findings will be useful pathological identifiers of CACH, and may provide clues to the pathogenesis of this disorder.

Neuropathy accompanying IgM? monoclonal gammopathy

SummaryA set of observations made on a patient with IgMλ monoclonal gammopathy and neuropathy implicate humoral immunity in the pathogenesis of the neuropathy.A sural nerve biopsy from the patient showed a characteristic increase in the width of the intraperiod lines. Deposits of μ-heavy chains and λ-light chains were found in myelin sheaths of the nerve biopsy. Immunohistochemically, it was demonstrated that μ-heavy chains and λ-light chains from the patients serum bound to myelin sheaths of normal peripheral nerves and to a lesser extent to myelin sheaths in the central nervous system (CNS). By immunoblots it was demonstrated that μ-heavy chains and λ-light chains from the patients serum bound to myelin associated glycoprotein but to no other antigens from the peripheral and central nervous systems. γ and α heavy chains and ϰ light chains from the patients serum were also shown to bind to myelin-associated glycoprotein but not as distinctly as the μ and λ chains. It is postulated that the monoclonal gammopathy may have arisen on the background of polyclonal autoimmune attack directed against myelin-assoiated glycoprotein.