Additivity and potentiation of IGF-I and GDNF in the complete rescue of postnatal motor neurons.

BACKGROUND: Both growth and survival of motor neurons may depend on multiple neurotrophic factors. Individually, insulin-like growth factor I (IGF-I) and glial cell line-derived neurotrophic factor (GDNF) are potent neurotrophic/survival factors for postnatal motor neurons. METHODS: We used an organotypic spinal cord model of glutamatergic degeneration in ALS to investigate whether IGF-I and GDNF interact to enhance motor neuron survival, their trophic effect on choline acetyltransferase (ChAT) activity, and their effect on neurite outgrowth. RESULTS: We show that the combination of IGF-I and GDNF at active doses (1) is additively neuroprotective, (2) completely rescues rat motor neurons from chronic glutamate-mediated toxicity, and (3) additively upregulates motor neuron ChAT activity. Further, IGF-I, which by itself does not promote neurite outgrowth in this model, potentiates the neurite promoting action of GDNF. CONCLUSION: The results predict that IGF-I combined with GDNF may provide a better therapy for the treatment of motor neuron disorders such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy.

A deceptive case of amyloid myopathy: clinical and magnetic resonance imaging features.

Amyloid myopathy is a well-described, increasingly recognized clinical entity. Similar to inflammatory myopathies, amyloid myopathy presents with proximal muscle weakness and can be associated with elevated levels of muscle enzymes. We report the case of a 58-year-old woman who, at presentation to her physician with proximal muscle weakness and congestive heart failure, was antinuclear antibody positive and had muscle biopsy findings "consistent with inflammatory myopathy." She was referred to Johns Hopkins University Medical Center with the diagnosis of polymyositis. Further investigation revealed a monoclonal gammopathy, a unique patterning of subcutaneous fat reticulation and hypodense bone marrow changes on magnetic resonance imaging (MRI), and an endocardial biopsy sample that was positive for light chain amyloid deposition. Paraffin sections of the muscle biopsy sample from the time of her original presentation were obtained, and Congo red staining showed diffuse amyloid deposition throughout the sample, but no inflammation. This case not only illustrates that proximal muscle weakness due to primary amyloid myopathy (as found in light chain amyloidosis and transthyretin amyloidosis) can mimic that of polymyositis, but also shows that unique findings on MRI can alert the clinician to the diagnosis of amyloidosis prior to muscle biopsy.

Delayed application of IGF-I and GDNF can rescue already injured postnatal motor neurons.

IGF-I, GDNF, and other neurotrophic factors, when applied at the time of injury, can protect postnatal motor neurons from slow glutamate injury in organotypic spinal cord. However, in human spinal cord diseases, motor neuron injury is already established when treatment could begin. We tested whether neurotrophic factors can protect already-injured motor neurons, and whether combinations of factors can further lengthen the therapeutic time window. Our data show that during a 7--8 week process of slow neurodegeneration either IGF-I or GDNF treatment, though delayed up to 4 weeks, still allowed substantial rescue of already injured motor neurons. However, the combination of both factors additively provided better neuroprotection than either factor alone, even after a 4-week delay. This proof of principle is relevant to the potential of IGF-I and GDNF as therapy for acquired disorders affecting motor neurons.

Preclinical testing of neuroprotective neurotrophic factors in a model of chronic motor neuron degeneration.

Many neurotrophic factors have been shown to enhance survival of embryonic motor neurons or affect their response to injury. Few studies have investigated the potential effects of neurotrophic factors on more mature motor neurons that might be relevant for neurodegenerative diseases. Using organotypic spinal cord cultures from postnatal rats, we have demonstrated that insulin-like growth factor-I (IGF-I) and glial-derived neurotrophic factor (GDNF) significantly increase choline acetyltransferase (ChAT) activity, but brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4/5), and neurotrophin-3 (NT-3) do not. Surprisingly, ciliary neurotrophic factor (CNTF) actually reduces ChAT activity compared to age-matched control cultures. Neurotrophic factors have also been shown to alter the sensitivity of some neurons to glutamate neurotoxicity, a postulated mechanism of injury in the neurodegenerative disease, amyotrophic lateral sclerosis (ALS). Incubation of organotypic spinal cord cultures in the presence of the glutamate transport inhibitor threo-hydroxyaspartate (THA) reproducibly causes death of motor neurons which is glutamate-mediated. In this model of motor neuron degeneration, IGF-I, GDNF, and NT-4/5 are potently neuroprotective, but BDNF, CNTF, and NT-3 are not. The organotypic glutamate toxicity model appears to be the best preclinical predictor to date of success in human clinical trials in ALS.

Pigment epithelium-derived factor (PEDF) protects motor neurons from chronic glutamate-mediated neurodegeneration.

Although pigment epithelium-derived factor (PEDF) is a neurotrophic factor that may aid the development, differentiation, and survival of adjacent neural retinae, the wider distribution of PEDF mRNA in the central nervous system suggested to us that this factor could have pleiotropic neurotrophic and neuroprotective effects on nonretinal neurons. We examined the distribution of PEDF mRNA and its transcript in the spinal cord. By immunohistochemistry and western blot analysis using an antihuman PEDF antiserum of known specificity, we found that PEDF protein is present in spinal cord, cerebrospinal fluid, and skeletal muscle and that its mRNA appears concentrated in motor neurons of the human spinal cord. These observations indicate that PEDF could have potential autocrine and paracrine effects on motor neurons, as well as being target-derived. We analyzed the pharmacologic utility of PEDF in a postnatal organotypic culture model of motor neuron degeneration and proved it is highly neuroprotective. The effect was biologically important, significantly sparing the spinal cord's gross organotypic morphological appearance and preserving motor neuron choline acetyltransferase (ChAT). PEDF alone did not increase ChAT, indicating that the observed effect is neuroprotective, not merely an upregulation of motor neuron ChAT. Further, PEDF preserved motor neuron number, proving a survival effect. We hypothesize that PEDF may play important roles in the survival and maintenance of spinal motor neurons in their neuroprotection against acquired insults in postnatal life. It should be developed further as a therapeutic strategy for motor neuron diseases such as amyotrophic lateral sclerosis (ALS).

Occult presentation of myotonia congenita in a 15-year-old athlete.

A case of myotonia congenita in an adolescent athlete was presented. Although this is a rare condition unknown to many treating physicians, the key to diagnosis was provocation of the patient's symptoms of muscle "tightening" and "cramping" during sustained exercise. The diagnosis would have been missed in routine office examinations with the patient at rest. The stereotypic generalized myotonic signs and symptoms were provoked after the patient was asked to play 20 minutes of basketball during one of his office evaluations. The provocative or postexercise examination was critical to the diagnosis as the resting office examination was completely normal. The diagnosis was subsequently confirmed by EMG and genetic testing. Myotonia congenita should be considered in the differential diagnosis of athletes with exercise-induced muscle "stiffness" or "cramping," particularly if the course is protracted and initial examinations are unremarkable. A provocative exercise period can be used to make the diagnosis. Once the diagnosis is established, appropriate pharmacologic treatment may improve symptoms and allow return to daily activity without restriction.

Neuroprotective utility and neurotrophic action of neurturin in postnatal motor neurons: comparison with GDNF and persephin.

Neurturin and persephin are recently discovered homologs of glial cell line-derived neurotrophic factor (GDNF). Here, we report that neurturin, like GDNF, increases the choline acetyltransferase activity of normal postnatal motor neurons, induces neurite outgrowth in spinal cord, and potently protects motor neurons from chronic glutamate-mediated degeneration. Persephin, in contrast, does not appear to have neurotrophic or neurite-promoting effects on mature motor neurons and may instead worsen the glutamate injury of motor neurons. This pattern in the TGF-beta family suggests certain receptor specificities, requiring at least the Ret/GFRalpha-1 receptor complex. The results predict potential benefit of neurturin, but not persephin, in the treatment of motor neuron disorders and spinal cord diseases.

Age-related biology and diseases of muscle and nerve.

Age-related biological changes in neurons and skeletal muscle commonly affect neuromuscular function and strongly influence the expression of neuromuscular disease. Of primary importance is the attrition of entire motor units, with resultant neurogenic atrophy of skeletal muscle. Other age-related processes are sensory neuron loss, distal axonal degeneration, axonal atrophy, accumulation of multiple mitochondrial DNA mutations in muscle, and physical inactivity and deconditioning. The decline for most of these begins in early life and proceeds steadily; the curious lack of an abrupt falloff with age is not yet accounted for by any theory of pathogenesis.

Sensory ataxic neuropathy as the presenting feature of a novel mitochondrial disease.

Four unrelated patients presented with a severe sensory ataxic neuropathy in association with dysarthria and chronic progressive external ophthalmoplegia. Electrophysiologic and pathologic studies showed severe axonal loss disproportionately affecting sensory nerves. Molecular genetic analysis revealed multiple mitochondrial DNA deletions in muscle and peripheral nerve. Sensory ataxic neuropathy may be the predominant and presenting manifestation of a mitochondrial disorder, and a mitochondrial etiology should be included in its differential diagnosis. The triad of sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO) may represent a novel mitochondrial disease associated with multiple mitochondrial DNA deletions.

A beta-subunit mutation in the acetylcholine receptor channel gate causes severe slow-channel syndrome.

Point mutations in the genes encoding the acetylcholine receptor (AChR) subunits have been recognized in some patients with slow-channel congenital myasthenic syndromes (CMS). Clinical, electrophysiological, and pathological differences between these patients may be due to the distinct effects of individual mutations. We report that a spontaneous mutation of the beta subunit that interrupts the leucine ring of the AChR channel gate causes an eightfold increase in channel open time and a severe CMS characterized by severe endplate myopathy and extensive remodeling of the postsynaptic membrane. The pronounced abnormalities in neuromuscular synaptic architecture and function, muscle fiber damage and weakness, resulting from a single point mutation are a dramatic example of a mutation having a dominant gain of function and of hereditary excitotoxicity.

Myology of the pharyngoesophageal segment: gross anatomic and histologic characteristics.

Although numerous studies have been performed on the function and dysfunction of the pharyngoesophageal segment, few studies have investigated features of the musculature in this area. Thus, the purpose of this study was to systematically exam. ine the structure (gross anatomy and histology) in this area and to relate these findings to the functions of the pharyngoesophageal segment. Twenty-one autopsy and surgery patients underwent careful measurement and observation of 1. the vertical (cephalad-caudad) height of the cricopharyngeus muscle (CP); 2. the presence or absence of Kilfian's dehiscence; and 3. the separation or blending of the CP with the upper esophageal circular muscles. Of the 21 subjects, muscle specimens were removed from 8 (4 autopsy, 4 surgical) to include a muscle strip from the upper esophageal circular muscles, CP, and inferior pharyngeal constrictor and submitted to a battery of histological and histochemical tests. Gross anatomic measurements of the vertical height of the CP were substantially longer than those reported elsewhere. Killian's dehiscence was shown to be present in fewer than one third of the specimens. Histology of these muscles also showed significant differences from the muscles discussed in other published reports, particularly when fresh and autopsy material were compared. These specialized muscles, therefore, require further detailed study.

Differential expression of PTP1D, a protein tyrosine phosphatase with two SH2 domains, in a slow and fast skeletal muscle fibers.

We show that PTP1D, a protein tyrosine phosphatase that contains two SH2 domains, is preferentially expressed in slow skeletal muscle fibers. Immunohistochemical staining using polyclonal antibodies against PTP1D demonstrated that PTP1D was expressed in a subpopulation of rodent muscle fibers. These fibers were identified as slow Type I fibers based on histochemical ATPase assays and slow myosin heavy chain expression. Northern and Western analyses showed that PTP1D levels were higher in predominantly slow muscles than in predominantly fast muscles. This differential expression of PTP1D in slow muscle fibers appeared by birth. In cultures of mouse myogenic cells, PTP1D was expressed after MyoD and myogenin and appeared in myotubes derived from embryonic, fetal, and postnatal myoblasts. Remarkably, PTP1D was organized into sarcomeres in a pattern coincident with myosin heavy chain, suggesting that PTP1D associates with a component of the thick filament. These results show that PTP1D is preferentially expressed in slow muscle fibers. We speculate that PTP1D may play a role in slow muscle fiber function and differentiation.

Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate.

Three glutamate transporters have been identified in rat, including astroglial transporters GLAST and GLT-1 and a neuronal transporter EAAC1. Here we demonstrate that inhibition of the synthesis of each glutamate transporter subtype using chronic antisense oligonucleotide administration, in vitro and in vivo, selectively and specifically reduced the protein expression and function of glutamate transporters. The loss of glial glutamate transporters GLAST or GLT-1 produced elevated extracellular glutamate levels, neurodegeneration characteristic of excitotoxicity, and a progressive paralysis. The loss of the neuronal glutamate transporter EAAC1 did not elevate extracellular glutamate in the striatum but did produce mild neurotoxicity and resulted in epilepsy. These studies suggest that glial glutamate transporters provide the majority of functional glutamate transport and are essential for maintaining low extracellular glutamate and for preventing chronic glutamate neurotoxicity.

Sensory nerve pathology in multifocal motor neuropathy.

The nosological status of multifocal motor neuropathy remains controversial. The clinical and electrodiagnostic hallmarks suggest selective motor fiber involvement. In this study, we asked to what extent sensory nerves might be involved pathologically in multifocal motor neuropathy. Examination of sensory nerve biopsy specimens from 11 patients did reveal pathological findings in all, but they were very mild. An increased number of thinly myelinated, large-caliber fibers was the unifying feature common to each specimen. By electron microscopy, each biopsy specimen had thinly myelinated fibers surrounded by minor onion bulbs. Active demyelination, though scant, was seen in 3 nerves. Myelinated fiber density was normal. Subperineurial edema and inflammation were not present. We conclude that multifocal motor neuropathy is not an exclusively motor abnormality, although it appears to be so clinically and electrophysiologically. The frequent, albeit mild, pathological abnormalities in sensory fibers suggest that the demyelinating pathophysiology also affects sensory fibers, but to a lesser degree than motor fibers. Some investigators maintain that multifocal motor neuropathy is within the spectrum of chronic inflammatory demyelinating polyneuropathy. The very mild degree of sensory fiber involvement, the absence of inflammation or edema, and the distinctive clinical features support the concept of multifocal motor neuropathy as distinct from chronic inflammatory demyelinating polyneuropathy.

Neuroprotective strategies in a model of chronic glutamate-mediated motor neuron toxicity.

A dramatic loss of glutamate transport has been observed in sporadic amyotrophic lateral sclerosis and has been postulated to contribute to the disease. Experimentally, this hypothesis was corroborated by mimicking the chronic loss of glutamate transport in postnatal rat spinal cord organotypic cultures through the use of glutamate transport inhibitors. This system is characterized by a relatively selective slow loss of ventral horn motor neurons resulting from glutamate transport inhibition. In this study, spinal cord organotypic cultures were used to test various drugs to evaluate their neuroprotective properties against this slow glutamate-mediated neurotoxicity The most potent neuroprotectants were drugs that altered glutamate neurotransmission, including non-NMDA receptor antagonists (GYKI-52466, PD144216, and PD13997) and drugs that could block presynaptic release or synthesis (riluzole and gabapentin). In addition, some antioxidants (U83836E and N-t-butyl-alpha-phenylnitrone) and inhibitors of nitric oxide synthesis (NG-monomethyl-L-arginine acetate) were modestly neuroprotective. The calcium endonuclease inhibitor aurintricarboxylic acid and the calcium release inhibitor dantrolene also provided partial motor neuron protection. However, several antioxidants and calcium channel antagonists had no excitotoxic neuroprotectant activity. This system provides a preclinical screening method for the burgeoning number of drugs postulated for clinical trials in motor neuron disease and a model to evaluate the mechanisms of chronic glutamate toxicity.

Selective loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis.

The pathogenesis of sporadic amyotrophic lateral sclerosis (ALS) is unknown, but defects in synaptosomal high-affinity glutamate transport have been observed. In experimental models, chronic loss of glutamate transport can produce a loss of motor neurons and, therefore, could contribute to the disease. With the recent cloning of three glutamate transporters, i.e., EAAC1, GLT-1, and GLAST, it has become possible to determine if the loss of glutamate transport in ALS is subtype specific. We developed C-terminal, antioligopeptide antibodies that were specific for each glutamate transporter. EAAC1 is selective for neurons, while GLT-1 and GLAST are selective for astroglia. Tissue from various brain regions of ALS patients and controls were examined by immunoblot or immunocytochemical methods for each transporter subtype. All tissue was matched for age and postmortem delay. GLT-1 immunoreactive protein was severely decreased in ALS, both in motor cortex (71% decrease compared with control) and in spinal cord. In approximately a quarter of the ALS motor cortex specimens, the loss of GLT-1 protein (90% decrease from control) was dramatic. By contrast, there was only a modest loss (20% decrease from control) of immunoreactive protein EAAC1 in ALS motor cortex, and there was no appreciable change in GLAST. The minor loss of EAAC1 could be secondary to loss of cortical motor neurons. As a comparison, glial fibrillary acidic protein, which is selectively localized to astroglia, was not changed in ALS motor cortex. Because there is no loss of astroglia in ALS, the dramatic abnormalities in GLT-1 could reflect a primary defect in GLT-1 protein, a secondary loss due to down regulation, or other toxic processes.

Autosomal dominant distal spinal muscular atrophy in four generations.

Distal spinal muscular atrophy is a rare lower motor neuron disorder that may be difficult to distinguish clinically from type II Charcot-Marie-Tooth disease. We report on clinical and pathologic findings in 13 members of a four-generation extended family with autosomal dominant distal spinal muscular atrophy. The patients developed a slowly progressive lower motor neuron disorder involving mainly the distal lower extremities; onset was from the second to fourth decades. Electromyography and muscle biopsy findings were indicative of motor denervation. Combined silver/cholinesterase/immunocytochemical staining of intramuscular nerve revealed abundant collateral axonal branching in mild disease but marked loss of terminal motor endplate innervation in the more severe state, suggesting decreased growth of motor axon collaterals with disease progression. Multipoint DNA linkage analysis showed that this family's disorder is not linked to the chromosome 5q11.2-13.3 spinal muscular atrophy locus.

Altered neurofilament phosphorylation and beta tubulin isotypes in Charcot-Marie-Tooth disease type 1.

Charcot-Marie-Tooth disease type 1 (CMT1) is associated with atrophy and degeneration of peripheral nerve axons in addition to prominent changes in the structure of Schwann cells. We have investigated the composition of the axonal cytoskeleton in sural nerve biopsies from patients with CMT1. Compared to controls, CMT1 nerves exhibited marked hypophosphorylation of neurofilament proteins and an increased relative abundance of beta tubulin isotypes 2 and 3. Biopsies from patients with other causes of neuropathy, matched to the CMT1 group for severity of axonal atrophy, exhibited an intermediate degree of neurofilament hypophosphorylation and no abnormality of tubulin isotypes. The axonal cytoskeleton in CMT1 resembles that of immature nerve fibers. A failure of normal Schwann cell-axon interaction in CMT1 may prevent full differentiation of the axonal cytoskeleton of myelinated nerve fibers.

Localization of neuronal and glial glutamate transporters.

The cellular and subcellular distributions of the glutamate transporter subtypes EAAC1, GLT-1, and GLAST in the rat CNS were demonstrated using anti-peptide antibodies that recognize the C-terminal domains of each transporter. On immunoblots, the antibodies specifically recognize proteins of 65-73 kDa in total brain homogenates. Immunocytochemistry shows that glutamate transporter subtypes are distributed differentially within neurons and astroglia. EAAC1 is specific for certain neurons, such as large pyramidal cortical neurons and Purkinje cells, but does not appear to be selective for glutamatergic neurons. GLT-1 is localized only to astroglia. GLAST is found in both neurons and astroglia. The regional localizations are unique to each transporter subtype. EAAC1 is highly enriched in the cortex, hippocampus, and caudate-putamen and is confined to pre- and postsynaptic elements. GLT-1 is distributed in astrocytes throughout the brain and spinal cord. GLAST is most abundant in Bergmann glia in the cerebellar molecular layer brain, but is also present in the cortex, hippocampus, and deep cerebellar nuclei.