Ralph W. Kuncl

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.

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.

Decreased Glutamate Transport by the Brain and Spinal Cord in Amyotrophic Lateral Sclerosis

BACKGROUND Amyotrophic lateral sclerosis (ALS) is a chronic degenerative neurologic disorder characterized by the death of motor neurons in the cerebral cortex and spinal cord. Recent studies have suggested that the metabolism of glutamate, a potentially neurotoxic amino acid, is abnormal in patients with ALS. We hypothesized that the high-affinity glutamate transporter is the site of the defect. METHODS We measured high-affinity, sodium-dependent glutamate transport in synaptosomes from neural tissue obtained from 13 patients with ALS, 17 patients with no neurologic disease, and 27 patients with other neuro-degenerative diseases (Alzheimers disease in 15 patients and Huntingtons disease in 12 patients). The groups were comparable with respect to age and the interval between death and autopsy. Synaptosomes were prepared from spinal cord, motor cortex, sensory cortex, visual cortex, striatum, and hippocampus. We also measured sodium-dependent transport of gamma-aminobutyric acid and phenylalanine in the synaptosomal preparations. RESULTS In patients with ALS, there was a marked decrease in the maximal velocity of transport for high-affinity glutamate uptake in synaptosomes from spinal cord (-59 percent, P less than 0.001), motor cortex (-70 percent, P less than 0.001), and somatosensory cortex (-39 percent, P less than 0.05), but not in those from visual cortex, striatum, or hippocampus. The affinity of the transporter for glutamate was not altered. No abnormalities in glutamate transport were found in synaptosomes from patients with other chronic neurodegenerative disorders. The transport of gamma-aminobutyric acid and phenylalanine was normal in patients with ALS. CONCLUSIONS ALS is associated with a defect in high-affinity glutamate transport that has disease, region, and chemical specificity. Defects in the clearance of extracellular glutamate because of a faulty transporter could lead to neurotoxic levels of extracellular glutamate and thus be pathogenic in ALS.

Colchicine Myopathy and Neuropathy

Although colchicine has been used for centuries, its neuromuscular toxicity in humans is largely unrecognized. In this report we describe a characteristic syndrome of myopathy and neuropathy and present 12 new cases of the condition. Colchicine myopathy may occur in patients with gout who take customary doses of the drug but who have elevated plasma drug levels because of altered renal function. It usually presents with proximal weakness and always presents with elevation of serum creatine kinase; both features remit within three to four weeks after the drug is discontinued. The accompanying axonal polyneuropathy is mild and resolves slowly. Electromyography of proximal muscles shows a myopathy that is marked by abnormal spontaneous activity. Because of these features, colchicine myoneuropathy is usually misdiagnosed initially, either as probable polymyositis or as uremic neuropathy. The myopathy is vacuolar, marked by accumulation of lysosomes and autophagic vacuoles unrelated to necrosis or to the mild denervation in distal muscles. The morphologic changes in muscle suggest that the pathogenesis involves disruption of a microtubule-dependent cytoskeletal network that interacts with lysosomes. Correct diagnosis may save patients with this disorder from inappropriate therapy.

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

Abstract: 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 post‐natal 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 PD139977) and drugs that could block presynaptic release or synthesis (riluzole and gabapentin). In addition, some antioxidants (U83836E and N‐t‐butyl‐α‐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.

Mutations in the sarcoglycan genes in patients with myopathy.

BACKGROUND Some patients with autosomal recessive limb-girdle muscular dystrophy have mutations in the genes coding for the sarcoglycan proteins (alpha-, beta-, gamma-, and delta-sarcoglycan). To determine the frequency of sarcoglycan-gene mutations and the relation between the clinical features and genotype, we studied several hundred patients with myopathy. METHODS Antibody against alpha-sarcoglycan was used to stain muscle-biopsy specimens from 556 patients with myopathy and normal dystrophin genes (the gene frequently deleted in X-linked muscular dystrophy). Patients whose biopsy specimens showed a deficiency of alpha-sarcoglycan on immunostaining were studied for mutations of the alpha-, beta-, and gamma-sarcoglycan genes with reverse transcription of muscle RNA, analysis involving single-strand conformation polymorphisms, and sequencing. RESULTS Levels of alpha-sarcoglycan were found to be decreased on immunostaining of muscle-biopsy specimens from 54 of the 556 patients (10 percent); in 25 of these patients no alpha-sarcoglycan was detected. Screening for sarcoglycan-gene mutations in 50 of the 54 patients revealed mutations in 29 patients (58 percent): 17 (34 percent) had mutations in the alpha-sarcoglycan gene, 8 (16 percent) in the beta-sarcoglycan gene, and 4 (8 percent) in the gamma-sarcoglycan gene. No mutations were found in 21 patients (42 percent). The prevalence of sarcoglycan-gene mutations was highest among patients with severe (Duchenne-like) muscular dystrophy that began in childhood (18 of 83 patients, or 22 percent); the prevalence among patients with proximal (limb-girdle) muscular dystrophy with a later onset was 6 percent (11 of 180 patients). CONCLUSIONS Defects in the genes coding for the sarcoglycan proteins are limited to patients with Duchenne-like and limb-girdle muscular dystrophy with normal dystrophin and occur in 11 percent of such patients.

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 cords 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).

Serum antibodies to GM1 ganglioside in amyotrophic lateral sclerosis

We report the presence of serum antibodies directed against GM1 ganglioside, a defined neural antigen, in many patients with amyotrophic lateral sclerosis (ALS). We examined serum from a series of patients with well-documented clinical diagnoses. Serum antibodies to GM1 ganglioside were measured using ELIS A assays. Our results showed that polyclonal IgM anti-GM1 antibodies were present at dilutions of 1:25 to 1:2,000 in 42 of 74 (57%) patients with ALS. The anti-GM1 antibodies were especially frequent in patients with prominent lower motor neuron signs (41/59; 69%). Few normal controls (2/23) and motor-sensory neuropathy patients (3/27) had similar antibodies. Anti-GM1 antibodies did occur in patients with nonneural autoimmune disorders. However, the anti-GM1 antibodies in these patients tended to differ from those in ALS based on an analysis of their light chain types. Further examination of the role and spectrum of serum antiganglioside antibody activity in motor neuron syndromes is warranted.

Head drop and camptocormia

The spectrum of bent spine disorders Head ptosis (drop) results from weakness of the neck extensor, or increased tone of the flexor muscles. It is characterised by marked anterior curvature or angulation of the cervical spine and is associated with various neuromuscular (table 1) and extrapyramidal disorders.12–15 Camptocormia or the bent spine syndrome was first described in hysterical soldiers in 1915 by the French neurologist Souques.16 Typically there is marked anterior curvature of the thoracolumbar spine. In some patients the spine is angulated forward, the arms propped against the thigh for support. More cases, all among soldiers, were reported during the first and second world wars. These patients responded well to psychotherapy. Recently camptocormia arising as a result of weakness or abnormality in the tone of the paraspinal muscles has been described (table 2). In contrast with other skeletal disorders of the spine such as kyphosis, the deformity in head ptosis and camptocormia is not fixed and is corrected by passive extension or lying in the supine position. It is not possible to straighten the neck or back voluntarily. The evaluation of these disorders can indeed be challenging and often no definite diagnosis is made, as illustrated by four cases of head ptosis and camptocormia seen by us at the Johns Hopkins Hospital. View this table: Table 1 Neuromuscular causes for head ptosis View this table: Table 2 Causes of camptocormia An 80 year old man developed head ptosis insidiously over a period of few weeks. A week before this he had an upper respiratory tract infection and also experienced transient sharp pain over the left and then the right shoulder. He had no diplopia, dysarthria, dysphagia, limb weakness, or fatiguability. Examination showed severe neck extensor weakness, Medical Research Council (MRC) grade 2. Muscle strength was normal in all other cranial, proximal, and distal limb muscles. Serum …

Reductions in acidic amino acids andN-acetylaspartylglutamate in amyotrophic lateral sclerosis CNS

Acidic excitatory amino acids have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). We now report that, in addition to selective regional reductions in endogenous aspartate and glutamate, N-acetylaspartate (NAA), and N-acetylaspartylglutamate (NAAG) are also decreased in the CNS, whereas the activity of N-acetylated-alpha-linked-amino dipeptidase (NAALADase) is increased. In cervical cord, the concentrations of aspartate and glutamate were decreased significantly in the ventral horn; NAA was decreased in the ventral horn, dorsal horn and ventral column, whereas NAAG was decreased in all regions of the cord examined, except the posterior column. NAALADase activity was increased in the ventral column. In motor cortex of ALS patients, aspartate and glutamate were decreased and NAALADase activity was increased in both gray and white matter; whereas NAAG was decreased in gray matter alone. None of these parameters was affected in the cerebral cortex of the Huntingtons patients. Of the markers examined, the alterations in the levels of NAAG most closely parallel the cellular neuropathology in ALS.