Date: Fri, 12 May 95 00:25:59 -0400 From: Bob Broedel To: als@huey.met.fsu.edu Subject: ALSD192 ALS-ON-LINE (Special MDA Issue) =============================================================== == == == ----------- ALS Interest Group ----------- == == ALS Digest (#192, 10 May 1995) == == == == ------ Amyotrophic Lateral Sclerosis (ALS) == == ------ Motor Neurone Disease (MND) == == ------ Lou Gehrig's disease == == ------ maladie de Charcot == == == == This e-mail list has been set up to serve the world-wide == == ALS community. That is, ALS patients, ALS researchers, == == ALS support/discussion groups, ALS clinics, etc. Others == == are welcome (and invited) to join. The ALS Digest is == == published (approximately) weekly. Currently there are == == 690+ subscribers. == == == == To subscribe, to unsubscribe, to contribute notes, == == etc. to ALS Digest, please send e-mail to: == == bro@huey.met.fsu.edu (Bob Broedel) == == Sorry, but this is *not* a LISTSERV setup. == == == == Bob Broedel; P.O. Box 20049; Tallahassee, FL 32316 USA == =============================================================== CONTENTS OF THIS ISSUE: 1 .. ALS - MDA Staff Research Report (1) ===== ALS - MDA Staff Research Report ========== Date : 11 May 95 17:34:17 EDT >From : Barry Goldberg <71154.330@compuserve.com> Subject: ALS MDA Staff Research Report Here's a rather lengthy report to MDA's field staff regarding ALS research. I thought it would be of interest. _____________________________________________________________________ "Amyotrophic Lateral Sclerosis (ALS)" -- MDA Research Staff Report 9 _____________________________________________________________________ AMYOTROPHIC LATERAL SCLEROSIS (ALS) OR MOTOR NEURON DISEASE (MND) -- UK & OTHER EUROPEAN COUNTRIES A-myo-trophic = without muscle nourishment. Lateral = lateral descending motor pathways in the spinal cord. Sclerosis = hardening due to replacement by connective tissue. Familial ALS is less common than sporadic form. Familial appears to affect SOD1 gene expression while evidence in sporadic ALS is glutamate accumulation to toxic levels and/or alteration of voltage gated calcium channels which may affect glutamate levels. Studies of dextromethorphan and others, riluzole, etc., block glutamate effect. Some additional evidence points to abnormality of a regulatory enzyme that induces alteration of a specific receptor in the spinal cord. NMDA receptor subtype decreases rapidly with development of ALS and some success in reversing this trend has occurred with an enzyme activator treatment. Also link of sporadic ALS due to autoimmunal disease antibodies attacking the nervous system and indications are the attack may be on various subunits of the voltage gated calcium channel. Increased calcium levels within neurons can kill them. -Siddique at NU located Familial ALS gene to chromosome 21q21.1-22.3 -Juvenile ALS located at 2q33 -Recently reported loci of ALS-dementia to 17q. -No treatment Drug trials -Deprenyl (aids Parkinson's) -Dextromethorphan -Interferes with glutamate which is toxic at high levels to motor neurons. -NGF (Nerve growth factor) -IGF-1 and CNTF enhance survival of motor neurons -May be an autoimmune disease thus trials with Cyclophisphamide and cyclosporine (suppress immune system) -Intravenous immunoglobulin infusion -Total lymphoid irradiation Causes?? -One theory of cause is toxic effects from environment either externally or internally kills motor neurons. -Some toxins in the environment can produce similar effects but may not actually cause ALS. -Glutamate may be one internal toxin (high levels) which may also affect astrocytes which nourish neurons. ALS patients show increased numbers of astrocytes and they are abnormal but this may be reacting to something abnormal in ALS. Astrocytes do balance glutamate levels. Glutamate associated with transmission and are essential to transmit cell to cell. SYMPTOMS -Weakening of muscles of swallowing, chewing and speech -Suspect cause is on chromosome 21 to a segment that is 5 units long -Too much glutamate found around neurons. Problem may be due to protein that should remove the glutamate, possibly by a transport system. -Most forms of ALS are not genetic but the Familial ALS (FALS) is genetic -May be both forms are due to a common biochemical defect. -An autoimmune response causes a protein abnormality similar to one caused by a genetic defect in familial ALS -DNA-liposome complex may lead to better therapy -Siddique at NU located Familial ALS gene to chromosome 21 -Brown's team at MIT identified the gene as SOD-1 = Superoxide dismutase 1, the protein. This protein keeps certain toxic substances from building up. -Probable now that ALS has several causes but may impinge upon the same protein production. -Clinical trials with Vit-C, E, and B-carotene. -Dextromethorphan as treatment blocks glutamate when in excess as in ALS type. -Riluzole also blocks glutamate SOD-1 -Superoxide dismutase 1 -Gene defect a cause of Familial ALS (FALS) -Gene on chromosome 21 -Cause of 30-50% of FALS -FALS only 10% of all ALS -At least one other gene is FALS type -Sporadic or non-genetic form of ALS is 90% of cases -Research now is focusing on that which would produce the same result as SOD1 defect -Sporadic and genetic ALS are clinically identical thus probably a common biochemical defect or pathway affected -Superoxide dismutase 1 -An enzyme that allows conversion of the toxic chemical called superoxide into hydrogen peroxide and oxygen. -Another theory is oxygen free radicals and hydroperoxides termed reactive oxygen species (ROS) are causal factors in aging. ROS produced by univalent reduction of dioxygen to generate sequentially superoxide anion radical and hydrogen peroxide. Hydrogen peroxide if not eliminated, generates the highly reactive hydroxyl free radical believed to be the main agent of oxidative damage. -Superoxide dismutase converts superoxide anion radical to hydrogen peroxide (H2O2) and in Drosophila m. catalase breaks down H2O2 to water and oxygen preventing production of the hydroxyl free radical. In higher animals, glutathione peroxidase removes H2O2. -At least 3 forms of the enzyme -Either an overactivity or underactivity of SOD1 would lead to buildup of toxic chemicals. Too much activity would increase levels of hydrogen peroxide which is also toxic. Too little activity causes an increase in superoxide also toxic. -Believe treatment will be done to either block or enhance SOD1 activity -Not known if Vit C, E or B-carotene will help as antioxidants. They are believed to protect cells from damage by free radicals &/or superoxides. Neurons generally regulate levels of vitamins. May be difficult to get enough Vit-C to help into the cells if admission is regulated. Appears Vit-E does not help either. Not known about B- carotene. -Looking for other genes for other forms of genetic ALS -In sporadic ALS, looking for what will cause a build up of superoxide or other related substances -Autoimmune attacks may cause buildup of superoxide like compounds Glutamate -A gene for a glutamate receptor was found within area of chromosome 21 which contained a gene for Familial ALS but now know that the flaw is in the SOD1 gene and not the receptor for glutamate -Neurotrophic factors also seem to protect neurons (CNTF, IGF1, etc) which may counter effect of superoxide -ALS shows later in life and only affects neurons. May be due to neurons do not replace themselves, no turnover, thus damage accumulates. Even so, when some die others take over so symptoms still do not show for some time. *** Kimura, et al., 1994 Ann. Neurol. -Possible autoimmune role in ALS. -Most ALS patients have immunoglobulins that bind to L type VGCC (voltage-gated Ca++ channel) and that levels of this anti-VGCC correlate with disease progression. -The ALS-IgG cause electrophysiological changes in VGCC binding to _1 subunit. Also shown to block binding of mAb specific for the channel. -Patients with LEMS (Lambert-Eaton) IgG bind to both the _1 and _- subunits of VGCC and affects both N and L type VGCC. -Possibility that upper and lower motor neurons have specific type of _-subunits making them more susceptible than other neuronal or other tissue types. -Also evidence of ligand binding effecting Ca++ channels and/or excitotoxicity as cause of neuronal death in that the uptake of glutamate is interfered with and it reaches toxic levels. *** Rainero, et al., Neurology 44:347:1994 -suggest FALS genetically heterogeneous -missense mutation of SOD1 chromosome 21 -autosomal dominant -FALS type 1 -early onset, short duration, autosomal dominant -rapid progression loss of motor function on mostly lower motor neurons -no dementia, parkinsonism or gynecomastia (male breast development) -high variability of onset -suggest oxidant stress involved in FALS -SOD1 catalyzes conversion of superoxide free radical anion to hydrogen peroxide plus dioxygen. Anion superoxide is a common product of both spontaneous and enzyme-catalyzed oxidation processes and is chemically active as both a reductant and an antioxidant. Anion peroxide reacts rapidly with nitric oxide (NO) and may interfere with the multiple functions exerted by NO in the CNS. A controlled trial of Riluzole in Amyotrophic Lateral Sclerosis Bensimon, G., et al. NEJM 330:(9):585-591:1994 ALS is a progressive and fatal neurodegenerative disorder associated with survival ranging from a few months to decades (mean of 37-49 months). Survival factors are age at onset, site of onset, duration of weakness and degree of clinical disabiltiy or respiratory function. Two forms of site of onset were tested, Bulbar site and limb muscle site. Bulbar site is the more serious form. Riluzole (2-amino-6-(trifluoromethoxy)benzothiazole, RP 54274) modulates the glutamatergic transmission. Overall, Riluzole reduced mortality by 38.6% at 12 months and 19.4% at 21 months (statistically significant) Treatment effect was greater survival in bulbar-onset disease (significant result) than in limb-onset disease (only a trend toward positive was seen). For each functional group score such as muscle strength, the rate of deterioration was less in the Riluzole group and was statistically significant Adverse side effects were asthenia (weakness), worsened spasticity, increases in alanine aminotransferase, asparatate aminotransferase and mild to moderate increase in blood pressure. All were tolerable but some patients did withdraw. Adverse effects do not outweigh therapy. Effect appears to be time related with strongest effect in 12 months versus 21 months. Riluzole has a positive effect on the rate of deterioration of muscle function suggesting interference with the disease process. Riluzole presynaptically inhibits the release of glutamic acid in the central nervous system and interferes postsynaptically with the effects of excitatory amino acids. It does not seem to interact competitively with any receptors of glutamic acid, but rather to antagonize the effects of such neurotransmitters indirectly, possibly by interacting with voltage-gated sodium channels or G proteins. *** RESPONSES to Riluzole study by Bensimon, et al. NEJM 331:(4):272-274:1994 * McKee, P. et al Included patients who did not meet inclusion criteria. Without this group, no statistical difference was significant. How did they differ from criteria? Would believe that riluzole or other agent would have affect more on late mortality than on early mortality. Believe differences may not reflect some biologic effect of riluzole. * MacRae, K.D. The conclusion that riluzole may improve survival in ALS of Bulbar onset is based on an analysis of a subgroup. Subgroup analysis should always be cautious. Randomization does not guarantee that groups will be similar in all relevant prognostic factors, some differences will occur especially with more prognostic factors being considered. Randomization only ensures absence of systematic allocation bias. With large groups, differences are usually small, but in small groups, any difference is much larger effect. In the bulbar patients, there are differences of prognostic outcome, age, duration of disease, bulbar-function score, and stiffness scale. These patients had a younger onset average, a longer duration of disease, higher bulbar function scores and higher mean on stiffness scale--all four factors indicate lower risk. Also, precise outcome needs to be considered. If outcome was death or tracheotomy as the end point, they are certainly different. Appears the subgroup analysis is very dubious. * Burgerman, R.S. Dr. Rowland states those with bulbar-onset have shorter life expectancy than limb weakness onset. Appears to treat this group as it were a different disease--never suggested before. May it be that 12 months is too short a follow-up period to detect a survival difference in those with a greater life expectancy? The limb weakness patients had not reached their end points (death or tracheotomy) at the end of the 12 months of this study. If a treatment is found, its survival effects may be seen first in those whose life expectancy is the shortest. * Rogawski, M.A. Riluzole is characterized as an antiglutamate agent, but its effect is not really known. Question results if only from the company sponsored laboratories. Claim Rhone-Poulenc Rorer does not allow distribution to private labs. * Murphy, J.R. There is a critical imbalance in the group with bulbar-onset concerning balancing base-line values for comparison in this study and the difference in survival seems to come from this difference. This is even more important in small groups. Would predict a second trial would show a negative effect. *** AUTHORS REPLY: * Bensimon, G., et al. The slight nonsignificant differences of MacRae and Murphy is variables at entry cannot be responsible for improvements in survival in the riluzole group. With factors for death or tracheotomy, the effect was greater. Agree with McKee that most of the treatment difference is seen before 12 months which could be a statistical artifact or evidence of a serious limitation of the treatment. But, drug efficacy often decreases over time and should not preclude any conclusions about mechanism of treatment effect. We are all ignorant of the disease process and risk factors. Only a much larger study can provide more information. * Rowland, L.P. This trial certainly had loose ends. As far as bulbar-onset patients there may be differences if onset begins with dysarthria (non-threatening) or dysphagia (life-threatening). Other treatment factors also enter in that a Dr. may want to start the tracheotomy put patient says no for some time. Tracheotomy was considered an end point equivalent to death in this study. Possible that unexpected benefit for bulbar-onset patients due to lack of suitable matching of treated and placebo groups for these symptoms. * Rhone-Poulenc Rorer (RPR) 35 requests from private labs for riluzole were granted. Labs on contract are only for support of equipment and supplies, the investigators do their own work. 65 publications of riluzole exist of which 27 are from RPR scientists. *** Ono, S., et al. Neurology 44:537-540:1994 Skin of ALS shows decreased content of collagen, alteration of cross-linkage of collagen and elastin, increased solubility of collagen, decreased glutamate and aspartate, collagen bundles in dermis are reduced in amount and loosely woven, significant negative correlation between duration of illness and diameter of fibrils (become less), significant amorphous material in ground substance separating collagen bundles and more of it with prolonged illness. Amorphous material not in normal skin but present in wound healing and scleroderma, in ALS the amount is greater than above and increases with duration of illness. Ground substance basically composed of macromolecules with bound water. Macromolecules in two classes GAGs and structural glycoproteins. GAGs form proteoglycans. In ALS, amorphous material shows GAGs greatly increased and that there is substantial lack of amino acids (proteins). Decreased diameter of collagen fibrils associated with duration of illness may be related to increased amorphous material related to duration. *** Camu, W., et al. Neurology 44:547:1994 CONJUGAL ALS. Environmental factors may be involved in ALS based upon: geographic clustering of sporadic cases, cases in common occupational environments, endemic areas of specific forms (Guam), and conjugal cases. Conjugal forms (both husband and wife) suggest environmental factors, food toxins or virus. Four cases reported in the past and now two more from the same geographical area of France (Languedoc- Roussillon). No common profile of conjugal ALS. In each family, husband and wife showed different expression of ALS and different progression of disease. Also, wide range of onset within the family suggest conjugal is not a distinct clinical entity. Such cases may occur by chance but chances are very low of two families from the same region. Without a toxic factor, the factor of geography suggests genetic origin. PROGRESSIVE BULBAR PALSY OF DUCHENNE -primarily affects chewing, swallowing and other bulbar functions -survival is shorter than others *** Smith, C.P., et al. Assignment of the gene coding for the human high-affinity glutamate transporter EAAC1 to 9p24: Potential role in dicarboxylic aminoaciduria and neurodegenerative disorders. Genomics 20:335-336:1994. -High-affinity glutamate transporters play an essential role in transporting glutamate across plasma membranes. This transport is crucial in terminating the action of the excitatory neurotransmitter glutamate and in maintaining extracellular glutamate concentration below neurotoxic levels. In small intestine and kidney, the transporter mediates net absorption of glutamate and aspartate across epithelial cells. An error in the glutamate transporter is thought to cause dicarboxylic aminoaciduria which shows as increased urinary excretion of glutamate and aspartate and is in general associated with neurologic and developmental abnormalities. EAAC1, a high affinity glutamate transporter also transports aspartate but no other amino acid and is found everywhere in the body especially in brain, intestine and kidney. Location of this gene is assigned to chromosome 9p24. This mutation in this gene could be associated with some forms of familial ALS besides SOD1 inheritance. *** Monyer, H., et al. Development and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12:529-540:1994 N-methyl-D-aspartate (NMDA) receptors constitute cation channels gated by the excitatory neurotransmitter L-glutamate and mediate signal transduction in central synapses. Properties include high Ca++ permeability, voltage-dependent Mg++ block and slow gating kinetics. Most lasting cellular effects of NMDA receptor activation are mediated by Ca++ entering through the channel. Intense or prolonged activation of the NMDA receptor can be lethal due to excessive Ca++ influx. Thus depending on strength, the same second messenger can change synaptic efficacy, alter cytoarchitecture or cause cell death. Have found different types of NMDA receptors. In development of rat CNS, NMDAR1 or NR1, is present at all stages in virtually all neurons. The four NR2 types differ in expression. NR2B and NR2D are prenatal, and NR2A and NR2C first occur about time of birth. Most peak at P20. Different parts will have different levels of the various receptor types. *** Smith, R.G., et al. Cytotoxicity of immunoglobulins from amyotrophic lateral sclerosis patients on a hybrid motoneuron cell line. Proc. Natl. Acad. Sci. 91:3393-3397:1994 Sporadic ALS may be due to activation of ligand-gated ion channels and excitotoxicity or from autoimmunity and antibody interaction with volage-gated Ca++ channels (VGCCs). Are IgG antibodies to VGCCs in sporadic but not familial ALS. They inhibit skeletal muscle L-type voltage-gated Ca++ currents. VGCCs implicated in regulating intracellular Ca++ homeostasis and neuronal survival, mitochondrial activity, oxidative injury, and excitotoxic ligand-gated ion channel function, such effects usually result from VGCC activation. The suggestion that neuronal VGCC activation and associated increased motoneuron Ca++ entry may result from interaction with IgG from ALS as shown in mice where ACh release was increased. A motoneuron-neuroblastoma hybrid cell line (VSC4.1) was developed that contains antagonistic binding sites for L-type VGCCs (dihydropyridines), N-type VGCCs (w-conotoxin) and P-type VGCCs (agatoxin IVa). Evidence here indicates ALS IgGs are cytotoxic for VSC4.1 cells when differentiated with cAMP and is Ca++ dependent and mediated by neuronal (N- and P-type) VGCCs. Cell loss by IgG is selective in both type and differentiatioin state of cells and type of disease from which IgGs come (ALS, LEMS). Differentiated cells with ALS IgG die in 24-48 hours appearing to make effect directly. Toxicity dependent upon cestracellular calcium and reducing extracellular Ca++ protects these cells from the IgG. Appears the IgG enhances Ca++ entry into the cells by activating the N- or P-type VGCCs of neurons which may affect other Ca++ entry channels. L-type VGCCs appear not to be involved. The ALS IgG may bind to epitopes common to all the VGCCs rather than to immunoglobulin specificity for a single VGCC subtype. Other cell varieties are not killed in this manner. Differences may lie in varied population size of channel type, functional differences during development, second-messenger differences, ability to buffer Ca++ or differing levels of Ca++ binding protein during development or cell type. LEMS IgG gave a comparable effect to that of ALS IgG with enhanced cytotoxicity with N-type VGCCs. *** Rothstein, J.D., et al. Chronic inhibition of superoxide dismutase produces apoptotic death of spinal neurons. Proc Natl Acad Sci 91:4155-4159:1994. Mutations in the Cu/Zn superoxide dismutase (SOD1) gene on chromosome 21 have been detected in some families with the autosomal dominant form of familial ALS where mutations reduce the activity of SOD1. Demonstrated that chronic reduction in SOD1 activity is toxic to motor neurons in vitro and may be directly toxic to motor neurons in vivo. It is likely that some factor other than SOD1 mutations themselves accounts for the selective degereration of motor neurons. Such a factor may be the excitotoxic influence of the neurotransmitter glutamate. Suggested that impaired glutamate transport or even baseline glutqmatergic activity, may target motor neurons and contribute to their loss in ALS. Other oxidative insults in motor neurons may also contribute. Possible that the SOD1 mutations in familial ALS patients are lethal because they confer some novel, cytotoxic function on the enzyme in addition to reducing its SOD activity. Chronic SOD1 inhibition appears to cause apoptotic death of neurons which is reparable by antioxidants in culture. Conversely, in some neuroal lines, overexpression of SOD1 diminishes apoptosis. Data sugest the hypothesis that motor neoron death may be apoptotic in familial ALS patients with SOD1 mutations. Some neurotophic factors can rescue primary neurons from apoptotic cell death. Preliminary observations suggest that motor neurons are more sensitive to oxygen radical toxicity than other neurons in the organotypic spinal cord culture. The neuroprotective effect of both a non-NMDA glutamate receptor antagonist and antioxidants in this model suggests that these agents may be beneficial in the treatment of ALS such as riluzole which alters glutamartergic neurotransmission. *** Dugan, L.L. and Choi, D.W. Excitotoxicity, free radicals, and cell membrane changes. Ann Neurol :35:S17-S21:1994 Excitotoxicity may interact with other pathophysiological processes to enhance neuronal injury. There is growing consensus that the injury processes in many neurological diseases may converge on a few key mechanisms, such as glutamate receptor-mediated excitotoxicity, free radical injury and deprivation of trophic factors. Excitotoxicity and the relationship between excitotoxicity and other injury mechanisms. The term excitotoxicity used to describe overactivation of putative excitatory neurotransmitter glutamate. Glutamate is stored in vesicles and when excess of it is released or uptake is blocked, extracellular concentrations can be significant leading to neuronal death. Energy depletion and reduced levels of ATP lead to release and accumulation of extracellular glutamate. Glutamate receptors are activated leading to an influx of Na+ and Ca++. Toxic intracellular pathways are initiated. This scheme shows ways to intervene in the process. Dying cells in culture release lactate dehydrogenase. A number of interventions that ameliorate neuronal injury act by limiting release of excitatory amino acids. In vitro, tetanus toxin (TnTx) blocks Ca++-dependent release of neurotransmitters including glutamate. Also hypothermia reduces release of glutamate thus protecting neurons. Hypothermia also reduces both the concentration of extracellular glutamate and tissue injury in CNS ischemia in vivo. Hypothermia is now being explored in various surgeries and CNS trauma. Adenosine receptor agonists also limit neurotransmitter release as in ischemia. The next point of intervention can be blockade of glutamate receptors limiting receptor overactivation. There are four types of glutamate receptors. NMDA (N-methyl-D-aspartate) receptors when activated by glutamate, allow entry of large amounts of Ca++ and Na+, this toxicity is quite rapid and fulminate (sudden, rapid, very intense). AMPA (_-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptors allow primarily Na+ entry which can cause secondary Ca++ entry, this death is slower than NMDA. G-protein-coupled metabotrophic receptors are linked to a number of second messenger system. Blockade of NMDA receptors protects neurons in ischemia and an antagonist to AMPA/kainate receptor (NBQX) reduced tissue injury in ischemia. Receptor antagonists are promising in stroke therapy. Originally, neuronal death from excitotoxicty was believed to result from depletion of cellular energy stores due to overexcitation on neurons. Later found that excitotoxicity depended critically on extracellular Ca++. One key link appears to be excess Ca++ influx. Attempts to define Ca++-dependent pathways are ongoing. A few of them may be that it includes activation of nitric oxide synthase (NOS), lipases (especially phospholipase A2, protein kinases, endonucleases and proteases. NOS is an enzyme with homology to P450 cytochrome c reductase. There are one inducible (i) and two constitutive forms (c, n) of NOS. All forms require calcium/calmodulin. nNOS and cNOS require binding of Ca++ to calmodulin for activation but iNOS has higher affinity for calmodulin and is active at resting Ca++ levels. NOS generates nitric oxide (NO) which is a endothelium-derived relaxing factor and regulator of vascular tone but NO is a free radical gas important in long-term potentiation and is produced by macrophages and microglia in antipathogenic reactions and NO may also release insulin. NO implicated as important in excitotoxic neuronal injury and inhibitors of NOS have showed to protect from NMDA induced cell death. NOS can generate superoxide radical and hydrogen peroxide directly. Both of these may react with metals such as iron to produce even more reactive radical species especially hydroxyl radicals. NO can combine with oxygen or the superoxide radical anion to produce another highly oxidizing molecule, peroxynitrite. Free radicals increase release of glutamate from hippocampal slices and inhibit glutamate uptake by glia. can be activated by Ca++ influx initiated by NMDA receptors. Phospholipase A2 hydrolyzes membrane lipids to release lysophospholipids and free fatty acids including arachidonic acid. Fatty acids can affect excitotoxicity in several ways. Arachidonic acid can be metabolized to active compound like prostaglandins, leukotrienes and HETE (hydroxyeicosatetraenoic acid) and free radicals can be generated as by products of these reactions. Aracidonic acid and other free fatty acids can alter the cell membrane fluidity which could affect function of ion channels, transporters and other membrane proteins. Phospholipase A2 can affect the physical state of the membrane as well. Besides affecting what has already been mentioned in the membrane, it can increase the open state of the NMDA receptor. A 10 minute exposure of cortical neurons in culture to NMDA caused significant fluidity. *** Wang, L.-Y., et al. Regulation of NMDA receptors in cultured hippocampal neurons by protein phosphatases 1 and 2A. Nature 369:230- 232:1994 Indications of which protein phosphatases can dephosphorylate glutamate or other ligand-gated channels. Regulation of NMDA receptors by endogenous protein phosphatases (PP), used calyculin A a potent inhibitor of PP1 and PP2A and a co-agonist of NMDA, glycine. Limited desensitization occurred and peak NMDA currents were potentiated with calyculin A and okadaic acid. NMDA receptors are regulated in hippocampal neurons by an endogenous protein phosphatase(s) that is identical to or is closely related to PP2A and/or PP1. Both of them are found in the CNS. PP1 is the principal protein phosphatase associated with postsynaptic densities and therefore with glutamate receptors. Serine/threonine phosphatases also modulate the activity of this ligand-gated channel. The phosphatases may directly dephosphorylate these receptors, or alternatively they may target substrates that could themselves in turn regulate NMDA receptors, or alternatively, they may target substrates that could themselves in turn regulate NMDA receptors. Cytoskeletal elements, including actin filaments, may modulate the activity of NMDA channels. The action of protein phosphatases, as well as their inhibitors, is a potentially important way to regulate NMDA receptors and excitatory synaptic transmission in the CNS. *** Wang, Y.T. & Salter, M.W. Regulation of NMDA receptors by tyrosine kinases and phosphatases. Nature 369:233-235:1994 Spinal dorsal horn neurons NMDA receptor mediated responses were evoked by applying selective NMDA receptor agonist, L-aspartate. Application of the PTK inhibitor genistein caused reduction of NMDA currents which was reversible and concentration dependent. Lavendustin A (another PTK) did the same. Activation of NMDA receptors causes a rise in intracellular Ca++. Genistein caused reduction of Ca++ response to 61.8% of control. NMDA receptors may be regulated by several endogenous PTKs. Another PTK, pp60c-src potentiated NMDA currents indicating that PTK activity causes potentiation of the currents and when genistein was applied beforehand, the effect was depressed. Mg+ is a NMDA channel blocker and blocks the current in a voltage-dependent manner. Regulation of NMDA currents by PTPs as well as PTKs by adding Sodium orthovanadate which potentiated NMDA currents and was reversible. NMDA receptors can be modulated by protein-tyrosine kinases and protein-tyrosine phosphatases. This modulation could be due to phosphorylation/dephosphorylation of the NMDA receptors themselves or of regulatory proteins associated with the receptors. *** Liebermann, D.N. & Mody, I. Regulation of NMDA channel function by endogenous Ca++-dependent phosphatase. Nature 369:235-239:1994 A specific serine/threonine phosphoprotein phosphatase appears to modulate the openings of native NMDA channels. Ca++ ions entering through the channel activate phosphatase 2B (calcineurin) which acts either directly or indirectly on the channel complex to diminish its activity. Calcineurin induction secondary to NMDA receptor activation may alter the state of microtubule and/or actin polymerization and thereby affect channel activity. Calcineurin is absent in neurons prior to postnatal day 4. In adults, Ca++-dependent phosphatases may ensure that Ca++ entry through NMDA channels adequately initiates Ca++-dependent second messenger actions while preventing pronounced intracellular Ca++ build up to excitotoxic cell damage. Loss of calcineurin as after ischemia could mean loss of negative feedback over increasing levels of Ca++ and may underlie the anoxia-induced selective potentiation of NMDA responses causing post-ischaemic damage. The degree of NMDA receptor activation by synaptically released or ambient levels of glutamate critically depends on channel kinetics making the phosphorylation/dephosphorylation-dependent control of channel openings a major component in regulating short-and long-term changes of neuronal excitabiltiy. PRESS RELEASE 6/17/94 MICE WITH HUMAN GENE TO SPEED ALS RESEARCH. *** Dr. Mark Gurney, Northwestern University, developed the transgenic mouse for familial ALS with the defective gene for SOD1. Research will be greatly accelerated with this model. *** Kew, J.J.M., et al. Cortical function in progressive lower motor neuron disorders and amyotrophic lateral sclerosis. Neurology 44:1101-1110:1994 Motor neuron disease is a disorder with onset in middle or late life with progressive neurogenic atrophy and pathologically by degeneration of anterior horn motor neurons in spinal cord. The most common variant is ALS and it is associated with additional degeneration of giant pyramidal (Betz) cells of the motor cortex, degeneration and gliosis of the corticospinal tracts and cell loss in the bulbar motor nuclei. ALS shows both upper and lower motor neuron signs an often with bulbar palsy. Patients may show proximal, symmetric pattern of wasting and weakness in the limbs resembling the childhood-onset spinal muscular atrophies, or may present with a chronic asymmetric neurogenic atrophy often confined to a limited number of segments of one upper limb. The latter form presents earlier than does classic ALS usually before 40, and prognosis is more favorable with survival up to 20 years. Sought to see effect of loss of large pyramidal neurons from cortex by measuring regional cerebral blood flow (rCBF) with a PET scanner. Found abnormalities of cortical function in ALS patients both in resting state and performance of upper limb movement. rCBF significantly reduced in ALS in all areas of the cortex that give rise to corticospinal projections and the somatotopic representation of the upper limb in primary sensorimotor cortex was abnormally expanded (beyond normal boundaries) during limb movement. Do not know if this somatotopic reorganization is related to cortical Betz cell loss and pyramidal tract disruption or to anterior horn cell loss. Progressive lower motor neuron degeneration (LMND) shows no clinical signs of upper motor neuron degeneration even when degree of upper limb weakness was the same in an ALS patient. Predicted if _- motor neuron loss alone is responsible for functional reorganization of the sensorimotor cortex during limb movement in ALS patients, similar reorganization should be observed when LMND patients are required to perform the same task. Results indicate that the abnormal pattern of resting-state rCBF in ALS patients reflects pyramidal cell loss from areas of the cortex projecting through the corticospinal tract combined with loss of cortico-cortical projections between the primary SMC and the premotor and parietal association areas. This is supported by anatomic studies on primates showing that these areas give rise to direct corticospinal projections and that the involved association areas receive direct projections from the motor cortex. ALS patients rCBF at rest is most reduced in primary SMC. Pathological examination shows this area most affected in ALS. Found rCBF at rest in supplementary motor and medial parietal cortex of LMND patients was intermediate between controls and ALS raises possibility at least some of the LMND patients may have had early, subclinical cortical involvement. Increase in rCBF in the hand/arm representation of the contralateral SMC in ALS during upper limb motion was not significantly different from that of controls but unexpectedly, there was an abnormal spread of activity into face representation of the primary SMC contralateral to the moving limb even without any facial or masticatory movements. Results suggest that the abnormal recruitment of these areas in ALS patients may represent cortical adaptation to the lesion. Seems clear that weakness due to _-motor neuron loss per se does not lead to reorganization of the sensorimotor cortex. Differences in SMC between LMND and ALS may be due to developing different strategies for movement between the groups and a differential use of proximal muscles may be possible. ALS showed the greater task difficulty. SII (second somatosensory cortex) not fully understood. Suggested involved with somesthesis, especially "active touch" and tactile discrimination and has a somatotopic body map. Projects direct corticospinal projections, connected with posterior insula and with its contralateral counterpart. Ipsilateral connections of SII to primary motor cortex (M1) may be reason for abnormal bilateral activation of this area in ALS. May be route to provide dorsal column cutaneous sensory input to M1 which may reflect abnormal SII activation in ALS to increase drive to M1 to compensate for an alteration in its output capacity, together with an increase in afferent feedback in generating the output. Supports view that SII may be "cortical loop" for afferent input to M1, the gain in which can be adjusted according to output requirements. *** Gurney, M.E., et al. Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science 264:17721775:1994 ALS is both sporadic and familial and causes degeneration of motor neurons in cortex, brainstem and spinal cord. It begins in adults as an asymmetric weakness in two or more limbs and progresses to complete paralysis. FALS is autosomal dominant and about 10% of all ALS. Of this 10%, only 20% have mutation in the Cu, Zn superoxide dismutase gene. FALS heterozygous for SOD mutations have 50-60% of normal level of SOD activity in RBCs and brain. Produced a transgenic mouse that expresses wild-type or mutant forms of human SOD. Two mutations were Ala4 cc: "Ray Harwood -- Data Basix: (602)721-1988" [RHarwood@Data.Basix.Com] --- MDA -- Working to find the cure for neuromuscular disease --- === = Barry ... thank you very much for letting us distribute this = important MDA document. We love you . rgds,bro === === end of als 192 ===