Biomedical subjects
Publications and source records attributed to S H Appel.
This rare myasthenic disorder is the first neurologic disease found to be caused by antibodies against an ion channel complex. It presents an unusual opportunity to clarify the pathobiology of so-called receptor diseases, as well as to gain better understanding of the normal biology of acetylcholine release and its effect on neuromuscular junctions.
BACKGROUND AND METHODS: Sporadic amyotrophic lateral sclerosis is a chronic, progressive degenerative disease of the motor neurons of the spinal cord and motor cortex. The cause is unknown. Recent electrophysiologic studies in animals indicate that immunoglobulins from patients with this disease alter presynaptic voltage-dependent calcium currents and calcium-dependent release of neurotransmitters. To determine whether similar interactions might be identified biochemically, we used an enzyme-linked immunosorbent assay (ELISA) to detect the reaction of serum IgG with purified complexes of L-type voltage-gated calcium channels from rabbit skeletal muscle. The results from patients with amyotrophic lateral sclerosis were compared with those obtained from patients with other types of motor neuron disease, patients with autoimmune and non-autoimmune neurologic diseases, and normal subjects. RESULTS: Serum samples from 36 of 48 patients with sporadic amyotrophic lateral sclerosis (75 percent) contained IgG that reacted with L-type calcium-channel protein, and serum reactivity on ELISA correlated with the rate of disease progression (Spearman rank-correlation coefficient, 0.62). Reactive serum was present in only 1 of 25 normal subjects and 1 of 35 control patients with no motor neuron disease. Antibodies to L-type voltage-gated calcium channels were identified in 6 of 9 patients with Lambert-Eaton syndrome, and in 3 of 15 patients with Guillain-Barré syndrome. CONCLUSIONS: Antibodies to L-type voltage-gated calcium channels are present in the serum of patients with amyotrophic lateral sclerosis, and antibody titers correlate with the rate of disease progression. Together with previous data, these results suggest a role for autoimmune mechanisms in the pathogenesis of sporadic amyotrophic lateral sclerosis.
In this study we determined the temporal association between the appearance of the dopamine transporter, measured by 1-[2-(diphenyl-methoxy)ethyl]-4-(3- phenylpropyl)-piperazine ([3H]-GBR12935), a potent and selective inhibitor of dopamine uptake, and other biochemical markers of dopaminergic nerve terminal growth into the developing striatum. [3H]-GBR12935 binding was minimally detected in the rudimentary striatum of embryonic day 14 rat brains, increased to 23% of the adult level by birth, and reached the adult level during the fifth postnatal week. This finding contrasts with a slower developmental increase in [3H]-dopamine uptake, a functional measure of the transporter. Tyrosine hydroxylase activity levels followed a developmental curve similar to that of [3H]-GBR12935 binding but did not reach adult levels until the 7th postnatal week. Dopamine content increased at a slower rate, being only 10% and 92% of the adult level at birth and postnatal week 8, respectively. These results indicate that the appearance of a structural, but not optimally functional, dopamine transporter may be the earliest detectable biochemical index of dopaminergic nerve terminal growth into the striatum during development.
To support a potential role for immune mechanisms in the destruction of substantia nigra (SN) neurons, guinea pigs were immunized with bovine mesencephalon containing SN neurons. After immunization no clinical signs of basal ganglia dysfunction appeared. However, pathological examination revealed evidence of neuronal damage in the SN in 8 of 17 guinea pigs immunized with bovine mesencephalon. No nigral pathology was noted in animals immunized with spinal cord gray matter or Freund's adjuvant alone. Accompanying the SN damage in mesencephalon-immunized guinea pigs was a 25% decrease in tyrosine hydroxylase activity in the SN and a 27% decrease in dopamine content in the striatum. Deposits of IgG were detected by immunohistochemical techniques in sections of SN from mesencephalon-immunized guinea pigs and in sections of human SN after exposure to serum from mesencephalon-immunized guinea pigs. These data document the antigenicity of SN and suggest the possibility that immune mechanisms can contribute to basal ganglia pathology.
Malnutrition has deleterious effects on immune functions, which predispose to an increased risk of infection. To study the effect of protein malnutrition on such immune functions and resistance to infection, we divided C57BL/6 mice into three groups: (1) control-standard diet, (2) protein-malnourished for 14 days, and (3) protein-malnourished for 14 days followed by standard diet for 3 days. The animals were further divided into subgroups: (1) an untreated group, (2) a muramyl dipeptide (MDP)-treated group, and (3) an interferon-gamma (IFN-gamma)-treated group before cecal ligation and puncture (CLP). Malnourished mice had significantly (P less than 0.05) lower body weight, serum albumin, spleen/body weight, percentage of splenic macrophages with Ia expression (%MOIa), increased splenic T suppressor cells, and greater mortality after CLP. Refeeding plus IFN-gamma or MDP significantly increased %MOIa (P less than 0.05) and also abrogated the increase in splenic lymphocytes seen in the malnourished animals. The increase in splenic suppressor T cells was not affected by refeeding or immunomodulation. Mortality after CLP was increased from 15% in the controls to 85% in the malnourished group and was significantly decreased by refeeding, MDP, and the combination of refeeding plus immunomodulators (P less than 0.05). These data show that 14-day malnutrition adversely affected the immune response to infection and increased mortality from CLP. Refeeding and immunomodulation restored macrophage Ia expression without CLP but not after the procedure, despite the significant reduction in mortality. The use of immunomodulation in protein malnourished conditions may serve as an adjuvant role to nutritional support.
The synthesis and release of 3H-acetylcholine was measured in hippocampal slices of adult rat brain following acute in vitro exposure to ethanolamine. Evoked release of 3H-acetylcholine was elevated by 60-70% but 3H-acetylcholine synthesis was unaffected. Other amino alcohols were also found to significantly increase evoked 3H-acetylcholine release. The effect may be stereochemically mediated since only one of four possible propanolamine configurations, R-alaninol, was active. The most potent compound tested was R-prolinol which showed an EC50 nearly 10-fold lower than that of either R-alaninol or ethanolamine; S-prolinol was inactive. Slices taken from adult rats which had been fed active compounds for two weeks also exhibited enhancements in evoked 3H-acetylcholine release. These results indicate that amino alcohols modulate acetylcholine release in the rat hippocampus.
Dissociated rat septal nucleus cells cultured in defined medium exhibited twofold increases in the maximal rates of sodium-dependent, high-affinity choline uptake and acetylcholine formation when grown in the presence of phosphoethanolamine. The effect was concentration-dependent (EC50 = 15 microM) and appeared to be associated with in vitro maturation of cholinergic neurons rather than with enhanced survival. Choline acetyltransferase, acetylcholinesterase, and choline kinase activities were unaffected by this treatment. The effect of phosphoethanolamine was specific for cholinergic neurons, because treatment with this compound did not alter the kinetic constants for high-affinity neuronal uptake of gamma-aminobutyric acid or dopamine. The action appeared to be mediated primarily through activation of the sodium-dependent, high-affinity transport mechanism for choline as opposed to alterations in the storage and release of acetylcholine.
1. The Vaseline-gap technique was used to record calcium currents (ICa) and charge movement in single cut fibres from normal human muscle. Experiments were carried out in 2 or 10 mM-extracellular Ca2+ concentration ([Ca2+]o) and at 17 or 27 degrees C. 2. The passive electrical properties of the fibres with this technique were: membrane resistance for unit length rm = 59.4 k omega cm; longitudinal resistance per unit length ri = 4.9 M omega/cm; longitudinal resistance per unit length under the Vaseline seals re = 438 M omega/cm; specific membrane resistance Rm = 1.176 k omega cm2; input capacitance = 5.53 nF; specific membrane capacitance = 8.9 microF/cm2. 3. The maximum amplitude of ICa at 17 degrees C was: in 2 mM [Ca2+]o, -0.42 microA/microF and in 10 mM [Ca2+]o, -1.44 microA/microF. At 27 degrees C and in 10 mM [Ca2+]o, it increased to -3.04 microA/microF. The calculated temperature coefficient (Q10) for the increase in amplitude from 17 to 27 degrees C was 2.1. 4. Ca2+ permeability (PCa) was calculated using the Goldman-Katz relation; in 2 mM [Ca2+]o at 17 degrees C, PCa = 1.26 x 10(-6) cm/s; in 10 mM [Ca2+]o at 17 degrees C, PCa = 2.23 x 10(-6) cm/s; in 10 mM [Ca2+]o at 27 degrees C, PCa = 4.03 x 10(-6) cm/s. 5. The activation curve calculated from the PCa was shifted by 10 mV to positive potentials when raising [Ca2+]o from 2 to 10 mM. Increasing the temperature did not change the curve. The mid-point potentials (Va 1/2) and steepness (k) of the activation curves were: at 17 degrees C, in 2 mM [Ca2+]o, Va 1/2 = -1.53 mV and k = 6.7 mV; in 10 mM [Ca2+]o, Va 1/2 = 9.96 mV and k = 6.8 mV; at 27 degrees C and 10 mM [Ca2+]o, Va 1/2 = 11.3 mV and k = 7.7 mV. The activation time constant in 10 mM [Ca2+]o reached a plateau at potentials positive to 10 mV, with a value of 93.8 ms at 17 degrees C and 17.4 ms at 27 degrees C. The calculated Q10 was 4.5. 6. The deactivation of the current was studied from tail currents at different membrane potentials in 10 mM [Ca2+]o.(ABSTRACT TRUNCATED AT 400 WORDS)
We investigated the role of the immune system in the pathogenesis of amyotrophic lateral sclerosis (ALS) by studying the long-term consequences of ALS immunoglobulin (Ig) application on the levator auris muscle of the mouse. We applied Ig from seven ALS patients, four disease controls, and a pool of normal Ig (6 mg of Ig in 2 weeks) by subcutaneous injection; removed the muscles 4 to 12 weeks after the beginning of treatment; and recorded both spontaneous and evoked release of transmitter. None of the control Ig induced changes in transmitter, whereas five of seven ALS Ig induced a significant increase in the rate of spontaneous release, and all ALS Ig produced significant changes in the quantal content of evoked release. In muscles treated with one of the ALS Igs, synaptic activity was completely absent. Cholinesterase and silver staining demonstrated intact neuromuscular junctions in the control Ig-treated muscles and also in many areas of ALS Ig-treated muscles. Axonal degeneration and denervation were present in most muscles treated with ALS Ig. There was complete denervation when no synaptic activity could be recorded. Thus, ALS Ig appears to lead to long-lasting effects at the neuromuscular junction, and such effects may be an early stage in the immune-mediated pathogenesis of ALS.
A dopaminergic neuroblastoma was derived using somatic cell fusion of rat embryonic mesencephalon cells and the murine neuroblastoma-glioma cell line N18TG2. The resulting interspecies hybrid, named MES23.5, has retained a stable phenotype and karyotype for a continuous culture period of 1 year. The hybrid exhibits several properties that suggest that the parent primary neurons originated in the substantia nigra. The cell line contains tyrosine hydroxylase, which is identifiable both by biochemical and immunological methods and synthesizes dopamine, but no other catecholamine. Additionally, the cell line expresses apparent voltage-gated CA2+ channels as measured by high-affinity omega-conotoxin binding. The MES23.5 omega-conotoxin receptors are of similar affinity class to those found in adult rat mesencephalon. No dihydropyridine receptors, as measured by PN200-100 ligand binding, are present. None of these properties are found in the N18TG2 parent. At least three neuronal features, namely, tyrosine hydroxylase, dopamine synthesis, and omega-conotoxin receptor expression, are quantitatively elevated after sustained treatment with cAMP analogs. The cell line expresses a complex range of neural properties found in the dopaminergic neurons of the substantia nigra, and may therefore be useful elucidating further details of their cell biology.
The Golgi apparatus (complex) is at the center stage of important functions of processing and transport of plasma membrane, lysosomal, and secreted proteins. The involvement of the Golgi apparatus in the pathogenesis of chronic degenerative diseases of neurons is virtually unknown. In the present study, fragmentation and atrophy of the Golgi apparatus of motor neurons in amyotrophic lateral sclerosis (ALS), has been detected with organelle specific antibodies. Approximately 30% of motor neurons in five ALS patients showed a fragmented Golgi apparatus whereas only about 1% of motor neurons from seven controls with neurologic or systemic disease showed a similar change. Morphometric studies are consistent with the hypothesis that the alteration of the Golgi apparatus is an early event in the pathogenesis of the neuronal degeneration in ALS. Immunocytochemical studies with antibodies against alpha tubulin, tau, and phosphorylated subunits of neurofilament polypeptides did not disclose differences in the staining of neurons with fragmented or normal Golgi apparatus, suggesting that the alteration of the organelle is not secondary to a gross lesion of the cytoskeleton. However, these observations do not rule out the hypothesis that the fragmentation of the Golgi apparatus is secondary to subtle changes of the polypeptides involved in the attachment of membranes of the organelle to the cytoskeleton.
Amyotrophic lateral sclerosis (ALS) is a devastating human disease of upper and lower motoneurons of unknown etiology. In support of the potential role of autoimmunity in ALS, two immune-mediated animal models of motoneuron disease have been developed that resemble ALS with respect to the loss of motoneurons, the presence of IgG within motoneurons and at the neuromuscular junction, and with respect to altered physiology of the motor nerve terminal. To provide direct evidence for the primary role of humoral immunity, passive transfer with immunoglobulins from the two animal models and human ALS was carried out. Mice injected with serum or immunoglobulins from the animal disease models and human ALS but not controls demonstrated IgG in motoneurons and at the neuromuscular junction. The mice also demonstrated an increase in miniature end-plate potential (mepp) frequency, with normal amplitude and time course and normal resting membrane potential, indicating an increased resting quantal release of acetylcholine from the nerve terminal. The ability to transfer motoneuron dysfunction with serum immunoglobulins provides evidence for autoimmune mechanisms in the pathogenesis of both the animal models and human ALS.
We studied trophic effects of angiotensin II, vasopressin, cholecystokinin, and oxytocin on explanted ventral spinal cord cultures from 13- and 14-day-old rat embryos. There was a significant neurite promoting effect of the spinal cord cultures by using angiotensin II, vasopressin, and cholecystokinin. Cholecystokinin had the most potent effect at any concentrations. The minimum effective concentration was 10(-8) M in angiotensin II and vasopressin and 10(-12) M in cholecystokinin, respectively. The effect of angiotensin II and vasopressin was dependent on concentrations. However, the rate and grade of neurite appearance did not correlate with the concentrations of cholecystokinin. Oxytocin had no neurotrophic effect at any concentrations. Our results demonstrated that angiotensin II, vasopressin and cholecystokinin have neurotrophic effects on the ventral spinal cord in cultures, and may be candidates for therapeutic trials of amyotrophic lateral sclerosis.
Guinea pigs inoculated with bovine spinal cord ventral horn homogenate develop a syndrome termed experimental autoimmune gray matter disease (EAGMD) characterized by extremity weakness, bulbar signs, and a loss of lower and upper motoneurons. To provide evidence for the role of autoimmune mechanisms, we have administered the immunosuppressant cyclophosphamide prior to and after gray matter immunization. Pretreatment with cyclophosphamide prevented the appearance of clinical signs of disease and decreased the loss of spinal cord motoneurons, the appearance of damaged motoneurons, and the antibody titer to motoneurons. Treatment 7 days after immunization attenuated the expression of disease. Treatment immediately after signs also improved the clinical and pathological findings. In all cyclophosphamide-treated animals there was less IgG within motoneurons and less inflammation. These results support the role for autoimmune mechanisms in motoneuron loss and degeneration in EAGMD.
1. The Vaseline-gap voltage clamp technique was used to record dihydropyridine (DHP)-sensitive Ca2+ currents (ICa) and charge movement in single cut fibres from the rat extensor digitorum longus (EDL) muscle. Amyotrophic lateral sclerosis (ALS) immunoglobulin G (IgG) action on ICa and charge movement has been characterized. 2. ALS IgG reduced ICa amplitude. The peak ICa of EDL fibres (mean +/- S.E.M.) at 0 mV, expressed as amperes per membrane capacitance, was -4.79 +/- 0.029 A F-1, while after 30 min incubation in ALS IgG it was -2.52 +/- 0.04 A F-1. IgG from healthy patients, and from patients with other diseases (familial ALS, myasthenia gravis, chronic relapsing inflammatory polyneuritis, multiple sclerosis and one sample from Lambert-Eaton syndrome, LES) did not affect ICa, while IgG from patients with Guillain-Barré syndrome and one other sample from a patient with LES affected the ICa in a similar way as ALS IgG. 3. The time constant of ICa activation (alpha m) at 0 mV was 44.8 +/- 1.4 ms in control, and 36.6 +/- 1.5 ms after an incubation of 30 min in ALS IgG. The steady-state activation curve (m infinity) was shifted to more positive potentials by ALS IgG. 4. The rate constants of activation (range -20 to 30 mV) were altered by ALS IgG: alpha m decreased while beta m increased. These data suggest that ALS IgG favours the permanence of the Ca2+ channels in the closed state. 5. The time constant of Ca2+ channels deactivation at -90 mV with a pre-pulse to 0 mV was 4.4 +/- 0.5 ms in control and 4.1 +/- 0.6 ms in ALS IgG. The relationship between the deactivation time constant and membrane potential was not significantly modified by ALS IgG. 6. ICa inactivation was not affected by ALS IgG. The potentials of half-inactivation were -32.1 and -36.6 mV in control and ALS IgG, respectively. Similarly, the rate constants of inactivation (alpha h and beta h) remained unaltered by ALS IgG. 7. We successfully blocked ICa with 100 microM-TMB-8 (3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester hydrochloride), without major effects on charge movement. We adopted this procedure to study charge movement. ALS IgG reduced charge movement without significant effects on the effective valence and voltage dependence. Qon and Qoff, the charges during and after the pulse, were similarly affected by ALS IgG. 8. The actions of ALS IgG on DHP-sensitive Ca2+ current and charge movement suggest an interaction between ALS IgG and some component of the DHP-receptor complex.
Amyotrophic lateral sclerosis (ALS) is a devastating human disease of upper and lower motoneurons. We studied the action of the immunoglobulin G (IgG) from ALS and disease control patients on dihydropyridine (DHP)-sensitive Ca2+ channels in single mammalian skeletal muscle fibers with the double Vaseline gap technique. The peak of the Ca2+ current (ICa) and the charge movement were reduced when the fibers were incubated in ALS IgG. These effects were lost when the IgG was boiled or adsorbed with skeletal tubular membranes. ALS IgG reduced skeletal muscle ICa in a similar fashion as nifedipine; the ICa blockade was voltage dependent, and the associated charge movement was reduced. These observations suggest that IgG from ALS patients reacts with the skeletal muscle DHP-sensitive Ca2+ channels or some associated regulatory moiety.
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