Parvalbumin overexpression alters immune-mediated increases in intracellular calcium, and delays disease onset in a transgenic model of familial amyotrophic lateral sclerosis.

Intracellular calcium is increased in vulnerable spinal motoneurons in immune-mediated as well as transgenic models of amyotrophic lateral sclerosis (ALS). To determine whether intracellular calcium levels are influenced by the calcium-binding protein parvalbumin, we developed transgenic mice overexpressing parvalbumin in spinal motoneurons. ALS immunoglobulins increased intracellular calcium and spontaneous transmitter release at motoneuron terminals in control animals, but not in parvalbumin overexpressing transgenic mice. Parvalbumin transgenic mice interbred with mutant SOD1 (mSOD1) transgenic mice, an animal model of familial ALS, had significantly reduced motoneuron loss, and had delayed disease onset (17%) and prolonged survival (11%) when compared with mice with only the mSOD1 transgene. These results affirm the importance of the calcium binding protein parvalbumin in altering calcium homeostasis in motoneurons. The increased motoneuron parvalbumin can significantly attenuate the immune-mediated increases in calcium and to a lesser extent compensate for the mSOD1-mediated 'toxic-gain-of-function' in transgenic mice.

Immune reactivity in a mouse model of familial ALS correlates with disease progression.

OBJECTIVE: The cause of motor neuron death in ALS is incompletely understood. This study aims to define the potential involvement of nonneuronal immune-inflammatory factors in the destruction of motor neurons in mutant superoxide dismutase-1 (SOD1) transgenic mice as a model of ALS. BACKGROUND: The presence of activated microglia, IgG and its receptor for Fc portion (FcgammaRI), and T lymphocytes in the spinal cord of both patients with ALS and experimental animal models of motor neuron disease strongly suggests that immune-inflammatory factors may be actively involved in the disease process. METHODS: The expression of immune-inflammatory factors was followed in both human mutant (G93A) SOD1 transgenic mice and human wild-type SOD1 transgenic mice, at different ages (40, 80, and 120 days). Fixed, frozen, free-floating sections of the lumbar spinal cord were stained with antibodies against CD11b, IgG, FcgammaRI, intercellular adhesion molecule-1 (ICAM-1), CD3, and glial fibrillary acidic protein. RESULTS: The earliest change observed was the upregulation of ICAM-1 in the ventral lumbar spinal cord of 40-day-old mutant SOD1 mice. IgG and FcgammaRI reactivities were detected on motor neurons as early as 40 days and on microglial cells at later stages. Microglial activation was first evident in the ventral horn at 80 days, whereas reactive astrocytes and T cells became most prominent in 120-day-old mutant SOD1 mice. CONCLUSION: The upregulation of proinflammatory factors during early presymptomatic stages as well as the expansion of immune activation as disease progresses in mutant SOD1 transgenic mice suggest that immune-inflammatory mechanisms could contribute to disease progression.

Ultrastructural evidence of calcium involvement in experimental autoimmune gray matter disease.

Experimental studies have suggested that increased calcium and inappropriate calcium handling by motoneurons might have a significant role in motoneuron degeneration. To further define the involvement of calcium in motoneuron loss we used the oxalate-pyroantimonate technique for calcium fixation and monitored the ultrastructural distribution of calcium in spinal motoneurons in experimental autoimmune gray matter disease (EAGMD). In cervical and hypoglossal motoneurons from animals with relatively preserved upper extremity and bulbar function, increased calcium precipitates were present in the cytoplasm as well as in mitochondria, endoplasmic reticulum and Golgi complex without significant morphologic alterations. In surviving lumbar motoneurons of animals with hindlimb paralysis, however, there was massive morphological destruction of intracellular organelles but no significant accumulation of calcium precipitates. These findings suggest that altered calcium homeostasis is involved in motoneuron immune-mediated injury with increased calcium precipitates early in the disease process and decreased to absent calcium precipitates later in the pathogenesis of motoneuron injury.

Experimental destruction of substantia nigra initiated by Parkinson disease immunoglobulins.

BACKGROUND: Increased levels of free radicals and oxidative stress may contribute to the pathogenesis of substantia nigra (SN) injury in Parkinson disease (PD), but the initiating etiologic factors remain undefined in most cases. OBJECTIVE: To determine the potential importance of immune mechanisms in triggering or amplifying neuronal injury, we assayed serum samples from patients with PD to determine the ability of IgG to initiate relatively specific SN injury in vivo. METHODS: IgG purified from the serum of 5 patients with PD and 10 disease control (DC) patients was injected into the right side of the SN in adult rats. Coronal sections were cut from the whole brain at the level of the stereotaxic injections, stained for tyrosine hydroxylase and with cresyl violet, and cellular profiles were counted in identical brain regions at the injection and contralateral sides. The ratio of cell profile counts of the corresponding injected and uninjected regions was used as an internal standard. RESULTS: Four weeks following injection of IgG, a 50% decrease in tyrosine hydroxylase-positive cellular profiles was noted on the injected sides compared with the contralateral sides of the same animals. Similarly, applied DC IgG caused only an 18% decrease. Cresyl violet staining revealed a 35% decrease in neuronal profiles of PD IgG injected into the SN pars compacta compared with the contralateral uninjected side, whereas DC IgG caused a minimal 10% decrease. Even at 4 weeks after the PD IgG injections, perivascular inflammation and significant microglial infiltration were present near injured SN pars compacta neurons. No cytotoxic effects of PD IgG were noted in choline acetyltransferase-positive neurons after stereotaxic injections into the medial septal region. Absorption of PD IgG with mesencephalic membranes and protein A agarose gel beads removed cytotoxicity, while absorption with liver membranes did not change the cytotoxicity. CONCLUSIONS: Our data suggest that PD IgG can initiate a relatively specific inflammatory destruction of SN pars compacta neurons in vivo and demonstrate the potential relevance of immune mechanisms in PD.

Intracellular calcium parallels motoneuron degeneration in SOD-1 mutant mice.

Transgenic mice with Cu,Zn superoxide dismutase (SOD-1) mutations provide a unique model to examine altered Ca homeostasis in selectively vulnerable and resistant motoneurons. In degenerating spinal motoneurons of G93 A SOD-1 mice, developing vacuoles were filled with calcium, while calcium was gradually depleted from the cytoplasm and intact mitochondria. In oculomotor neurons, no degenerative changes, vacuolization, or increased calcium were noted. Motor axon terminals of interosseus muscle gradually degenerated and intracellular calcium was depleted. Oculomotor terminals of mutant SOD-1 mice were smaller and exhibited no degenerative changes, but did exhibit unique membrane-enclosed organelles containing calcium. Spinal motoneurons of SOD-1 mice were shown to have fewer calcium binding proteins, such as parvalbumin, compared with oculomotor neurons. These data suggest that the SOD-1 mutation is associated with impaired calcium homeostasis in motoneurons in vivo, with increased likelihood of degeneration associated with higher levels of intracellular calcium and lower to absent levels of calbindin-D28K and/or parvalbumin, and decreased likelihood of degeneration associated with minimally changed calcium and ample calbindin-D28K and/or parvalbumin.

1Alpha, 25 dihydroxyvitamin D3-dependent up-regulation of calcium-binding proteins in motoneuron cells.

Our understanding of selective neuronal vulnerability as well as etiopathogenesis of sporadic neurodegenerative diseases is extremely limited. In ALS, altered calcium homeostasis appears to contribute significantly to selective neuronal injury. Further in ALS, the absence of calcium binding proteins (calbindin-D28K, parvalbumin, and calretinin) correlates with selective vulnerability and cell loss. In motoneuron cell culture models an ALS IgG-triggered and calcium-mediated destruction can be reversed by increased expression of calbindin-D28K following retroviral infection with calbindin-D28K cDNA. To increase calcium binding protein expression in motoneurons in vitro and in vivo, we have employed vitamin D3. Forty-eight hr treatment of differentiated VSC 4.1 cells with 0.1-30 nM 1,25 dihydroxyvitamin D3 induced a two-fold increase in the immunoreactivity for calbindin-D28K and parvalbumin. Injection of 80-120 ng, 1,25 dihydroxyvitamin D3 in the cerebral ventricles of adult rats also induced positive immunoreactivity for calcium binding proteins in ventral motoneurons which are completely devoid of such reactivity in the adult stage. These data suggest that analogs of 1,25 dihydroxyvitamin D3 may be useful tools in enhancing the expression of calcium binding proteins in the motor system and may have possible therapeutic value in neurodegenerative disease.

Amyotrophic lateral sclerosis immunoglobulins increase intracellular calcium in a motoneuron cell line.

A hybrid motoneuron cell line (VSC4.1) was used as a model system to study the relationship between alterations in intracellular calcium and subsequent cell death induced by immunoglobulin fractions purified from sera of patients with ALS. Using fluo-3 fluorescence imaging, immunoglobulins from 8 of 10 patients with ALS were found to induce transient increases in intracellular calcium ([Ca2+]i) in differentiated VSC4.1 cells. These transient [Ca2+]i increases required extracellular calcium entry through voltage-gated calcium channels sensitive to synthetic FTX and to high concentrations (>1 microM) of omega-agatoxin IVa. The incidence of transient [Ca2+]i increases induced by ALS immunoglobulins correlated with the extent of cytotoxicity induced by the same ALS immunoglobulins in parallel cultures of VSC4.1 cells. Furthermore, manipulations which blocked transient [Ca2+]i increases (addition of synthetic FTX or omega-agatoxin IVa) also inhibited the cytotoxic effects of ALS immunoglobulins. No transient calcium increases were observed in VSC4.1 cells following addition of immunoglobulins from 7 neurologic disease control patients. However, transient [Ca2+]i increases were observed following addition of immunoglobulins from 4 of 5 patients with myasthenia gravis (MG). The [Ca2+]i changes induced by MG immunoglobulins were not blocked by s-FTX, suggesting that they result from a different mechanism than those induced by ALS immunoglobulins. These results suggest that immunoglobulins from patients with ALS can induce transient increases in intracellular calcium in a motoneuron cell line, which may represent early events in the cascade of processes leading to injury and death of susceptible cells.

Autoimmunity and ALS.

Significant evidence has accrued suggesting that antibodies to voltage-gated calcium channel are observed in at least some patients with sporadic ALS (SALS) and that such antibodies alter the function of these ion channels in vitro and in vivo. Further, passive transfer of these immunoglobulin-containing fractions into mice produces changes at the neuromuscular junction that are very similar to changes observed in patients with SALS. These changes reflect local alterations in intracellular Ca2+ homeostasis and, in animal models, may also evidence early changes of motoneuron injury, such as Golgi apparatus swelling and fragmentation. Although not yet documented to induce motoneuron death in vivo, SALS immunoglobulins induce Ca(2+)-dependent apoptosis in a differentiated motoneuron hybrid cell line via a mechanism that involves oxidative injury. SALS immunoglobulin-mediated apoptosis in these cells is regulated by the presence of the same calcium-binding proteins that may modulate selective motoneuron vulnerability in SALS.

Expression of calbindin-D28K in motoneuron hybrid cells after retroviral infection with calbindin-D28K cDNA prevents amyotrophic lateral sclerosis IgG-mediated cytotoxicity.

Calbindin-D28K and/or parvalbumin appear to influence the selective vulnerability of motoneurons in amyotrophic lateral sclerosis (ALS). Their immunoreactivity is undetectable in motoneurons readily damaged in human ALS, and in differentiated motoneuron hybrid cells [ventral spinal cord (VSC 4.1 cells)] that undergo calcium-dependent apoptotic cell death in the presence of ALS immunoglobulins. To provide additional evidence for the role of calcium-binding proteins in motoneuron vulnerability, VSC 4.1 cells were infected with a retrovirus carrying calbindin-D28K cDNA under the control of the promoter of the phosphoglycerate kinase gene. Differentiated calbindin-D28K cDNA-infected cells expressed high calbindin-D28K and demonstrated increased resistance to ALS IgG-mediated toxicity. Treatment with calbindin-D28K antisense oligodeoxynucleotides, which significantly decreased calbindin-D28K expression, rendered these cells vulnerable again to ALS IgG toxicity.

Apoptosis induced by beta-N-oxalylamino-L-alanine on a motoneuron hybrid cell line.

It has been suggested that beta-N-oxalylamino-L-alanine, a non-protein amino acid present in the Lathyrus Sativus seeds, may play a role in the etiopathogenesis of neurolathyrism, a toxic form of motor neuron disease clinically characterized by a severe spastic paraparesis. In order to investigate the mechanisms of beta-N-oxalylamino-L-alanine-mediated cell death, we studied the effect of this neurotoxin as well as other excitatory amino acids agonists on the growth and survival of motoneuron hybrid ventral spinal cord 4.1 cells. beta-N-oxalylamino-L-alanine was toxic to ventral spinal cord 4.1 cells in a concentration-dependent fashion (0.5-10 mM). Among the excitatory amino acids tested, only glutamate (1-10 mM), quisqualate (1 mM) and, with less extent, beta-N-methylamino-L-alanine (10 mM) induced a significant reduction of cell survival. The effect of Lathyrus Sativus neurotoxin was a slow process, becoming apparent only after 24-48 h of incubation. Interestingly, a mathematical analysis applied to the time course and dose curve of beta-N-oxalylamino-L-alanine toxicity suggested that even for very low concentrations of the amino acid it is theoretically possible to predict a time-dependent effect. The cell death was not blocked by antagonists of N-methyl-D-aspartate or non-N-methyl-D-aspartate receptors; aurintricarboxylic acid and alpha-tocopherol gave a partial protection; cysteine (1 mM) prevented the toxic effect of both Lathyrus Sativus neurotoxin and glutamate as well as quisqualate. Morphologically, in the presence of either beta-N-oxalylamino-L-alanine, glutamate or quisqualate, ventral spinal cord 4.1 cells showed apoptotic features also confirmed by ISEL technique and agarose gel electrophoresis of genomic DNA. Thus, our results suggest that in ventral spinal cord 4.1 motoneuron hybrid cells, in the absence of functional synaptic excitatory amino acid receptors, beta-N-oxalylamino-L-alanine induces cell degeneration through an apoptotic mechanism, possibly mediated by a block of cystine/glutamate Xc antiporter.

The role of immune processes in amyotrophic lateral sclerosis pathogenesis.

Despite many efforts, the etiopathogenesis of ALS remains unknown. During the last decade evidence for an autoimmune involvement in motoneuron degeneration and death has remarkably increased. Multiple reports have documented significant expression of proteins associated with immune function in affected areas of ALS patients. Two animal models of immune-mediated motoneuron destruction have been developed that closely resemble clinical, electrophysiological and morphological features of human ALS. Inflammatory foci within the spinal cord, and IgG at the neuromuscular junction as well as within upper and lower motoneurons found in the animal models support the role of autoimmune mechanisms of motoneuron destruction in this model. IgG from ALS patients and from the animal models can passively transfer physiological changes at the neuromuscular junction in mice. That ALS IgG interact with calcium channels and induce an alteration of their function is now electrophysiologically and biochemically evident. Furthermore, it has been documented that motoneurons may be selectively vulnerable since they have a deficient calcium buffering capacity. Although further research efforts are necessary to elucidate the interaction of the ALS antibodies with the calcium channel function and how defective calcium handling by the motoneurons is important in their degeneration, the current data strongly suggest the involvement of autoimmune mechanisms in ALS etiopathogenesis.

Tomaculous neuropathy with unusual clinical aspects.

Tomaculous neuropathy represents the morphological substrate of the recurrent familial neuropathy with liability to pressure palsies. Some ultrastructural changes characterizing the tomaculous neuropathy can occur as incidental aspects in other different neuropathies. Few tomaculous neuropathy cases with clinical aspect of chronic polyneuropathy without paretic episodes have been mentioned in the literature. In the present work, we report four cases who offered the morphological surprise of a true tomaculous neuropathy with 15-37% of the teased fibres bearing tomaculae sized: 55-106 microns/20-23 microns, on the background of a demyelinating neuropathy with 25-56% of the teased fibres showing segmental de- or remyelination. The clinical and electrophysiological diagnoses of these 4 patients were: HSMN type I (2 cases), HSMN type VIII (polyneuropathy associated with a cerebello-extrapyramidal syndrome -1 case), and a neurogenic scapuloperoneal syndrome (1 case). The specificity of the tomaculous neuropathy is discussed.

Amyotrophic lateral sclerosis immunoglobulins increase Ca2+ currents in a motoneuron cell line.

The sporadic form of amyotrophic lateral sclerosis (ALS) is an idiopathic and eventually lethal disorder causing progressive degeneration of cortical and spinal motoneurons. Recent studies have shown that the majority of patients with sporadic ALS have serum antibodies that bind to purified L-type voltage-gated calcium channels and that antibody titer correlates with the rate of disease progression. Furthermore, antibodies purified from ALS patient sera have been found to alter the physiologic function of voltage-gated calcium channels in nonmotoneuron cell types. Using whole-cell patch-clamp techniques, immunoglobulins purified from sera of 5 of 6 patients with sporadic ALS are now shown to increase calcium currents in a hybrid motoneuron cell line, VSC4.1. These calcium currents are blocked by the polyamine funnel-web spider toxin FTX, which has previously been shown to block Ca2+ currents and evoked transmitter release at mammalian motoneuron terminals. These data provide additional evidence linking ALS to an autoimmune process and suggest that antibody-induced increases in calcium entry through voltage-gated calcium channels may occur in motoneurons in this disease, with possible deleterious effects in susceptible neurons.

The role of calcium-binding proteins in selective motoneuron vulnerability in amyotrophic lateral sclerosis.

The factors contributing to selective motoneuron loss in amyotrophic lateral sclerosis (ALS) remain undefined. To investigate whether calcium-binding proteins contribute to selective motoneuron vulnerability in ALS, we compared calbindin-D28K and parvalbumin immunoreactivity in motoneuron populations in human ALS, and in a ventral spinal cord hybrid cell line selectively vulnerable to the cytotoxic effects of ALS IgG. In human autopsy specimens, immunoreactive calbindin-D28k and parvalbumin were absent in motoneuron populations lost early in ALS (i.e., cortical and spinal motoneurons, lower cranial nerve motoneurons), while motoneurons damaged late or infrequently in the disease (i.e., Onuf's nucleus motoneurons, oculomotor, trochlear, and abducens nerve neurons) expressed markedly higher levels of immunoreactive calbindin-D28K and/or parvalbumin. Motoneuron-neuroblastoma VSC 4.1 hybrid cells lost immunoreactive calbindin-D28k and parvalbumin following dibutyryl-cyclic AMP-induced differentiation and were killed by IgG from ALS patients. Undifferentiated calbindin/parvalbumin-reactive VSC 4.1 cells were not killed, nor were other cell lines expressing high levels of calbindin-D28K and parvalbumin immunoreactivity (substantia nigra-neuroblastoma hybrid cells and N18TG2 neuroblastoma parent cells). These studies suggest that decreased calbindin-D28K and parvalbumin immunoreactivity may help explain the selective vulnerability of motoneurons in ALS.

Apoptotic cell death of a hybrid motoneuron cell line induced by immunoglobulins from patients with amyotrophic lateral sclerosis.

Apoptotic cell death has recently been implicated in diseases involving nonproliferating, terminally differentiated cells such as neurons. Previous experiments have documented that immunoglobulins from patients with amyotrophic lateral sclerosis (ALS) can kill motoneuron-neuroblastoma hybrid cells [ventral spinal cord 4.1 (VSC 4.1)] by a calcium-dependent process. Here, we studied the mechanism of ALS IgG-induced cell death. In the presence of ALS IgG the VSC 4.1 cells undergo cell shrinkage and membrane blebbing, which are morphological features of apoptotic cell death. The damaged cells can be identified by in situ end labeling of nicked DNA and biochemically show laddering on agarose gel electrophoresis. This ALS IgG-triggered process is prevented by cycloheximide, aurintricarboxylic acid, and zinc sulfate. These data demonstrate that immunoglobulins from patients with ALS are able to induce apoptosis in motoneuron hybrid cells and provide a potential mechanism for motoneuron degeneration in human ALS.

Cytotoxicity of immunoglobulins from amyotrophic lateral sclerosis patients on a hybrid motoneuron cell line.

Patients with amyotrophic lateral sclerosis possess antibodies (ALS IgGs) that bind to L-type skeletal muscle voltage-gated calcium channels (VGCCs) and inhibit L-type calcium current. To determine whether interaction of ALS IgGs with neuronal VGCCs might influence motoneuron survival, we used a motoneuron-neuroblastoma hybrid (VSC 4.1) cell line expressing binding sites for inhibitors of L-, N-, and P-type VGCCs. Using direct viable cell counts, quantitation of propidium iodide- and fluorescein diacetate-labeled cells, and lactate dehydrogenase release to assess cell survival, we document that ALS IgG kills 40-70% of cAMP-differentiated VSC 4.1 cells within 2 days. ALS IgG-mediated cytotoxicity is dependent on extracellular calcium and is prevented by peptide antagonists of N- or P-type VGCCs but not by dihydropyridine modulators of L-type VGCCs. Preincubating IgG with purified intact L-type VGCC or with isolated VGCC alpha 1 subunit also blocks ALS IgG-mediated cytotoxicity. These results suggest that ALS IgG may directly lead to motoneuron cell death by a mechanism requiring extracellular calcium and mediated by neuronal-type calcium channels.

Renaut bodies contain elastic fiber components.

Renaut bodies (RB) are fusiform endoneurial structures preferentially found at sites of nerve entrapment, often occupying more than 30% of the cross-sectional area of a nerve fascicle. Their composition and significance, however, are still incompletely understood. In this study, further evidence for the link between the appearance of RB and nerve entrapment is presented. Reanaut bodies were already found at the age of 1 year in the median nerve at the level of the wrist, i.e. in the carpal tunnel, a possible site of entrapment. Here, their number increased with age. Renaut bodies were absent, however, in fetal nerves at this site. Many of the cells in RB resembled perineurial cells or pericytes. They were stained with antibodies against vimentin and epithelial membrane antigen and were partially covered by a basal lamina reactive with antibodies against collagen IV, laminin, and s-laminin. Focally accumulated filaments and bundles of 30-40 nm collagen fibrils were major extracellular components of RB. The diameter of the filaments (8-12 nm) corresponded to the size of the microfibril, i.e. the oxytalan component of elastic fibers. Renaut bodies were intensely stained with antibodies against these microfibrils and several types of collagen glycoproteins. On the basis of these results, we conclude that RB are composed of cells that show perineurial differentiation. These cells produce an extracellular matrix highly enriched in elastic fiber components.

Chronic sensorimotor polyneuropathy associated with cerebello-extrapyramidal manifestations in a patient with tomaculous neuropathy.

Clinical, electrophysiological and morphological (sural nerve and gastrocnemius muscle biopsies) data of a 57-year-old man with a chronic sensorimotor polyneuropathy of Charcot-Marie-Tooth type associated with a progressive cerebello-extrapyramidal syndrome are reported. Patient's family data were negative. Nerve structural and ultrastructural examinations revealed the morphological picture of a tomaculous neuropathy. The association of different clinical syndromes and the specificity of the tomaculous neuropathy are discussed.

Neurotrophic trigeminal syndrome after pontine stroke.

The present study describes a patient, aged 72 yrs, with pontine crossed syndrome, displaying ipsilateral to the lesion a syndrome of cranial nerves V, VI, VII, VIII, on the right; a right neocerebellar syndrome; anhidrosis in the right hemibody; contralateral to the lesion: thermoalgic anesthesia with hyperhidrosis in the left hemibody. Concomitant with the pontine lesion, the patient developed torpid trophic ulcerations all over the right hemiface which, in the course of one year, became deformed, retractile, depigmented of hyperpigmented scars, the erosion being so deep at the level of the right nasal wing that the latter disappeared. This clinical picture developed in a patient with basilar invagination of 3 mm and left humeral micromelia. Eight cases of trigeminal torpid trophic ulceration associated with lesions of the brain stem are known till now, but association with vascular pontine lesion has not been described as yet. Etiopathogenic considerations are made.