Luis V. Colom

Role of potassium channels in amyloid-induced cell death

Abstract: Basal forebrain cholinergic neurons are severely depleted early in Alzheimers disease and appear particularly susceptible to amyloid β‐peptide (Aβ) toxicity in vivo. To model this effect in vitro, a cholinergic septal cell line (SN56) was exposed to Aβ. SN56 cells exhibited a tetraethylammonium (TEA)‐sensitive outward K+ current with delayed rectifier characteristics. Increases of 64% (±19; p < 0.02) and 44% (±12; p < 0.02) in K+ current density were noted 6–12 and 12–18 h following the addition of Aβ to SN56 cell cultures, respectively. Morphological observation and staining for cell viability showed that 25 ± 4 and 39 ± 4% of SN56 cells were dead after 48‐ and 96‐h exposures to Aβ, respectively. Perfusion of SN56 cells with 10–20 mM TEA blocked 71 ± 6 to 92 ± 2% of the outward currents, widened action potentials, elevated [Ca2+]i, and inhibited 89 ± 14 and 68 ± 14% of the Aβ toxicity. High [K+]o, which depolarizes cell membranes and increases [Ca2+]i, also protected SN56 cells from Aβ toxicity. This effect appeared specific since glucose deprivation of SN56 cells did not alter K+ current density and TEA did not protect these cells from hypoglycemic cell death. Furthermore, Aβ was toxic to a dopaminergic cell line (MES23.5) that expressed a K+ current with delayed rectifier characteristics; K+ current density was not altered by Aβ and MES23.5 cells were not protected by TEA from Aβ toxicity. In contrast, a noncholinergic septal cell line (SN48) that shows minimal outward K+ currents was resistant to the toxicity of Aβ. These data suggest that a K+ channel with delayed rectifier characteristics may play an important role in Aβ‐mediated toxicity for septal cholinergic cells.

Cell death induced by β-amyloid 1-40 in MES 23.5 hybrid clone: the role of nitric oxide and NMDA-gated channel activation leading to apoptosis

The molecular events associated with beta-amyloid-induced neuronal injury remain incompletely characterized. Using a substantia nigra/neuroblastoma hybrid cell line (MES 23.5) synthetic beta-amyloid 1-40 induced a time and dose-dependent apoptotic cell death which was characterized by cell shrinkage and fragmentation of DNA, and was inhibited by aurintricarboxylic acid (ATA), and cycloheximide (CHX). Following beta-amyloid 1-40 treatment, cyclic GMP, an index of NO synthesis, was increased in MES 23.5 cells. The NO scavenger hemoglobin, as well as the NO synthase inhibitors NG-monomethyl-L-arginine acetate (L-NMMA) and L-N5-(1-iminoethyl)ornithine hydrochloride (L-NI0) attenuated such increases. These same inhibitors and scavengers also significantly prevented cytotoxicity. beta-Amyloid also induced an early and transient increase in intracellular calcium as monitored with laser scanning confocal microscopy and Fluo-3 imaging. These induced calcium transients could be significantly blocked by the N-methyl-D-aspartic acid (NMDA) receptor antagonist MK-801. Pretreatment with MK-801 or removal of extracellular Ca2+ also reduced beta-amyloid-induced NO production and neurotoxicity. Furthermore, beta-amyloid neurotoxicity was greatly enhanced in the absence of Mg2+ or in the presence of glutamate or NMDA. These data suggest that beta-amyloid can lead to apoptotic cell death through a NO mediated process possibly triggered by Ca2+ entry through activated NMDA-gated channels.

Selective localization of cardiac SCN5A sodium channels in limbic regions of rat brain.

Voltage-dependent sodium channels have a critical role in membrane electrogenesis and repetitive firing in excitable cells. Members of this gene superfamily exhibit diffuse, tissue-specific expression in nervous, muscle and cardiac tissues. Tetrodotoxin (TTX) sensitivity and resistance to zinc blockade were thought to distinguish brain from heart sodium channels, but neuronal Na+ currents with heart-like properties have been found in brain.

Brainstem sites for the carbachol elicitation of the hippocampal theta rhythm in the rat

The effects of brainstem microinjections of carbachol on the hippocampal theta rhythm were examined in urethane anesthetized rats. The two most effective theta-eliciting sites with carbachol were the nucleus pontis oralis (RPO) and the acetylcholine-containing pedunculopontine tegmental nucleus (PPT) of the dorsolateral pontine tegmentum. RPO injections generated theta at mean latencies of 38.5±70.8 s and for mean durations of 12.9±5.1 min. Five of seven RPO injections gave rise to theta virtually instantaneously, i.e., before the completion of the injection. PPT injections generated theta at mean latencies of 1.7±1.1 min and for mean durations of 11.9±6.0 min. Injections rostral or caudal to RPO in the caudal midbrain reticular formation (RF) or the caudal pontine RF (nucleus pontis caudalis) generated theta at considerably longer latencies (generally greater than 5 min) or were without effect. Medullary RF injections essentially failed to alter the hippocampal EEG. The finding that theta was produced at very short latencies at RPO suggests that RPO, the putative brainstem source for the generation of theta, is modulated by a cholinergic input. The further demonstration that theta was also very effectively elicited with PPT injections suggests this acetylcholine-containing nucleus of the dorsolateral pons may be a primary source of cholinergic input to RPO in the generation of theta. The hippocampal theta rhythm is a major event of REM sleep. The present results are consistent with earlier work showing that each of the other major events of REM sleep, as well as the REM state, are cholinergically activated at the level of the pontine tegmentum.

Apoptotic Cell Death of a Hybrid Motoneuron Cell Line Induced by Immunoglobulins from Patients with Amyotrophic Lateral Sclerosis

Abstract: 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.

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 Ca (2+) 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 Ca2+-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. NEUROLOGY 1996;47(Suppl 2): S40-S46

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.

Modulation of septal cell activity by extracellular zinc.

ZINC released from axon terminals in the brain can interact with multiple membrane channels and receptors. However, the specific effects of these Zn2+-dependent interactions on physiological processes remains unclear. Because Zn2+-containing axon terminals are abundant in the septal region, we selected a septal cell line (SN56) to study the effects of Zn2+ on cell activity. Voltage-clamp recordings showed welldeveloped voltage-dependent Na+, Ca2+ and K+ currents. Micromolar concentrations of Zn2+ partially blocked Na+ and Ca2+ currents without affecting K+ currents. Current-clamp recordings showed that SN56 cells fire spontaneous and evoked action potentials. While most (≥ 83%) Na+ and Ca2+ currents were blocked with 1 μM tetrodotoxin (TTX) and 2 mM Co2+, action potentials persisted after either 1 μM TTX or 2 mM Co2+ application. In contrast, concentrations of Zn2+ (50–300 μM) that induced incomplete blockade (≤ 50%) of either Ca2+ and Na+ currents abolished action potential generation. These data show that simultaneous and partial blockade of Ca2+ and Na+ channels by Zn2+ inhibit SN56 cell activity. Because septal outputs extensively modulate the excitability of cortical and subcortical brain regions, Zn2+ inhibition of action potential generation in septal neurons could play an important physiological role in regulating brain activity.