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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.
Amyotrophic lateral sclerosis (ALS) is characterized by extensive loss of lower motoneurons in the spinal cord and brain stem, atrophy of the ventral roots, and degeneration of upper cortical motoneurons and the corticospinal tract (Hirano 1991; Leigh and Swash 1991). The specific etiologies and mechanisms leading to this pathological motoneuron injury and cell loss in ALS are still being resolved. Among the various proposals, increased intracellular calcium, increased glutamate excitotoxicity, and increased free radicals have received the most attention. Such perturbations could critically impair motoneuron structures such as mitochondria and/or neurofilaments, and compromise energy production and axoplasmic flow, impairing synaptic function. These mechanisms are not mutually exclusive, and increased intracellular calcium could be a common denominator.
In neurons, intracellular calcium is tightly regulated, and marked increases are associated with cell degeneration (Choi 1992). In ALS, calcium is increased within motor nerve terminals of biopsied human muscle specimens, as well as in motor nerve terminals of mice following passive transfer of ALS immunoglobulin G (IgG) (Engelhardt et al. 1995; Siklós et al. 1996; Pullen and Humphrey 2000). Calcium homeostasis is impaired in an immune-mediated model of motoneuron cell loss in guinea pigs (Engelhardt et al. 1991; Alexianu et al. 2000). Calcium is also increased in vesicular structures within spinal motoneurons of transgenic mice expressing mutant human Cu2+/Zn2+ superoxide dismutase (mSOD1), an enzyme involved in oxygen free radical metabolism (Deng et al. 1993; Rosen et al. 1993; Gurney et al. 1994; Siklós et al. 1998). Decreased function of a glial glutamate transporter in human ALS tissue and transgenic mSOD1 mice has suggested the possibility of increased glutamate excitotoxicity, a process believed to be mediated by increased intracellular calcium (Lin et al. 1998; Rothstein et al. 1992; Ferrante et al. 1997). Thus, regardless what may have initiated the process, increased intracellular calcium is present in motoneurons vulnerable to degeneration.
As increased intracellular calcium may be associated with degeneration in diverse neurodegenerative disorders, a key question is why motoneurons in ALS should be selectively vulnerable to an increased calcium load. A possible explanation may be related to their low expression of the calcium-binding proteins calbindin-D28K and/or parvalbumin (Celio 1990). Motoneurons relatively deficient in the calcium-binding proteins calbindin-D28K and/or parvalbumin (e.g. spinal and hypoglossal motoneurons) are lost early in ALS; whereas motoneurons expressing high levels of these proteins (cranial nerves III, IV, VI, and Onuf's nucleus motoneurons) are relatively spared (Ince et al. 1993; Alexianu et al. 1994; Elliott and Snider 1995). Extraocular motoneurons from mSOD1 transgenic mice, which express abundant levels of calbindin-D28K and/or parvalbumin, are less likely to degenerate as motoneuron disease develops (Siklós et al. 1998; Nimchinsky et al. 2000). Further, oculomotor neurons which possess ample parvalbumin in vivo, have five- to sixfold larger calcium ‘buffering’ capacity (Vanselow and Keller 2000) and specialized mechanisms to maintain calcium homeostasis compared with vulnerable spinal and hypoglossal motoneurons (Siklós et al. 1999). Thus, the inability to handle an increased Ca2+ load, associated with low levels of calbindin-D28K and/or parvalbumin may contribute to selective vulnerability of motoneurons in ALS.
To determine whether increased expression of parvalbumin in motoneurons can modulate calcium perturbations and influence calcium-dependent processes in vivo, we generated transgenic mice expressing rat parvalbumin within motoneurons under control of the rat calmodulin II (CaMII) promoter. We then determined the morphological and physiological effects in motoneurons of increased expression of parvalbumin on calcium increased by the intraperitoneal injection of ALS immunoglobulins. To test the functional effects of increased parvalbumin on mutant SOD1-mediated disease, parvalbumin transgenic mice were bred with mutant SOD1 (G93A) transgenic mice; and both disease onset and survival were monitored in progeny expressing both transgenes compared with progeny expressing only the mSOD1 transgene.
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The calcium-binding proteins calbindin-D28K and parvalbumin have been demonstrated to protect motoneurons against human mSOD1-mediated and ALS IgG-induced cell death in vitro (Ho et al. 1996; Roy et al. 1998), but effects in vivo and possible mechanism(s) of protection have not been characterized. In the present study, we demonstrated that parvalbumin is transcriptionally and translationally expressed in the spinal cords of mice possessing a parvalbumin transgene coupled with a CaMII promoter by in situ hybridization and immunohistochemistry, respectively. Furthermore, parvalbumin mRNA and protein were expressed in large spinal motoneurons that are particularly vulnerable to increased calcium concentrations and degeneration in ALS. This increased expression of parvalbumin in motoneurons completely blocked the ability of ALS immunoglobulins to increase calcium within motor axon terminals, as assayed by the oxalate–pyroantimonate morphological technique. Increased expression of parvalbumin also blocked the ability of ALS immunoglobulins to increase spontaneous acetylcholine release, a calcium-mediated physiological process monitored as MEPP frequency. Thus, spinal motoneurons in parvalbumin transgenic animals are able to prevent the increases in intracellular calcium induced by ALS immunoglobulins.
ALS immunoglobulins were used to increase intracellular calcium based on our demonstration that they could increase calcium currents in a differentiated motoneuron cell line (VSC4.1) in vitro, as assayed by whole-cell patch-clamp techniques (Mosier et al. 1995) as well as by fluo-3 fluorescence imaging (Colom et al. 1997). Transfection of this cell line with calbindin-D28K cDNA under control of a phosphoglycerate kinase promoter protected against Ca2+-dependent cytotoxicity (Ho et al. 1996). ALS immunoglobulins can also increase motoneuron calcium in axon terminals in vivo as monitored with the oxalate–pyroantimonate technique (Engelhardt et al. 1995; Pullen and Humphreys 2000). Further, mice injected with ALS sera or immunoglobulins demonstrate increased MEPP frequency with normal amplitude, time course, and resting membrane potential, indicating an increased resting quantal release of acetylcholine from the nerve terminal (Appel et al. 1991). These results have been replicated in our own laboratory (Mosier et al. 2000) as well as two other laboratories (O'Shaughnessy et al. 1998; Fratantoni et al. 2000). The oculomotor neuron, which is relatively resistant to degeneration in ALS and has ample parvalbumin expression, is also resistant to the calcium-increasing effects of ALS immunoglobulins (Mosier et al. 2000).
Calbindin-D28K and parvalbumin appear to exert their neuronal effects by altering intracellular calcium homeostasis, but their precise physiological functions remain to be clarified. In a transfected neuroblastoma cell line, parvalbumin overexpression suppressed large depolarization-induced increases in calcium by effectively buffering calcium in the intracellular environment (Dreessen et al. 1996). When transiently injected into primary rat dorsal root ganglion neurons, both calbindin-D28K and parvalbumin significantly attenuate depolarization-induced peak intracellular calcium concentrations, but neither protein changed the basal calcium level, or altered the inactivation or rate of run-down of the calcium current (Chard et al. 1993). However, calbindin-D28K, in addition to buffering calcium, can increase resting intracellular calcium levels, modulate both t- and l-type calcium currents, and influence calcium/ATPase pumps (Lledo et al. 1992).
In oculomotor neurons in vivo, endogenous calcium-binding proteins reduce the volume of local calcium elevations around open calcium channels, lower peak amplitudes of global calcium transients for a given influx, and prolong calcium recovery times for a given set of uptake and extrusion mechanisms (Vanselow and Keller 2000). These neurons, which possess ample parvalbumin, have a five- to sixfold larger calcium ‘buffering’ capacity (Vanselow and Keller 2000). They also possess specialized mechanisms to maintain calcium homeostasis compared with vulnerable spinal cord and hypoglossal motoneurons (Siklós et al. 1999).
We next examined whether increasing parvalbumin expression in spinal motoneurons could attenuate motoneuron dysfunction in mutant SOD1 transgenic mice (Gurney et al. 1994). We had previously demonstrated that calcium is increased in motor axon terminals of sporadic ALS patients as well as in spinal motoneurons of G93A mSOD1 mice (Siklós et al. 1996, 1998). In contrast, oculomotor neurons, with ample parvalbumin expression, did not have increased intracellular calcium and were relatively spared in mSOD1 mice (Siklós et al. 1998, 2000). Furthermore, using another mSOD1 ALS model, Nimchinsky et al. (2000) recently demonstrated that calcium-binding protein-containing neurons of the oculomotor nucleus are spared in mSOD1G86R transgenic mice, whereas calcium-binding protein deficient neurons, i.e. those of the facial nucleus, are severely affected.
In the present studies, transgenic expression of parvalbumin delayed the onset of disease in human mSOD1 transgenic mice by 17% and prolonged survival by 11%. This delay in the onset of disease is similar to the delay in onset noted with increased motoneuron expression of Bcl-2, or following the administration of vitamin E (a free radical scavenger), creatine, or a caspase inhibitor (Gurney et al. 1996; Kostic et al. 1997; Klivenyi et al. 1999; Li et al. 2000). These data contrast with the effects of riluzole (an inhibitor of glutamate release) or a dominant-negative inhibitor of interleukin-1β-converting enzyme (ICE), both of which prolonged survival without affecting onset (Friedlander et al. 1997; Kostic et al. 1997).
We also confirmed the observation that there is a significant loss of motoneurons in the mSOD1 (G93A) at end-stage disease. At 110–115 days of age, 45.4% of motoneurons were lost in mSOD1 transgenic mice compared with their wild-type controls. In our laboratory, mSOD1 animals at this age are moderately weakened. However, mSOD1/Parv animals showed no clinical signs of weakness and had 33.1% more motoneurons (p = 0.005) at this age when compared with mSOD1 transgenic mice. Thus, at our current level of parvalbumin over-expression, motoneurons continue to die in mSOD1/Parv transgenic mice, but do so at a slower rate. These data are in agreement with the observed onset/survival data.
The key finding in our study is the robust effect of parvalbumin expression within motoneurons in preventing the sustained increase in intracellular calcium induced by ALS immunoglobulins, and the more modest effects of parvalbumin expression on delaying onset of disease in mSOD1 transgenic mice. One possible explanation could be the higher PV transgene copy number in the ALS immunoglobulin experiments and the lower copy number in the mSOD1/Parv progeny. The former had 8–10 copies of the parvalbumin transgene, while the latter (hemizygous parvalbumin) mice had only 4–5 copies of the parvalbumin transgene. Studies are in progress to develop mSOD1/Parv mice with 8–10, and 16–20 copies of the PV transgene to test this hypothesis.
Alternatively, regardless of the extent of parvalbumin expression, increases in calcium in motoneurons in mSOD1 transgenic mice may be less sensitive to the level of parvalbumin expression than immune-mediated increases in calcium. In mSOD1 transgenic mice, the source of increased motoneuron calcium is not known, and increased intracellular calcium may be a later consequence of the toxic gain-of-function. Whether increased calcium is an early or a late event, the increases may have adverse effects on mitochondria, and may amplify neuronal injury. Mitochondria may act as a sink by sequestering calcium and effectively reduce cytosolic calcium concentrations (Nicholls and Ward 2000). Increased intramitochondrial calcium may in turn increase the generation of reactive oxygen species and alter the synthesis of many mitochondrial polypeptides (Borthwick et al. 1999).
Such mitochondrial alterations have been described as an early event in human mSOD1 transgenic models of ALS and could be the cause rather than the consequence of the increased intracellular calcium (Dal Canto and Gurney 1994; Wong et al. 1995; Carri et al. 1997; Kong and Xu 1998). At a critical mitochondrial calcium load, the mitochondria depolarize, leading to irreversible mitochondrial dysfunction and decreased ATP synthesis. By modulating calcium perturbations with calcium-binding proteins at an early stage, increased cytosolic calcium loads may be attenuated, and irreversible mitochondrial dysfunction may be delayed. Once mitochondria become sufficiently damaged and can no longer modulate intracellular calcium levels, it is unlikely that increased parvalbumin expression could significantly alter disease onset. This progression of mitochondrial injury to a stage incapable of modulating intracellular calcium may possibly explain the only modest delay in disease onset and survival. Nevertheless, the fact that increased parvalbumin expression did delay disease onset in the mSOD1 transgenic mice does suggest that, by decreasing voltage-dependent calcium channel current, enhancing Na+/Ca2+ ATPase activity, and/or by increasing Ca2+ buffering, parvalbumin may have a protective effect on motoneuron function and subsequent injury.