Selective vulnerability of motoneuron and perturbed mitochondrial calcium homeostasis in amyotrophic lateral sclerosis: implications for motoneurons specific calcium dysregulation

Abstract

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disorder characterized by the selective degeneration of defined subgroups of motoneuron in the brainstem, spinal cord and motor cortex with signature hallmarks of mitochondrial Ca(2+) overload, free radical damage, excitotoxicity and impaired axonal transport. Although intracellular disruptions of cytosolic and mitochondrial calcium, and in particular low cytosolic calcium ([Ca(2+)]c) buffering and a strong interaction between metabolic mechanisms and [Ca(2+)]i have been identified predominantly in motoneuron impairment, the causes of these disruptions are unknown. The existing evidence suggests that the mutant superoxide dismutase1 (mtSOD1)-mediated toxicity in ALS acts through mitochondria, and that alteration in cytosolic and mitochondria-ER microdomain calcium accumulation are critical to the neurodegenerative process. Furthermore, chronic excitotoxcity mediated by Ca(2+)-permeable AMPA and NMDA receptors seems to initiate vicious cycle of intracellular calcium dysregulation which leads to toxic Ca(2+) overload and thereby selective neurodegeneration. Recent advancement in the experimental analysis of calcium signals with high spatiotemporal precision has allowed investigations of calcium regulation in-vivo and in-vitro in different cell types, in particular selectively vulnerable/resistant cell types in different animal models of this motoneuron disease. This review provides an overview of latest advances in this field, and focuses on details of what has been learned about disrupted Ca(2+) homeostasis and mitochondrial degeneration. It further emphasizes the critical role of mitochondria in preventing apoptosis by acting as a Ca(2+) buffers, especially in motoneurons, in pathophysiological conditions such as ALS.

Keywords: Amyotrophic lateral sclerosis (ALS); Calcium buffering; Calcium dysregulation; ER-mitochondria calcium cycle (ERMCC); Mitochondria; Motoneuron; Multidrug therapy; Multifactorial disease; Selective vulnerability.

Figure 1

Mitochondrial structure of motor neurons in mutant SOD1 transgenic mice and calcium load in microdomains in a cell culture model of motoneuron disease. (A) a, Shows abnormalities like dilated cristae (asterisk) and leaking outer membrane (indicated with arrow) in mitochondrion. (A) b, Swollen dendritic mitochondria with dilated and disorganized cristae (adapted from ref. 31). (B-D) The simultaneous measurement of cytosolic calcium (Fura-2) and mitochondrial calcium (Rhod-2) concentrations in WT and G93A transfected SH-SY5Y cells during FCCP-evoked mitochondrial Ca²⁺ release. (B) The kinetic profile of the FCCP-evoked Ca²⁺ release in the WT transfected SH-SY5Y neuroblastoma cells; the cytosolic (Error bar green, black square trace) and mitochondrial (Error bar red, black circle trace) compartment were measured simultaneously. The trace represents the mean of 5 cells in focus stimulated with 2 μM FCCP. (C) The corresponding kinetic profile of the FCCP-evoked Ca²⁺ release in the G93A transfected SH-SY5Y neuroblastoma cells; the cytosolic (Error bar green, black square trace) and mitochondrial (Error bar red, black circle trace) compartment were measured simultaneously. The trace represents the mean of 5 cells in focus stimulated with 2 μM FCCP. FCCP-evoked [Ca²⁺]mito signals were smaller in amplitude and exhibited slower kinetics in G93A transfected SH-SY5Y cells compared to WT transfected cells and were altered from [Ca²⁺]i efflux. (D) A bar diagram of the cytosolic (green bar) and mitochondrial (red bar) fluorescence signals (F/F0) from WT (F/F0 = 0.1569 ± 0.0235 for [Ca²⁺]i and F/F0 = −0.1069 ± 0.0181 for [Ca²⁺] mito; hollow; N = 5, n = 17) and G93A (F/F0 = 0.1008 ± 0.0248 for [Ca²⁺]i and F/F0 = −0.0486 ± 0.0043 for[Ca²⁺]mito; striped pattern, N = 4; n = 17) transfected SH-SY5Y neuroblastoma cells. Values represent means ± SD, **p < 0.001. N = Number of experiments; n = Number of cells (adapted from ref. 94).

Figure 2

Ca ²⁺ homeostasis and its correlation with weakly and strongly buffered motoneurons under physiological and pathophysiological conditions. (A) The Ca²⁺ buffering capacity (K_S) of a cell, reflecting relative fraction of bound versus free Ca²⁺, can be calculated by using the ‘added buffer’ approach by linear one-compartment model. The recovery time of [Ca²⁺]_i elevations (τ) depends on the amount of endogenous buffer (S; denotes Ca²⁺-binding proteins), the amount of exogenous buffer (B; i.e. Fura-2) and the transport rate (γ) of Ca²⁺ across cellular membranes. K_B indicates the buffer capacity of the exogenous buffer (i.e. Fura-2). (B) Ca²⁺ homeostasis in weakly and strongly buffered MNs. The amplitude of Ca²⁺ transients is several times larger in weakly buffered cells (e.g. HMNS and SMNs) than in strongly buffered cells (e.g. oculomotor neurons), and the recovery time is significantly accelerated (τ). (C) Low Ca²⁺ buffering in ALS-vulnerable HMNs exposes mitochondria to higher Ca²⁺ loads compared to high-buffered cells. Under normal physiological conditions the neurotransmitter opens glutamate, NMDA and AMPA receptor channels along with VDCC with high glutamate release and reuptake by EAAT1 and EAAT2. This results in a small rise in intracellular calcium that can be buffered by the cell. In ALS disorder, the glutamate receptor channels possess high calcium conductivity and thereby high Ca²⁺ loads; increase the risk for mitochondrial damage. This triggers mitochondrial production of reactive oxygen species (ROS), which then inhibit glial EAAT2 function. This leads to further increase in glutamate concentrations in the synapse and further rises in postsynaptic calcium levels which contributes to the selective vulnerability of MNs in ALS. Low cytosolic Ca²⁺ buffering capacity promotes Ca²⁺ accumulation and formation of subcellular domains around influx sites (red), and thus facilitates the interaction of elevated calcium levels with intracellular organelles such as mitochondria (modified from refs. [62, 63, 73, 94, 118]).