TDP-43 toxicity proceeds via calcium dysregulation and necrosis in aging Caenorhabditis elegans motor neurons

Abstract

Amyotrophic lateral sclerosis (ALS) is a heterogeneous disease with either sporadic or genetic origins characterized by the progressive degeneration of motor neurons. At the cellular level, ALS neurons show protein misfolding and aggregation phenotypes. Transactive response DNA-binding protein 43 (TDP-43) has recently been shown to be associated with ALS, but the early pathophysiological deficits causing impairment in motor function are unknown. Here we used Caenorhabditis elegans expressing mutant TDP-43(A315T) in motor neurons and explored the potential influences of calcium (Ca(2+)). Using chemical and genetic approaches to manipulate the release of endoplasmic reticulum (ER) Ca(2+)stores, we observed that the reduction of intracellular Ca(2+) ([Ca(2+)]i) rescued age-dependent paralysis and prevented the neurodegeneration of GABAergic motor neurons. Our data implicate elevated [Ca(2+)]i as a driver of TDP-43-mediated neuronal toxicity. Furthermore, we discovered that neuronal degeneration is independent of the executioner caspase CED-3, but instead requires the activity of the Ca(2+)-regulated calpain protease TRA-3, and the aspartyl protease ASP-4. Finally, chemically blocking protease activity protected against mutant TDP-43(A315T)-associated neuronal toxicity. This work both underscores the potential of the C. elegans system to identify key targets for therapeutic intervention and suggests that a focused effort to regulate ER Ca(2+) release and necrosis-like degeneration consequent to neuronal injury may be of clinical importance.

Keywords: ALS; C. elegans; ER; TDP-43; calcium; necrosis.

Figure 1.

Genes regulating ER calcium release promote TDP-43 neuronal toxicity. A, Null mutations in cnx-1 or crt-1 suppress age-dependent paralysis caused by TDP-43^A315T compared with transgenic TDP-43^A315T controls. p < 0.0001 for TDP-43^A315T; cnx-1(nr2009) versus TDP-43^A315T; p = 0.0002 for TDP-43^A315T; cnx-1(nr2010) versus TDP-43^A315T; p < 0.0001 for TDP-43^A315T; crt-1(bz30) versus TDP-43^A315T; p < 0.0001 for TDP-43^A315T; crt-1(jh101) versus TDP-43^A315T. TDP-43^A315T, n = 114 ; TDP-43^A315T; cnx-1(nr2009), n = 76; TDP-43^A315T; cnx-1(nr2010), n = 98; TDP-43^A315T; crt-1(bz30), n = 90; and TDP-43^A315T; crt-1(jh101), n = 63. B, Mutations in cnx-1 or crt-1 reduce age-dependent neurodegeneration in TDP-43 ^A315T transgenics compared with TDP-43^A315T control animals. ***p < 0.001 versus TDP-43^A315T at day 9; ****p < 0.0001 versus TDP-43^A315T at day 9. C, Null mutations in unc-68 and itr-1 reduce TDP-43^A315T-mediated paralysis compared with control TDP-43^A315T transgenics. p < 0.0001 for either for TDP-43^A315T; unc-68(e540) or for TDP-43^A315T; itr-1(sa73) versus TDP-43^A315T. TDP-43^A315T, n = 90; TDP-43^A315T; itr-1(sa73), n = 88; and TDP-43^A315T; unc-68(e540), n = 84. D, Degeneration of motor neurons is reduced in adult day 9 TDP-43^A315T transgenics compared with controls. **p < 0.01 versus TDP-43^A315T at day 9. E, Western blotting with a human anti-TDP-43 antibody revealed comparable levels of protein expression in all strains.

Figure 2.

Pharmacological manipulation of [Ca²⁺]_i reduces TDP-43 neuronal toxicity. A, TDP-43^A315T transgenics treated with EGTA showed significantly less paralysis compared with untreated controls (p < 0.0001 versus TDP-43^A315T). The protective effect of EGTA was not additive to the suppression of TDP-43-mediated paralysis by mutation in either crt-1 or itr-1. p = 0.8470 versus TDP-43^A315T; crt-1(bz30); and p = 0.7817 versus TDP-43^A315T; itr-1(sa73). TDP-43^A315T, n = 90 ; TDP-43^A315T + EGTA, n = 181 ; TDP-43^A315T; itr-1(sa73), n = 90; TDP-43^A315T; itr-1(sa73) + EGTA, n = 90; TDP-43^A315T; crt-1(bz30), n = 90; TDP-43^A315T; crt-1(bz30) + EGTA, n = 90. B, Degeneration of motor neurons in TDP-43^A315T animals at day 9 of adulthood was reduced to comparable levels in TDP-43^A315T transgenics treated with EGTA alone or in combination with mutations in crt-1 or itr-1. ****p < 0.0001 versus TDP-43^A315T at day 9. C, Treatment with dantrolene suppressed TDP-43^A315T-mediated paralysis compared with untreated control animals (p = 0.0031 versus TDP-43^A315T). Suppression of TDP-43^A315T-mediated paralysis by crt-1 or itr-1 was not significantly different from these same mutant strains treated with dantrolene. TDP-43^A315T, n = 114 ; TDP-43^A315T + dantrolene, n = 100; TDP-43^A315T; itr-1(sa73), n = 88; TDP-43^A315T; itr-1(sa73) + dantrolene, n = 96; TDP-43^A315T; crt-1(bz30), n = 90; TDP-43^A315T; crt-1(bz30) + dantrolene, n = 90. D, Degeneration of motor neurons, in TDP-43^A315T animals at day 9 of adulthood was reduced to similar levels in TDP-43^A315T transgenics treated with dantrolene alone or in combination with mutations for crt-1 or itr-1. **p < 0.01 versus TDP-43^A315T at day 9; ***p < 0.001 versus TDP-43^A315T at day 9. E, TDP-43 protein expression was unchanged by culture conditions with EGTA or dantrolene.

Figure 3.

Disrupted Ca²⁺ homeostasis enhances wild-type TDP-43 toxicity. A, Transgenic worms expressing TDP-43^WT treated with either EGTA or dantrolene had increased rates of paralysis compared with untreated controls (p < 0.01 for dantrolene-treated worms versus untreated TDP-43^WT controls; p < 0.0001 for EGTA-treated worms versus untreated TDP-43^WT controls). TDP-43^WT, n = 90; TDP-43^WT + dantrolene, n = 90; TDP-43^WT + EGTA, n = 90. B, Treatment with dantrolene or EGTA increased neurodegeneration in adults on day 9; TDP-43^WT transgenics compared with untreated TDP-43^WT transgenics. *p < 0.05 versus TDP-43^WT at day 9, ****p < 0.0001 versus TDP-43^WT at day 9. C, Similar levels of TDP-43 proteins were detected by Western blotting in untreated TDP-43^WT transgenics compared with animals treated with EGTA or dantrolene.

Figure 4.

Forced release of ER Ca²⁺ stores enhances TDP-43 neuronal toxicity. A, Paralysis was enhanced in TDP-43^A315T; crt-1(bz30) and TDP-43^WT transgenics treated with thapsigargin compared with untreated transgenic controls. p < 0.001 for TDP-43^A315T; crt-1(bz30) animals versus those treated with thapsigargin; p < 0.001 for TDP-43^WT versus those treated with thapsigargin. TDP-43^WT, n = 98; TDP-43^WT + thapsigargin, n = 95; TDP-43^A315T; crt-1(bz30), n = 96; TDP-43^A315T; crt-1(bz30) + thapsigargin, n = 100. B, Thapsigargin enhanced neurodegeneration transgenics expressing TDP-43^WT at days 1, 5, and 9 of adulthood compared with untreated controls. The suppression of neurodegeneration in TDP-43^A315T; crt-1(bz30) animals was lost by thapsigargin treatment in adult day 9 transgenics. ***p < 0.001 versus TDP-43^WT at day 1; ****p < 0.0001 versus TDP-43^WT or TDP-43^A315T; crt-1(bz30). C, Thapsigargin did not affect TDP-43 protein expression in TDP-43^WT or TDP-43^A315T worms.

Figure 5.

Calpain and aspartyl proteases facilitate TDP-43 neuronal toxicity. A, Null mutations in tra-3 or asp-4 suppress age-dependent paralysis in TDP-43^A315T transgenics compared with TDP-43^A315T controls. Mutation in ced-3 had no significant effect on paralysis phenotypes compared with TDP-43^A315T. p < 0.0001 for TDP-43^A315T; tra-3(ok2207) or TDP-43^A315T; asp-4(ok2693) versus TDP-43^A315T transgenic controls. TDP-43^A315T, n = 90; TDP-43^A315T; tra-3(ok2207), n = 102; TDP-43^A315T; asp-4(ok2693), n = 79; TDP-43^A315T; ced-3(ok2734), n = 96. B, Neurodegeneration was significantly reduced in adult, day 9, TDP-43^A315T transgenics by tra-3 or asp-4 null mutations compared with TDP-43^A315T alone. A null mutation of ced-3 failed to suppress TDP-43^A315T neurodegeneration. ****p < 0.0001 versus TDP-43^A315T at day 9. C, The suppression of TDP-43^A315T-mediated paralysis by tra-3 or asp-4 was unaffected by the addition of thapsigargin. p < 0.0001 for TDP-43^A315T versus TDP-43^A315T; tra-3(ok2207) or TDP-43^A315T; asp-4(ok2693) with or without thapsigargin treatment. TDP-43^A315T, n = 90; TDP-43^A315T; tra-3(ok2207), n = 90; TDP-43^A315T; tra-3(ok2207) + thapsigargin, n = 90; TDP-43^A315T; asp-4(ok2693), n = 90; TDP-43^A315T; asp-4(ok2693) + thapsigargin, n = 90. D, Suppression of age-dependent neurodegeneration in TDP-43^A315T transgenics by tra-3 or asp-4 mutations was unchanged by thapsigargin treatment. ****p < 0.0001 versus TDP-43^A315T at day 9. E, Null mutations of tra-3, asp-4, or ced-3 did not affect TDP-43 protein expression. F, The calpain inhibitor MDL-28170 reduced paralysis in TDP-43^A315T transgenics. p < 0.001 for treated versus untreated TDP-43^A315T animals. TDP-43^A315T, n = 92; TDP-43^A315T + MDL-28170, n = 82.

Figure 6.

TDP-43-mediated motility defects require tra-3 and asp-4 in the nervous system. A, RNAi against tra-3 suppressed TDP-43^A315T-mediated paralysis. p < 0.05 for TDP-43^A315T treated with tra-3(RNAi) versus TDP-43^A315T treated with EV control RNAi. TDP-43^A315T + EV, n = 71; TDP-43^A315T + clp-1(RNAi), n = 71; TDP-43^A315T + clp-2(RNAi), n = 84; TDP-43^A315T + clp-4(RNAi), n = 79; TDP-43^A315T + tra-3(RNAi), n = 68; TDP-43^A315T + clp-7(RNAi), n = 69. B, RNAi against asp-4 suppressed TDP-43^A315T-mediated paralysis. p < 0.05 for TDP-43^A315T treated with asp-4(RNAi) versus TDP-43^A315T treated with EV control RNAi. TDP-43^A315T + EV, n = 71; TDP-43^A315T + asp-1(RNAi), n = 54; TDP-43^A315T + asp-3(RNAi), n = 66; TDP-43^A315T + asp-4(RNAi), n = 67; TDP-43^A315T + asp-6(RNAi), n = 59; TDP-43^A315T + asp-7(RNAi), n = 62; TDP-43^A315T + asp-10(RNAi), n = 68; TDP-43^A315T + asp-13(RNAi), n = 58. C, There were no significant differences in the rates of paralysis for TDP-43^A315T sensitized for intestine-specific RNAi by treatment with EV(RNAi), tra-3(RNAi), or asp-4(RNAi). TDP-43^A315T + EV, n = 77; TDP-43^A315T + tra-3(RNAi), n = 54; TDP-43^A315T + asp-4(RNAi), n = 59. D, There were no significant differences in the rates of paralysis for TDP-43^A315T sensitized for body wall muscle-specific RNAi by treatment with EV(RNAi), tra-3(RNAi), or asp-4(RNAi). TDP-43^A315T + EV, n = 64; TDP-43^A315T + tra-3(RNAi), n = 51; TDP-43^A315T + asp-4(RNAi), n = 60.

Figure 7.

Calcium homeostasis and protease genes do not suppress motility defects in TDP-43^WT animals. There was no significant suppression of TDP-43^WT motility defects by Ca²⁺ or protease gene mutations. p < 0.001 for TDP-43^A315T versus TDP-43^WT. TDP-43^A315T, n = 81; TDP-43^WT, n = 65; TDP-43^WT; crt-1(bz30), n = 71; TDP-43^WT; itr-1(sa73), n = 71; TDP-43^WT; unc-68(e540), n = 60; TDP-43^WT; cnx-1(nr2010), n = 61; TDP-43^WT; tra-3(ok2207), n = 71; TDP-43^WT; asp-4(ok2693), n = 115.

Figure 8.

Working model for TDP-43 and Ca²⁺-dependent necrosis-like neuronal toxicity. The chronic stress induced by protein misfolding and aggregation of mutant TDP-43 proteins may lead to inappropriate release of Ca²⁺ from ER stores into the cytoplasm. The resultant [Ca²⁺]_i increase is essential for downstream events, including activation of the Ca²⁺-regulated TRA-3 calpain protease, which in turn mediates leakage of killer aspartyl proteases (ASP-4), leading to neuronal dysfunction and cell death. Mutations affecting ER Ca²⁺ storage (calreticulin and calnexin) or ER calcium release (InsP3R and RYR calcium release channels) disrupt release and are therefore neuroprotective. Pharmacological reduction of [Ca²⁺]_i by EGTA or dantrolene is also neuroprotective, while a forced increase of [Ca²⁺]_i by thapsigargin enhances neuronal toxicity. Disabling the activity of calpain or aspartyl proteases also protects against TDP-43-associated neuronal dysfunction and degeneration.