ALS-associated ataxin 2 polyQ expansions enhance stress-induced caspase 3 activation and increase TDP-43 pathological modifications

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease caused by the loss of motor neurons. The degenerating motor neurons of ALS patients are characterized by the accumulation of cytoplasmic inclusions containing phosphorylated and truncated forms of the RNA-binding protein TDP-43. Ataxin 2 intermediate-length polyglutamine (polyQ) expansions were recently identified as a risk factor for ALS; however, the mechanism by which they contribute to disease is unknown. Here, we show that intermediate-length ataxin 2 polyQ expansions enhance stress-induced TDP-43 C-terminal cleavage and phosphorylation in human cells. We also connect intermediate-length ataxin 2 polyQ expansions to the stress-dependent activation of multiple caspases, including caspase 3. Caspase activation is upstream of TDP-43 cleavage and phosphorylation since caspase inhibitors block these pathological modifications. Analysis of the accumulation of activated caspase 3 in motor neurons revealed a striking association with ALS cases harboring ataxin 2 polyQ expansions. These findings indicate that activated caspase 3 defines a new pathological feature of ALS with intermediate-length ataxin 2 polyQ expansions. These results provide mechanistic insight into how ataxin 2 intermediate-length polyQ expansions could contribute to ALS--by enhancing stress-induced TDP-43 pathological modifications via caspase activation. Because longer ataxin 2 polyQ expansions are associated with a different disease, spinocerebellar ataxia 2, these findings help explain how different polyQ expansions in the same protein can have distinct cellular consequences, ultimately resulting in different clinical features. Finally, since caspase inhibitors are effective at reducing TDP-43 pathological modifications, this pathway could be pursued as a therapeutic target in ALS.

Figure 1.

Intermediate-length ataxin 2 polyQ expansions cause accumulation of a phosphorylated TDP-43 C-terminal fragment in HEK293 cells. A, HEK293 cells transfected with empty vector or ataxin 2 with increasing polyQ length (22Q, 31Q, or 39Q) were heat shocked for 1 h at 42°C, or control for 1 h at 37°C, then lysed into soluble and insoluble fractions. An accumulation of insoluble pTDP-43 C-terminal fragment was seen with ataxin 2 31Q compared with empty vector or ataxin 2 22Q and 39Q. B, Dephosphorylation of the insoluble fraction with dialysis and lambda phosphatase treatment removes the pTDP-43 band. C, Quantification from three independent experiments (pTDP-43 levels normalized to actin and compared with empty vector control; error bars represent SD; *p < 0.001).

Figure 2.

Intermediate-length ataxin 2 polyQ expansions enhance stress-induced accumulation of a phosphorylated TDP-43 C-terminal fragment in ALS patient-derived lymphoblast cell lines. A, ALS patient-derived lymphoblast cell lines expressing endogenous levels of normal or polyQ-expanded ataxin 2 were heat shocked for 1 h at 42°C or control for 1 h at 37°C, then lysed into soluble and insoluble fractions. Accumulation of an insoluble pTDP-43 C-terminal fragment was observed in cell lines with intermediate-length ataxin 2 polyQ expansions but not control or SCA2 polyQ-expanded cell lines. B, Quantification of three independent experiments (pTDP-43 levels normalized to actin and compared with pTDP-43 levels in 22Q control cell lines; error bars represent SD; *p < 0.01).

Figure 3.

Intermediate-length ataxin 2 polyQ expansions enhance caspase activation in response to cellular stress. Patient-derived lymphoblast cell lines with either ataxin 2 22Q or 29Q were heat shocked for 1 h at 42°C, or a cell line with ataxin 2 22Q was treated for 3 h with 1 μm staurosporine at 37°C. Cells were lysed into soluble and insoluble fractions. Lysates were immunoblotted with a panel of caspase antibodies to determine which caspases were activated. Caspase 3 and caspase 7 showed strong activation/cleavage in the ataxin 2 29Q cell line but not in the ataxin 2 22Q cell line. Caspase 8 did not show activation except in the staurosporine-treated cell line, and caspase 9 was only weakly activated in the 29Q cell line. Ataxin 2 appeared to be cleaved in the heat shocked 29Q cell line and in the staurosporine-treated 22Q cell line.

Figure 4.

Ataxin 2 intermediate-length polyQ expansions result in caspase 3 activation following heat-shock. A, HEK293T cells transfected with empty vector or ataxin 2 with increasing polyQ length (22Q, 31Q, or 39Q) were heat shocked for 1 h at 42°C, then analyzed for caspase 3 activity by immunoblot for cleaved caspase 3. B, ALS patient-derived lymphoblast cell lines with normal or polyQ-expanded ataxin 2 were heat shocked for 1 h at 42°C, then analyzed for caspase 3 activation by immunoblot. C, Quantification of three independent experiments (cleaved caspase 3 levels normalized to actin and compared with levels in 22Q control cell lines; error bars represent SD; *p < 0.01).

Figure 5.

Caspase 3 inhibition reduces accumulation of the pTDP-43 C-terminal fragment. A, HEK293T cells transfected with ataxin 2 of 22Q or 31Q were treated with vehicle alone or Z-DEVD-FMK for 24 h, heat shocked at 42°C for 1 h, and then lysed into soluble and insoluble fractions. Cells with ataxin 2 31Q showed an accumulation of pTDP-43 fragment compared with 22Q, but this accumulation is dissipated by inhibition of caspase 3. B, Quantification of three separate experiments (pTDP-43 levels normalized to actin and compared with pTDP-43 levels in 22Q control cell lines; error bars represent SD; *p < 0.05). C, ALS patient-derived lymphoblast cell lines with intermediate-length polyQ-expanded ataxin 2 were treated with vehicle alone or Z-DEVD-FMK for 24 h, then heat shocked at 42°C for 1 h (or control for 1 h at 37°C) and lysed into soluble and insoluble fractions. Accumulation of the pTDP-43 fragment seen with heat shock in ataxin 2 polyQ-expanded cells was reduced by caspase 3 inhibition. D, Quantification of two separate experiments from two separate cell lines (pTDP-43 levels normalized to actin and compared with pTDP-43 levels in nonheat-shocked cells; error bars represent SD; *p < 0.05).

Figure 6.

Accumulation of pTDP-43 and increased caspase 3 activation in differentiated BE(2)M17 cells expressing ataxin 2 with intermediate-length polyQ expansion. BE(2)-M17 neuroblastoma cells transfected with empty vector or ataxin 2 with increasing polyQ length (22Q, 31Q, or 39Q) were differentiated for 5 d and heat shocked for 1 h at 42°C, or control for 1 h at 37°C, then lysed into soluble and insoluble fractions. A, An accumulation of insoluble pTDP-43 C-terminal fragment was seen with ataxin 2 31Q compared with empty vector or ataxin 2 22Q and 39Q. There was also an increase in cleaved caspase 3 in cells expressing 31Q, but not 22Q or 39Q. B, Quantification from three independent experiments (pTDP-43 levels normalized to actin and compared with empty vector control; error bars represent SD; *p < 0.05). C, Quantification from three independent experiments (cleaved caspase 3 levels normalized to actin and compared with empty vector control; error bars represent SD; *p < 0.05).

Figure 7.

Caspase 3 activation in motor neurons of ALS patients with ataxin 2 intermediate-length polyQ expansions. Spinal cord sections of ALS patients with 22Q ataxin 2 or 27–31Q ataxin 2 were stained with cleaved caspase 3 antibody, and motor neurons were analyzed for staining and localization. Case numbers and ataxin 2 polyQ lengths are indicated. A, Immunofluorescence or light micrographs of cleaved caspase 3 staining in motor neurons (scale bar, 10 μm). B, Quantification of the percentage of motor neurons with cleaved caspase 3 staining (error bars represent SD, *p < 0.01). C, Quantification of the percentage of motor neurons counted with >3 cytoplasmic foci of cleaved caspase 3 (error bars represent SD, *p < 0.01, **p < 0.001). D, Table showing motor neuron counts from different patients.

Figure 8.

Phosphorylated TDP-43 and cleaved caspase 3 staining in motor neurons of ALS patients. Spinal cord sections of ALS patients with 22Q ataxin 2 (A) or 27–31Q ataxin 2 (B–E) were costained with pTDP-43 and cleaved caspase 3 antibodies. Case numbers and ataxin 2 polyQ lengths are indicated. Immunofluorescence or light micrographs show pTDP-43 and cleaved caspase 3 staining in motor neurons. A, 22Q ataxin 2 cases showed occasional nuclear cleaved caspase 3 staining with no correlation to the presence of pTDP-43 inclusions. B–E, Ataxin 2 polyQ-expanded motor neurons with cytoplasmic foci of cleaved caspase 3 showed little or no pTDP-43 preinclusions (B), Motor neurons with pTDP-43 preinclusions had fewer cleaved caspase 3 cytoplasmic foci (C, D), and motor neurons with pTDP-43 inclusions had rare or no cleaved caspase 3 foci (E). Top panels are light micrographs of pTDP-43 staining from ALS patients demonstrating similar TDP-43 pathology by chromogenic staining (scale bar, 10 μm).

Figure 9.

A model to explain the effect of ataxin 2 intermediate-length polyQ expansions on TDP-43 in response to cellular stress. A, In ALS patients with normal ataxin 2 polyQ length (22Q), TDP-43 is localized to the nucleus and ataxin 2 is diffuse in the cytoplasm. Upon cellular stress, ataxin 2 localizes to stress granules, TDP-43 partially leaves the nucleus and also associates with stress granules, accompanied by a slight activation of caspase 3. Following the stress, cells recover, stress granules dissolve, and TDP-43 returns to the nucleus. If the stress continues, stress granules persist, TDP-43 is cleared from the nucleus, cleaved and phosphorylated, and coalesces into large round or skein-like cytoplasmic aggregates. B, In cells with ALS-associated ataxin 2 intermediate-length polyQ expansions (27–34Q), the threshold for activating caspases in response to stress is lowered. Therefore, caspase 3 is strongly activated upon cellular stress. Increased caspase activation leads to enhanced TDP-43 cleavage and phosphorylation, which drives cytoplasmic aggregation and nuclear clearance. Because the kinetics of TDP-43 cytoplasmic aggregation may be different in ALS cases with and without ataxin 2 polyQ expansions, the morphologies of the TDP-43 aggregates at end stage might be different (Hart et al., 2012). C, In spinocerebellar ataxia 2 (SCA2), in which patients harbor long ataxin 2 polyQ expansions (>34Q), ataxin 2 can aggregate in the cytoplasm and this could cause toxic gain-of-function effects, potentially unrelated to the normal function in cellular stress responses, which might lead to distinct cellular phenotypes and clinical presentation.