Ubiquilin modifies TDP-43 toxicity in a Drosophila model of amyotrophic lateral sclerosis (ALS)

Abstract

TDP-43 (43-kDa TAR DNA-binding protein) is a major constituent of ubiquitin-positive cytosolic aggregates present in neurons of patients with amyotrophic lateral sclerosis (ALS) and ubiquitin-positive fronto-temporal lobar degeneration (FTLD-U). Inherited mutations in TDP-43 have been linked to familial forms of ALS, indicating a key role for TDP-43 in disease pathogenesis. Here, we describe a Drosophila melanogaster model of TDP-43 proteinopathy. Expression of wild-type human TDP-43 protein in Drosophila motor neurons led to motor dysfunction and dramatic reduction of life span. Interestingly, coexpression of ubiquilin 1, a previously identified TDP-43-interacting protein with suspected functions in autophagy and proteasome targeting, reduced steady-state TDP-43 expression but enhanced the severity of TDP-43 phenotypes. Finally, ectopically expressed TDP-43 was largely localized to motor neuron nuclei, suggesting that expression of wild-type TDP-43 alone is detrimental even in the absence of cytosolic aggregation. Our findings demonstrate that TDP-43 exerts cell-autonomous neurotoxicity in Drosophila and further imply that dose-dependent alterations of TDP-43 nuclear function may underlie motor neuron death in ALS.

FIGURE 1.

TDP-43 toxicity in the eye. A, Western blot analysis of transgenic protein expression in the TDP-43 and UBQLN lines used in this study, using the GMR-Gal4 driver. Each lane contains lysate from a single head. * indicates the expected size of TDP-43 cleavage products seen in cell culture (supplemental Fig. 2C). The neuronal marker elav is used as a loading control. B, external eye phenotype of control and TDP-43-expressing flies, using the indicated transgene combinations. Where possible, the same eye is shown for each genotype at age 2 and 23 days. C, TEM images of ommatidia from 7-day-old GMR-Gal4 and GMR-TDP43^L1 flies; the individual photoreceptors of an intact ommatidium are labeled R1–R7. D, TEM image of an ommatidium from a GMR-TDP43^L1 fly reared at 18 °C; a single rhabdomere is shown in the lower panel. Western blot (right) indicates relative TDP-43 expression at 18 °C as compared with 25 °C.

FIGURE 2.

UBQLN exacerbates TDP-43 motor neuron phenotypes. A, survival curves of D42-Gal4 control, D42-TDP43^L1, and D42-TDP43^L2 flies. B, survival curves of D42-TDP43^L2 and D42-TDP43^L2/UBQLN^L2 flies. See supplemental Table 1 for statistical analysis. C, quantification of pupal eclosion defects seen with TDP-43 and UBQLN transgenic flies. >300 pupae were scored for each genotype; both a complete lack of eclosion and incomplete eclosion were counted as eclosion failure. n.s., not significant. D, quantification of wing defects seen with TDP-43 and UBQLN transgenic flies. >200 adult flies were scored for each genotype. ***, p < 0.001. p values were calculated using the chi square statistic.

FIGURE 3.

UBQLN reduces the expression of, but does not coaggregate with, TDP-43 in vivo. A, upper, protein expression levels in GMR-UBQLN^L2 (left), GMR-TDP43^L2 (center), and GMR-TDP43^L2/UBQLN^L2 (right) flies. The reference band (Ref.) is a nonspecific band detected with α-TDP-43 antibody. Middle, quantification of three experiments showing the relative ratio of UBQLN to the reference band with and without TDP-43 coexpression. Lower, quantification of three experiments showing the relative ratio of TDP-43 to the reference band with and without UBQLN coexpression. Error bars represent S.D. *, p < 0.05. p values were calculated using Student's t test. n.s., not significant. B, localization of TDP-43 in motor neurons of D42-TDP43^L1 transgenic flies. Tissue sections were immunostained using α-TDP-43 (green) antibody and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Arrowheads indicate condensed chromatin. C, localization of TDP-43 and UBQLN in motor neurons of D42-TDP43^L1/UBQLN^L1 transgenic flies. Tissue sections were immunostained using α-TDP-43 (green) and α-UBQLN (red) antibodies. D, Western blot analysis of TDP-43 and UBQLN localization in transgenic flies after cellular fractionation, using GMR-TDP43^L1, GMR-UBQLN^L1, and GMR-TDP43^L1/UBQLN^L1 flies. C = cytosolic; N = nuclear; I = insoluble.