Pathogenic Ubqln2 gains toxic properties to induce neuron death

Abstract

Mutations in ubiquilin 2 (Ubqln2) is linked to amyotrophic lateral sclerosis and frontotemporal lobar degeneration. A foremost question regarding Ubqln2 pathogenesis is whether pathogenically mutated Ubqln2 causes neuron death via a gain or loss of functions. To better understand Ubqln2 pathobiology, we created Ubqln2 transgenic and knockout rats and compared phenotypic expression in these novel rat models. Overexpression of Ubqln2 with a pathogenic mutation (P497H substitution) caused cognitive deficits and neuronal loss in transgenic rats at the age of 130 days. In the transgenic rats, neuronal loss was preceded by the progressive formation of Ubqln2 aggregates and was accompanied by the progressive accumulation of the autophagy substrates p62 and LC3-II and the impairment of endosome pathways. In contrast, none of these pathologies observed in mutant Ubqln2 transgenic rats was detected in Ubqln2 knockout rats at the age of 300 days. Together, our findings in Ubqln2 transgenic and knockout rats collectively suggest that pathogenic Ubqln2 causes neuron death mainly through a gain of unrevealed functions rather than a loss of physiological functions.

Fig. 1

Mutant Ubqln2 was predisposed to aggregation in vitro and in vivo. a Western blotting revealed that mutant Ubqln2 was enriched in Triton X-100 insoluble fraction. HEK293 cells were transiently transfected with plasmids expressing wildtype (WT) or mutant (MUT, P497H substitution) human Ubqln2 that was tagged with FLAG. Triton X-100 soluble and insoluble fractions were isolated as described in “Materials and methods” and displayed in b. b Shows the procedure to separate insoluble from soluble Ubqln2 in cultured cells (dissolved in 7 M urea) and transgenic rats (dissolved in 1 % SDS). c Western blotting revealed that the full-length and C-terminal fragments of Ubqln2 were enriched in Triton X-100 insoluble fraction. Frontal cortex was dissected from CaMKα2-tTA single-transgenic (tTA: the control) and CaMKα2-tTA/TRE-hUBQLN2-P497H double-transgenic rats (P497H) at the age of 80 days. Immunoreactivity was detected with an antibody that recognizes both human and rat Ubqln2

Fig. 2

Mutant Ubqln2 aggressively formed inclusions in transgenic rats. a–p Immunostaining revealed that Ubqln2 progressively formed inclusions in the dentate gyrus (a–j, n–p) and cortex (k–m, q–s) in CaMKα2-tTA/TRE-hUBQLN2-P497H double-transgenic rats (P497H), but not in CaMKα2-tTA single-transgenic rats (tTA). Transgenic rats were deprived of Dox at birth to allow transgene expression and were examined at indicated ages (d days). Total Ubqln2 was detected with an antibody that recognized both human and rat Ubqln2 proteins (hrUB2), and rat Ubqln2 was detected with a rat-specific antibody (rUB2). q–s Confocal microscopy revealed that Ubqln2 inclusions were accumulated in the cytoplasm and nucleus of affected cells. Scale bars 100 μm (a–e) and 30 μm (f–p). t–u Immunoprecipitation (IP) followed by immunoblotting (IB) revealed that mutant human Ubqln2 (hUB2: P497H) bound to wildtype (WT) human (hUB2) and rat (rUB2) Ubqln2. Asterisk indicates IgG heavy chain. HEK293 cells were transfected with indicated plasmid and were analyzed 32 h after transfection

Fig. 3

Expression of mutant Ubqln2 in rats caused learning deficiency and neuronal death. a Barnes maze assay revealed spatial learning deficiency in CaMKα2-tTA/TRE-hUBQLN2-P497H double-transgenic rats (P497H) as compared to CaMKα2-tTA single-transgenic rats (tTA) at the age of 130 days. Rats were trained to locate an escape hole in a Barnes maze on the first day and then were examined daily for improvement in finding a fixed escape hole. Data are mean ± SD (n = 5). *p < 0.05. b–i Cresyl violet staining revealed neuronal loss in mutant Ubqln2 transgenic rats (P497H) as compared to the control rats (tTA). Rats of 130 days old were examined for neuronal loss in the dentate gyrus (b–e) and frontal cortex (f–i). j, k Unbiased stereological cell counting confirmed the loss of neurons in the dentate gyrus and frontal cortex of P497H rats. Data are mean ± SD (n = 4). *p < 0.05. l, m Golgi staining detected impaired neurons in P497H rats aging 130 days as compared to age-matched tTA rats. n–q Immunostaining revealed glial activation in P497H rats (o, q) as compared to tTA rats (n, p). The dentate gyrus of transgenic rats was examined for immunoreactivity with microglia marker Iba1 and astrocyte marker GFAP. Scale bars 100 μm (b–h, n–q), 30 μm (c–i), and 200 μm (l–m)

Fig. 4

Expression of mutant Ubqln2 led to p62 and LC3-II accumulation in vitro and in vivo. a Immunoblotting revealed that LC3-II accumulated in HEK293 cells expressing mutant Ubqln2. Wildtype and mutant human Ubqln2 proteins were tagged with FLAG. b Immunoblotting density was quantified and calculated as a ratio to the control. HEK293 cells were transfected with empty vector (CT), wildtype Ubqln2-expressing vector (WT), or mutant (P497H substitution) Ubqln2-expressing vector. Cells were treated with the proteasome inhibitor MG132 at 24 h after transfection and were harvested for analysis at 4 h after MG132 treatment. c Immunoblotting revealed accumulation of LC3-II and p62 in mutant Ubqln2 transgenic rats. Frontal cortex was examined for LC3-II and p62 expression. d–o Immunostaining revealed that p62 progressively accumulated in mutant Ubqln2 transgenic rats. All scale bars 30 μm

Fig. 5

Expression of mutant Ubqln2 in rats interfered with endosome pathways. a–l Immunostaining revealed that early endosomes labeled with EEA1 were decreased in mutant Ubqln2 transgenic rats. CaMKα2-tTA single-transgenic (tTA) and CaMKα2-tTA/TRE-hUBQLN2-P497H double-transgenic rats (P497H) were examined at indicated ages. m Immunoblotting revealed the expression of EEA1 and LAMP2a in mutant Ubqln2 transgenic rats. n–p Electromicroscopy revealed that vacuoles were accumulated in Ubqln2 transgenic rats. The microstructure of cortical neurons was examined for the transgenic control (tTA) and the mutant Ubqln2 transgenic (P497H) rats. Red arrowheads point to Golgi complex, blue arrowheads point to mitochondria, and yellow arrowheads point to vacuoles. Scale bars 30 μm (a–f) and 500 nm (n–p)

Fig. 6

Ubqln2 knockout rats displayed no detectable abnormality. a A strategy that was used to destroy the rat Ubqln2 gene. A pair of TALEN was constructed to cleave rat Ubqln2 within open reading frame (ORF) right after translational start site. TALEN induced a deletion of 19 nt within the ORF and thus introduced a premature stop codon, resulting in a truncated peptide. b Western blotting revealed the complete loss of Ubqln2 in homozygous knockout rats (M male, F female). The symbol asterisk indicates a nonspecific band. c The Open Field assay revealed no abnormality in mobility. Ubqln2 wildtype and knockout rats were examined weekly at indicated ages. Data are mean ± SD (n = 6, equal sex composition). d Barnes maze assay revealed no abnormality in spatial learning in Ubqln2 knockout rats at the age of 280 days. Rats were trained to locate an escape hole in a Barnes maze on the first day and then were examined daily for improvement in finding a fixed escape hole. Data are mean ± SD (n = 6). e–h Immunostaining revealed unaltered p62 and EEA1 expression in Ubqln2 knockout rats. i, j Nissl staining revealed the overall structure of dentate gyrus. All scale bars 30 μm