MSUT2 is a determinant of susceptibility to tau neurotoxicity

Abstract

Lesions containing abnormal aggregated tau protein are one of the diagnostic hallmarks of Alzheimer's disease (AD) and related tauopathy disorders. How aggregated tau leads to dementia remains enigmatic, although neuronal dysfunction and loss clearly contribute. We previously identified sut-2 as a gene required for tau neurotoxicity in a transgenic Caenorhabditis elegans model of tauopathy. Here, we further explore the role of sut-2 and show that overexpression of SUT-2 protein enhances tau-induced neuronal dysfunction, neurotoxicity and accumulation of insoluble tau. We also explore the relationship between sut-2 and its human homolog, mammalian SUT-2 (MSUT2) and find both proteins to be predominantly nuclear and localized to SC35-positive nuclear speckles. Using a cell culture model for the accumulation of pathological tau, we find that high tau levels lead to increased expression of MSUT2 protein. We analyzed MSUT2 protein in age-matched post-mortem brain samples from AD patients and observe a marked decrease in overall MSUT2 levels in the temporal lobe of AD patients. Analysis of post-mortem tissue from AD cases shows a clear reduction in neuronal MSUT2 levels in brain regions affected by tau pathology, but little change in regions lacking tau pathology. RNAi knockdown of MSUT2 in cultured human cells overexpressing tau causes a marked decrease in tau aggregation. Both cell culture and post-mortem tissue studies suggest that MSUT2 levels may influence neuronal vulnerability to tau toxicity and aggregation. Thus, neuroprotective strategies targeting MSUT2 may be of therapeutic interest for tauopathy disorders.

Figure 1.

SUT-2 overexpression exacerbates tau neurotoxicity. Overexpression of a SUT-2::GFP fusion protein alters tau-related phenotypes. (A) Diagram of the SUT-2::GFP transgene. (B) The SUT-2 transgene exacerbates tau induced neuronal dysfunction. T337 Tg has significantly higher dispersal rate than T337;SUT-2 double-transgenic animals (P < 0.0001). Non-transgenic control strains and the SUT-2::GFP transgene alone have similar locomotion with a dispersal rate of ∼15 mm/h. (C) SUT-2 overexpression exacerbates tau-mediated degeneration of GABAergic neurons. Comparison of T337;SUT-2::GFP double-transgenic animals with single T337 transgenic animals reveals a significant increase in neurodegenerative changes caused by SUT-2 overexpression (P < 0.0001). (D) SUT-2 protein overexpression drives accumulation of detergent-insoluble tau species as detected by extraction of tau protein using buffers of increasing solubilizing strength. Tau protein was sequentially extracted using high salt (RAB) and detergent-containing buffer (RIPA) to obtain detergent-insoluble tau protein from tau-transgenic animals with or without the SUT-2::GFP-expressing transgene.

Figure 2.

MSUT2 is a multi-isoform protein expressed in the nucleus of brain and cultured cells. (A) Immunoblotting demonstrates specificity of MSUT2 antibody in HEK293, mouse brain and human brain extracts. Note that the predicted human MSUT2 isoforms are 82, 65, 64 and 25 kDa. (B) MSUT2 immunofluorescence staining on HEK293. (C) MSUT2 staining on SH-SY5Y. (D) Localization of MSUT2 to SC35-positive nuclear speckles. (E) Lack of co localization between RNApol II and MSUT2. Scale bars are 10 µm.

Figure 3.

Tau expression increases MSUT2 protein levels, but has no effect on cellular localization. (A) Immunoblotting reveals an increase in MSUT2 protein in response to tau expression. (B) Immunofluorescence microscopy of MSUT2 (red) in nucleus of HEK293 cells. (C) HEK/tau cells overexpressing human wild-type (4R1N) tau. (D) Live mount image of non-transgenic C. elegans overexpressing SUT-2::GFP shows a nuclear speckle pattern as does (E) T337;SUT-2::GFP double-transgenic C. elegans. Scale bars are 10 µm.

Figure 4.

MSUT2 is decreased in AD brain samples from the temporal cortex. (A) MSUT2 levels in temporal cortex extracts were examined by immunoblotting with MSUT2-specific antibodies. Lysates from five age-matched controls and AD brains were compared by quantitative immunoblotting in triplicate. (B) Comparison of immunoblotting results shows a significant difference between control and AD brain samples (P = 0.0088).

Figure 5.

MSUT2 immunohistochemical staining is specifically reduced in hippocampal neurons but not ependymal cells of AD. Immunohistochemical staining reveals markedly reduced MSUT2 immunostaining of nuclei in hippocampal and parahippocampal neurons of AD patients (A) relative to controls (B). In contrast, nuclear staining is relatively unchanged in ependymal cells of AD (C) and controls (D).

Figure 6.

Knockdown of MSUT2 depletes aggregated and insoluble tau species. (A) Tau protein was sequentially extracted to obtain the detergent-insoluble tau protein from HEK/tau cells with or without PSI treatment and with or without MSUT2 RNAi knockdown. PSI treatment consisted of 2 μm PSI treatment for 18 h prior to harvest. MSUT2 RNAi involved treatment with chemical siRNA for 40 h prior to cell harvest. The total fraction was probed with MSUT2, actin and tau-specific antibodies. Three other fractions were generated by sequential extraction and probed using tau-specific antibodies. RAB contains tau solubilized by high salt; RIPA contains detergent-soluble tau, whereas FA contains the detergent-insoluble material FA-solubilized protein. (B–E) HEK/tau cells were subjected to siRNA treatment for 48 h (D and E) prior to 18 h treatment with 2 μm PSI (C and E). Fixed cells were double stained for tau (green) and MSUT2 (red) and examined with immunofluorescence microscopy.