XBP1 depletion precedes ubiquitin aggregation and Golgi fragmentation in TDP-43 transgenic rats

Abstract

Protein inclusion is a prominent feature of neurodegenerative diseases including frontotemporal lobar degeneration (FTLD) that is characterized by the presence of ubiquitinated TDP-43 inclusion. Presence of protein inclusions indicates an interruption to protein degradation machinery or the overload of misfolded proteins. In response to the increase in misfolded proteins, cells usually initiate a mechanism called unfolded protein response (UPR) to reduce misfolded proteins in the lumen of endoplasmic reticules. Here, we examined the effects of mutant TDP-43 on the UPR in transgenic rats that express mutant human TDP-43 restrictedly in the neurons of the forebrain. Over-expression of mutant TDP-43 in rats caused prominent aggregation of ubiquitin and remarkable fragmentation of Golgi complexes prior to neuronal loss. While ubiquitin aggregates and Golgi fragments were accumulating, neurons expressing mutant TDP-43 failed to up-regulate chaperones residing in the endoplasmic reticules and failed to initiate the UPR. Prior to ubiquitin aggregation and Golgi fragmentation, neurons were depleted of X-box-binding protein 1 (XBP1), a key player of UPR machinery. Although it remains to determine how mutation of TDP-43 leads to the failure of the UPR, our data demonstrate that failure of the UPR is implicated in TDP-43 pathogenesis.

Figure 1

Restricted expression of mutant TDP-43 in the neurons of rat forebrain. (A) Immunoblotting reveals regulated expression of human TDP-43 in the cortex (CT) and hippocampus [HP] of Camk2a-tTA/TRE-TDP43^M337V double transgenic rats. The double transgenic rats were given Dox during development and were deprived of Dox at the age of 35 days. Expression of mutant TDP-43 transgene was quickly recovered upon Dox withdrawal. * indicates a nonspecific band. (B, C) Immunostaining reveals expression of human TDP-43 in the cortex of Camk2a-tTA/TRE-TDP43^M337V double transgenic rat (C), but not in the tissues of Camk2a-tTA single transgenic rat (B). (D–L) Double-labeling immunofluorescence staining reveals that mutant human TDP-43 (hTDP43) was colocalized with the neuronal marker NeuN and was not colocalized with either the microglia marker Iba-1 (G–I) or the astrocyte marker GFAP (J–L). Scale bars: B and C, 100 µm; D–L, 30 µm.

Figure 2

Temporal expression of mutant TDP-43 causes a progressive loss of neurons and spatial memory in adult rats. (A, B) Immunostaining for human TDP-43 reveals that the restoration of transgene expression was tissue-dependent, with a quicker recovery in the hippocampus (B1–6) compared to the cortex (A1–6). Camk2a-tTA single (tTA) and Camk2atTA/TRE-TDP43^M337V double (M337V) transgenic rats were constantly given Dox until they were 35 days old. (C, D) Cresyl violet staining reveals that the onset of neuronal loss was closely correlated with the start of transgene expression in the cortex (C1–6) and hippocampus (D1–6). (E, F) Stereological cell counting reveals a remarkable loss of neurons in the dentate gyrus (E) and frontal cortex (F). Data are means + SEM (n = 6). * p < 0.01. (G) Barnes maze assay reveals a progressive loss of spatial memory in mutant TDP-43 transgenic rats (M337V) compared to the normal controls (tTA). Data are means + SEM (n = 12). Loss of spatial memory is used as a parameter defining the stages of disease progression in mutant TDP-43 transgenic rats. Scale bars: A1–6, B1–6 and D1–D6, 100 µm; C1–6, 30 µm.

Figure 3

Neurites and spines are lost prior to neuron loss in mutant TDP-43 transgenic rats. (A–J) Golgi staining reveals that the neurites were retracted before the soma of neurons was lost. The parietal cortex corresponding to the start of hippocampus was examined for Camk2a-tTA single (tTA) and Camk2a-tTA/TRE-TDP43^M337V double (M337V) transgenic rats at defined ages. Scale bars: A–E, 100 µm; F–J, 50 µm. (K, L) Spine densities and branch points were quantified for three neurons in the layer V of parietal cortex for individual rats. Data are means + SEM (n = 5). * p < 0.05.

Figure 4

Ubiquitin forms aggregates and Golgi apparatuses are fragmented in neurons expressing mutant TDP-43. (A, B) Representative photos show a gradual development of ubiquitin aggregates (A1–4) and Golgi fragments (B1–4) in the frontal cortex of Camk2atTA/ TRE-TDP43^M337V double transgenic rats (M337V), but not in the tissues of Camk2a-tTA single transgenic rats (tTA). Arrows point to neurons with fragmented Golgi apparatus. Scale bars: 10 µm. (C, D) Cortical neurons with ubiquitin aggregates (C) or fragmented Golgi were quantified for individual rats. Data are means + SEM (n = 5). (E–H) Confocal microscopy reveals that aggregated ubiquitin and fragmented Golgi occurred in neurons expressing mutant TDP-43 and that Cis-(stained of GM130) and Trans (stained of GLG1)-Golgi complexes were simultaneously fragmented and Golgi fragments were not colocalized with ubiquitin aggregates. Arrows point to fragmented Golgi complex. Z-stack images were taken at the same magnification and projected to show the profile of Golgi fragmentation (F–G). (I) Confocal microscopy reveals that aggregated mitochondria were not colocalized with ubiquitin inclusions. (J–L) Electromicroscopy reveals that damaged endoplasmic reticules and Golgi complexes of cortical neurons were swollen to form vacuoles as disease progressed. Nuclear envelope defined the boundary between the nucleus (N) and the cytoplasm (C). Arrows point to endoplasmic reticules and Golgi complexes. Arrowheads point to mitochondria. Vacuoles are marked with “V”. Scale bars: 500 nm.

Figure 5

Expression of XBP1 is downregulated in neurons and upregulated in reactive microglia in mutant TDP-43 transgenic rats. (A, B) Immunoblotting reveals expression of the genes responsive to endoplasmic reticule stress in the cortex of transgenic rats. Each lane was loaded with 20 µg of total proteins. Spliced (XBP1s) and unspliced (XBP1u) forms of XBP1 were upregulated in the total tissue homogenates of Camk2a-tTA/TRE-TDP43^M337V double transgenic rats (M337V) as compared to Camk2a-tTA single transgenic rats (tTA). (C1–L3) Confocal microscopy reveals that both XBP1s and XBP1u were downregulated in neurons expressing mutant TDP-43 and were upregulated in reactive microglia. Z-stack images were taken at the same magnification and were projected to show the profile of gene expression.

Figure 6

Glial cells are gradually activated in mutant TDP-43 transgenic rats. (A–J) Immunostaining of the microglial marker Iba-1 reveals remarkable activation of microglia in the tissues of Camk2a-tTA/TRE-TDP43^M337V double transgenic rats (M337V) as compared to those of Camk2a-tTA single transgenic rats (tTA). (K–T) Immunostaining of the astrocyte marker GFAP reveals a progressive activation of astrocytes in mutant TDP-43 transgenic rats. Scale bars: A–H and K–N, 100 µm; O–R, 50 µm; I–J and S–T, 25 µm.