Endoplasmic reticulum stress activates autophagy but not the proteasome in neuronal cells: implications for Alzheimer's disease

Abstract

Protein folding stress in the endoplasmic reticulum (ER) may lead to activation of the unfolded protein response (UPR), aimed to restore cellular homeostasis via transcriptional and post-transcriptional mechanisms. ER stress is also reported to activate the ER overload response (EOR), which activates transcription via NF-κB. We previously demonstrated that UPR activation is an early event in pre-tangle neurons in Alzheimer's disease (AD) brain. Misfolded and unfolded proteins are degraded via the ubiquitin proteasome system (UPS) or autophagy. UPR activation is found in AD neurons displaying both early UPS pathology and autophagic pathology. Here we investigate whether activation of the UPR and/or EOR is employed to enhance the proteolytic capacity of neuronal cells. Expression of the immunoproteasome subunits β2i and β5i is increased in AD brain. However, expression of the proteasome subunits is not increased by the UPR or EOR. UPR activation does not relocalize the proteasome or increase overall proteasome activity. Therefore proteasomal degradation is not increased by ER stress. In contrast, UPR activation enhances autophagy and LC3 levels are increased in neurons displaying UPR activation in AD brain. Our data suggest that autophagy is the major degradational pathway following UPR activation in neuronal cells and indicate a connection between UPR activation and autophagic pathology in AD brain.

Figure 1

Expression of β2i and β5i immunoproteasome subunits is increased in AD hippocampus. Representative immunohistochemical stainings for β2i (a–f) and β5i (g–l) in the hippocampal CA1 and CA4 region of a non-demented control Braak 1/0 (CA1 (a and g), CA4 (b and h)), AD Braak 3C (CA1 (c and i), CA4 (d and j)) and AD Braak 5C (CA1 (e and k), CA4 (f and l)) case. Reactivity to β2i is present in neurons, glial and endothelial cells, and increases with the Braak stage for NFTs. Double immunolabelling shows β5i expression in astrocytes using GFAP as a marker (m) and microglia using Iba1 as a marker (n) in AD hippocampus. Nuclei were counterstained with hematoxylin (blue). The GFAP/Iba1 and β5i signals were spectrally unmixed and are shown separately (m/n 1 and m/n 2, respectively) and as a merge with artificial colors (m/n 3: GFAP/Iba1: red; β5i: green). Scale bar: 50 μM

Figure 2

Effects of ER stress on proteasome β subunit expression. Differentiated SK-N-SH cells were treated with γIFN or Tm for 16 h. Relative mRNA and protein levels of proteasome subunits were determined using q-PCR and western blotting. (a) Normalized mRNA expression levels of the proteasome subunits α (HC5 and C7) and β (β1, β2, β5, β2i and β5i). (b) ER stress-responsive genes BiP and CHOP. Expression levels were normalized to GAPDH mRNA levels. Data are mean±S.D. from triplicate observations of a representative experiment. Asterisks indicate significant differences compared with control levels (P<0.05). (c) Western blot analysis with antibodies directed against the β5i, β2i and β2 subunits and the ER stress-responsive protein BiP. Asterisk indicates cross-reactivity of the BiP antibody with Hsc70. The expression levels of eEF2α are indicated as loading control. Blots from a representative experiment are shown

Figure 3

ER stress does not increase β5i promoter activity. (a) Representation of binding sites for ER stress-responsive transcription factors in the promoter regions of the β subunits of the proteasome. No classical ERSE-binding motifs are identified; in contrast putative NF-κB (p50–p65) binding sites are present in the proximal promoter region of all the immuno β subunits. The location of the binding site is indicated relative to the transcription start site. (b) Relative activity of β5i, β5i NF-κB-binding site mutant (β5i ΔNF-κB) and BiP promoters in HEK293 cells. Promoter activities were determined using a luciferase-renilla assay as described in Materials and methods in the presence or absence of γIFN or Tm at the indicated concentrations for 16 h. Data are mean±S.D. from triplicate observations of a representative experiment. Asterisks indicate significant differences compared with control (P<0.05)

Figure 4

ER stress does not influence β5i localization. Differentiated SK-N-SH cells were treated with γIFN or Tm for 16 h at the indicated concentrations compared with a non-treated control. Confocal pictures of the localization of β5i (left hand panel) and calnexin (middle panel) by immunofluorescence are shown. An overlay of β5i (red), calnexin (green) and DAPI nuclear counterstain (blue) is shown on the right. Scale bar: 20 μM

Figure 5

ER stress does not increase proteasome activity. Differentiated SK-N-SH cells were treated with indicated concentrations of Tm for 16 h and incubated with the Me₄BodipyFL proteasome activity probe for 5 h. (a) Visualization of probe binding after SDS-PAGE using a 2D proteomic imaging system shows probe binding to the β2 and β5 subunits predominantly (activity). The lower panel is a western blot of the same samples showing that β2 expression levels are not changed by the treatment (protein). (b) Quantification of the fluorescence intensity of the bands in the upper panel of (a), corrected for loading by the expression levels of eEF2α (not shown) and presented as percent of untreated control (c) cells. This reflects the relative activity of the β2 and β5 subunits following Tm treatment

Figure 6

ER stress induces autophagy and does not lead to increased polyubiquitination. Differentiated SK-N-SH cells were treated with ER stressors (Tm, Th and 2DG), γIFN and the proteasome inhibitor epoxomycin for 16 h and the protein levels were analyzed by western blotting. (a) Blots showing changes in the level of ubiquitinated (Ub) proteins (upper panel) and LC3-I and LC3-II (middle panel) following treatment with ER stressors, γIFN and epoxomycin. The expression levels of eEF2α are indicated as loading control. Blots from a representative experiment are shown. (b) LC3-II/LC3-I ratios determined from (a)

Figure 7

LC3 levels are increased in pPERK-positive neurons in the AD hippocampus. (a) Immunohistochemistry showing LC3 levels in hippocampal neurons of a representative AD (Braak 4C) case. A part of the CA1 and subiculum (sub) area is shown. Scale bar: 500 μM. (b and c) Magnification of CA1 (b) and subiculum (c) from (a), showing LC3 levels are increased in neurons with GVD. (d) Representative double immunohistochemistry staining showing LC3 levels are increased in neurons that show pPERK reactivity. Nuclei were counterstained with hematoxylin (blue). The LC3 and pPERK signals were spectrally unmixed and are shown separately (d1 and d2, respectively) and as a merge with artificial colors (d3: LC3 red; pPERK green). Scale bar (b and c) 25 μM

Figure 8

ER stress preferentially activates autophagy in neuronal cells. (a) Diagram showing the pathways investigated in this study by which ER stress may increase the proteolytic capacity of neuronal cells. ER stress could enhance proteolytic degradation by the proteasome by influencing the subunit composition via activation of the EOR. Alternatively, the UPR can be employed to enhance the expression levels of the constitutive subunits. In addition, regulation of the localization or activity of the proteasome may occur. In addition, the UPR may result in activation of autophagy. (b) Our data show that a disturbance of ER homeostasis resulting in activation of the UPR will activate the autophagy pathway. This can be due to a decreased capability to export misfolded proteins from the ER lumen under these conditions or may relate to the inability of the proteasome to degrade aggregated proteins. In AD neurons, decreased proteasomal degradation as well as a disruption in the autophagosomal/lysosomal system may eventually lead to neurodegeneration