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Protein folding stress in the endoplasmic reticulum (ER) may lead to activation of the unfolded protein response (UPR), aimed to restore proteostasis in the ER. Previously, we demonstrated that UPR activation is an early event in Alzheimer disease (AD) brain. In our recent work we investigated whether activation of the UPR is employed to enhance the capacity of the ubiquitin proteasome system or autophagy in neuronal cells. We showed that the levels, composition and activity of the proteasome are not regulated by the UPR. 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.
Alzheimer disease (AD) is a neurodegenerative disorder characterized by increased accumulation of protein aggregates: extracellular β-Amyloid aggregates and intracellular aggregates of hyperphosphorylated tau. Under physiological conditions, aberrant proteins are targeted for degradation by the ubiquitin proteasome system (UPS) or the autophagic-lysosomal system. Both systems are part of a complex network that maintains the protein homeostasis, also called proteostasis. The accumulation of protein aggregates in AD and many other neurodegenerative disorders indicates a severe disturbance in the proteostasis network. In the endoplasmic reticulum (ER) aberrant proteins are exported to the cytosol and subsequently degradated by the UPS; a process called ER-associated degradation (ERAD). In addition, ER stress conditions induce autophagy. Increased levels of aberrant proteins in the ER activate the unfolded protein response (UPR), a stress response aimed to restore proteostasis in the ER.
We have previously shown UPR activation in AD neurons that contain diffuse hyperphosphorylated tau, indicating that UPR activation is an early phenomenon in AD. UPR-positive neurons in the AD hippocampus are associated with UPS pathology, as they show ubiquitin positive-, p62-negative inclusion bodies and present with granulovacuolar degeneration (GVD), an indication of disturbed autophagic-lysosomal function. GVD consists of electron-dense granules surrounded by clear 2 to 4 µm vacuoles in the cytoplasm of hippocampal neurons. The UPR is activated in AD neurons, and may be a signal to enhance protein degradation in the affected neuron to prevent the formation of toxic aggregates. In our recent study we investigated the regulation of the UPS and autophagy during ER stress in vitro and in human AD brain.
ER stress has been reported to activate the ER overload response (EOR), which signals via NFκB. The constitutive β subunits of the proteasome can be exchanged for the immuno subunits β1i, -2i and -5i triggered by NFκB signaling. This could be a response to the accumulation of substrates that require a different proteolytic specificity in order to be degraded. Our data show that expression of the immunoproteasome subunits is increased in the AD hippocampus. Immunohistochemical analysis demonstrates that the β2i subunit is predominantly expressed in neurons and increases with the Braak stage, a pathological staging, which indicates the level of tau pathology. In contrast, neuronal β5i shows only a moderate increase in neurons in high Braak stages. However, there is a remarkable increase in the number of astrocytes and microglia that are immunoreactive for β5i in higher Braak stages.
We subsequently investigated whether ER stress affects proteasome subunit expression in a neuronal cell model. The mRNA levels of noncatalytic α subunits or the constitutive catalytic β subunits are not increased by induction of ER stress. The β2i and β5i mRNAs are upregulated, however, this does not result in increased protein levels. The immuno β subunit proximal promoters all contain a consensus NFκB site that is likely activated by the EOR if it is functional. Using a luciferase reporter approach, we showed that the β2i promoter is responsive to NFκB regulation. However, our data demonstrate that the activity of this promoter is not regulated by the UPR or EOR pathways of the ER stress response. In addition, β5i subcellular localization is not affected by the UPR. This indicates that UPR activation is not responsible for the increased immunoproteasome levels observed in AD, which of course does not imply that the presence of the immunoproteasome does not contribute to proteostasis. In order to directly investigate the effect of ER stress on total proteasome activity, a fluorescent proteasome activity probe was used that covalently binds the catalytic subunits of active proteasomes. This direct method is particularly appropriate to study activity during ER stress, when overall translation is inhibited. Despite an expected greater demand for degradation during ER stress, UPR activation has little to no effect on proteasome activity in our cell model. Interestingly, ubiquitinated proteins do not accumulate during ER stress, suggesting that autophagy suffices to degrade aberrant proteins under these conditions. This is in accordance with our previous work showing that complete proteasome inhibition induces the cytosolic heat shock response, but does not lead to robust activation of the UPR.
Corroborating other reports, we find that in neuronal cells LC3-II levels and the LC3-II/LC3-I ratio are increased by different ER stress inducers, indicating that autophagy becomes more active under conditions of ER stress. We show that hippocampal neurons in human brain have high LC3 levels and that these levels are increased in AD neurons that have activated the UPR. High LC3 levels can indicate a highly active autophagy system, and therefore this observation may suggest that autophagic activity is increased by ER stress in AD hippocampus. Because it is not possible to determine the autophagic state in postmortem brain material, an alternative explanation for the high LC3 levels may be impaired autophagic clearance, leading to LC3 accumulation. Strikingly, we found increased LC3 levels in neurons that show disturbed autophagy, demonstrated by the presence of GVD.
Our data indicate that autophagy is the major proteolytic pathway during ER stress in neuronal cells. We show that the levels, composition and activity of the proteasome are not regulated by the UPR or EOR. In contrast, induction of the UPR activates autophagy. The preference for autophagy may be due to overloading of the UPS during ER stress. Alternatively, higher amounts of misfolded proteins in the ER may more readily form aggregates, which cannot be exported. Under these conditions bulk degradation of parts of the ER is a more favorable pathway to restore homeostasis. Our data indicate a direct link between UPR activation, autophagy and the occurrence of autophagic pathology in human AD brain. We propose that during the disease a vicious cycle arises by increased demand from the UPR for autophagic clearance combined with decreased efficiency of autophagy itself as well as other parts of the proteostasis network due to the aging process (Fig. 1). Because the UPR is activated in neurons in which pathological tau is present, but not (yet) densely aggregated, the UPR-autophagy pathway may provide a target for early therapeutic intervention in AD.
Punctum to: Nijholt DA, de Graaf TR, van Haastert ES, Oliveira AO, Berkers CR, Zwart R, Ovaa H, Baas F, Hoozemans JJ, Scheper W. Endoplasmic reticulum stress activates autophagy but not the proteasome in neuronal cells—Implications for Alzheimer's disease. Cell Death Differ. 2011;18:1071–1081. doi: 10.1038/cdd.2010.176.