Neuron-specific resilience to proteostatic stress

Protein homeostasis or “proteostasis” is essential for cellular survival and particularly important for highly specialised post-mitotic cells such as neurons. Accordingly, neurons are expected to be more resilient to proteostatic stress than other cell types. However, the neuron-specific mechanisms that secure proteostatic stress resilience are currently unresolved. The objective of this thesis is to investigate neuron-specific resilience to proteostatic stress in the endoplasmic reticulum (ER), the cellular organelle that controls the synthesis, maturation and folding of secreted and membrane proteins. Failure in any of these processes in the ER results in the accumulation of unfolded or misfolded proteins, which leads to ER stress. A major response to ER stress is the activation the Unfolded Protein Response (UPR) that in mammalian cells consists of three intricate signalling pathways: the inositol requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)-pathway.

Chapter 2 investigates the resilience of neurons to PERK deficiency. Transient reduction of protein translation by PERK-mediated phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α) is a major proteostatic survival response during ER stress. Paradoxically, neurons are remarkably tolerant to PERK dysfunction, which suggests the existence of cell type-specific mechanisms that secure proteostatic stress resilience. In this chapter we employed cultured neurons and astrocytes to distinguish the cell autonomous neuronal response to ER stress. We demonstrate that PERK-deficient neurons, unlike astrocytes, retain the capacity to control translation during ER stress. Two molecular pathways – regulated by heme-regulated inhibitor (HRI) and the tRNA cleaving RNase angiogenin (ANG) - were identified that jointly drive translational control in PERK-deficient neurons.

In Chapter 3 the transmission of UPR activation is investigated in neuronal cells. We show that cell-to-cell transmission of the UPR from ER-stressed-donor cells to naive acceptor cells does not occur in cell culture. First, we employed pharmaca-based UPR induction to generate conditioned media (CM) from donor cells. However, we observed that carry-over of pharmaca to the acceptor cells via the CM was a major confounding factor. Hence, we genetically induced single pathways of the UPR, activated the full UPR by nutrient deprivation and expressed the heavy chain of immunoglobulin M (IgM) in donor cells, which all did not result in UPR transmission to acceptor cells. Moreover, also in direct co-culture of donor cells expressing the IgM heavy chain and fluorescent UPR reporter acceptor cells, no UPR transmission was observed.

Chapter 4 focusses on neuronal UPR-induced secretion. In this chapter we first determine the UPR-induced secretome of cultured primary mouse neurons using unbiased proteomics analysis and identified several members of the protein disulfide isomerase (PDI) family as transcriptional UPR targets that are secreted during ER stress. Next, we hypothesise that PDI proteins may provide an early tau-related cerebrospinal fluid (CSF) biomarker for neurodegenerative diseases. We demonstrated that intracellular tau aggregation induces the secretion of P4HB/PDIA1 in vitro. To investigate whether the CSF levels of PDI proteins are associated with tau pathology in the brain we analysed mass spectrometry proteomics data from control and AD patients and employed targeted immunodetection in a separate cohort. This demonstrated that CSF levels of PDI proteins correlate with total (t)-tau and phosphorylated (p)-tau levels in CSF. Furthermore, we demonstrate that PDI levels are selectively increased in the CSF of patients with tau-related dementia.

In this thesis we identify multiple novel routes neurons employ to counter proteostatic challenges. This creates a paradigm shift in the canonical stress pathways and provides crucial information for a better understanding of how proteostatic disturbance triggers neurodegeneration in a cell autonomous and cell non-autonomous manner.