Sustained translational repression by eIF2a-P mediates prion neurodegeneration

The mechanisms leading to neuronal death in neurodegenerative disease are poorly understood. Many of these disorders, including Alzheimer’s, Parkinson’s and prion diseases, are associatedwith the accumulation of misfolded disease-specific proteins. The unfolded protein response is a protective cellular mechanism triggered byrising levels ofmisfoldedproteins.Onearmof this pathwayresults in the transient shutdown of protein translation, through phosphorylation of the a-subunit of eukaryotic translation initiation factor, eIF2. Activation of the unfolded protein response and/or increased eIF2a-P levels are seen in patients with Alzheimer’s, Parkinson’s and prion diseases, but how this links to neurodegeneration is unknown. Here we show that accumulation of prion protein during prion replication causes persistent translational repression of global protein synthesis by eIF2a-P, associated with synaptic failure and neuronal loss in prion-diseased mice. Further, we show that promoting translational recovery in hippocampi of prion-infected mice is neuroprotective. Overexpression of GADD34, a specific eIF2a-P phosphatase, as well as reduction of levels of prion protein by lentivirally mediated RNA interference, reduced eIF2a-P levels. As a result, both approaches restored vital translation rates during prion disease, rescuing synaptic deficits and neuronal loss, thereby significantly increasing survival. In contrast, salubrinal, an inhibitor of eIF2a-P dephosphorylation, increased eIF2a-P levels, exacerbating neurotoxicity and significantly reducing survival in priondiseasedmice.Given the prevalence of proteinmisfolding and activation of the unfolded protein response in several neurodegenerative diseases, our results suggest thatmanipulation of common pathways such as translational control, rather thandisease-specific approaches, may lead to new therapies preventing synaptic failure and neuronal loss across the spectrum of these disorders. Neurodegenerative diseases pose an ever-increasing challenge for society and health care systems worldwide, but their molecular pathogenesis is still largely unknown and no curative treatments exist. Alzheimer’s (AD), Parkinson’s (PD) and prion diseases are separate clinical and pathological conditions, but it is likely they share common mechanisms leading to neuronal death. Mice with prion disease show misfolded prion protein (PrP) accumulation and develop extensive neurodegeneration (with profound neurological deficits), in contrast tomousemodels of ADor PD, inwhich neuronal loss is rare. Uniquely therefore, prion-infected mice allow access to mechanisms linking protein misfolding with neuronal death. Prion replication involves the conversion of cellular PrP, PrP, to its misfolded, aggregating conformer, PrP, a process leading ultimately to neurodegeneration. We have previously shown rescue of neuronal loss and reversal of early cognitive andmorphological changes in prion-infectedmice by depleting PrP in neurons, preventing prion replication and abrogating neurotoxicity. However, the molecular mechanisms underlying both the progression of disease, and those underlying recovery in PrP-depleted animals, were unknown. To understand these processes better, we now analysed the evolution of neurodegeneration in prion-diseased mice. We examined hippocampi fromprion-infected tg37miceused inour previous experiments, in which the time course of impairment and recovery are clearly defined. Hemizygous tg37 mice express mouse PrP at approximately three times wild-type levels and succumb to Rocky Mountain Laboratory (RML) prion infection within 12 weeks post infection (w.p.i.). They first develop behavioural signs with decreased burrowing activity at approximately 9w.p.i., after reduction in hippocampal synaptic transmission and first neuropathological changes. This is the windowof reversibilitywhen diseased neurons can still be rescued: PrP depletion up to 9w.p.i., but not later, rescues neurotoxicity, as by 10w.p.i. neuronal loss is established. We measured PrP levels, synapse number, levels of synaptic proteins and synaptic transmission in prion-infected mice weekly from 5w.p.i., and burrowing behaviour from 6w.p.i. We examined brains histologically and counted CA1 neurons. (Cohorts of at least 30 animals were used per group; biochemical and histological analyses were done on three mice per time point, burrowing behaviour on 12, n for other analyses is indicated in figure legends.) We found an early decline in synapse number in asymptomatic animals at 7w.p.i. to approximately 55% of control levels (Fig. 1a), despite unchanged levels of several preand postsynaptic marker proteins (Fig. 1b). Reduced synapse number with normal synaptic protein levels is likely to reflect impaired structural plasticity of synapses at this early stage of disease. At 9w.p.i., however, there was a sudden decline in synaptic protein levels to approximately 50% of control levels for several pre(SNAP-25 and VAMP-2) and postsynaptic (PSD-95 and NMDAR1) proteins (Fig. 1b and Supplementary Fig. 1b). This was associated with further decline in synapse number, and the critical reduction in synaptic transmission, both in amplitude of evoked excitatory postsynaptic currents (EPSCs) and in the number of spontaneousminiature EPSCs (mEPSCs) in CA1 neurons (Fig. 1c and Supplementary Fig. 1e). This was coincident with behavioural change (Fig. 1d) and first spongiform pathology (Supplementary Fig. 1d), and was rapidly followed by the onset of neurodegeneration, resulting in 50% reduction in hippocampal pyramidal neurons at 10w.p.i. (Fig. 1e). All animals developed overt motor signs and were terminally sick by 12w.p.i. The abrupt loss of synaptic proteins at 9w.p.i. appeared to be a critical factor in the evolution of disease, occurring when synapse number and transmission were already declining. This could result from increased degradation, or decreased synthesis. Prion infection in mice is known to impair the ubiquitin proteasome system, causing reduction—not increase—in protein degradation. We therefore asked if protein synthesis was reduced through translational control mechanisms. Given that total PrP levels rise during disease (Fig. 2a), and that PrP is synthesized in the endoplasmic reticulum, we examined the translational repression pathway of the unfolded protein response (UPR). Rising levels of unfolded proteins detected