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 associated with the accumulation of misfolded disease-specific proteins. The unfolded protein response is a protective cellular mechanism triggered by rising levels of misfolded proteins. One arm of this pathway results 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 1-4 , 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 5 , increased eIF2a-P levels, exacerbating neurotoxicity and significantly reducing survival in priondiseased mice. Given the prevalence of protein misfolding and activation of the unfolded protein response in several neurodegenerative diseases, our results suggest that manipulation of common pathways such as translational control, rather than disease-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 to mouse models of AD or PD, in which 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 C , to its misfolded, aggregating conformer, PrP Sc , a process leading ultimately to neurodegeneration 6 . We have previously shown rescue of neuronal loss and reversal of early cognitive and morphological changes in prion-infected mice by depleting PrP in neurons, preventing prion replication and ab...

Lipid transport between intracellular organelles is mediated by vesicular and nonvesicular transport mechanisms and is critical for maintaining the identities of different cellular membranes. Nonvesicular lipid transport between the endoplasmic reticulum (ER) and the Golgi complex has been proposed to affect the lipid composition of the Golgi membranes. Here, we show that the integral ER-membrane proteins VAP-A and VAP-B affect the structural and functional integrity of the Golgi complex. Depletion of VAPs by RNA interference reduces the levels of phosphatidylinositol-4-phosphate (PI4P), diacylglycerol, and sphingomyelin in the Golgi membranes, and it leads to substantial inhibition of Golgimediated transport events. These effects are coordinately mediated by the lipid-transfer/binding proteins Nir2, oxysterolbinding protein (OSBP), and ceramide-transfer protein (CERT), which interact with VAPs via their FFAT motif. The effect of VAPs on PI4P levels is mediated by the phosphatidylinositol/phosphatidylcholine transfer protein Nir2, which is required for Golgi targeting of OSBP and CERT and the subsequent production of diacylglycerol and sphingomyelin. We propose that Nir2, OSBP, and CERT function coordinately at the ER-Golgi membrane contact sites, thereby affecting the lipid composition of the Golgi membranes and consequently their structural and functional identities. INTRODUCTIONThe unique lipid compositions of the secretory and endocytic organelles are critical for maintaining their distinct structural and functional identities (van Meer, 2000). Their identities are maintained despite extensive interconnecting vesicular-trafficking pathways, and they require tight regulation of lipid sorting, metabolism, and transport (van Meer, 1993;Pomorski et al., 2001;van Meer and Sprong, 2004). Increasing lines of evidence suggest that lipid-transfer proteins (LTPs) play a major role in regulating the lipid composition of membranous organelles (Sprong et al., 2001;De Matteis et al., 2007). These proteins facilitate lipid transfer between a donor and acceptor membrane (Holthuis and Levine, 2005), and they usually contain dual-targeting determinants for different membrane compartments. LTPs have been proposed to efficiently transfer lipids at membrane contact sites (MCSs) (Holthuis and Levine, 2005;Levine and Loewen, 2006).MCSs or zones of close apposition between the ER membranes and other cellular membranes, including the plasma membrane (PM), the membranes of the vacuoles, mitochondria, peroxisomes, lipid droplets, late endosomes, lysosomes, and the Golgi apparatus, have been identified in all eukaryotes by morphological and biochemical studies (Shore and Tata, 1977;Levine, 2004;Levine and Loewen, 2006). More recently, electron tomography studies have identified close contacts (10 -20 nm) between the ER and the outer mitochondrial membrane (Perkins et al., 1997) and between a specialized trans-ER and the three trans-most cisternae of the Golgi complex (Ladinsky et al., 1999;Marsh et al., 2001Marsh et al., , 2004. This ...

In the healthy adult brain synapses are continuously remodelled through a process of elimination and formation known as structural plasticity1. Reduction in synapse number is a consistent early feature of neurodegenerative diseases2, 3, suggesting deficient compensatory mechanisms. While much is known about toxic processes leading to synaptic dysfunction and loss in these disorders2,3, how synaptic regeneration is affected is unknown. In hibernating mammals, cooling induces loss of synaptic contacts, which are reformed on rewarming, a form of structural plasticity4, 5. We have found that similar changes occur in artificially cooled laboratory rodents. Cooling and hibernation also induce a number cold-shock proteins in the brain, including the RNA binding protein, RBM36. The relationship of such proteins to structural plasticity is unknown. Here we show that synapse regeneration is impaired in mouse models of neurodegenerative disease, in association with the failure to induce RBM3. In both prion-infected and 5×FAD (Alzheimer-type) mice7, the capacity to regenerate synapses after cooling declined in parallel with the loss of induction of RBM3. Enhanced expression of RBM3 in the hippocampus prevented this deficit and restored the capacity for synapse reassembly after cooling. Further, RBM3 over-expression, achieved either by boosting endogenous levels through hypothermia prior to the loss of the RBM3 response, or by lentiviral delivery, resulted in sustained synaptic protection in 5×FAD mice and throughout the course of prion disease, preventing behavioural deficits and neuronal loss and significantly prolonging survival. In contrast, knockdown of RBM3 exacerbated synapse loss in both models and accelerated disease and prevented the neuroprotective effects of cooling. Thus, deficient synapse regeneration, mediated at least in part by failure of the RBM3 stress response, contributes to synapse loss throughout the course of neurodegenerative disease. The data support enhancing cold shock pathways as potential protective therapies in neurodegenerative disorders.