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.

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In this study, we examined the subcellular distribution and functions of LIMK1 in developing neurons. Confocal microscopy, subcellular fractionation, and expression of several epitope-tagged LIMK1 constructs revealed that LIMK1 is enriched in the Golgi apparatus and growth cones, with the LIM domain required for Golgi localization and the PDZ domain for its presence at neuritic tips. Overexpression of wild-type LIMK1 suppresses the formation of trans-Golgi derived tubules, and prevents cytochalasin D-induced Golgi fragmentation, whereas that of a kinase-defective mutant has the opposite effect. Transfection of wild-type LIMK1 accelerates axon formation and enhances the accumulation of Par3/Par6, insulin-like growth factor (IGF)1 receptors, and neural cell adhesion molecule (NCAM) at growth cones, while inhibiting the Golgi export of synaptophysin-containing vesicles. These effects were dependent on the Golgi localization of LIMK1, paralleled by an increase in cofilin phosphorylation and phalloidin staining in the region of the Golgi apparatus, and prevented by coexpression of constitutive active cofilin. The long-term overexpression of LIMK1 produces growth cone collapse and axon retraction, an effect that is dependent on its growth cone localization. Together, our results suggest an important role for LIMK1 in axon formation that is related with its ability to regulate Golgi dynamics, membrane traffic, and actin cytoskeletal organization.

Exocytotic incorporation of plasmalemmal precursor vesicles (PPVs) into the cell surface is necessary for neurite extension and is known to occur mainly at the growth cone. This report examines whether this is a regulated event controlled by growth factors. The Golgi complex and nascent PPVs of hippocampal neurons in culture were pulse-labeled with fluorescent ceramide. We studied the dynamics of labeled PPVs upon arrival at the axonal growth cone. In controls and cultures stimulated with brain-derived neurotrophic factor (BDNF), PPV clusters persisted in growth cones with a half-life(t1/2) of >14 minutes. Upon challenge with IGF-1,however, fluorescent elements cleared from the growth cones with a t1/2 of only 6 minutes. Plasmalemmal expansion was measured directly as externalization of membrane glycoconjugates in resealed growth cone particles (GCPs) isolated from fetal forebrain. These assays demonstrated that membrane expansion could be stimulated by IGF-1 in a dose-dependent manner but not by BDNF, even though intact, functional BDNF receptor was present on GCPs. Because both BDNF and IGF-1 are known to enhance neurite growth, but BDNF did not stimulate membrane expansion at the growth cone, we studied the effect of BDNF on the IGF-1 receptor. BDNF was found to cause the translocation of the growth-cone-specific IGF-1 receptor subunitβ gc to the distal axon, in a KIF2-dependent manner. We conclude that IGF-1 stimulates axonal assembly at the growth cone, and that this occurs via regulated exocytosis of PPVs. This mechanism is affected by BDNF only indirectly, by regulation of the βgc level at the growth cone.

This article shows a novel function for the MAP1B protein, related to the control of actin dynamics through interaction with Tiam1.

SummaryCooling and hypothermia are profoundly neuroprotective, mediated, at least in part, by the cold shock protein, RBM3. However, the neuroprotective effector proteins induced by RBM3 and the mechanisms by which mRNAs encoding cold shock proteins escape cooling-induced translational repression are unknown. Here, we show that cooling induces reprogramming of the translatome, including the upregulation of a new cold shock protein, RTN3, a reticulon protein implicated in synapse formation. We report that this has two mechanistic components. Thus, RTN3 both evades cooling-induced translational elongation repression and is also bound by RBM3, which drives the increased expression of RTN3. In mice, knockdown of RTN3 expression eliminated cooling-induced neuroprotection. However, lentivirally mediated RTN3 overexpression prevented synaptic loss and cognitive deficits in a mouse model of neurodegeneration, downstream and independently of RBM3. We conclude that RTN3 expression is a mediator of RBM3-induced neuroprotection, controlled by novel mechanisms of escape from translational inhibition on cooling.