Mutations in superoxide dismutase-1 (SOD1) cause familial amyotrophic lateral sclerosis (fALS). Recent evidence implicates adaptive responses to endoplasmic reticulum (ER) stress in the disease process via a pathway known as the unfolded protein response (UPR). Here, we investigated the contribution to fALS of X-box-binding protein-1 (XBP-1), a key UPR transcription factor that regulates genes involved in protein folding and quality control. Despite expectations that XBP-1 deficiency would enhance the pathogenesis of mutant SOD1, we observed a dramatic decrease in its toxicity due to an enhanced clearance of mutant SOD1 aggregates by macroautophagy, a cellular pathway involved in lysosome-mediated protein degradation. To validate these observations in vivo, we generated mutant SOD1 transgenic mice with specific deletion of XBP-1 in the nervous system. XBP-1-deficient mice were more resistant to developing disease, correlating with increased levels of autophagy in motoneurons and reduced accumulation of mutant SOD1 aggregates in the spinal cord. Post-mortem spinal cord samples from patients with sporadic ALS and fALS displayed a marked activation of both the UPR and autophagy. Our results reveal a new function of XBP-1 in the control of autophagy and indicate critical cross-talk between these two signaling pathways that can provide protection against neurodegeneration.[Keywords: Amyotrophic lateral sclerosis; unfolded protein response; endoplasmic reticulum stress; XBP-1; autophagy] Supplemental material is available at http://www.genesdev.org. Received June 10, 2009; revised version accepted August 19, 2009. Most neurodegenerative disorders-such as amyotrophic lateral sclerosis (ALS), Alzheimer's, and Parkinson's and Huntington's diseases-share a common pathology characterized by abnormal protein inclusions containing specific misfolded proteins (Matus et al. 2008). ALS is a progressive and deadly adult-onset motor neuron disease characterized by muscle weakness, spasticity, atrophy, paralysis, and premature death. The pathological hallmark of ALS is the selective degeneration of motoneurons in the spinal ventral horn, most brainstem nuclei, and the cerebral cortex (Boillee et al. 2006;Pasinelli and Brown 2006). ALS is more frequent in males, and the disease lacks a defined genetic component in a majority of ALS patients, so-called sporadic ALS (sALS), while ;10% of cases are familial (fALS). More than 100 mutations in the gene encoding superoxide dismutase-1 (SOD1) are linked to fALS and trigger its misfolding and abnormal aggregation, resulting in motoneuron dysfunction (Pasinelli and Brown 2006). Overexpression of human fALS-linked SOD1 mutations in transgenic mice recapitulates essential features of the human pathology, provoking agedependent protein aggregation, paralysis, and motor neuron degeneration. Since sALS and fALS affect the same neurons with similar pathology, therapeutics effective in mutant SOD1 mouse models may translate to sALS.The pathogenesis of mutant SOD1 toxicity remains unclear, and m...

Melanocytes reside within the basal layer of the human epidermis, where they attach to the basement membrane and replicate at a rate proportionate to that of keratinocytes, maintaining a lifelong stable ratio. In this study, we report that coculturing melanocytes with keratinocytes up-regulated CCN3, a matricellular protein that we subsequently found to be critical for the spatial localization of melanocytes to the basement membrane. CCN3 knockdown cells were dissociated either upward to the suprabasal layers of the epidermis or downward into the dermis. The overexpression of CCN3 increased adhesion to collagen type IV, the major component of the basement membrane. As the receptor responsible for CCN3-mediated melanocyte localization, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that acts as a collagen IV adhesion receptor. DDR1 knockdown decreased melanocyte adhesion to collagen IV and shifted melanocyte localization in a manner similar to CCN3 knockdown. These results demonstrate an intricate and necessary communication between keratinocytes and melanocytes in maintaining normal epidermal homeostasis.

Contextual memory formation relies on the induction of new genes in the hippocampus. A polymorphism in the promoter of the transcription factor XBP1 was identified as a risk factor for Alzheimer's disease and bipolar disorders. XBP1 is a major regulator of the unfolded protein response (UPR), mediating adaptation to endoplasmic reticulum (ER) stress. Using a phenotypic screen, we uncovered an unexpected function of XBP1 in cognition and behavior. Mice lacking XBP1 in the nervous system showed specific impairment of contextual memory formation and long-term potentiation (LTP), whereas neuronal XBP1s overexpression improved performance in memory tasks. Gene expression analysis revealed that XBP1 regulates a group of memory-related genes, highlighting brain-derived neurotrophic factor (BDNF), a key component in memory consolidation. Overexpression of BDNF in the hippocampus reversed the XBP1-deficient phenotype. Our study revealed an unanticipated function of XBP1 in cognitive processes that is apparently unrelated to its role in ER stress.

SummaryPerturbed neuronal proteostasis is a salient feature shared by both aging and protein misfolding disorders. The proteostasis network controls the health of the proteome by integrating pathways involved in protein synthesis, folding, trafficking, secretion, and their degradation. A reduction in the buffering capacity of the proteostasis network during aging may increase the risk to undergo neurodegeneration by enhancing the accumulation of misfolded proteins. As almost one‐third of the proteome is synthetized at the endoplasmic reticulum (ER), maintenance of its proper function is fundamental to sustain neuronal function. In fact, ER stress is a common feature of most neurodegenerative diseases. The unfolded protein response (UPR) operates as central player to maintain ER homeostasis or the induction of cell death of chronically damaged cells. Here, we discuss recent evidence placing ER stress as a driver of brain aging, and the emerging impact of neuronal UPR in controlling global proteostasis at the whole organismal level. Finally, we discuss possible therapeutic interventions to improve proteostasis and prevent pathological brain aging.

Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. Here we report the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA synergies with TMBIM6/BI-1 in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane. Cell Death and Differentiation (2012) 19, 1013-1026 doi:10.1038/cdd.2011; published online 13 January 2012Apoptosis is a conserved cell death mechanism essential for normal development and tissue homeostasis in multicellular organisms. In mammals, the BCL-2 family of proteins is a group of crucial upstream regulators of the caspase cascade, comprising both pro-and anti-apoptotic components. 1Although apoptosis is observed in most multicellular organisms, the BCL-2 family of proteins as a whole is poorly conserved in invertebrates including worms, flies, and other species.1,2 In fact, only two BCL-2 homologues are present in flies with controversial roles in programmed cell death. 3,4 A pioneering screening to identify human genes that prevents BAX toxicity in a yeast assay identified transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX inhibitor-1 (BI-1), as a new mammalian gene that negatively regulates apoptosis (reviewed in Robinson KS et al. and Reimers K et al. 5,6 ). Further studies demonstrated that TMBIM6/BI-1 is a six transmembrane-spanning protein, located at the endoplasmic reticulum (ER) that has a relevant role in preventing apoptosis. 5 Remarkably, bioinformatic analysis defined a putative family of at least six highly conserved orthologs of TMBIM6/BI-1containing the consensus motif UPF0005 with unknown function, a domain encoding for six to seven transmembrane-spanning regions. 7TMBIM family of proteins includes the founder member TMBIM6/BI-1, TMBIM1/RECS1 (responsive to centrifugal force and shear stress gene 1 protein), TMBIM2/LFG (life guard), TMBIM3/GRINA (glutamate receptor ionotropic NMDA protein 1), TMBIM4/GAAP (Golgi anti-apoptoticassociated protein), and TMBIM5/GHTIM (growth hormoneinducible transmembrane protein). The TMBIM family of proteins is highly conserved in mammals, zebrafish, and flies, with homol...

Tenascin-C (TNC) is highly expressed in melanoma; however, little is known about its functions. Recent studies indicate that TNC plays a role within the stem cell niche. We hypothesized that TNC creates a specific environment for melanoma cells to exhibit a stem cell-like phenotype, driving tumor growth and evading conventional therapies. TNC expression was strongly up-regulated in melanoma cells grown as 3D spheres (enriched for stem-like cells) when compared to adherent cells. Down-modulation of TNC by shRNA-lentiviruses significantly decreased the growth of melanoma spheres. The incidence of pulmonary metastases after intravenous injection of TNC knockdown cells was significantly lower in NOD/SCID IL2Rγnull mice compared to control cells. Melanoma spheres contain and increased number of side population (SP) cells, which exhibited stem cell characteristics and the potential for drug resistance due to their high efflux capacity. Knockdown of TNC dramatically decreased the SP fraction in melanoma spheres and lowered their resistance to doxorubicin treatment, likely due to the down-regulation of multiple ABC transporters, including ABCB5. These data suggest that TNC plays a critical role in melanoma progression by mediating protective signals in the therapy-resistant population of melanoma.

Neuronal proteostasis is maintained by the dynamic integration of different processes that regulate the synthesis, folding, quality control, and localization of proteins. The endoplasmic reticulum (ER) serves as a fundamental pillar of the proteostasis network, and is emerging as a key compartment to sustain normal brain function. The unfolded protein response (UPR), the main mechanism that copes with ER stress, plays a central role in the quality control of many ion channels and receptors, in addition to crosstalk with signaling pathways that regulate connectivity, synapse formation, and neuronal plasticity. We provide here an overview of recent advances in the involvement of the UPR in maintaining neuronal proteostasis, and discuss its emerging role in brain development, neuronal physiology, and behavior, as well as the implications for neurodegenerative diseases involving cognitive decline.

Although protein-folding stress at the endoplasmic reticulum (ER) is emerging as a driver of neuronal dysfunction in models of spinal cord injury and neurodegeneration, the contribution of this pathway to peripheral nerve damage remains poorly explored. Here we targeted the unfolded protein response (UPR), an adaptive reaction against ER stress, in mouse models of sciatic nerve injury and found that ablation of the transcription factor XBP1, but not ATF4, significantly delay locomotor recovery. XBP1 deficiency led to decreased macrophage recruitment, a reduction in myelin removal and axonal regeneration. Conversely, overexpression of XBP1s in the nervous system in transgenic mice enhanced locomotor recovery after sciatic nerve crush, associated to an improvement in key pro-regenerative events. To assess the therapeutic potential of UPR manipulation to axonal regeneration, we locally delivered XBP1s or an shRNA targeting this transcription factor to sensory neurons of the dorsal root ganglia using a gene therapy approach and found an enhancement or reduction of axonal regeneration in vivo, respectively. Our results demonstrate a functional role of specific components of the ER proteostasis network in the cellular changes associated to regeneration and functional recovery after peripheral nerve injury.