No abstract

Axons actively self-destruct following genetic, mechanical, metabolic, and toxic insults, but the mechanism of axonal degeneration is poorly understood. The JNK pathway promotes axonal degeneration shortly after axonal injury, hours before irreversible axon fragmentation ensues. Inhibition of JNK activity during this period delays axonal degeneration, but critical JNK substrates that facilitate axon degeneration are unknown. Here we show that superior cervical ganglion 10 (SCG10), an axonal JNK substrate, is lost rapidly from mouse dorsal root ganglion axons following axotomy. SCG10 loss precedes axon fragmentation and occurs selectively in the axon segments distal to transection that are destined to degenerate. Rapid SCG10 loss after injury requires JNK activity. The JNK phosphorylation sites on SCG10 are required for its rapid degradation, suggesting that direct JNK phosphorylation targets SCG10 for degradation. We present a mechanism for the selective loss of SCG10 distal to the injury site. In healthy axons, SCG10 undergoes rapid JNK-dependent degradation and is replenished by fast axonal transport. Injury blocks axonal transport and the delivery of SCG10, leading to the selective loss of the labile SCG10 distal to the injury site. SCG10 loss is functionally important: Knocking down SCG10 accelerates axon fragmentation, whereas experimentally maintaining SCG10 after injury promotes mitochondrial movement and delays axonal degeneration. Taken together, these data support the model that SCG10 is an axonalmaintenance factor whose loss is permissive for execution of the injury-induced axonal degeneration program.A xon loss is a devastating consequence of a wide range of neurological diseases. A hallmark of hereditary neuropathies, glaucoma, and diabetic neuropathy, axon loss also is found early in the progression of debilitating neurodegenerative diseases such as Alzheimer's and Parkinson disease (1, 2). Although the great length of many axons is essential to their function, it also makes them vulnerable to mechanical trauma and to neurotoxins such as chemotherapeutics that interfere with axonal transport (3). Current therapies for axonal degeneration target either the systemic diseases that lead to axon loss or the pain that results from axon dysfunction (4). Therapies targeting the axon breakdown process itself are notably absent. Elucidating the mechanism of axonal degeneration may help to develop such therapies.Axonal degeneration is an actively regulated process that is blocked by the overexpression of the Wallerian degeneration slow (Wld s ) fusion protein or its enzymatically active component NMNAT (5-10). Regulated protein degradation promotes the degeneration of injured axons (11), potentially via the degradation of labile axonal-maintenance factors. Rapid postinjury loss of axonal-maintenance factors is a likely mechanism for promoting axon degeneration. NMNAT2 is the first identified axonal-maintenance factor that is degraded soon after injury. Its loss triggers axonal degeneration, and forced express...

Individuals with neurofibromatosis type 1 (NF1) develop abnormalities of both neuronal and glial cell lineages, suggesting that the NF1 protein neurofibromin is an essential regulator of neuroglial progenitor function. In this regard, Nf1-deficient embryonic telencephalic neurospheres exhibit increased self-renewal and prolonged survival as explants in vivo. Using a newly developed brain lipid binding protein (BLBP)-Cre mouse strain to study the role of neurofibromin in neural progenitor cell function in the intact animal, we now show that neuroglial progenitor Nf1 inactivation results in increased glial lineage proliferation and abnormal neuronal differentiation in vivo. Whereas the glial cell lineage abnormalities are recapitulated by activated Ras or Akt expression in vivo, the neuronal abnormalities were Ras- and Akt independent and reflected impaired cAMP generation in Nf1-deficient cells in vivo and in vitro. Together, these findings demonstrate that neurofibromin is required for normal glial and neuronal development involving separable Ras-dependent and cAMP-dependent mechanisms.

Peripheral axons can re-extend robustly after nerve injury. Soon after a nerve crush regenerating axons grow through the nerve segment distal to the lesion in close proximity to distal axons that are still morphologically and molecularly preserved. Hence, following the progress of regenerating axons necessitates markers that can distinguish between regenerating and degenerating axons. Here, we show that axonal levels of superior cervical ganglion 10 (SCG10) is dynamically regulated after axonal injury and provides an efficient method to label regenerating axons. In contrast to the rapid loss of SCG10 in distal axons (Shin et al., 2012b), we report that SCG10 accumulates in the proximal axons within an hour after injury, leading to a rapid identification of the lesion site. The increase in SCG10 levels is maintained during axon regeneration after nerve crush or nerve repair and allows for more selective labeling of regenerating axons than the commonly used markers growth-associated protein 43 (GAP43) and YFP. SCG10 is preferentially expressed in regenerating sensory axons rather than motor axons in the sciatic nerve. In a mouse model of slow Wallerian degeneration, SCG10 labeling remains selective for regenerating axons and allows for a quantitative analysis of delayed regeneration in this mutant. Taken together, these data demonstrate the utility of SCG10 as an efficient and selective marker of sensory axon regeneration.

Accumulation of extracellular amyloid ␤ peptide (A␤), generated from amyloid precursor protein (APP) processing by ␤-and ␥-secretases, is toxic to neurons and is central to the pathogenesis of Alzheimer disease. Production of A␤ from APP is greatly affected by the subcellular localization and trafficking of APP. Here we have identified a novel intracellular adaptor protein, sorting nexin 17 (SNX17), that binds specifically to the APP cytoplasmic domain via the YXNPXY motif that has been shown previously to bind several cell surface adaptors, including Fe65 and X11. Overexpression of a dominant-negative mutant of SNX17 and RNA interference knockdown of endogenous SNX17 expression both reduced steady-state levels of APP with a concomitant increase in A␤ production. RNA interference knockdown of SNX17 also decreased APP half-life, which led to the decreased steadystate levels of APP. Immunofluorescence staining confirmed a colocalization of SNX17 and APP in the early endosomes. We also showed that a cell surface adaptor protein, Dab2, binds to the same YXNPXY motif and regulates APP endocytosis at the cell surface. Our results thus provide strong evidence that both cell surface and intracellular adaptor proteins regulate APP endocytic trafficking and processing to A␤. The identification of SNX17 as a novel APP intracellular adaptor protein highly expressed in neurons should facilitate the understanding of the relationship between APP intracellular trafficking and processing to A␤.Mounting evidence has demonstrated that proteolytic processing of the amyloid precursor protein (APP) 4 is central to the pathogenesis of Alzheimer disease (AD) (1, 2). Many reports have shown that APP processing to A␤ is greatly affected by the subcellular localization of APP, presumably because of the specific subcellular localizations of ␤-and ␥-secretases (3). Both transmembrane receptors and cytoplasmic adaptor proteins have been shown to interact with APP and affect its trafficking. The low-density lipoprotein receptor-related protein 1 (LRP1) increases APP endocytosis and A␤ production (4), whereas SorLA decreases APP processing to A␤ by shuttling APP away from endosomes (5). Several cell surface adaptor proteins, including Fe65, X11, and Dab1, bind to the NPXY motif within the APP cytoplasmic domain and regulate its trafficking and processing to A␤ (6 -8). By overexpression or knockdown, Fe65 has been shown to affect APP processing to A␤ (9, 10). Although Dab1 has been shown to affect APP processing and A␤ production (11), the function of its homologue Dab2 in APP trafficking and processing to A␤ has not been studied (8). These studies firmly establish that APP-interacting proteins can both positively and negatively affect A␤ production by altering APP trafficking through ␤-and ␥-secretase-containing compartments. Revealing the mechanisms by which intracellular trafficking of APP is regulated may permit the development of novel therapeutic approaches for AD.Sorting nexin 17 (SNX17) is a member of the sorting nexin family characteri...

Summary Injured peripheral neurons successfully activate a pro-regenerative transcriptional program to enable axon regeneration and functional recovery. How transcriptional regulators co-ordinate the expression of such program remains unclear. Here we show that hypoxia-inducible factor 1α (HIF-1α) controls multiple injury-induced genes in sensory neurons and contribute to the preconditioning lesion effect. Knockdown of HIF-1α in vitro or conditional knock out in vivo impairs sensory axon regeneration. The HIF-1α target gene Vascular Endothelial Growth Factor A (VEGFA) is expressed in injured neurons and contributes to stimulate axon regeneration. Induction of HIF-1α using hypoxia enhances axon regeneration in vitro and in vivo in sensory neurons. Hypoxia also stimulates motor neuron regeneration and accelerates neuromuscular junction re-innervation. This study demonstrates that HIF-1α represents a critical transcriptional regulator in regenerating neurons and suggests hypoxia as a tool to stimulate axon regeneration.

Following damage to a peripheral nerve, injury signaling pathways converge in the cell body to generate transcriptional changes that support axon regeneration. Here, we demonstrate that dual leucine zipper kinase (DLK), a central regulator of injury responses including axon regeneration and neuronal apoptosis, is required for the induction of the pro-regenerative transcriptional program in response to peripheral nerve injury. Using a sensory neuron-conditional DLK knockout mouse model, we show a time course for the dependency of gene expression changes on the DLK pathway after sciatic nerve injury. Gene ontology analysis reveals that DLK-dependent gene sets are enriched for specific functional annotations such as ion transport and immune response. A series of comparative analyses shows that the DLK-dependent transcriptional program is distinct from that promoted by the importin-dependent retrograde signaling pathway, while it is partially shared between PNS and CNS injury responses. We suggest that DLK-dependency might provide a selective filter for regeneration-associated genes among the injury-responsive transcriptome.

Axon regeneration is regulated by a neuron-intrinsic transcriptional program that is suppressed during development but that can be reactivated following peripheral nerve injury. Here we identifyProm1, which encodes the stem cell marker prominin-1, as a regulator of the axon regeneration program.Prom1expression is developmentally down-regulated, and the genetic deletion ofProm1in mice inhibits axon regeneration in dorsal root ganglion (DRG) cultures and in the sciatic nerve, revealing the neuronal role ofProm1in injury-induced regeneration. Elevating prominin-1 levels in cultured DRG neurons or in mice via adeno-associated virus-mediated gene delivery enhances axon regeneration in vitro and in vivo, allowing outgrowth on an inhibitory substrate.Prom1overexpression induces the consistent down-regulation of cholesterol metabolism-associated genes and a reduction in cellular cholesterol levels in a Smad pathway-dependent manner, which promotes axonal regrowth. We find that prominin-1 interacts with the type I TGF-β receptor ALK4, and that they synergistically induce phosphorylation of Smad2. These results suggest thatProm1and cholesterol metabolism pathways are possible therapeutic targets for the promotion of neural recovery after injury.