ABSTRACT Fusion of intracellular trafficking vesicles is mediated by the assembly of soluble N -ethylmaleimide-sensitive fusion protein receptors (SNAREs) to form membrane-bridging complexes. Also required for SNARE-mediated membrane fusion are Sec1/Munc18-family (SM) proteins, SNARE chaperones that can function as templates to catalyze SNARE complex assembly. In the paradigmatic structure of an SM–SNARE complex, Munc18-1 bound to the Qa-SNARE syntaxin 1, the SNARE protein is trapped in an autoinhibited closed conformation that prevents it from entering into SNARE complexes. Here, we present the structure of a second SM–Qa-SNARE complex, Vps45–Tlg2. Strikingly, Vps45 holds Tlg2 in an open conformation, with its SNARE motif disengaged from its three-helical Habc domain and its linker region unfolded. The domain 3a helical hairpin of Vps45 is unfurled, exposing the presumptive R-SNARE binding site to allow template complex formation. Tlg2 has a pronounced tendency to self-associate via its SNARE motif, and we demonstrate that Vps45 can rescue Tlg2 oligomers into stoichiometric Vps45–Tlg2 complexes. Our findings demonstrate that SM proteins can engage Qa-SNAREs using at least two different modes, one in which the SNARE is closed and one in which it is open.
After peripheral nerve injury, neurotrophins play a key role in the regeneration of damaged axons that can be augmented by exercise, although the distinct roles played by neurons and Schwann cells are unclear. In this study, we evaluated the requirement for the neurotrophin, brain-derived neurotrophic factor (BDNF), in neurons and Schwann cells for the regeneration of peripheral axons after injury. Common fibular or tibial nerves in thy-1-YFP-H mice were cut bilaterally and repaired using a graft of the same nerve from transgenic mice lacking BDNF in Schwann cells ( BDNF −/− ) or wild-type mice (WT). Two weeks postrepair, axonal regeneration into BDNF −/− grafts was markedly less than WT grafts, emphasizing the importance of Schwann cell BDNF. Nerve regeneration was enhanced by treadmill training posttransection, regardless of the BDNF content of the nerve graft. We further tested the hypothesis that training-induced increases in BDNF in neurons allow regenerating axons to overcome a lack of BDNF expression in cells in the pathway through which they regenerate. Nerves in mice lacking BDNF in YFP + neurons (SLICK) were cut and repaired with BDNF −/− and WT nerves. SLICK axons lacking BDNF did not regenerate into grafts lacking Schwann cell BDNF. Treadmill training could not rescue the regeneration into BDNF −/− grafts if the neurons also lacked BDNF. Both Schwann cell- and neuron-derived BDNF are thus important for axon regeneration in cut peripheral nerves.
Fusion of intracellular trafficking vesicles is mediated by the assembly of SNARE proteins into membrane-bridging complexes. SNARE-mediated membrane fusion requires Sec1/Munc18-family (SM) proteins, SNARE chaperones that can function as templates to catalyze SNARE complex assembly. Paradoxically, the SM protein Munc18-1 traps the Qa-SNARE protein syntaxin-1 in an autoinhibited closed conformation. Here we present the structure of a second SM–Qa-SNARE complex, Vps45–Tlg2. Strikingly, Vps45 holds Tlg2 in an open conformation, with its SNARE motif disengaged from its Habc domain and its linker region unfolded. The domain 3a helical hairpin of Vps45 is unfurled, exposing the presumptive R-SNARE binding site to allow template complex formation. Although Tlg2 has a pronounced tendency to form homo-tetramers, Vps45 can rescue Tlg2 tetramers into stoichiometric Vps45–Tlg2 complexes. Our findings demonstrate that SM proteins can engage Qa-SNAREs using at least two different modes, one in which the SNARE is closed and one in which it is open.
After peripheral nerve injury, neurotrophins play a key role in the regeneration of damaged axons that can be augmented by exercise, although the distinct roles played by neurons and Schwann cells are unclear. In this study, we evaluated the requirement for the neurotrophin, brain-derived neurotrophic factor (BDNF), in neurons and Schwann cells for the regeneration of peripheral axons after injury. Common fibular or tibial nerves in thy-1-YFP-H mice were cut bilaterally and repaired using a graft of the same nerve from transgenic mice lacking BDNF in Schwann cells (BDNF(-/-)) or wild-type mice (WT). Two weeks postrepair, axonal regeneration into BDNF(-/-) grafts was markedly less than WT grafts, emphasizing the importance of Schwann cell BDNF. Nerve regeneration was enhanced by treadmill training posttransection, regardless of the BDNF content of the nerve graft. We further tested the hypothesis that training-induced increases in BDNF in neurons allow regenerating axons to overcome a lack of BDNF expression in cells in the pathway through which they regenerate. Nerves in mice lacking BDNF in YFP(+) neurons (SLICK) were cut and repaired with BDNF(-/-) and WT nerves. SLICK axons lacking BDNF did not regenerate into grafts lacking Schwann cell BDNF. Treadmill training could not rescue the regeneration into BDNF(-/-) grafts if the neurons also lacked BDNF. Both Schwann cell- and neuron-derived BDNF are thus important for axon regeneration in cut peripheral nerves.
The three homologs of insulin like growth factor‐II (IGF‐II) mRNA binding protein (IMP) play an essential role in the posttranscriptional regulation of gene expression in nervous tissue. IMP1/ZBP1 (zipcode binding protein) regulates the localization and translation of specific mRNAs allowing for axon guidance and regeneration. Additionally, previous studies show that IMP1 and IMP3 expression decreases dramatically after birth, whereas IMP2 is sustained in in brain, liver and other organs throughout life (Leeds et al., 1997; Mueller‐Pillasch et al., 1999; Nielsen et al., 1999; Hansen et al., 2004; Gu et al., 2004; Hammer et al., 2005). Of the three homologs, IMP2 is least understood. Our pilot data implicates that IMP2 may play a role in axon regeneration by localizing specific mRNAs. Understanding IMP2 expression pattern is fundamental to further investigation of its functions. We hypothesized that IMP2 is present in both the central and peripheral nervous systems throughout life. Using a custom made IMP2‐specific antibody, along with immunohistochemistry, Western Blot, and primary dorsal root ganglion (DRG) culture we examined the expression of IMP2 in the spinal cord and DRG of the mouse at embryonic, postnatal, and adult stages of life. We found that IMP2 expression is present in both the spinal cord and DRG through all developmental stages of life. Ongoing experiments are focused on the role of IMP2 in axon regeneration in the adult nervous system.
Insulin ‐ like growth factor‐II (IGF‐II) mRNA‐binding protein‐2 (IMP2) is one of the three homologs (IMP1–3) that play important roles in the posttranscriptional regulation of gene expression in several tissues. IMP1/ZBP1 (zipcode binding protein) has been shown to play important roles in axon guidance and regeneration by regulating the localization and translation of specific mRNAs. However, the function of IMP2 is least understood, largely because an isoform‐specific antibody is not available, which makes the conventional techniques to locate protein expression not feasible. We custom made an IMP2‐specific antibody. We used Western blot and immunocytochemistry to test its specificity on cultured cells following over expression of IMP 1–3 isoforms, respectively. Using this IMP2‐specific antibody, we examined IMP2 expression in the mouse nervous system. We found that IMP2 expression in the nervous system is sustained postnatally, unlike that of IMP1 and IMP3. Ongoing experiments are aimed at further understanding IMP2 expression patterns during injury and assessment of its role to facilitate mRNA localization during axon regeneration in the adult nervous system. This work is supported by the Department of Anatomy, PCOM and Center for Chronic Disorders of Aging (CCDA).
