Yasuko Nakamura

The mitochondrial outer membrane protein Mitochondrial Fission Factor (Mff) plays a key role in both physiological and pathological fission. It is well established that at stressed or functionally impaired mitochondria, PINK1 recruits the ubiquitin ligase Parkin which ubiquitinates Mff and other mitochondrial outer membrane proteins to facilitate the removal of defective mitochondria and maintain the integrity of the mitochondrial network. Here we show that, in addition to this clearance pathway, Parkin also ubiquitinates Mff in a PINK1-dependent manner under non-stressed conditions to regulate constitutive Mff turnover. We further show that removing Parkin via shRNA-mediated knockdown does not completely prevent Mff ubiquitination under these conditions, indicating that at least one other ubiquitin ligase contributes to Mff proteostasis. These data suggest that that Parkin plays a role in physiological maintenance of mitochondrial membrane protein composition in unstressed cells through constitutive low-level activation.

Cannabinoid type one receptor (CB1R) is only stably surface expressed in axons, where it downregulates neurotransmitter release. How this tightly regulated axonal surface polarity is established and maintained is unclear. To address this question, we used time-resolved imaging to determine the trafficking of CB1R from biosynthesis to mature polarised localisation in cultured rat hippocampal neurons. We show that the secretory pathway delivery of CB1R is axonally biased and that surface expressed CB1R is more stable in axons than in dendrites. This dual mechanism is mediated by the CB1R C-terminus and involves the Helix 9 (H9) domain. Removal of the H9 domain increases secretory pathway delivery to dendrites and decreases surface stability. Furthermore, CB1R^ΔH9 is more sensitive to agonist-induced internalisation and less efficient at downstream signalling than CB1R^WT. Together, these results shed new light on how polarity of CB1R is mediated and indicate that the C-terminal H9 domain plays key roles in this process.

Interruption of blood supply to the heart is a leading cause of death and disability. However, the molecular events that occur during heart ischemia, and how these changes prime consequent cell death upon reperfusion, are poorly understood. Protein SUMOylation is a post-translational modification that has been strongly implicated in the protection of cells against a variety of stressors, including ischemia-reperfusion. In particular, the SUMO2/3-specific protease SENP3 has emerged as an important determinant of cell survival after ischemic infarct. Here, we used the Langendorff perfusion model to examine changes in the levels and localisation of SUMOylated target proteins and SENP3 in whole heart. We observed a 50% loss of SENP3 from the cytosolic fraction of hearts after preconditioning, a 90% loss after ischemia and an 80% loss after ischemia-reperfusion. To examine these effects further, we performed ischemia and ischemia-reperfusion experiments in the cardiomyocyte H9C2 cell line. Similar to whole hearts, ischemia induced a decrease in cytosolic SENP3. Furthermore, shRNA-mediated knockdown of SENP3 led to an increase in the rate of cell death upon reperfusion. Together, our results indicate that cardiac ischemia dramatically alter levels of SENP3 and suggest that this may a mechanism to promote cell survival after ischemia-reperfusion in heart.

Retromer is an evolutionarily conserved endosomal trafficking complex that mediates the retrieval of cargo proteins from a degradative pathway for sorting back to the cell surface. To promote cargo recycling, the core retromer trimer of VPS (vacuolar protein sorting)26, VPS29 and VPS35 recognises cargo either directly, or through an adaptor protein, the most well characterised of which is the PDZ [postsynaptic density 95 (PSD95), disk large, zona occludens] domain-containing sorting nexin SNX27. Neuroligins (NLGs) are postsynaptic trans-synaptic scaffold proteins that function in the clustering of postsynaptic proteins to maintain synaptic stability. Here, we show that each of the NLGs (NLG1–3) bind to SNX27 in a direct PDZ ligand-dependent manner. Depletion of SNX27 from neurons leads to a decrease in levels of each NLG protein and, for NLG2, this occurs as a result of enhanced lysosomal degradation. Notably, while depletion of the core retromer component VPS35 leads to a decrease in NLG1 and NLG3 levels, NLG2 is unaffected, suggesting that, for this cargo, SNX27 acts independently of retromer. Consistent with loss of SNX27 leading to enhanced lysosomal degradation of NLG2, knockdown of SNX27 results in fewer NLG2 clusters in cultured neurons, and loss of SNX27 or VPS35 reduces the size and number of gephyrin clusters. Together, these data indicate that NLGs are SNX27–retromer cargoes and suggest that SNX27–retromer controls inhibitory synapse number, at least in part through trafficking of NLG2.

Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABA_ARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABA_ARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents.Gabra2–1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. SurvivingGabra2-1 mice show anxiety and elevations in electroencephalogramδpower, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development.

Kainate receptors (KARs) are glutamate-gated ion channels that play fundamental roles in regulating neuronal excitability and network function in the brain. After being cloned in the 1990s, important progress has been made in understanding the mechanisms controlling the molecular and cellular properties of KARs, and the nature and extent of their regulation of wider neuronal activity. However, there have been significant recent advances towards understanding KAR trafficking through the secretory pathway, their precise synaptic positioning, and their roles in synaptic plasticity and disease. Here we provide an overview highlighting these new findings about the mechanisms controlling KARs and how KARs, in turn, regulate other proteins and pathways to influence synaptic function.