ABSTRACT
Amphisomes are organelles of the autophagy pathway that result from the fusion of autophagosomes with late endosomes. While biogenesis of autophagosomes and late endosomes occurs continuously at axon terminals, non-degradative roles of autophagy at boutons are barely described. Here, we show that in neurons BDNF/TrkB traffick in amphisomes that signal locally at presynaptic boutons during retrograde transport to the soma. This is orchestrated by the Rap GTPase-activating (RapGAP) protein SIPA1L2, which connects TrkB amphisomes to a dynein motor. The autophagosomal protein LC3 regulates RapGAP activity of SIPA1L2 and controls retrograde trafficking and local signaling of TrkB. Following induction of presynaptic plasticity, amphisomes dissociate from dynein at boutons enabling local signaling and promoting transmitter release. Accordingly, sipa1l2 knockout mice show impaired BDNF-dependent presynaptic plasticity. Taken together, the data suggest that in hippocampal neurons, TrkB-signaling endosomes are in fact amphisomes that during retrograde transport have local signaling capacity in the context of presynaptic plasticity.
Subject(s)
Autophagosomes/metabolism , Brain-Derived Neurotrophic Factor/metabolism , Endosomes/metabolism , GTPase-Activating Proteins/metabolism , Membrane Glycoproteins/metabolism , Microtubule-Associated Proteins/metabolism , Neuronal Plasticity/genetics , Neurons/metabolism , Presynaptic Terminals/metabolism , Protein-Tyrosine Kinases/metabolism , Animals , Axonal Transport , Axons/metabolism , Dyneins/metabolism , GTPase-Activating Proteins/genetics , Hippocampus , Mice , Mice, Knockout , Protein TransportABSTRACT
BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent and lethal cancers worldwide and mortality mostly results from loco-regional recurrence and metastasis. Despite its significance, our knowledge on molecular, cellular and environmental mechanisms that drive disease pathogenesis remains largely elusive, and there are limited therapeutic options, with only negligible clinical benefit. METHODS: We applied global gene expression profiling with samples derived from a recently established mouse model for oral cancer recurrence and identified a list of genes with differential expression between primary and recurrent tumors. RESULTS: One differentially expressed gene codes for Myb-binding protein 1a (MYBBP1A), which is known as a transcriptional co-regulator that physically interacts with nuclear transcription factors, such as NFκB and p53. We confirmed significantly reduced MYBBP1A protein levels on tissue sections of recurrent mouse tumors compared to primary tumors by immunohistochemistry, and found aberrant MYBBP1A protein levels also in tumor samples of HNSCC patients. Interestingly, silencing of MYBBP1A expression in murine SCC7 and in human HNSCC cell lines elicited increased migration but decreased cell growth. CONCLUSION: We provide experimental evidence that MYBBP1A is an important molecular switch in the regulation of tumor cell proliferation versus migration in HNSCC and it will be a major challenge for the future to proof the concept whether regulation MYBBP1A expression and/or function could serve as a novel option for anti-cancer therapy.
Subject(s)
Carcinoma, Squamous Cell/genetics , Mouth Neoplasms/genetics , Neoplasm Recurrence, Local/genetics , Nuclear Proteins/genetics , Nucleocytoplasmic Transport Proteins/genetics , Animals , Blotting, Western , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Cell Movement , Cell Proliferation , DNA-Binding Proteins , Disease Models, Animal , Gene Expression Profiling , Humans , Immunohistochemistry , Mice , Mouth Neoplasms/metabolism , Mouth Neoplasms/pathology , Neoplasm Recurrence, Local/metabolism , Neoplasm Recurrence, Local/pathology , Nuclear Proteins/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , RNA-Binding Proteins , Real-Time Polymerase Chain Reaction , Transcription FactorsABSTRACT
Spine-associated RapGAP 2 (SPAR2) is a novel GTPase activating protein (GAP) for the small GTPase Rap that shows significant sequence homology to SPAR, a synaptic RapGAP that was reported to regulate spine morphology in hippocampal neurons. SPAR2, like SPAR, interacts with the recently described synaptic scaffolding protein ProSAP-interacting protein (ProSAPiP), which in turn binds to the PDZ domain of ProSAP/Shank post-synaptic density proteins. In subcellular fractionation experiments, SPAR2 is enriched in synaptosomes and post-synaptic density fractions indicating that it is a synaptic protein. Furthermore, we could show using in vitro GAP assays that SPAR2 has GAP activity for Rap1 and Rap2. Expression in COS-7 cells, however, revealed different actin-binding properties of SPAR2 and SPAR. Additionally, over-expression of SPAR2 in cultured hippocampal neurons did not affect spine morphology as it was reported for SPAR. In situ hybridization studies also revealed a differential tissue distribution of SPAR and SPAR2 with SPAR2 transcripts being mainly expressed in cerebellar and hippocampal granule cells. Moreover, in the cerebellum SPAR2 is developmentally regulated with a peak of expression around the period of synapse formation. Our results imply that SPAR2 is a new RapGAP with specific functions in cerebellar and hippocampal granule cells.
