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.

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.

The Ras oncogene is known to activate three major MAPK pathways, ERK, JNK, p38 and exert distinct cellular phenotypes, that is, apoptosis and invasion through the Ras-MKK3-p38-signaling cascade. We attempted to identify the molecular targets of this pathway that selectively govern the invasive phenotype. Stable transfection of NIH3T3 fibroblasts with MKK3 act cDNA construct revealed similar p38-dependent in vitro characteristics observed in Ha-Ras EJ -transformed NIH3T3 cells, including enhanced invasiveness and anchorage-independent growth correlating with p38 phosphorylation status. To identify the consensus downstream targets of the Ras-MKK3-p38 cascade involved in invasion, in vitro invasion assays were used to isolate highly invasive cells from both, MKK3 and Ha-Ras EJ transgenic cell lines. Subsequently a genome-wide transcriptome analysis was employed to investigate differentially regulated genes in invasive Ha-Ras EJ -and MKK3 act -transfected NIH3T3 fibroblasts. Using this phenotype-assisted approach combined with system level protein-interaction network analysis, we identified FOXM1, PLK1 and CDK1 to be differentially regulated in invasive Ha-Ras EJ -NIH3T3 and MKK3 act -NIH3T3 cells. Finally, a FOXM1 RNAknockdown approach revealed its requirement for both invasion and anchorage-independent growth of Ha-Ras EJand MKK3 act -NIH3T3 cells. Together, we identified FOXM1 as a key downstream target of Ras and MKK3-induced cellular in vitro invasion and anchorage-independent growth signaling.

BackgroundHead 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.MethodsWe 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.ResultsOne 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.ConclusionWe 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.

Recently, increased expression of the submaxillary gland androgen-regulated protein 3A (SMR3A) was found in recurrent tumors of an orthotopic floor-of-mouth mouse tumor model after surgery. However, SMR3A expression in the pathogenesis of human malignancy and its correlation with the clinical outcome have not been addressed so far. We analyzed tissue microarrays with specimens from oropharyngeal squamous cell carcinoma (OPSCC) patients (n = 157) by immunohistochemistry and compared SMR3A expression with clinical and pathological features by statistical analysis. Strong SMR3A expression was found in almost 36 % of all primary OPSCCs. Although, SMR3A protein levels were not associated with any clinical or histopathological feature tested, univariate Kaplan-Meier analysis revealed a significant correlation between high SMR3A protein expression and poor progression-free (p = 0.02) and overall survival (p = 0.03). Furthermore, high SMR3A expression was an independent marker for poor clinical outcome [HR (SMR3A(high) vs. SMR3(low)) = 2.32; 95 % CI = 1.03-5.23] concerning overall survival in a multivariate analysis of OPSCC patients with surgery as primary therapy (n = 100). Our data demonstrate for the first time increased SMR3A protein expression in the pathogenesis of OPSCC, which serves as an unfavorable risk factor for patient survival.

2 SummaryAmphisomes are transient organelles that derive from fusion of autophagosomes with late endosomes. They rapidly transform into degradative autolysosomes, whereas non-degradative roles of the autophagic pathway have been barely described. Here we show that in neurons BDNF/TrkB receptor bearing Rab7 / Light chain 3 (LC3) -positive amphisomes signal at presynaptic boutons during retrograde trafficking to the soma. Local signaling and inward transport essentially require the Rap GTPase-activating (RapGAP) protein SIPA1L2, which directly binds to TrkB and Snapin to connect TrkB-containing amphisomes to dynein.Association with LC3 regulates the RapGAP activity of SIPA1L2 and thereby retrograde trafficking. Following induction of presynaptic plasticity amphisomes dissociate from dynein at boutons, and this enables local signaling and promotes transmitter release. Accordingly, sipa1l2 knockout mice show impaired BDNF-dependent presynaptic plasticity. Collectively, the data suggest that TrkB-signaling endosomes are in fact amphisomes that during retrograde transport have local signaling capacity in the context of presynaptic plasticity.3 TrkB signaling endosomes that carry axonal neurotrophic signals are generated at axon terminals and constitute an important long-range retrograde signaling mechanism conveying information of synaptic activity (1). Upon binding to BDNF, BDNF/TrkB have been shown to be internalized either via pinocytosis (2,3) or in a clathrin-dependent manner (4) to specialized vesicular compartments and sorted to diverse pathways by yet unknown mechanisms. TrkB signaling is required for different aspects of neuronal function including the expression of presynaptic LTP at mossy fiber (MF) synapses (5). By binding to different adaptor proteins, TrkB activates three major molecular cascades: the Ras-MAPK pathway, the phosphatidyl inositol-3 (PI3)-kinase cascade and the phospholipase C-γ1 pathway (6).Rap-1 based signaling ensures sustained ERK activation since prenylated Rap1 is associated to TrkB containing endosomes. These are long-lived and persist during transport from nerve terminals to the cell soma (7, 8 9,10). SIPA1L2 (also known as SPAR2) is a member of the SIPA1L family of neuronal RapGAPs ( Fig. S1) (11). The protein is most abundant in granule cells of the dentate gyrus (DG) and cerebellum and shows RapGAP activity for the small GTPases Rap1 and 2 (12). Here, we report that SIPA1L2 binds directly to TrkB, and links the receptor tyrosine kinase to a Dynein motor for retrograde trafficking via a direct interaction with the adaptor protein Snapin.Interestingly, SIPA1L2 concurrently associates via LC3b to Rab7-positive amphisomes and binding of LC3b promotes RapGAP activity. The amphisome trafficks retrogradely along axons, it stops at presynaptic boutons and both motility and signaling are controlled by SIPA1L2 whose RapGAP activity reduces the velocity of amphisome transport. Presynaptic long-term potentiation (LTP) induces a Protein kinase A (PKA)-dependent dissociation of the SIPA1L2/Snap...