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2 Acta Neurobiologiae Experimentalis

2 2017

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Role of Rab11 in the mTOR-mediated autophagy in the dendritic spines

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Janusz-Kaminska A. , Switon K. , Jaworski J.

Acta Neurobiologiae Experimentalis

2017

tom 77

nr Suppl.1

INTRODUCTION: Rab11a is a protein that belongs to the small GTPase superfamily. It is known to be involved in the endocytic recycling. In neurons, it was demonstrated to participate in TrkB receptor translocation to the postsynaptic density in the chemical long-term potentiation (c-LTP) upon BDNF stimulation and in AMPA receptor recycling. Recycling endosomes were also shown to contribute to early steps in autophagy, and Rab11 depletion inhibited autophagosome formation in human HEK293A cells. Lack of mTOR-driven autophagy was implied to cause defective dendritic spine pruning and faulty synaptic plasticity in autistic spectrum disorders. AIM(S): We sought to investigate how Rab11 strikes a balance between endocytic recycling and autophagy in synaptic plasticity via mTOR dependent pathway. METHOD(S): So far, we have performed live imaging of primary hippocampal neurons on the confocal spinning disc microscope to estimate the mobility of Rab11-mCherry in the dendritic spines. By super-resolution immunofluorescence imaging we have investigated colocalization of Rab11a with early autophagy marker Atg9a, recycling endosome marker syntaxin13 and Hook 1 protein in the dendritic spines. We have also co-immunoprecipitated EGFP-Rab11 with myc-mTOR. RESULTS: We have shown that Rab11 vesicles decrease their mobility upon mTOR inhibition in the dendritic spines of primary hippocampal neurons. We have also confirmed that EGFP-Rab11 is pulled down with myc-mTOR in the immunoprecipitation experiment. Preliminary analysis indicated increased Rab11 colocalization with autophagy markers upon mTOR inhibition. CONCLUSIONS: Altogether our results point to the potential role of Rab11 in the mTOR-dependent autophagy in the synaptic plasticity. FINANCIAL SUPPORT: This research was funded by Polish National Science Centre Sonata Bis (2012/07/E/ NZ3/00503).

Kainic acid causes mTOR activation and translocation from cytosol to the nucleus – Live imaging study

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Macias M. , Janusz-Kaminska A. , Tarkowski B. , Gozdz A. , Jaworski J.

Acta Neurobiologiae Experimentalis

2017

tom 77

nr Suppl.1

INTRODUCTION: The mechanistic target of rapamycin (mTOR) is a protein kinase, which integrates eukaryotic cell growth, metabolism and external stimuli. Most research link mTOR with control of protein translation but recent studies revealed additional mTOR function in cell nucleus. Previously, we showed that phosphorylation of mTOR (Ser2448; P-mTOR) is upregulated in neuronal nucleus upon kainate (KA) induced status epilepticus. Whether other stimuli have the same effect on nuclear mTOR phosphorylation and if increased nuclear import of mTOR contributes to this phenomenon remained unknown. Also it was not known if nuclear transport of other proteins affects mTOR signaling. AIM(S): To analyze effects of neuronal activity on nuclear translocation of mTOR and its nuclear activity. To analyze importance of nuclear transport for mTOR signaling. METHOD(S): Cultured hippocampal neurons were treated with: KA, BDNF; NMDA and chemical LTP (cLTP) protocol or TTX. mTOR activity was measured with FRET method. mTOR nuclear translocation was assessed using FRAP. Nuclear import was blocked with importazole. Immunofluorescence of P-S6 protein was used as a marker of mTOR activity. RESULTS: We found that KA, BDNF, NMDA and cLTP caused nuclear upregulation of P-mTOR. However, TTX or cLTD had no effect. FRAP and FRET revealed that mTOR activity due to KA treatment is first observed in cytosol and then in nucleus, where mTOR is translocated upon treatment. Blocking nuclear import silenced mTOR activity in response to KA and inhibited P-mTOR upregulation in the nucleus. CONCLUSIONS: Our experiments showed that increased neuronal activity upregulates nuclear P-mTOR and increases nuclear activity of mTOR due to nuclear translocation of the kinase. FINANCIAL SUPPORT: The research was supported by PNSC grants no. 2012/05/B/NZ3/00429 and 2012/07/E/ NZ3/00503.