Pathway sensor-based functional genomics screening identifies modulators of neuronal activity

Neuronal signal transduction shapes brain function and malfunction may cause mental disorders. Despite the existence of functional genomics screens for proliferation and toxicity, neuronal signalling has been difficult to address so far. To overcome this limitation, we developed a pooled screening assay which combines barcoded activity reporters with pooled genetic perturbation in a dual-expression adeno-associated virus (AAV) library. With this approach, termed pathScreener, we comprehensively dissect signalling pathways in postmitotic neurons. This overcomes several limitations of lentiviral-based screens. By applying first a barcoded and multiplexed reporter assay, termed cisProfiler, we identified the synaptic-activity responsive element (SARE) as top performance sensor of neuronal activity. Next, we targeted more than 4,400 genes and screened for modulatory effects on SARE activity in primary cortical neurons. We identified with high replicability many known genes involved in glutamatergic synapse-to-nucleus signalling of which a subset was validated in orthogonal assays. Several others have not yet been associated with the regulation of neuronal activity such as the hedgehog signalling members Ptch2 and Ift57. This assay thus enhances the toolbox for analysing regulatory processes during neuronal signalling and may help identifying novel targets for brain disorders

Role of SNX16 in the Dynamics of Tubulo-Cisternal Membrane Domains of Late Endosomes

In this paper, we report that the PX domain-containing protein SNX16, a member of the sorting nexin family, is associated with late endosome membranes. We find that SNX16 is selectively enriched on tubulo-cisternal elements of this membrane system, whose highly dynamic properties and formation depend on intact microtubules. By contrast, SNX16 was not found on vacuolar elements that typically contain LBPA, and thus presumably correspond to multivesicular endosomes. We conclude that SNX16, together with its partner phosphoinositide, define a highly dynamic subset of late endosomal membranes, supporting the notion that late endosomes are organized in distinct morphological and functional regions. Our data also indicate that SNX16 is involved in tubule formation and cholesterol transport as well as trafficking of the tetraspanin CD81, suggesting that the protein plays a role in the regulation of late endosome membrane dynamics

The interplay between endosomal sorting of the epidermal growth factor receptor and signaling

The epidermal growth factor (EGF)-induced removal of the EGF receptor (EGFR) from the plasma membrane and its endocytic downregulation is a major negative feedback mechanism controlling the intensity and duration of receptor signaling. Different mechanisms of ligand-accelerated endocytosis, rapid ubiquitination of activated EGFR, and sorting of the receptor into multivesicular bodies for lysosomal degradation, are the underlying principles of EGFR downregulation. The physical degradation of the EGFR is thought to protect cells from excessive stimulation. In addition, sequestering the receptor into intralumenal vesicles of endosomes, thereby uncoupling the intracellular tyrosine kinase domain from its downstream effectors, is proposed to contribute to signal attenuation. On the other hand, endosomal EGFR can be active and is able to compensate for signaling initiated at the plasma membrane. How the pool of active endosomal receptor is regulated, and to what extend it contributes to the biological response, has not been investigated conclusively to date. In this study, we aimed to dissect the precise contribution of endocytic sorting events to the EGF response. Consequences of perturbations in EGFR sorting, particularly upon interfering with clathrin- and dynamin-dependent endocytosis, after knockdown of CBL ubiquitin ligases, and of depletion of endosomal sorting complex required for transport (ESCRT) subunits, were investigated in detail. The activation status of signaling components was determined, and a reporter assay was set up to measure EGF-dependent transcriptional activation in living cells. The induction of endogenous target genes downstream of the EGFR-MAPK (mitogen-activated protein kinase) cascade was quantified by real-time RT-PCR, microarray analysis, and by utilizing the recently developed NanoString technology