CREB3L2 MODULATES NEURITE OUTGROWTH THROUGH THE REGULATION OF RAB5 GTPase IN NGF-DIFFERENTIATED PC12 CELLS

FUNES CHABÁN, MACARENA; ROZÉS-SALVADOR, VICTORIA; SAMPIERI, L; DI GIUSTO, PABLO; ALVAREZ, C

On line

Congreso; Congreso SAIB 2021; 2021

SAIB

CREB3L2 is a member of the CREB3 family of transcription factors localized in the endoplasmic reticulum (ER) membrane. Upon activation, it is transported from the ER to the Golgi where is cleaved and the N-terminal domain is translocated to the nucleus. CREB3L2 is expressed in several cell types such as hepatic stellate cells, chondroblasts and human B-cells, among others. It is linked to the regulation of the secretory pathway performing functions on growth, survival, and maintenance of the cell phenotype. Although CREB3L2 is expressed in different cell types of the nervous system, its participation in neuronal processes, such as differentiation, remains poorly explored. In our laboratory, PC12 cells treated with nerve growth factor (NGF) were used to study neuronal differentiation. We have previously shown (SAIB 2019) that NGF increased not only CREB3L2 mRNA and protein levels, but also GM130, GalNAc-T2 and Rab1b, which are proteins of the secretory pathway. In this work, loss- and gain-of-function experiments were carried out to analyze the role of CREB3L2 during PC12 cells differentiation. Results showed that shCREB3L2-transfected cells displayed Golgi fragmentation and both the total neurite length and the longest neurite was increased relative to control. In contrast, overexpression of CREB3L2 full length (CREB3L2FL) induced the opposite effect in neurite phenotype, suggesting that CREB3L2 could be associated with the activity of membrane trafficking pathway-related proteins. To address this hypothesis, PC12 differentiation was analyzed after disturbing ER-Golgi or endosomal transport by overexpressing Rab1b or Rab5 constructs, respectively. Overexpression of wild-type Rab1b increased neurite outgrowth relative to control cells, whereas the opposite effect was observed upon transfection with dominant negative Rab1b (Rab1b N121I). Interestingly, the neurite outgrowth was impaired by overexpression of wild-type Rab5, whereas it was promoted by the dominant negative Rab5 construct, Rab5 S34N. These last results agree with previous studies1 and are similar to those observed in CREB3L2 gain- and loss-of-function. To evaluate whether CREB3L2 levels affects Rab5 expression, PC12 cells were transiently transfected with shCREB3L2, CREB3L2FL and treated with NGF. Quantitative immunofluorescence analysis indicated that shCREB3L2-transfected PC12 cells have a decreased expression of Rab5. On the other hand, in CREB3L2 overexpressing cells, Rab5 levels were higher than control cells. Taken together, the data indicate that CREB3L2 modulates NGF-induced PC12 cell differentiation and strongly suggest that Rab5 GTPase is one of CREB3L2 targets .