Semaphorin 3C preserves survival and induces neuritogenesis of cerebellar granule neurons in culture

Semaphorins (sema) constitute a family of molecules sharing a common extracellular domain (semaphorin domain). This family includes several types of secreted and membrane-associated molecules that are grouped into eight subclasses (subclasses 1–7 and viral semaphorins). Subclass 3 semaphorins are secreted molecules involved in axonal guidance, mainly through repulsive gradients and induction of growth cone collapse. More recently sema 3 molecules have been identified as positive factors in dependence of the type of neurons. Besides their axonal guidance function, some semaphorins have been implicated in apoptosis and survival. We investigated the effect of sema3C on survival and neurite outgrowth of rat cerebellar granule neurons (CGNs) in culture. 3T3 cells were stably transfected with sema3C. Several clonal lines were established and tested for their neuritogenic activity and one, S3C-8, was selected for the bulk of experiments. S3C-8 was co-cultured with CGNs. Sema3C enhanced CGN viability as assessed in co-cultures of CGNs with monolayers of S3C-8 in comparison with co-cultures of CGNs with control mock-transfected 3T3 cells. Moreover sema3C induced neuritogenesis of cultured CGNs, which express neuropilin-1 and -2. S3C-8 cells, overexpressing sema3C, were significantly more neuritogenic for CGN than poly l-lysine (PLL), a positive substrate for CGNs, as assessed by the measurement of the length of neurites and confirmed by Tau expression along the time of culture. CGNs co-cultured with S3C-8, showed up-regulation of the expression of axonal microtubule-associated proteins (MAPs) such as Tau, phosphorylated MAP2C and mode I-phosphorylated MAP1B compared with neurons cultured on control 3T3 cells. We also found increased expression of a specific marker of neuronal cell bodies and dendrites, high molecular weight MAP2 (HMW-MAP2). Interestingly, there was no accompanying up-regulation of a marker enriched within the neuronal somatodendritic domain, mode II-phosphorylated MAP1B. These data support the idea that secreted sema3C favors survival and neuritogenesis of cultured CGNs.This work was supported by Spanish DGCYT, grant PB 94–0040 and CAM, grant ref # CAM 08.5/0039/2000. MTM was supported by Spanish CSIC and Neurofarma contracts. M. J. Martín-Bermejo was supported by Neurofarma contracts.Peer reviewe