Klf9 is necessary and sufficient for Purkinje cell survival in organotypic culture.

During their phase of developmental programmed cell death (PCD), neurons depend on target-released trophic factors for survival. After this period, however, they critically change as their survival becomes target-independent. The molecular mechanisms underlying this major transition remain poorly understood. Here, we investigated, which transcription factors (TFs) might be responsible for the closure of PCD. We used Purkinje cells as a model since their PCD is restricted to the first postnatal week in the mouse cerebellum. Transcriptome analysis of Purkinje cells during or after PCD allowed the identification of Krüppel like factor 9 (Klf9) as a candidate for PCD closure, given its high increase of expression at the end of the 1st postnatal week. Klf9 function was tested in organotypic cultures, through lentiviral vector-mediated manipulation of Klf9 expression. In absence of trophic factors, the Purkinje cell survival rate is of 40%. Overexpression of Klf9 during PCD dramatically increases the Purkinje cell survival rate from 40% to 88%, whereas its down-regulation decreases it to 14%. Accordingly, in organotypic cultures of Klf9 knockout animals, Purkinje cell survival rate is reduced by half as compared to wild-type mice. Furthermore, the absence of Klf9 could be rescued by Purkinje cell trophic factors, Insulin growth factor-1 and Neurotrophin3. Altogether, our results ascribe a clear role of Klf9 in Purkinje cell survival. Thus, we propose that Klf9 might be a key molecule involved in turning off the phase of Purkinje PCD.

Trim17, a novel E3 ubiquitin-ligase, initiates neuronal apoptosis.

Accumulating data indicate that the ubiquitin-proteasome system controls apoptosis by regulating the level and the function of key regulatory proteins. In this study, we identified Trim17, a member of the TRIM/RBCC protein family, as one of the critical E3 ubiquitin ligases involved in the control of neuronal apoptosis upstream of mitochondria. We show that expression of Trim17 is increased both at the mRNA and protein level in several in vitro models of transcription-dependent neuronal apoptosis. Expression of Trim17 is controlled by the PI3K/Akt/GSK3 pathway in cerebellar granule neurons (CGN). Moreover, the Trim17 protein is expressed in vivo, in apoptotic neurons that naturally die during post-natal cerebellar development. Overexpression of active Trim17 in primary CGN was sufficient to induce the intrinsic pathway of apoptosis in survival conditions. This pro-apoptotic effect was abolished in Bax(-/-) neurons and depended on the E3 activity of Trim17 conferred by its RING domain. Furthermore, knock-down of endogenous Trim17 and overexpression of dominant-negative mutants of Trim17 blocked trophic factor withdrawal-induced apoptosis both in CGN and in sympathetic neurons. Collectively, our data are the first to assign a cellular function to Trim17 by showing that its E3 activity is both necessary and sufficient for the initiation of neuronal apoptosis.