Enhancement of dopaminergic properties and protection mediated by neuronal activation of Ras in mouse ventral mesencephalic neurones

Abstract

The poor differentiation and survival of dopaminergic neurones are practical constraints in their therapeutic applications. Here we explored the role of neuronally activated Ras in ventral mesencephalon-derived neurospheres generated from synRas mouse embryos. The expression of Val12 Ha-Ras transgene and enhanced Ras activity was evident after differentiation of the neurospheres with a corresponding activating phosphorylation of mitogen-activated protein kinase. Phosphorylation of Akt/PKB, the target kinase of phosphoinositide 3-kinase, along with phosphorylation of Bad and CREB were enhanced in synRas-derived differentiated neurosphere cultures. Furthermore, increased Nurr1 expression was associated with elevated numbers of dopaminergic neurones in synRas-derived cultures compared with the wild-type. Correspondingly, tyrosine hydroxylase promoter assays revealed enhanced transcriptional activation of the promoter in synRas-derived cultures. synRas-derived dopaminergic neurones were greatly resistant to degeneration induced by various noxious stimuli. Consistently, the transgenic expression of activated Ras attenuated the adverse 6-hydroxydopamine effects on dopaminergic neurones. Dopaminergic neurones derived from both wild-type and synRas cultures expressed voltage-gated potassium and sodium currents, fired action potentials and exhibited electrical network activity. Thus, expression of the transgene promotes survival and enhances differentiation towards a dopaminergic cell fate without altering their basic electrical properties. Our results suggest that intracellular cell therapy mimicking trophic signalling may offer potential benefit in models of human disease associated with dopamine neurone dysfunction.

Ras Homolog Enriched in Brain (Rheb) Enhances Apoptotic Signaling*

“…The RBD of c-Raf kinase was expressed and purified as described previously (34–36). Western blot analysis was also performed essentially as described previously (17). …”

“…Vast evidence exists for Ras activity acting as a protective agent both in non-neuronal (13) and neuronal systems (14–17). Furthermore, transgenic activation of neuronal Ras in the brain prevents degeneration in several lesion paradigms (17–19). Similar to Ras, Rheb signaling is also directly correlated with the promotion of survival.…”

Exercise can rescue recognition memory impairment in a model with reduced adult hippocampal neurogenesis

“…In order to gain further insights into the regulation of newly generated neurons we have used here the synapsin I promoter to express permanently activated Ha-Val12 Ras in mice (named synRas). In a number of experimental paradigms this transgenic activation of Ras in brain neurons prevented lesion-induced neuronal degeneration during postnatal development and in the adult brain (Heumann et al, 2000; Felderhoff-Mueser et al, 2004; Chakrabarty et al, 2007; Makwana et al, 2009). Consistently, also in normal newborn hippocampal neurons there is enhanced cell survival in synRas mice.…”

Role of Neuronal Ras Activity in Adult Hippocampal Neurogenesis and Cognition

“…This difference presumably reveals direct and indirect actions of the transgene protein product. On the one hand, H-Ras-transgene expression shields nerve cells from lesion-induced degeneration (Heumann et al, 2000; Chakrabarty et al, 2007; Makwana et al, 2009). There is previous evidence indicating that intracellular H-Ras activation has a cell-autonomous effect in dissociated single neurons as shown by inhibition of neurotrophin-induced survival after intracellular application of Ras-neutralizing monoclonal antibody or its non-crosslinking F ab -fragments (Borasio et al, 1989, 1993; Heumann, 1994) Accordingly, enhanced survival of newborn neurons might reflect the neuroprotective action of elevated intracellular H-Ras activity although it cannot be excluded that H-Ras-activated neurons secrete factors other than BDNF, which also contribute to survival effects in synRas animals.…”

“…Thus, decreased early precursor cell proliferation must represent an indirect effect of the activated H-Ras transgene protein. Its expression has been shown to be repressed in nestin-positive neuronal precursors (Chakrabarty et al, 2007). This in turn points to the presence of signals derived from the existing hippocampal circuitry as a secondary consequence of H-Ras-transgene expression in differentiated neurons (see “Working Hypothesis” in red in Figure 2C).…”