Although the primary purpose of these investigations was to identify and characterize the antiapoptotic effect of MLK inhibition in vivo
, we observed that the kinase-dead dominant-negative form of DLK also had trophic effects on DA neurons of the SN. These effects were observed at the level of the cell soma, and included an increase in neuron size and the activity of TH within the nigra. These observations have precedents in studies performed in tissue culture. Harris et al. (2002)
observed that the MLK inhibitor CEP1347 prevented not only the cell death but also the decreases in protein synthesis and cell size that occur after withdrawal of NGF. Strikingly, CEP1347 not only prevented soma atrophy but also reversed it (Harris et al., 2002
). Subsequent observations by Wang et al. (2005)
demonstrated that treatment with a CEP1347 analog, CEP11004, induced increased expression of TrkA and activation of the signaling kinase Akt. These investigators postulated that inhibition of the MLKs mediated the changes in TrkA and Akt, but they recognized that a non-MLK-related mechanism could be at work. Indeed, Roux et al. (2002)
also observed trophic effects of CEP1347, associated with activation of Akt, but they postulated that these effects were independent of MLK inhibition. Our results support the proposal of Wang et al. that inhibition of the MLKs alone may be sufficient.
It is uncertain why a kinase-dead dominant-negative form of DLK had trophic effects on DA neurons, whereas an LZ alone construct did not. We doubt that this difference can be attributed to quantitative differences between the two constructs in their blockade of DLK, because DN-DLK-LZ was produced at a higher titer and had a more pronounced effect in protecting DA neurons from cell death. We postulate that the different effects of the two dominant negatives are more likely to be attributable to qualitative differences in their mechanisms of action. The DN-DLK-LZ construct blocks homodimerization of DLK molecules and thereby prevents autophosphorylation and activation (Nihalani et al., 2000
). DLK-LZ does not have dominant-negative effects on MLK3 kinase activity (Nihalani et al., 2000
). DN-DLK(K152A), however, remains capable of homodimerization and heterodimerization, and may have a broader spectrum of dominant-negative effects.
The DN-DLK(K152A) and DN-DLK-LZ dominant-negative forms had equal efficacy in their ability to block apoptosis in DA neurons in the 6OHDA model. For both dominant negatives, the antiapoptotic effect provided a lasting enhanced survival. Although DN-DLK-LZ provided a greater degree of survival than DN-DLK(K152A), the parameters of these in vivo
experiments do not permit a conclusion that DN-DLK-LZ is inherently more effective. These results support the interpretation of previous studies of CEP1347 and CEP11004 that their antiapoptotic effects are likely to be primarily attributable to inhibition of the MLKs rather than their apparently independent ability to stimulate the antiapoptotic kinase Akt (Roux et al., 2002
). Although previous studies with these compounds in rodent models of parkinsonism (for review, see Silva et al., 2005a
) do not permit analysis of molecular specificity, because of their broad inhibitory effects within the MLK family, the present study suggests that DLK may play a specific role. Whereas observations of the antiapoptotic effect of DN-DLK(K152A) alone would not permit such a conclusion, because of its known interactions with other MLKs, including MLK3 (Xu et al., 2001
), similar observations with DN-DLK-LZ do support a specific role, given its molecular specificity (Nihalani et al., 2000
). The role of DLK in mediating apoptosis in this context is unlikely to be attributable to a unique ability, among the MLKs, to mediate cell death. Observations in vitro
in rat cells have demonstrated the ability of other members of the MLK family to mediate apoptosis (Xu et al., 2001
). It is more likely that the much greater relative abundance of DLK in rodent SN (Ganguly et al., 2004
) is responsible for its preponderant role. Furthermore, although we were unable to demonstrate a protective effect of DN-MLK3(K144R) in the presence of confirmed protein expression (), we did not quantify the level of expression in comparison with that of the DLK forms. Therefore, we cannot definitively exclude a possible role for MLK3 in cell death in this model.
In addition to its antiapoptotic effect, DN-DLK-LZ prevented atrophy of dopaminergic neurons after 6OHDA lesion. This trophic effect appears to be distinct from that of DN-DLK(K152A) on normal, unlesioned neurons, because DN-DLK-LZ did not demonstrate such effects. This ability of DN-DLK-LZ to either prevent or reverse atrophy of these neurons after lesion is similar to the ability of CEP1347 to both prevent and reverse atrophy of sympathetic neurons after NGF withdrawal (Harris et al., 2002
The neuroprotective effects of DN-DLK(K152A) and DN-DLK-LZ did not, however, include preservation of the nigrostriatal axonal projections. This result was disappointing and unexpected, given our previous demonstration of a striking protective effect of CEP11004 on striatal dopaminergic fibers in postnatal 6OHDA model (Ganguly et al., 2004
). Other investigators had also previously demonstrated protection of dopaminergic axons by blockade of down-stream targets in the MAPK cascade, including c-jun in an axotomy model (Crocker et al., 2001
) and c-jun N-terminal kinase (JNK) in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of parkinsonism (Xia et al., 2001
). This result is not, however, unlike those of others who failed to achieve axonal protection despite effective preservation of nigral dopaminergic cell bodies because of blockade of PCD pathways (Eberhardt et al., 2000
; Hayley et al., 2004
; Silva et al., 2005b
). This result supports the concept that the molecular pathways that mediate axonal degeneration are likely to be distinct from those of PCD, which mediate destruction of the cell soma (Raff et al., 2002
; Coleman, 2005
Transduction of SN DA neurons with dominant-negative forms of DLK was not entirely effective in blocking cell death induced by 6OHDA. In DN-DLK(K152A) and DN-DLK-LZ injected mice, there was persistent apoptosis resulting in a loss of 45 and 25%, respectively, of DA neurons. In the case of DN-DLK-LZ, the more effective of the two, this loss seemed to be simply attributable to the level of transduction efficiency. Among DN-DLK-LZ-injected mice, a mean of 71% of DA neurons were successfully transduced (range, 60–84%). This level of transduction is not significantly different from the percent survival achieved (75 ± 3%).
The canonical model for mediation of PCD by MAPK cascade signaling has been that phosphorylation and activation of JNK by upstream kinases leads to activation of c-jun, which, in turn, results in the transcriptional activation of cell death mediators (for review, see Silva et al., 2005a
). However, there is now much evidence indicating that other JNK targets, in addition to c-jun, play a role in PCD. JNK can phosphorylate and diminish the antiapoptotic effects of Bcl-2 and Bcl-XL
(Maundrell et al., 1997
; Kharbanda et al., 2000
), and it can activate the proapoptotic effects of Bad, Bim, and Bmf (Donovan et al., 2002
; Lei and Davis, 2003
). In addition, a non-c-jun-dependent mechanism of cell death induction by JNK3 has been observed in an axotomy model in vivo
(Keramaris et al., 2005
). Conversely, phosphorylation of c-jun is not universally associated with cell death; c-jun also appears to play a vital role in axonal regeneration (Raivich et al., 2004
; Waetzig et al., 2006
). However, our analysis of the relationships between transduction efficiency, phosphorylation of c-jun at the cellular and population levels, and cell loss suggest that DLK and its downstream target JNK act in this model to induce cell death primarily by the phosphorylation and activation of c-jun. At the population level, percent transduction efficiencies for SN DA neurons (71 ± 6%) were not significantly different from the percent reduction in phospho-c-jun profiles induced by 6OHDA (62 ± 13%). At a cellular level, the presence of phosphorylated c-jun in AAV DN-DLK-LZ transduced neurons was exceedingly rare. Given that JNK is the principal kinase for c-jun in tissue (Kyriakis and Avruch, 2001
), these results suggest that dominant-negative blockade of DLK in this context is highly likely to have also blocked JNK activation. In experiments with AAV DN-DLK-LZ, the percentage of non-transduced neurons, the residual percentage of cells expressing phosphorylated c-jun, and the percentage of neurons that died were not significantly different. These similar percentages suggest that it is unlikely that JNK acts substantially by any c-jun-independent mechanism to induce cell death.
The principal conclusion from these studies is that specific inhibition of DLK in this model blocks the phosphorylation of c-jun and thereby prevents neuronal death. In fact, our results suggest that the chief limiting factor for prevention of neuron death is transduction efficiency. These results add to the growing evidence that MLK inhibition provides protection from neuronal death (for review, see Wang et al., 2004
). Although enthusiasm for this approach has been diminished by the negative results of a clinical trial in PD patients that failed to demonstrate an ability of CEP1347 to slow clinical progression (Waldmeier et al., 2006
), these results cannot be taken as proof of failure of the MLK inhibition approach. One reason is that it is unknown whether kinase inhibition was achieved (Burke, 2007
). A viral vector-based approach offers the advantages that intracellular blockade of MLKs is likely to be achieved, and molecular and regional specificity are possible. We therefore conclude that viral vector-based inhibition of cell death kinases merits consideration as a neuroprotective approach to PD.