Apoptosis in dopamine neurons during development 
As in most neural systems, apoptosis occurs in dopamine neurons of the substantia nigra (SN) during normal development (Janec & Burke, 1993; Oo & Burke, 1997). This naturally occurring cell death (NCD) event occurs largely postnatally, and is biphasic. The magnitude of the NCD event in dopamine neurons is regulated by interactions with their target, the striatum, as would be predicted by classic neurotrophic theory (reviewed in Burke, 2004). Our Laboratory is engaged in efforts to identify the endogenous neurotrophic factors in brain which regulate NCD in SN dopamine neurons.

One possible candidate for a striatal target-derived neurotrophic factor for SN dopamine neurons is glial cell line-derived neurotrophic factor (GDNF). Its mRNA and protein are highly expressed in postnatal striatum (Cho et al, 2003; Oo et al, 2005). mRNA for its receptor, GFRα1, is highly expressed in SN dopamine neurons (Cho et al, 2004), and receptor protein is expressed within dopaminergic terminals in the striatum (Cho et al, 2004). Because conventional GDNF and GFRα1 null mice die shortly after birth, before most of the NCD event has occurred, they are not useful in studying the role of GDNF in regulating NCD in SN dopamine neurons. We have used a neutralizing antibody approach to demonstrate that during development, intrastriatal “knock down” of GDNF augments NCD (Oo et al, 2003). Conversely, intrastriatal injection of GDNF suppresses it. In conformance with classic neurotrophic theory, sustained overexpression of GDNF selectively in the striatum, using a regionally selective transgenic approach, reduces the NCD event among SN dopamine neurons and increases the surviving number after the first phase of NCD (Kholodilov et al, 2004). However, this increase does not persist into adulthood, suggesting that other factors must be required. We are currently investigating the nature of these additional factors.

Naturally occurring cell death in neurons is regulated not only by target-derived neurotrophic factors, but also by local factors, released either by adjacent cells (paracrine) or by the cells themselves (autocrine). There is some evidence that brain-derived neurotrophic factor (BDNF) may play such a role for SN dopamine neurons, and we are investigating that possibility.

Apoptosis in models of Parkinson’s Disease
Apoptosis occurs in dopamine neurons not only during normal development, but also in models of PD. However, the occurrence of apoptosis in neurotoxin-induced models depends on the methods used. We have shown that the neurotoxin 6-hydroxydopamine (6OHDA) induces classic apoptosis in dopamine neurons in both immature and adult rodents when injected into the striatum (Marti et al, 1997; Marti et al, 2002), but not when injected directly into the substantia nigra (Jeon et al, 1995). The widely studied neurotoxin MPTP does not induce apoptosis when injected in an acute regimen (Jackson et al, 1995), but does in a chronic regimen (Tatton & Kish, 1997). While the field is changing rapidly, there unfortunately is not yet a transgenic mouse model, based on a known genetic cause of PD, in which death of dopamine neurons has been robustly and consistently observed. There may be a sufficient abundance of dopamine neuron death in viral vector-based genetic models of PD, and we are currently exploring these approaches.

One concern about targeting PCD in attempting to provide neuroprotection in PD is that blocking these pathways too far “downstream” may protect neurons such that they remain viable, but are non-functional (see Burke 2007a, 2007b). For this reason, we have focused our efforts on approaches which target PCD as far “upstream” as possible. There has been considerable interest in the transcription factor c-jun as a mediator of PCD, following its phosphorylation and activation by c-jun N-terminal kinase (JNK) (reviewed in Silva & Burke, 2005). We have shown that inhibition of c-jun phosphorylation with an inhibitor of the mixed lineage kinases (Figure 1) prevents apoptosis in dopamine neurons (Ganguly et al, 2004).



We have also shown that similar neuroprotection can be achieved using molecular approaches with dominant negative forms of the dual leucine zipper kinase (DLKs) administered by viral vector transfer (unpublished).

Another concern about efforts to target PCD in providing neuroprotection is that death pathways are so diverse and redundant, that simply blocking one is unlikely to be effective. For this reason, we have an interest in the survival signaling kinase Akt, which has been shown to block apoptosis by acting on numerous pathways at multiple levels (Figure 2) (reviewed in Burke, 2007b).



We have found that a constitutively active form of Akt, myristoylated Akt (Myr-Akt) not only inhibits apoptosis in the adult mouse 6OHDA model, but also has striking trophic effects on dopamine neurons in both adult and aged mice (Figure 3) (Ries et al, 2006).



One of our current major interests is in exploring the mechanisms whereby Akt provides these neuroprotective and neurotrophic effects.