4. ANÁLISIS EINTERPRETACIÓN DE RESULTADOS
4.3 Comprobación de hipótesis
The previous chapter demonstrated the importance of the caspase family of proteases in mediating MPP^ toxicity for dopaminergic neurones in vitro, and this has also been demonstrated by a number of other groups both in vivo and in vitro
(discussed in detail in Chapter 4, above). As has been discussed, the upstream pathways which may lead to caspase activation are unclear. Recent studies have implicated activation of the stress activated MAP kinases in MPP^/MPTP toxicity; in vivo, several reports have demonstrated neuroprotective effects of JNK pathway inhibition against MPTP toxicity. MPTP treatment in vivo results in phosphorylation of JNK and the upstream kinase MKK-4 (Saporito et al., 2000). C EP-1347, which inhibits the JNK pathway at the level of mixed lineage kinase, prevents degeneration of mesencephalic dopaminergic neurones and loss of striatal innervation in MPTP treated mice and primates (Saporito et al., 1999, 2000). Mice expressing the JNK binding domain of JNK inhibitory protein-1 following adenoviral gene transfer are also resistant to MPTP toxicity in vivo, and have increased numbers of nigral dopaminergic neurones, increased levels of striatal dopamine and metabolites and behavioural benefit; decreased levels of caspase 3 activation were also observed in the nigra (Xia et al., 2001).
As well as the JNK pathway, a number of models of cellular stress activate the p38 pathway. Recently, it has been demonstrated that inhibition of this pathway protects MPTP treated mouse NT2a cells (DeGirolamo et al., 2002); another report has implicated the p38 pathway in MPP^ toxicity, though increased generation of H2O2 (Du et al., 2001). In this chapter the contribution of the JNK
and p38 pathways to MPP^ toxicity in vitro were evaluated using selective pathway inhibitors, to evaluate the extent of the neuroprotective effect observed with these inhibitors, and to determine whether these pathways are linked to downstream caspase activation.
Activation of the JNK signalling pathway has been reported to lead to caspase activation and apoptosis in a number of paradigms of cellular stress. Evidence for a role of JNK activation in MPP^ toxicity for dopaminergic neurones in this culture system comes from the demonstration of increased levels of phosphorylated c-jun, a transcription factor phosphorylated by JNK but not p38, in MPP^ treated dopaminergic neurones. These data confirm previous observations in mesencephalic neurones exposed to MPP^ (Gearan et al., 2001); conflicting data have, however, been reported in the dopaminergic cell line MN9D (Choi et al., 1999, 2001). That the maximum increase in P-jun levels preceded the onset of expression of apoptotic morphology indicates that c-jun phosphorylation precedes apoptotic execution. The decreased levels at timepoints where apoptosis increases may reflect a loss of neurones which have activated P- jun; the persistence of increased numbers of P-jun immunoreactive neurones in cultures co-treated with caspase inhibitor and MPP^ reinforces this hypothesis.
Both C EP-1347 and the JNK/p38 inhibitors evaluated gave significant partial neuroprotection from MPP^ toxicity in mesencephalic dopaminergic neurones; in this culture system, then, the JNK pathway is clearly involved in the dopaminergic degeneration. When a selective p38 inhibitor was used, the results were markedly different.
In this culture system, p38 inhibition using selective inhibitors was not sufficient to spare dopaminergic neurones. The three p38 inhibitors described had little effect on the number of remaining TH-immunoreactive neurones following MPP^ lOpM treatment. Compound 2, the most extensively tested of the selective p38 inhibitors had no effect on the number of apoptotic TH-immunoreactive neurones, nor on the expression of active caspase 3 in TH-immunoreactive neurones at 500nM concentration, significant neuroprotection was observed above this concentration, in the range where some JNK inhibition might be expected. Thus there is little evidence that p38 inhibition spares dopaminergic neurones, indicating that activation of the JNK pathway and consequent caspase activation is of more importance in mediating MPP^ toxicity within these purified neuronal cultures.
As previously discussed, there are a number of well defined intracellular signalling pathways which can activate the caspase cascade; these include death receptor ligation, release of mitochondrial pro-apoptotic factors into the cytoplasm either through opening of the mitochondrial permeability transition pore or through pores formed by pro-apoptotic Bcl-2 family members, and direct activation of initiator caspases (discussed in Chapter 1 and Chapter 4, above).
The JNK pathway has been shown to be involved in activation of pathways through at least two of these mechanisms. Activation of the JNK pathway is reported to induce expression of the cell death inducing cytokine fas ligand (Le- Niculescu et ah, 1999); fas ligand in the cell membrane binds to the death receptor fas, which recruits a number of intracellular transducer proteins and activates a caspase cascade involving activation of caspase 8 and ultimately caspase 3. There is evidence in vivo that MPTP treatment causes activation of caspase 8, and inhibition of this pathway is neuroprotective (Hartmann et al., 2001, Viswanath et al., 2001). The JNK pathway has recently been shown to induce apoptosis in |3- amyloid peptide (Morishima et al., 2001), and a novel neurotoxic amyloid fragment P-amyloid 17-40 (Wei et al., 2002), treated cortical neurones through induction of fas ligand - this route may also be a candidate pathway for coupling activation of JNK to the caspase cascade. The JNK pathway also interacts with the Bcl-2 family of pro- and anti-apoptotic factors. Activation of the JNK pathway is reported to cause translocation of Bid and Bim, pro-apoptotic BH3 domain only members of the Bcl-2 family, from the cytoplasm to the mitochondrial membrane (Harris and Johnson, 2001). Once located in the mitochondrial membrane, these factors act as accessory factors to facilitate the pore formation by factors such as Bax, allowing cytochrome c release. Transgenic mice lacking Bax show reduced susceptibility to MPTP/MPP'*’ toxicity (Vila et al., 2001). This pore forming activity of Bax is prevented by anti-apoptotic Bcl-2 (Antonsson et al., 1997), and neurones from Bcl-2 overexpressing mice are resistant to MPTP/MPP^ toxicity in vivo and in vitro (Yang et al., 1998, Offen et
al., 1998). Cytochrome c released from mitochondria forms a complex with APAF-1 and dATP, which activates caspase 9 and then caspase 3 (discussed in detail in Chapter 1 and Chapter 4). Cytochrome c has been shown to be released from mitochondria in cerebellar granule neurones treated with MPP^ (Du et al.,
1997), and it was demonstrated in the previous chapter that inhibition of caspase 9
in vitro partially attenuates MPP^ toxicity in vitro. Furthermore, overexpression of X-linked inhibitor of apoptosis (lAP) protein, an endogenous inhibitor of caspase 9, attenuates MPTP toxicity in vivo. Thus, this cascade is another possible pathway coupling JNK activation to caspase cleavage. It will be of interest to further examine the relative contribution to MPP^ mediated apoptosis made by such JNK activated pathways, though it seems likely that these are not the only pathways activated by MPP^.
In this regard, it is interesting that both of the JNK inhibitors tested caused only a partial sparing of dopaminergic neurones. The JNK/p38 inhibitor abolished the expression of P-jun at both 24 and 48 hour timepoints, yet decreased the number of apoptotic nuclei in those TH-immunoreactive neurones remaining in the culture after 48 hours by only half. Similar results were observed when the expression of active caspase 3 was evaluated, with JNK/p38 inhibition causing only a 50% decrease in the MPP^ mediated increase in active caspase 3 expression in TH- immunoreactive neurones at 48 hours. These data would indicate that under these culture conditions, inhibition of the JNK pathway alone is not sufficient to spare all dopaminergic neurones from MPP^ toxicity, nor to prevent all the cleavage and activation of caspase 3. Together these results imply that imply that another
mechanism leading to caspase activation may be involved. W hether this alternative route to apoptosis is normally activated concurrently with JNK phosphorylation or is a compensatory mechanism activated upon JNK inhibition is not clear. An obvious candidate pathway for the alternative route to caspase activation and apoptosis in MPP^ treated neurones is mitochondrial permeability transition pore opening and subsequent release of pro-apoptotic factors into the cytoplasm. This was discussed in some detail in Chapter 4, above. This is an interesting area for future research.
Although the data presented here appear to indicate that JNK inhibition may be of limited effectiveness in preventing all MPTP/MPP^ related cell death, it may have therapeutic benefit in Parkinson’s disease. C EP-1347 is currently undergoing clinical trials for Parkinson’s disease. There is evidence that human PD nigrae express phosphorylated c-jun. In a very interesting recent paper, another possible link to JNK activation in PD was postulated. In this paper (Hashimoto et al, 2002) it was shown that a-synuclein overexpression protected a neuronal cell line from oxidative stress through inactivation of the JNK pathway; synuclein inhibited JNK pathway activity by increasing expression and activity of JIP-1. These data are fascinating, given that there are mutations in the a-synuclein gene which are associated with early onset Parkinson’s disease; it will be of great interest to see whether mutant synuclein has reduced JNK pathway inhibitory activity. Another potentially interesting area for further research is the potential for JNK inhibitors to increase survival of primary dopaminergic neurones prior to transplantation; both caspase (Schierle et al., 1999) and the p38/JNK inhibitor
SB203580 (Zawada et al., 2001) increase survival of dopaminergic neurones in transplantation models. In vitro, Compound 1 increased survival of mesencephalic dopaminergic neurones either when applied continuously from plating for up to 7 days, or when cultures were pretreated for timepoints up to 2 hours prior to plating (data not shown). This then may represent another potential use for JNK inhibition in PD.
Thus, the data in this chapter demonstrate that in this culture model, treatment with MPP^ induces phosphorylation of c-jun which appears to precede apoptotic cell death; this cell death can be inhibited by inhibition of JNK, of both JNK and p38, but not by p38 inhibition alone. The activation of the JNK pathway is upstream of caspase activation, and this activation can be inhibited by JNK inhibition, but not by p38 inhibition alone. The JNK pathway, however, is unlikely to be the only pathway activated by MPP^ treatment, as complete inhibition of c-jun phosphorylation using a JNK inhibitor decreased but did not abolish either caspase 3 activation, TH-immunoreactive cell loss or expression of apoptotic profiles.
Thus, there is strong evidence that the JNK pathway is involved in the apoptotic response to MPTP/MPP^ in both in vitro and in vivo models. The next chapter describes a series of experiments carried out to evaluate whether the JNK pathway might be implicated the loss of TH-immunoreactive neurones in another in vivo