The results outlined in this manuscript show that the sensitivity of DA levels in the striatum to the toxicity induced by a low dose regimen of MPTP is significantly reduced in NL mice compared to either WT or HET mice. These data also indicate that one functional caspase 2 gene is sufficient to incur normal sensitivity to MPTP.
Following treatment with MPTP, the levels of TH immunoreactive protein were also significantly less affected in the striata of NL versus WT mice. These data, which represent an independent index of the integrity of the nigrostriatal pathway, provide collaborative evidence that the nigrostriatal dopaminergic pathway of NL mice is significantly less sensitive to the selective neurotoxic effects of MPTP.
In order to increase the probability of detecting potentially small protective effects, the total dosages of MPTP administered to the mice in the initial experiments were kept intentionally low, i.e., roughly one-fourth of that administered in a frequently employed dosing regimen (Mayer et al.,1986
). Others have shown that the loss of dopaminergic neurons in the SN following treatment with MPTP is dose-related with a cumulative dosage of 40 mg/kg resulting in roughly an 11 percent decrease (Jackson-Lewis et al.,1995
). In our initial in vivo studies the time course of our treatment regimen was the same but the cumulative dosage of 20 mg/kg MPTP was half that given by these other investigators. Therefore, it seems likely that the reduction of striatal DA observed in our initial studies is more the result of toxic effects at the level of the nerve terminals rather than the death of cells bodies. However, it is yet unclear whether the toxic effects observed in these particular experiments reflect damage to the nerve terminal itself or to the enzymatic machinery involved in the synthesis of DA in the terminals (Przedborski and Jackson-Lewis,1998
The apparently reduced sensitivity to MPTP toxicity in caspase 2 NL versus WT mice cannot be explained by an altered metabolism of this drug as our results show that clearance of the active metabolite MPP+ from the striatum is comparable in both WT and caspase 2 NL mice.
When the neurotoxic effects of MPP+, the active metabolite of MPTP, were studied in primary cultures of mesencephalic neurons; cells isolated from 8–10 day-old NL mouse pups were again shown to be more resistant to the toxicity of MPP+ compared to cells prepared for similarly aged WT mice. These data show that MPP+ produced a markedly greater loss of TH+ neurons in cultures prepared from WT compared to NL mice. Interestingly, there was also a significantly greater preservation of TH+ neurites in the NL-derived cultures while these structures were either absent or more rudimentary in cultures prepared from WT embryos. These data suggest that, at least for neonatal neurons, a homozygous knockout of the caspase 2 gene reduces the sensitivity of both dopaminergic nerve processes and cell bodies to MPTP-associated toxicity.
The results of the studies with primary cultures of mesencephalic neurons along with those obtained from in vivo studies of dopaminergic-related parameters in the striatum suggest, although does not prove, the intriguing possibility that caspase 2 may play a heretofore unsuspected role in the loss of functionally active dopaminergic terminals, which is induced by MPTP-induced toxicity. This possibility is consistent with an earlier report that caspase-related mechanisms may be involved in the degeneration of nerve terminals (Mattson et al.,1998
An examination of nuclear morphology of the TH+ neurons in mesencephalic cultures indicated that death by apoptosis is responsible for at least a significant portion of the disappearance of these cells following exposure to MPP+. This result is consistent with a previous study which also reported apoptotic cell death following treatment with MPP+ in mesencephalic cultures prepared from rat embryos (Furuya et al.,2004
). Our results also show that MPP+ induced significantly less apoptosis in TH+ neurons derived from NL as opposed to WT mouse pups. These latter results provide further evidence that (1) a homozygous knockout of caspase 2 reduces the sensitivity of nigrostriatal dopaminergic pathway to MPTP-induced toxicity and (2) at least in mesencephalic TH+ neurons the knockout of this gene protects dopaminergic cell bodies as well as nerve terminals.
Analysis by unbiased stereology showed that the numbers of TH+ neurons in the SN of untreated NL mice were significantly less that those observed in this same brain region of untreated WT mice. When TH immunopositive protein levels were measured in the VMB, the levels of this parameter in NL mice were actually significantly greater than those observed in WT mice. This increase in TH protein in the VMB may reflect a compensatory response to the presence of a fewer number of TH+ neuron in the SN of the NL mice. With the caveat that the dissection of the VMB likely includes at least a portion of the neurons of the nigrotegmental as well as the nigrostriatal pathway, these data taken together suggest that the reduced number of TH+ neurons determined by stereology reflect a true reduction of nigrostriatal neurons rather than a failure to detect nigrostriatal neurons which were deficient in TH immunoreactive protein. The reduced numbers of TH+ neurons in the SN of the NL mice suggest that caspase 2 may play a role in the development or maturation of the normal constituent of dopaminergic neurons in the SN, but the elucidation of the underlying mechanism will require future investigation.
To determine whether a knockout of caspase 2 could significantly affect the sensitivity of nigral dopaminergic cell bodies in vivo to MPTP-induced toxicity, a dosing regimen was employed which had been previously shown by others to result in an apoptotic-like death of TH+ neurons in the SN of C57BL6 mice (Tatton and Kish,1997
). Treatment with this MPTP regimen induced a significant decrease in the numbers of TH+ neurons in the SN of WT mice compared to untreated WT mice. Although we did not also count the number of Nissl-stained neurons in the SN, the fact that the MPTP-regimen employed was similar to that previously shown to induce nigral neuron cell death suggests that the MPTP-induced decrease in TH+ neurons observed in the WT animals is likely due, at least in part, to the cell death. By contrast, this same MPTP treatment did not significantly reduce the numbers of TH+ neurons in the SN of NL mice when compared to untreated NL mice. This latter observation is consistent with our other data which demonstrated (1) a reduced sensitivity of the in vivo nigral dopaminergic pathway to MPTP-induced neurotoxicity and (2) that primary cultures of mesencephalic TH+ neurons prepared from early postnatal caspase 2 NL mice were less sensitive to MPP+.
Interestingly, the stereological data indicate that the numbers of TH+ neurons observed in both the untreated NL and the MPTP-treated NL mice were not significantly different from those observed in the MPTP-treated WT mice. One possible explanation for these data is that normally there are at least two populations of TH+ neurons in the SN, one more sensitive and a second less sensitive to MPTP-induced toxicity. If this were true and if one were to speculate further that caspase 2 plays a preferential role in the development of the population of these neurons which are more sensitive to MPTP, then the population less sensitive to MPTP might predominant in NL mice. This could explain why (1) the number of TH+ neurons are reduced in the SN of untreated NL mice and (2) these neurons in NL mice show reduced sensitivity to MPTP-induced toxicity.
Increased activation of caspase 2 has been observed in association with cell death in cell lines exposed to MPP+ (Bando et al.,2005
;Chee et al.,2005
), and in one case the cell death was partially prevented by pretreatment with a non-selective caspase 2 antagonist (Chee et al.,2005
). However, to our knowledge, the results summarized in this manuscript provide the first evidence for a functional role for caspase 2 in an intact animal model of MPTP-induced neurotoxicity.
Other observations also suggest that caspase 2 may play an important role in at least some neurodegenerative diseases. For example, caspase 2 may play an important role in the neuronal cell loss associated with Huntington’s Chorea (Hermel et al.,2004
). Further, primary cultures of sympathetic neurons isolated from homozygous caspase 2 null mice were resistant to the neurotoxic effects of β-amyloid (Troy et al.,2000
). Caspase 2 is also the initiator caspase in the apoptotic cell death induced by treatment of primary cultures of neonatal cerebral cortical neurons with rotenone (Tiwari et al.,2011
In summary, the results of this study suggest that homozygous caspase 2 null mice show resistance to the neurotoxic effects of low dosages of MPTP. Further, the knockout of both copies of the caspase 2 gene is necessary to produce this protective effect. In vivo, this increased resistance is best reflected in parameters associated with nigrostriatal dopaminergic cell processes, i.e., striatal DA and TH immunoreactive protein level. However, the TH+ neurons in the SN of NL mice also appear to be more resistant to MPTP. These in vivo observations are also supported by the in vitro data which show that both TH+ cell processes and cell bodies in caspase 2 NL mice are more resistant to the neurotoxin. Although suggestive, the results of these studies do not conclusively show that caspase 2 is normally part of the MPTP-induced cell death pathway. As noted, some evidence provided by these studies suggest that caspase 2 may play a role in the development of the normal number of TH+ neurons in the SN. It is possible, therefore, that the reduced sensitivity of the dopaminergic nigrostriatal pathway to MPTP-induced toxicity observed in caspase null mice may be more the result of the developmental selection of a population of MPTP-resistant neurons rather than due to the removal of caspase 2 as an important factor in mediating MPTP-induced damage to this neuronal pathway. Studies to resolve these alternative possibilities are underway.