Actin and apoptosis

The mammalian homologue o f the C. elegans ced-3 gene is CPP32 (Caspase-3)

[Xue et a l 1996]. This gene is specifically expressed in the trigeminal ganglia,

facio-acoustic ganglion complex, and DRGs o f mouse as early as E l 0.5 days. Caspase-3 like proteases are activated during apoptosis of DRG neurons in the absence o f NGF and serum [Mukasa et al. 1997]. Mashima et al. showed that

caspase-3 specifically cleaves the actin protein into two (ISKand 3OK) fragments

in vitro as well as in vivo [Mashima et al. 1995] [Mashima et al. 1997]. Actin is

the major component o f cytoskeleton. Cytoskeletal degradation has indeed been identified as an early event of apoptosis [Guenal et a l 1997] [van Engeland et a l

1997]. Disruption o f micro-filaments by cytochalasins [Rubtsova et a l 1998] or

depolymerization o f actin by thymosine piO [Hall 1995] will induce apoptotic events. This would suggest that disruption o f the cytoskeleton could be a key factor in the initiation o f apoptosis.

In addition to being one of the basic elements of cytoskeleton, actin is also an inhibitor o f the endonucleolysis which is mediated by DNasel [Kayalar et a l

i.e., pynknosis, cytoplasmic shrinkage and intemucleosomal DNA degradation which is typical for apoptosis [Polzar et a l 1993]. Thus, disruption in actin

biogenesis and function could, in addition to regulating apoptotic events, mediate its effect through its interaction with DNasel. This, in turn, could also impact on cell viability.

Nascent actin has been shown to bind with a specific heat shock protein (or chaperon) hsp27. This association is important during gastrulation; neural tube development and the migration o f neural tube derived cell lineage’s including those that give rise to the DRG [Walsh et a l 1997]. Although these early

developmental events might not be wholly consistent with that of the phenotype associated with the rat mutation, other studies have shown that disruption o f this association can lead to apoptotic events in the neural retina [Tezel and Wax

2000].

The missense mutation in Cct4 of m f rat resulted in cysteine to tyrosine change.

The free energy o f the disulfide bond (S-S) bridging two cysteines, is much stronger than the hydrogen bond (S-OH) between cysteine and tyrosine. The distance between the two cysteines is less than the distance between a cysteine and a tyrosine. This may further destabilise the molecule in the absence o f enhanced stability afforded by the formation of a disulphide bridge that could form between adjacent cysteines. Therefore, we can speculate that the mutation in Cct4 will cause the distortion of the normal 3-D structure o f this subunit as

well as the whole CCT protein. As the actins are the major substrate o f CCT and the apical domain o f Cct4 is one o f the binding sites for nascent actin protein

prior to folding, this change is predicted to reduce the affinity o f actin to the CCT ring complex resulting in either degradation of actin or diminishing in its

polymerisation. The aberration might also influence the binding o f ATPase to CCT protein, which cleaves the ATP to provide the energy for protein folding. Therefore, the abnormalities in actin may eventually cause the destruction o f the cytoskeleton and start the process of neuronal apoptosis.

4.2.2 2 Independent role in neuronal apoptosis

In addition to the role of the TCP-1 complex in protein folding, individual subunits can also act independently. As shown in the chick embryo sympathetic neurons, over-expression o f TCP-16 (CCT4) accelerated the neuronal apoptosis induced by dopamine treatment [Zilkha-Falb et a l 2000]. Other subunits did not

produce this effect. The authors suggest that over-expression o f CCT4 in

sympathetic neurons accelerated dopamine induced neuronal apoptosis. The use o f antisense oligonucleotide treatment to inhibit CCT4 expression significantly reduced dopamine induced neuronal death in sympathetic neurons. These results suggest that the CCT4 protein is probably a positive mediator in the process of neuronal apoptosis. This study therefore demonstrated that in the process of inducing neuronal death, CCT4 played a role unrelated to the whole CCT protein. Also this study supports other research demonstrating that Cet subunits can act independently. Wu-Baer et a l purified a cofactor SRB, also known as

human CCT4, which worked with an elongation factor l a and polypyrimidine tract binding protein to facilitate the binding o f RNA polymerase II and TRP-185

to TAR RNA (in HIV-1) to increase gene expression [Wu-Baer et al. 1996].

Another study showed that the a , y, Ç, and 0 subunits can form smaller

complexes than those of the CCT complex and act as a microtubule-associated protein [Roobol et a l 1999]. These studies suggest that either as an independent

subunit or as a component of the TCP-1, the CCT4 protein may play an important role in the process o f neuronal apoptosis, which is the pathological hallmark o f m f disease.

4. 3 Conclusion

The pathological defect associated with the m f mutation suggests that the sensory

neuropathy is due to a loss of neurons in DRG. At this stage, it is unclear

whether this apoptosis is due to a developmental defect involving the ‘wiring’ of the peripheral nervous system mediated through a defect in actin. It may be that actin is either having a direct impact on apoptosis or an indirect effect because the axons are failing to make “survival contact”. It is also possible that the apoptosis is due to a general problem with protein folding that is unrelated to actin. Alternatively, the excess apoptosis may be due to some independent function o f the CCT4 protein itself. Whatever the mechanism, it is clear that a mutation in Cct4 can have serious pathological/clinical consequences in rat and

possibly humans as well. Clearly more work needs to be done in order to understand the molecular mechanism that underlies that pathological/clinical defect.

Chapter 5