Search

Human cells are exposed to numerous exogenous and endogenous insults every day. Unlike other molecules, DNA cannot be replaced by resynthesis, hence damage to DNA can have major consequences for the cell. The DNA damage response contains overlapping signalling networks that repair DNA and hence maintain genomic integrity, and aberrant DNA damage responses are increasingly described in neurodegenerative diseases. Furthermore, DNA repair declines during aging, which is the biggest risk factor for these conditions. If unrepaired, the accumulation of DNA damage results in death to eliminate cells with defective genomes. This is particularly important for postmitotic neurons because they have a limited capacity to proliferate, thus they must be maintained for life. Neuronal death is thus an important process in neurodegenerative disorders. In addition, the inability of neurons to divide renders them susceptible to senescence or re-entry to the cell cycle. The field of cell death has expanded significantly in recent years, and many new mechanisms have been described in various cell types, including neurons. Several of these mechanisms are linked to DNA damage. In this review, we provide an overview of the cell death pathways induced by DNA damage that are relevant to neurons and discuss the possible involvement of these mechanisms in neurodegenerative conditions.

BackgroundMutations in the CCNF gene encoding cyclin F are associated with sporadic and familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but the underlying pathophysiological mechanisms are unknown. Proper functioning of the endoplasmic reticulum (ER) is essential for physiological cellular function. MethodsWe used human neuroblastoma SH-SY5Y and human embryonic kidney HEK293T cell lines and mouse primary neurons-overexpressing two familial ALS cyclin F mutants to examine whether mutant ALS/FTD-associated cyclin F perturbs key functions of the ER and Golgi compartments. Specific cellular assays were used to examine ER-Golgi transport (VSVGts045), the budding of vesicles from ER membranes and ER-associated degradation (ERAD). Immunocytochemistry was used to examine the morphology of the Golgi and ER-exit sites, and to detect ER stress and apoptosis. Western blotting was used to examine the content of vesicles budding from ER membranes and the interaction between Sec 31 and cyclin F. Flow cytometry was used to examine cell death.Results We demonstrated that mutant cyclin F inhibited protein transport from the ER to Golgi apparatus by a mechanism involving aberrant vesicle sorting from the ER. It also impeded ER-associated degradation, whereby misfolded ER proteins are ubiquitinated and degraded by the proteasome. This was associated with induction of ER stress and Golgi fragmentation, leading to apoptosis. Conclusion Together, these results demonstrate that ER dysfunction is a pathogenic pathway associated with ALS/FTD-variant cyclin F.

Several components of the Wnt signaling pathway have in recent years been linked to the nuclear pore complex. β-catenin, the primary transducer of Wnt signals from the plasma membrane to the nucleus, has been shown to transiently associate with different FG-repeat containing nucleoporins (Nups) and to translocate bidirectionally through pores of the nuclear envelope in a manner independent of classical transport receptors and the Ran GTPase. Two key regulators of β-catenin, IQGAP1 and APC, have also been reported to bind specific Nups or to locate at the nuclear pore complex. The interaction between these Wnt signaling proteins and different Nups may have functional implications beyond nuclear transport in cellular processes that include mitotic regulation, centrosome positioning and cell migration, nuclear envelope assembly/disassembly, and the DNA replication checkpoint. The broad implications of interactions between Wnt signaling proteins and Nups will be discussed in the context of cancer.

The adenomatous polyposis coli (APC) tumour suppressor gene is mutated in the majority of colon cancers. APC is a multi-domain protein whose distribution at different subcellular locations correlates with unique cellular processes. Our laboratory has focused on the link between APC subcellular location and function, and has characterized pathways for the trafficking of APC both into and out of the nucleus. Antibody specificity is an important factor in the determination of APC localization, and in this chapter we outline a strategy for the unambiguous detection of APC using a combination of biochemical and cell biology approaches.

Adenomatous polyposis coli protein (APC) translocates to, and stabilizes, the plus-ends of microtubules. In microtubule-dependent cellular protrusions, APC frequently accumulates in peripheral clusters at the basal membrane. APC targeting to membrane clusters is important for cell migration, but the localization mechanism is poorly understood. In this study, we performed deletion mapping and defined a minimal sequence (amino acids 1-2226) that efficiently targets APC to membrane clusters. This sequence lacks DLG-1 and EB1 binding sites, suggesting that these partners are not absolutely required for APC membrane targeting. A series of APC sequences were transiently expressed in cells and compared for their ability to compete endogenous APC at the membrane; potent inhibition of endogenous APC targeting was elicited by the Armadillo- (binds KAP3A, B56alpha, and ASEF) and beta-catenin-binding domains. The Armadillo domain was predicted to inhibit APC membrane localization through sequestration of the kinesin-KAP3A complex. The role of beta-catenin in APC membrane localization was unexpected but affirmed by overexpressing the APC binding sequence of beta-catenin, which similarly reduced APC membrane staining. Furthermore, we used RNA interference to show that loss of beta-catenin reduced APC at membrane clusters in migrating cells. In addition, we report that transiently expressed APC-yellow fluorescent protein co-localized with beta-catenin, KAP3A, EB1, and DLG-1 at membrane clusters, but only beta-catenin stimulated APC anchorage at the membrane. Our findings identify beta-catenin as a regulator of APC targeting to membrane clusters and link these two proteins to cell migration.