CDR1 is associated with the paraneoplastic neurodegenerative disease PCD, and increased levels of CDR1 expression are observed in ovarian cancer (10). However, the functional properties of the protein in the brain and in cancer are unknown. Using a combination of cancer cells and brain tissue, we have investigated the subcellular localization of CDR1 and its potential interaction partners. In ovarian cancer cells, we found that the majority of CDR1 is localized to mitochondria with a clustered distribution along mitochondrial tubules. Using PLA, we found that CDR1 is closely associated with the outer mitochondrial membrane. CDR1 colocalized with mitofusin 1 – a transmembrane GTPase that mediates mitochondrial fusion (24). This colocalization was seen in cancer cells in culture, the rat cerebellum, and the Purkinje cell culture. In the human cerebellum however only partial colocalization was seen. In human cerebellum, anti-CDR1 antibodies stained small clusters in the Purkinje cell soma but did not colocalize with any of the mitochondrial proteins or organelle markers tested.
According to the MitoMiner database of mitochondrial localization data (25), CDR1 is not predicted to have a mitochondrial targeting sequence at its N-terminus. However, some proteins interact with the outer mitochondrial membrane in a dynamic manner. Post-translational modifications can regulate this interaction. The CDR1 protein has at least three high probability sites for O-linked glycosylation (26). Furthermore, we found that CDR1 antibodies identify two distinct bands on western blot, and only the higher molecular weight band was present in the mitochondrial fraction. It is therefore possible that post-translational modifications like glycosylation contribute to the association of CDR1 with mitochondria. Several mitochondria-associated proteins have been found to have glycosylated isoforms (27). For example, O-linked glycosylation modulates the recruitment of the mitochondrial fission protein Drp1 (28) and regulates mitochondrial transport in neurons through glycosylation of the adaptor protein TRAK (also known as Milton) (29).
We also found that CDR1 is localized to filamentous structures, but these were only prominent in a subset of the cancer cells. A majority of these structures consisted of the intermediate filament protein vimentin. Some colocalization was also seen with cytokeratin 8, and it is likely that other cytokeratins would yield similar results. Only some of the ovarian cells in the culture expressed vimentin and cytokeratin 8 and showed overlapping CDR1 staining. This differential expression of the intermediate filaments may explain why only some of the cells had filamentous structures stained by CDR1. Vimentin expression is a marker for the epithelial-to-mesenchymal transition (EMT), a process by which cells alter their gene expression programme resulting in increased motility and invasive capabilities (29). The EMT also affects mitochondrial dynamics. Wu et al. (30) found that EMT induces mitochondrial fusion through upregulation of mitofusin 1. As we found that CDR1 colocalizes with mitofusin 1, it is possible that the expression of CDR1 is affected by the EMT in cancer cells.
Colocalization between CDR1 and intermediate filaments was also observed in the Purkinje cell cultures and in human cerebellar sections. There was little vimentin staining in Purkinje cells, instead CDR1 colocalized with neurofilaments and to a lesser extent with GFAP. GFAP is expressed in astrocytes and certain other glial cells, whereas neurofilaments provide structural support for neurons, especially for radial growth of myelinated axons. In contrast to our observation in cerebellar sections, CDR1 antibodies showed only weak staining of the Purkinje cells in culture. This is probably due to the different age of the Purkinje cells in culture (harvested on embryonic day 18; matured for three weeks) and cerebellar sections (harvested from adults). CDR1 may therefore be expressed age-dependently in the Purkinje cells, which is the case for other Purkinje-related proteins (30).
The dual localization of CDR1 on mitochondria and on cytoskeletal filaments suggests that it functions in both structures. Mitochondria depend on interactions with the cytoskeleton to accumulate at regions with high energy demands, such as the leading edge of cancer cells during cell migration and matrix invasion (31) and axonal domains like growth cones and presynaptic terminals (32, 33). Long-range mitochondrial transport is primarily mediated by microtubules, whereas actin filaments mediate shorter range movements and anchoring, important in growth cones and dendritic spines (34, 35). The outer mitochondrial membrane GTPase Miro and the motor adaptor TRAK are key mediators of mitochondrial transportation through coupling of mitochondria to motor proteins (34). We used PLA to investigate the association between CDR1 and this transport machinery in the cancer cells and found that CDR1 colocalized with Miro2, which is critical for actin-dependent transport (34). We also observed CDR1 within actin-rich structures at the periphery of the cancer cells. To ensure proper distribution of mitochondria, there is extensive crosstalk between the transportation and fusion-fission machinery, and the Miro proteins have been found to interact with the mitofusin proteins (34, 36). Since we found that CDR1 is colocalized with mitofusin 1, it is therefore likely that CDR1 is part of a protein complex important for mitochondrial dynamics.
Due to their apolarity, intermediate filaments cannot provide directional cues for mitochondrial transport (37). Instead, intermediate filaments like vimentin and neurofilaments anchor the mitochondria within the cytoplasm (38, 39), and this affects mitochondrial morphology, motility, and distribution (38–41). Intermediate filaments bind to mitochondria via intermediate filament-associated proteins. As we found CDR1 both at the outer mitochondrial membrane and on vimentin and neurofilaments, we hypothesise that CDR1 is involved in the interaction between mitochondria and intermediate filaments. A similar function has been shown for the cytolinker protein plectin 1b, which tethers mitochondria to intermediate filaments and thereby affects the shape and formation of the mitochondrial network (42). Like CDR1, plectin 1b colocalizes with both mitochondria and vimentin (42). Furthermore, plectin 1b was found to be associated with the mitochondrial fusion machinery, as plectin 1b deficiency caused an upregulation of mitofusin 2 (43). Taken together, the colocalization of CDR1 with key proteins involved in mitochondrial transport and fusion and with intermediate filaments suggests that CDR1 is involved in the subcellular distribution of mitochondria in cells. As highly energy-demanding cells, cancer cells and neurons may therefore benefit from upregulating the expression of CDR1.
Disruption of normal mitochondrial function is a common pathological feature of neurodegenerative diseases. Deficiency of key regulators of mitochondrial transport and fusion-fission impairs mitochondrial distribution, causing depletion of mitochondria from dendrites and axons and subsequent loss of dendritic spines and synapses (44–46). For example, neurons with reduced expression of the mitofusin proteins have dysfunctional mitochondria and altered Ca2+ homeostasis, which leads to increased excitotoxicity and neuronal death (47). Levels of expression of mitofusin 1 are decreased in patients with Alzheimer’s disease (48), amyotrophic lateral sclerosis (49), and Huntington’s disease (50). Our findings linking CDR1 with mitofusin 1 suggest that CDR1 may be affected by or may contribute to the mitochondrial dysfunction in neurodegeneration. Interestingly, expression of CDR1 is also dysregulated in Alzheimer’s disease (20), Huntington’s disease (22), and prion disease (21), and mitochondrial dysfunction is also likely to contribute to the pathogenesis of PCD (51).