CLU has long been implicated in AD, however, its exact role in AD pathogenesis and disease development remains unclear. Our study demonstrates a central role for CLU proteins in facilitating Aβ25−35 induced cell death and neurite damage in human neurons in vitro.
Previous studies have examined the cytoprotective role of secreted CLU and the pro-apoptotic role of non-glycosylated CLU proteins (21,23,37,49). However, these studies have focussed on the role of CLU proteins in cancer pathways in immortal cancer cell lines and few studies have directly manipulated the expression of non-glycosylated CLU proteins in human neurons to explore their importance in neurodegeneration. In contrast to previous work, we demonstrate that stress induced by Aβ25−35 does not induce increased expression of non-glycosylated CLU in human neurons. Therefore, the previously identified pro-apoptotic role of non-glycosylated CLU proteins may not be universal and may be dependent on factors such as the type of cell and source of stress. Yang et al., demonstrated that overexpression of non-glycosylated CLU proteins in cancer cells sensitizes cells to stress and induces more death in basal conditions (36). This is not observed in human neurons where we have demonstrated that exon 2 -/- neurons displayed similar levels of cell death in basal conditions, indicating that increased expression of non-glycosylated CLU did not result in increased neuronal death. Exon 2 -/- iPSC-neurons displayed reduced sensitivity to Aβ25−35 induced cell death and neurite damage, suggesting that it is the glycosylated version of CLU that facilitate Aβ25−35 toxicity. We have therefore demonstrated that non-glycosylated CLU proteins likely do not play a critical role in mediating neurodegeneration in human neurons induced by Aβ25−35.
Although typically considered a secreted protein, glycosylated CLU proteins have been shown to localise within cells (22,29,33), however, the mechanisms leading to intracellular retention of sCLU have not been described. Numerous studies have demonstrated that trafficking of glycosylated CLU proteins is altered by stress in a variety of cell types/lines (22,30–33,38), resulting in reduced secretion and increased intracellular retention of CLU. Additionally, CLU-AD mutations and Aβ25−35 treatment of rodent neurons have been shown to alter CLU trafficking, providing a potential mechanistic role of CLU in mediating neurodegeneration (29,34).
To date, few studies have attempted to dissect whether stress induced alterations in CLU trafficking act to facilitate cell stress or act to protect cells from further damage. Gregory et al. demonstrated altered CLU trafficking by ER stress in N2a cells resulting in increased binding of intracellular CLU and TDP-43, which reduced both the aggregation and toxicity of TDP-43, thus suggesting altered CLU trafficking in this context provides a protection to cells (33). In comparison, previous work by our group found evidence that altered CLU trafficking facilitates Aβ25−35 induced cell death in rodent neurons and that Aβ25−35 increased intracellular CLU retention and CLU knockdown provided protection from Aβ25−35 induced neurodegeneration (29). It is therefore likely that stress-induced CLU trafficking alterations and subsequent increases in intracellular CLU as well as reduced secretion can be both protective and detrimental to cells dependent on the type of stress and cell. Understanding the importance of glycosylated CLU proteins is particularly important since several studies have identified plasma CLU as a promising marker for AD (50); higher plasma CLU levels are associated with increased hippocampal atrophy and increased rate of clinical progression (51,52).
Our data clearly show that loss of glycosylated CLU protein in human neurons in vitro provides partial protection against Aβ25−35 induced toxicity, which we postulate may arise from ECM remodelling (Fig. 5). Although cell death was still induced by Aβ25−35 in exon 2 -/- neurons, it was significantly less than control neurons and neurite damage was absent in exon 2 -/- neurons. What remains unclear is whether the observed protection arises because of the loss of intracellular glycosylated CLU proteins or the loss of secreted glycosylated CLU proteins, or both. Although previous studies have suggested a pro-apoptotic role of non-glycosylated CLU proteins in cells (36,37), little is really known of their physiological and pathological functions in cells. We demonstrate an increase in the abundance of non-glycosylated CLU proteins in exon 2 -/- neurons, which is not altered by Aβ25−35 treatment. It would be crucial to determine if protection observed in exon 2 -/- is conferred by the increased abundance of these CLU proteins.
It is therefore crucial to determine the roles played by intracellular and secreted CLU proteins in Aβ25−35 induced toxicity to determine the exact roles played by CLU proteins in Aβ25−35 induced cell death and neurite retraction and the underlying mechanisms. Several studies have shown that CLU proteins bind directly to Aβ peptides (17,30) to regulate Aβ uptake and clearance by astrocytes (30). This role is attributed to secreted glycosylated CLU proteins. We hypothesise that in control neurons in response to Aβ25−35 treatment, secreted CLU is taken back into cells, potentially as a method of protecting cells. However, this role may facilitate toxicity if concentrations of Aβ25−35 peptides is high in the cellular media. sCLU proteins may bind to Aβ25−35 peptides and facilitate the uptake of CLU- Aβ25−35 complexes into neurons, thereby promoting the entrance of Aβ25−35 peptides into cells. The intracellular accumulation of Aβ25−35 peptides can then induce damage to neurons. Thus, in this context CLU is facilitating Aβ25−35 toxicity. Further interrogation of this relationship is required. Several receptors for CLU have been identified, including the megalin receptor (18,28), but this interaction is not well described and has not been analysed in iPSC-neurons. In exon 2 -/- neurons, the absence of secreted CLU may result in reduced Aβ25−35 uptake into neurons, resulting in a lower concentration of Aβ25−35 peptides accumulating in the neurons and thus, less cell damage. Since we described only partial protection in exon 2 -/- neurons, other mechanisms must also facilitate the uptake of Aβ25−35 peptides and Aβ25−35 toxicity in human neurons, in addition to CLU proteins.
Changes to the ECM were demonstrated in exon 2 -/- neurons and we postulate that ECM remodelling contributes to the partial protection observed in exon 2 -/- neurons to Aβ25−35 providing evidence for targeting the ECM as a therapeutic option for AD and neurodegeneration. The ECM is composed of a range of diverse proteins that serve a multitude of functions (53). Those include regulation of cell death and survival (54), albeit this is not clearly described in neurons, synaptic function (55,56) and neurite outgrowth (57,58), all three of which are significantly altered in AD and by Aβ25−35. ECM remodelling is associated with several diseases including AD (59–62). Numerous ECM proteins have been described as upregulated or downregulated in AD brains (62). For example, Collagen V and fibronectin are upregulated in the cerebral cortex of AD patients in early stages of disease progression and may arise from a reduction in proteolytic activity and may contribute to early changes in the AD brain (63). Several ECM proteins play a crucial role in regulating synaptic transmission and thus, changes to these proteins may result in a disruption in synaptic function and result in abnormal synaptic activity, and impaired learning and memory (64,65). ECM proteins have also been shown to alter Aβ toxicity. Heparan and chondroitin sulfate proteoglycans bind to Aβ peptides reducing Aβ25−35 induced toxicity in hippocampal cultures and PC12 cells (66,67). Indeed, reducing the expression of chondroitin sulfate proteoglycans enhances Aβ1−42 toxicity in neurons (68). Other ECM proteins have been shown to promote Aβ aggregation and plaque formation (69,70). Most recently, a large scale deep multi-layer analysis study of AD brains identified the ECM to strongly correlate with AD traits (71),
providing strength to our observations that the ECM may play a key role in mediating Aβ25−35 induced toxicity in human neurons. More work needs to be done to fully understand the role of the ECM in AD since both, protective and faciliatory roles have been identified. In our study it is apparent that the ECM directly contributes to toxicity induced by Aβ25−35 in vitro in human iPSC-neurons since ECM remodelling is associated with neuroprotective properties to iPSC-neurons. Given the varied functions of ECM proteins in neurons, it is essential to identify the key proteins that are central to the partial protection observed in exon 2 -/- neurons to determine if they can be utilised as a therapeutic target to provide neuroprotection.