Introduction
Protein misfolding, aggregation and deposition are unifying features of a wide range of neurodegenerative diseases [
1]. The ability of neurons to manage the burden of misfolded proteins and to resist their accumulation into insoluble protein deposits depends critically on the functioning of molecular chaperones. Previous studies have demonstrated that elevation of chaperone levels can protect against neurotoxicity resulting from the effects of pathological protein misfolding in cell culture and in transgenic animal models [
2,
3]. Most chaperones are localised within intracellular compartments, although some are secreted into the extracellular environment. Prominent amongst such extracellular chaperones is clusterin (CLU), which is present in both plasma and cerebrospinal fluid (CSF). CLU is a cytoprotective chaperone whose expression level is increased in response to a diverse range of stresses including heat, pro-apoptotic insults, oxidative stress, ionising radiation, and proteotoxicity [
4‐
6]. It has been linked to a correspondingly diverse group of clinical disorders associated with protein misfolding including Alzheimer’s disease (AD) [
7], amyloidotic cardiomyopathy [
8] and familial amyloidotic polyneuropathy [
9]. CLU binds promiscuously to a wide range of misfolded client proteins and either sequesters them into stable, soluble complexes (in the case of proteins forming amorphous aggregates) or inhibits the formation and accumulation of toxic amyloid assemblies [
10,
11]. Clusterin is a particularly potent chaperone and can inhibit protein aggregation at molar ratios of chaperone:client protein that are significantly lower than those needed by other chaperones [
12,
13]. Extracellular CLU-client protein complexes are susceptible to endocytic clearance by monocytes and hepatocytes, and this action is thought to form a central plank in the operation of a co-ordinated system to maintain extracellular protein homeostasis (proteostasis) [
14].
Whilst CLU is predominantly a secreted protein, multiple independent studies have now clearly established that during conditions of ER stress, CLU secretion is inhibited, and full-length CLU exits the secretory system to enter the cytosol. Firstly, the translocation of mature CLU from the ER/Golgi into the cytosol of human astrocytoma U251 cells in response to ER stress was shown by two complementary biochemical and immunofluorescence approaches [
15]. Subsequently it was demonstrated that the ER-resident chaperone BiP (GRP78) facilitates the translocation of CLU to the cytosol during ER stress in human prostate cancer LNCaP cells [
16]. In addition, it was recently shown that during ER stress in PC3 and LNCaP cells, cytoplasmic CLU interacts with LC3 to promote autophagy [
17]. While the mechanism by which CLU translocates from the ER lumen to the cytosol in response to stress is yet to be elucidated, it appears that the release may form a part of a larger strategy in which many different chaperones are released from the ER to the cytosol to defend the cell against intracellular stresses. This has been suggested by the demonstration that, in response to cytosolic foreign DNA or inhibition of the proteasome, BiP is released to the cytosol [
18]. Other earlier studies have also suggested that CLU might be involved in stress responses within the cell. For example, CLU accumulates in cells when either lysosomal degradation or the proteasome are specifically inhibited [
19], and CLU is itself ubiquitinated during ER stress (a process that can only occur in the cytosol) [
15]. Furthermore, other studies have suggested that CLU promotes ubiquitination and proteosomal degradation of the cytosolic proteins, COMMD1 and IkB [
20] and the lysosome-mediated degradation of the cell-surface copper transport proteins, ATP7A and ATP7B [
21,
22]. Collectively, the previous literature points to a potential involvement of CLU, especially during ER stress, in facilitating the degradation of specific cell proteins via both of the primary cell degradative pathways, the proteasome and autophagy.
The pathologies of many neurodegenerative diseases are known to involve both ER stress and cytotoxic protein aggregation [
23]. Consequently, we examined whether increased expression of the unusually potent chaperone CLU could provide protection against intraneuronal protein aggregation and proteotoxicity, especially under conditions of ER stress. As a primary model, we studied the aggregation of the 43 kDa transactive response DNA-binding protein (TDP-43), which is implicated in the pathology of amyotrophic lateral sclerosis (ALS), where it translocates from its normal location in the nucleus and forms insoluble inclusions in the cytoplasm. We investigated the ability of CLU to influence the aggregation and clearance of TDP-43 in vitro, in neuronal cells and in transgenic
Drosophila melanogaster. The results indicate that CLU can interact with TDP-43 and potently inhibit its aggregation, and that in a neuronal cell model undergoing ER stress, CLU reduces the numbers of cytoplasmic TDP-43 inclusions. We further show in transgenic
Drosophila that co-expression of CLU reduces cytoplasmic mislocalization of TDP-43 in motor neurons, protects against loss of locomotor activity and significantly extends lifespan. Lastly, we showed that in
Drosophila photoreceptor cells, CLU gave ER stress-dependent protection against proteotoxicity arising from both Huntingtin-Q128 and mutant (R406W) human tau.
Discussion
Although the precise pathway of release remains to be determined, multiple studies now strongly suggest that ER stress triggers the release of post-translationally modified CLU from the ER to the cytoplasm [
15‐
17] (see also Additional file
1: Figure S1). This may well explain the many previous reports that, under a variety of disease conditions, CLU occurs in cellular locations outside the secretory system, including the co-localization of CLU with intracellular tau tangles in Alzheimer’s disease [
44], α-synuclein rich Lewy bodies in Parkinson’s disease [
45], and inclusion bodies in myofibrillar myopathies [
46]. It has also been reported that in response to chemotherapeutic drug treatment, CLU relocates to mitochondria and interacts with Bax to inhibit apoptosis [
47]. There is abundant evidence from transgenic mouse models of ALS, neuropathological post mortem studies, and genetic studies that ER stress is a significant component in ALS pathology [
48,
49]. While certainly not in themselves definitive, the results of our own immunohistochemical analyses of ALS patient spinal cord tissues are consistent with CLU also being released from the ER in ALS affected neurons (Additional file
1: Figure S2).
Furthermore, the inappropriate mislocalization and accumulation of TDP-43 in cytoplasmic inclusions is a unifying pathology in frontotemporal dementia (FTD) and familial and sporadic ALS cases. It is seen, with the exception of cases resulting from SOD1 mutations [
50], in most post-mortem cases of FTD and ALS [
51], and correlates well with synaptic pathology and cognitive deficits [
52]. Thus, a therapeutic strategy that protects against TDP-43 proteotoxicity could provide benefits in most cases of ALS. Therefore, in the present study we used a variety of complementary approaches, including in vitro, cell and whole animal models, to evaluate whether the unusually potent and multifunctional chaperone CLU can affect the aggregation, cytoplasmic mislocalization and proteotoxicity of TDP-43.
We showed using both a C-terminal peptide corresponding to the most aggregation-prone region of TDP-43, and full length in vitro translated TDP-43-tGFP, that CLU potently inhibits the in vitro aggregation of TDP-43 and maintains it in a soluble form (Fig.
2). These results strongly suggest that CLU can directly interact with TDP-43, as there is no known mechanism by which a chaperone can inhibit protein aggregation without directly interacting with the client protein. The ability of CLU to inhibit the aggregation of the TDP-43
286-331 peptide even at molar ratios of CLU:peptide as low as 1:1000 could arise from the fact that only a very small fraction of the soluble peptide molecules may be in a misfolded conformation at any one time, enabling CLU to interact specifically with these species to stabilize them and prevent them acting as aggregation nuclei [
10]. We also showed using three different transfected cell models (N2a, SH-SY5Y and U251), that during ER stress, but not otherwise, CLU co-localized with cytoplasmic TDP-43-GFP inclusions (Fig.
1, Additional file
1: Figure S3). The treatments used to induce ER stress in the cell models (MG132, Tg and A23187) have been widely used in other studies [
15‐
18] and we confirmed that even an 18 h exposure of N2a cells to 10 uM MG132 had negligible effects on cell viability (Additional file
1: Figure S4). In N2a cells, we showed that over-expression of CLU specifically and significantly reduced the numbers of inclusions within these cells (Fig.
2c). Co-immunoprecipitation analyses demonstrated that CLU can physically associate with TDP-43
CTF-GFP in N2a cell lysates (Fig.
1b), suggesting that once in the cytosol, CLU would be capable of forming stable complexes with TDP-43.
In transgenic
Drosophila we showed that overexpression of TDP-43 induced both ER stress and the retention of CLU within both photoreceptor and motor neurons (Fig.
3). Under these conditions, CLU was shown to co-localize with TDP-43 first in the cytoplasm and then, at later stages in development, in the nuclei of adult
Drosophila motor neurons, effectively restoring the normal nuclear distribution of TDP-43 (Fig.
4). In this whole animal model, the effects of enforced CLU expression were to generate a dramatic recovery of the loss of locomotor activity associated with TDP-43 proteotoxicity, and a significant extension of lifespan (Fig.
5). These striking effects are specific to CLU expression as we showed that the co-expression of GFP in TDP-43-expressing
Drosophila did not alter either the level of TDP-43 expression or the median survival (Additional file
1: Figure S7). Finally, we showed that enforced CLU expression could also protect against the proteotoxicity of Htt-Q72 and R406W tau expressed in
Drosophila photoreceptor neurons, but only when ER stress was chemically induced (Fig.
6).
In both the neuronal cell and
Drosophila models studied here, ER stress was a consistent requirement for CLU to provide protection against protein mislocalization and toxicity. We cannot exclude the suggestion that over-expression of CLU might act to reduce the levels of experimentally induced ER stress, and this might account in part for the protective effect of CLU expression. However, even if this were correct, our results in transgenic
Drosophila strongly suggest that this is not the only mechanism by which CLU over-expression can protect against the cytoplasmic accumulation of toxic proteins. Notably, the level of cytoplasmic accumulation/toxicity of Htt-Q72/R406W tau expressed in cells of the
Drosophila eye is unaffected by either DTT treatment alone or CLU co-expression alone, but is significantly reduced only when the two conditions are combined (Fig.
6). Furthermore, in transfected N2a cells, we only measure the release of CLU to the cytosol following treatments to induce ER stress, again strongly suggesting that CLU over-expression does not ablate the induction of ER stress. All our findings are consistent with a model in which ER stress induces release of CLU to the cytosol where it exerts effects that act to reduce net TDP-43 accumulation. There are a several putative mechanisms by which cytosolic CLU could exert the observed effects on TDP-43 mislocalization, aggregation, toxicity and clearance. These include (i) the well characterized chaperone activity of CLU [
11,
12,
53] allowing it to directly interact with TDP-43 to form stable, soluble complexes, thereby inhibiting its further aggregation to form cytoplasmic inclusions, (ii) CLU may interact with misfolded species of TDP-43 to neutralize their toxicity, as is the case in its interactions with other toxic protein oligomers [
12,
54,
55], and (iii) CLU, which is ubiquitinated once in the cytosol [
15], may direct soluble CLU-TDP-43 complexes towards the proteasome or autophagy for degradation. Both the proteasome and autophagy are known to act to clear TDP-43 inclusions [
56] and there is a growing body of evidence implicating CLU as a substrate of both these major cellular degradative pathways and a control element of autophagy [
15,
19‐
22]. It is also interesting to note in this context that Cha-Molstad et al. have recently reported that in response to MG132-mediated inhibition of the proteasome, or cytosolic foreign DNA, the ER-resident chaperone BiP is released to the cytosol [
18]. We speculate therefore that the release of CLU to the cytosol might form a part of a larger strategy in which multiple chaperones are released from the ER to the cytosol to defend the cell against intracellular stresses.
An important aspect of motor neurons is that they have a very limited heat shock response owing to their impaired ability to activate the key molecular heat shock response molecule, HSF1 [
57]. This results in a high threshold for induction of the heat shock response in motor neurons conferring an element of vulnerability. The results presented in this study provide a clear demonstration in a whole organism model that during ALS disease-relevant conditions the enforced expression of CLU provides significant protection against the TDP-43-mediated proteotoxicity underpinning pathology, to substantially enhance motor neuron survival, reduce locomotor deficits and extend lifespan. We have shown previously that constitutively intracellular chaperones are able to rescue TDP-43 associated neurotoxicity [
14], however this is the first instance, to our knowledge, where a normally secreted chaperone can exert similar protective effects.
In protein misfolding diseases such as ALS and Alzheimer’s disease (AD) there are multiple proteins misfolding in both the intra- and extracellular environments. For example, pathological, extracellular amyloid beta accumulation is often seen in post-mortem ALS and AD cases exhibiting intracellular accumulation of TDP-43 or tau [
58]. It appears feasible that CLU can combat protein misfolding in both the intra- and extracellular compartments and may alter its subcellular localization dependent upon the environmental stress. Previous studies attempting to harness the therapeutic benefits of chaperones in protein misfolding diseases have used Hsp90 inhibitors, such as 17-AAG, which can upregulate small heat shock proteins in the intracellular compartment. 17-AAG can rescue the neurotoxicity associated with the pathological accumulation of TDP-43, polyglutamine, alpha synuclein, mutant androgen receptor, tau and amyloid beta [
59‐
63]. However, 17-AAG also upregulates CLU expression [
64], potentially contributing to the neuroprotective effects afforded by the therapeutic intervention in these models. Apart from ALS, many other neurodegenerative diseases including Alzheimer’s, Parkinson’s and Huntington’s diseases, also feature ER stress associated with pathology [
23]. Therefore, it is conceivable that further studies investigating temporal changes in CLU levels and localization associated with pathologies could reveal new avenues for developing therapeutic strategies to address currently incurable and highly devastating diseases.