Background
α-Synuclein (aSyn) is a cytosolic and presynaptic protein strongly implicated in the pathogenesis of neurodegenerative disorders. Point mutations in the corresponding gene,
SNCA, as well as over-expression of the wild-type variant due to locus multiplication, cause autosomal-dominant forms of Parkinson disease (PD) [
1‐
3]. Furthermore, accumulation of aggregated, insoluble aSyn is a hallmark of many other neurodegenerative diseases, including sporadic PD, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), the Lewy body variant of Alzheimer's disease, and
PANK2-linked neurodegeneration. Collectively, these disorders are referred to as synucleinopathies [
4‐
7]. These observations from human studies and related insights from multiple vertebrate and invertebrate animal models of
SNCA over-expression (reviewed in: [
8]) demonstrate that both wild-type and mutant forms of aSyn can induce neurodegeneration [
9‐
13].
Given that aSyn inclusions are a pre-requisite feature of synucleinopathies, the processing of aSyn has been examined extensively in both
ex vivo and
in vivo models. These investigations have focused either on post-translational modifications of aSyn [
14] or on mechanisms of degradation. Initially, a key role had been postulated for the ubiquitin proteasome pathway (UPP) in the degradation of aSyn, because mutations in two UPP-related genes,
Parkin and
UchL-1 have been shown to influence PD risk [
15‐
18] and because molecular, cellular and animal studies linked these genes to UPP-dependent processing of aSyn [
19‐
21]. However, growing evidence has indicated that the lysosome, as well as the proteasome, can mediate degradation of aSyn [
22,
23]. In general, proteins are sequestered within lysosomes by one of three known methods,
i.e., macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) (reviewed in [
24]). Of these, it appears that aSyn can be a substrate for both macroautophagy and CMA [
25‐
28].
Regardless of the exact autophagic pathway by which aSyn enters the lysosome, it is assumed that it undergoes rapid degradation by a proteolytic enzyme (or enzyme complex, referred to as 'synucleinase/s'). Cathepsins are lysosomal proteases whose enzymatic activity is conferred by critical residues,
e.g., serine, cysteine or aspartic acid. Cathepsin D (CathD) is a major lysosomal aspartyl protease composed of two disulfide-linked polypeptide chains, both produced from a single protein precursor [
29]. Interestingly, CathD deficiency and its enzymatic inactivation in either humans, sheep, dogs or mice results in an early-onset, progressive and ultimately fatal neurodegeneration, which has been classified as one of several 'neuronal ceroid lipofuscinoses' (NCL) [
30‐
34].
In vitro experiments indicated that the treatment of recombinant aSyn with CathD resulted in partial aSyn proteolysis [
35]. Recently, Sevlever
et al. confirmed a proteolytic effect of CathD in in vitro digestion studies and extended their work to lysosomal fractions using human neuroblastoma cells over-expressing
SNCA cDNA [
36].
Here, we first examined the ability of CathD to regulate both wild-type and mutant aSyn in a dopaminergic cell culture system, and then examined the brains of several CathD-deficient mammals with NCL for evidence of misprocessing of endogenous aSyn. Finally, we tested the effects of ctsd expression on neuronal aSyn toxicity in a well-described Drosophila model of synucleinopathy. Our data indicate that ectopically expressed CathD enhances degradation of wild-type and mutant aSyn proteins, and that absence or deficiency of CathD promotes aSyn aggregation and toxicity in vivo.
Discussion
This report summarizes complementary investigations into the role of a key lysosomal enzyme, CathD, in the processing of aSyn. The in vivo results from three mammalian and one insect species described here provide additional insights into the pathogenetic effects of CathD deficiency, and identify in CathD a candidate for the much sought after 'synucleinase' activity in vivo.
Our initial set of experiments focused on a dopaminergic neuroblastoma cell culture model over-expressing aSyn together with CathD (Fig.
1).
CTSD expression reduced aSyn concentrations in a manner that was dependent on the level of newly synthesized CathD (Fig.
1A and
1B). This effect could not be explained by a loss of cell viability, as the integrity of our culture model was unchanged from untransfected and vector-transfected cells in two standard viability assays. Similarly, the effect of
CTSD cDNA could not be explained by a competition effect with the
SNCA plasmid for the protein synthesis machinery, because co-expression of aSyn with Cathepsin L (CathL) or Cathepsin E (CathE) under the same conditions did not cause a reduction of aSyn concentrations (data not shown).
The simplest explanation for the effects of
CTSD expression on aSyn is that CathD-mediated degradation of aSyn within the lysosome was enhanced (see alternative explanations below). For this explanation to be valid, aSyn must have entered the lysosome by either macroautophagy or CMA, or both, given that CathD is thought to be active only at low pH. Several investigators have shown that the Asp98 and Gln99 residues of aSyn facilitate recognition by the Hsc70 chaperone and subsequent binding of the aSyn/Hsc70 complex to the Lamp2a receptor during CMA [
26,
28,
52]. According to Cuervo
et al., Ala30Pro and Ala53Thr mutants of aSyn are able to bind to isolated lysosomal membranes but unable to translocate into the lysosomal lumen for degradation [
28]. The fact that in our cell culture system, the D98A/Q99A double mutant of aSyn, as well as the Ala30Pro, Glu46Lys and Ala53Thr mutants of aSyn were degradable by exogenous CathD (Fig.
1C) suggests that, at the collection time point of 24 hours, the cells did not entirely rely on CMA as a means of transporting aSyn into lysosomes, and that, rather, macroautophagy could be equally, or even more important than CMA [
26,
52]. Likewise, changing the phosphorylation state of human aSyn at Ser129 did not alter the proteolytic synucleinase activity exhibited by CathD in our MES23.5 cell model. Of note, only select neurons in sheep cingulate cortex deficient for lysosomal CathD activity (but not in mouse or human brain) revealed evidence for abnormal anti-Ser129-aSyn reactivity (Fig.
7 and data not shown). Although the observed species difference could be caused by a variety of factors, our cellular data argue that Ser129 phosphorylation of aSyn is not a universal pre-requisite for CathD-induced aSyn degradation.
Western blotting analyses of our MES23.5 cell lysates demonstrated that the CathD-induced reduction in aSyn occurred without the concomitant appearance of either low-molecular weight (LMW) or HMW species, as examined 24 hours after transfection. Of note, we had previously confirmed by Western blotting that our Abs are capable of recognizing truncated aSyn species, (
i.e., 10 to 12.5 kDa; see Additional file
2, Fig. S2) [
46]. In particular, the appearance of LMW species of aSyn would have indicated incomplete proteolysis of aSyn, potentially at the C-terminus. Such LMW aSyn-ΔC species are prone to aggregate formation and have an enhancing effect on aSyn-mediated toxicity [
46,
53‐
55]. Previous studies by Dufty
et al. [
56] showed that calpain-mediated digestion of aSyn could induce the formation of aSyn-ΔC species and formation of HMW species. Recently, Sevlever
et al. confirmed a proteolytic effect of both enzymes, calpain and CathD, in
in vitro biochemical studies and extended their work to purified lysosomes from
SNCA-expressing neuroblastoma cells [
36].
In our
SNCA and
CTSD over-expression model, we observed a maximum effect of CathD-induced reduction of excess aSyn levels that plateaued at ≤ 80 per cent of vector-transfected cells (Fig.
1B). We concluded from these findings that a distinct pool of aSyn species (e.g., synaptic vesicle-associated) is not amenable to CathD-mediated aSyn degradation. To explore whether over-expression of wt
CTSD also reduces the concentration of endogenous aSyn, we carried out additional Western blotting and ELISA experiments in rodent MES23.5 and human SY5Y neuroblastoma cells. In the background of normal lysosomal function and normal aSyn levels, we saw no consistent, further reduction of endogenous aSyn levels by ectopic CathD expression (not shown).
To examine the relevance of endogenous CathD expression on endogenous aSyn processing
in vivo, we next examined brain tissue from homozygous
ctsd-/- mice [
41]. We detected a consistent decrease in soluble aSyn proteins in homozygous CathD deficient mouse brains
versus those of wt animals using Western blotting and ELISA (Figs.
2 and
3). This result was verified by quantitative 2D gel electrophoresis, which showed a significant reduction of the 16 kDa aSyn spot in
ctsd-/- mice (data not shown). Our quantitative ELISA showed a comparable decrease in the amount of soluble aSyn in NP-40 extracts (18.6 per cent) and in SDS-extracts (20.0 per cent) of
ctsd-/- mice compared with controls (Fig.
3). This observation was in agreement with our immunohistochemistry results from the same mice that exhibited some reduction in aSyn staining in the neuropil of
ctsd-/- mice, particularly within the thalamus (Fig.
5). Previously, we have confirmed that CathD deficient mice show a decrease in pre-synaptic markers in the ventral posterior nucleus of the thalamus, and a > 30 per cent loss of neurons in its corresponding target somatosensory cortex [
34]. Because aSyn is a predominantly pre-synaptic protein, we surmised that the reduction of soluble aSyn concentration recorded in our
ctsd-/- mice resulted – in part – from pre-synaptic abnormalities.
However, in parallel to the reduction of soluble aSyn throughout the neuropil, we detected aSyn accumulation-carrying neurons within many brain regions of 24-day old
ctsd-/- mice. This finding was substantiated by our observation of increased levels of insoluble species of oligomeric aSyn in the formic acid extracts of
ctsd-/- mice (15%; Fig.
4). (Of note, our current ELISA protocol is incompatible with the quantification of aSyn in the presence of formic acid). We concluded from these collective findings that the presence of murine CathD is important for the prevention of aSyn misprocessing and that its absence facilitates the formation of aSyn aggregation
in vivo. In accordance, we speculate that the neuronal accumulation of murine aSyn in
ctsd-/- mice may represent early precursor lesions to classical Lewy inclusions.
We next examined the role of mutant CathD protein expression on sheep aSyn levels (Fig.
6). Homozygous missense mutation in the ovine
CTSD gene (Gln934Ala) essentially abolishes the enzyme's proteolytic activity (residual level, < 5 per cent), while heterozygotes retain 40 per cent activity and are phenotypically normal [
50]. The affected newborn lambs are weak, tremble and are unable to stand. When sacrificed shortly after birth, their brains are smaller and display mild cortical thinning, demyelination, neuronal loss, reactive astrocytosis and macrophage infiltration, while visceral organs are spared. Classical NCL-type changes and widespread ubiquitin-positive inclusions within neurons were readily detected in the homozygous animals (Fig.
6). In contrast to the more extensive neuropil disruption and synucleinopathy seen in
ctsd-/- mouse brain, we detected more select neuronal changes in homozygous CathD-mutant sheep, with abnormal anti-aSyn staining in the cingulate cortex and in the axons of deep white matter tracts. Intriguingly, synucleinopathy of the cingulate cortex is a hallmark of human dementia with Lewy bodies and advanced PD cases [
6]. Unfortunately, no frozen material was available to determine whether insoluble, HMW oligomeric species of ovine aSyn can be found biochemically in the cingulate cortex.
Humans with congenital NCL are affected at birth and survive only for days. Two mutations in the human
CTSD gene have been described to underlie a subset of congenital NCL cases [
30,
57]. Both mutations inactivate the CathD enzyme, in that the insertion mutation, c.764dupA, results in a premature stop-codon and rapid degradation of the protein, while the c.299C > T mutation leads to the production of an inactive but stable protein
in vivo. The brains of these infants show extreme cortical atrophy, loss of neurons and myelin, as well as generalized gliosis [
30,
51,
57]. In brain specimens of two affected siblings from a NCL pedigree linked to
CTSD and of one unrelated patient with congenital NCL, anti-aSyn staining of the neuropil was strongly reduced throughout the brain (Fig.
7). Accumulation of aSyn was particularly pronounced in the thalamus and basal ganglia, where the neuronal soma appeared to be loaded with aSyn aggregates, thereby morphologically resembling Lewy bodies of the forebrain; however, in contrast to classical Lewy inclusions these lesions were negative for ubiquitin and phospho-Ser129-specific reactivity in the affected newborns (not shown). Throughout the brains from these three infants, smaller aSyn-positive grains appeared to localize to neurites (Fig.
7B and
7C). Therefore, although the degree of brain atrophy and neuronal loss was much more severe in human infants with CathD deficiency than in
ctsd-/- mice, the aSyn-reactive histopathologies were very similar between the two species, possibly because of the complete loss of CathD protein expression in both.
Regarding the topographic distribution of aSyn accumulation within the three species of CathD-deficient mammals, it is interesting to note that the latter is markedly different from that of lipofuscin deposition in NCL diseases. These autofluorescent accumulations are generally widespread within cortical neurons in humans and sheep affected by CathD deficiency and in the thalamus of ctsd-/- mice, while the observed aSyn accumulations occurred predominantly in the thalamus and basal ganglia in humans, in the cingulate cortex of affected sheep, and in the cerebral cortex of ctsd-/- mice.
Finally, our experiments using the established
Drosophila model of PD provided evidence for a modulatory effect of endogenous CathD expression on aSyn toxicity
in vivo (Fig.
8). Deletion of the homologous CathD-encoding gene promoted toxicity of ectopically expressed human aSyn in the fly retina; these findings suggest that differences in the expression level of CathD (and by inference, of other synucleinase-conferring proteases) may lower the threshold for synucleinopathy in other brain regions and species, too.
Importantly, our data do not demonstrate a direct interaction between CathD as an enzyme and aSyn as its substrate. Although such a relationship was suggested by
in vitro experiments in one study [
35], purification of lysosomes from co-transfected MES23.5 cells and detailed imaging analyses will be required to further test this hypothesis. Intriguingly, aSyn transgenic mice show a decrease in aSyn inclusions when immunized with recombinant aSyn, and this decrease was associated with a concomitant increase in CathD levels as well as co-localization of CathD and aSyn [
58]. Stefanis
et al. [
59] also noted co-localization of a pool of aSyn with CathD in their PC12 cell line-based experiments.
Indirect effects on aSyn accumulation by CathD deficiency are also possible. For example,
CTSD gene expression could activate another cathepsin or non-cathepsin protease, and this second enzyme may process aSyn for degradation. In this regard, it is interesting to note that CathD possesses activity that is similar to pepsin, and that pepsin can proteolytically activate Cathepsin F (CathF; [
60]); intriguingly, in our cell culture system, over-expression of CathF was able to reduce human aSyn levels, too, with an efficiency similar to that of CathD, whereas over-expression of CathE and CathL was ineffective (V.C., M.G.S., unpublished observations). It is noteworthy that mice deficient in CathF develop a late-onset neurological phenotype with gait disturbance, hind leg weakness and tremors, which is associated with brain and spinal cord pathology [
61]; it will be interesting to discern whether these mice also display biochemical and/or immunohistochemical evidence of synucleinopathy.
Another possible mechanism for interaction between the
CTSD and
SNCA genes
in vivo is a change in lysosomal constituency downstream of CathD protein expression. Perturbations in the activities of several lysosomal sphingolipid-metabolising enzymes have been documented in CathD-mutant sheep [
31] and dogs [
32], while
ctsd-/- mice accumulate phospho- and glyco-sphingolipids, including gangliosides [
62]. Since aSyn is a lipid- and ganglioside-binding protein [
63‐
65], and since lipids can regulate the oligomerization of aSyn [
45,
66], it is plausible that dysregulated lipid metabolism is a mechanism by which CathD deficiency leads to aSyn misprocessing. CathD is also known to exhibit activities that are unrelated to its enzymatic function [
67‐
69]. It is therefore possible that non-proteolytic effects of CathD are essential in aSyn processing, akin to its effects on the ras-MAPK pathway [
70]. Our data on aSyn processing in the cell soma and neurites of mutant sheep brain, where CathD is over-expressed but enzymatically disabled, would rather argue against this theory. However, one cannot exclude with certainty that the homozygous mutation in CathD did not also compromise its non-enzymatic functions, although computational modeling did not predict the induction of a conformational change in the sheep mutant (J.T., data not shown).
Finally, our collective findings reported herein are in remarkable agreement with many (but not all) of the findings in the related report by Qiao and coworkers [
71], who independently discovered CathD as a critical enzyme in the metabolism of neuronal aSyn using both
in vitro and
in vivo experiments. Of note, Qiao
et al. examined offspring of the same CathD-deficient mouse model that we employed [
41], and observed a reduction of truncated aSyn species in
ctsd-null mice, which we did not detect in our protocol. In addition to possible technical and Ab-related differences between the two reports, the slightly younger age of our animals at the time of euthanasia (post-natal day 24
versus 25) may also have contributed to this discrepancy.