Lysosomes are the primary degradative components responsible for clearing intracellular waste products and damaged proteins, their proper activity is vital for the well-being of the cell.1
The enzymatic degradation that occurs in lysosomes is highly dependent on the lysosomal acidic pH, which is maintained by the vacuolar ATPase (v-ATPase) proton pumps. Impaired lysosomal activity was initially observed in heredity lysosome storage diseases, and recent studies have demonstrated a tight link between lysosomes and neurodegenerative diseases. Of particular interest is the role of lysosomes in Alzheimer’s disease (AD): A decline in lysosomal activity is observed in aging brains, and defects in lysosomal acidification are associated with typical AD pathology of fibrillogenic β amyloid (Aβ) deposits.2-5
Accumulation of Aβ plaques is a key hallmark in AD pathogenesis. The 40- or 42-residue Aβ peptides that make up the plaques are generated by sequential proteolysis of the amyloid precursor protein (APP) by β-secretase, βeta-site APP Cleaving Enzyme 1 (BACE1), and presenilin-dependent γ-secretase.6,7
Reducing the accumulation of Aβ deposition is thus believed to be a useful therapeutic strategy. Disruption in lysosomal acidification resulted in enhanced Aβ pathology and reduced cognitive ability in AD mouse models,2-5
giving rise to the hypothesis that restoring lysosomal acidity reverse AD symptoms.
Glycogen synthase kinase-3 (GSK-3) is an evolutionary conserved serine/threonine kinase expressed as two isozymes, GSK-3α and GSK-3β. GSK-3 is emerging an important drug target in AD therapy. Excessive phosphorylation of GSK-3 targets such as the microtubule-associated protein tau, collapsin response mediator proteins (CRMPs) and β-catenin is implicated in mechanisms contributing to AD pathogenesis.8-10
Indeed, treatment with GSK-3 inhibitors reverses AD symptoms in various animal models.11
An initial study connected GSK-3α isozyme with Aβ production via enhanced γ-secretase-mediated APP proteolysis.12
To gain further insights into the role of GSK-3 in Aβ pathology, we used the “5XFAD” mouse model. These mice co-express a total of five familial AD mutations in APP and presenilin-1 (PS1) and develop massive cerebral Aβ loads.13
We treated these mice nasally with L803-mts, a selective, substrate-competitive GSK-3 inhibitor developed in our laboratory. We found that treatment with L803-mts reduces Aβ pathology and ameliorates cognitive deficits.14
We also showed that L803-mts restores the lysosomal acidification that was severely impaired in the brains of the 5XFAD mice.14
This effect was independent of autophagy indicating that lysosomes play a major role in the catabolic disposal of Aβ loads under these conditions.
Recent studies implicated PS1 in controlling lysosomal acidification.15
We asked whether inhibition of GSK-3 can “repair” lysosomal malfunction caused by dysfunctional PS1. We treated MEF cells deficient in presenilin proteins (MEF-PS1/2−/−
with L803-mts. After the treatment cells were stained with LysoTracker Red, a dye that accumulates in acidified organelles, and imaged by confocal microscopy. L803-mts increased the number of acidified lysosomes and intensity of staining as compared with control untreated cells (). We next examined the levels of Cathepsin D (CatD), a principle lysosomal protease that is activated in the acidified lysosomal environment. Immunofluorescence analysis with anti-CatD antibody showed a low level, diffuse signal in the untreated cells. In contrast, L803-mts increased CatD signal (). To examine whether CatD was more active in L803-mts treated cells, cells were stained with pepstatin A BODIPY, which binds specifically to the active form of CatD. The BODIPY signal was enhanced by L803-mts, confirming that L803-mts restored lysosomal acidification in these cells. We conclude that GSK-3 and PS1 likely operate via similar mechanisms that impair lysosomal acidification, perhaps through disrupted glycosylation of v-ATPase V0a1 subunit; this glycosylation is critically important for v-ATPase assembly in the lysosome membrane.15
Another important conclusion from this study is that GSK-3 inhibition should provide benefit in treating conditions associated with defective PS1.
Figure 1. Inhibition of GSK-3 restores impaired lysosomal acidification caused by disrupted PS proteins. MEF-PS1/2−/− cells were treated with L803-mts (40 μM, 6 h) and screened by the following lysosomal markers: live-cell (more ...)
Additional work that was published in parallel to our publication demonstrated the role of GSK-3 in regulating Aβ pathology, but suggested different mechanisms that involved either reduction in β-site APP cleaving enzyme-1, BACE1, expression17
or enhancement in APP processing via lysosome biogenesis.18
Altogether, GSK-3 is clearly a prominent factor that contributes to accumulation of Aβ loads in the AD brain. The mechanisms involved are likely dependent on the cellular context including the levels and/or activities of additional factors that contribute to Aβ pathology such as APP, PS1 and lysosomes.