Autophagy Modulation as a Treatment of Amyloid Diseases

Abstract

Amyloids are fibrous proteins aggregated into toxic forms that are implicated in several chronic disorders. More than 30 diseases show deposition of fibrous amyloid proteins associated with cell loss and degeneration in the affected tissues. Evidence demonstrates that amyloid diseases result from protein aggregation or impaired amyloid clearance, but the connection between amyloid accumulation and tissue degeneration is not clear. Common examples of amyloid diseases are Alzheimer's disease (AD), Parkinson's disease (PD) and tauopathies, which are the most common forms of neurodegenerative diseases, as well as polyglutamine disorders and certain peripheral metabolic diseases. In these diseases, increased accumulation of toxic amyloid proteins is suspected to be one of the main causative factors in the disease pathogenesis. It is therefore important to more clearly understand how these toxic amyloid proteins accumulate as this will aide in the development of more effective preventive and therapeutic strategies. Protein homeostasis, or proteostasis, is maintained by multiple cellular pathways-including protein synthesis, quality control, and clearance-which are collectively responsible for preventing protein misfolding or aggregation. Modulating protein degradation is a very complex but attractive treatment strategy used to remove amyloid and improve cell survival. This review will focus on autophagy, an important clearance pathway of amyloid proteins, and strategies for using it as a potential therapeutic target for amyloid diseases. The physiological role of autophagy in cells, pathways for its modulation, its connection with apoptosis, cell models and caveats in developing autophagy as a treatment and as a biomarker is discussed.

Keywords: Alzheimer’s disease; Huntington’s disease; Parkinson’s disease; Tau protein; amyloid; autophagy; beta amyloid; clearance; lysosome; polyglutamine; toxicity; α-synuclein.

Figure 1

Regulation of autophagy mediated degradation: Autophagosome formation begins when phagophore is produced from ER initiated by either AMPK activation or mTOR inhibition by nutrient starvation. Once autophagosome formation is initiated, the ULK1 complex (composed of Atg13, Ulk2, Atg101 and Fip200) is phosphorylated. Vps4 complex is positively regulated and the developing phagophore elongates. Beclin-1 together with Ambra-1, Bif-1 and UVRAG activate Vps34, generates P13P—which then facilitates Atg16L1-Atg5-Atg12 recruitment. Beclin-1 mediated autophagosome generation can be inhibited by Bcl-2. P13P binds with WIPI1 and WIP2 and promotes phagophore elongation. LC3-I is lipidated to form LC3-II. LC3 additionally anchors adaptor proteins p62 and NBR-1 onto the phagophore for substrate targeting, and phagophore closes to become an autophagosome. Fusion of autophagosome and lysosome create the autolysosome, which digests the substrates and recycles back into the cytoplasm.

Figure 2

Clearance of APP-C99 and Aβ in an AD neuronal cell model. Clearance of APP-C99 and Aβ was assessed in MC65, a human derived cell line that produces Aβ from a stably transfected precursor protein APP fragment C99. MC65 cells were grown without tetracycline for two days to activate APP-C99 and Aβ production, followed by spiking with tetracycline to block APP-C99/Aβ production. After spiking with tetracycline (1 µg/mL), cell lysates were collected at 6 and 24 h to measure levels of Aβ (B), APP-C99 (C), LC3I (D) LC3II (E), Tau protein (F), and phosphorylated Tau protein (ser199) (G) by western immunoblotting analysis. Representative image of the Aβ clearance assay is shown here (A). APP-C99 and Aβ levels were ~50% lower at 24 h in cells spiked with tetracycline compared to tetracycline depleted cells (** p < 0.001, n = 4), suggesting clearance. Aβ producing MC65 cells (tetracycline depleted) showed increased accumulation of Tau protein, phosphorylated Tau protein (ser199) at 6 h and autophagy markers LC3I and LC3II (* p < 0.005, n = 4) at both 6 and 24 h, suggesting increased autophagosome synthesis and autophagy activation.