Our compound screen has identified a pathway regulating autophagy that provides many possibilities for therapeutic intervention (). Previous studies have shown that the clearance of mutant huntingtin exon 1 (both EGFP-tagged or HA-tagged), mutant full-length huntingtin and A53T or A30P α-synuclein is largely mediated by autophagy8, 13, 14, 20
. Conversely, wild-type full-length or exon 1 huntingtin and wild-type α-synuclein have very low dependencies on autophagy for their clearance8, 14, 20
. Consistent with these expectations, a range or autophagy-inducing drugs have no obvious effects on wild-type huntingtin exon 1 or wild-type α-synuclein clearance10, 14
. Similarly, the drugs acting on the pathway we have identified in this study have no obvious effects on the clearance of wild-type huntingtin exon 1 (EGFP-HDQ23) or wild-type α-synuclein (Supplementary Figs. 2g, h
Our data suggest that raised cAMP levels act via Epac and PLC-ε to enhance calpain activity, which retards the clearance of autophagy substrates (Figs.
, , and Supplementary Figs.
3b, d, f, j-l
online). The Gsα
release pathway operates in neurons and neuronal cells32, 47
. Importantly, this pathway can also be inhibited in neuronal cells by Gi
stimulation (as would occur with clonidine)34
. Likewise, L-type Ca2+
channel agonists slow the clearance of mutant proteins by blocking autophagy through calpain activation (Figs.
, and Supplementary Figs. 2a-c, e, f
provides a link between these two pathways as it is activated following calpain cleavage leading to increased cAMP levels ( and Supplementary Figs. 6a, d, e
online). Interestingly, autophagosome synthesis can be induced and mutant huntingtin aggregation can be significantly inhibited by genetic knockdown of Gsα
( and Supplementary Fig. 6f
online). This is the first demonstration of the importance of the Epac pathway in autophagy regulation. Previous studies in yeast have focused on PKA and many of the mammalian studies that implicated PKA were performed before the Epac pathway was discovered48, 49
. The validity of the Epac pathway is consistent with the known activities of four additional compounds we identified in our screen that inhibit A53T α-synuclein clearance and enhance EGFP-HDQ74 aggregation: FPL61476 (30
) has Ca2+
channel agonist activity, quinine sulphate and tolazamide are ATP-sensitive K+
channel antagonists, and rolipram (31
) inhibits phophodiesterase 4 (leading to increased levels of cAMP) (Supplementary Figs. 2e, f
It is possible that changes in IP3
levels may have additional effects on autophagy. These are considered in the context of previous literature in the Supplementary Discussion
online, which also deals with issues of cell-type pathway specificity.
Our data provides a possible link between excitotoxicity, which results from increased Ca2+
entry into neurons via glutamate receptors, and enhanced intracytosolic protein aggregation due to impaired autophagy. Excitotoxicity is believed to contribute to a number of neurodegenerative diseases, including HD and Alzheimer’s disease (AD)6, 7
, and it is notable that the proteins that aggregate in many of these diseases are autophagy substrates1, 8, 15
. Calpain inhibition of autophagy may also contribute to the link between β-amyloid toxicity (which may elevate cytosolic Ca2+
levels) and tau accumulation in AD, as insoluble tau accumulates when autophagy is blocked15
. Finally, it is tempting to speculate that our data may also go some way to explaining why apoptosis and autophagy generally do not coexist, as many apoptotic (and necrotic) cell death processes are associated with, or stimulated by, raised cytosolic Ca2+
Calpain activation has been proposed to contribute to HD pathogenesis by promoting huntingtin cleavage - the toxicity of the HD mutation appears to be exposed or enhanced after cleavage4, 5
. As the huntingtin fragments we used are N-terminal to the calpain cleavage sites at residues 469 and 536 (Supplementary Fig. 1a
online), the cumulative data strongly argue that calpain inhibition will reduce mutant huntingtin fragment levels by impairing both cleavage (production) and by enhancing degradation (). In addition to these effects that result in lowered levels of the toxic protein, calpain inhibition may have other benefits in neurodegenerative diseases, including protection against cell death ().
Our data suggest that drugs acting on this mTOR-independent pathway may have added efficacy for neurodegenerative diseases in combination with rapamycin, providing a new direction for combinatorial treatment of disorders like HD by enhancing autophagy through two different routes ( and Supplementary Figs. 6i, j
online). Combination therapy with more moderate calpain and mTOR inhibition may be safer for long-term treatment compared to using higher doses of either compound that result in more severe inhibition of a single pathway. This strategy may allow a larger safety window before toxic effects from non-autophagy-related effects of each drug are seen.
The discovery that autophagy induction can be mediated by Ca2+
channel blockers is particularly exciting, as these drugs are safer and better tolerated than rapamycin - for instance, verapamil has been used for decades to treat hypertension with minimal side effects. Our findings may have broad applicability, as autophagy appears to regulate the clearance of a range of intra-cytosolic aggregate-prone proteins associated with neurodegeneration and various intracellular bacteria1