Most adult-onset neurodegenerative diseases are characterized by the formation of protein inclusions inside neurons. Frequently, these inclusions or aggregates are intracytoplasmic, as is the case in Parkinson’s disease, Alzheimer’s disease (which also has extracellular aggregates) and Huntington’s disease. When these types of conditions are caused by Mendelian mutations, the mutant protein causes disease typically via a toxic gain of function, which is associated with its propensity to aggregate42
. Hence, strategies that can limit the toxic load by enhancing the removal of such proteins may have therapeutic potential.
Most neurodegenerative disease-associated proteins that form intracytoplasmic aggregates are autophagy substrates43–45
. Inhibition of autophagosome formation or autophagosome–lysosome fusion slows the clearance of both full-length mutant huntingtin (the protein that causes Huntington’s disease) and its more toxic aminoterminal fragments, whereas the wild-type counterparts of these species are still efficiently cleared46
. The clearance of these proteins is delayed when autophagy is impaired and, most importantly, their turnover is enhanced when autophagy is upregulated. Indeed, autophagy upregulation decreases the toxic accumulation of different mutant proteins, such as mutant huntingtin (causing Huntington’s disease), mutant ataxin 3 (causing spinocerebellar ataxia type 3) and mutant α-synuclein (causing Parkinson’s disease), and has beneficial effects on disease-associated phenotypes in cell culture as well as Drosophila melanogaster
, zebrafish and mouse models43–45,47–50
. In addition to facilitating the removal of toxic proteins, autophagy upregulation may reduce neuronal susceptibilities to caspase activation and apoptosis51
Conversely, conditional knockouts of key autophagy genes in mouse neurons lead to aggregate formation and neurotoxicity52,53
. Furthermore, defective autophagy results in the accumulation of mitochondria with an abnormal membrane potential, which are more likely to release pro-apoptotic molecules, as this population of mitochondria is believed to be selectively targeted by mitophagy54
. Autophagy may be impaired in neurodegenerative diseases. For instance, autophagy may be impaired at both the levels of autophagosome degradation55
and autophagosome formation56
in Alzheimer’s disease, although these effects may vary according to the relevant genotype of the patients or the stage of the disease.
The accumulation of α-synuclein is a hallmark of Parkinson’s disease, and an excess of this protein is sufficient to cause this condition. Interestingly, excess α-synuclein impairs autophagosome biogenesis in cell cultures and in vivo57
, whereas increased autophagy is observed when levels of this protein are reduced58
. Mutations in PTEN-induced putative kinase 1 (PINK1) and the E3 ubiquitin ligase parkin (also known as PARK2) cause recessive forms of Parkinson’s disease. Recent data suggest that these proteins work together to mediate the clearance of dysfunctional mitochondria via mitophagy59
. As disease-associated mutations have impaired mitophagy-related activity, this supports previous assertions that mitochondrial dysfunction may contribute to these forms of Parkinson’s disease. However, the relevance of mitophagy to the common sporadic forms of Parkinson’s disease is still unclear. Furthermore, impaired autophagy is implicated in a rare degenerative form of epilepsy caused by mutations in laforin, called Lafora epilepsy60
, as well as in forms of motor neuron disease caused by mutations in dynactin61,62
. It is thus likely that neurodegenerative diseases associated with mutations that impair autophagy will have an increased propensity towards aggregate formation and cellular toxicity, and that some of the neuronal stress may be due to defective mitophagy.
Therapeutic strategies and challenges
Autophagy defects can occur at different stages of the pathway in different diseases, and this may influence treatment strategies. Defects in autophagosome formation may be amenable to drugs that enhance autophagosome biogenesis. For example, laforin mutations (causing Lafora epilepsy) impair autophagosome formation by enhancing mTOR activity; mTOR inhibitors (for example, rapamycin) may therefore be beneficial in this context60
. Similarly, nitric oxide induction — a frequent occurrence in neurodegenerative diseases — blocks autophagosome formation and this effect can be reversed in Huntington’s disease models by N
-arginine methyl ester (l
-NAME), which inhibits nitric oxide generation63
. Indeed, l
-NAME induces autophagy, enhances the degradation of mutant huntingtin and alleviates toxicity in in vivo
. However, it may not be beneficial to induce autophagosome formation in certain disease settings: if the mutation or disease prevents the delivery of autophagosomes to lysosomes (which occurs if there are mutations in the dynein apparatus)62
; if the mutation results in impaired lysosome activity, which is observed in various lysosomal storage diseases64,65
; or in familial Alzheimer’s disease caused by presenilin 1 mutations55
. In such cases, increasing autophagosome formation will not necessarily enhance autophagic substrate degradation, and may result in cellular membrane build-up, as the newly formed autophagosome would not be efficiently delivered to lysosomes.
When the initial studies were performed to examine the possibility of upregulating autophagy to enable the clearance of intracytoplasmic aggregation-prone proteins, the only known pharmacological method of inducing autophagy chronically was using rapamycin. However, the side effects of rapamycin (which are unrelated to autophagy) may make it unattractive for use in pre-symptomatic patients who may require long-term therapy. Various screens have identified pathways and compounds that regulate autophagy independently of mTOR. For instance, imidazoline receptor agonists such as clonidine and rilmenidine induce autophagy and have protective effects in cell culture, D. melanogaster
and zebrafish models of Huntington’s disease49
. Rilmenidine also has protective effects in a mouse model of Huntington’s disease66
. Rilmenidine is a safe, centrally acting antihypertensive drug that lowers blood pressure by activating imidazoline receptors in the brain (which are widely distributed) — the same receptors it acts on to induce autophagy.
To date, there have been no reports of deleterious effects associated with specific autophagy upregulation in vivo
. Indeed, rapamycin prolongs lifespan in D. melanogaster
and in rodents67–69
and, at least in D. melanogaster
, these effects are largely autophagy-dependent67
. Although it is still not known whether specific upregulation of autophagy is beneficial in mice, deletion of the polyglutamine tract in the wild-type huntingtin protein induces autophagy in mice. This zero glutamine allele is associated with enhanced lifespan in otherwise wildtype mice; it increases lifespan and decreases motor symptoms in a knock-in mouse model of Huntington’s disease50
. From a therapeutic perspective, however, constitutive autophagy induction may not be necessary. The therapeutic regimes with rapamycin and rilmenidine in mice have used dosing protocols that are likely to result in pulsatile upregulation of autophagosome formation, with periods of normality between dosing51,66
. Thus, intermittent upregulation of autophagy may be effective and associated with fewer side effects in patients.
One major challenge when considering clinical trials for neurodegenerative diseases will be the feasibility of monitoring autophagic flux (not simply autophagosome numbers) in the correct tissue at the correct time. This will be a major challenge in the brain, but it will also be complex in tissues that are likely to be used for obtaining biopsy samples, such as tumours. Simply assaying autophagy in white blood cells or other easily accessible tissues may not be informative as there may be issues associated with tissue access for drugs (for example, the blood–brain barrier or tumour cores) as well as different receptors on different cells; therefore, certain compounds that act in the brain may have no effects in white blood cells.