Autophagy in Huntington disease and huntingtin in autophagy

Review

Autophagy in Huntington disease and huntingtin in autophagy Dale D.O. Martin, Safia Ladha, Dagmar E. Ehrnhoefer, and Michael R. Hayden Centre for Molecular Medicine and Therapeutics (CMMT), Department of Medical Genetics, Child and Family Research Institute, University of British Columbia, Vancouver, BC, Canada

Autophagy is an important biological process that is essential for the removal of damaged organelles and toxic or aggregated proteins by delivering them to the lysosome for degradation. Consequently, autophagy has become a primary target for the treatment of neurodegenerative diseases that involve aggregating proteins. In Huntington disease (HD), an expansion of the polyglutamine (polyQ) tract in the N-terminus of the huntingtin (HTT) protein leads to protein aggregation. However, HD is unique among the neurodegenerative proteinopathies in that autophagy is not only dysfunctional but wild type (wt) HTT also appears to play several roles in regulating the dynamics of autophagy. Herein, we attempt to integrate the recently described novel roles of wtHTT and altered autophagy in HD.

The vast majority of HD patients are heterozygous for the HTT mutation (mHTT), and therefore have a functional copy of wtHTT. HTT is essential for embryonic development, and adult-onset loss of total HTT leads to neurodegeneration, indicating an essential role in neuronal health [4–7]. While many efforts have been made to understand and develop novel therapeutics to promote pathways that can clear mHTT and its aggregates, it may be important to do so selectively without interfering with wtHTT levels. We describe here the importance of protein clearance in neurodegenerative diseases with a focus on HD. This review addresses the emerging role and cyclical nature of HTT degradation, and potential regulation, of autophagy.

Huntington disease HD is a devastating neurological disease that is characterized by loss of motor control and cognitive ability and, ultimately, death. The area of the brain most affected by the disease is the striatum, which plays a key role in initiating and controlling movements of the body, limbs and eyes [1]. HD is an autosomal dominant disease caused by a CAG expansion that encodes a polyQ repeat at the N-terminus of HTT [2]. Many factors have been implicated in HD including alterations in calcium handling, IGF signaling, vesicle transport, endoplasmic reticulum (ER) maintenance, and autophagy. The polyQ tract promotes the formation of toxic oligomers and aggregates, although it is still under debate whether these large aggregates are toxic or not [3]. As in Parkinson disease (PD), aggregates may act as sinks for aggregating proteins while the intermediate oligomers are toxic [3]. An expansion of the polyQ tract in HTT to greater than 36Q causes disease, and the greater the number of tandem repeats, the younger the age of onset. Ultimately, HD leads to death typically within 10–20 years after the appearance of the first diagnosable symptoms, with an average age of onset of about 35–40 years, although juvenile cases have been detected as early as 5 years of age. Currently, there is no disease-modifying therapy available for people suffering from HD.

Protein clearance in neurodegeneration The cell has two main pathways for clearance and removal of toxic proteins that are distinct, but linked; autophagy and the ubiquitin protein system (UPS)/unfolded protein response (UPR) [3]. Autophagy is divided into three main types based on how the cargo is delivered to the lysosome – microautophagy, chaperone-mediated autophagy (CMA); and macroautophagy. Microautophagy involves the translocation of cytoplasmic materials into the lysosome by direct engulfment by the lysosomal membrane. The molecular components that participate in this autophagic process in mammals remain unknown [8]. In CMA, individual soluble proteins that contain a KFERQ-like motif are specifically selected by chaperone proteins, unfolded, and translocated across the lysosomal membrane [9]. Internalization of substrate proteins by this pathway is attained through the coordinated function of chaperones on both sides of the lysosomal membrane and by a membrane protein (the lysosome-associated membrane protein type 2A or LAMP-2A) that acts both as a receptor and as an essential component of the translocation complex [8]. Macroautophagy, hereafter referred to as autophagy, was first described as a bulk clearance mechanism that is activated during starvation and consists of three main stages: autophagosome formation, maturation, and fusion with lysosomes [10] (Figure 1A). During autophagosome formation, an isolation membrane forms that can engulf portions of cytoplasm containing proteins and whole organelles [10]. Once formed, these autophagosomes are transported along microtubules to finally fuse with lysosomes

Corresponding authors: Martin, D.D.O. ([email protected]); Hayden, M.R. ([email protected]). Keywords: autophagy; Huntington disease; myristoylation; neurodegeneration; posttranslational modifications; trafficking; therapies; caspases. 0166-2236/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tins.2014.09.003

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Trends in Neurosciences, January 2015, Vol. 38, No. 1

Review

Trends in Neurosciences January 2015, Vol. 38, No. 1

(A)

Iniaon

Elongaon

Cargo loading

Maturaon

Transport and fusion

p62 p62 p62

Lysosome

p62

Omegasome Isolaon Lipidaon membrane

Key:

Autophagosome

LC3-I

LC3-II Recycled to LC3-I

LC3-II PI3P

Autophagolysosome

(B)

Iniaon

Elongaon

Cargo loading defect

Maturaon

Transport failure

p62

Lysosome

mHTT p62

mHTT p62

Omegasome

p62

Isolaon Lipidaon membrane

Autophagosome

p62

p62

Accumulaon of empty autophagosomes

TRENDS in Neurosciences

Figure 1. Autophagy is altered in Huntington disease (HD). (A) Autophagy involves the formation of double-membraned vesicles that incorporate damaged organelles and toxic or aggregated proteins, and fuse with the lysosome for degradation. (B) In HD it has been shown that autophagy is affected at several steps including a defect in cargo loading, trafficking of autophagosomes, and decreased fusion between autophagosomes and lysosomes leading to a build-up of toxic materials in the cytoplasm and empty autophagosomes.

leading to degradation of the autophagic cargo by lysosomal proteases [10]. Although autophagy was initially described as a bulk degradation mechanism, selective forms of autophagy have recently been identified that are specific for mitochondria, ribosomes, ER, or aggregated proteins such as mHTT; these are known as mitophagy [11], ribophagy [12], ERphagy [13], and aggrephagy [14,15], respectively. The autophagy process can be regulated through the serine/threonine kinase mTOR, which suppresses autophagosome formation under nutrient-rich conditions, while its inhibition by starvation signals leads to increased autophagy [16]. Pharmacological inhibition of mTOR by drugs such as rapamycin can constitutively increase autophagic flux [16]. Additionally, mTOR-independent pathways have also been identified that can be modulated pharmacologically to alter autophagic flux. For example, autophagosome formation is upregulated by increased levels of beclin1, which is freed from its inhibitory interaction with Bcl-2 upon different types of cellular stress

[17]. Furthermore, high intracellular calcium levels can inhibit autophagy through the activation of calpains and G-stimulatory protein a with a subsequent increase of cAMP levels [18]. The UPS is the second main form of protein degradation in mammalian cells, and its targets are marked for degradation through the attachment of ubiquitin. Typically, the UPS is responsible for the degradation of short-lived, soluble proteins, and cannot efficiently degrade bulky oligomeric and aggregated proteins such as mHTT [19]. HTT is subject to ubiquitination at amino acids K6, K9, and K15, which leads to its degradation and lowers the toxicity of mHTT [20–22]. However, this UPS-mediated degradation mechanism is thought to become impaired during the course of the disease, allowing mHTT to accumulate into insoluble, ubiquitin-containing aggregates in vitro and in vivo [23–25]. While ubiquitination can serve as a signal for degradation by the UPS, it has become apparent that it can fulfill a plethora of functions such as mediating protein transport 27

Review to different subcellular localizations and the regulation of enzymatic activity or degradation by autophagy [19,26]. The downstream function of ubiquitination is thereby often determined by the type of linkage of ubiquitin molecules to the protein substrate (mono- or polyubiquitination, and site-specific lysine linkages of ubiquitin) [19,26]. K48-, K11-, and K63-linked ubiquitin chains accumulate in brains from HD patients as well as mouse models, indicating that global changes in the ubiquitin system and UPS impairment are a consistent feature of HD pathology [27–31]. Interestingly, K63 ubiquitin linkage is also associated with a selective autophagic degradation mechanism [19,26]. K63-ubiquitinated autophagic cargo is subsequently recognized by autophagy receptors such as p62 or optineurin, both of which have been shown to bind HTT [32–35]. Autophagy receptor proteins such as p62 bind to LC3 (microtubule-associated protein 1 light chain 3), an essential component of autophagosomes, and thereby link ubiquitinated cargo with the nascent autophagosome [19,26]. Consequently, mature autophagosomes containing cargo, together with p62 and LC3 proteins, fuse with lysosomes leading to the degradation of internalized proteins [36–39]. p62 can also polymerize and form a shell around mHTT inclusions, which leads to the autophagic degradation of aggregates (also known as aggrephagy) [36,40]. The adaptor protein autophagy-linked FYVE protein (ALFY; also known as WD repeat and FYVE domaincontaining 3, WDFY3) has a central role in the autophagic degradation of mHTT aggregates and acts as a scaffold for aggregates, p62, and the autophagosome [15]. Selective autophagy thereby contributes to the clearance of mHTT but it is also an important pathway for the clearance of other proteins involved in neurodegenerative disease such as tau, a-synuclein, ataxin-3, and SOD1 [32,41–43]. Many of these proteins can be substrates for both the UPS and autophagy, but progressive proteasomal impairment and the formation of protein aggregates in the disease state leads to a shift toward autophagic degradation [8]. Upregulating the autophagy pathway and preventing dysfunction may therefore be a promising therapeutic strategy for several neurodegenerative diseases including HD (Box 1). Catch 22: autophagy is required for clearance of aggregated proteins, but mHTT interrupts the process Mechanistic alterations in HD Many neurodegenerative diseases are characterized by abnormally low autophagic flux, leading to the accumulation of autophagy substrates and toxicity [44]. However, human and rodent HD samples display an increase in the number of autophagosomes while maintaining appropriate (or even higher) levels of flux compared to wt controls [45–47]. In particular, mHTT contributes to the activation of the autophagy pathway in cell, rodent, and human HD tissues by sequestering and inactivating mTOR, leading to an induction of autophagy [48]. However, it was shown that the autophagosomes, while increased in abundance, are in fact devoid of contents owing to a deficit in cargo recognition [47]. Hence, despite an increase in the initiation of autophagy and the formation of autophagic vacuoles, aggregated proteins (such 28


Box 1. Autophagy as a therapeutic target for HD Given that aggregation-prone proteins such as HTT are preferentially degraded by autophagy, much effort is being devoted to developing therapies that seek to upregulate macroautophagy in an attempt to clear mHTT. Studies using multiple mammalian cell, fly, and rodent models have shown an improvement in HD phenotypes following treatment with various autophagy inducers ([105] for detailed review on the state of current autophagy therapeutics in HD). First among these was rapamycin/CCI-779, an mTOR inhibitor, which was found to attenuate neurodegeneration in both a fly and mouse model of HD and improved behavior and motor performance in the mouse model [48]. Although such mTOR-dependent small molecules are not ideal, owing to a host of negative side-effects due to the wide range of mTOR-related signaling pathways and cellular processes, this provided a proof of principle that the general induction of autophagy confers therapeutic benefit. Consequently, mTOR-independent compounds such as lithium, trehalose, and rilmenidine have yielded beneficial results through the upregulation of autophagy and clearance of mHTT. Given that these two classes of drugs possess additive effects [106], it has been proposed that a combination therapy involving both mTOR-dependent and -independent compounds would enable the greatest extent of mHTT clearance [105]. Such treatment would likely be most efficacious if administered pre-symptomatically, at which point the likelihood of consistent mHTT turnover and prevention of aggregate overload is highest. Given the potential role of HTT in regulating autophagy, further work is required to identify therapeutic targets that could restore HTT function and normalize defects observed in key autophagy processes such as autophagosome transport, fusion to lysosomes, aberrant protease cleavage, and altered post-translational modification. Despite the fact that general autophagy inducers have shown to be beneficial, combination therapies that also include drugs that work toward correcting the specific autophagy-related mechanistic dysfunctions observed in HD may lead to the best therapeutic outcomes.

as HTT) and damaged organelles are not degraded, and these continue to accumulate in the cytoplasm, thereby contributing to toxicity (Figure 1B). The mechanism whereby cargo loading is restricted is not yet known [47]. Such mechanistic defects in the autophagy pathway could conceivably lead to a negative feedback loop, where the presence of mHTT results in defective autophagymediated degradation and accumulation of aggregated protein, after which the cell responds with a further compensatory upregulation of autophagy. The end-result is an accumulation of mHTT and subsequent neurotoxicity. The mechanistic defects in autophagy in HD are exacerbated by the effect of mHTT on vesicle trafficking. mHTT disrupts autophagosome motility and subsequently prevents autophagosome fusion with lysosomes, further promoting an increase in autophagosomes [49] (Figure 1B). In addition, mHTT shows a decreased interaction with the optineurin/Rab8 complex, leading to dysfunctional autophagosome/lysosome dynamics [50]. It is still unclear at which point during the pathogenesis such defects dominate. For example, fusion dynamics may be affected early, and then be overcome by alternate pathways, after which failure of autophagy machinery to recognize mHTT may result in toxicity or vice versa. Such deficits might need to be corrected if an increase in autophagy is to be used as a therapeutic strategy for clearing mHTT. Furthermore, while macroautophagy is the most commonly studied autophagic mechanism, additional autophagic pathways play a role in HD. As mentioned earlier,

Review


mitophagy refers to the selective degradation of dysfunctional mitochondria [51,52]; because mHTT expression is associated with impaired mitochondrial health [53] and a concomitant increase in reactive oxygen species (ROS) and energy deprivation [54], upregulation of mitophagy might be beneficial. CMA is upregulated in different cellular and mouse models of HD [55], and this could be a mechanism to compensate for the dysfunction of both the UPS and macroautophagy. While the HTT protein contains 3 KFERQ-like motifs, which are a prerequisite for degradation by CMA, the full-length protein cannot be degraded through this mechanism, although fragments encompassing HTT exon 1 (both wt and mutant) are susceptible to degradation by CMA [55]. Taken together, it is clear that there is a complex interplay between multiple forms of autophagy in HD, and that the numerous effects of polyQ-expanded HTT may be at multiple points along autophagy pathways. In light of these findings, while efforts to upregulate autophagic flux alone may be beneficial, correcting the specific mechanistic defects in addition to enhancing flux may offer the best possibility of efficiently clearing mHTT and attenuating toxicity (Box 1). Altered gene expression of the autophagy and ubiquitination pathways in HD patients Owing to the transient nature of autophagic induction, studying autophagy at the protein level in mouse brains can be very difficult; this may explain some of the conflicting results in the literature regarding levels of various autophagy markers [56,57]. In addition, although many studies look at specific regions of the brain, these tissues still contain mixed cell populations, which may also confound the results if autophagy is not consistent between cell types. Changes in the expression levels of autophagy proteins have been observed in HD striatum [57]. While a

more recent study suggests that these changes occur primarily in oligodendrocytes [58], significant neuronal death is already observed at the disease stage analyzed. It is therefore expected that neuron-specific dysregulation of autophagic pathways would occur significantly earlier in the pathogenesis, but this is difficult to assess in human patients owing to the lack of accessibility of relevant brain tissues. In the caudate nucleus from HD patients, mRNA expression of LC3A, ULK2, and LAMP2 is significantly increased, while PTEN-induced kinase 1 (PINK1), WDFY3 or ALFY, and FK506-binding protein 1A (FKBP1A) are significantly decreased (Table 1). LC3A and ULK2 are involved in early stages of autophagy induction and autophagosome formation. Their increased expression correlates with literature suggesting that early stages of autophagy are increased in HD [45–47]. Conversely, FKBP1A acts an inhibitor of mTor [59]. While this would suggest that mTor activity may be increased, and autophagy would be decreased, the upregulation of LC3A and ULK2 may compensate and lead to the observed net increase in autophagy in HD. In addition, it is possible that an upregulation of autophagy may be occurring through mTor-independent pathways in these tissues. Furthermore, the increase in LAMP2 mRNA is consistent with increased levels of LAMP2 protein and concomitant induction of CMA in HD patient cells and mouse models [55]. Of particular note are the significant decreases in ALFY and PINK1. As mentioned, ALFY has recently been shown to be an important scaffolding protein for the removal of aggregated proteins, particularly HTT, via basal aggrephagy [14,15]. Therefore, a significant decrease in ALFY protein in the striatum may lead to increases in aggregated proteins and toxicity. Finally, knockdown of PINK1 expression induces mitochondrial fragmentation and induces autophagy and mitophagy [60].

Table 1. Autophagy and ubiquitination pathways gene expressions altered in oligodendrocytes and neurons of the caudate from HD patients [58] Probe set Neurons 204540_at

logFC a

Gene Symbol

Gene Name

Function

0.16

EEF1A2

202073_at

0.40

OPTN

Eukaryotic translation elongation factor 1a2 Optineurin

217797_at

0.26

UFC1

Ubiquitin-fold modifier conjugating enzyme 1

Lysosomal variant stabilizes the translocation complex in CMA [107] Multifunctional protein involved in vesicle trafficking and aggregation clearance via autophagy [108] Member of the ubiquitin-like protein family involved in ER stress-induced apoptosis [109]

Oligodendrocytes 224378_x_at

0.79

MAP1LC3A

Microtubule-associated protein 1 light chain 3a

Involved in autophagosome biogenesis and cargo recruitment [107]

232011_s_at 227219_x_at 200821_at

0.68 0.51 0.32

MAP1LC3A MAP1LC3A LAMP2

The splice-variant LAMP2A is essential in CMA [55]

212598_at

0.53

WDFY3

204063_s_at 209018_s_at

0.34 0.92

ULK2 PINK1

210186_s_at

0.90

FKBP1A

202316_x_at

0.42

UBE4B

Lysosomal-associated membrane protein 2 WD repeat and FYVE domain-containing 3 Unc-51-like kinase 2 PTEN induced putative kinase 1 FK506 binding protein 1A, 12 kDa Ubiquitination factor E4B

Scaffolding adaptor protein for aggrephagy (also known as ALFY) [14] Initiator of macroautophagy [110] Involved in the recruitment of parkin for mitophagy [60] Inhibitor of mTor [59] Negative regulator of p53 [111]

a

logFC, logarithm of fold change.

29

Review HD is associated with increased mitochondrial fragmentation [61] and inefficient incorporation of mitochondria into autophagosomes [49], suggesting that the decreases in PINK1 may exacerbate the dysfunctional cargo loading in HD cells. In addition, changes in the expression of genes involved in ubiquitination pathways such as UFC1, and UBE4B also provide further evidence for the dysregulation of the UPS in HD. In addition to gene expression changes, genetic alterations such as mutations in key autophagy genes can also contribute to the pathogenesis of HD. ATG7 (autophagyrelated protein 7) is required for lipidation of LC3, and complete loss of ATG7 is associated with neurodegeneration [3]. A polymorphism in ATG7 is associated with an earlier age of onset in HD [62], further suggesting that proper regulation of autophagy is essential for clearing mHTT. The fox in charge of the henhouse: HTT regulates autophagy HTT plays a role in the trafficking of autophagosomes Although it is known that autophagy is dysfunctional in HD [47,63], new studies now suggest that wtHTT may also have a role in regulating autophagy. In neurons, autophagosomes are constitutively generated under basal conditions at distal axons, and are trafficked retrogradely to the cell body [64]. Silencing of either HTT or its interactor, HTT-associated protein 1 (HAP1), blocks retrograde transport of autophagosomes along the axon [49]. HAP1 depletion is also associated with decreased net run-speeds of autophagosomes in both retrograde and anterograde transport. Retrograde transport was also dependent on the interaction between HTT and the microtubule motor-protein dynein. Indeed, dynein mutations or dysfunction impair fusion between autophagosomes and lysosomes, resulting in mHTT accumulation and enhanced toxicity [65]. Of note, disrupting autophagosomal transport by HTT depletion also leads to an accumulation of autophagosomes with non-degraded cargo including mitochondria [49]. Similar effects are observed in the presence of mHTT, which leads to inefficient clearance of mHTT and cargo, suggesting that HTT may regulate its own clearance. HTT has also been shown to play a role in fast axonal transport, but this function is inhibited by mHTT at various stages ([66] gives a more detailed review on HTT in fast axonal transport). In particular, Akt-mediated phosphorylation of HTT promotes anterograde transport through kinesin-1 recruitment [66]. The HTT/HAP1 complex promotes robust retrograde transport of autophagosomes [49], but because HAP1 is also involved in anterograde transport, mHTT may lock the motor protein kinesin in place, essentially acting in a tug-of-war. Dephosphorylation of HTT stimulates dynein-dependent retrograde transport [66]. These results strongly point to a role for wtHTT in the trafficking of autophagosomes along microtubules. HTT promotes autophagosome formation More recently, we identified an autophagy-inducing domain within HTT, spanning amino acids 553–586, that is regulated by myristoylation at G553 [67] (Figure 2). Using 30


a truncated form of HTT (HTT1–588–YFP) [68], we have shown that HTT is post-translationally myristoylated at G553 following caspase cleavage at D552 [67], and may play a role in regulating autophagy. Myristoylation promotes membrane binding and involves the non-reversible addition of a 14 carbon fatty acid to N-terminal glycines [69], either co-translationally on the nascent polypeptide following the removal of the initiator methionine or posttranslationally after exposure of internal glycine residues following caspase-mediated cleavage [70,71]. When expressed in multiple cell lines, myristoylated HTT553– 585 fused to GFP (myr-HTT553–585–EGFP) promotes the formation of autophagosomes, but not when myristoylation was blocked (G553A-HTT553–585–GFP) [67]. Expression of myr-HTT553–585–EGFP is also associated with elevated levels of LC3-II, indicating an increase in autophagic flux [67]. The myristoylation-dependent autophagy induced by myr-HTT provides a novel link between autophagy and HD [67]. In common with many other post-translational modifications of HTT, post-translational myristoylation is also significantly decreased in mHTT compared to wtHTT, suggesting decreased membrane association and altered autophagy induction in the presence of the expanded polyQ tract. This may contribute to the already decreased fusion between autophagosomes and lysosomes, and to impaired cargo loading that leads to a build-up of cytotoxic materials and promotes the HD phenotype [47,50]. Interestingly, it is predicted that HTT may have evolved from several yeast autophagy proteins, including ATG23, ATG11, and Vac8 (vacuolar protein 8), that are required for selective vacuolar targeting in the yeast autophagy pathway [72]. The autophagy-inducing domain HTT553–586 also partially aligns with the autophagy regulatory domain of ATG14L, the Barkor/Atg14(L) autophagosome-targeting sequence (BATS) domain [67]. The BATS domain senses membrane curvature via its amphipathic a-helix and induces the formation of autophagosome remarkably similar to myr-HTT553–586 [67,73]. We propose that myrHTT553–586 acts as a membrane curvature-sensing domain that induces the formation of autophagosome, similarly to the BATS domain – by sensing and inducing membrane curvature at the ER as cleavage of HTT progresses. This is a novel and unexpected role for HTT in autophagosome formation and processing. Autophagy cargo is delivered to autophagosomes by autophagy receptors that link their cargo to LC3. They bind to LC3 through short linear motifs known as LC3interacting repeats (LIRs) [74]. A recent prediction analysis tool predicts 11 LIR motifs within full-length HTT [74] (Figure 2). Of note, only one LIR motif was predicted within the first 600 amino acids of the N-terminus of HTT [74]. Typically, shorter N-terminal fragments of mHTT are more toxic and generate more robust phenotypes in HD models [75]. The majority of the predicted LIR motifs are concentrated in the regions predicted to share homology with Vac8 (HTT aa807–1653) and ATG11 (HTT aa1743–3144) (Figure 2) [72]. This may suggest that shorter forms of HTT are more toxic owing to decreased association with LC3. In particular, blocking caspase cleavage at D586 has been shown to prevent HD in the

Review


2 Caspase-6

1 C

Polyglutamine tract C

N

N

Full-length HTT

C

3

N

Lysosome

7 Autophagolysosome

N

6

4 Caspase-3

Autophagosomes

5 NMT

mHTT

HTT553-585

HS-CoA TRENDS in Neurosciences

Figure 2. An autophagy-inducing domain within huntingtin (HTT) is released by caspase-cleavage and is dependent on post-translational myristoylation. HTT (1) has been shown to be cleaved at D586 by caspase-6 in the nucleus (2). Subsequent processing by caspase-2 or -3 at D552 exposes an N-terminal glycine at position 553 (3,4), which is post-translationally myristoylated (5) by N-myristoyltransferase (NMT). Myristoylation was shown to direct HTT553–586–EGFP to the endoplasmic reticulum (ER) (6) and to promote the formation of autophagosomes which ultimately fuse with the lysosome (7). Myristoylation at G553 is decreased in mutant (m)HTT. The myristoylated HTT peptide is predicted to detect the highly curved membranes of the endoplasmic reticulum (ER) where it inserts the myristate moiety into the membrane (6). As the peptide accumulates, it may increase membrane curvature to promote the formation of autophagosomes. Inhibiting autophagosome–lysosome fusion did not increase levels of myristoylated-HTT553–586–EGFP, suggesting that it does not accumulate within the autophagosome.

YAC128 mouse model [76]. Rendering mHTT resistant to caspase 6-mediated cleavage may therefore provide a means to keep the predicted autophagy domains together and mediate increased clearance of mHTT. Marked for destruction: modulation of autophagic clearance of mHTT through post-translational modifications (PTMs) Ubiquitination and SUMOylation differentially regulate mHTT clearance by autophagy HTT is subject to many different PTMs, some of which are differentially regulated in the disease context [77] (Figure 3). Ubiquitination at residues K6, K9, and K15 in particular is associated with HTT degradation, and SUMOylation of these lysines competes with ubiquitination, leading to increased toxicity in the presence of the HD mutation [22] (Figure 3). wtHTT is preferentially ubiquitinated through K48 linkage, which is considered the ‘classical’ signal for proteasomal degradation [78]. Conversely, mHTT, and in particular mHTT fragments, show preferential ubiquitination through K63 of ubiquitin, and this is correlated with increased stability and aggregation [78]. It would be interesting to determine whether these K63-ubiquitinated mHTT fragments are bound by p62, and

would, therefore, be targets for autophagy, while K48 ubiquitinated wtHTT would be expected to be a better UPS substrate. mHTT may thus regulate its own aggrephagy based on K63 and K48 ubiquitination, similar to the aggregation-prone protein synphilin-1 [79], resulting in differential recruitment of autophagy proteins. The switch between HTT SUMOylation and ubiquitination at K6, K9, and K15 is regulated through phosphorylation at S13 and S16, and it has been shown that these phosphorylation events are crucial determinants of mHTT toxicity and degradation [80,81]. Increased expression of the IKK (inhibitor of kB kinase) complex mediating S13/ S16 phosphorylation can be achieved through the inhibition of histone deacetylases (HDACs 1 and 3), which also upregulates several genes in the ubiquitination pathway and leads to increased autophagic and proteasomal HTT degradation [82]. In line with these findings, genetic reduction of HDAC4, as well as treatment with the HDAC4 destabilizing agent suberanilohydroxamic acid (SAHA), are beneficial in a mouse model of HD [83,84]. Acetylation at K444 is another PTM that has been shown to increase the autophagic clearance of mHTT (Figure 3), and this effect is mediated through increased binding of acetylated mHTT to p62 [85]. However, it remains unclear whether 31

Review


ATG23 homology 1

Vac8 homology

586 807

LIR

LIR

K9 ubi/SUMO

ATG11 homology 1653

LIR

LIR LIR

1743

3144 amino acids

LIR

LIR

LIR LIR

K15 ubi/SUMO

K6 ubi/SUMO S13 P

N

S16 P

HEAT2

HEAT3

C

HEAT4

HEAT1

Ac K444

✂ ✂ D552 casp2/3

G553 Myr

D586 casp6

LIR sequence PEFQKL SVYLKL PSYLKL FEYIEV EMFILV

locaon 119–124 959–964 1234–1239 1479–1484 1586–1591

LIR sequence LTWLIV LMYVTL KVFQTL RDWVML

locaon 1916–1921 2719–2724 3018–3023 3033–3038

D586 TRENDS in Neurosciences

Figure 3. Post-translational modifications (PTMs) of huntingtin HTT regulate its degradation as well as its role in autophagy. At the N-terminus, HTT is subject to either ubiquitination (ubi) or SUMOylation (SUMO) at K6, K9, and K15, and this process is regulated by phosphorylation (P) at S13 and S16. These PTMs, as well as HTT acetylation (Ac) at K444, regulate the autophagic degradation of HTT. Caspase (casp) cleavage at D553 and D586, as well as myristoylation (Myr) at G553, are important for the role of HTT in regulating autophagy. All these PTMs are altered in the presence of the HD mutation and modulate the toxicity of mHTT. Sites of predicted homology between HTT and yeast autophagy genes are included, as well as the novel sites of predicted LC3-interacting repeats (LIR) motifs which may be important for the interaction between HTT and LC3. Additional abbreviations: ATG11/23, autophagy-related protein 11/23; HEAT, huntingtin, elongation factor 3, protein phosphatase, and lipid kinase TOR1 domain; Vac8, vacuolar protein 8.

acetylation at K444 increases this interaction through preferential K63 ubiquitination of HTT or via a direct increase in the binding affinity. mHTT meets at the crossroad between apoptosis and autophagy Multiple studies have shown that autophagy and apoptosis, most notably caspase activation, can influence each other and are often activated sequentially as a response to cellular stress [86,87]. While autophagy can be an initial response to deal with low-level insults by degrading damaged proteins and organelles, or by providing additional nutrients during starvation, apoptotic mechanisms typically take over once a threshold level of stress is reached from which recovery is not possible and the cell is destined to die [86]. However, both caspase activation and autophagic protein clearance can play physiological roles in the CNS, where low levels of caspase activity can lead to synaptic and axonal pruning, and memory and learning deficits [88–92], whereas basal autophagy contributes to axonal homeostasis and synaptic plasticity [93–97]. While caspases and autophagy therefore contribute to similar neuronal housekeeping functions, it is unclear so far how these two systems interact in the absence of stress. We have shown previously that caspase-6 cleaves HTT at amino acid D586 (Figure 3), which is associated with mHTT toxicity [76]. In addition, mHTT feeds forward to further activate caspase-6, and this may increase toxicity and cell death [98]. Interestingly, it has been shown that the ablation of caspase-6 in an HD mouse model leads to significantly decreased levels of p62 and HTT while levels 32

of the autophagosome-associated LC3-II increase; this suggests that autophagy is activated in the absence of caspase-6 [99]. However, a more in-depth analysis of autophagic pathways in caspase-6 knockout animals would be necessary to determine whether the absence of caspase-6 alone constitutively upregulates autophagy or whether the presence of mHTT is necessary. Both scenarios are possible because caspase-6 is known to cleave multiple proteins of the autophagic machinery including beclin, ATG3, and p62 [100]. The ablation of caspase-6 might, therefore, increase the availability of these proteins and elevate levels of basal autophagy. In addition, caspase-6 cleaves many cytoskeletal proteins such as tau and a-tubulin [101], and this could lead to impaired trafficking of autophagosomes and reduced autophagosome–lysosome fusion, and thus impair the autophagic process. A similar nonapoptotic role in the regulation of autophagy has been described recently for caspase-2, which negatively regulates autophagy induction under both basal and stress conditions [102]. The increase in caspase activity may also generate more of the myristoylated HTT553–586 fragment that could further promote autophagosome formation, but in the context of decreased cargo loading and autophagosome and lysosome fusion, this would further perpetuate the dysregulation of autophagy in HD, much like pouring more water into a clogged sink. It will be interesting to investigate whether caspase-6 cleavage, myristoylation, ubiquitination, and acetylation of HTT work together to influence its autophagic clearance, or whether these events represent separate pathways that each may contain targets for therapeutic intervention. Outstanding questions are listed in Box 2.

Review


Box 2. Outstanding questions Is HTT a scaffold for autophagy? HTT interacts with several regulators of autophagy involved in autophagosome biogenesis and transport. It has also been suggested that HTT may have evolved from yeast autophagy genes Vac8, ATG11, and ATG23 (Figure 3). In addition, HTT contains many predicted LIR repeats, and depletion of HTT interrupts vesicle trafficking and alters autophagy. Given that HTT essentially links vesicle trafficking and autophagy, it is tempting to suggest that wtHTT may act as a scaffold for autophagy, and that HD is not simply due to a gain-of-function mHTT but is also the result of loss of wtHTT function in autophagy. Interestingly, point mutations or depletion of multiple autophagy or trafficking proteins, such as dynein, lead to neurodegenerative diseases that share characteristics with HD [66]. It will be important to parse apart the roles of the different autophagy domains and LIR repeats to determine their contribution to autophagy under basal conditions as well as in HD. Does proteolysis of HTT release alternative autophagy domains? Because HTT may contain multiple autophagy domains, and because it has been shown that caspase cleavage may release and activate at least one hidden autophagy domain (myr-HTT553–586), it is interesting to think that other autophagy domains may be differentially released by caspase-mediated cleavage. Consequently, blocking caspase cleavage at D586 may mediate its protective effect by retaining alternative domains. For instance, blocking cleavage at D586 may promote cleavage and myristoylation at D552 and G553, respectively, thereby generating an extended C-terminal autophagy domain containing the newly identified myristoylated domain and the ATG23 and ATG11 domains. It will be important to determine how this extended domain affects autophagy in the HD mouse model.

Concluding remarks Overall, evidence is building that suggests HTT is not simply a passive passenger in autophagy but that it may also be an important regulator of autophagy acting as a scaffold for autophagosome transport and biogenesis. What makes this so compelling is the fact that mutations in several motor proteins and autophagy regulators also lead to neurodegenerative disease with HD-like phenotypes [66,103]. In particular, similarly to HTT, dynein is involved in autophagy, ER structure, mitotic spindle orientation [103], and retrograde transport [49]. Loss of dynein results in death, and specific mutations in dynein lead to a toxic gain of function that promotes mild but similar phenotypes to HD [104]. HD is typically described as a toxic gain of function by mHTT, but a loss-of-function mechanism of wtHTT that involves autophagy may also contribute significantly to the disease. Acknowledgments We thank CMMT Medical Illustrator Intern Erin Kenzie for providing figures. D.D.O.M. holds a Canadian Institutes of Health Research (CIHR), Michael Smith Foundation for Health Research Postdoctoral Fellowship, and the Bluma Tischler Postdoctoral Fellowship from the University of British Columbia. S.L. holds a CIHR Doctoral Award. M.R.H. is a University Killam Professor and holds a Canada Research Chair in Human Genetics. This work was funded by CIHR grants (MOP 84438 and GPG-102165) to M.R.H.

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How is myristoylation affected in HD and autophagy? As described herein, myristoylation of HTT at G553 was decreased in mHTT compared to wtHTT. This may be due to steric hindrance by the expanded polyQ if caspase-mediated cleavage does not lead to complete dissociation of the proteolytic cleavage products. Alternatively, mHTT may alter N-myristoyltransferase (NMT) activity or levels. mHTT has been shown to decrease the activity of its own palmitoylacyltransferase HIP14, which results in decreased palmitoylation, another form of fatty acylation of mHTT, and increased aggregation and toxicity [112]. Because NMT activity is essential for cell viability and is highly expressed in brain, it will be important to measure both NMT levels and activity in HD brain [70,113]. In addition, NMTs are also subject to caspase cleavage, and this appears to alter their specificity for substrates [114]. The increase in caspase activity in HD may lead to increased cleavage of NMT and altered myristoylation of HTT and other NMT substrates. During induction of autophagy, many proteins are recruited to membranes and fatty acylation, including palmitoylation and myristoylation, may play an important role in this process. Do levels of caspase-6 directly influence autophagy? Caspase-6 activity is increased in HD, as well as in other neurodegenerative diseases such as Alzheimer disease [115]. While caspase-6 may modulate autophagy through its cleavage of HTT, it has several additional substrates, some of which are autophagy proteins, such as beclin, ATG3, and p62, as well as several cytoskeletal proteins [101]. It is conceivable that the increased activation of caspase-6 in neurodegenerative conditions, and even in normal aging, leads to a progressive decrease in autophagy. It will be interesting to determine whether the ablation or inhibition of caspase-6 have effects on autophagic pathways independent of the expression of mHTT.

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