Mechanisms of Aggresome Biogenesis Ubiquitination, Transport, & Maintenance

Neurodegenerative diseases are universally marked by the accumulation of misfolded protein. Neurons respond to these proteostatic disturbances by sequestering, and thus inactivating, toxic misfolded proteins into a perinuclear organelle called the aggresome. The aggresome can be subsequently degraded in bulk by autophagy, a process termed aggrephagy. The formation of protein aggregates has historically been considered a spontaneous and unregulated process, but emerging research has instead discovered a diverse cohort of regulatory proteins that mediate protein aggregation. Chaperones are the first proteins to respond to misfolded proteins, and do so by recognizing the aberrant exposure of hydrophobic domains. When chaperones are unable to correctly refold proteins, their substrates are transferred to ubiquitin ligating machinery to catalyze polyubiquitination. Although ubiquitin chains typically direct proteins towards proteasomes, severe proteotoxic stress can overwhelm, or even directly inhibit, proteasomes. As an alternative to proteasomal degradation, misfolded proteins are redirected towards the mitotic organizing center (MTOC) and, following retrograde transport by dynein, are packaged and sequestered within an intermediate filament (IF) cage to form the aggresome. The biogenesis of the aggresome is thus a highly regulated event, and a better understanding of the mechanisms facilitating this process will provide critical insight into neurodegenerative disease. ____________________________________________________ 1 MECHANISMS OF AGGRESOME BIOGENESIS AUTHOR MANUSCRIPT Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 28 December 2018 © 2018 by the author(s). Distributed under a Creative Commons CC BY license. doi:10.20944/preprints201812.0332.v1


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The author has declared no conflicts of interest The author received no funding for article preparation

HIGHLIGHTS :
• Perinuclear aggresomes serve a critical role in maintaining proteostasis.
• HDAC6 and BAG3 are the primary adaptors for retrograde transport of misfolded proteins.
• Aggresomes are constructed by p62, which also mediates its autophagic clearance.
• Undegradable aggresomes aggravate protein misfolding and can cause other pathologies.

How p62 Aggregates the Aggresome
Once misfolded proteins have been retrogradely transported and delivered to the MTOC, they must be efficiently packaged and sequestered for aggrephagy.The essential role of p62 (SQSTM1) in aggrephagy is well understood, where it functions as an adaptor between LC3, on autophagosomes, and ubiquitin, on aggregates.Known as an autophagic cargo adaptor, p62 serves to localize and bridge the nucleating autophagosome to its substrate (Figure 3).In addition to aggrephagy, accumulating evidence also suggests that p62 is essential for the initial formation of the aggresome.The formation of aggresomes complex activated p38, while BAG3 or HSP70 deletion potently suppressed p38 activation.Proteasome inhibition has also been previously linked to p38 activation ( Ito et al, 2002 ).DRiP accumulation and proteasome inhibition, both common proteostatic stressors, are thus capable of activating p38 which subsequently activates p62.In addition to phosphorylation, p62 is also extensively ubiquitinated during proteostatic stress.Peng at al recently documented an essential ubiquitin modification of p62 at lysine 420 that is required for the disinhibition of p62's UBA domain ( Peng et al, 2017 ).Following proteasome inhibition, K420 ubiquitin modifications accumulate on p62, drastically enhancing its affinity for K63 ubiquitin chains.
Research by Lee et al confirmed p62's K420 ubiquitination and its relationship to aggregation ( Lee et al, 2017 ).This modification was catalyzed by the KEAP1/Cullin3 E3 ubiquitin ligase and was essential for aggregate formation following p62 upregulation (Figure 3).The phosphorylation of p62 by p38, and ubiquitination by KEAP1/Cullin3, are at least two critical modifications of p62, but many more have been documented.

Figure 2 Figure 3 Figure 1
Figure 2 Retrograde Transport Of Peripheral Microaggregates.PMAs (red) are recognized by folding chaperones like HSP70 and HSP110.E3 ubiquitin ligases CHIP, PARKIN, and Hrd1 catalyze K63 linked ubiquitination (purple) of aggregates.VCP and RuvbL1/2 extract misfolded proteins from larger aggregates.Deubiquitinases ataxin-3 and UCH-L1 cleave ubiquitin linkages to recruit and activate HDAC6, which then mediates dynein retrograde transport.DRiPs are also an important source of misfolded protein, and are prepared for retrograde transport by the BAG3/HSP70 or CED complexes.
PARKIN is another E3 ubiquitin ligase important in the recognition, ubiquitination, and targeting of PMAs to the aggresome (Figure2).PARKIN, which can associate with and be activated by CHIP( Imai et al, 2002 ), colocalizes with aggresomes and PMAs, and loss of function mutations result in familial forms of Parkinson's Disease( Olzmann et al, 2007 ).The loss of PARKIN results in the accumulation of either soluble misfolded protein species or PMAs, both of which are characteristic of defects in the transport of pre-aggresome aggregates ( Chung et al, 2001 Yang et al, 2007 ).In addition, PD patients with mutations in PARKIN fail to form characteristic Lewy Bodies suggesting PARKIN is involved in directing alpha-synuclein to aggresomes in human disease ( Johanson et al, 2018 Lim et al, 2005 ).During MG132 induced proteasome inhibition, or other UPS dysfunctions, PARKIN mediated ubiquitination can facilitate the formation of the aggresome and has been demonstrated to direct various proteins including DJ-1, SOD1, synphilin-1, and TDP-43 to the aggresome ( Olzmann et al, 2007 Hebron et al, 2012 Olzmann et al, 2015 Yung et al, 2015 ).In order to promote aggresome formation, however, PARKIN itself must escape an autoinhibited conformation, which is coordinated by the PINK1 kinase.When localized to depolarized mitochondria, PINK1 is unable to translocate into the mitochondrial matrix, and instead forms a mitochondrial outer membrane-anchored kinase that phosphorylates ubiquitin and PARKIN.PARKIN not only requires PINK1 phosphorylation to escape an autoinhibited conformation but also requires phosphorylated ubiquitin for efficient substrate polyubiquitination (Reviewed in Pickrell et al, 2015 ).Thus PARKIN dependent aggresome biogenesis must also be contingent on the presence of depolarized mitochondria, otherwise PARKIN would, presumably, not be active.Although CHIP and PARKIN are the most well studied E3 ubiquitin ligases implicated in aggresome biogenesis, there are many others.For example, ER-localized E3 ubiquitin ligase Hrd1 has recently been shown to be critical to the formation of aggresomes composed of misfolded OPTN (Figure 2; Mao et al, 2017 ).Hrd1 has also been implicated in the targeting of Amyloid Precursor Protein to aggresomes via the ER-Associated Degradation pathway ( Kaneko et al, 2010 ).Many E3 ubiquitin ligases function redundantly in the targeting proteins to the proteasome, and thus their role in targeting proteins to the aggresome probably overlaps as well.Importantly, the targeting of ubiquitinated substrate to aggresomes, as opposed to the proteasome, appears to be facilitated primary by K63 linked ubiquitin chains -a modification that is indeed catalyzed by PARKIN1, CHIP, and Hrd1 ( Tan et al, 2008 Olzmann et al, 2008 Chin et al, 2010 ).Unlike the canonical K48 linked ubiquitin chains that consistently send proteins to the proteasome, K63 linked ubiquitin chains actually impede this process by physically occupying lysine residues, and instead promote the formation of large macromolecular protein complexes (not unlike an aggresome).However, a direct mechanistic link between proteotoxic stress (such as proteasome inhibition) and K63 ubiquitination has not been clearly identified.Research by Lim et al recently sought to understand this relationship and discovered that the stress induced expression of UBC13, an E2 ubiquitin ligase specific for K63 linkages, was capable of shifting PARKIN's catalytic activity from K48 to K63 ubiquitin linkages ( Lim et al, 2013 ).Interestingly, CHIP has also been shown to bind UBC13 and this promotes the catalysis of K63 ubiquitin chains, but its role in aggresome formation has not been specifically investigated ( Zhang et al, 2005 Slotman et al, 2012 ).Although it is then tempting to suggest proteasome inhibition triggers the transcription of aggresome-promoting genes like UBC13, research has suggested that neither transcription or translation affects proteasome inhibition-induced aggresome formation ( Meriin et al, 2012 ).Regardless of how proteasome inhibition alters the ubiquitin landscape, the catalysis of K63 ubiquitin linkages alone is still not sufficient to redirect misfolding proteins towards aggresomes, and additional modifications are required.The retrograde transport of PMAs probably requires some degree of disaggregation or separation from their associated complexes, otherwise these misfolded cargoes may be too physically large for efficient transport.The disaggregation of peripheral aggregates prior to transport is achieved principally by the HSP70 chaperone in association with its cognate cochaperone HSP110 (Figure 2; Rampelt et al, 2012 Reviewed in Nillegoda et al, 2018 ).At least two other proteins have been implicated in this process; VCP and RuvbL1/2 (Figure 2).Both proteins are of the AAA-ATPase family and, in concert with various chaperones, are responsible for the ATP dependent extraction of ubiquitinated proteins from membranes or aggregates, followed by entrance into the proteasome or linkage to dynein for retrograde transport.In a recent high throughput screening experiment by Zaarur et al, the RuvbL1 and RuvbL2 homologs were discovered as two critical proteins in the formation of aggresomes in response to proteasomal inhibition.siRNA mediated knockdown of RuvbL1 and RuvbL2 reduced the percent of MG132 treated HeLa cells with aggresomes from ~80% to ~40%.( Zaarur et al, 2015

Figure 2 ;
VCP has indeed been characterized as a kind of ubiquitin "signaling hub", in part due to its ability to bind numerous ubiquitin ligases and DUBs, including Hrd1 and ataxin-3, and its role in numerous ubiquitin-related processes( Stach et al, 2017Meyer et al, 2014 ).While localized to peripheral microaggregates, VCP and RuvbL1/2 probably function to extract, linearize, and facilitate the remodeling of ubiquitinated cargo in order to prime them for retrograde transport.MICROAGGREGATE TRANSPORTHDAC6 & BAG3 Facilitate Retrograde TransportOnce misfolded proteins have been ubiquitinated and extracted from their initial aggregate or membrane, they are retrogradely transported by dynein, the microtubule motor protein.The critical importance of dynein, as opposed to other potential mechanisms of transport, is drawn from the observation that dynein inhibition (or microtubule depolymerization) effectively and consistently blocks aggresome formation( Johnston et al, 2002 ).Misfolded proteins probably do not bind to dynein directly, but are instead linked to dynein through adaptors, most notably HDAC6 (Figure2).Originally discovered as histone deacetylase, HDAC6 has emerged as a central protein in the formation of aggresomes.By harboring both a ubiquitin and dynein binding domain, HDAC6 functions as a dynein adaptor for the transport of ubiquitinated misfolded proteins towards the MTOC.The importance of this mechanism has been illustrated in multiple models of aggresome formation, and, in general, HDAC6 inhibition or deletion results in ~50% less aggresome positive cells and reduced aggresome size ( Kowaguchi et al, 2003 Kalveram et al, 2008 Su et al, 2011 ).Recently, in a mouse model of microglial inflammation induced Tau aggregation, researchers Tseng et al discovered HDAC6 was essential in mediating the aggregation of Tau in vivo ( Tseng et al, 2017 ).The importance of HDAC6's deacetylase activity in aggresome formation is controversial, but may also promote aggresome formation through the deacetylation of tubulin ( Jiang et al, 2008 ).Mechanistic details of how HDAC6 links dynein and ubiquitinated cargo have been complicated by structural observations of HDAC6's ubiquitin-binding domain, which does not actually bind to anchored ubiquitin chains, but instead binds free ubiquitin moieties by a C-terminal diglycine motif ( Ouyang et al, 2011 Turcu et al, 2006 ).In order to reconcile this contradiction, researchers have proposed the possibility that deubiquitinases could cleave ubiquitin moieties in order to bind and activate HDAC6.The ataxin-3 deubiquitinase was an attractive candidate due to its colocalization with both aggresomes and PMA structures.In addition, ataxin-3 can form a complex with VCP and HDAC6 proteins, and also has an apparent preference for editing K63 linked ubiquitin chains ( Burnett et al, 2005 ).Wang et al demonstrated that ataxin-3 can indeed cleave K63 linked ubiquitin moieties to activate proximal HDAC6 proteins, and deletion or mutation of ataxin-3 disrupts aggresome formation (Figure 2; Wang et al, 2011 ).The UCH-L1 deubiquitinase has also been implicated in the activation of HDAC6, and thus aggresome formation, by cleaving ubiquitin chains on misfolded protein ( Yu et al, 2017 ).Besides the deubiquitination of HDAC6 substrate, another important regulator of HDAC6 is the nucleocytoplasmic shuttling protein RanBPM.Researchers Salemi et al serendipitously discovered DNA damage induced the nuclear export and accumulation of RanBPM into HDAC6 positive aggresomes ( Salemi et al, 2014 ).Knockdown of RanBPM following etoposide treatment, which induces double-stranded DNA breaks, reduced aggresome positive cells from 38% in controls to 18%, and HDAC6 positive aggresomes from 19% to 6%.Subsequent analysis revealed RanBPM associates with HDAC6 following DNA damage (or proteasome inhibition) and promotes aggresome formation, but it was not clear how RanBMP's interaction with HDAC6 facilitated this process.Although HDAC6 has received the most attention regarding microaggregate transport, it is important to note that HDAC6 deletion only partially inhibits aggresome formation suggesting there are multiple mechanisms of aggresome transport.Indeed, emerging research has implicated several additional pathways in the transport of misfolded proteins towards the MTOC.A non-canonical mechanism of misfolded protein transport has been discovered that occurs independent of ubiquitination and is dependent on the BAG3 protein.BAG3 was originally implicated by its colocalization with PMAs and aggresomes, and studies since have shown BAG3 knockdown inhibits aggresome formation, and BAG3 overexpression sensitizes cells to aggresome formation ( Gamerdinger et al, 2010 Minoia et al, 2013 Guilburt et al, 2018 Hut et al, 2018 ).This understanding of BAG3 has led to the development of BAG3 inhibitors, which are currently being investigated as a treatment of cystic fibrosis, and function by preventing the aggresomal targeting of mutant CFTR ( Reilly et al, 2017 ).In Alzheimer's Disease, recent research has suggested that BAG3 may instead be insufficiently activated.Work by Fu et al utilized high throughput experiments to probe the selective vulnerability of excitatory neurons in Alzheimer's Disease, and discovered a profound link to insufficient BAG3 expression, resulting in impaired aggresome formation ( Fu et al, 2019 ).Following overexpression, BAG3 induced the perinuclear sequestration and eventual autophagic disposal of tau in vivo.BAG3 has been repeatedly linked to aggresome formation and could be therapeutically utilized to either promote, or inhibit, aggresome formation.BAG3 promotes aggresome formation by functioning as a stress-induced co-chaperone in complex with the HSP70 chaperone.BAG3 binds to the ATPase domain of the HSP70 folding chaperone effectively inhibiting its folding activity and promoting aggresome formation.Pharmacological inhibition of chaperones, including HSP70, does indeed promote aggresome formation ( Gedalya et al, 2011 Muchowski et al, 2000 Miyata et al, 2013 ).By inhibiting HSP70 refolding activity, BAG3 might promote the unloading of HSP70 clients onto dynein for retrograde transport.BAG3 facilitates this process by interacting with dynein, either via a C-terminal proline-rich domain or through the 14-3-3 adaptor protein ( Xu et al, 2013 ).Additionally, BAG3 appears to compete with its homolog BAG1 for the same HSP70 binding domain.By displacing BAG1, which directs HSP70 bound substrate to the proteasome, BAG3 redirects HSP70 substrate to the aggresome( Minoia et al, 2013 ).Since BAG3 expression is uniquely stress-induced, this may provide a mechanism by which proteostatic stressors like MG132 redirect HSP70 substrate towards aggresomes.BAG3 and HSP70 have also been captured in complex with CHIP, and this interaction was shown to promote aggresome formation( Zhang et al, 2011  ).BAG3 appears to be an important mediator in the retrograde transport of misfolded protein, possibly by bridging HSP70 and CHIP associated substrates to dynein for transport to the aggresomes.More recently, BAG3 has been implicated in the preferential binding and targeting of defective ribosomal products (DRIPs) to aggresomes (Figure 2).DRiPs are dysfunctional and misfolded peptides associated with aberrant translation, such as the peptides produced during nonsense-mediated decay of mRNA.These defective peptides are normally degraded through the UPS but accumulate during proteasomal inhibition and, even under basal conditions, represent about 12-15% of all newly synthesized protein ( Wang et al, 2013 ).In a series of experiments, Meriin et al show that BAG3 binds DRiPs in an HSP70 dependent manner, and the loss of ribosomal quality control machinery nearly completely blocks BAG3/HSP70 dependent aggresome formation ( Meriin et al, 2018 ).For example, shRNA mediated knockdown of the ribosome-associated E3 ubiquitin ligase LTN1, or the ribosome-associated chaperones NACA/NACB, blocked the interaction between DRiPs and BAG3/HSP70 complexes and reduced the number of aggresome-positive cells from ~50% to ~5%.These results suggest that the ribosome quality control proteins may provide the source of misfolded proteins that BAG3/HSP70 recognize for transport to the aggresome.Recent work by Park et al elucidated a similar mechanism, whereby DRiPs are recognized by the CED complex, composed of CTIF, eEF1A1, and the dynein subunit DCTN1, and then retrogradely transported to the aggresome (Figure 2; Park et al, 2017 ).Loss of CTIF resulted in the dispersion of aggregates outside of aggresomes during MG132 treatment, consistent with defects in efficient transport.The recognition of DRIPs by BAG3 or the CED complex, and subsequent targeting to aggresomes, is likely an extension of ribosomal surveillance programs, but specific for the defective protein product derived from the initial translation of aberrant mRNA.Since certain mRNA species, such as those containing expanded CAG repeats, are inefficiently translated, DRiPs could potentially be a major source of misfolded protein in certain neurodegenerative diseases ( Jain et al, 2017 Marti, 2016 ).Besides HDAC6, BAG3, and CED, one report also suggests the dynein subunit NDEL1 can directly bind and transport ubiquitinated cargo to the aggresome ( Wan et al, 2012 ).The only negative regulator of aggresome formation that has been elucidated is, interestingly, the Rheb GTPase (Zhou et al, 2009 ).Activated GTP bound Rheb potently inhibited aggresome formation and resulted in widespread dispersion of normally aggresome-sequestered proteins, characteristic of defects in transport.Since Rheb is typically inactivated during starvation, this may explain how glucose deprivation induces aggresome formation, an effect observed in cardiomyocytes ( Marambio et al, 2010 ).Future research will need to carefully assess the relative contribution of each mechanism to the aggresome formation in neurons, especially in animals models of disease.Recent research by Lu et al has demonstrated, using super resolution microscopy, that polyglutamine aggregates reach the aggresome primarily through simple diffusion rather than active transport (Figure 2; Lu et al, 2018 ).According to their analysis only 11% of peripheral aggregates are colocalized with HDAC6, and only 3% appear to be directionally transported.These discoveries are in stark contrast to studies that have reported an essential role of microtubules and dynein for aggresome formation.Using mathematical models Lu et al propose that the initial nucleation of aggresomes requires active transport, such as those described here, but subsequent aggresome growth is driven by random diffusion.Furthermore, Lu et al discovered small immature aggresomes were primarily composed of newly synthesized protein, supporting theories positing DRiPs as a critical source of aggresome protein.This exciting research highlights the utility of super resolution microscopy and its potential to revolutionize our understanding of cellular processes.
is dependent on p62 expression, promoted by p62 overexpression, and highly colocalized with p62, illustrating the indispensability of p62 in aggresome formation( Gal et  al, 2007 Bjørkøy et al, 2005 Pankiv et al, 2007 ).Additionally, p62 expression appears to be highly sensitive to proteasome inhibition, suggesting aggresome formation and p62 expression are coordinated by the same proteostatic pressures.A recent study by Sha et al found proteasome inhibition in neuroblastoma cells rapidly upregulated p62 and GABARAP1 expression, an effect contingent on the Nrf1 transcription factor (Figure 3; Sha et al, 2018 ).Consistent with these results, previous research has shown neuron-specific Nrf1 knockout mice accumulate soluble nonaggregated ubiquitinated proteins ( Kobayashi et al, 2011 ).A similar phenotype of ubiquitinated cargo accumulation results following p62 deletion, and thus could be functionally related.The inhibition of the proteasome, which is accompanied by p62 upregulation and aggresome formation, may also be facilitated by p62 itself.By forming a complex with the ubiquitin-specific protease USP10, p62 potently inhibits proteasomes resulting in aggresome formation ( Takahashi et al, 2018 ).Thus the physiological inhibition of proteasomes, which is frequently observed in neurodegenerative disease and well known to induce aggresome formation, could actually be a regulated and cytoprotective mechanism used to upregulate p62.As a working model, perhaps USP10/p62 complexes inhibit the proteasome triggering Nrf1 dependent p62 upregulation and, ultimately, facilitates aggresome biogenesis.Once p62 expression has been sufficiently activated, how exactly does it promote aggregation?P62's critical role in facilitating protein aggregation appears to be dependent on its ubiquitin binding domain UBA, and its protein binding PB1 domain ( Zaffagnini et al, 2018 Cabe et al, 2018 ).The UBA domain of p62 is required for the localization of p62 to ubiquitinated aggregates, and has a preference for the K63 linked ubiquitin chains that are enriched at aggresomes ( Seibenhener et al, 2004 ).As discussed above, these K63 ubiquitin chains are generated by E3 ubiquitin ligases such as Hrd1, CHIP, and PARKIN, each of which localizes to ____________________________________________________ 11 MECHANISMS OF AGGRESOME BIOGENESIS AUTHOR MANUSCRIPT Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 21 January 2019 Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 21 January 2019 doi:10.20944/preprints201812.0332.v2aggresomes.During mitophagy, PARKIN is essential for the mitochondrial localization of both p62 and HDAC6 by generating K63 linked ubiquitin chains, and thus a similar mechanism could occur at aggresomes ( Lee et al, 2010 ).These K63 ubiquitin chains may then be released by various DUBs, which are indeed catalytically active at aggresomes, and thereby allow additional K63 chains to be generated ( Juenemann et al, 2018 ).For example, Nunduri et al have described an essential role for Poh1, a proteasome-bound deubiquitinase, in aggrephagy, where it functions to cleave and release K63 linked ubiquitin chains that then activate HDAC6, but could also recruit p62 (Figure 3; Nanduri et al, 2015 ).Similarly, the inhibition of other proteasome-associated DUBs by the small molecule b-AP15 induces cancer cell death by preventing cytoprotective aggresome formation, an effect that could be related to deficient p62 or HDAC6 activation ( Hillert et al, 2018 ).It seems plausible, then, that various K63 ubiquitin ligases, in conjunction with deubiquitinases, coordinate the local saturation of K63 ubiquitin chains near aggresomes to recruit p62 en mass.Recruitment of p62, driven by its UBA domain, is then followed by PB1 domain dependent oligomerization.PB1 domains facilitate protein interactions by binding other PB1 domains thereby promoting protein polymerization.The capacity for p62 to oligomerize is exemplified in autophagy-deficient cells, where the inability to turnover p62 by autophagy leads to its accumulation and aggregation driven by PB1 domain interactions ( Wang et al, 2016 Sukseree et al, 2018 ).PB1 dependent oligomerization is critical for p62's ability to stably bind ubiquitin and LC3B, and thereby initiate aggrephagy ( Wurzer et al, 2015 ).In the context of aggresome formation, p62's PB1 domain probably co-aggregates with aggresomes and promotes liquid-liquid phase separation.Supporting this notion, the PB1 domain of p62 is essential to p62's localization to aggresomes, and its deletion prevents the formation of aggresomes ( Cabe et al, 2018 ).Furthermore, the presence of polyubiquitinated proteins, or even unanchored free K63 ubiquitin chains, enhances PB1 dependent p62 aggregation ( Herhaus et al, 2018 ).P62 oligomerization at aggresomes may also be promoted by other autophagic cargo receptors such as ALFY, discovered to relocalize to aggresomes in a p62 dependent manner and facilitate aggresome formation ( Clausen et al, 2010 ).Thus increased local concentrations of p62 at aggresomes, and the abundant presence of ubiquitinated proteins, both promote p62 oligomerization.Additionally, if PMA transport is indeed principally driven by diffusion, as described in Lu et al, then perhaps p62 could function to capture and then facilitate the absorption of PMAs into the aggresome.In sum, p62 facilitates aggresome formation by first localizing to aggregates through preferentially binding of K63 linked ubiquitin chains followed by regulated oligomerization, perhaps stabilizing the growing aggresome and priming it for aggrephagy.Several post-translational modifications have been identified that greatly enhance p62's propensity to localize to and stabilize aggresomes.In particular, the phosphorylation of serine 272 and threonine 269 by the p38 MAP kinase is essential for p62 mediated aggresome formation, and p38 deletion dramatically reduced aggresome formation by nearly 90% (Figure 3; Zhang et al, 2018 Rubio et al, 2014 ).In support of these findings, Meriin et al discovered that, following proteasome inhibition, the DRiP-associated BAG3/HSP70 protein localization and repair, aggresomal sequestration of ubiquitin could starve cells of ubiquitin resulting in a chronically defective DNA damage response( Yehuda et al, 2017 ).Indeed, cells harboring aggresomes are depleted of ubiquitin and deficient in histone ubiquitination modifications.This potential mechanism of cytotoxicity is intriguing since a large body of research has centered around the relationship between neuronal DNA damage and various neurodegenerative diseases (Reviewed in Madabhushi et al, 2014 ).Aggresomes sequester other critical components of the ubiquitin protein system as well, including proteasomes and folding chaperones, potentially fueling a positive feedback cycle exacerbating protein misfolding (Figure 3; Guo et al, 2018 ).Supporting this notion, the presence of an aggresome does accelerate the formation of PMAs, which could be explained by the sequestration of machinery capable of thwarting or disaggregating PMAs (Figure 3; Sahl et al, 2016 ).Moreover, by forming at the MTOC, the presence of aggresomes has also been linked to the disruption of the centrosome and the nuclear membrane.The centrosome is critical to the formation of cilia and thus, not surprisingly, the accumulation of misfolded proteins around this organelle severely disrupts the formation and function of cilia (Figure 3; Iqbal et al, 2017 Lam et al, 2013 ).Aberrant cilia, like DNA damage, has been proposed to contribute to neurodegenerative processes including dysfunctional metabolic and autophagic signaling ( Brown & Witman, 2014 Kaliszewski et al, 2015 ).Since olfactory neurons are uniquely reliant on cilia for sensory transduction, aggresome mediated ciliary defects would, in theory, manifest initially as olfactory sensory deficits, which is indeed among the first clinical symptoms of various neurodegenerative diseases.Centrosomes also periodically spawn "satellites", large macromolecular complexes that crawl up and down microtubules to regulate activities such as autophagy and stress signaling (Reviewed in Joachim & Tooze, 2018 and Vertii et al, 2016 ).Centriolar satellites might become incorrigibly entangled within the intricate mesh of misfolded protein as they navigate through aggresomes.And finally, the aggresome's perinuclear proximity has been linked to nuclear membrane distortions.The physiological effect of nuclear membrane abnormalities has been extensively studied in progerias resulting from mutations in the nuclear membrane support protein lamin A. These mutations disrupt transcriptional programs, chromatin organization, and various signaling pathways (Reviewed in Gruenbaum & Foisner, 2015 ).In HD, nuclear membrane abnormalities from perinuclear huntingtin aggregates have been linked to aberrant cell cycle signaling and nucleocytoplasmic shuttling defects (Figure 3; Liu et al, 2014 , Rosa et al, 2017 ).In summary, the proteostatic relief offered by aggresomes may only be temporary as abnormalities in DNA, cilia, or nuclear membranes eventually culminate in cell death.Although great strides have been made in our understanding of aggresome biogenesis and disposal, many essential questions remain.Most importantly, are aggresomes between diseases truly the same structure and should they be treated as such?As the repertoire of proteins and pathways by which aggresomes are formed expands, the prospect of a single unified model of aggresome biogenesis breaks down.Despite this, there are several general mechanics conserved between aggresomes in neurodegenerative diseases, such as the importance of p62, ubiquitin, autophagy, or chaperones.A better understanding of aggresomes may be the key to deciphering the fundamental cellular dysfunction arising from protein misfolding, and could ultimately explain the pleiotropic nature of cellular pathology in neurodegenerative disease.