InteractiveFly: GeneBrief

Histone deacetylase 6: Biological Overview | References

Gene name - Histone deacetylase 6

Synonyms -

Cytological map position - 13B6-13B6

Function - enzyme

Keywords - protein degradation, stress response, compensatory autophagy, 'histone deacetylase'

Symbol - HDAC6

FlyBase ID: FBgn0026428

Genetic map position - X: 15,226,988..15,234,388 [+]

Classification - protein deacetylase and zf-UBP

Cellular location - cytoplasmic

NCBI links: EntrezGene

HDAC6 orthologs: Biolitmine
Recent literature
Perry, S., Kiragasi, B., Dickman, D. and Ray, A. (2017). The role of Histone Deacetylase 6 in synaptic plasticity and memory. Cell Rep 18: 1337-1345. PubMed ID: 28178513
Histone deacetylases (HDACs) have been extensively studied as drug targets in neurodegenerative diseases, but less is known about their role in healthy neurons. This study tested zinc-dependent HDACs using RNAi in Drosophila melanogaster and found memory deficits with RPD3 and HDAC6. HDAC6 was found to be required in both the larval and adult stages for normal olfactory memory retention. Neuronal expression of HDAC6 rescues memory deficits, and the N-terminal deacetylase (DAC) domain is required for this ability. This suggests that deacetylation of synaptic targets associated with the first DAC domain, such as the active-zone scaffold Bruchpilot, is required for memory retention. Finally, electrophysiological experiments at the neuromuscular junction reveal that HDAC6 mutants exhibit a partial block of homeostatic plasticity, suggesting that HDAC6 may be required for the stabilization of synaptic strength. The learning deficit observed in HDAC6 mutants could be a behavioral consequence of these synaptic defects.
Beaver, M., Bhatnagar, A., Panikker, P., Zhang, H., Snook, R., Parmar, V., Vijayakumar, G., Betini, N., Akhter, S. and Elefant, F. (2020). Disruption of Tip60 HAT mediated neural histone acetylation homeostasis is an early common event in neurodegenerative diseases. Sci Rep 10(1): 18265. PubMed ID: 33106538
Epigenetic dysregulation is a common mechanism shared by molecularly and clinically heterogenous neurodegenerative diseases (NDs). Histone acetylation homeostasis, maintained by the antagonistic activity of histone acetyltransferases (HATs) and histone deacetylases (HDACs), is necessary for appropriate gene expression and neuronal function. Disruption of neural acetylation homeostasis has been implicated in multiple types of NDs including Alzheimer's disease (AD), yet mechanisms underlying alterations remain unclear. This study shows that like AD, disruption of Tip60 HAT/HDAC2 balance with concomitantm epigenetic repression of common Tip60 target neuroplasticity genes occurs early in multiple types of Drosophila ND models such as Parkinson's Disease (PD), Huntington's Disease (HD) and Amyotrophic Lateral Sclerosis (ALS). Repressed neuroplasticity genes show reduced enrichment of Tip60 and epigentic acetylation signatures at all gene loci examined with certain genes showing inappropriate HDAC2 repressor enrichment. Functional neuronal consequences for these disease conditions are reminiscent of human pathology and include locomotion, synapse morphology, and short-term memory deficits. Increasing Tip60 HAT levels specifically in the mushroom body learning and meory center in the Drosophila brain protects against locomotion and short-term memory function deficits in multiple NDs. Together, these results support a model by which Tip60 protects against neurological impairments in different NDs via similar modes of action.
Zhang, H., Karisetty, B. C., Bhatnagar, A., Armour, E. M., Beaver, M., Roach, T. V., Mortazavi, S., Mandloi, S. and Elefant, F. (2020). Tip60 protects against amyloid-beta-induced transcriptomic alterations via different modes of action in early versus late stages of neurodegeneration. Mol Cell Neurosci 109: 103570. PubMed ID: 33160016
Alzheimer's disease (AD) is an age-related neurodegenerative disorder hallmarked by amyloid-β (Aβ) plaque accumulation, neuronal cell death, and cognitive deficits that worsen during disease progression. Histone acetylation dysregulation, caused by an imbalance between reduced histone acetyltransferases (HAT) Tip60 and increased histone deacetylase 2 (HDAC2) levels, can directly contribute to AD pathology. However, whether such AD-associated neuroepigenetic alterations occur in response to Aβ peptide production and can be protected against by increasing Tip60 levels over the course of neurodegenerative progression remains unknown. This study profiled Tip60 HAT/HDAC2 dynamics and transcriptome-wide changes across early and late stage AD pathology in the Drosophila brain produced solely by human amyloid-β(42). Early Aβ(42) induction leads to disruption of Tip60 HAT/HDAC2 balance during early neurodegenerative stages preceding Aβ plaque accumulation that persists into late AD stages. Correlative transcriptome-wide studies reveal alterations in biological processes were classified as transient (early-stage only), late-onset (late-stage only), and constant (both). Increasing Tip60 HAT levels in the Aβ(42) fly brain protects against AD functional pathologies that include Aβ plaque accumulation, neural cell death, cognitive deficits, and shorter life-span. Strikingly, Tip60 protects against Aβ(42)-induced transcriptomic alterations via distinct mechanisms during early and late stages of neurodegeneration. These findings reveal distinct modes of neuroepigenetic gene changes and Tip60 neuroprotection in early versus late stages in AD that can serve as early biomarkers for AD, and support the therapeutic potential of Tip60 over the course of AD progression.

A prominent feature of late-onset neurodegenerative diseases is accumulation of misfolded protein in vulnerable neurons. When levels of misfolded protein overwhelm degradative pathways, the result is cellular toxicity and neurodegeneration. Cellular mechanisms for degrading misfolded protein include the ubiquitin-proteasome system (UPS), the main non-lysosomal degradative pathway for ubiquitinated proteins, and autophagy, a lysosome-mediated degradative pathway. The UPS and autophagy have long been viewed as complementary degradation systems with no point of intersection. This view has been challenged by two observations suggesting an apparent interaction: impairment of the UPS induces autophagy in vitro, and conditional knockout of autophagy in the mouse brain leads to neurodegeneration with ubiquitin-positive pathology. It is not known whether autophagy is strictly a parallel degradation system, or whether it is a compensatory degradation system when the UPS is impaired; furthermore, if there is a compensatory interaction between these systems, the molecular link is not known. This study shows that autophagy acts as a compensatory degradation system when the UPS is impaired in Drosophila melanogaster, and that histone deacetylase 6 (HDAC6; Barlow, 2001), a microtubule-associated deacetylase that interacts with polyubiquitinated proteins (Kawaguchi, 2003), is an essential mechanistic link in this compensatory interaction. Compensatory autophagy was induced in response to mutations affecting the proteasome and in response to UPS impairment in a fly model of the neurodegenerative disease spinobulbar muscular atrophy. Autophagy compensates for impaired UPS function in an HDAC6-dependent manner. Furthermore, expression of HDAC6 os sufficient to rescue degeneration associated with UPS dysfunction in vivo in an autophagy-dependent manner. This study suggests that impairment of autophagy (for example, associated with ageing or genetic variation) might predispose to neurodegeneration. Moreover, these findings suggest that it may be possible to intervene in neurodegeneration by augmenting HDAC6 to enhance autophagy (Pandey, 2007).

DTS7 is a temperature sensitive, dominant negative mutant of the β2 subunit of the proteasome (Smyth, 1999). Using the UAS/GAL4 system12, DTS7 expression was targeted to the Drosophila eye to cause tissue-restricted proteasome impairment. At 22°C, proteasome function is intact and eye morphology was normal (Fig. 1a). However, at 28°C substantial degeneration of the retina occurs owing to proteasome impairment. To investigate the role of HDAC6 in the setting of misfolded protein stress, transgenic flies were generated expressing wild-type Drosophila HDAC6 as well as wild-type and mutant versions of human HDAC6. Expression of either Drosophila HDAC6 or human HDAC6 strongly suppresses the degenerative phenotype associated with proteasome impairment. However, expression of a catalytically dead mutant of human HDAC6 (H216A;H611A) failed to modify the degenerative phenotype, indicating that the deacetylase function of HDAC6 is required for suppression. To assess the role of endogenous HDAC6, RNAi knockdown was used. Targeted knockdown of Drosophila HDAC6 did not noticeably alter eye morphology on its own, but strongly enhanced degeneration when the proteasome was impaired. HDAC6 did not modify the rough eye phenotype caused by ectopic expression of the positive regulator of cell death reaper, indicating that HDAC6 is not a general suppressor of cell death pathways. Ectopic expression of Drosophila HDAC3 and Drosophila HDAC11 did not suppress degeneration caused by proteasome impairment, indicating that this is not a general response of HDACs (Pandey, 2007).

Impaired UPS function has been implicated in a broad array of neurodegenerative disorders, but in vivo evidence is lacking. Spinobulbar muscular atrophy (SBMA) is an inherited neurodegenerative disease that is caused by polyglutamine (polyQ) repeat expansion in the androgen receptor (AR). Like most adult-onset neurodegenerative diseases, SBMA pathology features accumulation of ubiquitin-positive protein aggregates in vulnerable neurons. To develop a Drosophila model of SBMA, transgenic flies were generated expressing full-length human AR with 12-121 glutamine repeats using the UAS/GAL4 system. Flies expressing polyQ-expanded AR recapitulate key features of human SBMA, including ligand-dependent, polyQ length-dependent degeneration (Pandey, 2007).

To evaluate UPS function in this fly model of SBMA, transgenic flies were generated expressing a fluorescent reporter of UPS function. CL1-GFP is a fusion protein created by introducing a degradation signal to otherwise stable green fluorescent protein (GFP). This protein is rapidly degraded by the UPS, and its steady state levels reflect the functional status of this pathway. When stable GFP was expressed in eye imaginal discs from third-instar larvae, a robust fluorescent signal was detected by confocal microscopy. In contrast, eye imaginal discs from control flies expressing the CL1-GFP reporter emitted a low fluorescent signal, reflecting an active UPS. To test the ability of the UPS reporter flies to detect proteasome impairment in vivo, CL1-GFP was co-expressed in the eye with DTS7. At 22°C, CL1-GFP reporter levels remained low in eye imaginal discs co-expressing DTS7, consistent with normal proteasome function. In contrast, at 28°C, there was a significant increase in the CL1-GFP signal, demonstrating the ability of the reporter to detect proteasome impairment associated with a degenerative phenotype in vivo. UPS reporter RNA levels were not altered by the conditions used in these experiments (Pandey, 2007).

The CL1-GFP reporter in SBMA flies. In AR121 flies not exposed to ligand, fluorescent signal from the UPS reporter remained low, indicating that proteasome function was normal despite high expression of polyQ-expanded AR. However, flies reared on dihydrotestosterone (DHT), the natural ligand of AR, exhibited a significant increase in reporter signal, indicating proteasome impairment in association with induction of toxicity. The ligand-dependent nature of this finding indicates that UPS impairment is not merely a consequence of overexpressed AR121. Proteasome impairment by AR expression is a polyQ length-dependent phenomenon, because no impairment was observed in flies expressing AR12. The finding of proteasome impairment in SBMA flies is consistent with a prior report that polyQ toxicity in vivo is enhanced by proteasome mutations (Pandey, 2007).

The determination that there is impairment of the UPS in SBMA flies led to an examination of the ability of HDAC6 to modify the degenerative phenotype in this model of human neurodegenerative disease. Consistent with the results using proteasome mutant flies, ectopic expression of either Drosophila or human HDAC6 suppressed the ligand-dependent degenerative phenotype in flies expressing polyQ-expanded AR. Expression of the catalytically dead mutant of human HDAC6 (H216A;H611A) failed to modify the degenerative phenotype, indicating that the deacetylase function of HDAC6 is also required for suppression of polyQ toxicity. Knockdown of endogenous HDAC6 with RNAi enhanced ligand-dependent degeneration in AR52 flies. Thus, endogenous HDAC6 also plays a role in protecting cells from polyQ toxicity (Pandey, 2007).

Induction of autophagy and sequestration of polyQ-expanded AR in autophagic vacuoles has been reported in vitro (Taylor, 2003). Induction of autophagy in vitro in response to proteasome impairment has also been described. To determine whether autophagy is induced in vivo when the UPS is impaired, ultrastructural evaluation was performed by transmission electron microscopy (TEM) in the DTS7 and SBMA flies. In both cases, a significant increase was found in morphological features of autophagy. These included autophagic vacuoles such as early autophagosomes in which membranes surrounded cytoplasmic components, more mature autophagic vacuoles, multilamellar bodies and multivesicular bodies (Pandey, 2007).

To assess the role of autophagy when the UPS is impaired, autophagy was inhibited by RNAi knockdown of the autophagy genes atg6 and atg12. Knockdown of either atg6 or atg12 did not affect eye morphology, indicating that the Drosophila eye can tolerate reduced autophagy when UPS function is intact, at least in 1-day-old flies. In contrast, knocking down either atg6 or atg12 strongly enhanced the rough eye phenotype associated with UPS impairment in DTS7 flies reared at 28°C and in AR52 flies reared on DHT. From these data, it can be inferred that the autophagy induced by UPS impairment is compensatory (Pandey, 2007).

It was hypothesized that ectopic expression of HDAC6 suppressed degeneration by promoting autophagic degradation of aberrant protein. Thus, AR levels were examined in vivo, and it was determined that expression of HDAC6 leads to lower steady state levels of polyQ-expanded AR in vivo, whereas inhibition of autophagy by knockdown of atg6 or atg12 resulted in higher steady state levels. These altered steady state levels occurred despite no significant change in RNA levels, suggesting that HDAC6 accelerates the rate of AR degradation. To investigate this further, the inducible Geneswitch expression system was adapted to monitor protein turnover. In elav-GS;UAS-AR52 flies, no expression was detected before exposure to the inducing agent RU486. To induce expression, starved flies were fed sucrose media containing RU486 for one hour, which resulted in a pulse of expression that became detectable within 2 h, peaked after approximately 10 h, and then gradually decayed with a half-life of 100 min. In elav-GS;UAS-AR52;UAS-dHDAC6 flies, there was a parallel induction of AR52 expression, but an accelerated rate of decay, with a half-life of 50 min. Importantly, co-expression of Drosophila HDAC6 accelerated the turnover of not only AR52 monomers, but also high molecular weight aggregates that were trapped in the stacking gel (Pandey, 2007).

It was determined that treatment with rapamycin suppressed degeneration caused by either proteasome impairment or polyQ toxicity. This finding is consistent with a prior report, in which rapamycin suppressed degeneration in fly and mouse models of Huntington's disease. Rescue by rapamycin has been attributed to inhibition of TOR and induction of autophagy, although a role for other TOR-regulated pathways could not be excluded. This study found that knockdown of atg12 blocked the ability of rapamycin to suppress degeneration when the proteasome was impaired, verifying that rapamycin rescue is autophagy-dependent. Importantly, it was also determined that knockdown of Drosophila HDAC6 blocks the ability of rapamycin to suppress degeneration, indicating that Drosophila HDAC6 is essential in order for induction of autophagy to compensate for proteasome impairment. Furthermore, it was determined that the ability of Drosophila HDAC6 to suppress degeneration was autophagy-dependent, since rescue was blocked by knockdown of atg12. Thus, HDAC6 is integral to rescue of degeneration by autophagy and essential for autophagy to compensate for impaired UPS function (Pandey, 2007).

These findings extend previous studies in three important ways. First, it was determined that induction of autophagy is sufficient to rescue degeneration associated with UPS impairment, dramatically illustrating the compensatory relationship between autophagy and the UPS. Second, it was determined that HDAC6 activity is essential for autophagy to compensate for impaired UPS function. Finally, it was determined that ectopic expression of HDAC6 alone is sufficient to rescue degeneration caused by proteasome mutations and polyQ toxicity, and does so in an autophagy-dependent manner. These observations are consistent with a mechanism in which HDAC6 facilitates turnover of aberrant proteins by autophagy, lowering their steady state levels and mitigating toxicity. It was recently determined that overexpression of HDAC6 also suppressed degenerative phenotypes in additional models of neurodegenerative disease, including flies expressing pathologic A fragments and other polyQ-expanded proteins. Thus, the HDAC6-mediated pathway of protein clearance may have broad relevance to degenerative proteopathies (Pandey, 2007).

Although the current study indicates that the mechanism of HDAC6 rescue involves accelerated turnover of misfolded protein by autophagy, further study is required to determine the precise details of how this occurs. The mechanism could involve modulation of HSP90 activity, since this chaperone is a substrate of HDAC6 deacetylase activity (Kovacs, 2005). Alternatively, HDAC6 may be involved in shuttling polyubiquitinated substrates to a location conducive to engulfment by autophagosomes, consistent with a known role for HDAC6 in the formation of aggresomes in vitro (Kawaguchi, 2003). A third possibility is that HDAC6 may contribute to the transport of lysosomes to the site of autophagy, as suggested by the observation that HDAC6 knockdown results in dispersal of lysosomes away from the microtubule organizing centre (Iwata, 2005). Elucidating the precise role of HDAC6 in linking autophagy and the UPS promises substantial insights into cellular management of misfolded protein (Pandey, 2007).

The deacetylase HDAC6 is a novel critical component of stress granules involved in the stress response

An essential part of the cellular response to environmental stress is a reversible translational suppression, taking place in dynamic cytoplasmic structures called stress granules (SGs). In a study carried out in murine cultured cells, it was discovered that HDAC6, a cytoplasmic deacetylase that acts on tubulin and HSP90 and also binds ubiquitinated proteins with high affinity, is a novel critical SG component. HDAC6 interacts with another SG protein, G3BP (Ras-GTPase-activating protein SH3 domain-binding protein 1), and localizes to SGs under all stress conditions tested. Pharmacological inhibition or genetic ablation of HDAC6 abolishes SG formation. Intriguingly, it was found that the ubiquitin-binding domain of HDAC6 is essential and that SGs are strongly positive for ubiquitin. Moreover, disruption of microtubule arrays or impairment of motor proteins also prevents formation of SGs. These findings identify HDAC6 as a central component of the stress response, and suggest that it coordinates the formation of SGs by mediating the motor-protein-driven movement of individual SG components along microtubules (Kwon, 2007).

Reversible protein acetylation has emerged in recent years as one of the major forms of protein modifications whose importance has been particularly well documented in the case of the N-terminal histone tails, and of a few transcription factors such as p53 and STAT3. Acetylation and deacetylation are catalyzed by histone acetylases (HATs) and histone deacetylases (HDACs). HDAC6 is a unique class II deacetylase that contains two catalytic domains and also a C-terminal zinc finger domain (ZnF-UBP) binding with high-affinity free ubiquitin as well as mono- and polyubiquitinated proteins. HDAC6 is actively maintained in the cytoplasm (Verdel, 2000; Bertos, 2004), where it is found partly associated with the microtubule network. HDAC6 can deacetylate tubulin as well as the microtubule network in vivo (Hubbert, 2002; Matsuyama, 2002; Zhang, 2003). HDAC6 associates with the chaperone-like AAA ATPase p97/VCP, a protein that is critical for proteasomal degradation of misfolded proteins. Thereby, the ratio of HDAC6 and p97/VCP modulates the levels of polyubiquitinated aggregates (Boyault, 2006). HDAC6 also facilitates the clearance of misfolded ubiquitinated proteins, promoting their accumulation in an aggresome, and protects cells from apoptosis following stress induced by misfolded proteins (Kawaguchi, 2003). At the same time, HDAC6 also controls the induction of heat-shock proteins in response to the accumulation of ubiquitinated protein aggregates (Boyault, 2007). Furthermore, HDAC6 can deacetylate the chaperone Hsp90 and regulate its activity (Bali, 2005; Kovacs, 2005). Consequently, these different biochemical functions of HDAC6 impinge on diverse cellular processes. For example, HDAC6 function was found to be necessary for the formation of an immune synapse between antigen-presenting cells and T lymphocytes and also for nuclear translocation and transcription activation by the glucocorticoid receptor. Mice lacking HDAC6 are viable despite having highly elevated tubulin acetylation in multiple organs; in addition, they exhibit a moderately impaired immune response and also a mild phenotype in the bone (Kwon, 2007 and references therein).

One of the most immediate responses to cellular stress is a reversible block of mRNA translation, triggered by phosphorylation of the translation initiation factor eIF2α under the action of several stress-sensing kinases such as PKR or PERK. Thereby, translationally stalled mRNAs are sequestered in dynamic cytoplasmic structures called stress granules (SGs). These granules represent a complex assembly of initiation factors, such as eIF3 or eIF4E; proteins involved in translation control, such as T-cell intracellular antigen (TIA-1) or Fragile X mental retardation protein (FMR1); proteins implicated in RNA remodeling or degradation, such as HuR, tristetraproline (TTP), or Staufen; as well as 40S ribosome subunits. In addition, SGs also contain various polyadenylated mRNAs whose translation has been arrested. However, mRNAs encoding stress-induced proteins, such as heat-shock proteins, are excluded from SGs and are spared from translational inhibition. It is thought that SGs are sites where triage takes place in order to direct RNAs to degradation in processing bodies (PBs), or to recycle mRNAs for translation. In addition, very recent evidence suggests that parts of the microRNA pathway may also take place in SGs that contain Argonaute proteins and microRNAs such as let-7 (Kwon, 2007 and references therein).

This study reports the identification of G3BP-1 (RasGTPase-activating protein SH3 domain-binding protein 1, hereafter G3BP), an SG component, as a novel protein interacting with HDAC6 in vivo and in vitro. This protein is conserved between species, and orthologs are found in Drosophila, humans, and mice. G3BP has been implicated in modulating Ras activity and the cell cycle, by binding to the RasGAP protein. The precise function of G3BP is not understood yet, but it appears to be essential in the mouse where inactivation of the G3BP gene leads to embryonic lethality and growth retardation. G3BP has attracted attention recently as it was found to increase decay of the c-Myc RNA and to localize to SGs. This study shows that HDAC6 is recruited to SGs and that pharmacological HDAC inhibition leads to impaired SG assembly. Indeed, HDAC6-deficient mouse embryo fibroblasts (MEFs) fail to form SGs, although they exhibit normal phosphorylation of eIF2α in response to stress. Furthermore, inactivating mutations in the catalytic domains or in the ZnF-UBP domain of HDAC6 impair SG assembly. Moreover, SG formation is abolished by disruption of microtubule arrays or by impairment of dynein motor proteins. HDAC6 is required for the cells to recover from oxidative stress: In the absence of intact HDAC6 function, cells that have been treated with arsenite undergo apoptosis. Based on these results, it is proposed that HDAC6 is a central component of the stress response, regulating SG formation and potentially contributing to the control of RNA metabolism and translation (Kwon, 2007).

HDAC6 suppresses age-dependent ectopic fat accumulation by maintaining the proteostasis of PLIN2 in Drosophila

Age-dependent ectopic fat accumulation (EFA) in animals contributes to the progression of tissue aging and diseases such as obesity, diabetes, and cancer. However, the primary causes of age-dependent EFA remain largely elusive. This study characterized the occurrence of age-dependent EFA in Drosophila and identified HDAC6, a cytosolic histone deacetylase, as a suppressor of EFA. Loss of HDAC6 leads to significant age-dependent EFA, lipid composition imbalance, and reduced animal longevity on a high-fat diet. The EFA and longevity phenotypes are ameliorated by a reduction of the lipid-droplet-resident protein PLIN2. HDAC6 was found to be associated physically with the chaperone protein dHsc4/Hsc70 to maintain the proteostasis of PLIN2. These findings indicate that proteostasis collapse serves as an intrinsic cue to cause age-dependent EFA. This study suggests that manipulation of proteostasis could be an alternative approach to the treatment of age-related metabolic diseases such as obesity and diabetes (Yan, 2017).

Age-dependent EFA occurs in mammals as a hallmark of aging and contributes to age-related tissue deterioration and dysfunction. This study used a Drosophila model to assess the molecular basis of age-dependent EFA formation. Age-dependent EFA appears mainly in the thoracic jump muscles of adult flies in an age-dependent manner. Further, proteostatic regulators, dHDAC6 and dHs4, are identified to suppress age-dependent EFA. The genetic and biochemical data indicate that dHDAC6 maintains the proteostasis of lipid droplet protein PLIN2 by modulating the acetylation level of dHsc4. The dHDAC6-dHsc4-PLIN2 axis links proteostasis to fat metabolism during aging. These results also highlight that it is the protein quality rather than the protein quantity of PLIN2 that controls age-dependent EFA (Yan, 2017).

PLIN2, belonging to the PAT family, is an lipid droplet (LD) coating protein that has been shown to play important roles in the formation and turnover of LDs in non-adipose tissues such as the skeletal muscle, pancreas, gonads, and gut. PLIN2 accumulates in human muscle with age and is associated with muscle weakness, obesity, and diabetes. Since the activity of both ubiquitin-proteasome and lysosome weakens during aging, it is plausible to infer that the increase in PLIN2 protein levels in aged individuals are caused by lowered activity of either ubiquitin-proteasome or lysosome. The results demonstrate that the degradation of PLIN2 is mediated by dHDAC6 through chaperone dHsc4-assisted autophagy but not macro-autophagy, and that the quality but not the quantity of PLIN2 plays an important role in EFA formation and tissue dysfunction during aging. The substrates of chaperone Hsc70/dHsc4 exhibit a consensus pentapeptide KFERQ motif, and Hsc70 has been reported to mediate the degradation of PAT family proteins, PLIN2 and PLIN3, in mouse. This study excluded the possibility that dHsc4/Hsc70 mediates the degradation of PLIN2 through the CMA machinery based on the following evidence: First, no conserved KREFQ motif specific for CMA degradation was found in Drosophila PLIN2; second, a mutant form of Drosophila PLIN2 was made in the non-canonical KREFQ motif region, which did not show any decreased degradation rate; Third, CMA degradation in mammals requires the lysosome receptor LAMP2A, however, Lamp1, the Drosophila homolog of LAMP2A, is not involved in age-dependent EFA. Therefore, it is speculated that dHsc4/Hsc70 may mediate the degradation of PLIN2 through chaperone-assisted selective autophagy, which involves co-chaperones and ubiquitination to degrade mainly insoluble proteins. Supporting this hypothesis, a significant amount of ubiquitinated aggregates were detected to accumulate on the surface of LDs in the jump muscles of dHDAC6 mutants, which colocalize with PLIN2. On the other hand, several co-chaperones (Dnaj-1, HspB8, Dnaj-2, mrj, and CG5001) and the E3 ligase CHIP were tested, but none of them were required for the chaperone-assisted selective autophagy process to lead to EFA. It is speculated that there may be another unknown co-chaperone(s) that functions with dHDAC6/dHsc4 in Drosophila (Yan, 2017).

Recently, studies show that PLIN2 is associated with the progression of age-related diseases, such as insulin resistance, fatty liver, type 2 diabetes, sarcopenia, and cancer. All the diseases reported thus far that are associated with PLIN2 are linked to aging, implying that the changes in PLIN2 during aging might have a pivotal contribution to the severity of these age-related diseases. This study assessed the changes in soluble and insoluble PLIN2 protein levels during aging and showed that only the insoluble PLIN2 protein level was increased and associated with the increase in age-dependent EFA in the jump muscle. The results suggest a possibility of improving the proteostasis of PLIN2 as an efficient way to ameliorate the progressive defects of age-related metabolic diseases (Yan, 2017).

Another question is how increased insoluble PLIN2 can cause increased EFA in aging muscle. Insoluble proteins exhibit hydrophobic aggregation properties and LDs containing a hydrophobic core are prone to act as an anchoring site for hydrophobic proteins. Thus, it is proposed that insoluble PLIN2 is prone to be anchored on the LDs to sequester more hydrophobic lipases. Anchored insoluble PLIN2 or insoluble PLIN2 aggregates prevent triglyceride lipases from reaching the LD surface to mediate lipid breakdown. In support of this hypothesis, the data show that increased LD accumulation in the jump muscle of the dHDAC6 mutant could not be reverted by overexpression of lipases such as Bmm or dHSL. However, more investigations are needed to explore precisely how the proteostasis of PLIN2 affects LD turnover in the aging process and whether the proteostasis of PLIN2 may also be involved in other physiological processes (Yan, 2017).

The maintenance of proteostasis in organelles, such as the endoplasmic reticulum, mitochondrion, and the nucleus, involves specialized cellular compartments. Mitochondrial proteostasis requires mitochondrial chaperones, ATFS-1 signaling, and GCN2 signaling to activate mitochondrial unfolded protein response (UPRmt); whereas nuclear proteostasis requires nuclear envelope, nuclear pore complexes, and transport pathways. In Drosophila, proteostasis of the muscles controls systemic aging and requires Foxo/4E-BP signaling and Activin signaling. As the primary site of lipid metabolism, LDs are considered as dynamic organelles, but little is known about how proteostasis of LDs is maintained. This study identified that the chaperone dHsc4 and the deacetylase dHDAC6 play key roles in maintaining LD proteostasis. More importantly, proteostasis of the LD protein PLIN2 links the proteostasis network to age-dependent EFA (Yan, 2017).

Age-dependent EFA appears mainly in the tubular jump muscle, but not in the fibrous indirect flight muscle, which is a large part of adult thoracic muscles, indicating LD accumulation is more sensitive to aging in jump muscle than in indirect flight muscle. Age-dependent EFA was also detected in other regions of an aging fly particularly in the posterior midgut and the tip region of testis. However, EFA in these tissues appears to be regulated in a non-tissue autonomous manner, since muscle-specific expression of dHDAC6 in the dHDAC6 mutants reverted the EFA-increase phenotype not only in the jump muscle but also in the posterior midgut and the testis. Recently, fat accumulation has also been shown to occur in other conditions, such as in the niche glia of stem cells of larval CNS under oxidant/oxidative stress (Bailey, 2015) and in the adult pigment cells of mitochondrial mutants (Liu, 2015). LD formation in the niche glia functions as a protective organelle to sequester polyunsaturated fatty acids and to reduce the levels of reactive oxygen species, whereas LD accumulation in adult pigment cells appears to increase lipid peroxidation and to promote neurodegenerative disease (Bailey, 2015; Liu, 2015). LD accumulation in either the niche glia cells or the pigment cells can be reverted by overexpression of triglyceride lipases, indicating that the LDs formed in such conditions are an 'active' organelle. However, the LD accumulation in the aging jump muscle described in this study could not be reverted by overexpression of lipases, implying that the LDs in aging jump muscle seem to be a 'steady' organelle. This 'steady' LD formation can only be ameliorated by improving the proteostasis of LD-resident protein PLIN2. LD accumulation could also be induced by altering some of the fat-metabolism-related genes, but not in an age-dependent manner; thus, age-dependent EFA occurs in a distinct way contributing to the dysfunction of muscle aging. This study suggests that improvement of proteostasis of PLIN2 may be a new approach to ameliorate age-related metabolic diseases such as obesity (Yan, 2017).

The microtubule regulator ringer functions downstream from the RNA repair/splicing pathway to promote axon regeneration

Promoting axon regeneration in the central and peripheral nervous system is of clinical importance in neural injury and neurodegenerative diseases. Both pro- and anti-regeneration factors are being identified. Previous work has shown that the Rtca mediated RNA repair/splicing pathway restricts axon regeneration by inhibiting the nonconventional splicing of Xbp1 mRNA under cellular stress. However, the downstream effectors remain unknown. Through transcriptome profiling this study has shown that the tubulin polymerization-promoting protein (TPPP) ringmaker/ringer is dramatically increased in Rtca-deficient Drosophila sensory neurons, which is dependent on Xbp1. Ringer is expressed in sensory neurons before and after injury, and is cell-autonomously required for axon regeneration. While loss of ringer abolishes the regeneration enhancement in Rtca mutants, its overexpression is sufficient to promote regeneration both in the peripheral and central nervous system. Ringer maintains microtubule stability/dynamics with the microtubule-associated protein Futsch/MAP1B, which is also required for axon regeneration. Furthermore, ringer lies downstream from and is negatively regulated by the microtubule-associated deacetylase HDAC6, which functions as a regeneration inhibitor. Taken together, these findings suggest that Ringer acts as a hub for microtubule regulators that relays cellular status information, such as cellular stress, to the integrity of microtubules in order to instruct neuroregeneration (Monahan Vargas, 2020).

In recent years, several strategies have shown efficacy augmenting nerve regeneration in various experimental models. Unfortunately, therapeutic interventions to promote nerve regeneration and functional recovery still do not exist. Previous work has also helped shape the approach researchers have taken toward better understanding regeneration and drawing connections between successful paradigms. This study reports a link between two cellular mechanisms that are essential for regeneration: RNA processing and microtubule dynamics (Monahan Vargas, 2020).

In Drosophila, sensory dendritic arborization (da) neurons show differential regenerative potentials between the periphery and the central nervous system (CNS), resembling that of mammalian neurons. Moreover, distinct subclasses of da neurons also regenerate differently. A previous study developed a two-photon-based axon injury model that assays class III (C3da) and class IV (C4da) da neurons to identify and analyze targets that enhance regeneration. Using this model, Rtca (RNA 3'-terminal phosphate cyclase), an RNA-binding protein (RBP), was identified as an inhibitor of axon regeneration. Rtca is involved in stress induced Xbp1 mRNA splicing, and its knockout or neuronal knockdown promotes axon regeneration both in the peripheral nervous system (PNS) and CNS. However, its downstream effectors and signaling mechanisms remain unexplored. RBPs are increasingly shown to regulate complex cellular processes associated with neurodegenerative diseases and regeneration. This study reports the results from transcriptome profiling revealing that a microtubule associated protein, Ringer (also known as Ringmaker, which is the fly homolog of the mammalian tubulin polymerization-promoting proteins [TPPPs]), is strongly increased following Rtca removal (Monahan Vargas, 2020).

Microtubules and the cytoskeletal network are essential for neuronal function and are paramount to an axon's ability to respond to guidance cues, transport proteins and organelles, grow, survive, and regenerate. Microtubule-binding small molecules and microtubule-associated proteins (MAPs) that regulate microtubule dynamics are attractive therapeutic targets to augment axon regeneration. Ringer belongs to the brain-specific protein, p25α, also known as the TPPP protein family. TPPPs regulate tubulin polymerization and are implicated in neurodegenerative disorders such as α-synucleinopathies and Multiple System Atrophy. Drosophila has only one TPPP ortholog, Ringer, and it directly binds tubulin, promotes microtubule bundling and polymerization in vitro, and is critical for microtubule stabilization and developmental axon growth. This study shows that transcription of ringer is negatively regulated by Rtca via Xbp1. ringer was found to function as a neuronal intrinsic promoter of axon regeneration, working in concert with other MAPs, specifically Futsch/MAP1B and HDAC6, which have been previously shown to be integral for axonal health and integrity. The results reveal MAPs as important arbiters of axon regeneration, and ringer (TPPP homologs) is proposed as an attractive therapeutic target for promoting axon regeneration (Monahan Vargas, 2020).

RBPs have been shown to be crucial in regulating complex cellular processes such as mRNA editing, transport and local translation. Aberrant processing of RNA is present in neuronal diseases and injury. How these processes are affected after nervous system trauma and their regulation during neural repair are poorly understood. Previous work has identified Rtca, an RNA-binding protein regulating RNA repair and splicing, as a potential damage sensor that inhibits axon regeneration. Rtca LOF enhances axon regeneration in both fly and mammalian neurons. To better understand its underlying mechanism, RNA-seq was performed to assess the transcriptome of Rtca mutant neurons; ringer transcripts were found to be highly expressed. Ringer is a MAP homologous to the mammalian tubulin polymerization-promoting proteins (TPPPs), in particular TPPP3 or TPPP1, which has been shown to be a regulator of axonal microtubule organization by promoting microtubule polymerization, assembly, and stability both in vitro and in vivo. This study has revealed a connection between the injury-evoked RNA repair/splicing system and the MAP ringer; it is proposed that Rtca suppresses Xbp1 via nonconventional mRNA splicing, which in turn reduces ringer expression to inhibit axon regeneration. Furthermore, evidence is provided for an association between Futsch and HDAC6, additional MAPs capable of regulating microtubule stability and posttranslational modifications. Ringer is also inhibited by HDAC6, and it cooperates with Futsch to relay a cellular stress signal to the microtubule network. In addition, these data suggest that Rtca and Xbp1 likely have additional downstream effectors independent of ringer, and that Futsch likely receives additional inputs, in parallel to Ringer, to support axonal regeneration. Future studies to directly monitor microtubule dynamics in Rtca LOF mutants will help further validate this model and offer clues to the identity of additional players in this pathway (Monahan Vargas, 2020).

The capacity of an axon to regenerate depends on both the external environment and cell-intrinsic mechanisms, which ultimately converge onto axonal microtubules. MAPs have become popular targets for augmenting nerve regeneration given the importance of microtubule stability and polymerization in both the nascent axon and the regenerating axon's growth cone. As an axon elongates, microtubules engorge the growth cone to fill it with microtubule mass. As the growth cone advances, microtubules bundle and consolidate within the nascent axon to provide structure and support. Ringer has been shown to be essential for proper microtubule bundling. Microtubules are inherently polarized because newly added tubulin dimers only assemble and disassemble at the 'plus' end of the lattice, whereas the minus end of a microtubule is highly stabilized with special tubulin variants, abundant post translational modifications (e.g., acetylation of α-tubulin), and minus-end associating proteins. Therefore, a single microtubule can be thought of as having two general domains; a plus-end that is labile (i.e., where dynamic instability occurs) and a minus end that is stable and resists depolymerization. Microtubule stabilization prevents depolymerization and favors microtubule growth, which is beneficial for the axon's growth cone to advance. Inducing microtubule stabilization using extremely low doses of the drugs paclitaxel or epithilones has resulted in significant augmentation of nerve regeneration in vivo. The results of this study demonstrated a loss of microtubule acetylation in whole-cell lysate and specifically within the proximal axon of injured neurons in ringer mutants. This is in line with the function of Ringer, which has been associated with microtubule polymerization and stability. Future experiments to dynamically track Ringer proteins in accordance with microtubule polymerization during axon regeneration, and an extensive investigation of microtubule posttranslational modifications following axotomy are warranted (Monahan Vargas, 2020).

Futsch, a MAP1B homolog, was recently shown to associate with ringer. Together, Ringer and Futsch were found to regulate synapse formation at neuromuscular junctions via a microtubule-based mechanism. It can be inferred that Ringer and Futsch may help promote the formation of a growth cone rather than a retracting dystrophic end within injured axons, similar to its maintenance of synaptic integrity. Ringer mutation led to a decrease in futsch mRNAs and immunolabeling, suggesting a role in regulating futsch transcription, localization, and protein levels. Both ringer and futsch mutations impaired axon regeneration, albeit futsch had a more dramatic effect, suggesting that futsch may contribute to additional signaling independent of ringer. While heterozygous mutants for futsch and ringer did not have a reduction in regeneration, transheterozygotes of ringer and futsch mutations exhibited a similar reduction in regeneration as ringer mutants alone. Coimmunoprecipitation experiments showed that ringer, futsch, and tubulin physically interact and form a molecular complex, and that Ringer facilitates Futsch binding to tubulin. Epistasis analysis further demonstrated that overexpression of Futsch failed to rescue the reduced axon regeneration in ringer mutants, while overexpression of futsch is sufficient to promote axon regeneration despite the absence of futsch. Importantly, this study found that microtubule turnover is faster in injured versus uninjured axons, and that futsch LOF dysregulates microtubule dynamics, accelerating its turnover after injury. Taken together, sthe data suggest that Ringer and Futsch cooperate in the same complex with tubulin, to maintain microtubule dynamics/stability, and that both are critical to the ability of sensory neurons to regenerate. Futsch is phosphorylated by GSK3 and sustained GSK3 activity promotes axon regeneration and increases the pool of dynamic microtubule mass, which further leads to a speculation that futsch might be regulated by additional signaling pathways (Monahan Vargas, 2020).

Elucidating how microtubule stability properties are altered following an injury and the MAPs responsible for mediating those changes may identify novel therapeutic targets. This study found that acetylation properties were altered by ringer mutations and, therefore, attempts were made to explore the role HDAC6, the primary tubulin deacetylase, may play in instructing regeneration. HDAC6 knockout and pharmacological inhibition increased regeneration in C3da neurons, a subtype of sensory neurons incapable of regeneration in WT flies. Previous studies have shown that HDAC6 inhibition and deletion leads to the hyperacetylation of microtubules. Early studies found that HDAC6 was neuroprotective after a CNS injury and associated these findings with HDAC6's role in transcriptional regulation. However, more recent studies found that HDAC6 is neuroprotective in a manner that was associated with its deacetylation of microtubules. Other studies have shown that HDAC6 is essential for healthy axonal transport and influences MAP-microtubule interactions. This study showd that HDAC6 LOF leads to increased protein levels of ringer and futsch, likely through posttranscriptional mechanisms. It may also be possible that HDAC6 knockout affects microtubule-binding kinetics and the protein localization of Ringer and Futsch (i.e., concentrated versus diffuse). Augmented regeneration following HDAC6 knockout was lost with a ringer mutation. These results, along with the changes observed in Ac-Tub levels, suggest an interaction between HDAC6 and Ringer, where Ringer may function to either directly or indirectly restrict HDAC6 deacetylase activity with respect to α tubulin acetylation. This is likely, given that Ringer has been shown to regulate microtubule bundling and stability, which are associated with highly acetylated domains of microtubules. Ringer may be essential to protecting highly acetylated and stable microtubule domains from HDAC6 deacetylation by occluding its interaction with α tubulin or directly blocking deacetylase activity. This would be consistent with in vitro studies suggesting that mammalian TPPP modulates microtubule acetylation by binding to HDAC6 and inhibiting its activity. Alternatively, HDAC6 could inhibit TPPP nucleation by binding to TPPP and preventing its association to tubulin. Furthermore, HDAC6 can also physically modify kinases shown to negatively interrupt TPPP function such as ERK2. This network hypothesis could help explain an underlying positive feedback loop regulating microtubule stability: Increase of TPPP would inhibit HDAC6 leading to an enhancement of acetylated, potentially stable microtubule; in contrast, modification of kinases by HDAC6 could lead to kinase activation and downstream phosphorylation of TPPP, limiting its microtubule binding activity. It is believed that HDAC6 and ringer are involved in a pathway that ultimately affects the stability and dynamics of microtubules. Future studies will explore whether Ringer and HDAC6 expression, along with posttranslational modifications of tubulin, can predict the regenerative potential of da sensory neurons. C4da neurons show only ~75% regeneration and it is proposed that the other 25% will show differences in the expression of MAPs and microtubule posttranslational modifications, specifically acetylation of α-tubulin (Monahan Vargas, 2020).

The future treatments for nerve regeneration will most likely be combinatorial, with a need to address the extrinsic and intrinsic barriers to regeneration. This study has identified a link between RNA repair/splicing and microtubule organization via a damage-evoked mechanism involving Rtca and Ringer. Further evidence is presented that therapeutic targets capable of augmenting nerve regeneration ultimately converge on microtubules. Microtubules are a bottleneck to regeneration and identifying intrinsic signaling cascades that regulate microtubule dynamics using fly genetics will reveal pathways critical to microtubule-mediated nerve regeneration. Given the complexity of MAPs and the increasing number of candidate proteins, utilizing the fly injury model system allows screening for promising targets that warrant an investigation into their mammalian homologs with in vitro and in vivo mammalian nerve injury models. Excitingly, the zebrafish homolog of TPPP3 was recently shown to promote axon regeneration in Mauthner cells and is regulated at the transcript level by microRNA 133b. This corroborates the current findings, leading to the proposal that ringer/TPPP is tightly regulated and may function as a relay station at multiple levels. Moreover, HDAC6 was also recently shown to be inhibitory in a regeneration screen performed in C. elegans. In summary, this study has identified a RNA repair/splicing pathway that up-regulates the MAP Ringer, which interacts with other MAPs associated with microtubule stability/dynamics and tubulin posttranslational modifications, processes that are evolutionarily conserved and promising targets for regenerative therapies (Monahan Vargas, 2020).


Search PubMed for articles about Drosophila Hdac6

Bailey, A. P., Koster, G., Guillermier, C., Hirst, E. M., MacRae, J. I., Lechene, C. P., Postle, A. D. and Gould, A. P. (2015). Antioxidant Role for Lipid Droplets in a Stem Cell Niche of Drosophila. Cell 163(2): 340-353. PubMed ID: 26451484

Bali, P., et al. (2005). Inhibition of histone deacetylase 6 acetylates and disrupts the chaperone function of heat shock protein 90: A novel basis for antileukemia activity of histone deacetylase inhibitors. J. Biol. Chem. 280: 26729-26734. PubMed ID: 15937340

Barlow, A. L., et al. (2001). dSIR2 and dHDAC6: two novel, inhibitor-resistant deacetylases in Drosophila melanogaster. Exp. Cell Res. 265(1): 90-103. PubMed ID: 11281647

Bertos, N. R., Gilquin, B., Chan, G. K., Yen, T. J., Khochbin, S. and Yang, X. J. (2004). Role of the tetradecapeptide repeat domain of human histone deacetylase 6 in cytoplasmic retention. J. Biol. Chem. 279: 48246-48254. PubMed ID: 15347674

Boyault, C., Gilquin, B., Zhang, Y., Rybin, V., Garman, E., Meyer-Klaucke, W., Matthias, P., Muller, C. W. and Khochbin, S. (2006). HDAC6-p97/VCP controlled polyubiquitin chain turnover. EMBO J. 25: 3357-3366. PubMed ID: 16810319

Boyault, C., et al. (2007). HDAC6 controls major cell response pathways to cytotoxic accumulation of protein aggregates. Genes Dev. 21: 2172-2181. PubMed ID: 17785525

Hubbert, C., Guardiola, A., Shao, R., Kawaguchi, Y., Ito, A., Nixon, A., Yoshida, M., Wang, X. F., and Yao, T. P. (2002). HDAC6 is a microtubule-associated deacetylase. Nature 417: 455-458. PubMed ID: 12024216

Iwata, A., Riley, B. E., Johnston, J. A. and Kopito, R. R. (2005). HDAC6 and microtubules are required for autophagic degradation of aggregated huntingtin. J. Biol. Chem. 280: 40282-40292. PubMed ID: 16192271

Kawaguchi, Y. et al. (2003). The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress. Cell 115: 727-738. PubMed ID: 14675537

Kovacs, J. J. et al. (2005). HDAC6 regulates Hsp90 acetylation and chaperone-dependent activation of glucocorticoid receptor. Mol. Cell 18: 601-607. PubMed ID: 15916966

Kwon, S., Zhang, Y. and Matthias, P. (2007). The deacetylase HDAC6 is a novel critical component of stress granules involved in the stress response. Genes Dev. 21(24): 3381-94. PubMed ID: 18079183

Liu, L., Zhang, K., Sandoval, H., Yamamoto, S., Jaiswal, M., Sanz, E., Li, Z., Hui, J., Graham, B. H., Quintana, A. and Bellen, H. J. (2015). Glial lipid droplets and ROS induced by mitochondrial defects promote neurodegeneration. Cell 160(1-2): 177-190. PubMed ID: 25594180

Matsuyama, A., Shimazu, T., Sumida, Y., Saito, A., Yoshimatsu, Y., Seigneurin-Berny, D., Osada, H., Komatsu, Y., Nishino, N., Khochbin, S., et al. (2002). In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation. EMBO J. 21: 6820-6831. PubMed ID: 12486003

Monahan Vargas, E. J., Matamoros, A. J., Qiu, J., Jan, C. H., Wang, Q., Gorczyca, D., Han, T. W., Weissman, J. S., Jan, Y. N., Banerjee, S. and Song, Y. (2020). The microtubule regulator ringer functions downstream from the RNA repair/splicing pathway to promote axon regeneration. Genes Dev 34(3-4): 194-208. PubMed ID: 31919191

Pandey, U. B., et al. (2007). HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS. Nature 447(7146): 859-63. PubMed ID: 17568747

Smyth, K. A. and Belote, J. M. (1999). The dominant temperature-sensitive lethal DTS7 of Drosophila melanogaster encodes an altered 20S proteasome -type subunit. Genetics 151: 211-220. PubMed ID: 9872961

Taylor, J. P. et al. (2003). Aggresomes protect cells by enhancing the degradation of toxic polyglutamine-containing protein. Hum. Mol. Genet. 12: 749-757. PubMed ID: 12651870

Verdel, A., Curtet, S., Brocard, M.P., Rousseaux, S., Lemercier, C., Yoshida, M., and Khochbin, S. (2000). Active maintenance of mHDA2/mHDAC6 histone-deacetylase in the cytoplasm. Curr. Biol. 10: 747-749. PubMed ID: 10873806

Yan, Y., Wang, H., Hu, M., Jiang, L., Wang, Y., Liu, P., Liang, X., Liu, J., Li, C., Lindstrom-Battle, A., Lam, S. M., Shui, G., Deng, W. M. and Jiao, R. (2017). HDAC6 suppresses age-dependent ectopic fat accumulation by maintaining the proteostasis of PLIN2 in Drosophila. Dev Cell 43(1): 99-111.e115. PubMed ID: 28966044

Zhang, Y., Li, N., Caron, C., Matthias, G., Hess, D., Khochbin, S. and Matthias, P. (2003). HDAC-6 interacts with and deacetylates tubulin and microtubules in vivo. EMBO J. 22: 1168-1179. PubMed ID: 12606581

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date revised: 15 April 2020

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