Nuclear translocation of AMPK-��1 potentiates striatal neurodegeneration in Huntington��s disease

Adenosine monophosphate–activated protein kinase (AMPK) is a major energy sensor that maintains cellular energy homeostasis. Huntington’s disease (HD) is a neurodegenerative disorder caused by the expansion of CAG repeats in the huntingtin (Htt) gene. In this paper, we report that activation of the α1 isoform of AMPK (AMPK-α1) occurred in striatal neurons of humans and mice with HD. Overactivation of AMPK in the striatum caused brain atrophy, facilitated neuronal loss, and increased formation of Htt aggregates in a transgenic mouse model (R6/2) of HD. Such nuclear accumulation of AMPK-α1 was activity dependent. Prevention of nuclear translocation or inactivation of AMPK-α1 ameliorated cell death and down-regulation of Bcl2 caused by mutant Htt (mHtt). Conversely, enhanced expression of Bcl2 protected striatal cells from the toxicity evoked by mHtt and AMPK overactivation. These data demonstrate that aberrant activation of AMPK-α1 in the nuclei of striatal cells represents a new toxic pathway induced by mHtt.     

Essential sequence of the N-terminal cytoplasmic localization-related domain of huntingtin and its effect on huntingtin aggregates

Huntington’s disease (HD) is caused by abnormal CAG repeat expansion in the 5′-end of the Huntingtin (HTT) gene. In addition to the canonical C-terminal full-length huntingtin (htt) nuclear export signal, a cytoplasmic localization-related domain (CLRD) in the N-terminus of htt has recently been reported. Here, we analyzed this domain by introducing deletion and substitution mutations in a truncated N-terminal htt protein and subsequently monitored htt expression, aggregation and subcellular localization by immunocytochemistry and Western blot analysis. We demonstrated that Htt_4–17 was the essential sequence for htt cytoplasmic localization. We also found that the subcellular distribution of htt was altered when Htt_1–17 was mutated to contain amino acids of different charges, suggesting a structural requirement of Htt_1–17 for the cytoplasmic localization of htt. Deletion of the first three amino acids did not affect its association with mitochondria. We observed that defective cytoplasmic localization resulted in a reduction of total htt aggregates and increased nuclear aggregates, indicating that the subcellular distribution of the protein might influence the aggregation process. These studies provide new insight into the molecular mechanism of htt aggregation in HD.     

N-alpha-acetylation of Huntingtin protein increases its propensity to aggregate

Huntington’s disease (HD) is a neurodegenerative disorder caused by a poly-CAG expansion in the first exon of the HTT gene, resulting in an extended poly-glutamine tract in the N-terminal domain of the Huntingtin (Htt) protein product. Proteolytic fragments of the poly-glutamine–containing N-terminal domain form intranuclear aggregates that are correlated with HD. Post-translational modification of Htt has been shown to alter its function and aggregation properties. However, the effect of N-terminal Htt acetylation has not yet been considered. Here, we developed a bacterial system to produce unmodified or N-terminally acetylated and aggregation-inducible Htt protein. We used this system together with biochemical, biophysical, and imaging studies to confirm that the Htt N-terminus is an in vitro substrate for the NatA N-terminal acetyltransferase and show that N-terminal acetylation promotes aggregation. These studies represent the first link between N-terminal acetylation and the promotion of a neurodegenerative disease and implicates NatA-mediated Htt acetylation as a new potential therapeutic target in HD.     

Does Huntingtin play a role in selective macroautophagy?

The accumulation of protein aggregates in neurons appears to be a basic feature of neurodegenerative disease. In huntington disease (HD), a progressive and ultimately fatal neurodegenerative disorder caused by an expansion of the polyglutamine repeat within the protein huntingtin (Htt), the immediate proximal cause of disease is well understood. However, the cellular mechanisms which modulate the rate at which fragments of Htt containing polyglutamine accumulate in neurons is a central issue in the development of approaches to modulate the rate and extent of neuronal loss in this disease. We have recently found that Htt is phosphorylated by the kinase IKK on serine (s) 13, activating its phosphorylation on S16 and its acetylation and poly-SUMOylation, modifications that modulate its clearance by the proteasome and lysosome in cells.¹ In the discussion here I suggest that Htt may have a normal function in the lysosomal mechanism of selective macroautophagy involved in its own degradation which may share some similarity with the yeast cytoplasm to vacuole targeting (Cvt) pathway. Pharmacologic activation of this pathway may be useful early in disease progression to treat HD and other neurodegenerative diseases characterized by the accumulation of disease proteins.Key words: Huntington disease, Huntingtin, polyglutamine, autophagy, IKKAn age-related reduction in protein clearance mechanisms has been implicated in the pathogenesis of neurodegenerative diseases including the polyglutamine (polyQ) repeat diseases, Alzheimer disease (AD), Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). These diseases are each associated with the accumulation of insoluble protein aggregates in diseased neurons. Huntington Disease (HD), caused by an expansion of the polyQ repeat in the protein Huntingtin (Htt), is one such disease of aging in which mutant Htt inclusions form in striatal and cortical neurons as disease progresses. Clarification of the mechanisms of Htt clearance is paramount to finding therapeutic targets to treat HD that may be broadly useful in the treatment of these currently incurable neurodegenerative diseases.     

Interaction with polyglutamine-expanded huntingtin alters cellular distribution and RNA processing of huntingtin yeast two-hybrid protein A (HYPA)

Huntington disease (HD) is an autosomal inherited disorder that causes the deterioration of brain cells. The polyglutamine (polyQ) expansion of huntingtin (Htt) is implicated in the pathogenesis of HD via interaction with an RNA splicing factor, Htt yeast two-hybrid protein A/forming-binding protein 11 (HYPA/FBP11). Besides the pathogenic polyQ expansion, Htt also contains a proline-rich region (PRR) located exactly in the C terminus to the polyQ tract. However, how the polyQ expansion influences the PRR-mediated protein interaction and how this abnormal interaction leads to the biological consequence remain elusive. Our NMR structural analysis indicates that the PRR motif of Htt cooperatively interacts with the tandem WW domains of HYPA through domain chaperoning effect of WW1 on WW2. The polyQ-expanded Htt sequesters HYPA to the cytosolic location and then significantly reduces the efficiency of pre-mRNA splicing. We propose that the toxic gain-of-function of the polyQ-expanded Htt that causes dysfunction of cellular RNA processing contributes to the pathogenesis of HD.     

The Predicted Structure of the Headpiece of the Huntingtin Protein and Its Implications on Huntingtin Aggregation

We have performed simulated tempering molecular dynamics simulations to study the thermodynamics of the headpiece of the Huntingtin (Htt) protein (N17^Htt). With converged sampling, we found this peptide is highly helical, as previously proposed. Interestingly, this peptide is also found to adopt two different and seemingly stable states. The region from residue 4 (L) to residue 9 (K) has a strong helicity from our simulations, which is supported by experimental studies. However, contrary to what was initially proposed, we have found that simulations predict the most populated state as a two-helix bundle rather than a single straight helix, although a significant percentage of structures do still adopt a single linear helix. The fact that Htt aggregation is nucleation dependent infers the importance of a critical transition. It has been shown that N17^Htt is involved in this rate-limiting step. In this study, we propose two possible mechanisms for this nucleating event stemming from the transition between two-helix bundle state and single-helix state for N17^Htt and the experimentally observed interactions between the N17^Htt and polyQ domains. More strikingly, an extensive hydrophobic surface area is found to be exposed to solvent in the dominant monomeric state of N17^Htt. We propose the most fundamental role played by N17^Htt would be initializing the dimerization and pulling the polyQ chains into adequate spatial proximity for the nucleation event to proceed.     

Reduction of Htt inclusion formation in strains of Saccharomyces cerevisiae deficient in certain DNA repair functions: a statistical analysis of phenotype

Saccharomyces cerevisiae has been used as a model system to examine the aggregation of the huntingtin protein (Htt), a well-established marker in the pathology of the triplet expansion disorder Huntington's disease (HD). Several genetic backgrounds, such as Deltahsp104, have proven to be refractory to inclusion formation through a process yet to be fully elucidated. These results have prompted a wide-ranging search for other mutant strains that exhibit a lower level of Htt aggregation. A novel assay system in which Htt is expressed as a fusion protein containing eGFP enables an analysis of aggregation and the factors that suppress it. We have examined several strains that are devoid of certain mismatch repair genes and find that some of these support a reduced level of inclusion body formation. We apply a detailed and stringent statistical analysis to the results obtained for all yeast strains that exhibit a definable phenotype. Such analyses should be useful and applicable to other in vivo analyses of related phenomena.     

Complete suppression of Htt fibrilization and disaggregation of Htt fibrils by a trimeric chaperone complex

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded CAG trinucleotide repeat in the huntingtin gene (HTT). Molecular chaperones have been implicated in suppressing or delaying the aggregation of mutant Htt. Using in vitro and in vivo assays, we have identified a trimeric chaperone complex (Hsc70, Hsp110, and J‐protein) that completely suppresses fibrilization of HttExon1Q₄₈. The composition of this chaperone complex is variable as recruitment of different chaperone family members forms distinct functional complexes. The trimeric chaperone complex is also able to resolubilize Htt fibrils. We confirmed the biological significance of these findings in HD patient‐derived neural cells and on an organismal level in Caenorhabditis elegans. Among the proteins in this chaperone complex, the J‐protein is the concentration‐limiting factor. The single overexpression of DNAJB1 in HEK293T cells is sufficient to profoundly reduce HttExon1Q₉₇ aggregation and represents a target of future therapeutic avenues for HD.     

Degradation of mutant huntingtin via the ubiquitin/proteasome system is modulated by FE65

An unstable expansion of the polyglutamine repeat within exon 1 of the protein Htt (huntingtin) causes HD (Huntington's disease). Mounting evidence shows that accumulation of N-terminal mutant Htt fragments is the source of disruption of normal cellular processes which ultimately leads to neuronal cell death. Understanding the degradation mechanism of mutant Htt and improving its clearance has emerged as a new direction in developing therapeutic approaches to treat HD. In the present study we show that the brain-enriched adaptor protein FE65 is a novel interacting partner of Htt. The binding is mediated through WW-polyproline interaction and is dependent on the length of the polyglutamine tract. Interestingly, a reduction in mutant Htt protein level was observed in FE65-knockdown cells, and the process requires the UPS (ubiquitin/proteasome system). Moreover, the ubiquitination level of mutant Htt was found to be enhanced when FE65 is knocked down. Immunofluroescence staining revealed that FE65 associates with mutant Htt aggregates. Additionally, we demonstrated that overexpression of FE65 increases mutant Htt-induced cell death both in vitro and in vivo. These results suggest that FE65 facilitates the accumulation of mutant Htt in cells by preventing its degradation via the UPS, and thereby enhances the toxicity of mutant Htt.     

Beta conformation of polyglutamine track revealed by a crystal structure of Huntingtin N-terminal region with insertion of three histidine residues

Huntington disease is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine (polyQ) expansion (> 35Q) in the first exon (EX1) of huntingtin protein (Htt). mHtt protein is thought to adopt one or more toxic conformation(s) that are involved in pathogenic interactions in cells . However, the structure of mHtt is not known. Here, we present a near atomic resolution structure of mHtt36Q-EX1. To facilitate crystallization, three histidine residues (3H) were introduced within the Htt36Q stretch resulting in the sequence of Q₇HQHQHQ₂₇. The Htt36Q3H region adopts α-helix, loop, β-hairpin conformations. Furthermore, we observed interactions between the backbone of the Htt36Q3H β-strand with the aromatic residues mimicking putative-toxic interactions with other proteins. Our findings support previous predictions that the expanded mHtt-polyQ region adopts a β-sheet structure. Detailed structural information about mHtt improves our understanding of the pathogenic mechanisms in HD and other polyQ expansion disorders and may form the basis for rational design of small molecules that target toxic conformations of disease-causing proteins.     

Normal Aging Modulates the Neurotoxicity of Mutant Huntingtin

Aging likely plays a role in neurodegenerative disorders. In Huntington''s disease (HD), a disorder caused by an abnormal expansion of a polyglutamine tract in the protein huntingtin (Htt), the role of aging is unclear. For a given tract length, the probability of disease onset increases with age. There are mainly two hypotheses that could explain adult onset in HD: Either mutant Htt progressively produces cumulative defects over time or “normal” aging renders neurons more vulnerable to mutant Htt toxicity. In the present study, we directly explored whether aging affected the toxicity of mutant Htt in vivo. We studied the impact of aging on the effects produced by overexpression of an N-terminal fragment of mutant Htt, of wild-type Htt or of a β-Galactosidase (β-Gal) reporter gene in the rat striatum. Stereotaxic injections of lentiviral vectors were performed simultaneously in young (3 week) and old (15 month) rats. Histological evaluation at different time points after infection demonstrated that the expression of mutant Htt led to pathological changes that were more severe in old rats, including an increase in the number of small Htt-containing aggregates in the neuropil, a greater loss of DARPP-32 immunoreactivity and striatal neurons as assessed by unbiased stereological counts.The present results support the hypothesis that “normal” aging is involved in HD pathogenesis, and suggest that age-related cellular defects might constitute potential therapeutic targets for HD.     

Huntingtin aggregation kinetics and their pathological role in a Drosophila Huntington's disease model

Huntington's disease is a neurodegenerative disorder resulting from expansion of a polyglutamine tract in the Huntingtin protein. Mutant Huntingtin forms intracellular aggregates within neurons, although it is unclear whether aggregates or more soluble forms of the protein represent the pathogenic species. To examine the link between aggregation and neurodegeneration, we generated Drosophila melanogaster transgenic strains expressing fluorescently tagged human huntingtin encoding pathogenic (Q138) or nonpathogenic (Q15) proteins, allowing in vivo imaging of Huntingtin expression and aggregation in live animals. Neuronal expression of pathogenic Huntingtin leads to pharate adult lethality, accompanied by formation of large aggregates within the cytoplasm of neuronal cell bodies and neurites. Live imaging and Fluorescence Recovery After Photobleaching (FRAP) analysis of pathogenic Huntingtin demonstrated that new aggregates can form in neurons within 12 hr, while preexisting aggregates rapidly accumulate new Huntingtin protein within minutes. To examine the role of aggregates in pathology, we conducted haplo-insufficiency suppressor screens for Huntingtin-Q138 aggregation or Huntingtin-Q138-induced lethality, using deficiencies covering ~80% of the Drosophila genome. We identified two classes of interacting suppressors in our screen: those that rescue viability while decreasing Huntingtin expression and aggregation and those that rescue viability without disrupting Huntingtin aggregation. The most robust suppressors reduced both soluble and aggregated Huntingtin levels, suggesting toxicity is likely to be associated with both forms of the mutant protein in Huntington's disease.     

Herp Promotes Degradation of Mutant Huntingtin: Involvement of the Proteasome and Molecular Chaperones

In neurodegenerative diseases, pathogenic proteins tend to misfold and form aggregates that are difficult to remove and able to induce excessive endoplasmic reticulum (ER) stress, leading to neuronal injury and apoptosis. Homocysteine-induced endoplasmic reticulum protein (Herp), an E3 ubiquitin ligase, is an important early marker of ER stress and is involved in the ubiquitination and degradation of many neurodegenerative proteins. However, in Huntington’s disease (HD), a typical polyglutamine disease, whether Herp is also involved in the metabolism and degradation of the pathogenic protein, mutant huntingtin, has not been reported. Therefore, we studied the relationship between Herp and N-terminal fragments of huntingtin (HttN-20Q and HttN-160Q). We found that Herp was able to bind to the overexpressed Htt N-terminal, and this interaction was enhanced by expansion of the polyQ fragment. Confocal microscopy demonstrated that Herp was co-localized with the HttN-160Q aggregates in the cytoplasm and tightly surrounded the aggregates. Overexpression of Herp significantly decreased the amount of soluble and insoluble HttN-160Q, promoted its ubiquitination, and inhibited its cytotoxicity. In contrast, knockdown of Herp resulted in more HttN-160Q protein, less ubiquitination, and stronger cytotoxicity. Inhibition of the autophagy-lysosomal pathway (ALP) had no effect on the function of Herp. However, blocking the ubiquitin-proteasome pathway (UPP) inhibited the reduction in soluble HttN-160Q caused by Herp. Interestingly, blocking the UPP did not weaken the ability of Herp to reduce HttN-160Q aggregates. Deletions of the N-terminal of Herp weakened its ability to inhibit HttN-160Q aggregation but did not result in a significant increase in its soluble form. However, loss of the C-terminal led to a significant increase in soluble HttN-160Q, but Herp still maintained the ability to inhibit aggregate formation. We further found that the expression level of Herp was significantly increased in HD animal and cell models. Our findings suggest that Herp is a newly identified huntingtin-interacting protein that is able to reduce the cytotoxicity of mutant huntingtin by inhibiting its aggregation and promoting its degradation. The N-terminal of Herp serves as the molecular chaperone to inhibit protein aggregation, while its C-terminal functions as an E3 ubiquitin ligase to promote the degradation of misfolded proteins through the UPP. Increased expression of Herp in HD models may be a pro-survival mechanism under stress.     

Degradation of huntingtin mediated by a hybrid molecule composed of IAP antagonist linked to phenyldiazenyl benzothiazole derivative

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by aggregation of mutant huntingtin (mHtt), and removal of mHtt is expected as a potential therapeutic option. We previously reported protein knockdown of Htt by using hybrid small molecules (Htt degraders) consisting of BE04, a ligand of ubiquitin ligase (E3), linked to probes for protein aggregates. Here, in order to examine the effect of changing the ligand, we synthesized a similar Htt degrader utilizing MV1, an antagonist of the inhibitor of apoptosis protein (IAP) family (a subgroup of ubiquitin E3 ligases), which is expected to have a higher affinity and specificity for IAP, as compared with BE04. The MV1-based hybrid successfully induced interaction between Htt aggregates and IAP, and reduced mHtt levels in living cells. Its mode of action was confirmed to be the same as that of the BE04-based hybrid. However, although the affinity of MV1 for IAP is greater than that of BE04, the efficacy of Htt degradation by the MV1-based molecule was lower, suggesting that linker length between the ligand and probe might be an important determinant of efficacy.     

Calretinin interacts with huntingtin and reduces mutant huntingtin‐caused cytotoxicity

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by an expansion of CAG trinucleotide repeats encoding for polyglutamine (polyQ) in the huntingtin (Htt) gene. Despite considerable effort, the mechanisms underlying the toxicity of the mutated Htt protein remains largely uncertain. To identify novel therapeutic targets, we recently employed the approach of tandem affinity purification and discovered that calretinin (Cr), a member of the EF‐hand family of calcium‐binding proteins, is preferentially associated with mHtt, although it also interacts with wild‐type Htt. These observations were supported by coimmunoprecipitation and by colocalization of Cr with mHtt in neuronal cultures. Over‐ expression of Cr reduced mHtt‐caused cytotoxicity in both non‐neuronal and neuronal cell models of HD, whereas knockdown of Cr expression in the cells enhanced mHtt‐caused neuronal cell death. In addition, over‐expression of Cr was also associated with reduction of intracellular free calcium and activation of Akt. These results suggest that Cr may be a potential therapeutic target for treatment of HD.     

N-terminal Huntingtin Knock-In Mice: Implications of Removing the N-terminal Region of Huntingtin for Therapy

The Huntington’s disease (HD) protein, huntingtin (HTT), is a large protein consisting of 3144 amino acids and has conserved N-terminal sequences that are followed by a polyglutamine (polyQ) repeat. Loss of Htt is known to cause embryonic lethality in mice, whereas polyQ expansion leads to adult neuronal degeneration. Whether N-terminal HTT is essential for neuronal development or contributes only to late-onset neurodegeneration remains unknown. We established HTT knock-in mice (N160Q-KI) expressing the first 208 amino acids of HTT with 160Q, and they show age-dependent HTT aggregates in the brain and neurological phenotypes. Importantly, the N-terminal mutant HTT also preferentially accumulates in the striatum, the brain region most affected in HD, indicating the importance of N-terminal HTT in selective neuropathology. That said, homozygous N160Q-KI mice are also embryonic lethal, suggesting that N-terminal HTT alone is unable to support embryonic development. Using Htt knockout neurons, we found that loss of Htt selectively affects the survival of developing neuronal cells, but not astrocytes, in culture. This neuronal degeneration could be rescued by a truncated HTT lacking the first 237 amino acids, but not by N-terminal HTT (1–208 amino acids). Also, the rescue effect depends on the region in HTT known to be involved in intracellular trafficking. Thus, the N-terminal HTT region may not be essential for the survival of developing neurons, but when carrying a large polyQ repeat, can cause selective neuropathology. These findings imply a possible therapeutic benefit of removing the N-terminal region of HTT containing the polyQ repeat to treat the neurodegeneration in HD.     

Prothymosin-α interacts with mutant huntingtin and suppresses its cytotoxicity in cell culture

Huntington disease (HD), a fatal neurodegenerative disorder, is caused by a lengthening of the polyglutamine tract in the huntingtin (Htt) protein. Despite considerable effort, thus far there is no cure or treatment available for the disorder. Using the approach of tandem affinity purification we recently discovered that prothymosin-α (ProTα), a small highly acidic protein, interacts with mutant Htt (mHtt). This was confirmed by co-immunoprecipitation and a glutathione S-transferase (GST) pull-down assay. Overexpression of ProTα remarkably reduced mHtt-induced cytotoxicity in both non-neuronal and neuronal cell models expressing N-terminal mHtt fragments, whereas knockdown of ProTα expression in the cells enhanced mHtt-caused cell death. Deletion of the central acidic domain of ProTα abolished not only its interaction with mHtt but also its protective effect on mHtt-caused cytotoxicity. Additionally, overexpression of ProTα inhibited caspase-3 activation but enhanced aggregation of mHtt. Furthermore, when added to cultured cells expressing mHtt, the purified recombinant ProTα protein not only entered the cells but it also significantly suppressed the mHtt-caused cytotoxicity. Taken together, these data suggest that ProTα might be a novel therapeutic target for treating HD and other polyglutamine expansion disorders.