A stress sensitive ER membrane-association domain in Huntingtin protein defines a potential role for Huntingtin in the regulation of autophagy

We have recently published the precise definition of an aminoterminal membrane association domain in huntingtin, capable of targeting to the endoplasmic reticulum and late endosomes as well as autophagic vesicles. In response to ER stress induced by several pathways, huntingtin releases from membranes and rapidly translocates into the nucleus. Huntingtin is then capable of nuclear export and re-association with the ER in the absence of stress. This release is inhibited when huntingtin contains the polyglutamine expansion seen in Huntington's disease. As a result, mutant huntingtin expressing cells have a perturbed ER and an increase in autophagic vesicles. Here, we discuss the potential function of the huntingtin protein as an ER sentinel, potentially regulating autophagy in response to ER stress. We compare these recent findings to the well characterized mammalian target of rapamycin, mTor, a protein described over a decade ago as related to huntingtin structurally by leucine-rich, repetitive HEAT sequences. Since then, the described functional similarities between Huntingtin and mTor are striking, and this new information about huntingtin's direct association with autophagic vesicles indicates that this structural similarity may extend to functional similarities having an impact upon ER functionality and autophagy.     

Huntingtin is required for ER-to-Golgi transport and for secretory vesicle fusion at the plasma membrane

Huntingtin is a large membrane-associated scaffolding protein that associates with endocytic and exocytic vesicles and modulates their trafficking along cytoskeletal tracks. Although the progression of Huntington’s disease is linked to toxic accumulation of mutant huntingtin protein, loss of wild-type huntingtin function might also contribute to neuronal cell death, but its precise function is not well understood. Therefore, we investigated the molecular role of huntingtin in exocytosis and observed that huntingtin knockdown in HeLa cells causes a delay in endoplasmic reticulum (ER)-to-Golgi transport and a reduction in the number of cargo vesicles leaving the trans-Golgi network. In addition, we found that huntingtin is required for secretory vesicle fusion at the plasma membrane. Similar defects in the early exocytic pathway were observed in primary fibroblasts from homozygous Htt^140Q/140Q knock-in mice, which have the expansion inserted into the mouse huntingtin gene so lack wild-type huntingtin expression. Interestingly, heterozygous fibroblasts from a Huntington’s disease patient with a 180Q expansion displayed no obvious defects in the early secretory pathway. Thus, our results highlight the requirement for wild-type huntingtin at distinct steps along the secretory pathway.KEY WORDS: Exocytosis, Huntingtin, ER, Golgi, Vesicle fusion     

Huntingtin phosphorylation acts as a molecular switch for anterograde/retrograde transport in neurons

The transport of vesicles in neurons is a highly regulated process, with vesicles moving either anterogradely or retrogradely depending on the nature of the molecular motors, kinesins and dynein, respectively, which propel vesicles along microtubules (MTs). However, the mechanisms that determine the directionality of transport remain unclear. Huntingtin, the protein mutated in Huntington's disease, is a positive regulatory factor for vesicular transport. Huntingtin is phosphorylated at serine 421 by the kinase Akt but the role of this modification is unknown. Here, we demonstrate that phosphorylation of wild-type huntingtin at S421 is crucial to control the direction of vesicles in neurons. When phosphorylated, huntingtin recruits kinesin-1 to the dynactin complex on vesicles and MTs. Using brain-derived neurotrophic factor as a marker of vesicular transport, we demonstrate that huntingtin phosphorylation promotes anterograde transport. Conversely, when huntingtin is not phosphorylated, kinesin-1 detaches and vesicles are more likely to undergo retrograde transport. This also applies to other vesicles suggesting an essential role for huntingtin in the control of vesicular directionality in neurons.     

Regulation of intracellular accumulation of mutant Huntingtin by Beclin 1

Intracellular accumulation of mutant Huntingtin with expanded polyglutamine provides a context-dependent cytotoxicity critical for the pathogenesis of Huntington disease (Everett, C. M., and Wood, N. W. (2004) Brain 127, 2385-2405). Here we demonstrate that the accumulation of mutant Huntingtin is highly sensitive to the expression of beclin 1, a gene essential for autophagy. Moreover, we show that the accumulated mutant Huntingtin recruits Beclin 1 and impairs the Beclin 1-mediated long lived protein turnover. Thus, sequestration of Beclin 1 in the vulnerable neuronal population of Huntington disease patients might further reduce Beclin 1 function and autophagic degradation of mutant Huntingtin. Finally, we demonstrate that the expression of beclin 1 decreases in an age-dependent fashion in human brains. Because beclin 1 gene is haploid insufficient in regulating autophagosome function (Qu, X., Yu, J., Bhagat, G., Furuya, N., Hibshoosh, H., Troxel, A., Rosen, J., Eskelinen, E. L., Mizushima, N., Ohsumi, Y., Cattoretti, G., and Levine, B. (2003) J. Clin. Invest. 112, 1809-1820; Yue, Z., Jin, S., Yang, C., Levine, A. J., and Heintz, N. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 15077-15082), we propose that the age-dependent decrease of beclin 1 expression may lead to a reduction of autophagic activity during aging, which in turn promotes the accumulation of mutant Htt and the progression of the disease.     

Polyglutamine expansion in huntingtin alters its interaction with phospholipids

Huntingtin has an expanded polyglutamine tract in patients with Huntington's disease. Huntingtin localizes to intracellular and plasma membranes but the function of huntingtin at membranes is unknown. Previously we reported that exogenously expressed huntingtin bound pure phospholipids using protein-lipid overlays. Here we show that endogenous huntingtin from normal ( Hdh ^7Q/7Q) mouse brain and mutant huntingtin from Huntington's disease ( Hdh ^140Q/140Q) mouse brain bound to large unilamellar vesicles containing phosphoinositol (PI) PI 3,4-bisphosphate, PI 3,5-bisphosphate, and PI 3,4,5-triphosphate [PI(3,4,5)P3]. Huntingtin interactions with multivalent phospholipids were similar to those of dynamin. Mutant huntingtin associated more with phosphatidylethanolamine and PI(3,4,5)P3 than did wild-type huntingtin, and associated with other phospholipids not recognized by wild-type huntingtin. Wild-type and mutant huntingtin also bound to large unilamellar vesicles containing cardiolipin, a phospholipid specific to mitochondrial membranes. Maximal huntingtin-phospholipid association required inclusion of huntingtin amino acids 171–287. Endogenous huntingtin recruited to the plasma membrane in cells that incorporated exogenous PI 3,4-bisphosphate and PI(3,4,5)P3 or were stimulated by platelet-derived growth factor or insulin growth factor 1, which both activate PI 3-kinase. These data suggest that huntingtin interacts with membranes through specific phospholipid associations and that mutant huntingtin may disrupt membrane trafficking and signaling at membranes.     

Huntingtin associates with acidic phospholipids at the plasma membrane

We have identified a domain in the N terminus of huntingtin that binds to membranes. A three-dimensional homology model of the structure of the binding domain predicts helical HEAT repeats, which emanate a positive electrostatic potential, consistent with a charge-based mechanism for membrane association. An amphipathic helix capable of inserting into pure lipid bilayers may serve to anchor huntingtin to the membrane. In cells, N-terminal huntingtin fragments targeted to regions of plasma membrane enriched in phosphatidylinositol 4,5-bisphosphate, receptor bound-transferrin, and endogenous huntingtin. N-terminal huntingtin fragments with an expanded polyglutamine tract aberrantly localized to intracellular regions instead of plasma membrane. Our data support a new model in which huntingtin directly binds membranes through electrostatic interactions with acidic phospholipids.     

Mutant Huntingtin Impairs Post-Golgi Trafficking to Lysosomes by Delocalizing Optineurin/Rab8 Complex from the Golgi Apparatus

Huntingtin regulates post-Golgi trafficking of secreted proteins. Here, we studied the mechanism by which mutant huntingtin impairs this process. Colocalization studies and Western blot analysis of isolated Golgi membranes showed a reduction of huntingtin in the Golgi apparatus of cells expressing mutant huntingtin. These findings correlated with a decrease in the levels of optineurin and Rab8 in the Golgi apparatus that can be reverted by overexpression of full-length wild-type huntingtin. In addition, immunoprecipitation studies showed reduced interaction between mutant huntingtin and optineurin/Rab8. Cells expressing mutant huntingtin produced both an accumulation of clathrin adaptor complex 1 at the Golgi and an increase of clathrin-coated vesicles in the vicinity of Golgi cisternae as revealed by electron microscopy. Furthermore, inverse fluorescence recovery after photobleaching analysis for lysosomal-associated membrane protein-1 and mannose-6-phosphate receptor showed that the optineurin/Rab8-dependent post-Golgi trafficking to lysosomes was impaired in cells expressing mutant huntingtin or reducing huntingtin levels by small interfering RNA. Accordingly, these cells showed a lower content of cathepsin D in lysosomes, which led to an overall reduction of lysosomal activity. Together, our results indicate that mutant huntingtin perturbs post-Golgi trafficking to lysosomal compartments by delocalizing the optineurin/Rab8 complex, which, in turn, affects the lysosomal function.     

Hypothesis: Huntingtin may function in membrane association and vesicular trafficking.

Huntington's disease is a progressive neurodegenerative genetic disorder that is caused by a CAG triplet-repeat expansion in the first exon of the IT15 gene. This CAG expansion results in polyglutamine expansion in the 350 kDa huntingtin protein. The exact function of huntingtin is unknown. Understanding the pathological triggers of mutant huntingtin, and distinguishing the cause of disease from downstream effects, is critical to designing therapeutic strategies and defining long- and short-term goals of therapy. Many studies that have sought to determine the functions of huntingtin by determining huntingtin's protein-protein interactions have been published. Through these studies, huntingtin has been seen to interact with a large number of proteins, and is likely a scaffolding protein for protein-protein interactions. Recently, using imaging, integrative proteomics, and cell biology, huntingtin has been defined as a membrane-associated protein, with activities related to axonal trafficking of vesicles and mitochondria. These functions have also been attributed to some huntingtin-interacting proteins. Additionally, discoveries of a membrane association domain and a palmitoylation site in huntingtin reinforce the fact that huntingtin is membrane associated. In Huntington's disease mouse and fly models, axonal vesicle trafficking is inhibited, and lack of proper uptake of neurotrophic factors may be an important pathological trigger leading to striatal cell death in Huntington's disease. Here we discuss recent advances from many independent groups and methodologies that are starting to resolve the elusive function of huntingtin in vesicle transport, and evidence that suggests that huntingtin may be directly involved in membrane interactions.     

Converging pathways in the occurrence of endoplasmic reticulum (ER) stress in Huntington's disease

A variety of neurological diseases including Huntington's disease (HD), Alzheimer's disease and Parkinson's disease share common neuropathology, primarily featuring the presence of abnormal protein inclusions containing specific misfolded proteins. Mutations leading to expansion of a poly-glutamine track in Huntingtin cause HD, and trigger its misfolding and aggregation. Recent evidence indicates that alterations in the secretory pathway, in particular the endoplasmic reticulum (ER), are emerging features of HD. Although it is not clear how cytoplasmic/nuclear located mutant Huntingtin alters the function of the ER, several reports indicate that mutant Huntingtin affects many essential processes related to the secretory pathway, including inhibition of ER-associated degradation, altered ER/Golgi vesicular trafficking and axonal transport, disrupted autophagy and abnormal ER calcium homeostasis. All these alterations are predicted to have a common pathological outcome associated to disturbance of protein folding and maturation pathways at the ER, generating chronic ER stress and neuronal dysfunction. Here, we review recent evidence involving ER stress in HD pathogenesis and discuss possible therapeutic strategies to target organelle function in the context of disease.     

Autophagy Induced by Calcium Phosphate Precipitates Involves Endoplasmic Reticulum Membranes in Autophagosome Biogenesis

Calcium can play an important role in the regulation of autophagy. We previously reported that exogenously introduced calcium in the form of calcium phosphate precipitates (CPP) induces autophagy. Here we showed that CPP-induced autophagy required the classical autophagic machinery, including the autophagosome initiating molecules FIP200 and Beclin 1, as well as molecules involved in the autophagosome membrane extension, Atg4, Atg5 and Atg3. On the other hand, Atg9 seemed to place a restriction on CPP-induced autophagy. Loss of Atg9 led to enhanced LC3 punctation and enhanced p62 degradation. CPP-induced autophagy was independent of mTOR and reactive oxygen species. It also did not affect MAP kinase activation and ER stress. DFCP1 is an ER-resident molecule that binds to phosphatidylinositol 3-phosphate. CPP activated DFCP1 punctation in a class III phosphatidylinositol-3-kinase and calcium dependent manner, and caused the association of DFCP1 puncta with the autophagosomes. Consistently, ER membranes, but not Golgi or mitochondrial membranes, colocalized with CPP-induced LC3 positive autophagosomes. These data suggest that CPP-induced autophagosome formation involves the interaction with the ER membrane.     

Regulation of ER stress-induced macroautophagy by protein kinase C

The endoplasmic reticulum (ER) is the primary site for folding and quality control for proteins destined to the cell surface and intracellular organelles. A variety of cellular insults alter ER homeostasis to disrupt protein folding, cause the accumulation of misfolded protein and activate an autophagic response. However, the molecular signaling pathways required for ER stress-induced autophagy are largely unknown. Recently, we discovered that a novel-type protein kinase C family member (PKCθ) is required for ER stress-induced autophagy. We shown that ER stress, in a Ca²⁺-dependent manner, induces PKCθ phosphorylation within the activation loop and localization with LC3-II in punctate cytoplasmic structures. Pharmacological inhibition, siRNA-mediated knockdown, or transdominant-negative mutant expression of PKCθ block the ER stress-induced autophagic response. PKCθ activation is not required for autophagy induced by amino acid starvation, and PKCθ activation in response to ER stress does not require either the mTOR kinase or the unfolded protein response signaling pathways. Herein, we review and discuss the significance of these findings with respect to regulation of autophagy in response to ER stress.

Addendum to: Sakaki K, Wu J, Kaufman RJ. Protein kinase C-θ is required for autophagy in response to stress in the endoplasmic reticulum. J Biol Chem 2008; 283:15370-80.     

Interaction of huntingtin fragments with brain membranes--clues to early dysfunction in Huntington's disease

Abstract Huntingtin is a large, multi-domain protein of unknown function in the brain. An abnormally elongated polyglutamine stretch in its N-terminus causes Huntington's disease (HD), a progressive neurodegenerative disorder. Huntingtin has been proposed to play a functional role in membrane trafficking via proteins involved in endo- and exocytosis. Here, we supply evidence for a direct association between huntingtin and membranes. In the brains of R6/2 mice with HD pathology, a 64 kDa N-terminal huntingtin fragment accumulated in postsynaptic membranes during the pre-symptomatic period of 4-8 weeks of age. In addition, an oligomeric fragment of approximately 200 kDa was detected at 8 weeks of age. Simultaneous progressive changes in distribution of amphiphysin, synaptojanin, and subunits of NMDA- and AMPA-receptors provide a strong indication of dysfunctional synaptic trafficking. Composition of the major phospholipids in the synaptic membranes was unaffected. In vitro, large unilamellar vesicles of brain lipids readily associated with soluble N-terminal huntingtin exon 1 fragments and stimulated fibrillogenesis of mutant huntingtin aggregates. Moreover, interaction of both mutant and wild-type huntingtin exon 1 fragments with brain lipids caused bilayer perturbation, mediated through a proline-rich region adjacent to the polyglutamines. This suggests that lipid interactions in vivo could influence misfolding of huntingtin and may play an early role in HD pathogenesis.     

The reciprocal interaction between autophagic dysfunction and ER stress in adipose insulin resistance

Autophagy, a predominantly cytoprotective process, is an important regulator in diabetic metabolism and endoplasmic reticulum (ER) stress responses. However, the interaction and biological significance between autophagic imbalance and ER stress involved in insulin resistance remain not fully elucidated. In the present study, when compared with normal glucose tolerance (NGT) subjects, enhanced ER stress and pronounced protein and mRNA levels of the autophagic genes such as Atg7, LC3A, and LC3B were evident in adipose tissue of patients with type 2 diabetes. An increased number of autophagosomes and elevated autophagy flux in adipose explants incubated with lysomoal inhibitor were also observed in type 2 diabetes. In addition, adipocytes differentiation was significantly repressed by exogenous ER stress and defective autophagy in vitro. Tunicamycin-induced ER stress in adipocytes can trigger autophagic response and insulin insensitivity that was partially attributed to the upregulation of IRE1-JNK pathway, whereas autophagy deficiency resulted in ER stress and impaired insulin signaling, further supporting the crucial roles of autophagy in ER stress and insulin resistance. Moreover, disturbance of autophagy and insulin sensitivity induced by tunicamycin can be effectively corrected by the addition of osteocalcin in an NFκB-dependent manner in vitro. In conclusion, our results demonstrated a reciprocal functional interaction among autophagy, ER stress, and insulin signaling in adipose tissue of type 2 diabetes and adipocytes, supporting an adaptive role of autophagy-dependent mechanism in response to ER stress-induced insulin resistance in type 2 diabetes.     

Autophagosome formation is initiated at phosphatidylinositol synthase‐enriched ER subdomains

The autophagosome, a double‐membrane structure mediating degradation of cytoplasmic materials by macroautophagy, is formed in close proximity to the endoplasmic reticulum (ER). However, how the ER membrane is involved in autophagy initiation and to which membrane structures the autophagy‐initiation complex is localized have not been fully characterized. Here, we were able to biochemically analyze autophagic intermediate membranes and show that the autophagy‐initiation complex containing ULK and FIP200 first associates with the ER membrane. To further characterize the ER subdomain, we screened phospholipid biosynthetic enzymes and found that the autophagy‐initiation complex localizes to phosphatidylinositol synthase (PIS)‐enriched ER subdomains. Then, the initiation complex translocates to the ATG9A‐positive autophagosome precursors in a PI3P‐dependent manner. Depletion of phosphatidylinositol (PI) by targeting bacterial PI‐specific phospholipase C to the PIS domain impairs recruitment of downstream autophagy factors and autophagosome formation. These findings suggest that the autophagy‐initiation complex, the PIS‐enriched ER subdomain, and ATG9A vesicles together initiate autophagosome formation.     

Huntingtin inhibits caspase-3 activation

Huntington's disease results from a mutation in the HD gene encoding for the protein huntingtin. The function of huntingtin, although beginning to be elucidated, remains largely unclear. To probe the prosurvival function of huntingtin, we modulate levels of wild-type huntingtin in a number of cellular and in vivo models. Huntingtin depletion resulted in caspase-3 activation, and overexpression of huntingtin resulted in caspase-3 inhibition. Additionally, we demonstrate that huntingtin physically interacts with active caspase-3. Interestingly, mutant huntingtin binds active caspase-3 with a lower affinity and lower inhibitory effect on active caspase-3 than does wild-type huntingtin. Although reduction of huntingtin levels resulted in caspase-3 activation in all conditions examined, the cellular response was cell-type specific. Depletion of huntingtin resulted in either overt cell death, or in increased vulnerability to cell death. These data demonstrate that huntingtin inhibits caspase-3 activity, suggesting a mechanism whereby caspase-mediated huntingtin depletion results in a detrimental amplification cascade leading to further caspase-3 activation, resulting in cell dysfunction and cell death.     

The vacuolar transporter chaperone (VTC) complex is required for microautophagy

Microautophagy involves direct invagination and fission of the vacuolar/lysosomal membrane under nutrient limitation. This occurs by an autophagic tube, a specialized vacuolar membrane invagination that pinches off vesicles into the vacuolar lumen. In this study we have identified the VTC (vacuolar transporter chaperone) complex as required for microautophagy. The VTC complex is present on the ER and vacuoles and at the cell periphery. On induction of autophagy by nutrient limitation the VTC complex is recruited to and concentrated on vacuoles. The VTC complex is inhomogeneously distributed within the vacuolar membranes, showing an enrichment on autophagic tubes. Deletion of the VTC complex blocks microautophagic uptake into vacuoles. The mutants still form autophagic tubes but the production of microautophagic vesicles from their tips is impaired. In line with this, affinity-purified antibodies to the Vtc proteins inhibit microautophagic uptake in a reconstituted system in vitro. Our data suggest that the VTC complex is an important constituent of autophagic tubes and that it is required for scission of microautophagic vesicles from these tubes.