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 amino-terminal 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.

Addendum to: Atwal RS, Xia J, Pinchev D, Taylor J, Epand RM, Truant R. Huntingtin has a membrane association signal that can modulate huntingtin aggregation, nuclear entry and toxicity. Hum Mol Genet 2007; 16:2600-15.     

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.     

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.     

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.     

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.     

Restoration of ER proteostasis attenuates remote apoptotic cell death after spinal cord injury by reducing autophagosome overload

The pathogenic mechanisms that underlie the progression of remote degeneration after spinal cord injury (SCI) are not fully understood. In this study, we examined the relationship between endoplasmic reticulum (ER) stress and macroautophagy, hereafter autophagy, and its contribution to the secondary damage and outcomes that are associated with remote degeneration after SCI. Using a rat model of spinal cord hemisection at the cervical level, we measured ER stress and autophagy markers in the axotomized neurons of the red nucleus (RN). In SCI animals, mRNA and protein levels of markers of ER stress, such as GRP78, CHOP, and GADD34, increased 1 day after the injury, peaking on Day 5. Notably, in SCI animals, the increase of ER stress markers correlated with a blockade in autophagic flux, as evidenced by the increase in microtubule-associated protein 2 light chain 3 (LC3-II) and p62/SQSTM1 (p62) and the decline in LAMP1 and LAMP2 levels. After injury, treatment with guanabenz protected neurons from UPR failure and increased lysosomes biogenesis, unblocking autophagic flux. These effects correlated with greater activation of TFEB and improved neuronal survival and functional recovery—effects that persisted after suspension of the treatment. Collectively, our results demonstrate that in remote secondary damage, impairments in autophagic flux are intertwined with ER stress, an association that contributes to the apoptotic cell death and functional damage that are observed after SCI.Subject terms: Cell death in the nervous system, Neurodegeneration, Molecular neuroscience     

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.     

Protein kinase Ctheta is required for autophagy in response to stress in the endoplasmic reticulum

Autophagy is an evolutionally conserved process for the bulk degradation of cytoplasmic proteins and organelles. Recent observations indicate that autophagy is induced in response to cellular insults that result in the accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER). However, the signaling mechanisms that activate autophagy under these conditions are not understood. Here, we report that ER stress-induced autophagy requires the activation of protein kinase C (PKC), a member of the novel-type PKC family. Induction of ER stress by treatment with either thapsigargin or tunicamycin activated autophagy in immortalized hepatocytes as monitored by the conversion LC3-I to LC3-II, clustering of LC3 into dot-like cytoplasmic structures, and electron microscopic detection of autophagosomes. Pharmacological inhibition of PKC or small interfering RNA-mediated knockdown of PKC prevented the autophagic response to ER stress. Treatment with ER stressors induced PKC phosphorylation within the activation loop and localization of phospho-PKC to LC3-containing dot structures in the cytoplasm. However, signaling through the known unfolded protein response sensors was not required for PKC activation. PKC activation and stress-induced autophagy were blocked by chelation of intracellular Ca(2+) with BAPTA-AM. PKC was not activated or required for autophagy in response to amino acid starvation. These observations indicate that Ca(2+)-dependent PKC activation is specifically required for autophagy in response to ER stress but not in response to amino acid starvation.     

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.     

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.     

The Metastasis Suppressor,N-myc Downstream-regulated Gene 1 (NDRG1), Inhibits Stress-induced Autophagy in Cancer Cells

N-myc downstream regulated gene 1 (NDRG1) is a potent metastasis suppressor with an undefined role in the stress response. Autophagy is a pro-survival pathway and can be regulated via the protein kinase-like endoplasmic reticulum kinase (PERK)/eIF2α-mediated endoplasmic reticulum (ER) stress pathway. Hence, we investigated the role of NDRG1 in stress-induced autophagy as a mechanism of inhibiting metastasis via the induction of apoptosis. As thiosemicarbazone chelators induce stress and up-regulate NDRG1 to inhibit metastasis, we studied their effects on the ER stress response and autophagy. This was important to assess, as little is understood regarding the role of the stress induced by iron depletion and its role in autophagy. We observed that the chelator, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), which forms redox-active iron and copper complexes, effectively induced ER stress as shown by activation of the PERK/eIF2α pathway. Dp44mT also increased the expression of the autophagic marker, LC3-II, and this was dependent on activation of the PERK/eIF2α axis, as silencing PERK prevented LC3-II accumulation. The effect of Dp44mT on LC3-II expression was at least partially due to iron-depletion, as this effect was also demonstrated with the classical iron chelator, desferrioxamine (DFO), and was not observed for the DFO-iron complex. NDRG1 overexpression also inhibited basal autophagic initiation and the ER stress-mediated autophagic pathway via suppression of the PERK/eIF2α axis. Moreover, NDRG1-mediated suppression of the pro-survival autophagic pathway probably plays a role in its anti-metastatic effects by inducing apoptosis. In fact, multiple pro-apoptotic markers were increased, whereas anti-apoptotic Bcl-2 was decreased upon NDRG1 overexpression. This study demonstrates the role of NDRG1 as an autophagic inhibitor that is important for understanding its mechanism of action.     

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.     

Endoplasmic reticulum stress triggers autophagy

Eukaryotic cells have evolved strategies to respond to stress conditions. For example, autophagy in yeast is primarily a response to the stress of nutrient limitation. Autophagy is a catabolic process for the degradation and recycling of cytosolic, long lived, or aggregated proteins and excess or defective organelles. In this study, we demonstrate a new pathway for the induction of autophagy. In the endoplasmic reticulum (ER), accumulation of misfolded proteins causes stress and activates the unfolded protein response to induce the expression of chaperones and proteins involved in the recovery process. ER stress stimulated the assembly of the pre-autophagosomal structure. In addition, autophagosome formation and transport to the vacuole were stimulated in an Atg protein-dependent manner. Finally, Atg1 kinase activity reflects both the nutritional status and autophagic state of the cell; starvation-induced autophagy results in increased Atg1 kinase activity. We found that Atg1 had high kinase activity during ER stress-induced autophagy. Together, these results indicate that ER stress can induce an autophagic response.     

Endoplasmic Reticulum Stress-Induced Autophagy Provides Cytoprotection from Chemical Hypoxia and Oxidant Injury and Ameliorates Renal Ischemia-Reperfusion Injury

We examined whether endoplasmic reticulum (ER) stress-induced autophagy provides cytoprotection from renal tubular epithelial cell injury due to oxidants and chemical hypoxia in vitro, as well as from ischemia-reperfusion (IR) injury in vivo. We demonstrate that the ER stress inducer tunicamycin triggers an unfolded protein response, upregulates ER chaperone Grp78, and activates the autophagy pathway in renal tubular epithelial cells in culture. Inhibition of ER stress-induced autophagy accelerated caspase–3 activation and cell death suggesting a pro-survival role of ER stress-induced autophagy. Compared to wild-type cells, autophagy-deficient MEFs subjected to ER stress had enhanced caspase–3 activation and cell death, a finding that further supports the cytoprotective role of ER stress-induced autophagy. Induction of autophagy by ER stress markedly afforded cytoprotection from oxidants H₂O₂ and tert-Butyl hydroperoxide and from chemical hypoxia induced by antimycin A. In contrast, inhibition of ER stress-induced autophagy or autophagy-deficient cells markedly enhanced cell death in response to oxidant injury and chemical hypoxia. In mouse kidney, similarly to renal epithelial cells in culture, tunicamycin triggered ER stress, markedly upregulated Grp78, and activated autophagy without impairing the autophagic flux. In addition, ER stress-induced autophagy markedly ameliorated renal IR injury as evident from significant improvement in renal function and histology. Inhibition of autophagy by chloroquine markedly increased renal IR injury. These studies highlight beneficial impact of ER stress-induced autophagy in renal ischemia-reperfusion injury both in vitro and in vivo.