Conophylline Protects Cells in Cellular Models of Neurodegenerative Diseases by Inducing Mammalian Target of Rapamycin (mTOR)-independent Autophagy

Macroautophagy is a cellular response that leads to the bulk, nonspecific degradation of cytosolic components, including organelles. In recent years, it has been recognized that autophagy is essential for prevention of neurodegenerative diseases, including Parkinson disease (PD) and Huntington disease (HD). Here, we show that conophylline (CNP), a vinca alkaloid, induces autophagy in an mammalian target of rapamycin-independent manner. Using a cellular model of PD, CNP suppressed protein aggregation and protected cells from cell death caused by treatment with 1-methyl-4-phenylpyridinium, a neurotoxin, by inducing autophagy. Moreover, in the HD model, CNP also eliminated mutant huntingtin aggregates. Our findings demonstrate the possible use of CNP as a therapeutic drug for neurodegenerative disorders, including PD and HD.     

Protein aggregation and pathogenesis of Huntington's disease: mechanisms and correlations

The formation of insoluble protein aggregates is a hallmark of Huntington's disease (HD) and related neurodegenerative disorders, such as dentatorubral pallidoluysian atrophy (DRPLA), spinal bulbar muscular atrophy (SBMA) and the spinocerebellar ataxia (SCA) type 1, 2, 3, 6 and 7. These disorders are caused by an expanded polyglutamine (polyQ) tract in otherwise unrelated proteins. They are characterized by late-onset, selective neuropathology, a pathogenic polyQ threshold and a relationship between polyQ length and disease progression. Thus, molecular models of HD and related glutamine-repeat disorders must account for these characteristic features. During the last three years, considerable effort has been invested in the development of in vitro and in vivo model systems to study the mechanisms of protein aggregation in glutamine-repeat disorders and its potential effects on disease progression and neurodegeneration. A selection of these studies is reviewed here. Furthermore, the correlation between aggregate formation and development of HD is discussed.     

Quantitative connection between polyglutamine aggregation kinetics and neurodegenerative process in patients with Huntington's disease

ABSTRACT: BACKGROUND: Despite enormous progress in elucidating the biophysics of aggregation, no cause-and-effect relationship between protein aggregation and neurodegenerative disease has been unequivocally established. Here, we derived several risk-based stochastic kinetic models that assess genotype/phenotype correlations in patients with Huntington's disease (HD) caused by the expansion of a CAG repeat. Fascinating disease-specific aspects of HD include the polyglutamine (polyQ)-length dependence of both age at symptoms onset and the propensity of the expanded polyQ protein to aggregate. In vitro, aggregation of polyQ peptides follows a simple nucleated growth polymerization pathway. Our models that reflect polyQ aggregation kinetics in a nucleated growth polymerization divided aggregate process into the lengthdependent nucleation and the nucleation-dependent elongation. In contrast to the repeatlength dependent variability of age at onset, recent studies have shown that the extent of expansion has only a subtle effect on the rate of disease progression, suggesting possible differences in the mechanisms underlying the neurodegenerative process. RESULTS: Using polyQ-length as an index, these procedures enabled us for the first time to establish a quantitative connection between aggregation kinetics and disease process, including onset and the rate of progression. Although the complexity of disease process in HD, the time course of striatal neurodegeneration can be precisely predicted by the mathematical model in which neurodegeneration occurs by different mechanisms for the initiation and progression of disease processes. Nucleation is sufficient to initiate neuronal loss as a series of random events in time. The stochastic appearance of nucleation in a cell population acts as the constant risk of neuronal cell damage over time, while elongation reduces the risk by nucleation in proportion to the increased extent of the aggregates during disease progression. CONCLUSIONS: Our findings suggest that nucleation is a critical step in gaining toxic effects to the cell, and provide a new insight into the relationship between polyQ aggregation and neurodegenerative process in HD.     

Modeling Huntington disease in Drosophila

Huntington disease (HD) is an inherited neurodegenerative disorder caused by a polyglutamine (polyQ) expansion in the huntingtin (Htt) gene. Despite years of research, there is no treatment that extends life for patients with the disorder. Similarly, little is known about which cellular pathways that are altered by pathogenic Huntingtin (Htt) protein expression are correlated with neuronal loss. As part of a longstanding effort to gain insights into HD pathology, we have been studying the protein in the context of the fruitfly Drosophila melanogaster. We generated transgenic HD models in Drosophila by engineering flies that carry a 12-exon fragment of the human Htt gene with or without the toxic trinucleotide repeat expansion. We also created variants with a monomeric red fluorescent protein (mRFP) tag fused to Htt that allows in vivo imaging of Htt protein localization and aggregation. While wild-type Htt remains diffuse throughout the cytoplasm of cells, pathogenic Htt forms insoluble aggregates that accumulate in neuronal soma and axons. Aggregates can physically block transport of numerous organelles along the axon. We have also observed that aggregates are formed quickly, within just a few hours of mutant Htt expression. To explore mechanisms of neurodegeneration in our HD model, we performed in vivo and in vitro screens to search for modifiers of viability and pathogenic Htt aggregation. Our results identified several novel candidates for HD therapeutics that can now be tested in mammalian models of HD. Furthermore, these experiments have highlighted the complex relationship between aggregates and toxicity that exists in HD.     

Polyglutamine pathogenesis.

An increasing number of neurodegenerative disorders have been found to be caused by expanding CAG triplet repeats that code for polyglutamine. Huntington's disease (HD) is the most common of these disorders and dentatorubral-pallidoluysian atrophy (DRPLA) is very similar to HD, but is caused by mutation in a different gene, making them good models to study. In this review, we will concentrate on the roles of protein aggregation, nuclear localization and proteolytic processing in disease pathogenesis. In cell model studies of HD, we have found that truncated N-terminal portions of huntingtin (the HD gene product) with expanded repeats form more aggregates than longer or full length huntingtin polypeptides. These shorter fragments are also more prone to aggregate in the nucleus and cause more cell toxicity. Further experiments with huntingtin constructs harbouring exogenous nuclear import and nuclear export signals have implicated the nucleus in direct cell toxicity. We have made mouse models of HD and DRPLA using an N-terminal truncation of huntingtin (N171) and full-length atrophin-1 (the DRPLA gene product), respectively. In both models, diffuse neuronal nuclear staining and nuclear inclusion bodies are observed in animals expressing the expanded glutamine repeat protein, further implicating the nucleus as a primary site of neuronal dysfunction. Neuritic pathology is also observed in the HD mice. In the DRPLA mouse model, we have found that truncated fragments of atrophin-1 containing the glutamine repeat accumulate in the nucleus, suggesting that proteolysis may be critical for disease progression. Taken together, these data lead towards a model whereby proteolytic processing, nuclear localization and protein aggregation all contribute to pathogenesis.     

Double-strand RNA dependent protein kinase (PKR) is involved in the extrastriatal degeneration in Parkinson's disease and Huntington's disease

Double-strand RNA dependent protein kinase (PKR) plays an important role in control of cell death. We previously reported that activation of PKR is associated with hippocampal neuronal loss in Alzheimer's disease (AD). Recent studies have reported that Parkinson's (PD) and Huntington's (HD) disease brains displayed progressive hippocampal neuronal loss in extrastriatal degeneration. However, association between PKR and hippocampal neuronal loss in PD and HD brains is not known. In this report, brain tissues from patients with PD and HD displayed strong induction of phosphorylated-PKR (p-PKR) in hippocampal neurons. Immunoblotting analysis also demonstrated that levels of nuclear p-PKR in the hippocampus affected by these diseases were increased compared with age-matched disease controls. These results suggest that a close association exists between PKR and extrastriatal degeneration in PD and HD pathology.     

Quantitative connection between polyglutamine aggregation kinetics and neurodegenerative process in patients with Huntington’s disease

Background

Despite enormous progress in elucidating the biophysics of aggregation, no cause-and-effect relationship between protein aggregation and neurodegenerative disease has been unequivocally established. Here, we derived several risk-based stochastic kinetic models that assess genotype/phenotype correlations in patients with Huntington??s disease (HD) caused by the expansion of a CAG repeat. Fascinating disease-specific aspects of HD include the polyglutamine (polyQ)-length dependence of both age at symptoms onset and the propensity of the expanded polyQ protein to aggregate. In vitro, aggregation of polyQ peptides follows a simple nucleated growth polymerization pathway. Our models that reflect polyQ aggregation kinetics in a nucleated growth polymerization divided aggregate process into the length-dependent nucleation and the nucleation-dependent elongation. In contrast to the repeat-length dependent variability of age at onset, recent studies have shown that the extent of expansion has only a subtle effect on the rate of disease progression, suggesting possible differences in the mechanisms underlying the neurodegenerative process.

Results

Using polyQ-length as an index, these procedures enabled us for the first time to establish a quantitative connection between aggregation kinetics and disease process, including onset and the rate of progression. Although the complexity of disease process in HD, the time course of striatal neurodegeneration can be precisely predicted by the mathematical model in which neurodegeneration occurs by different mechanisms for the initiation and progression of disease processes. Nucleation is sufficient to initiate neuronal loss as a series of random events in time. The stochastic appearance of nucleation in a cell population acts as the constant risk of neuronal cell damage over time, while elongation reduces the risk by nucleation in proportion to the increased extent of the aggregates during disease progression.

Conclusions

Our findings suggest that nucleation is a critical step in gaining toxic effects to the cell, and provide a new insight into the relationship between polyQ aggregation and neurodegenerative process in HD.     

Mutant huntingtin promotes the fibrillogenesis of wild-type huntingtin: a potential mechanism for loss of huntingtin function in Huntington's disease

Aggregation of huntingtin (htt) in neuronal inclusions is associated with the development of Huntington's disease (HD). Previously, we have shown that mutant htt fragments with polyglutamine (polyQ) tracts in the pathological range (>37 glutamines) form SDS-resistant aggregates with a fibrillar morphology, whereas wild-type htt fragments with normal polyQ domains do not aggregate. In this study we have investigated the co-aggregation of mutant and wild-type htt fragments. We found that mutant htt promotes the aggregation of wild-type htt, causing the formation of SDS-resistant co-aggregates with a fibrillar morphology. Conversely, mutant htt does not promote the fibrillogenesis of the polyQ-containing protein NOCT3 or the polyQ-binding protein PQBP1, although these proteins are recruited into inclusions containing mutant htt aggregates in mammalian cells. The formation of mixed htt fibrils is a highly selective process that not only depends on polyQ tract length but also on the surrounding amino acid sequence. Our data suggest that mutant and wild-type htt fragments may also co-aggregate in neurons of HD patients and that a loss of wild-type htt function may contribute to HD pathogenesis.     

Structural and morphological characterization of aggregated species of α-synuclein induced by docosahexaenoic acid

The interaction of brain lipids with α-synuclein may play an important role in the pathogenesis of Parkinson disease (PD). Docosahexaenoic acid (DHA) is an abundant fatty acid of neuronal membranes, and it is presents at high levels in brain areas with α-synuclein inclusions of patients with PD. In animal models, an increase of DHA content in the brain induces α-synuclein oligomer formation in vivo. However, it is not clear whether these oligomeric species are the precursors of the larger aggregates found in Lewy bodies of post-mortem PD brains. To characterize these species and to define the role of fatty acids in amyloid formation, we investigated the aggregation process of α-synuclein in the presence of DHA. We found that DHA readily promotes α-synuclein aggregation and that the morphology of these aggregates is dependent on the ratio between the protein and DHA. In the presence of a molar ratio protein/DHA of 1:10, amyloid-like fibrils are formed. These fibrils are morphologically different from those formed by α-synuclein alone and have a less packed structure. At a protein/DHA molar ratio of 1:50, we observe the formation of stable oligomers. Moreover, chemical modifications, methionine oxidations, and protein-lipid adduct formations are induced by increasing concentrations of DHA. The extent of these modifications defines the structure and the stability of aggregates. We also show that α-synuclein oligomers are more toxic if generated in the presence of DHA in dopaminergic neuronal cell lines, suggesting that these species might be important in the neurodegenerative process associated with PD.     

Arfaptin 2 regulates the aggregation of mutant huntingtin protein

Huntington's disease (HD) is an inherited neurodegenerative disorder. Here we demonstrate that expression of arfaptin 2/POR1 (partner of Rac1) in cultured cells induces the formation of pericentriolar and nuclear aggregates, which morphologically resemble mutant huntingtin aggregates characteristic of HD. Endogenous arfaptin 2 localizes to aggregates induced by expression of an abnormal amino-terminal fragment of huntingtin that contains polyglutamine (polyQ) expansions. A dominant inhibitory mutant of arfaptin 2 inhibits aggregation of mutant huntingtin, but not in the presence of proteasome inhibitors. Using cell-free biochemical assays, we show that arfaptin 2 inhibits proteasome activity. Finally, we show that expression of arfaptin 2 is increased at sites of neurodegeneration and the protein localizes to huntingtin aggregates in HD transgenic mouse brains. Our data suggest that arfaptin 2 is involved in regulating huntingtin protein aggregation, possibly by impairing proteasome function.     

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.     

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.     

Early alterations of autophagy in Huntington disease-like mice

In a recent study, we reported in vivo evidence of early and sustained alterations of autophagy markers in a novel knock-in mouse model of Huntington disease (HD). The novel model is derived from selective breeding of HdhQ150 knock-in mice to generate mice with ~200 CAG/polyglutamine repeats (HdhQ200). HdhQ200 knockin mice exhibit an accelerated and more robust motor phenotype than the parent line with detectable abnormalities at 50 weeks and substantial impairments at 80 weeks. Heterozygous HdhQ200 knock-in mice accumulate htt aggregates as cytoplasmic aggregation foci (AF) as early as 9 weeks of age followed by striatal neuronal intranuclear inclusions (NIIs) by 20 weeks. By 40 weeks, striatal AF are perinuclear and immunoreactive for ubiquitin and the autophagosome marker LC3. Increased LC3-II protein expression is noted at 9 weeks and sustained throughout the disease course, and is paralleled by increased expression of p62. Early and sustained expression of: autophagy-related proteins in this genetically precise mouse model of HD suggests that alteration of autophagic flux is an important and early component of neuronal response to polyglutamine expanded huntingtin.     

A light-inducible protein clustering system for in vivo analysis of α-synuclein aggregation in Parkinson disease

Neurodegenerative disorders refer to a group of diseases commonly associated with abnormal protein accumulation and aggregation in the central nervous system. However, the exact role of protein aggregation in the pathophysiology of these disorders remains unclear. This gap in knowledge is due to the lack of experimental models that allow for the spatiotemporal control of protein aggregation, and the investigation of early dynamic events associated with inclusion formation. Here, we report on the development of a light-inducible protein aggregation (LIPA) system that enables spatiotemporal control of α-synuclein (α-syn) aggregation into insoluble deposits called Lewy bodies (LBs), the pathological hallmark of Parkinson disease (PD) and other proteinopathies. We demonstrate that LIPA-α-syn inclusions mimic key biochemical, biophysical, and ultrastructural features of authentic LBs observed in PD-diseased brains. In vivo, LIPA-α-syn aggregates compromise nigrostriatal transmission, induce neurodegeneration and PD-like motor impairments. Collectively, our findings provide a new tool for the generation, visualization, and dissection of the role of α-syn aggregation in PD.

How do alpha-synuclein aggregates contribute to neuronal damage in Parkinson’s disease? To help address this question, this study presents a new optogenetic-based experimental model that allows for the induction and real-time monitoring of alpha-synuclein clustering in vivo.     

TRIM family proteins: roles in proteostasis and neurodegenerative diseases

Neurodegenerative diseases (NDs) are a diverse group of disorders characterized by the progressive degeneration of the structure and function of the central or peripheral nervous systems. One of the major features of NDs, such as Alzheimer''s disease (AD), Parkinson''s disease (PD) and Huntington''s disease (HD), is the aggregation of specific misfolded proteins, which induces cellular dysfunction, neuronal death, loss of synaptic connections and eventually brain damage. By far, a great amount of evidence has suggested that TRIM family proteins play crucial roles in the turnover of normal regulatory and misfolded proteins. To maintain cellular protein quality control, cells rely on two major classes of proteostasis: molecular chaperones and the degradative systems, the latter includes the ubiquitin-proteasome system (UPS) and autophagy; and their dysfunction has been established to result in various physiological disorders including NDs. Emerging evidence has shown that TRIM proteins are key players in facilitating the clearance of misfolded protein aggregates associated with neurodegenerative disorders. Understanding the different pathways these TRIM proteins employ during episodes of neurodegenerative disorder represents a promising therapeutic target. In this review, we elucidated and summarized the diverse roles with underlying mechanisms of members of the TRIM family proteins in NDs.     

Controlling the mass action of alpha-synuclein in Parkinson's disease

Parkinson's disease (PD) is an age-related neurodegenerative disease with unknown etiology. Growing evidence from genetic, pathologic, animal modeling, and biochemical studies strongly support the theory that abnormal aggregation of alpha-synuclein plays a critical role in the pathogenesis of PD. Protein aggregation is an alternative folding process that competes with the native folding pathway. Whether or not a protein is subject to the aggregation process is determined by the concentration of the protein as well as thermodynamic properties inherent to each polypeptide. An increase in cellular concentration of alpha-synuclein has been associated with the disease in both familial and sporadic forms of PD. Thus, maintenance of the intraneuronal steady state levels of alpha-synuclein below the critical concentration is a key challenge neuronal cells are facing. Expression of the alpha-synuclein gene is under the control of environmental factors and aging, the two best-established risk factors for PD. Studies also suggest that the degradation of this protein is mediated by proteasomal and autophagic pathways, which are two mechanisms that are related to the pathogenesis of PD. Recently, vesicle-mediated exocytosis has been suggested as a novel mechanism for disposal of neuronal alpha-synuclein. Relocalization of the protein to specific compartments may be another method for increasing its local concentration. Regulation of the neuronal steady state levels of alpha-synuclein has significant implications in the development of PD, and understanding the mechanism may disclose potential therapeutic targets for PD and other related diseases.     

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.     

Reticulon 3 attenuates the clearance of cytosolic prion aggregates via inhibiting autophagy

Autophagy plays an important role in targeting cellular proteins, protein aggregates and organelles for degradation for cell survival. Autophagy dysfunction has been extensively described in neurodegenerative conditions linked to protein misfolding and aggregation. However, the role of autophagy in the prion disease process is unclear. Here, we show that when expressed in mouse neuroblastoma N2a cells, cytoplasmic PrP (cyPrP) aggregates lead to endoplasmic reticulum stress (ER stress), activation of reticulon 3 (RTN3), impairment of ubiquitin-proteasome system (UPS), induction of autophagy and apoptosis. RTN3 belongs to the reticulon family with the highest expression in the brain and RTN3 is often activated under ER stress. To assess the function of RTN3 in pathological conditions involving cyPrP protein misfolding, we knocked down the expression of RTN3 in cyPrP-transfected cells; unexpectedly, the inhibition of expression of RTN3 enhances the induction of autophagy resulted from cyPrP aggregates, and the process is mediated by the enhanced interaction between Bcl-2 and Beclin1 promoted by RTN3, which enhances Bcl-2-mediated inhibition of Beclin 1-dependent autophagy. Furthermore, down-regulation of RTN3 promoted the clearance of cyPrP aggregates, allowed the activity of the UPS to resume and alleviated ER stress; ultimately, apoptosis due to the cyPrP aggregates was inhibited. Together, these data suggest that RTN3 negatively regulates autophagy to block the clearance of cyPrP aggregates and provide a clue regarding the potential to induce autophagy for the treatment of prion disease and other neurodegenerative diseases such as Parkinson disease (PD), Alzheimer disease (AD) and Huntington disease (HD).     

Dendritic spine loss and neurodegeneration is rescued by Rab11 in models of Huntington's disease

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by expansion of a polyglutamine tract in the huntingtin protein (htt) that mediates formation of intracellular protein aggregates. In the brains of HD patients and HD transgenic mice, accumulation of protein aggregates has been causally linked to lesions in axo-dendritic and synaptic compartments. Here we show that dendritic spines - sites of synaptogenesis - are lost in the proximity of htt aggregates because of functional defects in local endosomal recycling mediated by the Rab11 protein. Impaired exit from recycling endosomes (RE) and association of endocytosed protein with intracellular structures containing htt aggregates was demonstrated in cultured hippocampal neurons cells expressing a mutant htt fragment. Dendrites in hippocampal neurons became dystrophic around enlarged amphisome-like structures positive for Rab11, LC3 and mutant htt aggregates. Furthermore, Rab11 overexpression rescues neurodegeneration and dramatically extends lifespan in a Drosophila model of HD. Our findings are consistent with the model that mutant htt aggregation increases local autophagic activity, thereby sequestering Rab11 and diverting spine-forming cargo from RE into enlarged amphisomes. This mechanism may contribute to the toxicity caused by protein misfolding found in a number of neurodegenerative diseases.