Heather L. Montie

Cerebral ischemia and the unfolded protein response

We review studies of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) following cerebral ischemia and reperfusion (I/R). The UPR is a cell stress program activated when misfolded proteins accumulate in the ER lumen. UPR activation causes: (i) a PERK‐mediated phosphorylation of eIF2α, inhibiting protein synthesis to prevent further accumulation of unfolded proteins in the ER and (ii) upregulation of genes coding for ER‐resident enzymes and chaperones and others, via eIF2α(p), and ATF6 and IRE1 activation. UPR‐induced transcription increases capacity of the ER to process misfolded proteins. If ER stress and the UPR are prolonged, apoptosis ensues. Multiple forms of ER stress have been observed following brain I/R. The UPR following brain I/R is not isomorphic between in vivo I/R models and in vitro cell culture systems with pharmacological UPR induction. Although PERK and IRE1 are activated in the initial hours of reperfusion, total PERK decreases, ATF6 is not activated, and there is delayed appearance of UPR‐induced mRNAs. Thus, multiple damage mechanisms associated with brain I/R alter UPR expression and contribute to a pro‐apoptotic phenotype in neurons. Insights resulting from these studies will be important for the development of therapies to halt neuronal death following brain I/R.

Cytoplasmic Retention of Polyglutamine-Expanded Androgen Receptor Ameliorates Disease via Autophagy in a Mouse Model of Spinal and Bulbar Muscular Atrophy

The nucleus is the primary site of protein aggregation in many polyglutamine diseases, suggesting a central role in pathogenesis. In SBMA, the nucleus is further implicated by the critical role for disease of androgens, which promote the nuclear translocation of the mutant androgen receptor (AR). To clarify the importance of the nucleus in SBMA, we genetically manipulated the nuclear localization signal of the polyglutamine-expanded AR. Transgenic mice expressing this mutant AR displayed inefficient nuclear translocation and substantially improved motor function compared with SBMA mice. While we found that nuclear localization of polyglutamine-expanded AR is required for SBMA, we also discovered, using cell models of SBMA, that it is insufficient for both aggregation and toxicity and requires androgens for these disease features. Through our studies of cultured motor neurons, we further found that the autophagic pathway was able to degrade cytoplasmically retained expanded AR and represents an endogenous neuroprotective mechanism. Moreover, pharmacologic induction of autophagy rescued motor neurons from the toxic effects of even nuclear-residing mutant AR, suggesting a therapeutic role for autophagy in this nucleus-centric disease. Thus, our studies firmly establish that polyglutamine-expanded AR must reside within nuclei in the presence of its ligand to cause SBMA. They also highlight a mechanistic basis for the requirement for nuclear localization in SBMA neurotoxicity, namely the lack of mutant AR removal by the autophagic protein degradation pathway.

Prolonged translation arrest in reperfused hippocampal cornu Ammonis 1 is mediated by stress granules

Global brain ischemia and reperfusion cause phosphorylation of the alpha subunit of eukaryotic initiation factor 2alpha, a reversible event associated with neuronal translation inhibition. However, the selective vulnerability of cornu Ammonis (CA) 1 pyramidal neurons correlates with irreversible translation inhibition. Phosphorylation of eukaryotic initiation factor 2alpha also leads to the formation of stress granules, cytoplasmic foci containing, in part, components of the 48S pre-initiation complex and the RNA binding protein T cell internal antigen-1 (TIA-1). Stress granules are sites of translationally inactive protein synthesis machinery. Here we evaluated stress granules in rat hippocampal formation neurons after 10 min global brain ischemia and 10 min, 90 min or 4 h of reperfusion by double-labeling immunofluorescence for two stress granule components: small ribosomal subunit protein 6 and TIA-1. Stress granules in CA3, hilus and dentate gyrus, but not CA1, increased at 10 min reperfusion and returned to control levels by 90 min reperfusion. Dynamic changes in the nuclear distribution of TIA-1 occurred in resistant neurons. At 4 h reperfusion, small ribosomal subunit protein 6 was solely localized within stress granules only in CA1 pyramidal neurons. Both TIA-1 and small ribosomal subunit protein 6 levels decreased approximately 50% in hippocampus homogenates. Electron microscopy showed stress granules to be composed of electron dense bodies 100-200 nm in diameter, that were not membrane bound, but were associated with endoplasmic reticulum. Alterations in stress granule behavior in CA1 pyramidal neurons provide a definitive mechanism for the continued inhibition of protein synthesis in reperfused CA1 pyramidal neurons following dephosphorylation of eukaryotic initiation factor 2alpha.

SIRT1 Modulates Aggregation and Toxicity through Deacetylation of the Androgen Receptor in Cell Models of SBMA

Posttranslational protein modifications can play a major role in disease pathogenesis; phosphorylation, sumoylation, and acetylation modulate the toxicity of a variety of proteotoxic proteins. The androgen receptor (AR) is substantially modified, in response to hormone binding, by phosphorylation, sumoylation, and acetylation; these modifications might thus contribute to DHT-dependent polyglutamine (polyQ)-expanded AR proteotoxicity in spinal and bulbar muscular atrophy (SBMA). SIRT1, a nuclear protein and deacetylase of the AR, is neuroprotective in many neurodegenerative disease models. Our studies reveal that SIRT1 also offers protection against polyQ-expanded AR by deacetylating the AR at lysines 630/632/633. This finding suggested that nuclear AR acetylation plays a role in the aberrant metabolism and toxicity of polyQ-expanded AR. Subsequent studies revealed that the polyQ-expanded AR is hyperacetylated and that pharmacologic reduction of acetylation reduces mutant AR aggregation. Moreover, genetic mutation to inhibit polyQ-expanded AR acetylation of lysines 630/632/633 substantially decreased its aggregation and completely abrogated its toxicity in cell lines and motor neurons. Our studies also reveal one means by which the AR acetylation state likely modifies polyQ-expanded AR metabolism and toxicity, through its effect on DHT-dependent AR stabilization. Overall, our findings reveal a neuroprotective function of SIRT1 that operates through its deacetylation of polyQ-expanded AR and highlight the potential of both SIRT1 and AR acetylation as powerful therapeutic targets in SBMA.

An Interdomain Interaction of the Androgen Receptor Is Required for Its Aggregation and Toxicity in Spinal and Bulbar Muscular Atrophy

Polyglutamine expansion within the androgen receptor (AR) causes spinal and bulbar muscular atrophy (SBMA) and is associated with misfolded and aggregated species of the mutant AR. We showed previously that nuclear localization of the mutant AR was necessary but not sufficient for SBMA. Here we show that an interdomain interaction of the AR that is central to its function within the nucleus is required for AR aggregation and toxicity. Ligands that prevent the interaction between the amino-terminal FXXLF motif and carboxyl-terminal AF-2 domain (N/C interaction) prevented toxicity and AR aggregation in an SBMA cell model and rescued primary SBMA motor neurons from 5α-dihydrotestosterone-induced toxicity. Moreover, genetic mutation of the FXXLF motif prevented AR aggregation and 5α-dihydrotestosterone toxicity. Finally, selective androgen receptor modulators, which prevent the N/C interaction, ameliorated AR aggregation and toxicity while maintaining AR function, highlighting a novel therapeutic strategy to prevent the SBMA phenotype while retaining AR transcriptional function.

Disrupting SUMOylation Enhances Transcriptional Function and Ameliorates Polyglutamine Androgen Receptor-Mediated Disease

Expansion of the polyglutamine (polyQ) tract within the androgen receptor (AR) causes neuromuscular degeneration in individuals with spinobulbar muscular atrophy (SBMA). PolyQ AR has diminished transcriptional function and exhibits ligand-dependent proteotoxicity, features that have both been implicated in SBMA; however, the extent to which altered AR transcriptional function contributes to pathogenesis remains controversial. Here, we sought to dissociate effects of diminished AR function from polyQ-mediated proteotoxicity by enhancing the transcriptional activity of polyQ AR. To accomplish this, we bypassed the inhibitory effect of AR SUMOylation (where SUMO indicates small ubiquitin-like modifier) by mutating conserved lysines in the polyQ AR that are sites of SUMOylation. We determined that replacement of these residues by arginine enhances polyQ AR activity as a hormone-dependent transcriptional regulator. In a murine model, disruption of polyQ AR SUMOylation rescued exercise endurance and type I muscle fiber atrophy; it also prolonged survival. These changes occurred without overt alterations in polyQ AR expression or aggregation, revealing the favorable trophic support exerted by the ligand-activated receptor. Our findings demonstrate beneficial effects of enhancing the transcriptional function of the ligand-activated polyQ AR and indicate that the SUMOylation pathway may be a potential target for therapeutic intervention in SBMA.

Autophagy and access: Understanding the role of androgen receptor subcellular localization in SBMA

Ridding neurons of toxic misfolded proteins is a critical feature of many neurodegenerative diseases. We have recently reported that lack of access of nuclear polyglutamine-expanded androgen receptor (AR) to the autophagic degradation pathway is a critical point in pathogenesis. When mutant AR is contained within the cytoplasm, it can be degraded by autophagy, resulting in amelioration of its toxic effects, as has been observed in other polyglutamine expansion diseases involving cytoplasmic mutant proteins. However, we have also found that pharmacological induction of autophagy protects SBMA motor neurons from the toxic effects of even nuclear localized mutant AR, albeit without affecting mutant nuclear AR levels. Thus, we have further investigated the mechanism by which autophagy elicits therapeutic benefit in cell culture. We found that endogenous autophagy only slightly alters nuclear mutant AR aggregation compared to substantial effects on cytoplasmic AR aggregation. Interestingly, pharmacological activation of mTOR-dependent autophagy did not significantly alter nuclear AR aggregation, whereas we observed that it protects SBMA motor neurons. Our findings indicate that therapeutic intervention to induce autophagy represents a potential potent benefit for SBMA, and that it likely does so by protecting SBMA motor neurons independent of a direct effect on mutant AR.

Inhibition of Stat5a/b Enhances Proteasomal Degradation of Androgen Receptor Liganded by Antiandrogens in Prostate Cancer

Although poorly understood, androgen receptor (AR) signaling is sustained despite treatment of prostate cancer with antiandrogens and potentially underlies development of incurable castrate-resistant prostate cancer. However, therapies targeting the AR signaling axis eventually fail when prostate cancer progresses to the castrate-resistant stage. Stat5a/b, a candidate therapeutic target protein in prostate cancer, synergizes with AR to reciprocally enhance the signaling of both proteins. In this work, we demonstrate that Stat5a/b sequesters antiandrogen-liganded (MDV3100, bicalutamide, flutamide) AR in prostate cancer cells and protects it against proteasomal degradation in prostate cancer. Active Stat5a/b increased nuclear levels of both unliganded and antiandrogen-liganded AR, as demonstrated in prostate cancer cell lines, xenograft tumors, and clinical patient-derived prostate cancer samples. Physical interaction between Stat5a/b and AR in prostate cancer cells was mediated by the DNA-binding domain of Stat5a/b and the N-terminal domain of AR. Moreover, active Stat5a/b increased AR occupancy of the prostate-specific antigen promoter and AR-regulated gene expression in prostate cancer cells. Mechanistically, both Stat5a/b genetic knockdown and antiandrogen treatment induced proteasomal degradation of AR in prostate cancer cells, with combined inhibition of Stat5a/b and AR leading to maximal loss of AR protein and prostate cancer cell viability. Our results indicate that therapeutic targeting of AR in prostate cancer using antiandrogens may be substantially improved by targeting of Stat5a/b. Mol Cancer Ther; 14(3); 713–26. ©2014 AACR.

Identification of novel polyglutamine-expanded aggregation species in spinal and bulbar muscular atrophy.

Polyglutamine-repeat disorders are part of a larger family of neurodegenerative diseases characterized by protein misfolding and aggregation. In spinal and bulbar muscular atrophy (SBMA), polyglutamine expansion within the androgen receptor (AR) causes progressive debilitating muscular atrophy and lower motor neuron loss in males. Although soluble polyglutamine-expanded aggregation species are considered toxic intermediates in the aggregation process, relatively little is known about the spectrum of structures that are formed. Here we identify novel polyglutamine-expanded AR aggregates that are SDS-soluble and bind the toxicity-predicting antibody 3B5H10. Soluble, 3B5H10-reactive aggregation species exist in low-density conformations and are larger by atomic force microscopy, suggesting that they may be less compact than later-stage, insoluble aggregates. We demonstrate disease-relevance in vivo and draw correlations with toxicity in vitro. This article is part of a Special Issue entitled SI: Neuroprotection.

The Cell and Molecular Biology of Neurodegenerative Diseases: An Overview

In this research topic, the primary focus is on understanding the cellular and molecular mechanisms in the pathogenesis of different neurodegenerative disorders. These include Alzheimer’s disease (AD), Parkinson’s disease (PD), and polyglutamine (polyQ) expansion diseases. To date, no cure exists for these disorders and it is paramount that research efforts continue to focus on understanding the molecular underpinnings behind these disorders. This will enable better symptom-directed therapeutics and perhaps even curative treatments to be developed. Throughout this topic, it becomes evident that there are common cellular pathways that are altered in these disorders, including protein, mitochondrial, and transcriptional homeostasis. In the case of AD, it has become widely accepted that AD is a synaptopathy, meaning that there is a loss or damage of synapses. This damage to synapses leads to altered neuronal circuitry. The neuron-specific, post-synaptic protein, Arc, has gained recent attention for its contribution in the regulation of memory consolidation. Kerrigan and Randall (1) discuss how alterations of Arc protein in the brains of AD patients and animal models of AD may be a clue as to how synaptic transmission is altered in AD, and how this cellular pathway may be of interest for therapeutic development. The next three reviews discuss the molecular events underlying PD and how the normal function of specific proteins associated with PD can help shed light on the causes of familial and sporadic PD. Lim and Zhang (2) outline a range of studies that implicate aberrations in mitochondrial function and protein homeostasis, with oxidative stress as the possible link between these two. A review from Dr. Edward A. Fon’s group complements this discussion by focusing on the structure and function of Parkin, PINK1, and DJ-1 as they relate to PD (3). The second review from Dr. Fon’s group digs even deeper into the role of Parkin and PINK1 in mitophagy in neurons. They discuss the importance of research initiatives to better define the roles of these two proteins in mitophagy and, in particular, within the context of a neuronal setting (4). The next five reviews focus on polyQ expansion diseases. Almeida et al. (5) provide a structural and functional view of trinucleotide repeats and encoded homopeptide expansions, emphasizing polyQ expansions and their role in inducing the self-assembly, aggregation, and functional alterations of the protein, leading to neuronal toxicity and cell death. These authors focus on ataxin-3 and huntingtin (Htt), the main protein implicated in Machado–Joseph Disease (MJD) and Huntington’s disease (HD) respectively. Drs. Durcan and Fon, also focus on ataxin-3 and its function as a deubiquitinating enzyme (6). These authors have recently identified ataxin-3’s E3 ubiquitin ligase partners to be parkin and CHIP. As MJD patients often present with PD symptoms, the fact that parkin’s activity is regulated by ataxin-3-mediated deubiquitination is a critical link for this phenotype. As mutant, but not wild-type ataxin-3 promotes clearance of parkin via the autophagy pathway, there seems to be a possibility that increased turnover of parkin contributes to pathogenesis in MJD. Moreover, ataxin-3 also induces a reduction in CHIP levels. In light of these findings, the authors discuss the implications for the role of mutant ataxin-3’s effect upon Parkin and CHIP levels in understanding the molecular processes involved in SCA3 and perhaps other neurodegenerative disorders. Moumne et al. (7) discuss the role of transcriptional disruption in HD, specifically, as it relates to the transcriptional repressor, R element-1 silencing transcription factor (REST). REST is a transcriptional repressor of neuronal survival factors and normally associates with wild-type Htt. However, there is an aberrant alteration of cytoplasmic retention of the transcriptional repressor REST by mutant Htt. The authors go on to describe studies that have implicated this aberrant effect of mutant Htt on REST function to regulate neuronal genes and how this may impact pathogenesis in HD. Du et al. (8) focus on depression, the major psychiatric manifestation of HD. They discuss potential mechanisms of pathogenesis identified from animal models and compare depression in HD patients with non-HD persons, asking the question; Does HD-related depression differ from non-HD persons? They also go on to discuss some molecular and cellular mechanisms which may contribute to depression in HD. Beitel et al. (9) discuss spinal and bulbar muscular atrophy (SBMA), a polyQ expansion disease caused by the expansion of a CAG tract in the androgen receptor (AR) gene. This review summarizes all of the aspects of AR metabolism, from posttranslational modifications, to protein degradation and transcriptional function that have been implicated in SBMA pathogenesis. Spencer et al. (10) offer a commentary on Western Pacific Amyotrophic lateral sclerosis (ALS) – parkinsonism-dementia complex (PDC) that plagues the island populations of Chamorros on Guam, Japanese in Honshu Island’s Kii Peninsula, and Papuan New Guineans in Irian Jaya, Indonesia. It is a spectrum disorder believed to be to be triggered by a toxin in the seed of the neurotoxic cycad plant. This toxin is thought to induce a prototypical neurodegenerative disorder linked to DNA damage and aberrant proteogenesis. The final review discusses how cellular surfaces modulate protein aggregation related to neurodegeneration. The interaction of proteins with liquid/surface interfaces is a fundamental phenomenon with potential implications for protein-misfolding diseases. Burke et al. (11) provide an overview of what is known about the influence of (sub) cellular surfaces in driving protein aggregation and/or stabilizing specific aggregate forms and how further understanding of such could provide new insights into toxic mechanisms associated with these diseases. Lastly, the methods paper by Lange et al. (12) describes a detailed method for culturing embryonic dorsal root ganglion neurons for Seahorse Extracellular Flux XF24 analysis. This is a procedure used to measure the relative state of glycolytic and aerobic metabolism in live cells, in order to assess mitochondrial function. As changes in mitochondrial dynamics and function contribute to multiple neurodegenerative diseases, this method outlined herein is of significant interest to this topic.