Reduced free ubiquitin levels and proteasome activity in cultured neurons and brain tissues treated with amyloid beta aggregates.
ABSTRACT: Neurodegenerative diseases are characterized by progressive cognitive decline and the loss of neurons in the central nervous system; many are also characterized by abnormal aggregation of misfolded proteins. Ubiquitin (Ub) is a eukaryotic protein that plays pivotal roles in protein degradation and cellular signaling. Ubiquitinated aggregates are observed in neurodegenerative diseases; this ultimately results in reduced levels of available or free Ub. However, it remains unclear whether neurotoxicity arises from the aggregates or a deficiency of free Ub. To investigate this, we treated primary neurons of mouse embryonic brains with amyloid beta (A?) 42 and found that free Ub levels were decreased and cell viability was reduced in A?42-treated neurons. As reduced levels of free Ub are closely related to impaired function of the proteasome, we evaluated proteasome activity and found that proteasome activity was reduced upon treatment of primary neurons and mouse brain slices with A?42. Therefore, we conclude that proteotoxic stress from A?42 treatment reduced the levels of available Ub and decreased proteasome activity, resulting in inflammatory stress and apoptosis of neurons.
Project description:A hallmark of most neurodegenerative diseases, including those caused by polyglutamine expansion, is the formation of ubiquitin (Ub)-positive protein aggregates in affected neurons. This finding suggests that the Ub system may be involved in common mechanisms underlying these otherwise unrelated diseases. Here we report the finding of ataxin-3 (Atx-3), whose mutation is implicated in the neurodegenerative disease spinocerebellar ataxia type 3, in a bioinformatics search of the human genome for components of the Ub system. We show that wild-type Atx-3 is a Ub-binding protein and that the interaction of Atx-3 with Ub is mediated by motifs homologous to those found in a proteasome subunit. Both wild-type Atx-3 and the otherwise unrelated Ub-binding protein p62/Sequestosome-1 have been shown to be sequestered into aggregates in affected neurons in several neurodegenerative diseases, but the mechanism for this recruitment has remained unclear. In this article, we show that functional Ub-binding motifs in Atx-3 and p62 proteins are required for the localization of both proteins into aggregates in a cell-based assay that recapitulates several features of polyglutamine disease. We propose that the Ub-mediated sequestration of essential Ub-binding protein(s) into aggregates may be a common mechanism contributing to the pathogenesis of neurodegenerative diseases.
Project description:Spatial control of RhoGTPase-inactivating GAP components remains largely enigmatic. We describe a brain-specific RhoGAP splice variant, BARGIN (BGIN), which comprises a combination of BAR, GAP, and partial CIN phosphatase domains spliced from adjacent SH3BP1 and CIN gene loci. Excision of BGIN exon 2 results in recoding of a 42-amino acid N-terminal stretch. The partial CIN domain is a poly-ubiquitin (poly-Ub)-binding module that facilitates BGIN distribution to membranous and detergent-insoluble fractions. Poly-Ub/BGIN interactions support BGIN-mediated inactivation of a membranous Rac1 population, which consequently inactivates membrane-localized Rac1 effector systems such as reactive oxygen species (ROS) generation by the Nox1 complex. Given that Ub aggregate pathology and proteotoxicity are central themes in various neurodegenerative disorders, we investigated whether BGIN/Rac1 signaling could be involved in neurodegenerative proteotoxicity. BGIN/Ub interactions are observed through colocalization in tangle aggregates in the Alzheimer's disease (AD) brain. Moreover, enhanced BGIN membrane distribution correlates with reduced Rac1 activity in AD brain tissue. Finally, BGIN contributes to Rac1 inhibition and ROS generation in an amyloid precursor protein (APP) proteotoxicity model. These results suggest that BGIN/poly-Ub interactions enhance BGIN membrane distribution and relay poly-Ub signals to enact Rac1 inactivation, and attenuation of Rac1 signaling is partially dependent on BGIN in a proteotoxic APP context.
Project description:Conditions causing an increase in misfolded or aberrant proteins can impair the activity of the ubiquitin/proteasome system (UPS). This observation is of particular interest, given the fact that proteotoxic stress is closely associated with a large variety of disorders. Although impairment of the UPS appears to be a general consequence of proteotoxic insults, the underlying mechanisms remain enigmatic. Here, we show that heat shock-induced proteotoxic stress resulted in conjugation of ubiquitin to detergent-insoluble protein aggregates, which coincided with reduced levels of free ubiquitin and impediment of ubiquitin-dependent proteasomal degradation. Interestingly, whereas soluble proteasome substrates returned to normal levels after a transient accumulation, the levels of an aggregation-prone substrate remained high even when the free ubiquitin levels were restored. Consistently, overexpression of ubiquitin prevented accumulation of soluble but not aggregation-prone substrates in thermally stressed cells. Notably, cells were also unable to resume degradation of aggregation-prone substrates after treatment with the translation inhibitor puromycin, indicating that selective accumulation of aggregation-prone proteins is a consistent feature of proteotoxic stress. Our data suggest that the failure of the UPS to clear aggregated proteins in the aftermath of proteotoxic stress episodes may contribute to the selective deposition of aggregation-prone proteins in conformational diseases.
Project description:The development of drugs directly interfering with neurodegeneration has proven to be astonishingly difficult. Alternative therapeutic approaches could result from a better understanding of the supportive function of glial cells for stressed neurons. Therefore, here, we investigated the mechanisms involved in the endogenous neuro-defensive activity of astrocytes. A well-established model of postmitotic human dopaminergic neurons (LUHMES cells) was used in the absence ('LUHMES' mono-culture) or presence ('co-culture') of astrocytes. Inhibition of the LUHMES proteasome led to proteotoxic (protein aggregates; ATF-4 induction) and oxidative (GSH-depletion; NRF-2 induction) stress, followed by neuronal apoptosis. The presence of astrocytes attenuated the neuronal stress response, and drastically reduced neurodegeneration. A similar difference between LUHMES mono- and co-cultures was observed, when proteotoxic and oxidative stress was triggered indirectly by inhibitors of mitochondrial function (rotenone, MPP+). Human and murine astrocytes continuously released glutathione (GSH) into the medium, and transfer of glia-conditioned medium was sufficient to rescue LUHMES, unless it was depleted for GSH. Also, direct addition of GSH to LUHMES rescued the neurons from inhibition of the proteasome. Both astrocytes and GSH blunted the neuronal ATF-4 response and similarly upregulated NRF-1/NFE2L1, a transcription factor counter-regulating neuronal proteotoxic stress. Astrocyte co-culture also helped to recover the neurons' ability to degrade aggregated poly-ubiquitinated proteins. Overexpression of NRF-1 attenuated the toxicity of proteasome inhibition, while knockdown increased toxicity. Thus, astrocytic thiol supply increased neuronal resilience to various proteotoxic stressors by simultaneously attenuating cell death-related stress responses, and enhancing the recovery from proteotoxic stress through upregulation of NRF-1.
Project description:The olfactory bulb is one of the most vulnerable brain regions in age-related proteinopathies. Proteinopathic stress is mitigated by the heat shock protein (Hsp) family of chaperones. Here, we describe age-related decreases in Hsc70 in the olfactory bulb of the female rat and higher levels of Hsp70 and Hsp25 in middle and old age than at 2-4 months. To model proteotoxic and oxidative stress in the olfactory bulb, primary olfactory bulb cultures were treated with the proteasome inhibitors lactacystin and MG132 or the pro-oxidant paraquat. Toxin-induced increases were observed in Hsp70, Hsp25, and Hsp32. To determine the functional consequences of the increase in Hsp70, we attenuated Hsp70 activity with two mechanistically distinct inhibitors. The Hsp70 inhibitors greatly potentiated the toxicity of sublethal lactacystin or MG132 but not of paraquat. Although ubiquitinated protein levels were unchanged with aging in vivo or with sublethal MG132 in vitro, there was a large, synergistic increase in ubiquitinated proteins when proteasome and Hsp70 functions were simultaneously inhibited. Our study suggests that olfactory bulb cells rely heavily on Hsp70 chaperones to maintain homeostasis during mild proteotoxic, but not oxidative insults, and that Hsp70 prevents the accrual of ubiquitinated proteins in these cells. The olfactory bulb is affected in the early phases of many age-related neurodegenerative disorders. Here, we described the impact of aging on multiple heat shock proteins (Hsps), such as Hsp70, in the female rat olfactory bulb in vivo. Using multiple proteasome and Hsp70 inhibitors (see schematic), we found that proteotoxicity elicited a compensatory increase in Hsp70 in primary olfactory bulb cells in vitro. Hsp70 then reduced the proteotoxic buildup of ubiquitinated proteins and robustly protected against cell death according to three independent viability assays. Thus, olfactory bulb neurons can mount impressive natural adaptations to proteotoxic injury, perhaps explaining why neurodegenerative disorders are so delayed in onset and so slow to progress.
Project description:A hallmark of amyotrophic lateral sclerosis (ALS) pathology is the accumulation of ubiquitylated protein inclusions within motor neurons. Recent studies suggest the sequestration of ubiquitin (Ub) into inclusions reduces the availability of free Ub, which is essential for cellular function and survival. However, the dynamics of the Ub landscape in ALS have not yet been described. Here, we show that Ub homeostasis is altered in a cell model of ALS induced by expressing mutant SOD1 (SOD1A4V). By monitoring the distribution of Ub in cells expressing SOD1A4V, we show that Ub is present at the earliest stages of SOD1A4V aggregation, and that cells containing SOD1A4V aggregates have greater ubiquitin-proteasome system (UPS) dysfunction. Furthermore, SOD1A4V aggregation is associated with the redistribution of Ub and depletion of the free Ub pool. Ubiquitomics analysis indicates that expression of SOD1A4V is associated with a shift of Ub to a pool of supersaturated proteins, including those associated with oxidative phosphorylation and metabolism, corresponding with altered mitochondrial morphology and function. Taken together, these results suggest that misfolded SOD1 contributes to UPS dysfunction and that Ub homeostasis is an important target for monitoring pathological changes in ALS.This article has an associated First Person interview with the first author of the paper.
Project description:Neurodegeneration is characterized by protein aggregate deposits and mitochondrial malfunction. Reduction in Tom40 (translocase of outer membrane 40) expression, a key subunit of the translocase of the outer mitochondrial membrane complex, led to accumulation of ubiquitin (Ub)-positive protein aggregates engulfed by Atg8a-positive membranes. Other macroautophagy markers were also abnormally accumulated. Autophagy was induced but the majority of autophagosomes failed to fuse with lysosomes when Tom40 was downregulated. In Tom40 RNAi tissues, autophagosome-like (AL) structures, often not sealed, were 10 times larger than starvation induced autophagosomes. Atg5 downregulation abolished Tom40 RNAi induced AL structure formation, but the Ub-positive aggregates remained, whereas knock down of Syx17, a gene required for autophagosome-lysosome fusion, led to the disappearance of giant AL structures and accumulation of small autophagosomes and phagophores near the Ub-positive aggregates. The protein aggregates contained many mitochondrial preproteins, cytosolic proteins, and proteasome subunits. Proteasome activity and ATP levels were reduced and the ROS levels was increased in Tom40 RNAi tissues. The simultaneous inhibition of proteasome activity, reduction in ATP production, and increase in ROS, but none of these conditions alone, can mimic the imbalanced proteostasis phenotypes observed in Tom40 RNAi cells. Knockdown of ref(2)P or ectopic expression of Pink1 and park greatly reduced aggregate formation in Tom40 RNAi tissues. In nerve tissues, reduction in Tom40 activity leads to aggregate formation and neurodegeneration. Rather than diminishing the neurodegenerative phenotypes, overexpression of Pink1 enhanced them. We proposed that defects in mitochondrial protein import may be the key to linking imbalanced proteostasis and mitochondrial defects. ABBREVIATIONS:AL: autophagosome-like; Atg12: Autophagy-related 12; Atg14: Autophagy-related 14; Atg16: Autophagy-related 16; Atg5: Autophagy-related 5; Atg6: Autophagy-related 6; Atg8a: Autophagy-related 8a; Atg9: Autophagy-related 9; ATP: adenosine triphosphate; Cas9: CRISPR associated protein 9; cDNA: complementary DNA; COX4: Cytochrome c oxidase subunit 4; CRISPR: clustered regularly interspaced short palindromic repeats; Cyt-c1: Cytochrome c1; DAPI: 4,6-diamidino-2-phenylindole dihydrochloride; Dcr-2: Dicer-2; FLP: Flippase recombination enzyme; FRT: FLP recombination target; GFP: green fluorescent protein; GO: gene ontology; gRNA: guide RNA; Hsp60: Heat shock protein 60A; HDAC6: Histone deacetylase 6; htt: huntingtin; Idh: Isocitrate dehydrogenase; IFA: immunofluorescence assay; Irp-1A: Iron regulatory protein 1A; kdn: knockdown; Marf: Mitochondrial assembly regulatory factor; MitoGFP: Mitochondrial-GFP; MS: mass spectrometry; MTPAP: mitochondrial poly(A) polymerase; Nmnat: Nicotinamide mononucleotide adenylyltransferase; OE: overexpression; Pink1/PINK1: PTEN-induced putative kinase 1; polyQ: polyglutamine; PRKN: parkin RBR E3 ubiquitin protein ligase; Prosα4: proteasome α4 subunit; Prosβ1: proteasome β1 subunit; Prosβ5: proteasome β5 subunit; Prosβ7: proteasome β7 subunit; ref(2)P: refractory to sigma P; RFP: red fluorescent protein; RNAi: RNA interference; ROS: reactive oxygen species; Rpn11: Regulatory particle non-ATPase 11; Rpt2: Regulatory particle triple-A ATPase 2; scu: scully; sicily: severe impairment of CI with lengthened youth; sesB: stress-sensitive B; Syx17: Syntaxin17; TEM: transmission electron microscopy; ttm50: tiny tim 50; Tom: translocase of the outer membrane; Tom20: translocase of outer membrane 20; Tom40: translocase of outer membrane 40; Tom70: translocase of outer membrane 70; UAS: upstream active sequence; Ub: ubiquitin; VNC: ventral nerve cord; ZFYVE1: zinc finger FYVE-type containing 1.
Project description:In Alzheimer's disease proteasome activity is reportedly downregulated, thus increasing it could be therapeutically beneficial. The proteasome-associated deubiquitinase USP14 disassembles polyubiquitin-chains, potentially delaying proteasome-dependent protein degradation. We assessed the protective efficacy of inhibiting or downregulating USP14 in rat and mouse (Usp14axJ) neuronal cultures treated with prostaglandin J2 (PGJ2). IU1 concentrations (HIU1>25μM) reported by others to inhibit USP14 and be protective in non-neuronal cells, reduced PGJ2-induced Ub-protein accumulation in neurons. However, HIU1 alone or with PGJ2 is neurotoxic, induces calpain-dependent Tau cleavage, and decreases E1~Ub thioester levels and 26S proteasome assembly, which are energy-dependent processes. We attribute the two latter HIU1 effects to ATP-deficits and mitochondrial Complex I inhibition, as shown herein. These HIU1 effects mimic those of mitochondrial inhibitors in general, thus supporting that ATP-depletion is a major mediator of HIU1-actions. In contrast, low IU1 concentrations (LIU1≤25μM) or USP14 knockdown by siRNA in rat cortical cultures or loss of USP14 in cortical cultures from ataxia (Usp14axJ) mice, failed to prevent PGJ2-induced Ub-protein accumulation. PGJ2 alone induces Ub-protein accumulation and decreases E1~Ub thioester levels. This seemingly paradoxical result may be attributed to PGJ2 inhibiting some deubiquitinases (such as UCH-L1 but not USP14), thus triggering Ub-protein stabilization. Overall, IU1-concentrations that reduce PGJ2-induced accumulation of Ub-proteins are neurotoxic, trigger calpain-mediated Tau cleavage, lower ATP, E1~Ub thioester and E1 protein levels, and reduce proteasome activity. In conclusion, pharmacologically inhibiting (with low or high IU1 concentrations) or genetically down-regulating USP14 fail to enhance proteasomal degradation of Ub-proteins or Tau in neurons.
Project description:Nearly all neurodegenerative diseases are associated with abnormal accumulation of ubiquitin (Ub) conjugates within neuronal inclusion bodies. To directly test the hypothesis that depletion of cellular Ub is sufficient to cause neurodegeneration, we have disrupted Ubb, one of four genes that supply Ub in the mouse. Here, we report that loss of Ubb led to a progressive degenerative disorder affecting neurons within the arcuate nucleus of the hypothalamus. This neurodegenerative cytopathology was accompanied by impaired hypothalamic control of energy balance and adult-onset obesity. Ubb was highly expressed in vulnerable hypothalamic neurons and total Ub levels were selectively reduced in the hypothalamus of Ubb-null mice. These findings demonstrate that maintenance of adequate supplies of cellular Ub is essential for neuronal survival and establish that decreased Ub availability is sufficient to cause neuronal dysfunction and death.
Project description:The ubiquitin-proteasome system (UPS) and macroautophagy (autophagy) are central to normal proteostasis and interdependent in that autophagy is known to compensate for the UPS to alleviate ensuing proteotoxic stress that impairs cell function. UPS and autophagy dysfunctions are believed to have a major role in the pathomechanisms of neurodegenerative disease. Here we show that continued 26S proteasome dysfunction in mouse brain cortical neurons causes paranuclear accumulation of fragmented dysfunctional mitochondria, associated with earlier recruitment of Parkin and lysine 48-linked ubiquitination of mitochondrial outer membrane (MOM) proteins, including Mitofusin-2. Early events also include phosphorylation of p62/SQSTM1 (p62) and increased optineurin, as well as autophagosomal LC3B and removal of some mitochondria, supporting the induction of selective autophagy. Inhibition of the degradation of ubiquitinated MOM proteins with continued 26S proteasome dysfunction at later stages may impede efficient mitophagy. However, continued 26S proteasome dysfunction also decreases the levels of essential autophagy proteins ATG9 and LC3B, which is characterised by decreases in their gene expression, ultimately leading to impaired autophagy. Intriguingly, serine 351 phosphorylation of p62 did not enhance its binding to Keap1 or stabilise the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor in this neuronal context. Nrf2 protein levels were markedly decreased despite transcriptional activation of the Nrf2 gene. Our study reveals novel insights into the interplay between the UPS and autophagy in neurons and is imperative to understanding neurodegenerative disease where long-term proteasome inhibition has been implicated.