Mahogunin ring finger 1 confers cytoprotection against mutant SOD1 aggresomes and is defective in an ALS mouse model
Graphical abstract
Introduction
Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset progressive neurodegenerative disorder that is characterized by the selective death of motor neurons in the spinal cord and cerebral cortex (Bruijn et al., 2004). Dominant mutations in the copper-zinc superoxide dismutase 1(SOD1) gene encoding the SOD1 enzyme are one of the frequent causes of familial ALS (Bruijn et al., 2004, Raoul et al., 2002, Wong et al., 1995). The overexpression of the human SOD1 gene carrying ALS-linked mutations in mice reproduces the major pathological hallmark of ALS (Gurney et al., 1994). Mutant SOD1 proteins, which are highly ubiquitinated, form insoluble abnormal inclusions with components of the ubiquitin proteasome system (UPS) and autophagy pathway in motor neurons (Banerjee et al., 2010, Cheroni et al., 2009).
The key mechanism of motor neuron death that is linked to mutant SOD1 aggregates remains poorly understood. Recently, it has been shown that supersaturation of the cellular protein is linked to neurodegeneration and aging (Ciryam et al., 2013). Because the UPS facilitates the degradation of mutant SOD1 proteins, the overexpression of mutant SOD1 proteins decreases the elimination capacity of the proteasome system (Allen et al., 2003, Cheroni et al., 2009, Hoffman et al., 1996, Urushitani et al., 2002). Numerous studies have suggested that mutant SOD1 aggregates lead to several critical cellular dysfunctions, such as UPS alterations, endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and oxidative stress (Cheroni et al., 2009, Kikuchi et al., 2006, Lin and Beal, 2006).
Despite knowing that abnormal SOD1 protein aggregation results in a toxic gain of function and contributes to ALS disease progression, it is still not clear how neuronal cells survive under such devastating dysfunctions. One possible cellular response is autophagy. Several lines of evidence have suggested that autophagy activation alleviates mutant SOD1-linked toxic insults (Wong and Cuervo, 2010). SOD1G93A-transgenic mice show numerous microtubule-associated protein 1A/1B-light chain 3 (LC-3)-labeled autophagic vacuoles in the spinal motor neurons (Li et al., 2008). In addition, it has been shown that p62/SQSTM1 recognizes mutant SOD1 proteins through an autophagy-linked lysosomal pathway and promotes their clearance (Gal et al., 2009, Zhang et al., 2007).
Aberrant function of Mahogunin ring finger-1 (MGRN1), a RING domain-containing E3 ubiquitin ligase, causes late-onset spongiform neurodegeneration in mice (He et al., 2003). MGRN1, a unique E3 ligase which catalyzes multi-monoubiquitination to the substrate, and is likely to be involved in the cellular quality control machinery through proteasome-independent pathway (Chhangani and Mishra, 2013, Chhangani et al., 2014b, Kim et al., 2007). Mice lacking MGRN1 function exhibit dysregulation of the mitochondrial pathway and neurodegeneration (Sun et al., 2007). Downregulation of MGRN1 disturbs endo-lysosome molecular trafficking of epidermal growth factor and null mice of MGRN1 indicate that MGRN1-deficient neurons are more prone for high vulnerable risk in comparison of other cells due to abnormal endosomal trafficking (Kim et al., 2007). Recently it has also been demonstrated that MGRN1 interacts with transmembrane (CtmPrP) and toxic (cyPrP) prion disease proteins; depletion of MGRN1 affects lysosomal morphology and leads to neurodegeneration likely due to such improper sequestration (Chakrabarti and Hegde, 2009). MGRN1 interacts with several proteins such as expanded polyglutamine proteins (Chhangani et al., 2014b), molecular chaperone (Chhangani and Mishra, 2013), α-tubulin (Srivastava and Chakrabarti, 2014), MC1R/MC2R (Cooray et al., 2011, Perez-Oliva et al., 2009), cyPrP/CtmPrP (Chakrabarti and Hegde, 2009), TSG 101 (Kim et al., 2007), and NEDD4 (Gunn et al., 2013). Under such crucial biological interactions it is plausible that a loss of function or depletion of MGRN1 can cause multifactorial defects in cells and generate various pathological states in important physiological events. As recently we reviewed (Upadhyay et al., 2015) and previous studies also indicated strong implications of MGRN1 in neuro-pathobiological mechanisms. How misfolded or aggregated proteins affect physiological function of MGRN1 and disturb normal localization at the site of proper recruitment is a crucial question. However, the role of MGRN1 in neurodegenerative diseases has not been clarified. Although abnormal protein accumulation is a prominent feature of neurodegenerative diseases, how the sequestration or loss of function of MGRN1 contributes to neurodegeneration is not known.
In our present study, we found MGRN1 dysregulation in cellular and mouse models of ALS. MGRN1 interacts with normal and mutant SOD1 proteins in cells. The motor neurons showed diminished MGRN1 cytoplasmic labeling in presymptomatic mutant SOD1 mice. MGRN1 was partially recruited to mutant SOD1 inclusions, which were positive for p62 and Lamp2 in the spinal motor neurons of ALS mice. Finally, MGRN1 alleviates the cytotoxicity mediated by abnormal inclusions of mutant SOD1 and thus, exerts a cytoprotective effect. Overall, these findings suggest that an MGRN1-mediated protein quality control mechanism alleviates the mutant SOD1-linked proteotoxicity likely through autophagic pathway.
Section snippets
Materials
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Bafilomycin A1 (Baf), chloroquine, cycloheximide, MGRN1-specific siRNA oligonucleotides, and all cell culture reagents were obtained from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). The protein G-agarose beads were obtained from Roche Applied Science (Indianapolis, IN, USA). The TRIzol reagent, Lipofectamine®2000, OptiMEM, and reverse transcription-polymerase chain reaction (RT-PCR) kits were purchased from Life Technologies
Dysregulation of MGRN1 in ALS-linked transgenic mice prior to neurodegeneration
To test whether the endogenous MGRN1 E3 ubiquitin ligase was dysregulated by mutant SOD1 proteins, we first examined the expression levels of MGRN1 in cultured cells expressing mutant forms of SOD1 proteins. We observed that the endogenous levels of MGRN1 were depleted in the cells expressing mutant SOD1G37R and SOD1G85R proteins and not in the cells expressing wild-type SOD1 protein (Fig. 1A). Mutant SOD1-overexpressing cells showed about a 0.4-fold decrease in the endogenous levels of MGRN1 (
Discussion
Our present study shows that the MGRN1 E3 ubiquitin ligase interacted with both wild-type and mutant SOD1 proteins in cells. The overexpression of MGRN1 facilitated the degradation of mutant SOD1 proteins. Moreover, mutant SOD1 proteins dysregulated endogenous MGRN1 levels and overexpression of MGRN1 alleviated their proteotoxic insults. Taken together, the findings of all of these studies support our current conclusion that MGRN1 is a novel cytoprotective factor to alleviate mutant
Disclosure statement
The authors do not have any actual or potential conflicts of interests to disclose.
Acknowledgment
This work was supported by an Innovative Young Biotechnologist Award (IYBA) from the Department of Biotechnology Government of India (BT/06/IYBA/2012) (to A.M.); INSA-JSPS Joint Research Project Programme (IA/INSA-JSPS Project 2013-2016/4097) (to A.M. and K.Y.); Grants-in-Aid for Scientific Research (23111006) and Scientific Research (B) (26293208) from the Ministry for Education, Culture and Sports, Science and Technology, Japan; Grant-in-Aid for Research on rare and intractable diseases, the
References (48)
Analysis of the cytosolic proteome in a cell culture model of familial amyotrophic lateral sclerosis reveals alterations to the proteasome, antioxidant defenses, and nitric oxide synthetic pathways
J. Biol. Chem.
(2003)Autophagy in neurodegenerative disorders: pathogenic roles and therapeutic implications
Trends Neurosci.
(2010)ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions
Neuron
(1997)- et al.
Functional depletion of mahogunin by cytosolically exposed prion protein contributes to neurodegeneration
Cell
(2009) Mahogunin ring finger 1 suppresses misfolded polyglutamine aggregation and cytotoxicity
Biochim. Biophys. Acta
(2014)Widespread aggregation and neurodegenerative diseases are associated with supersaturated proteins
Cell Rep.
(2013)Proteasome inhibition enhances the stability of mouse Cu/Zn superoxide dismutase with mutations linked to familial amyotrophic lateral sclerosis
J. Neurol. Sci.
(1996)Degradation of amyotrophic lateral sclerosis-linked mutant Cu,Zn-superoxide dismutase proteins by macroautophagy and the proteasome
J. Biol. Chem.
(2006)Toxicity of familial ALS-linked SOD1 mutants from selective recruitment to spinal mitochondria
Neuron
(2004)Mutated human SOD1 causes dysfunction of oxidative phosphorylation in mitochondria of transgenic mice
J. Biol. Chem.
(2002)
UBE3A/E6-AP regulates cell proliferation by promoting proteasomal degradation of p27
Neurobiol. Dis.
Dorfin ubiquitylates mutant SOD1 and prevents mutant SOD1-mediated neurotoxicity
J. Biol. Chem.
Mahogunin ring finger-1 (MGRN1) E3 ubiquitin ligase inhibits signaling from melanocortin receptor by competition with Galphas
J. Biol. Chem.
Motoneuron death triggered by a specific pathway downstream of Fas. Potentiation by ALS-linked SOD1 mutations
Neuron
Mitochondrial dysfunction precedes neurodegeneration in mahogunin (Mgrn1) mutant mice
Neurobiol. Aging
Membrane filter assay for detection of amyloid-like polyglutamine-containing protein aggregates
Methods Enzymol.
Histological evidence of protein aggregation in mutant SOD1 transgenic mice and in amyotrophic lateral sclerosis neural tissues
Neurobiol. Dis.
An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria
Neuron
Interaction between familial amyotrophic lateral sclerosis (ALS)-linked SOD1 mutants and the dynein complex
J. Biol. Chem.
Onset and progression in inherited ALS determined by motor neurons and microglia
Science
Unraveling the mechanisms involved in motor neuron degeneration in ALS
Annu. Rev. Neurosci.
Functional alterations of the ubiquitin-proteasome system in motor neurons of a mouse model of familial amyotrophic lateral sclerosis
Hum. Mol. Genet.
Misfolded proteins recognition strategies of E3 ubiquitin ligases and neurodegenerative diseases
Mol. Neurobiol.
Mahogunin ring finger-1 (MGRN1) suppresses chaperone-associated misfolded protein aggregation and toxicity
Sci. Rep.
Cited by (15)
Pulse-Chase Proteomics of the App Knockin Mouse Models of Alzheimer's Disease Reveals that Synaptic Dysfunction Originates in Presynaptic Terminals
2021, Cell SystemsCitation Excerpt :For detection of proteins in large aggregates, dot blot analysis was performed. Equal amount of proteins from amyloid enriched fractions (see preparation above) were loaded on the nitrocellulose membranes as dots followed by standard immunoblotting procedure (Chhangani et al., 2016). The following primary antibodies were used for Western and dot blots
Ubiquitin biology in neurodegenerative disorders: From impairment to therapeutic strategies
2020, Ageing Research ReviewsCitation Excerpt :Moreover, inducible subunits of proteasome including, LMP2, LMP7 and MECL-1 get overexpressed early during disease pathogenesis and generates peptides crucial for major histocompatibility class-I presentation and subsequent immunity in the SOD1 mice model of ALS (Johnston-Carey et al., 2016). Likewise, alteration in E3 ligases- TRAF6 and Mahogunin ring finger 1 (MGRN1) conferred mutant SOD1 linked cytotoxic protein aggregates in the ALS mouse model (Chhangani et al., 2016; Semmler et al., 2019). Conversely, the role of UPS has also been demonstrated in the clearance of ALS pathological proteins.
Axon-Seq Decodes the Motor Axon Transcriptome and Its Modulation in Response to ALS
2018, Stem Cell ReportsCitation Excerpt :Multiple transcripts that are important for axon function were downregulated or absent in SOD1G93A axons, including Dbn1, which is important for axon initiation, growth, and guidance (McIntyre et al., 2010; Sharma et al., 2012); Nrp1, a semaphorin receptor involved in both axon guidance and subcellular target recognition (Telley et al., 2016); and Mgrn1, a ubiquitin ligase, which appears important for mitochondrial function and neuronal survival (Upadhyay et al., 2016). Mgrn1 is downregulated in SOD1 mice and is recruited to SOD1-positive inclusions (Chhangani et al., 2016). Nrp1 acts as an axonal attractant during development (Chauvet et al., 2007) and is important for limb innervation (Huettl et al., 2011).
Copper homeostasis and the ubiquitin proteasome system
2023, MetallomicsMahogunin Ring Finger 1 regulates pigmentation by controlling the pH of melanosomes in melanocytes and melanoma cells
2022, Cellular and Molecular Life Sciences