Ubiquilin-2 Research Articles

UBQLN2 and HSP70 participate in Parkin-mediated mitophagy by facilitating outer mitochondrial membrane rupture.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two aging-related neurodegenerative diseases that share common key features, including aggregation of pathogenic proteins, dysfunction of mitochondria, and impairment of autophagy. Mutations in ubiquilin 2 (UBQLN2), a shuttle protein in the ubiquitin-proteasome system (UPS), can cause ALS/FTD, but the mechanism underlying UBQLN2-mediated pathogenesis is still uncertain. Recent studies indicate that mitophagy, a selective form of autophagy which is crucial for mitochondrial quality control, is tightly associated with neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and ALS. In this study, we show that after Parkin-dependent ubiquitination of damaged mitochondria, UBQLN2 is recruited to poly-ubiquitinated mitochondria through the UBA domain. UBQLN2 cooperates with the chaperone HSP70 to promote UPS-driven degradation of outer mitochondrial membrane (OMM) proteins. The resulting rupture of the OMM triggers the autophagosomal recognition of the inner mitochondrial membrane receptor PHB2. UBQLN2 is required for Parkin-mediated mitophagy and neuronal survival upon mitochondrial damage, and the ALS/FTD pathogenic mutations in UBQLN2 impair mitophagy in primary cultured neurons. Taken together, our findings link dysfunctional mitophagy to UBQLN2-mediated neurodegeneration.

Ion Mobility Mass Spectrometry Unveils Global Protein Conformations in Response to Conditions that Promote and Reverse Liquid-Liquid Phase Separation.

Liquid-liquid phase separation (LLPS) is a process by which biomacromolecules, particularly proteins, condense into a dense phase that resembles liquid droplets. Dysregulation of LLPS is implicated in disease, yet the relationship between protein conformational changes and LLPS remains difficult to discern. This is due to the high flexibility and disordered nature of many proteins that phase separate under physiological conditions and their tendency to oligomerize. Here, we demonstrate that ion mobility mass spectrometry (IM-MS) overcomes these limitations. We used IM-MS to investigate the conformational states of full-length ubiquilin-2 (UBQLN2) protein, LLPS of which is driven by high-salt concentration and reversed by noncovalent interactions with ubiquitin (Ub). IM-MS revealed that UBQLN2 exists as a mixture of monomers and dimers and that increasing salt concentration causes the UBQLN2 dimers to undergo a subtle shift toward extended conformations. UBQLN2 binds to Ub in 2:1 and 2:2 UBQLN2/Ub complexes, which have compact geometries compared to free UBQLN2 dimers. Together, these results suggest that extended conformations of UBQLN2 are correlated with UBQLN2's ability to phase separate. Overall, delineating protein conformations that are implicit in LLPS will greatly increase understanding of the phase separation process, both in normal cell physiology and disease states.

Wild-type and pathogenic forms of ubiquilin 2 differentially modulate components of the autophagy-lysosome pathways

Missense mutations of ubiquilin 2 (UBQLN2) have been identified to cause X-linked amyotrophic lateral sclerosis (ALS). Proteasome-mediated protein degradation is reported to be impaired by ALS-associated mutations of UBQLN2. However, it remains unknown how these mutations affect autophagy-lysosome protein degradation, which consists of macroautophagy (MA), microautophagy (mA), and chaperone-mediated autophagy (CMA). Using a CMA/mA fluorescence reporter we found that overexpression of wild-type UBQLN2 impairs CMA. Conversely, knockdown of endogenous UBQLN2 increases CMA activity, suggesting that normally UBQLN2 negatively regulates CMA. ALS-associated mutant forms of UBQLN2 exacerbate this impairment of CMA. Using cells stably transfected with wild-type or ALS-associated mutant UBQLN2, we further determined that wild-type UBQLN2 increased the ratio of LAMP2A (a CMA-related protein) to LAMP1 (a lysosomal protein). This could represent a compensatory reaction to the impairment of CMA by wild-type UBQLN2. However, ALS-associated mutant UBQLN2 failed to show this compensation, exacerbating the impairment of CMA by mutant UBQLN2. We further demonstrated that ALS-associated mutant forms of UBQLN2 also impair MA, but wild-type UBQLN2 does not. These results support the view that ALS-associated mutant forms of UBQLN2 impair both CMA and MA which may contribute to the neurodegeneration observed in patients with UBQLN2-mediated ALS.

Axon guidance genes modulate neurotoxicity of ALS-associated UBQLN2.

Mutations in the ubiquitin (Ub) chaperone Ubiquilin 2 (UBQLN2) cause X-linked forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) through unknown mechanisms. Here, we show that aggregation-prone, ALS-associated mutants of UBQLN2 (UBQLN2ALS) trigger heat stress-dependent neurodegeneration in Drosophila. A genetic modifier screen implicated endolysosomal and axon guidance genes, including the netrin receptor, Unc-5, as key modulators of UBQLN2 toxicity. Reduced gene dosage of Unc-5 or its coreceptor Dcc/frazzled diminished neurodegenerative phenotypes, including motor dysfunction, neuromuscular junction defects, and shortened lifespan, in flies expressing UBQLN2ALS alleles. Induced pluripotent stem cells (iPSCs) harboring UBQLN2ALS knockin mutations exhibited lysosomal defects while inducible motor neurons (iMNs) expressing UBQLN2ALS alleles exhibited cytosolic UBQLN2 inclusions, reduced neurite complexity, and growth cone defects that were partially reversed by silencing of UNC5B and DCC. The combined findings suggest that altered growth cone dynamics are a conserved pathomechanism in UBQLN2-associated ALS/FTD.

UBQLN2 restrains the domesticated retrotransposon PEG10 to maintain neuronal health in ALS.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor neuron dysfunction and loss. A portion of ALS cases are caused by mutation of the proteasome shuttle factor Ubiquilin 2 (UBQLN2), but the molecular pathway leading from UBQLN2 dysfunction to disease remains unclear. Here, we demonstrate that UBQLN2 regulates the domesticated gag-pol retrotransposon 'paternally expressed gene 10 (PEG10)' in human cells and tissues. In cells, the PEG10 gag-pol protein cleaves itself in a mechanism reminiscent of retrotransposon self-processing to generate a liberated 'nucleocapsid' fragment, which uniquely localizes to the nucleus and changes the expression of genes involved in axon remodeling. In spinal cord tissue from ALS patients, PEG10 gag-pol is elevated compared to healthy controls. These findings implicate the retrotransposon-like activity of PEG10 as a contributing mechanism in ALS through the regulation of gene expression, and restraint of PEG10 as a primary function of UBQLN2.

Ubiquilin-2 regulates pathological alpha-synuclein

The key protein implicated in Parkinson’s disease and other synucleinopathies is α-synuclein, and a post-translationally modified form of the protein, phosphorylated at serine 129 (pS129), is a principal component in Lewy bodies, a pathological hallmark of PD. While altered proteostasis has been implicated in the etiology of Parkinson’s disease, we still have a limited understanding of how α-synuclein is regulated in the nervous system. The protein quality control protein Ubiquilin-2 (UBQLN2) is known to accumulate in synucleinopathies, but whether it directly regulates α-synuclein is unknown. Using cellular and mouse models, we find that UBQLN2 decreases levels of α-synuclein, including the pS129 phosphorylated isoform. Pharmacological inhibition of the proteasome revealed that, while α-synuclein may be cleared by parallel and redundant quality control pathways, UBQLN2 preferentially targets pS129 for proteasomal degradation. Moreover, in brain tissue from human PD and transgenic mice expressing pathogenic α-synuclein (A53T), native UBQLN2 becomes more insoluble. Collectively, our studies support a role for UBQLN2 in directly regulating pathological forms of α-synuclein and indicate that UBQLN2 dysregulation in disease may contribute to α-synuclein-mediated toxicity.

Silencing UBQLN2 Enhances the Radiosensitivity of Esophageal Squamous Cell Carcinoma (ESCC) via Activating p38 MAPK.

Ubiquilin 2 (UBQLN2) is an adaptor of ubiquitinated proteins and the proteasome. The potential role of UBQLN2 in carcinogenesis has been demonstrated. However, its role in modulating the radiosensitivity of cancer is not clear. Here, we explored the radiosensitizing effect of silencing UBQLN2 on esophageal squamous cell carcinoma (ESCC) and its mechanisms. We analyzed the prognostic role of UBQLN2 in the ESCC patient cohort from the Cancer Genomic Atlas (TCGA) database and our hospital. We also conducted a series of experiments in vivo and in vitro to investigate the effect of silencing UBQLN2 on ESCC radiosensitivity and its mechanisms. UBQLN2 is highly expressed in ESCC tissues and positively correlated with poor overall survival (OS). The knockdown of UBQLN2 dramatically increased the radiosensitivity of ESCC cells. Mechanically, UBQLN2 suppression substantially upregulated p38 mitogen-activated protein kinases (MAPK). The p38 MAPK inhibitor SB203580 could reverse the radiation-enhancing effect induced by UBQLN2 knockdown. The direct interaction between UBQLN2 and p38 MAPK was confirmed by co-immunoprecipitation (CO-IP) assay. Furthermore, silencing UBQLN2 also inhibited the expression of phosphorylated DNA-dependent protein kinase catalytic subunit (p-DNA-PKcs) after irradiation. Finally, the xenografted tumor experiment confirmed the radiosensitizing effect of silencing UBQLN2 on ESCC in vivo. Our results suggest that silencing UBQLN2 enhances the radiosensitivity of ESCC by activating p38 MAPK, and UBQLN2 may be a potential target to enhance the radiosensitivity of ESCC.

An Optimized Stress Granule Detection Method: Investigation of UBQLN2 Effect on Stress Granule Formation.

Stress granules (SGs) are cytosolic ribonucleoprotein granules that form via a liquid-liquid phase separation in response to environmental stresses such as heat, oxidative, and osmotic changes. Due to the condensation of low complexity, hydrophobic regions in core SG components in these highly dynamic granules, defects in SG maintenance and formation have been linked to toxic aggregate formation in neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia. However, efforts to dissect mechanisms regulating SG formation and maintenance have been limited by methods of tracking protein-SG localization. Here we describe a method for detecting and quantifying recruitment of cytosolically enriched proteins to SGs by indirect immunofluorescence microscopy. Using this method, we tracked the transient recruitment of the cytosolically enriched ubiquitin-like protein, ubiquilin 2 (UBQLN2), and a number of other factors into SGs, demonstrating its utility (Alexander et al., Proc Natl Acad Sci U S A 115:E11485-E11494, 2018).

Damaged DNA Is an Early Event of Neurodegeneration in Induced Pluripotent Stem Cell-Derived Motoneurons with UBQLN2P497H Mutation.

Ubiquilin-2 (UBQLN2) mutations lead to familial amyotrophic lateral sclerosis (FALS)/and frontotemporal dementia (FTLD) through unknown mechanisms. The combination of iPSC technology and CRISPR-mediated genome editing technology can generate an iPSC-derived motor neuron (iPSC-MN) model with disease-relevant mutations, which results in increased opportunities for disease mechanism research and drug screening. In this study, we introduced a UBQLN2-P497H mutation into a healthy control iPSC line using CRISPR/Cas9, and differentiated into MNs to study the pathology of UBQLN2-related ALS. Our in vitro MN model faithfully recapitulated specific aspects of the disease, including MN apoptosis. Under sodium arsenite (SA) treatment, we found differences in the number and the size of UBQLN2+ inclusions in UBQLN2P497H MNs and wild-type (WT) MNs. We also observed cytoplasmic TAR DNA-binding protein (TARDBP, also known as TDP-43) aggregates in UBQLN2P497H MNs, but not in WT MNs, as well as the recruitment of TDP-43 into stress granules (SGs) upon SA treatment. We noted that UBQLN2-P497H mutation induced MNs DNA damage, which is an early event in UBQLN2-ALS. Additionally, DNA damage led to an increase in compensation for FUS, whereas UBQLN2-P497H mutation impaired this function. Therefore, FUS may be involved in DNA damage repair signaling.

Multi-omics profiling identifies a deregulated FUS-MAP1B axis in ALS/FTD-associated UBQLN2 mutants.

Ubiquilin-2 (UBQLN2) is a ubiquitin-binding protein that shuttles ubiquitinated proteins to proteasomal and autophagic degradation. UBQLN2 mutations are genetically linked to the neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). However, it remains elusive how UBQLN2 mutations cause ALS/FTD. Here, we systematically examined proteomic and transcriptomic changes in patient-derived lymphoblasts and CRISPR/Cas9-engineered HeLa cells carrying ALS/FTD UBQLN2 mutations. This analysis revealed a strong up-regulation of the microtubule-associated protein 1B (MAP1B) which was also observed in UBQLN2 knockout cells and primary rodent neurons depleted of UBQLN2, suggesting that a UBQLN2 loss-of-function mechanism is responsible for the elevated MAP1B levels. Consistent with MAP1B's role in microtubule binding, we detected an increase in total and acetylated tubulin. Furthermore, we uncovered that UBQLN2 mutations result in decreased phosphorylation of MAP1B and of the ALS/FTD-linked fused in sarcoma (FUS) protein at S439 which is critical for regulating FUS-RNA binding and MAP1B protein abundance. Together, our findings point to a deregulated UBQLN2-FUS-MAP1B axis that may link protein homeostasis, RNA metabolism, and cytoskeleton dynamics, three molecular pathomechanisms of ALS/FTD.

Effects of Modulating Multivalent Ligand Binding Accessibility & Affinity on Liquid‐Liquid Phase Separation

Elucidating the molecular rules of ligand‐induced phase transitions is essential to determining the regulation of the formation and disassembly of biomolecular condensates. Ubiquitin‐binding shuttle protein Ubiquilin‐2 (UBQLN2) undergoes liquid‐liquid phase separation (LLPS) in vitro and colocalizes into stress‐induced cytoplasmic puncta in vivosuch as stress granules.[1] A recent study from our lab indicated that ubiquitin (Ub), and compact polyUb chains such as K48‐ & K11‐Ub4 can inhibit UBQLN2 LLPS by binding to phase separation‐driving UBQLN2 stickers whereas more extended chains such as K63‐ & M1‐Ub4 can promote UBQLN2 phase separation over a large Ub:UBQLN2 range by acting as a multivalent ligand scaffold.[2] Using a designed tetrameric ubiquitin construct (HOTag6‐Ub) that resembles a multi‐monoubiquitinated substrate, we further probed how alterations to the stickers and spacers of the HOTag6‐Ub ligand can modulate UBQLN2 phase behavior. Using turbidity and microscopy experiments, we obtained phase diagrams for LLPS driven by heterotypic interactions between UBQLN2 and HOTag6‐Ub. We discovered that, by changing the length and flexibility of the linker that tethers the Ub units to HOTag6, we can tune UBQLN2 LLPS. Single‐ and double‐point mutations to the Ub hydrophobic binding surface (I44 & V70 “stickers”) decreased the binding affinity between Ub and UBQLN2, thereby leading to changes in the shape of HOTag6‐Ub/UBQLN2 phase diagrams. Using nuclear magnetic resonance spectroscopy (NMR), microscopy and UV‐Vis spectroscopic methods we demonstrated that linker conformation, linker flexibility, and interactions with the Ub binding surface are driving factors of UBQLN2/HOTag6‐Ub co‐phase separation. Overall, our findings highlight how multivalent ligands can tune phase separation behavior of protein systems.[1] Dao, T. P., Kolaitis, R.‐M., Kim, H. J., O’Donovan, K., Martyniak, B., Colicino, E., Hehnly, H., Taylor, J. P., and Castañeda, C. A. (2018). Ubiquitin Modulates Liquid‐Liquid Phase Separation of UBQLN2 via Disruption of Multivalent Interactions. Mol. Cell 69, 965‐978.[2] Dao, T. P., Yang, Y., Cosgrove, M. S., Hopkins, J. B., Ma, W., Castaneda, C. A. (2021). Mechanistic insights into the enhancement or inhibition of phase separation by polyubiquitin chains of different lengths or linkages. bioRxiv 2021.11.12.467822; doi: https://doi.org/10.1101/2021.11.12.467822.

Disrupting the Balance of Protein Quality Control Protein UBQLN2 Accelerates Tau Proteinopathy.

Tau protein accumulation drives toxicity in several neurodegenerative disorders. To better understand the pathways regulating tau homeostasis in disease, we investigated the role of ubiquilins (UBQLNs)-a class of proteins linked to ubiquitin-mediated protein quality control (PQC) and various neurodegenerative diseases-in regulating tau. Cell-based assays identified UBQLN2 as the primary brain-expressed UBQLN to regulate tau. UBQLN2 efficiently lowered wild-type tau levels regardless of aggregation, suggesting that UBQLN2 interacts with and regulates tau protein under normal conditions or early in disease. Moreover, UBQLN2 itself proved to be prone to accumulation as insoluble protein in male and female tau transgenic mice and the human tauopathy progressive supranuclear palsy. Genetic manipulation of UBQLN2 in a tauopathy mouse model demonstrated that a physiological UBQLN2 balance is required for tau homeostasis. UBQLN2 overexpression exacerbated phosphorylated tau pathology and toxicity in mice expressing P301S mutant tau, whereas P301S mice lacking UBQLN2 showed significantly reduced phosphorylated tau. Further studies support the view that an imbalance of UBQLN2 perturbs ubiquitin-dependent PQC and autophagy. We conclude that changes in UBQLN2 levels, whether because of pathogenic mutations or secondary to disease states, such as tauopathy, contribute to proteostatic imbalances that exacerbate neurodegeneration.SIGNIFICANCE STATEMENT We defined a role for the protein quality control protein Ubiquilin-2 (UBQLN2), in age-related neurodegenerative tauopathies. This group of disorders is characterized by the accumulation of tau protein aggregates, which differ when UBQLN2 levels are altered. Given the lack of effective disease-modifying therapies for tauopathies and the function of UBQLN2 in handling various disease-linked proteins, we explored the role of UBQLN2 in regulating tau. We found that UBQLN2 reduced tau levels in cell models but behaved differently in mouse brain, where it accelerated mutant tau pathology and tau-mediated toxicity. A better understanding of the diverse functions of regulatory proteins like UBQLN2 can elucidate some of the causative factors in neurodegenerative disease and outline new routes to therapeutic intervention.

UBQLN2 regulation of α-synuclein in synucleinopathies.

The protein quality control protein ubiquilin-2 (UBQLN2) is implicated in synucleinopathies due to its accumulation in Lewy body diseases. However, little is known about how it may interact with and clear α-synuclein (α-syn). This study aimed to define the role of UBQLN2 in handling α-syn. To evaluate whether UBQLN2 regulates α-syn, we measured levels of α-synuclein in HEK-293 cells transiently expressing or deleted of UBQLN2. To evaluate whether UBQLN2 regulates α-syn clearance in the nervous system in vivo, we used western blot to measure total α-syn or phosphorylated human α-syn (pS129) levels in multiple transgenic mouse lines including: UBQLN2 overexpressing mice (Ub2-hi), UBQLN2 knock-out mice (Ub2-KO), and A53T α-syn mice crossed to either Ub2-hi or Ub2-KO mice. To assess changes in UBQLN2 solubility in synucleinopathies we measured levels of UBQLN2 by Western blot in PBS-soluble versus sarkosyl-soluble brain lysates from PD and LBD human brains and from A53T mouse brains. In vitro, UBQLN2 significantly decreased levels of soluble α-syn. In vivo, endogenous insoluble α-syn levels are decreased in Ub2-hi mice, while total endogenous α-syn levels are significantly increased in Ub2-KO mice. Total α-syn and pS129 levels were unchanged in A53TxUb2-hi mice versus A53T controls, but were significantly increased in A53TxUb2-KO mice. Solubility studies revealed increased insoluble UBQLN2 levels in human LBD and mouse A53T brains. While UBQLN2 is known to colocalize with a-syn in disease, our results support a functional role for UBQLN2 in regulating α-syn levels. Further, we show that UBQLN2 solubility is altered in synucleinopathies. In disease, a change in UBQLN2 solubility may indicate a loss of its ability to handle a-syn, contributing to a-syn toxicity. Ongoing studies seek to elucidate the mechanism by which UBQLN2 handles, and potentially clears, α-syn.

Amyotrophic lateral sclerosis (ALS) linked mutation in Ubiquilin 2 affects stress granule assembly via TIA-1.

AimsThe ubiquilin‐like protein ubiquilin 2 (UBQLN2) is associated with amyotrophic lateral sclerosis and frontotemporal degeneration (ALS/FTD). The biological function of UBQLN2 has previously been shown to be related to stress granules (SGs). In this study, we aimed to clarify the regulatory relationship between UBQLN2 and SGs.MethodsIn this study, we transfected UBQLN2‐WT or UBQLN2‐P497H plasmids into cell lines (HEK293T, HeLa), and observed the process of SG dynamics by immunofluorescence. Meanwhile, immunoblot analyses the protein changes of stress granules related components.ResultsWe observed that ubiquilin 2 colocalizes with the SG component proteins G3BP1, TIA‐1, ATXN2, and PABPC1. In cells expressing WT UBQLN2 or P497H mutants, in the early stages of SG formation under oxidative stress, the percentage of cells with SGs and the number of SGs per cell decreased to varying degrees. Between WT and mutant, there was no significant difference in eIF2α activity after stress treatment. Interestingly, the UBQLN2 P497H mutant downregulates the level of TIA‐1. In addition, the overexpression of the UBQLN2 P497H mutant inhibited the phosphorylation of 4E‐BP1 and affected the nucleoplasmic distribution of TDP‐43.ConclusionsUbiquilin 2 colocalizes with the SG component proteins G3BP1, TIA‐1, ATXN2, and PABPC1. It participates in regulating SG dynamics. And UBQLN2 mutation affects the assembly of stress granules by regulating TIA‐1. In addition, the overexpression of the UBQLN2 P497H mutant inhibited the phosphorylation of 4E‐BP1 and affected the nuclear and cytoplasmic distribution of TDP‐43. These provide new insights into the role of UBQLN2 in oxidative stress and the pathogenesis of ALS.

ALS-linked mutations impair UBQLN2 stress-induced biomolecular condensate assembly in cells.

Mutations in ubiquilin-2 (UBQLN2), a ubiquitin-binding shuttle protein involved in several protein quality control processes, can lead to amyotrophic lateral sclerosis (ALS). We previously found that wild-type UBQLN2 forms dynamic, membraneless biomolecular condensates upon cellular stress, and undergoes liquid-liquid phase separation in vitro. However, the impact of ALS-linked mutations on UBQLN2 condensate formation in cells remains unknown. Here, we overexpress mCherry-fused UBQLN2 with five patient-derived ALS-linked mutations and employ live-cell imaging and photokinetic analysis to investigate how each of these mutations impact stress-induced UBQLN2 condensate assembly and condensate material properties. Unlike endogenous UBQLN2, exogenously introduced UBQLN2 forms condensates distinct from stress granules. Both wild-type and mutant UBQLN2 condensates are generally cytoplasmic and liquid-like. However, mutant UBQLN2 forms fewer stress-induced UBQLN2 condensates than wild-type UBQLN2. Exogenously expressed P506T UBQLN2 forms the lowest number of stress-induced condensates of all UBQLN2 mutants, and these condensates are significantly smaller than those of wild-type UBQLN2. Fluorescence recovery after photobleaching (FRAP) analysis of UBQLN2 condensates revealed higher immobile fractions for UBQLN2 mutants, especially P506T. P497S and P497H mutations differentially impact condensate properties, demonstrating that the effects of ALS-linked mutations are both position- and amino acid-dependent. Collectively, our data show that disease mutations hinder assembly and alter viscoelastic properties of stress-induced UBQLN2 condensates, potentially leading to aggregates commonly observed in ALS.

Human Ubiquilin 2 and TDP-43 copathology drives neurodegeneration in transgenic Caenorhabditis elegans.

Amyotrophic lateral sclerosis (ALS) is a debilitating, fatal neurodegenerative disease that causes rapid muscle wasting. It shares a spectrum of symptoms and pathology with frontotemporal lobar degeneration (FTLD). These diseases are caused by aberrant activity of a set of proteins including TDP-43 and UBIQUILIN-2 (UBQLN2). UBQLN2 encodes a ubiquitin-like adaptor protein involved in the ubiquitin-proteasome protein degradation pathway. Mutations in the PXX domain of UBQLN2 cause familial ALS. UBQLN2 aggregates in skein-like inclusions with other ALS and FTLD associated proteins including TDP-43 and ubiquitin. To facilitate further investigation of UBQLN2-mediated mechanisms of neurodegeneration, we made Caenorhabditis elegans transgenic lines pan-neuronally expressing human UBQLN2 cDNAs carrying either the wild-type UBQLN2 sequence or UBQLN2 with ALS causing mutations. Transgenic animals exhibit motor dysfunction accompanied by neurodegeneration of GABAergic motor neurons. At low levels of UBQLN2 expression, wild-type UBQLN2 causes significant motor impairment and neurodegeneration that is exacerbated by ALS associated mutations in UBQLN2. At higher levels of UBQLN2 expression, both wild-type and ALS mutated versions of UBQLN2 cause severe impairment. Molecular genetic investigation revealed that UBQLN2 dependent locomotor defects do not require the involvement of the endogenous homolog of TDP-43 in C. elegans (tdp-1). However, co-expression of wild-type human TDP-43 exacerbates UBQLN2 deficits. This model of UBQLN2-mediated neurodegeneration may be useful for further mechanistic investigation into the molecular cascades driving neurodegeneration in ALS and ALS-FTLD.

Impaired 26S Proteasome Assembly Precedes Neuronal Loss in Mutant UBQLN2 Rats.

Proteasomal dysfunction is known to be associated with amyotrophic lateral sclerosis and frontotemporal degeneration (ALS/FTD). Our previous reports have shown that a mutant form of ubiquilin-2 (UBQLN2) linked to ALS/FTD leads to neurodegeneration accompanied by accumulations of the proteasome subunit Rpt1 in transgenic rats, but the precise pathogenic mechanisms of how this mutation impairs the proteasome remains to be elucidated. Here, we reveal that this UBQLN2 mutation in rats disrupted the proteasome integrity prior to neurodegeneration, that it dissociated the 26S proteasome in vitro, and that its depletion did not affect 26S proteasome assembly. During both disease progression and in an age-dependent manner, we found that proteasome subunits were translocated to the nucleus, including both of the 20S core particles (PSMA1 and PSMB7) and the 19S regulatory particles (Rpt1 and Rpn1), suggesting that defective proteasome function may result from the proteasome-subunit mislocalization. Taken together, the present data demonstrate that impaired proteasome assembly is an early event in the pathogenesis of UBQLN2-associated neurodegeneration in mutant UBQLN2 rats.

Ubiquilin-2 differentially regulates polyglutamine disease proteins

Divergent protein context helps explain why polyglutamine expansion diseases differ clinically and pathologically. This heterogeneity may also extend to how polyglutamine disease proteins are handled by cellular pathways of proteostasis. Studies suggest, for example, that the ubiquitin-proteasome shuttle protein Ubiquilin-2 (UBQLN2) selectively interacts with specific polyglutamine disease proteins. Here we employ cellular models, primary neurons and mouse models to investigate the potential differential regulation by UBQLN2 of two polyglutamine disease proteins, huntingtin (HTT) and ataxin-3 (ATXN3). In cells, overexpressed UBQLN2 selectively lowered levels of full-length pathogenic HTT but not of HTT exon 1 fragment or full-length ATXN3. Consistent with these results, UBQLN2 specifically reduced accumulation of aggregated mutant HTT but not mutant ATXN3 in mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 3 (SCA3), respectively. Normally a cytoplasmic protein, UBQLN2 translocated to the nuclei of neurons in HD mice but not in SCA3 mice. Remarkably, instead of reducing the accumulation of nuclear mutant ATXN3, UBQLN2 induced an accumulation of cytoplasmic ATXN3 aggregates in neurons of SCA3 mice. Together these results reveal a selective action of UBQLN2 toward polyglutamine disease proteins, indicating that polyglutamine expansion alone is insufficient to promote UBQLN2-mediated clearance of this class of disease proteins. Additional factors, including nuclear translocation of UBQLN2, may facilitate its action to clear intranuclear, aggregated disease proteins like HTT.

UBQLN2 Promotes the Production of Type I Interferon via the TBK1-IRF3 Pathway.

Mutations of Ubiquilin 2 (UBQLN2) or TANK-binding kinase 1 (TBK1) are associated with amyotrophic lateral sclerosis and frontotemporal degeneration (ALS/FTD). However, the mechanisms whereby UBQLN2 or TBK1 mutations lead to ALS and FTD remain unclear. Here, we explored the effect of UBQLN2 on TBK1 in HEK-293T cells or in CRISPR–Cas9-mediated IRF3 and IRF7 knockout (KO) cells. We found an interaction between TBK1 and UBQLN2, which was affected by ALS/FTD-linked mutations in TBK1 or UBQLN2. Co-expression of UBQLN2 with TBK1 elevated the protein level of TBK1 as well as the phosphorylation of TBK1 and IRF3 in a UBQLN2 dose-dependent manner, and this phosphorylation was reduced by mutant UBQLN2. In addition, the cellular production of IFN1 and related pro-inflammatory cytokines was substantially elevated when UBQLN2 and TBK1 were co-expressed, which was also decreased by mutant UBQLN2. Functional assay revealed that mutant UBQLN2 significantly reduced the binding affinity of TBK1 for its partners, including IRF3, (SQSTM1)/p62 and optineurin (OPTN). Moreover, complete loss of IRF3 abolished the induction of IFN1 and related pro-inflammatory cytokines enhanced by UBQLN2 in HEK-293T cells, whereas no significant change in IRF7 knockout cells was observed. Thus, our findings suggest that UBQLN2 promotes IRF3 phosphorylation via TBK1, leading to enhanced IFN1 induction, and also imply that the dysregulated TBK1-IRF3 pathway may play a role in UBQLN2-related neurodegeneration.

Neurofilament Proteins as Prognostic Biomarkers in Neurological Disorders.

Neurofilaments: light, medium, and heavy (abbreviated as NF-L, NF-M, and NF-H, respectively), which belong to Type IV intermediate filament family (IF), are neuron-specific cytoskeletal components. Neurofilaments are axonal structural components and integral components of synapses, which are important for neuronal electric signal transmissions along the axons and post-translational modification. Abnormal assembly of neurofilaments is found in several human neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), infantile spinal muscular atrophy (SMA), and hereditary sensory-motor neuropathy (HSMN). In addition, those pathological neurofilament accumulations are known in α-synuclein in Parkinson's disease (PD), Aβ and tau in Alzheimer's disease (AD), polyglutamine in CAG trinucleotide repeat disorders, superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TDP43), neuronal FUS proteins, optineurin (OPTN), ubiquilin 2 (UBQLN2), and dipeptide repeat protein (DRP) in amyotrophic lateral sclerosis (ALS). When axon damage occurs in central nervous disorders, neurofilament proteins are released and delivered into cerebrospinal fluid (CSF), which are then circulated into blood. New quantitative analyses and assay techniques are well-developed for the detection of neurofilament proteins, particularly NF-L and the phosphorylated NF-H (pNF-H) in CSF and serum. This review discusses the potential of using peripheral blood NF quantities and evaluating the severity of damage in the nervous system. Intermediate filaments could be promising biomarkers for evaluating disease progression in different nervous system disorders.