Pathogenic mutations in UBQLN2 exhibit diverse aggregation propensity and neurotoxicity.

The ubiquitin-adaptor protein UBQLN2 promotes degradation of several aggregate-prone proteins implicated in neurodegenerative diseases. Missense UBQLN2 mutations also cause X-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Previously we demonstrated that the liquid-like properties of UBQLN2 molecular assemblies are altered by a specific pathogenic mutation, P506T, and that the propensity of UBQLN2 to aggregate correlated with neurotoxicity. Here, we systematically assess the effects of multiple, spatially distinct ALS/FTD-linked missense mutations on UBQLN2 aggregation propensity, neurotoxicity, phase separation, and autophagic flux. In contrast to what we observed for the P506T mutation, no other tested pathogenic mutant exhibited a clear correlation between aggregation propensity and neurotoxicity. These results emphasize the unique nature of pathogenic UBQLN2 mutations and argue against a generalizable link between aggregation propensity and neurodegeneration in UBQLN2-linked ALS/FTD.

Neuronal Puncta/Aggregate Formation by WT and Mutant UBQLN2.

Protein aggregates are a common feature of nearly all neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). Here we describe a method to quickly and accurately measure protein aggregation in cells expressing a fluorescently tagged aggregation-prone protein. This unbiased method obviates the need for manual scoring and facilitates the identification of factors governing protein self-assembly and its downstream consequences for cell heath.

RTL8 promotes nuclear localization of UBQLN2 to subnuclear compartments associated with protein quality control.

The brain-expressed ubiquilins (UBQLNs) 1, 2 and 4 are a family of ubiquitin adaptor proteins that participate broadly in protein quality control (PQC) pathways, including the ubiquitin proteasome system (UPS). One family member, UBQLN2, has been implicated in numerous neurodegenerative diseases including ALS/FTD. UBQLN2 typically resides in the cytoplasm but in disease can translocate to the nucleus, as in Huntington's disease where it promotes the clearance of mutant Huntingtin. How UBQLN2 translocates to the nucleus and clears aberrant nuclear proteins, however, is not well understood. In a mass spectrometry screen to discover UBQLN2 interactors, we identified a family of small (13 kDa), highly homologous uncharacterized proteins, RTL8, and confirmed the interaction between UBQLN2 and RTL8 both in vitro using recombinant proteins and in vivo using mouse brain tissue. Under endogenous and overexpressed conditions, RTL8 localizes to nucleoli. When co-expressed with UBQLN2, RTL8 promotes nuclear translocation of UBQLN2. RTL8 also facilitates UBQLN2's nuclear translocation during heat shock. UBQLN2 and RTL8 colocalize within ubiquitin-enriched subnuclear structures containing PQC components. The robust effect of RTL8 on the nuclear translocation and subnuclear localization of UBQLN2 does not extend to the other brain-expressed ubiquilins, UBQLN1 and UBQLN4. Moreover, compared to UBQLN1 and UBQLN4, UBQLN2 preferentially stabilizes RTL8 levels in human cell lines and in mouse brain, supporting functional heterogeneity among UBQLNs. As a novel UBQLN2 interactor that recruits UBQLN2 to specific nuclear compartments, RTL8 may regulate UBQLN2 function in nuclear protein quality control.

Development of a specific live-cell assay for native autophagic flux.

Autophagy is an evolutionarily conserved pathway mediating the breakdown of cellular proteins and organelles. Emphasizing its pivotal nature, autophagy dysfunction contributes to many diseases; nevertheless, development of effective autophagy modulating drugs is hampered by fundamental deficiencies in available methods for measuring autophagic activity or flux. To overcome these limitations, we introduced the photoconvertible protein Dendra2 into the MAP1LC3B locus of human cells via CRISPR/Cas9 genome editing, enabling accurate and sensitive assessments of autophagy in living cells by optical pulse labeling. We used this assay to perform high-throughput drug screens of four chemical libraries comprising over 30,000 diverse compounds, identifying several clinically relevant drugs and novel autophagy modulators. A select series of candidate compounds also modulated autophagy flux in human motor neurons modified by CRISPR/Cas9 to express GFP-labeled LC3. Using automated microscopy, we tested the therapeutic potential of autophagy induction in several distinct neuronal models of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In doing so, we found that autophagy induction exhibited discordant effects, improving survival in disease models involving the RNA binding protein TDP-43, while exacerbating toxicity in neurons expressing mutant forms of UBQLN2 and C9ORF72 associated with familial ALS/FTD. These studies confirm the utility of the Dendra2-LC3 assay, while illustrating the contradictory effects of autophagy induction in different ALS/FTD subtypes.

Shared and divergent phase separation and aggregation properties of brain-expressed ubiquilins.

The brain-expressed ubiquilins, UBQLNs 1, 2 and 4, are highly homologous proteins that participate in multiple aspects of protein homeostasis and are implicated in neurodegenerative diseases. Studies have established that UBQLN2 forms liquid-like condensates and accumulates in pathogenic aggregates, much like other proteins linked to neurodegenerative diseases. However, the relative condensate and aggregate formation of the three brain-expressed ubiquilins is unknown. Here we report that the three ubiquilins differ in aggregation propensity, revealed by in-vitro experiments, cellular models, and analysis of human brain tissue. UBQLN4 displays heightened aggregation propensity over the other ubiquilins and, like amyloids, UBQLN4 forms ThioflavinT-positive fibrils in vitro. Measuring fluorescence recovery after photobleaching (FRAP) of puncta in cells, we report that all three ubiquilins undergo liquid-liquid phase transition. UBQLN2 and 4 exhibit slower recovery than UBQLN1, suggesting the condensates formed by these brain-expressed ubiquilins have different compositions and undergo distinct internal rearrangements. We conclude that while all brain-expressed ubiquilins exhibit self-association behavior manifesting as condensates, they follow distinct courses of phase-separation and aggregation. We suggest that this variability among ubiquilins along the continuum from liquid-like to solid informs both the normal ubiquitin-linked functions of ubiquilins and their accumulation and potential contribution to toxicity in neurodegenerative diseases.

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.

Monitoring Neuronal Survival via Longitudinal Fluorescence Microscopy.

Standard cytotoxicity assays, which require the collection of lysates or fixed cells at multiple time points, have limited sensitivity and capacity to assess factors that influence neuronal fate. These assays require the observation of separate populations of cells at discrete time points. As a result, individual cells cannot be followed prospectively over time, severely limiting the ability to discriminate whether subcellular events, such as puncta formation or protein mislocalization, are pathogenic drivers of disease, homeostatic responses, or merely coincidental phenomena. Single-cell longitudinal microscopy overcomes these limitations, allowing the researcher to determine differences in survival between populations and draw causal relationships with enhanced sensitivity. This video guide will outline a representative workflow for experiments measuring single-cell survival of rat primary cortical neurons expressing a fluorescent protein marker. The viewer will learn how to achieve high-efficiency transfections, collect and process images enabling the prospective tracking of individual cells, and compare the relative survival of neuronal populations using Cox proportional hazards analysis.

Mutant UBQLN2 promotes toxicity by modulating intrinsic self-assembly.

UBQLN2 is one of a family of proteins implicated in ubiquitin-dependent protein quality control and integrally tied to human neurodegenerative disease. Whereas wild-type UBQLN2 accumulates in intraneuronal deposits in several common age-related neurodegenerative diseases, mutations in the gene encoding this protein result in X-linked amyotrophic lateral sclerosis/frontotemporal dementia associated with TDP43 accumulation. Using in vitro protein analysis, longitudinal fluorescence imaging and cellular, neuronal, and transgenic mouse models, we establish that UBQLN2 is intrinsically prone to self-assemble into higher-order complexes, including liquid-like droplets and amyloid aggregates. UBQLN2 self-assembly and solubility are reciprocally modulated by the protein's ubiquitin-like and ubiquitin-associated domains. Moreover, a pathogenic UBQLN2 missense mutation impairs droplet dynamics and favors amyloid-like aggregation associated with neurotoxicity. These data emphasize the critical link between UBQLN2's role in ubiquitin-dependent pathways and its propensity to self-assemble and aggregate in neurodegenerative diseases.

Inhibition of PIP4Kγ ameliorates the pathological effects of mutant huntingtin protein.

The discovery of the causative gene for Huntington's disease (HD) has promoted numerous efforts to uncover cellular pathways that lower levels of mutant huntingtin protein (mHtt) and potentially forestall the appearance of HD-related neurological defects. Using a cell-based model of pathogenic huntingtin expression, we identified a class of compounds that protect cells through selective inhibition of a lipid kinase, PIP4Kγ. Pharmacological inhibition or knock-down of PIP4Kγ modulates the equilibrium between phosphatidylinositide (PI) species within the cell and increases basal autophagy, reducing the total amount of mHtt protein in human patient fibroblasts and aggregates in neurons. In two Drosophila models of Huntington's disease, genetic knockdown of PIP4K ameliorated neuronal dysfunction and degeneration as assessed using motor performance and retinal degeneration assays respectively. Together, these results suggest that PIP4Kγ is a druggable target whose inhibition enhances productive autophagy and mHtt proteolysis, revealing a useful pharmacological point of intervention for the treatment of Huntington's disease, and potentially for other neurodegenerative disorders.

The Proline/Arginine Dipeptide from Hexanucleotide Repeat Expanded C9ORF72 Inhibits the Proteasome.

An intronic hexanucleotide repeat expansion (HRE) mutation in the C9ORF72 gene is the most common cause of familial ALS and frontotemporal dementia (FTD) and is found in ∼7% of individuals with apparently sporadic disease. Several different diamino acid peptides can be generated from the HRE by noncanonical translation (repeat-associated non-ATG translation, or RAN translation), and some of these peptides can be toxic. Here, we studied the effects of two arginine containing RAN translation products [proline/arginine repeated 20 times (PR20) and glycine/arginine repeated 20 times (GR20)] in primary rat spinal cord neuron cultures grown on an astrocyte feeder layer. We find that PR20 kills motor neurons with an LD50 of 2 µM, but in contrast to the effects of other ALS-causing mutant proteins (i.e., SOD or TDP43), PR20 does not evoke the biochemical signature of mitochondrial dysfunction, ER stress, or mTORC down-regulation. PR20 does result in a time-dependent build-up of ubiquitylated substrates, and this is associated with a reduction of flux through both autophagic and proteasomal degradation pathways. GR20, however, does not have these effects. The effects of PR20 on the proteasome are likely to be direct because (1) PR20 physically associates with proteasomes in biochemical assays, and (2) PR20 inhibits the degradation of a ubiquitylated test substrate when presented to purified proteasomes. Application of a proteasomal activator (IU1) blocks the toxic effects of PR20 on motor neuron survival. This work suggests that proteasomal activators have therapeutic potential in individuals with C9ORF72 HRE.

Motor neuron disease, TDP-43 pathology, and memory deficits in mice expressing ALS-FTD-linked UBQLN2 mutations.

Missense mutations in ubiquilin 2 (UBQLN2) cause ALS with frontotemporal dementia (ALS-FTD). Animal models of ALS are useful for understanding the mechanisms of pathogenesis and for preclinical investigations. However, previous rodent models carrying UBQLN2 mutations failed to manifest any sign of motor neuron disease. Here, we show that lines of mice expressing either the ALS-FTD-linked P497S or P506T UBQLN2 mutations have cognitive deficits, shortened lifespans, and develop motor neuron disease, mimicking the human disease. Neuropathologic analysis of the mice with end-stage disease revealed the accumulation of ubiquitinated inclusions in the brain and spinal cord, astrocytosis, a reduction in the number of hippocampal neurons, and reduced staining of TAR-DNA binding protein 43 in the nucleus, with concomitant formation of ubiquitin+ inclusions in the cytoplasm of spinal motor neurons. Moreover, both lines displayed denervation muscle atrophy and age-dependent loss of motor neurons that correlated with a reduction in the number of large-caliber axons. By contrast, two mouse lines expressing WT UBQLN2 were mostly devoid of clinical and pathological signs of disease. These UBQLN2 mouse models provide valuable tools for identifying the mechanisms underlying ALS-FTD pathogenesis and for investigating therapeutic strategies to halt disease.

Signature changes in ubiquilin expression in the R6/2 mouse model of Huntington's disease.

Ubiquilin proteins have been implicated in the cause and the pathology of neurodegenerative diseases. In the R6/2 mouse model of Huntington's disease (HD), ubiquilin levels decline during disease progression. Restoration of their levels by transgenic expression of ubiquilin-1 extends survival. Here we provide a comprehensive assessment of the expression and localization of all four ubiquilin proteins in both normal and R6/2-affected mice brains, using antibodies specific for each protein. Ubiquilin-1, 2 and 4 proteins were detected throughout the brain, with increased expression seen in the hippocampus and cerebellum. Ubiquilin-3 expression was not detected. All three ubiquilins expressed in the brain were found in Htt inclusions. Their expression changed during development and disease. Ubiquilin-1 and ubiquilin-2 protein levels decreased from 6 to 18 weeks of mouse development, independent of disease. Ubiquilin-1 and ubiquilin-4 protein levels also changed during HD disease progression. Ubiquilin-4 proteins that are normally expressed in the brain were lost and instead replaced by a novel 115 kDa higher molecular weight immunoreactive band. Taken together, our results demonstrate that all ubiquilin proteins are involved in HD pathology and that distinct changes in the signature of ubiquilin-4 expression could be useful for monitoring end-stage of HD disease.

Ubiquilin-1 overexpression increases the lifespan and delays accumulation of Huntingtin aggregates in the R6/2 mouse model of Huntington's disease.

Huntington's Disease (HD) is a neurodegenerative disorder that is caused by abnormal expansion of a polyglutamine tract in huntingtin (htt) protein. The expansion leads to increased htt aggregation and toxicity. Factors that aid in the clearance of mutant huntingtin proteins should relieve the toxicity. We previously demonstrated that overexpression of ubiqulin-1, which facilitates protein clearance through the proteasome and autophagy pathways, reduces huntingtin aggregates and toxicity in mammalian cell and invertebrate models of HD. Here we tested whether overexpression of ubiquilin-1 delays or prevents neurodegeneration in R6/2 mice, a well-established model of HD. We generated transgenic mice overexpressing human ubiquilin-1 driven by the neuron-specific Thy1.2 promoter. Immunoblotting and immunohistochemistry revealed robust and widespread overexpression of ubiquilin-1 in the brains of the transgenic mice. Similar analysis of R6/2 animals revealed that ubiquilin is localized in huntingtin aggregates and that ubiquilin levels decrease progressively to 30% during the end-stage of disease. We crossed our ubiquilin-1 transgenic line with R6/2 mice to assess whether restoration of ubiquilin levels would delay HD symptoms and pathology. In the double transgenic progeny, ubiquilin levels were fully restored, and this correlated with a 20% increase in lifespan and a reduction in htt inclusions in the hippocampus and cortex. Furthermore, immunoblots indicated that endoplasmic reticulum stress response that is elevated in the hippocampus of R6/2 animals was attenuated by ubiquilin-1 overexpression. However, ubiquilin-1 overexpression neither altered the load of htt aggregates in the striatum nor improved motor impairments in the mice.

Pattern of ubiquilin pathology in ALS and FTLD indicates presence of C9ORF72 hexanucleotide expansion.

C9ORF72-hexanucleotide repeat expansions and ubiquilin-2 (UBQLN2) mutations are recently identified genetic markers in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We investigate the relationship between C9ORF72 expansions and the clinical phenotype and neuropathology of ALS and FTLD. Genetic analysis and immunohistochemistry (IHC) were performed on autopsy-confirmed ALS (N = 75), FTLD-TDP (N = 30), AD (N = 14), and controls (N = 11). IHC for neurodegenerative disease pathology consisted of C9ORF72, UBQLN, p62, and TDP-43. A C9ORF72 expansion was identified in 19.4 % of ALS and 31 % of FTLD-TDP cases. ALS cases with C9ORF72 expansions frequently showed a bulbar onset of disease (57 %) and more rapid disease progression to death compared to non-expansion cases. Staining with C9ORF72 antibodies did not yield specific pathology. UBQLN pathology showed a highly distinct pattern in ALS and FTLD-TDP cases with the C9ORF72 expansion, with UBQLN-positive cytoplasmic inclusions in the cerebellar granular layer and extensive UBQLN-positive aggregates and dystrophic neurites in the hippocampal molecular layer and CA regions. These UBQLN pathologies were sufficiently unique to allow correct prediction of cases that were later confirmed to have C9ORF72 expansions by genetic analysis. UBQLN pathology partially co-localized with p62, and to a minor extent with TDP-43 positive dystrophic neurites and spinal cord skein-like inclusions. Our data indicate a pathophysiological link between C9ORF72 expansions and UBQLN proteins in ALS and FTLD-TDP that is associated with a highly characteristic pattern of UBQLN pathology. Our study indicates that this pathology is associated with alterations in clinical phenotype, and suggests that the presence of C9ORF72 repeat expansions may indicate a worse prognosis in ALS.