Counter-regulation of RNA stability by UPF1 and TDP43.

RNA quality control is crucial for proper regulation of gene expression. Disruption of nonsense mediated mRNA decay (NMD), the primary RNA decay pathway responsible for the degradation of transcripts containing premature termination codons (PTCs), can disrupt development and lead to multiple diseases in humans and other animals. Similarly, therapies targeting NMD may have applications in hematological, neoplastic and neurological disorders. As such, tools capable of accurately quantifying NMD status could be invaluable for investigations of disease pathogenesis and biomarker identification. Toward this end, we assemble, validate, and apply a next-generation sequencing approach (NMDq) for identifying and measuring the abundance of PTC-containing transcripts. After validating NMDq performance and confirming its utility for tracking RNA surveillance, we apply it to determine pathway activity in two neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) characterized by RNA misprocessing and abnormal RNA stability. Despite the genetic and pathologic evidence implicating dysfunctional RNA metabolism, and NMD in particular, in these conditions, we detected no significant differences in PTC-encoding transcripts in ALS models or disease. Contrary to expectations, overexpression of the master NMD regulator UPF1 had little effect on the clearance of transcripts with PTCs, but rather restored RNA homeostasis through differential use and decay of alternatively poly-adenylated isoforms. Together, these data suggest that canonical NMD is not a significant contributor to ALS/FTD pathogenesis, and that UPF1 promotes neuronal survival by regulating transcripts with abnormally long 3'UTRs.

RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia.

RNA methylation at adenosine N6 (m6A) is one of the most common RNA modifications, impacting RNA stability, transport, and translation. Previous studies uncovered RNA destabilization in amyotrophic lateral sclerosis (ALS) models in association with accumulation of the RNA-binding protein TDP43. Here, we show that TDP43 recognizes m6A RNA and that RNA methylation is critical for both TDP43 binding and autoregulation. We also observed extensive RNA hypermethylation in ALS spinal cord, corresponding to methylated TDP43 substrates. Emphasizing the importance of m6A for TDP43 binding and function, we identified several m6A factors that enhance or suppress TDP43-mediated toxicity via single-cell CRISPR-Cas9 in primary neurons. The most promising modifier-the canonical m6A reader YTHDF2-accumulated within ALS spinal neurons, and its knockdown prolonged the survival of human neurons carrying ALS-associated mutations. Collectively, these data show that m6A modifications modulate RNA binding by TDP43 and that m6A is pivotal for TDP43-related neurodegeneration in ALS.

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.

A CRMP4-dependent retrograde axon-to-soma death signal in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal non-cell-autonomous neurodegenerative disease characterized by the loss of motor neurons (MNs). Mutations in CRMP4 are associated with ALS in patients, and elevated levels of CRMP4 are suggested to affect MN health in the SOD1G93A -ALS mouse model. However, the mechanism by which CRMP4 mediates toxicity in ALS MNs is poorly understood. Here, by using tissue from human patients with sporadic ALS, MNs derived from C9orf72-mutant patients, and the SOD1G93A -ALS mouse model, we demonstrate that subcellular changes in CRMP4 levels promote MN loss in ALS. First, we show that while expression of CRMP4 protein is increased in cell bodies of ALS-affected MN, CRMP4 levels are decreased in the distal axons. Cellular mislocalization of CRMP4 is caused by increased interaction with the retrograde motor protein, dynein, which mediates CRMP4 transport from distal axons to the soma and thereby promotes MN loss. Blocking the CRMP4-dynein interaction reduces MN loss in human-derived MNs (C9orf72) and in ALS model mice. Thus, we demonstrate a novel CRMP4-dependent retrograde death signal that underlies MN loss in ALS.

TDP43 nuclear export and neurodegeneration in models of amyotrophic lateral sclerosis and frontotemporal dementia.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are progressive neurodegenerative disorders marked in most cases by the nuclear exclusion and cytoplasmic deposition of the RNA binding protein TDP43. We previously demonstrated that ALS-associated mutant TDP43 accumulates within the cytoplasm, and that TDP43 mislocalization predicts neurodegeneration. Here, we sought to prevent neurodegeneration in ALS/FTD models using selective inhibitor of nuclear export (SINE) compounds that target exportin-1 (XPO1). SINE compounds modestly extend cellular survival in neuronal ALS/FTD models and mitigate motor symptoms in an in vivo rat ALS model. At high doses, SINE compounds block nuclear egress of an XPO1 cargo reporter, but not at lower concentrations that were associated with neuroprotection. Neither SINE compounds nor leptomycin B, a separate XPO1 inhibitor, enhanced nuclear TDP43 levels, while depletion of XPO1 or other exportins had little effect on TDP43 localization, suggesting that no single exporter is necessary for TDP43 export. Supporting this hypothesis, we find overexpression of XPO1, XPO7 and NXF1 are each sufficient to promote nuclear TDP43 egress. Taken together, our results indicate that redundant pathways regulate TDP43 nuclear export, and that therapeutic prevention of cytoplasmic TDP43 accumulation in ALS/FTD may be enhanced by targeting several overlapping mechanisms.

Spontaneous age-related neurite branching in Caenorhabditis elegans.

The analysis of morphological changes that occur in the nervous system during normal aging could provide insight into cognitive decline and neurodegenerative disease. Previous studies have suggested that the nervous system of Caenorhabditis elegans maintains its structural integrity with age despite the deterioration of surrounding tissues. Unexpectedly, we observed that neurons in aging animals frequently displayed ectopic branches and that the prevalence of these branches increased with time. Within age-matched populations, the branching of mechanosensory neurons correlated with decreased response to light touch and decreased mobility. The incidence of branching was influenced by two pathways that can affect the rate of aging, the Jun kinase pathway and the insulin/IGF-1 pathway. Loss of Jun kinase signaling, which slightly shortens lifespan, dramatically increased and accelerated the frequency of neurite branching. Conversely, inhibition of the daf-2 insulin/IGF-1-like signaling pathway, which extends lifespan, delayed and suppressed branching, and this delay required DAF-16/FOXO activity. Both JNK-1 and DAF-16 appeared to act within neurons in a cell-autonomous manner to influence branching, and, through their tissue-specific expression, it was possible to disconnect the rate at which branching occurred from the overall rate of aging of the animal. Old age has generally been associated with the decline and deterioration of different tissues, except in the case of tumor cell growth. To our knowledge, this is the first indication that aging can potentiate another form of growth, the growth of neurite branches, in normal animals.

Disease-associated mutant ubiquitin causes proteasomal impairment and enhances the toxicity of protein aggregates.

Protein homeostasis is critical for cellular survival and its dysregulation has been implicated in Alzheimer's disease (AD) and other neurodegenerative disorders. Despite the growing appreciation of the pathogenic mechanisms involved in familial forms of AD, much less is known about the sporadic cases. Aggregates found in both familial and sporadic AD often include proteins other than those typically associated with the disease. One such protein is a mutant form of ubiquitin, UBB+1, a frameshift product generated by molecular misreading of a wild-type ubiquitin gene. UBB+1 has been associated with multiple disorders. UBB+1 cannot function as a ubiquitin molecule, and it is itself a substrate for degradation by the ubiquitin/proteasome system (UPS). Accumulation of UBB+1 impairs the proteasome system and enhances toxic protein aggregation, ultimately resulting in cell death. Here, we describe a novel model system to investigate how UBB+1 impairs UPS function and whether it plays a causal role in protein aggregation. We expressed a protein analogous to UBB+1 in yeast (Ub(ext)) and demonstrated that it caused UPS impairment. Blocking ubiquitination of Ub(ext) or weakening its interactions with other ubiquitin-processing proteins reduced the UPS impairment. Expression of Ub(ext) altered the conjugation of wild-type ubiquitin to a UPS substrate. The expression of Ub(ext) markedly enhanced cellular susceptibility to toxic protein aggregates but, surprisingly, did not induce or alter nontoxic protein aggregates in yeast. Taken together, these results suggest that Ub(ext) interacts with more than one protein to elicit impairment of the UPS and affect protein aggregate toxicity. Furthermore, we suggest a model whereby chronic UPS impairment could inflict deleterious consequences on proper protein aggregate sequestration.

MITF links differentiation with cell cycle arrest in melanocytes by transcriptional activation of INK4A.

Cell cycle exit is required for proper differentiation in most cells and is critical for normal development, tissue homeostasis, and tumor suppression. However, the mechanisms that link cell cycle exit with differentiation remain poorly understood. Here, we show that the master melanocyte differentiation factor, microphthalmia transcription factor (MITF), regulates cell cycle exit by activating the cell cycle inhibitor INK4A, a tumor suppressor that frequently is mutated in melanomas. MITF binds the INK4A promoter, activates p16(Ink4a) mRNA and protein expression, and induces retinoblastoma protein hypophosphorylation, thereby triggering cell cycle arrest. This activation of INK4A was required for efficient melanocyte differentiation. Interestingly, MITF was also required for maintaining INK4A expression in mature melanocytes, creating a selective pressure to escape growth inhibition by inactivating INK4A. These findings demonstrate that INK4A can be regulated by a differentiation factor, establish a mechanistic link between melanocyte differentiation and cell cycle exit, and potentially explain the tissue-specific tendency for INK4A mutations to occur in melanoma.