Rudich, Paige et al. (2017) International Worm Meeting "Novel translational products encoded by disease-associated GC-rich repeat expansions cause toxicity in C. elegans."

Rudich, Paige, Lamitina, Todd, Snoznik, Carley

[International Worm Meeting, 2017]

Expanded GC-rich repeats cause many age-onset neurodegenerative diseases. An emerging mechanism that may underlie disease pathology is an unusual type of protein translation called Repeat Associated non-ATG (RAN) translation, which specifically targets these GC-rich repeats. RAN translation requires extended GC-rich repeats and occurs independent of a canonical start codon, allowing translation in all three reading frames. Antisense RNA from these GC-rich repeats also gives rise to RAN protein products causing up to six distinct protein products from one repeat expansion. Most of these RAN proteins have never been studied in any biological system. Defining the role of RAN proteins in neurodegenerative diseases and their potential mechanisms of toxicity represents an exciting new translational research opportunity. We developed RAN protein models in C. elegans for two GC-rich expansion repeat diseases - C9orf72-associated Amyotrophic Lateral Sclerosis (ALS) and Huntington's Disease (HD). To separate RAN protein toxicity from RNA-derived toxicity, we generated 'pure' protein models by synthesizing codon-varied constructs that preserve RAN protein sequence but eliminate repeat-derived RNA structures. We used canonical ATG-dependent protein translation to bypass RAN translation and a C-terminal GFP or RFP tag to examine the subcellular localization. In our C. elegans ALS model, the arginine-containing dipeptides Pro-Arg (PR) and Gly-Arg (GR) exhibited age-onset toxicity when expressed in multiple cell types, including motor neurons. PR and GR exhibited nuclear localization that was necessary for toxicity. PR and GR did not form protein aggregates. Age-onset toxicity of PR, but not GR, was influenced by mutations that alter the rate of ageing, suggesting differences in the toxicity mechanisms of these related RAN peptides. In our HD model, initial studies suggest that HD RAN proteins exhibit distinct cell biological properties from the canonical polyGln (Q) HD protein. Currently, we are comparing the toxicity of these new HD RAN products to the well-described toxicity of polyQ. Understanding how different RAN peptides contribute to HD and to ALS is vital for developing appropriate treatments for these diseases. Moreover, comparing RAN proteins within a single experimental paradigm may reveal common themes that facilitate RAN protein toxicity and generate a more integrated understanding of the role of RAN-derived proteins in neurodegenerative diseases.

Rudich, Paige et al. (2021) International Worm Meeting "Identification of common lifespan-modulating genes through genomic comparison of diverse long-lived genetic mutants"

Soo, Sonja, Rudich, Paige, Van Raamsdonk,, Jeremy, Mistry, Meeta, Senchuk, Megan

[International Worm Meeting, 2021]

As the age of the world population rises, there is a concordant rise in age-associated diseases such as cancer, cardiovascular disease, and neurodegeneration. Understanding the aging process at the molecular level may aid development of preventative treatments for these diseases, as targeting lifespan-modulating pathways can be protective in models of age-onset diseases. However, lifespan-modulating pathways have wide ranging cellular effects, beyond delaying aging. To advance our understanding of genetic pathways contributing to longevity, and identify novel genetic targets to test in models of neurodegenerative disease, we identified "common" genes similarly modulated in a variety of long-lived genetic mutants. We performed RNA sequencing on nine long-lived genetic mutants representing six different lifespan-modulating pathways: insulin-IGF1 signaling (daf-2), dietary restriction (eat-2), germline deficiency (glp-1), reduced mechanosensation (osm-5), reduced translation (ife-2), elevated mitochondrial reactive oxygen species (sod-2), and weak mitochondrial impairment (nuo-6, isp-1, clk-1). There is a significant overlap in differentially expressed genes between almost all pairs of long-lived mutants. Thus, diverse lifespan-modulating pathways converge onto overlapping genetic targets. The genes upregulated by the lifespan-modulating pathways fall primarily into two disparate groups: genes upregulated in eat-2, osm-5, and ife-2 worms, and genes upregulated in daf-2, sod-2, isp-1, and nuo-6 worms. The two gene groups are enriched for different tissue expression and different molecular pathways, further supporting that these genes extend lifespan through distinct means. To identify "common" genes potentially shared across these groups, we selected for genes similarly modulated in at least six of the nine mutants. We identified 196 "common" upregulated genes enriched for genes involved in immunity and metabolism, and 62 "common" downregulated genes enriched for genes associated with translation initiation. We will determine if the overarching common genes are required for enhanced longevity and then examine if the group-specific genes are required for longevity in their respective mutants. Developing a clearer understanding of the specific genes and pathways which directly modulate lifespan will advance our understanding of the aging process, and help to develop preventative treatments for our aging population.

Soo, Sonja et al. (2021) International Worm Meeting "Genetic basis of enhanced stress resistance in long-lived mutants"

Van Raamsdonk, Jeremy, Mistry, Meeta, Rudich, Paige, Soo, Sonja

[International Worm Meeting, 2021]

In order to gain insight into the relationship between stress resistance and longevity, we directly compared resistance to multiple external stressors (heat, oxidative stress, bacterial pathogens, osmotic stress, and anoxia) with the magnitude of lifespan extension in nine long-lived C. elegans mutants representative of different pathways of lifespan extension. Furthermore, we have used RNA sequencing to analyse gene expression in each of these mutants to identify genes and pathways responsible for the enhanced resistance to stress. We find that all of the long-lived mutants examined have increased resistance to one or more type of stress. Resistance to each of the types of stress resistance examined exhibited a positive, significant correlation with lifespan, with bacterial pathogen resistance showing the strongest relationship. All of the long-lived mutants examined show upregulation of at least on stress response pathway, but differ in which pathway shows the greatest enrichment. We used RNA sequencing data to identify which genes are most highly correlated with each type of stress resistance. In comparing these results to genes that are most highly correlated with longevity, we observed a highly significant overlap suggesting that the same genetic pathways are involved in both. This was especially true for genes correlated with bacterial pathogen resistance, which showed an 84% overlap with genes correlated with lifespan. Overall, our results demonstrate a strong correlation between stress resistance and longevity, and indicate that the same genes contribute to both phenotypes.

Snoznik, Carley et al. (2021) International Worm Meeting "The nuclear ubiquitin ligase adaptor SPOP is a conserved regulator of C9orf72 dipeptide toxicity"

Medvedeva, Valentina, Mojsilovic-Petrovic, Jelena, Kalb, Robert, Snoznik, Carley, Rudich, Paige, Lamitina, Todd, Oosten, James

[International Worm Meeting, 2021]

A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Unconventional translation of the C9orf72 repeat produces dipeptide repeat proteins (DPRs). Previously, we showed that the DPRs (PR)50 and (GR)50 are highly toxic when expressed in C. elegans and this toxicity depends on nuclear localization of the DPR. In an unbiased genome-wide RNAi screen for suppressors of (PR)50 toxicity, we identified 12 genes that consistently suppressed either the developmental arrest and/or paralysis phenotype evoked by (PR)50 expression. All of these genes have vertebrate homologs and 7/12 contain predicted nuclear localization signals. One of these genes was spop-1, the C. elegans homolog of SPOP, a nuclear localized E3 ubiquitin ligase adaptor only found in metazoans. spop-1 is also required for (GR)50 toxicity and functions in a genetic pathway that includes cul-3, which is the canonical E3 ligase partner for SPOP. Genetic or pharmacological inhibition of SPOP in mammalian primary spinal cord motor neurons suppressed DPR toxicity without affecting DPR expression levels. SPOP is commonly mutated in prostate, endometrial, and renal cancer. We find that CRISPR/Cas9 knock-in of two of the most common cancer-causing SPOP missense mutations also protect against (PR)50 toxicity, suggesting that similar mechanisms mediate the roles of SPOP in cancer and C9 toxicity. Finally, we find that genetic inhibition of bet-1, the C. elegans homolog of the known SPOP ubiquitination targets BRD2/3/4, suppresses the protective effect of spop-1 mutations. Together, these data suggest a model in which SPOP promotes the DPR-dependent ubiquitination and degradation of BRD proteins. We speculate the pharmacological manipulation of this pathway, which is currently underway for multiple cancer subtypes, could also represent a novel entry point for therapeutic intervention to treat C9 FTD/ALS.

Campos, Juliane et al. (2021) International Worm Meeting "Mild impairment of mitochondrial function increases longevity and pathogen resistance through ATFS-1-driven activation of p38-regulated innate immunity"

Blackwell, Keith, Mistry, Meeta, Ferreira, Julio, Campos, Juliane, Wu, Ziyun, Rudich, Paige, Van Raamsdonk, Jeremy, Soo, Sonja

[International Worm Meeting, 2021]

While mitochondrial function is essential for life in all multicellular organisms, a mild impairment of mitochondrial function can extend longevity. By understanding the molecular mechanisms involved, these pathways might be targeted to promote healthy aging. In studying two long-lived mitochondrial mutants in C. elegans, we found that disrupting subunits of the mitochondrial electron transport chain resulted in upregulation of genes involved in innate immunity, which we found to be dependent on not only the canonical p38-mediated innate immune signaling pathway but also on the mitochondrial unfolded protein response. Both of these pathways are absolutely required for the increased resistance to bacterial pathogens and extended longevity of the long-lived mitochondrial mutants, as is the FOXO transcription factor DAF-16. This work demonstrates that both the p38-mediated innate immune signaling pathway and the mitochondrial unfolded protein response can act on the same innate immunity genes to promote resistance to bacterial pathogens, and that input from the mitochondria can extend longevity by signaling through these two pathways. Combined, this indicates that multiple evolutionarily conserved genetic pathways controlling innate immunity also function to modulate lifespan.

Puleo, Noah et al. MicroPubl Biol

Lamitina, Todd, Puleo, Noah

[MicroPubl Biol, 2020]

Neurodegenerative diseases caused by short expansive repeats like the (CAG) in Huntingtons disease (Orr 2012) or the (GGGGCC) repeat in C9orf72-associated Amyotrophic lateral sclerosis (ALS)/Frontotemporal dementia (FTD) (DeJesus-Hernandez et al. 2011) undergo an unusual type of translation called repeat associated non-AUG-dependent (RAN) translation (Cleary and Ranum 2014). Interestingly, RAN translation occurs without an AUG start codon (Cleary and Ranum 2014). This allows for the (GGGGCC) repeat mutation to be translated, even though it is located in the intron between exon 1 and exon 2 of the C9orf72 gene, which would normally be spliced out and degraded (DeJesus-Hernandez et al. 2011). Translation of the repeat occurs in all 3 reading frames, leading to the production of three distinct dipeptide repeat proteins (DPRs). RAN translation begins within the (GGGGCC) repeat, but the exact translation initiation site remains unclear. However, RAN translation does not stop at the end of the repeat and will continue to translate the intronic sequence until it reaches a stop codon. This means that each of the distinct DPRs will be fused to peptides encoded in the downstream intron sequence. Because the DPRs are derived from intron sequence that is spliced out of the mature C9orf72 mRNA, none of these intron-derived DPR fusion peptides are incorporated into the normal C9orf72 protein. While it is known that the DPR fusion peptides are made in patients, the precise sequences of the DPR fusion peptides that they produce is not currently known. Therefore, questions about where precisely RAN translation initiates, how many repeats are produced, and whether the number of repeats produced are uniform or heterogenous remain important but unresolved questions.There is also a C9orf72 antisense transcript, which contains the complementary repeat sequence (GGCCCC). This antisense transcript also undergoes RAN translation to produce another three DPRs (Zu et al. 2013). Therefore, a single DNA repeat expansion in one gene gives rise to six distinct DPRs. These DPRs form p62 positive/pTDP-43 negative inclusions that are distinct hallmarks of C9orf72-associated ALS/FTD (Cleary and Ranum 2014). Our laboratory as well as others have shown that two of these DPRs, proline-arginine (PR) and glycine-arginine (GR) are highly toxic (Kwon et al. 2014; Wen et al. 2014; Rudich et al. 2017), however the mechanisms of toxicity are poorly defined.In order to study the mechanisms that cause C9orf72-associated ALS/FTD PR and GR toxicity, we utilized the Caenorhabditis elegans model system. With short lifespans (3-4 weeks), a conserved neuromuscular system, and a genome that encodes ~20,000 genes with many conserved human homologs, the C. elegans model system is highly relevant for the study of aging and age-related diseases like ALS (Olsen et al. 2006). To study how PR and GR cause toxicity in C. elegans, we created animals expressing codon-optimized (PR)50-GFP and (GR)50-GFP (Rudich et al. 2017). With this approach, we are able to observe the effects of a single DPR at a time, without additional contributions from either the loss of the C9orf72 gene expression, introduction of the G4C2 repeat containing RNA, or the other five RAN translated DPRs. Therefore, this is a pure DPR model. Our laboratory has previously shown (PR)50 and (GR)50 to be toxic by causing a decrease in motility (paralysis) and arrested growth, when expressed in muscle (Rudich et al. 2017). Nuclear localization of these two DPRs was also discovered to be necessary and sufficient for toxicity in C. elegans (Rudich et al. 2017)...