ABSTRACTThe degeneration of neurons in patients with amyotrophic lateral sclerosis (ALS) is commonly associated with accumulation of misfolded, insoluble proteins. Heat shock proteins (HSPs) are central regulators of protein homeostasis as they fold newly synthesized proteins and refold damaged proteins. Heterozygous loss-of- function mutations in theDNAJC7gene that encodes an HSP co-chaperone were recently identified as a cause for rare forms of ALS, yet the mechanisms underlying pathogenesis remain unclear. Using mass spectrometry, we found that the DNAJC7 interactome in human motor neurons (MNs) is enriched for RNA binding proteins (RBPs) and stress response chaperones. MNs generated from iPSCs with the ALS-associated mutation R156X inDNAJC7exhibit increased insolubility of its client RBP HNRNPU and associated RNA metabolism alterations. Additionally, DNAJC7 haploinsufficiency renders MNs increasingly susceptible to proteotoxic stress and cell death as a result of an ablated HSF1 stress response pathway. Critically, expression of HSF1 in mutant DNAJC7 MNs is sufficient to rescue their sensitivity to proteotoxic stress, while postmortem ALS patient cortical neurons exhibit a reduction in the expression of HSF1 pathway genes. Taken together, our work identifies DNAJC7 as a crucial mediator of HNRNPU function and stress response pathways in human MNs and highlights HSF1 as a therapeutic target in ALS.

Background: Obesity-which develops from environmental and biological factors-is disproportionately more prevalent among individuals of African ancestry. Part of this difference may be due to DNA variants specific to African ancestry. Recent observational studies have reported an association between obesity and the common APOL1 G1 and G2 DNA variants, specific to individuals of sub-Saharan African ancestry. Compared to the G0 major allele, the G1 and G2 risk variants (RV) are known to increase the risk of kidney disease in African-ancestry patients and have been extensively studied in the renal context. However, no functional validation has established whether the APOL1 RV are also causal in obesity. The aim of this study is to address the hypothesis that APOL1 RV are causal in the pathogenesis of obesity independent of kidney disease. Methods: Because APOL1 expression is primate-specific, we leveraged transgenic G0 and APOL1-RV mice (n = 58), which were fed standard chow or high-fat diet (HFD) for 16 weeks. The mice underwent measurements of body mass, body composition by Echo-MRI, and tail cuff BP. Blood, urine, kidneys, perigonadal adipose tissue (AT), and bone-marrow derived macrophages (BMDMs) were examined for cardiometabolic traits. Results: Concordant with single-cell RNA-seq data of human AT (GSE176067) revealing APOL1 expression in multiple cell types, robust APOL1 protein expression was detected in AT of the APOL1 transgenic mice. Although no differences in body mass were seen among males, female APOL1-RV mice had higher body mass than G0 females in both the standard chow (p = 0.01) and HFD groups (p = 0.04). Both male and female mice on standard chow did not exhibit genotype-specific differences in body composition, but G0 female mice on HFD had significantly higher lean:fat mass ratio than female APOL1-RV mice (7.7 vs. 3.8, p = 0.04). In addition, female APOL1-RV mice on standard chow had significantly higher systolic BP than G0 females (129.5 vs. 107.6 mmHg, p = 0.04), while female APOL1-RV mice on HFD had more albuminuria than G0 females (43.4 vs. 22.7 mg/g, p = 0.01). Although qPCR assays of kidney and AT were unrevealing, APOL1-RV BMDMs modeling inflamed AT macrophages of obesity expressed significantly higher levels of Tnf than G0 when stimulated with interferon-γ. Conclusion: This is the first study to report a causal link between APOL1 kidney disease variants and obesity, seen more prominently in female transgenic mice.

UPF1-mediated decay entails several mRNA surveillance pathways that play a crucial role in cellular homeostasis. However, the precise role of UPF1 in postmitotic neurons remains unresolved, as does its activity in amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease characterized by TDP-43 pathology and disrupted mRNA metabolism. Here, we used human iPSC-derived spinal motor neurons (MNs) to identify mRNAs subject to UPF1 degradation by integrating RNA-seq before and after UPF1 knockdown with RIP-seq to identify RNAs that co-immunoprecipitate with the active form of phosphorylated UPF1. We define a stringent set of bona fide UPF1 targets in MNs that are functionally enriched for autophagy and structurally enriched for GC-rich and long 3' UTRs but not for premature termination codon (PTC)-containing transcripts. TDP-43 depletion in iPSC-derived MNs reduces UPF1 phosphorylation and consequently post-transcriptional upregulation of UPF1 targets, suggesting that TDP-43 dysfunction compromises UPF1-mediated mRNA surveillance. Intriguingly, our datasets reveal that UPF1 and TDP-43 regulate alternative polyadenylation and 3'UTR length of mRNAs associated with synaptic and axonal function, a process that we find to be compromised in ALS models in vitro and ALS patient tissue. Our study provides a comprehensive description of UPF1-mediated mRNA decay activity in neurons, reveals overlapping roles between UPF1 and TDP-43 in regulating 3'UTR length, and offers novel insight into the intricate interplay between RNA metabolism and neurodegeneration in ALS.

Background While many women with rheumatoid arthritis (RA) improve during pregnancy and others worsen, there are no biomarkers to predict this improvement or worsening. In our unique RA pregnancy cohort that includes a pre-pregnancy baseline, we have examined pre-pregnancy gene co-expression networks to identify differences between women with RA who subsequently improve during pregnancy and those who worsen. Methods Blood samples were collected before pregnancy (T0) from 19 women with RA and 13 healthy women enrolled in our prospective pregnancy cohort. RA improvement/worsening between T0 and 3rd trimester was assessed by changes in the Clinical Disease Activity Index (CDAI). Pre-pregnancy expression profiles were examined by RNA sequencing and differential gene expression analysis. Weighted gene co-expression network analysis (WGCNA) was used to identify co-expression modules correlated with the improvement/worsening of RA during pregnancy and to assess their functional relevance. Results Of the 19 women with RA, 14 improved during pregnancy (RA improved ) while 5 worsened (RA worsened ). At the T0 baseline, however, the mean CDAI was similar between the two groups. WGCNA identified one co-expression module related to B cell function that was significantly correlated with the worsening of RA during pregnancy and was significantly enriched in genes differentially expressed between the RA improved and RA worsened groups. A neutrophil-related expression signature was also identified in the RA improved group at the T0 baseline. Conclusion The pre-pregnancy gene expression signatures identified represent potential biomarkers to predict the subsequent improvement/worsening of RA during pregnancy, which has important implications for the personalized treatment of RA during pregnancy.

Background To evaluate our hypotheses that, when rheumatoid arthritis (RA) flares postpartum, gene expression patterns are altered compared to (a) healthy women, (b) RA women whose disease activity is low or in remission postpartum, and (c) pre-pregnancy expression profiles. Methods Twelve women with RA and five healthy women were included in this pilot study. RA disease activity and postpartum flare were assessed using the Clinical Disease Activity Index (CDAI). Total RNA from frozen whole blood was used for RNA sequencing. Differential gene expression within the same women (within-group) over time, i.e., postpartum vs. third trimester (T3) or pre-pregnancy (T0), were examined, using a significance threshold of q < 0.05 and fold-change ≥ 2. Results Nine of the women with RA experienced a flare postpartum (RA Flare ), while three had low disease activity or were in remission (RA NoFlare ) during that time frame. Numerous immune-related genes were differentially expressed postpartum (vs. T3) during a flare. Fold-changes in expression from T3 to postpartum were mostly comparable between the RA Flare and healthy groups. At 3 months postpartum, compared to healthy women, several genes were significantly differentially expressed only among the RA Flare women, and not among the RA NoFlare women. Some of these genes were among those whose “normal” expression was significantly modulated postpartum, and the postpartum expression patterns were significantly altered during the RA flare. There were also some genes that were significantly differentially expressed in RA Flare compared to both healthy and RA NoFlare women, even though their expression was not significantly modulated postpartum. Furthermore, while postpartum expression profiles were similar to those at pre-pregnancy among healthy women, significant differences were found between those time points among the RA Flare women. Conclusions The large majority of gene expression changes between T3 and 3 months postpartum among RA women who flared postpartum reflected normal postpartum changes also seen among healthy women. Nonetheless, during a postpartum flare, a set of immune-related genes showed dysregulated expression compared to healthy women and women with RA whose disease activity was low or in remission during the same time frame, while other genes demonstrated significant differences in expression compared to RA pre-pregnancy levels.

Objective. To assess whether gene expression signatures associated with rheumatoid arthritis (RA) before pregnancy differ between women who improve or worsen during pregnancy, and to determine whether these expression signatures are altered during pregnancy when RA improves or worsens. Methods. Clinical data and blood samples were collected before pregnancy (T0) and at the third trimester (T3) from 11 women with RA and 5 healthy women. RA disease activity was assessed using the Clinical Disease Activity Index (CDAI). At each timepoint, RA-associated gene expression signatures were identified using differential expression analysis of RNA sequencing profiles between women with RA and healthy women. Results. Of the women with RA, 6 improved by T3 (RA improved) , 3 worsened (RA worsened) ,and 2 were excluded. At T0, mean CDAI scores were similar in both groups (RA improved 11.2 ± 9.8; RA worsened 13.8 ± 6.7; Wilcoxon rank-sum test: P = 0.6). In the RA improved group, 89 genes were differentially expressed at T0 ( q < 0.05 and fold change ≥ 2) compared to healthy women. When RA improved at T3, 65 of 89 (73%) of these genes no longer displayed RA-associated expression. In the RA worsened group, a largely different RA gene expression signature (429 genes) was identified at T0. When RA disease activity worsened at T3, 207 of 429 (48%) genes lost their differential expression, while an additional 151 genes became newly differentially expressed. Conclusion. In our pilot dataset, pre-pregnancy RA expression signatures differed between women who subsequently improved or worsened during pregnancy, suggesting that inherent genomic differences may influence how pregnancy affects disease activity. Further, these RA signatures were altered during pregnancy as disease activity changed.

In addition to canonical open reading frames (ORFs), thousands of translated small ORFs (containing less than 100 codons) have been identified in untranslated mRNA regions (UTRs) across eukaryotes. Small ORFs in 5' UTRs (upstream (u)ORFs) often repress translation of the canonical ORF within the same mRNA. However, the function of translated small ORFs in the 3' UTRs (downstream (d)ORFs) is unknown. Contrary to uORFs, we find that translation of dORFs enhances translation of their corresponding canonical ORFs. This translation stimulatory effect of dORFs depends on the number of dORFs, but not the length or peptide they encode. We propose that dORFs represent a new, strong, and universal translation regulatory mechanism in vertebrates.

mRNA translation decodes nucleotide into amino acid sequences. However, translation has also been shown to affect mRNA stability depending on codon composition in model organisms, although universality of this mechanism remains unclear. Here, using three independent approaches to measure exogenous and endogenous mRNA decay, we define which codons are associated with stable or unstable mRNAs in human cells. We demonstrate that the regulatory information affecting mRNA stability is encoded in codons and not in nucleotides. Stabilizing codons tend to be associated with higher tRNA levels and higher charged/total tRNA ratios. While mRNAs enriched in destabilizing codons tend to possess shorter poly(A)-tails, the poly(A)-tail is not required for the codon-mediated mRNA stability. This mechanism depends on translation; however, the number of ribosome loads into a mRNA modulates the codon-mediated effects on gene expression. This work provides definitive evidence that translation strongly affects mRNA stability in a codon-dependent manner in human cells.