Idiopathic Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, which is associated with neuroinflammation and reactive gliosis. The underlying cause of PD and the concurrent neuroinflammation are not well understood. In this study, we utilize human and murine neuronal lines, stem cell-derived dopaminergic neurons, and mice to demonstrate that three previously identified genetic risk factors for PD, namely SATB1, MIR22HG, and GBA, are components of a single gene regulatory pathway. Our findings indicate that dysregulation of this pathway leads to the upregulation of glucocerebrosides (GluCer), which triggers a cellular senescence-like phenotype in dopaminergic neurons. Specifically, we discovered that downregulation of the transcriptional repressor SATB1 results in the derepression of the microRNA miR-22-3p, leading to decreased GBA expression and subsequent accumulation of GluCer. Furthermore, our results demonstrate that an increase in GluCer alone is sufficient to impair lysosomal and mitochondrial function, thereby inducing cellular senescence. Dysregulation of the SATB1-MIR22-GBA pathway, observed in both PD patients and normal aging, leads to lysosomal and mitochondrial dysfunction due to the GluCer accumulation, ultimately resulting in a cellular senescence-like phenotype in dopaminergic neurons. Therefore, our study highlights a novel pathway involving three genetic risk factors for PD and provides a potential mechanism for the senescence-induced neuroinflammation and reactive gliosis observed in both PD and normal aging.

This file contains descriptions of all of the bioinformatic analyses used in the paper.

Distribution of allele fraction of inherited and somatic variants before and after filtering. This figure is similar to Fig. 4a, but includes a plotting of all somatic variants before filtering (black), which contains a large number of false positive errors.

The following file contains Tables S1-12. The contents of these tables are referenced through the manuscript and contain the basis for the results summarized in the paper. The list of tables is as follows: S1. CNV. This table contains all the copy number gains and losses identified in the CTCs. S2. SNV. This table contains all of the somatic SNVs identified in the CTCs. S3. Indel. This table contains all of the somatic insertions and deletions identified in the CTCs. S4. NA12877 somatic. This table contains all of the somatic SNVs identified in NA12877. S5. Allele fraction. This table contains allele fraction calculations and compartment number filtering calculations. S6. Sanger and Miseq. This table contains all of the Sanger sequencing and Miseq validation for the 77 somatic SNVs validated in this study. S7. Mutational spectrum. This table contains the mutation spectrum analysis and the comparison of the CTC mutation spectrum to other tumor types. S8. Non-coding annotation. This table contains a list of potentially important somatic noncoding variants. S9. Splicing. This table contains somatic splicing variants. S10. MHC-II. This table contains the potential somatic coding changes which would result in MHC-II binding antigens. S11. Two hit. This table contains genes with two potential inactivating variants, this can include inherited and somatic variants. S12. Phasing. This table contains phased somatic and/or inherited variants that potentially inactivate a gene through a compound heterozygous mechanism.

Copy number analysis based on read coverage in all batches. Read data is plotted starting from Chr1 and ending at ChrX. Each sample is labeled on the right side of the plot. All CTC samples identify chr1q amplification, and chr13 and chr16q losses.

Mutational spectrum of CTCs. The percent of particular base changes (labeled on the right of the figure) for different sets of mutations from CTCs, NA12877, and breast cancer from TCGA (BRCA) data are shown. Founder mutations and mutations found in 5 cells have a pattern very similar to that seen in breast cancer.

Small molecule-induced cell fate transitions are characterized by low efficiency and slow kinetics. An optimized chemical reprogramming approach now facilitates the robust and rapid conversion of somatic cells to pluripotent stem cells, unlocking exciting avenues to study and manipulate human cell identity.

Introduction: The Yale Fatty Liver Disease Program (YFLDP) is a patient care model incorporating weight management with standard hepatology care for targeted treatment of nonalcoholic fatty liver disease (NAFLD). Weight management is the mainstay treatment for NAFLD, with 10% total body weight loss (TBWL) leading to disease improvement. Bariatric surgery is an effective treatment for obesity and associated metabolic diseases, though only an estimated 1% of eligible patients receive referral nationwide. We studied YFLDP patient referral rates to bariatric surgery, clinical and surgical characteristics, and weight loss outcomes. Methods: We retrospectively extracted demographic, surgical, and weight loss outcome data of adult YFLDP patients from 10/2015 to 5/2022. Standard eligibility criteria included body mass index (BMI) >40 kg/m2 or >35 kg/m2 with an obesity-associated comorbidity. Actual 1-year weight loss outcomes were compared to predicted weight-loss outcomes calculated using the MBSAQIP Bariatric Surgical Risk/Benefit Calculator and assessed with a 2-tailed t-test. Results: There were 1,166 patients, of whom 596 (51.1%) were eligible for bariatric surgery (Table). Of these, 150 (25.3%) received referral, 32 patients (21.3%) underwent surgery (22 received sleeve gastrectomy (SG) and 10 had a Roux-en-Y gastric bypass [RYGB]). Patients who received surgery were more likely female and upper class compared to those who continued with lifestyle/medication management. Patients receiving SG and RYGB had mean TBWL 21.5% and 20.0%, respectively, while patients who continued medication/lifestyle management had TBWL of 3%. There was no significant difference comparing actual vs predicted final BMI (P=0.12), actual vs predicted weight loss (P=0.13), and actual vs predicted TBWL (P=0.13) post-bariatric surgery for all procedures (Figure). Conclusion: Integration of weight management with hepatology medical care is an effective model for NAFLD weight loss treatment. Patients receive referral for bariatric surgical evaluation at a rate 25 times greater than the national average, and those that receive surgery have comparable weight loss to that predicted in all patients receiving these surgeries, and on average exceeded the treatment threshold expected to treat NAFLD. Further study to identify patients who would most benefit from bariatric surgery would help to inform better patient selection and proactive referral efforts.Figure 1.: Predicted vs actual bariatric surgical outcomes in patients referred for surgery. (A) Actual vs Predicted Final Body Mass Index (BMI) post Bariatric Surgery: Comparison between YFLDP (Yale Fatty Liver Disease Program) patients’ actual BMI post bariatric surgery vs predicted BMI post bariatric surgery using the MBSAQIP Bariatric Surgical Risk/Benefit Calculator. There was no statistically significant difference between actual and predicted BMI (P=0.12). (B) Total Body Weight Loss (TBWL) in Patients given Bariatric Surgery Referral: Comparison between YFDLP patients’ total body weight % reduction in patients given a bariatric surgery referral who either underwent a gastric bypass or sleeve gastrectomy or did not have surgery and continued with lifestyle/medication management. Patients who underwent surgery had a statistically significant percent reduction in TBWL compared to patients managed with only lifestyle/medications. Error bars represent standard error. ***P< 0.001 Table 1. - Demographic and surgical characteristics of Yale Fatty Liver Disease Program patients who were referred for bariatric surgery Patient Characteristics Referred, Underwent Bariatric Surgery (+)(n=32) Referred, Did Not Undergo Bariatric Surgery (-)(n=118) P Sex n (%) Male 6 (19) 35 (30) 0.22 Female 26 (81) 83 (70) Age years (SD) 49 (10.3) 51 (12.98) 0.60 Medicare Qualifying n (%) 2 (6) 19 (12.98) 0.15 Race n (%) White 22 (69) 86 (73) 0.29 Black 4 (13) 15 (13) Asian 1 (3) 0 (0) Other/Declined 5 (15) 17 (14) Ethnicity n (%) Hispanic 6 (19) 27 (23) 0.62 Non-Hispanic 26 (81) 91 (77) Insurance Status n (%) Uninsured 0 (0) 0 (0) 0.045 Medicaid 14 (44) 31 (26) Medicare 2 (6) 27 (23) Private 16 (50) 60 (51) Median House Income by Zip Code n (%) < $50k 7 (22) 29 (25) 0.48 $50k-100k 19 (59) 75 (63) >$100k 6 (19) 14 (12) Anti-Obesity Medications n (%) Lifetime 16 (47) 38 (32) 0.78 Since YFLDP 10 (31) 29 (25) BMI Reduction mean (SD) RYGB (n=10) 8.3 (5.4) Predicted 1.3 (3.9) < 0.001 SG (n=22) 9.4 (6.1) < 0.001 TBWL % (SE) RYGB (n=10) 20.5 (3.5) Predicted 3 (0.8) < 0.001 SG (n=22) 21 (2.73) < 0.001 (+) Surgery denotes patients who underwent bariatric surgery after referral. (-) Surgery denotes patients who did not undergo bariatric surgery after referral. Patients who underwent bariatric surgery had statistically significant greater reduction in mean BMI compared to patients who did not have surgery. SD is standard deviation, SE is standard error, BMI is body mass index (kg/m2), RYGB is Roux-en-Y gastric bypass, SG is sleeve gastrectomy, TBWL is total body weight loss.

Chimeric antigen receptors (CARs) repurpose natural signaling components to retarget T cells to refractory cancers but have shown limited efficacy in persistent, recurrent malignancies. Here, we introduce “CAR Pooling,” a multiplexed approach to rapidly identify CAR designs with clinical potential. Forty CARs with signaling domains derived from a range of immune cell lineages were evaluated in pooled assays for their ability to stimulate critical T cell effector functions during repetitive stimulation that mimics long-term tumor antigen exposure. Several domains were identified from the tumor necrosis factor (TNF) receptor family that have been primarily associated with B cells. CD40 enhanced proliferation, whereas B cell–activating factor receptor (BAFF-R) and transmembrane activator and CAML interactor (TACI) promoted cytotoxicity. These functions were enhanced relative to clinical benchmarks after prolonged antigen stimulation, and CAR T cell signaling through these domains fell into distinct states of memory, cytotoxicity, and metabolism. BAFF-R CAR T cells were enriched for a highly cytotoxic transcriptional signature previously associated with positive clinical outcomes. We also observed that replacing the 4-1BB intracellular signaling domain with the BAFF-R signaling domain in a clinically validated B cell maturation antigen (BCMA)–specific CAR resulted in enhanced activity in a xenotransplant model of multiple myeloma. Together, these results show that CAR Pooling is a general approach for rapid exploration of CAR architecture and activity to improve the efficacy of CAR T cell therapies.

Single cell RNA sequencing has the potential to elucidate transcriptional programs underlying key cellular phenotypes and behaviors. However, many cell phenotypes are incompatible with indiscriminate single cell sequencing because they are rare, transient, or can only be identified by imaging. Existing methods for isolating cells based on imaging for single cell sequencing are technically challenging, time-consuming, and prone to loss because of the need to physically transport single cells. Here, we developed See-N-Seq, a method to rapidly screen cells in microwell plates in order to isolate RNA from specific single cells without needing to physically extract each cell. Our approach involves encapsulating the cell sample in a micropatterned hydrogel with spatially varying porosity to selectively expose specific cells for targeted RNA extraction. Extracted RNA can then be captured, barcoded, reverse transcribed, amplified, and sequenced at high-depth. We used See-N-Seq to isolate and sequence RNA from cell-cell conjugates forming an immunological synapse between T-cells and antigen presenting cells. In the hours after synapsing, we found time-dependent bifurcation of single cell transcriptomic profiles towards Type 1 and Type 2 helper T-cells lineages. Our results demonstrate how See-N-Seq can be used to associate transcriptomic data with specific functions and behaviors in single cells.

Mitochondria and chloroplasts are organelles with high iron demand that are particularly susceptible to iron-induced oxidative stress. Despite the necessity of strict iron regulation in these organelles, much remains unknown about mitochondrial and chloroplast iron transport in plants. Here, we propose that Arabidopsis ferroportin 3 (FPN3) is an iron exporter that is dual-targeted to mitochondria and chloroplasts. FPN3 is expressed in shoots, regardless of iron conditions, but its transcripts accumulate under iron deficiency in roots. fpn3 mutants cannot grow as well as the wild type under iron-deficient conditions and their shoot iron levels are lower compared with the wild type. Analyses of iron homeostasis gene expression in fpn3 mutants and inductively coupled plasma mass spectrometry (ICP-MS) measurements show that iron levels in the mitochondria and chloroplasts are increased relative to the wild type, consistent with the proposed role of FPN3 as a mitochondrial/plastid iron exporter. In iron-deficient fpn3 mutants, abnormal mitochondrial ultrastructure was observed, whereas chloroplast ultrastructure was not affected, implying that FPN3 plays a critical role in the mitochondria. Overall, our study suggests that FPN3 is essential for optimal iron homeostasis.

Plants use intricate mechanisms to adapt to changing iron conditions because iron is essential and also one of the most limiting nutrients for plant growth. Furthermore, iron is potentially toxic in excess and must be tightly regulated. Previously, we showed that chromatin remodeling via histone 3 lysine 27 trimethylation (H3K27me3) modulates the expression of FIT-dependent genes under iron deficiency in roots. This study builds on our previous findings, showing that H3K27me3 also modulates iron regulation in shoots. In the clf mutant, which lacks the predominant H3K27 tri-methyltransferase, we detected increased iron translocation to shoots under iron deficiency as compared to wild type. Transcriptomic analysis of shoots also revealed differential expression of genes consistent with higher iron levels in clf shoots than wild type shoots under iron-deficient conditions. In addition, we verify that YSL1 and IMA1, two genes involved in signaling iron status from shoots to roots, are direct targets of H3K27me3 and reveal iron-dependent deposition of H3K27me3 on these loci. This study contributes to a better understanding of the molecular mechanisms behind iron regulation in plants, as the effect of PRC2-mediated H3K27me3 on iron homeostasis genes expressed in the shoots has not been previously reported to our knowledge.

Genes encoding cell-surface proteins control nervous system development and are implicated in neurological disorders. These genes produce alternative mRNA isoforms which remain poorly characterized, impeding understanding of how disease-associated mutations cause pathology. Here we introduce a strategy to define complete portfolios of full-length isoforms encoded by individual genes. Applying this approach to neural cell-surface molecules, we identify thousands of unannotated isoforms expressed in retina and brain. By mass spectrometry we confirm expression of newly-discovered proteins on the cell surface in vivo. Remarkably, we discover that the major isoform of a retinal degeneration gene, CRB1 , was previously overlooked. This CRB1 isoform is the only one expressed by photoreceptors, the affected cells in CRB1 disease. Using mouse mutants, we identify a function for this isoform at photoreceptor-glial junctions and demonstrate that loss of this isoform accelerates photoreceptor death. Therefore, our isoform identification strategy enables discovery of new gene functions relevant to disease.

Cellular senescence is a mechanism used by mitotic cells to prevent uncontrolled cell division. As senescent cells persist in tissues, they cause local inflammation and are harmful to surrounding cells, contributing to aging. Generally, neurodegenerative diseases, such as Parkinson's, are disorders of aging. The contribution of cellular senescence to neurodegeneration is still unclear. SATB1 is a DNA binding protein associated with Parkinson's disease. We report that SATB1 prevents cellular senescence in post-mitotic dopaminergic neurons. Loss of SATB1 causes activation of a cellular senescence transcriptional program in dopamine neurons both in human stem cell-derived dopaminergic neurons and in mice. We observed phenotypes that are central to cellular senescence in SATB1 knockout dopamine neurons in vitro and in vivo. Moreover, we found that SATB1 directly represses expression of the pro-senescence factor p21 in dopaminergic neurons. Our data implicate senescence of dopamine neurons as a contributing factor in the pathology of Parkinson's disease.

Iron is an essential micronutrient for nearly all organisms, but excessive iron can lead to the formation of cytotoxic reactive oxygen species. Therefore, iron acquisition and homeostasis must be tightly regulated. Plants have evolved complex mechanisms to optimize their use of iron, which is one of the most limiting nutrients in the soil. In particular, transcriptional regulation is vital for regulating iron in plants, and much work has revealed the role of transcription factors on this front. Our study adds novel insights to the transcriptional regulation of iron homeostasis in plants by showing that chromatin remodeling via histone 3 lysine 27 trimethylation (H3K27me3) modulates the expression of FIT-dependent genes under iron deficiency. We provide evidence that FIT-dependent iron acquisition genes, IRT1 and FRO2, as well as FIT itself are direct targets of PRC2-mediated H3K27me3. In the clf mutant, which lacks the predominant H3K27 tri-methyltransferase, induction of FIT, FRO2, IRT1, and other FIT-regulated genes in roots is significantly higher under iron deficient conditions than in wild type. Furthermore, we observe that clf mutants are more tolerant to iron deficiency than wild type, indicating that gene expression levels appear to be limiting the plants ability to access iron. We propose that H3K27me3 attenuates the induction of FIT-target genes under iron deficiency and hypothesize that this may serve as a mechanism to restrict the maximum level of induction of iron acquisition genes to prevent iron overload.

For adaptive behavior, an organism must identify and assign subjective value to salient sensory information, but what stimuli are salient could change depending upon the local features of the environment. Insects such as fruit flies (Drosophila), for example, rely on olfactory cues to locate food and oviposition sites. But not all Drosophila species find the same stimuli to be salient: for example, four geographically isolated populations of Drosophila mojavensis, which feed and oviposit on necrotic cacti, show olfactory-driven behavioral preferences for host cacti specific to the local environment of each population 1, 2. We wondered whether visual features specific to certain environments could drive divergent visuomotor responses. We compared the visuomotor reflexes of D. melanogaster, a cosmopolitan generalist found in moderately dense visual environments, with D. mojavensis, a cactophilic specialist found in comparatively sparse visual landscapes. We found that, like D. melanogaster, D. mojavensis steer towards long vertical stripes, such as landscape features [3], but in contrast to D. melanogaster’s aversion to small objects [3], D. mojavensis find small objects attractive or of neutral value.