Toxic PR Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Target LC Domain Polymers.

Two complementary approaches were used in search of the intracellular targets of the toxic PR poly-dipeptide encoded by the repeat sequences expanded in the C9orf72 form of amyotrophic lateral sclerosis. The top categories of PRn-bound proteins include constituents of non-membrane invested cellular organelles and intermediate filaments. PRn targets are enriched for the inclusion of low complexity (LC) sequences. Evidence is presented indicating that LC sequences represent the direct target of PRn binding and that interaction between the PRn poly-dipeptide and LC domains is polymer-dependent. These studies indicate that PRn-mediated toxicity may result from broad impediments to the dynamics of cell structure and information flow from gene to message to protein.

Employing core regulatory circuits to define cell identity.

The interplay between extrinsic signaling and downstream gene networks controls the establishment of cell identity during development and its maintenance in adult life. Advances in next-generation sequencing and single-cell technologies have revealed additional layers of complexity in cell identity. Here, we review our current understanding of transcription factor (TF) networks as key determinants of cell identity. We discuss the concept of the core regulatory circuit as a set of TFs and interacting factors that together define the gene expression profile of the cell. We propose the core regulatory circuit as a comprehensive conceptual framework for defining cellular identity and discuss its connections to cell function in different contexts.

Single-cell transcriptomic signatures and gene regulatory networks modulated by Wls in mammalian midline facial formation and clefts.

Formation of highly unique and complex facial structures is controlled by genetic programs that are responsible for the precise coordination of three-dimensional tissue morphogenesis. However, the underlying mechanisms governing these processes remain poorly understood. We combined mouse genetic and genomic approaches to define the mechanisms underlying normal and defective midfacial morphogenesis. Conditional inactivation of the Wnt secretion protein Wls in Pax3-expressing lineage cells disrupted frontonasal primordial patterning, cell survival and directional outgrowth, resulting in altered facial structures, including midfacial hypoplasia and midline facial clefts. Single-cell RNA sequencing revealed unique transcriptomic atlases of mesenchymal subpopulations in the midfacial primordia, which are disrupted in the conditional Wls mutants. Differentially expressed genes and cis-regulatory sequence analyses uncovered that Wls modulates and integrates a core gene regulatory network, consisting of key midfacial regulatory transcription factors (including Msx1, Pax3 and Pax7) and their downstream targets (including Wnt, Shh, Tgfß and retinoic acid signaling components), in a mesenchymal subpopulation of the medial nasal prominences that is responsible for midline facial formation and fusion. These results reveal fundamental mechanisms underlying mammalian midfacial morphogenesis and related defects at single-cell resolution.

Single-cell causal network inferred by cross-mapping entropy.

Gene regulatory networks (GRNs) reveal the complex molecular interactions that govern cell state. However, it is challenging for identifying causal relations among genes due to noisy data and molecular nonlinearity. Here, we propose a novel causal criterion, neighbor cross-mapping entropy (NME), for inferring GRNs from both steady data and time-series data. NME is designed to quantify 'continuous causality' or functional dependency from one variable to another based on their function continuity with varying neighbor sizes. NME shows superior performance on benchmark datasets, comparing with existing methods. By applying to scRNA-seq datasets, NME not only reliably inferred GRNs for cell types but also identified cell states. Based on the inferred GRNs and further their activity matrices, NME showed better performance in single-cell clustering and downstream analyses. In summary, based on continuous causality, NME provides a powerful tool in inferring causal regulations of GRNs between genes from scRNA-seq data, which is further exploited to identify novel cell types/states and predict cell type-specific network modules.

MR-GGI: accurate inference of gene-gene interactions using Mendelian randomization.

BACKGROUND: Researchers have long studied the regulatory processes of genes to uncover their functions. Gene regulatory network analysis is one of the popular approaches for understanding these processes, requiring accurate identification of interactions among the genes to establish the gene regulatory network. Advances in genome-wide association studies and expression quantitative trait loci studies have led to a wealth of genomic data, facilitating more accurate inference of gene-gene interactions. However, unknown confounding factors may influence these interactions, making their interpretation complicated. Mendelian randomization (MR) has emerged as a valuable tool for causal inference in genetics, addressing confounding effects by estimating causal relationships using instrumental variables. In this paper, we propose a new statistical method, MR-GGI, for accurately inferring gene-gene interactions using Mendelian randomization. RESULTS: MR-GGI applies one gene as the exposure and another as the outcome, using causal cis-single-nucleotide polymorphisms as instrumental variables in the inverse-variance weighted MR model. Through simulations, we have demonstrated MR-GGI's ability to control type 1 error and maintain statistical power despite confounding effects. MR-GGI performed the best when compared to other methods using the F1 score on the DREAM5 dataset. Additionally, when applied to yeast genomic data, MR-GGI successfully identified six clusters. Through gene ontology analysis, we have confirmed that each cluster in our study performs distinct functional roles by gathering genes with specific functions. CONCLUSION: These findings demonstrate that MR-GGI accurately inferences gene-gene interactions despite the confounding effects in real biological environments.

Multiomics Evaluation of Human iPSCs and iPSC-Derived Neurons.

Human induced pluripotent stem cells (iPSCs) can be differentiated into neurons, providing living human neurons to model brain diseases. However, it is unclear how different types of molecules work together to regulate stem cell and neuron biology in healthy and disease states. In this study, we conducted integrated proteomics, lipidomics, and metabolomics analyses with confident identification, accurate quantification, and reproducible measurements to compare the molecular profiles of human iPSCs and iPSC-derived neurons. Proteins, lipids, and metabolites related to mitosis, DNA replication, pluripotency, glycosphingolipids, and energy metabolism were highly enriched in iPSCs, whereas synaptic proteins, neurotransmitters, polyunsaturated fatty acids, cardiolipins, and axon guidance pathways were highly enriched in neurons. Mutations in the GRN gene lead to the deficiency of the progranulin (PGRN) protein, which has been associated with various neurodegenerative diseases. Using this multiomics platform, we evaluated the impact of PGRN deficiency on iPSCs and neurons at the whole-cell level. Proteomics, lipidomics, and metabolomics analyses implicated PGRN's roles in neuroinflammation, purine metabolism, and neurite outgrowth, revealing commonly altered pathways related to neuron projection, synaptic dysfunction, and brain metabolism. Multiomics data sets also pointed toward the same hypothesis that neurons seem to be more susceptible to PGRN loss compared to iPSCs, consistent with the neurological symptoms and cognitive impairment from patients carrying inherited GRN mutations.

A genome-wide computational approach to define microRNA-Polycomb/trithorax gene regulatory circuits in Drosophila.

Characterization of gene regulatory networks is fundamental to understanding homeostatic development. This process can be simplified by analyzing relatively simple genomes such as the genome of Drosophila melanogaster. In this work we have developed a computational framework in Drosophila to explore for the presence of gene regulatory circuits between two large groups of transcriptional regulators: the epigenetic group of the Polycomb/trithorax (PcG/trxG) proteins and the microRNAs (miRNAs). We have searched genome-wide for miRNA targets in PcG/trxG transcripts as well as for Polycomb Response Elements (PREs) in miRNA genes. Our results show that 10% of the analyzed miRNAs could be controlling PcG/trxG gene expression, while 40% of those miRNAs are putatively controlled by the selected set of PcG/trxG proteins. The integration of these analyses has resulted in the predicted existence of 3 classes of miRNA-PcG/trxG crosstalk interactions that define potential regulatory circuits. In the first class, miRNA-PcG circuits are defined by miRNAs that reciprocally crosstalk with PcG. In the second, miRNA-trxG circuits are defined by miRNAs that reciprocally crosstalk with trxG. In the third class, miRNA-PcG/trxG shared circuits are defined by miRNAs that crosstalk with both PcG and trxG regulators. These putative regulatory circuits may uncover a novel mechanism in Drosophila for the control of PcG/trxG and miRNAs levels of expression. The computational framework developed here for Drosophila melanogaster can serve as a model case for similar analyses in other species. Moreover, our work provides, for the first time, a new and useful resource for the Drosophila community to consult prior to experimental studies investigating the epigenetic regulatory networks of miRNA-PcG/trxG mediated gene expression.

Male-pronuclei-specific granulin facilitates somatic cells reprogramming via mitigating excessive cell proliferation and enhancing lysosomal function.

Critical reprogramming factors resided predominantly in the oocyte or male pronucleus can enhance the efficiency or the quality of induced pluripotent stem cells (iPSCs) induction. However, few reprogramming factors exist in the male pronucleus had been verified. Here, we demonstrated that granulin (Grn), a factor enriched specifically in male pronucleus, can significantly improve the generation of iPSCs from mouse fibroblasts. Grn is highly expressed on Day 1, Day 3, Day 14 of reprogramming induced by four Yamanaka factors and functions at the initial stage of reprogramming. Transcriptome analysis indicates that Grn can promote the expression of lysosome-related genes, while inhibit the expression of genes involved in DNA replication and cell cycle at the early reprogramming stage. Further verification determined that Grn suppressed cell proliferation due to the arrest of cell cycle at G2/M phase. Moreover, ectopic Grn can enhance the lysosomes abundance and rescue the efficiency reduction of reprogramming resulted from lysosomal protease inhibition. Taken together, we conclude that Grn serves as an activator for somatic cell reprogramming through mitigating cell hyperproliferation and promoting the function of lysosomes.

Brachyury controls Ciona notochord fate as part of a feed-forward network.

The notochord is a defining feature of the chordates. The transcription factor Brachyury (Bra) is a key regulator of notochord fate but here we show that it is not a unitary master regulator in the model chordate Ciona Ectopic Bra expression only partially reprograms other cell types to a notochord-like transcriptional profile and a subset of notochord-enriched genes is unaffected by CRISPR Bra disruption. We identify Foxa.a and Mnx as potential co-regulators, and find that combinatorial cocktails are more effective at reprogramming other cell types than Bra alone. We reassess the network relationships between Bra, Foxa.a and other components of the notochord gene regulatory network, and find that Foxa.a expression in the notochord is regulated by vegetal FGF signaling. It is a direct activator of Bra expression and has a binding motif that is significantly enriched in the regulatory regions of notochord-enriched genes. These and other results indicate that Bra and Foxa.a act together in a regulatory network dominated by positive feed-forward interactions, with neither being a classically defined master regulator.

Cis-regulatory dissection of cone development reveals a broad role for Otx2 and Oc transcription factors.

The vertebrate retina is generated by retinal progenitor cells (RPCs), which produce >100 cell types. Although some RPCs produce many cell types, other RPCs produce restricted types of daughter cells, such as a cone photoreceptor and a horizontal cell (HC). We used genome-wide assays of chromatin structure to compare the profiles of a restricted cone/HC RPC and those of other RPCs in chicks. These data nominated regions of regulatory activity, which were tested in tissue, leading to the identification of many cis-regulatory modules (CRMs) active in cone/HC RPCs and developing cones. Two transcription factors, Otx2 and Oc1, were found to bind to many of these CRMs, including those near genes important for cone development and function, and their binding sites were required for activity. We also found that Otx2 has a predicted autoregulatory CRM. These results suggest that Otx2, Oc1 and possibly other Onecut proteins have a broad role in coordinating cone development and function. The many newly discovered CRMs for cones are potentially useful reagents for gene therapy of cone diseases.

Neurodevelopmental effects of genetic frontotemporal dementia in young adult mutation carriers.

While frontotemporal dementia has been considered a neurodegenerative disease that starts in mid-life or later, it is now clearly established that cortical and subcortical volume loss is observed more than a decade prior to symptom onset and progresses with ageing. To test the hypothesis that genetic mutations causing frontotemporal dementia have neurodevelopmental consequences, we examined the youngest adults in the GENFI cohort of pre-symptomatic frontotemporal dementia mutation carriers who are between 19 and 30 years of age. Structural brain differences and improved performance on some cognitive tests were found for MAPT and GRN mutation carriers relative to familial non-carriers, while smaller volumes were observed in C9orf72 repeat expansion carriers at a mean age of 26 years. The detection of such early differences supports potential advantageous neurodevelopmental consequences of some frontotemporal dementia-causing genetic mutations. These results have implications for the design of therapeutic interventions for frontotemporal dementia. Future studies at younger ages are needed to identify specific early pathophysiologic or compensatory processes that occur during the neurodevelopmental period.

Galectin-3 is upregulated in frontotemporal dementia patients with subtype specificity.

INTRODUCTION: Neuroinflammation is a major contributor to the progression of frontotemporal dementia (FTD). Galectin-3 (Gal-3), a microglial activation regulator, holds promise as a therapeutic target and potential biomarker. Our study aimed to investigate Gal-3 levels in patients with FTD and assess its diagnostic potential. METHODS: We examined Gal-3 levels in brain, serum, and cerebrospinal fluid (CSF) samples of patients with FTD and controls. Multiple linear regressions between Gal-3 levels and other FTD markers were explored. RESULTS: Gal-3 levels were increased significantly in patients with FTD, mainly across brain tissue and CSF, compared to controls. Remarkably, Gal-3 levels were higher in cases with tau pathology than TAR-DNA Binding Protein 43 (TDP-43) pathology. Only MAPT mutation carriers displayed increased Gal-3 levels in CSF samples, which correlated with total tau and 14-3-3. DISCUSSION: Our findings underscore the potential of Gal-3 as a diagnostic marker for FTD, particularly in MAPT cases, and highlights the relation of Gal-3 with neuronal injury markers.

Unveiling New Genetic Variants Associated with Age at Onset in Alzheimer's Disease and Frontotemporal Lobar Degeneration Due to C9orf72 Repeat Expansions.

Alzheimer's disease (AD) and Frontotemporal lobar degeneration (FTLD) represent the most common forms of neurodegenerative dementias with a highly phenotypic variability. Herein, we investigated the role of genetic variants related to the immune system and inflammation as genetic modulators in AD and related dementias. In patients with sporadic AD/FTLD (n = 300) and GRN/C9orf72 mutation carriers (n = 80), we performed a targeted sequencing of 50 genes belonging to the immune system and inflammation, selected based on their high expression in brain regions and low tolerance to genetic variation. The linear regression analyses revealed two genetic variants: (i) the rs1049296 in the transferrin (TF) gene, shown to be significantly associated with age at onset in the sporadic AD group, anticipating the disease onset of 4 years for each SNP allele with respect to the wild-type allele, and (ii) the rs7550295 in the calsyntenin-1 (CLSTN1) gene, which was significantly associated with age at onset in the C9orf72 group, delaying the disease onset of 17 years in patients carrying the SNP allele. In conclusion, our data support the role of genetic variants in iron metabolism (TF) and in the modulation of the calcium signalling/axonal anterograde transport of vesicles (CLSTN1) as genetic modulators in AD and FTLD due to C9orf72 expansions.

Mutational Landscape of Alzheimer's Disease and Frontotemporal Dementia: Regional Variances in Northern, Central, and Southern Italy.

Alzheimer's Disease (AD) and Frontotemporal Dementia (FTD) are the two major neurodegenerative diseases with distinct clinical and neuropathological profiles. The aim of this report is to conduct a population-based investigation in well-characterized APP, PSEN1, PSEN2, MAPT, GRN, and C9orf72 mutation carriers/pedigrees from the north, the center, and the south of Italy. We retrospectively analyzed the data of 467 Italian individuals. We identified 21 different GRN mutations, 20 PSEN1, 11 MAPT, 9 PSEN2, and 4 APP. Moreover, we observed geographical variability in mutation frequencies by looking at each cohort of participants, and we observed a significant difference in age at onset among the genetic groups. Our study provides evidence that age at onset is influenced by the genetic group. Further work in identifying both genetic and environmental factors that modify the phenotypes in all groups is needed. Our study reveals Italian regional differences among the most relevant AD/FTD causative genes and emphasizes how the collaborative studies in rare diseases can provide new insights to expand knowledge on genetic/epigenetic modulators of age at onset.

Gene expression variability across cells and species shapes the relationship between renal resident macrophages and infiltrated macrophages.

BACKGROUND: Two main subclasses of macrophages are found in almost all solid tissues: embryo-derived resident tissue macrophages and bone marrow-derived infiltrated macrophages. These macrophage subtypes show transcriptional and functional divergence, and the programs that have shaped the evolution of renal macrophages and related signaling pathways remain poorly understood. To clarify these processes, we performed data analysis based on single-cell transcriptional profiling of renal tissue-resident and infiltrated macrophages in human, mouse and rat. RESULTS: In this study, we (i) characterized the transcriptional divergence among species and (ii) illustrated variability in expression among cells of each subtype and (iii) compared the gene regulation network and (iv) ligand-receptor pairs in human and mouse. Using single-cell transcriptomics, we mapped the promoter architecture during homeostasis. CONCLUSIONS: Transcriptionally divergent genes, such as the differentially TF-encoding genes expressed in resident and infiltrated macrophages across the three species, vary among cells and include distinct promoter structures. The gene regulatory network in infiltrated macrophages shows comparatively better species-wide consistency than resident macrophages. The conserved transcriptional gene regulatory network in infiltrated macrophages among species is uniquely enriched in pathways related to kinases, and TFs associated with largely conserved regulons among species are uniquely enriched in kinase-related pathways.

To stripe or not to stripe: the origin of a novel foliar pigmentation pattern in monkeyflowers (Mimulus).

The origin of phenotypic novelty is one of the most challenging problems in evolutionary biology. Although genetic regulatory network rewiring or co-option has been widely recognised as a major contributor, in most cases how such genetic rewiring/co-option happens is completely unknown. We have studied a novel foliar pigmentation pattern that evolved recently in the monkeyflower species Mimulus verbenaceus. Through genome-wide association tests using wild-collected samples, experimental crosses of laboratory inbred lines, gene expression analyses, and functional assays, we identified an anthocyanin-activating R2R3-MYB gene, STRIPY, as the causal gene triggering the emergence of the discrete, mediolateral anthocyanin stripe in the M. verbenaceus leaf. Chemical mutagenesis revealed the existence of upstream activators and repressors that form a 'hidden' prepattern along the leaf proximodistal axis, potentiating the unique expression pattern of STRIPY. Population genomics analyses did not reveal signatures of positive selection, indicating that nonadaptive processes may be responsible for the establishment of this novel trait in the wild. This study demonstrates that the origin of phenotypic novelty requires at least two separate phases, potentiation and actualisation. The foliar stripe pattern of M. verbenaceus provides an excellent platform to probe the molecular details of both processes in future studies.

Accumulation of TMEM106B C-terminal fragments in neurodegenerative disease and aging.

Several studies using cryogenic electron microscopy (cryo-EM) techniques recently reported the isolation and characterization of novel protein filaments, composed of a C-terminal fragment (CTF) of the endolysosomal transmembrane protein 106B (TMEM106B), from human post-mortem brain tissue with various neurodegenerative conditions and normal aging. Genetic variation in TMEM106B is known to influence the risk and presentation of several neurodegenerative diseases, especially frontotemporal dementia (FTD) caused by mutations in the progranulin gene (GRN). To further elucidate the significance of TMEM106B CTF, we performed immunohistochemistry with antibodies directed against epitopes within the filament-forming C-terminal region of TMEM106B. Accumulation of TMEM106B C-terminal immunoreactive (TMEM-ir) material was a common finding in all the conditions evaluated, including frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP), Alzheimer's disease, tauopathies, synucleinopathies and neurologically normal aging. TMEM-ir material was present in a wide range of brain cell types and in a broad neuroanatomical distribution; however, there was no co-localization of TMEM-ir material with other neurodegenerative proteins in cellular inclusions. In most conditions, the presence and abundance of TMEM-ir aggregates correlated strongly with patient age and showed only a weak correlation with the TMEM106B haplotype or the primary pathological diagnosis. However, all patients with FTD caused by GRN mutations were found to have high levels of TMEM-ir material, including several who were relatively young (< 60 years). These findings suggest that the accumulation of TMEM106B CTF is a common age-related phenomenon, which may reflect lysosomal dysfunction. Although its significance in most neurodegenerative conditions remains uncertain, the consistent finding of extensive TMEM-ir material in cases of FTLD-TDP with GRN mutations further supports a pathomechanistic role of TMEM106B and lysosomal dysfunction in this specific disease population.

Shared brain transcriptomic signature in TDP-43 type A FTLD patients with or without GRN mutations.

Frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) is a complex heterogeneous neurodegenerative disorder for which mechanisms are poorly understood. To explore transcriptional changes underlying FTLD-TDP, we performed RNA-sequencing on 66 genetically unexplained FTLD-TDP patients, 24 FTLD-TDP patients with GRN mutations and 24 control participants. Using principal component analysis, hierarchical clustering, differential expression and coexpression network analyses, we showed that GRN mutation carriers and FTLD-TDP-A patients without a known mutation shared a common transcriptional signature that is independent of GRN loss-of-function. After combining both groups, differential expression as compared to the control group and coexpression analyses revealed alteration of processes related to immune response, synaptic transmission, RNA metabolism, angiogenesis and vesicle-mediated transport. Deconvolution of the data highlighted strong cellular alterations that were similar in FTLD-TDP-A and GRN mutation carriers with NSF as a potentially important player in both groups. We propose several potentially druggable pathways such as the GABAergic, GDNF and sphingolipid pathways. Our findings underline new disease mechanisms and strongly suggest that affected pathways in GRN mutation carriers extend beyond GRN and contribute to genetically unexplained forms of FTLD-TDP-A.

A Systematic Review of the Genetics and Pathology of Psychosis in Frontotemporal Dementia.

OBJECTIVES: Frontotemporal dementia (FTD) patients frequently present with psychosis, which complicates diagnosis and management. In this study, we aim to examine the relationship between psychosis and the most common genetic mutations predisposing to FTD, and in the different pathological subtypes of FTD. DESIGN: We conducted a systematic review, searching the literature up to December 2022, and reviewed 50 articles that met our inclusion criteria. From the reviewed articles, we extracted and summarized data regarding the frequency of psychosis and patient characteristics in each major genetic and pathological subtype of FTD. RESULTS: Among FTD patients with confirmed genetic mutations or pathological diagnosss, the frequency of psychosis was 24.2%. Among the genetic mutation carriers, C9orf72 mutation carriers had the highest frequency of psychosis (31.4%), whereas GRN (15.0%) and MAPT (9.2%) mutation carriers had lower frequencies of psychosis. MAPT mutation carriers notably developed psychosis at a younger age compared to other genetic groups. The most common psychotic symptoms were delusions among C9orf72 carriers and visual hallucinations among GRN mutation carriers. Among the pathological subtypes, 30% of patients with FUS pathology, 25.3% of patients with TDP-43 pathology, and 16.4% of patients with tau pathology developed psychosis. In the TDP-43 group, subtype B pathology was the most common subtype reported in association with psychosis. CONCLUSION: Our systematic review suggests a high frequency of psychosis in specific subgroups of FTD patients. Further studies are required to understand the structural and biological underpinnings of psychosis in FTD.

Pathogenic variants in the Longitudinal Early-onset Alzheimer's Disease Study cohort.

INTRODUCTION: One goal of the Longitudinal Early-onset Alzheimer's Disease Study (LEADS) is to investigate the genetic etiology of early onset (40-64 years) cognitive impairment. Toward this goal, LEADS participants are screened for known pathogenic variants. METHODS: LEADS amyloid-positive early-onset Alzheimer's disease (EOAD) or negative early-onset non-AD (EOnonAD) cases were whole exome sequenced (N = 299). Pathogenic variant frequency in APP, PSEN1, PSEN2, GRN, MAPT, and C9ORF72 was assessed for EOAD and EOnonAD. Gene burden testing was performed in cases compared to similar-age cognitively normal controls in the Parkinson's Progression Markers Initiative (PPMI) study. RESULTS: Previously reported pathogenic variants in the six genes were identified in 1.35% of EOAD (3/223) and 6.58% of EOnonAD (5/76). No genes showed enrichment for carriers of rare functional variants in LEADS cases. DISCUSSION: Results suggest that LEADS is enriched for novel genetic causative variants, as previously reported variants are not observed in most cases. HIGHLIGHTS: Sequencing identified eight cognitively impaired pathogenic variant carriers. Pathogenic variants were identified in PSEN1, GRN, MAPT, and C9ORF72. Rare variants were not enriched in APP, PSEN1/2, GRN, and MAPT. The Longitudinal Early-onset Alzheimer's Disease Study (LEADS) is a key resource for early-onset Alzheimer's genetic research.