Biology subjects

Friedrich, C.

Publications and source records attributed to Friedrich, C..

14 recordsLinked to original sources

bioRxiv2026

Scalable proteomics with multiDIA resolves proteoforms in neurodegenerative disease

Deep proteome characterization has traditionally required multidimensional chromatographic fractionation, long acquisition times and large sample amounts, thus limiting scalability. Here, we introduce multiDIA, which combines independent digestions with multiple proteases and data-independent acquisition (DIA) to increase sequence coverage without extensive chromatographic fractionation. By generating complementary and overlapping peptide representations of proteins, multiDIA identifies about 270,000 distinct peptides per sample, including 3,649 phosphosites. This is more than twice the coverage of unfractionated workflows and exceeds the coverage of multidimensional fractionation workflows that use 10-fold more material and time. We demonstrate the approach in Parkinson's disease (PD) brain samples, showing that multiDIA substantially expands sequence coverage while retaining the sensitivity, reproducibility, and scalability of DIA. This enabled the identification of disease-associated proteoform changes undetectable by single-protease DIA and the quantification of many disease-relevant post-translational modifications. MultiDIA thus enables deep, high-confidence proteome characterization from limited sample quantities without the throughput and sensitivity constraints of multidimensional fractionation.

systems biology↗

bioRxiv2026

A single-nucleus multiomic and spatial atlas of gene regulation in human spermatogenesis

Gametogenesis, the production of oocytes and sperm, ensures the faithful transmission of genetic material to the next generation. In males, spermatogenesis occurs within the testis, where germ cells progress through a highly ordered developmental programme in close association with supporting somatic cells. Defects in this process cause infertility, yet the gene regulatory mechanisms coordinating normal and dysfunctional human spermatogenesis remain incompletely defined. Here we generate a single-nucleus multiomic and spatial atlas of human spermatogenesis by profiling chromatin accessibility and gene expression in the same nuclei and integrating these data with spatial transcriptomics of intact testicular tissue. We infer high-confidence gene regulatory networks that resolve stage-specific activity of known and novel candidate regulators across germline and somatic compartments, and map spatially restricted signalling interactions within the seminiferous tubule niche. Integration with infertility-associated genetic variation links non-coding risk loci to candidate enhancers and target genes in cell-type-specific regulatory contexts. Finally, profiling clinical cryptozoospermia samples as in vivo perturbations supports the ability of this network to capture downstream transcriptional consequences of disease-associated regulatory disruption. Together, these data provide a spatially resolved regulatory framework for human spermatogenesis and a foundation for interpreting the molecular basis of male infertility.

genomics↗

bioRxiv2025

STAT3 operates as an inflammation-dependent transcriptional switch

Signal transducer and activator of transcription 3 (STAT3) is a key regulator of immune cell function, but its role in lymphocytes remains incompletely understood. Here, we show that STAT3 has a context-dependent function in antiviral natural killer (NK) cells, either promoting or impairing adaptive NK cell responses dependent on the level of inflammation. STAT3 is recruited to distinct genomic sites under homeostatic versus inflammatory environments, where it drives different transcriptional programs. Through this re-localization, STAT3 regulates downstream transcription factors MYB and BLIMP-1 in an inflammation-dependent manner to shape NK cell differentiation under homeostasis and during infection. Thus, STAT3 acts as a transcriptional switch that integrates cytokine signals to control lymphocyte adaptation to different environments. This mechanism highlights how therapeutic interventions targeting STAT3 can result in different outcomes depending on the degree of inflammation. HIGHLIGHTS- STAT3 exerts a context-dependent role on adaptive NK cells during viral infection - STAT3 modulates IL-15 signaling by competing with STAT5 and regulating MYB - Homeostatic versus inflammatory cytokines relocate STAT3 to distinct genomic sites - Downstream transcription factors MYB and BLIMP-1 govern STAT3-dependent differentiation

immunology↗

bioRxiv2025

Pro-inflammatory role of granzyme K producing bystander CD8+ T cells in acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous group of blood malignancies with a 5-year survival rate below 30%, highlighting the urgent need for more effective therapeutic strategies. T cell-based immunotherapies have demonstrated remarkable success in solid tumors, yet the role of CD8+ T cells in AML remains unclear. In this study, we analyzed the composition, antigenic specificity, and function of CD8+ T cells in paired blood and bone marrow samples from AML patients. While we did not identify exhausted CD8+ T cells as seen in solid tumors, we observed a distinct population of functional CD69+ CD8+ T cells specifically enriched in the bone marrow. These cells primarily recognized non-tumor antigens, including epitopes derived from Epstein-Barr virus (EBV) and cytomegalovirus (CMV). Notably, this bystander CD8+ T cell population showed high expression of Granzyme K, a cytokine found in the bone marrow of AML patients. Granzyme K did not induce leukemic cell death but instead promoted the secretion of IL-8, a pro-inflammatory cytokine known to play a detrimental role in AML pathology. Rather than mounting an anti-tumor response, these CD8+ T cells contribute to a pro-inflammatory environment that may exacerbate AML progression and severity. These findings provide a rationale for exploring therapeutic strategies aimed at inhibiting pro-inflammatory CD8+ T cells and targeting Granzyme K activity in association with actual therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/669682v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1fc9798org.highwire.dtl.DTLVardef@4491b1org.highwire.dtl.DTLVardef@1b8feadorg.highwire.dtl.DTLVardef@58515d_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Ubiquitin-Proteasome System Dysregulation in Alzheimer's Disease Impacts Protein Abundance

Alzheimers disease (AD) is a relentlessly progressive, fatal neurodegenerative disorder that results in widespread protein dysfunctions. However, the full extent of aberrant proteomic changes in AD and their impact remains unknown, in part, because of the challenges of comprehensively measuring the proteome. Here, we used plexDIA, an approach that provides deep proteomic coverage and high throughput by parallelizing the acquisition of peptides and samples, to characterize proteomic changes in AD. Using human dorsolateral prefrontal cortex tissue, we identified 281 differentially abundant proteins in AD. By systematically analyzing compartment and protein complex-specific shifts in protein abundance, we identified an AD-specific decrease in levels of the 20S proteasome, the catalytic core of the cells primary protein degradation pathway. This alteration was accompanied by widespread decreases in proteasome subunit stoichiometries. Many proteasome substrate proteins were negatively correlated with 20S levels and increased in AD, suggesting that reduced 20S levels leads to abnormal protein accumulation. By analyzing proteins increased in AD, we identify key properties of such proteins: They have fast degradation rates, they contain signal sequences that allow them to be targeted for proteasomal degradation, and they are targeted by quality control pathways that recognize mislocalized proteins. Changes in these gene products at the protein and mRNA levels were highly discordant, providing additional evidence for increased protein abundance driven by impaired clearance. We also identified coherent sets of ubiquitin system enzymes, proteins that target substrates for proteasomal degradation, whose levels robustly discriminate AD from non-AD samples. One subset exhibited consistent increases in AD, while another, which contained the tau E3 ligase Cul5, exhibited consistent decreases, revealing complex changes in the ubiquitin system in AD. Taken together, our results suggest that decreased ubiquitin-proteasome system capacity and impaired clearance of short-lived and mislocalized proteins contribute substantially to proteopathic burden in AD.

neuroscience↗

bioRxiv2025

PSMtags improve peptide sequencing and throughput in sensitive proteomics

Mass spectrometry-based proteomics enables comprehensive characterization of protein abundance, function, and interactions. Label-free approaches are simple to implement but challenging to scale to thousands of samples per day. Multiplexed techniques, such as plexDIA, can address these limitations but remain restricted by the lack of mass tags optimized for data-independent acquisition (DIA) workflows. Here, we present a systematic approach screening a library of 576 compounds that identifies several small molecules that, when conjugated to peptides, improve their detection and sequence identification by mass spectrometry. The lead molecule, PSMtag, substantially increases the detection of fragment b-ions, which increases the confidence of sequence identification and enhances de novo sequencing. PSMtags allow 9-plexDIA, using only stable isotopes of carbon, oxygen and nitrogen. As a result, it allows simultaneously increasing proteome coverage and sample throughput for plexDIA workflows without compromising quantitative accuracy. We demonstrate 240 samples-per-day with 9-plexDIA, while acquiring 28,359 protein data points in the same time label-free methods acquire 4,340. Our approach constitutes an expandable framework for designing mass tags to overcome existing limitations in multiplexed proteomics and provides plexDIA reagents capable of analyzing over 1,000 samples per day when using 10 minute runs. By facilitating higher throughput and improved identification, this innovation holds significant potential for accelerating proteomic studies across diverse biological and clinical applications.

bioengineering↗

bioRxiv2024

Humanized in vivo bone marrow models orchestrate multi-lineage human hematopoietic cell development

Hematopoiesis develops in the bone marrow (BM) where multiple interactions regulate differentiation and preservation of hematopoietic stem/progenitor cells (HSPCs). Although murine BM has been extensively analyzed, the human BM microenvironment remains less understood. Immune-deficient murine models have enabled the analysis of molecular and cellular regulation of human HSPCs, which remains limited as human hematopoietic cells develop in xenogenic microenvironments. In this study, we thoroughly characterized a humanized (h) in vivo BM model, based on mesenchymal stromal cell (MSC) differentiation (called hOssicles (hOss)), and hematopoietic cell compartments generated 3 months post-transplant of CD34+ cells using single-cell RNA sequencing and cellular barcoding. Serial isolation of MSCs and HSPCs from hOss and transplant experiments revealed the dynamic nature of these hBM niches. hOss altered human hematopoietic development by modulating myeloid/lymphoid cell production and HSPC levels. Clonal tracking highlighted hematopoietic cell cross-talks between the murine BM and hOss, indicating the multipotent or more restricted lineage origin of human hematopoiesis shared in the BM sites.

cell biology↗

bioRxiv2023

AXDND1 is required to balance spermatogonial commitment and for sperm tail formation in mice and humans

Dynein complexes are large, multi-unit assemblies involved in many biological processes including male fertility via their critical roles in protein transport and axoneme motility. Previously we identified a pathogenic variant in the dynein gene AXDND1 in an infertile man. Subsequently we identified an additional four potentially compound heterozygous variants of unknown significance in AXDND1 in two additional infertile men. We thus tested the role of AXDND1 in mammalian male fertility by generating a knockout mouse model. Axdnd1-/- males were sterile at all ages but could undergo one round of histologically complete spermatogenesis. Subsequently, a progressive imbalance of spermatogonial commitment to spermatogenesis over self-renewal occurred, ultimately leading to catastrophic germ cell loss, loss of blood-testis barrier patency and immune cell infiltration. Sperm produced during the first wave of spermatogenesis were immotile due to abnormal axoneme structure, including the presence of ectopic vesicles and abnormalities in outer dense fibres and microtubule doublet structures. Sperm output was additionally compromised by a severe spermiation defect and abnormal sperm individualisation. Collectively, our data highlight the essential roles of AXDND1 as a regulator of spermatogonial commitment to spermatogenesis and during the processes of spermiogenesis where it is essential for sperm tail development, release and motility.

cell biology↗

bioRxiv2022

A novel lineage of RORγt+Aire+ antigen presenting cells promotes peripheral generation of intestinal regulatory T cells and tolerance during early life

Establishing and maintaining tolerance to self- or innocuous foreign antigens is vital for preservation of organismal health. Within the thymus, medullary thymic epithelial cells (mTECs) expressing AutoImmune Regulator, Aire, play a critical role in self-tolerance through deletion of autoreactive T cells and promotion of thymic regulatory T (Treg) cell development. Within weeks of birth, a separate wave of Treg cell differentiation occurs in the periphery, upon exposure to dietary and commensal microbiota derived antigens, yet the cell types responsible for the generation of peripheral Treg (pTreg) cells are not known. Here we identified a new class of ROR{gamma}t+ antigen-presenting cells (APC), dubbed Thetis cells (TCs), with transcriptional features of both mTECs and dendritic cells (DCs), comprising 4 major sub-groups (TC I-IV). We uncovered a developmental wave of TCs within intestinal lymph nodes during a critical early life window, coincident with the wave of pTreg cell differentiation. While TC I and III expressed the signature mTEC nuclear factor Aire, TC IV lacked Aire expression and were enriched for molecules required for pTreg generation, including the TGF-{beta} activating integrin v{beta}8. Loss of either MHCII or Itgb8 expression by TCs led to a profound impairment in intestinal pTreg differentiation, with onset of intestinal inflammation. In contrast, MHCII expression by ROR{gamma}t+ group 3 innate lymphoid cells (ILC3) and classical DCs was neither sufficient nor required for pTreg generation, further implicating TCs as the tolerogenic ROR{gamma}t+ APC with an essential early life function. Our studies reveal parallel pathways for establishment of tolerance to self and foreign antigen within the thymus and periphery, marked by involvement of shared cellular and transcriptional programs.

immunology↗

bioRxiv2021

Hematopoietic differentiation is characterized by a transient peak of cell-to-cell gene expression variability in normal and pathological conditions.

Hematopoietic differentiation has been metaphorically represented as linear trajectories with discrete steps from hematopoietic stem cells to mature cells. While the transcriptional state of cells at the beginning or at the end of these trajectories are well described from bulk analysis, what happens in the intermediate states has remained elusive until the use of single cell approaches. Applying Shannon entropy to measure cell-to-cell variability among cells at the same stage of differentiation, we observed a transient peak of gene expression variability in all the hematopoietic differentiation pathways. Strikingly, genes with the highest entropy variation in a given differentiation pathway matched genes known as pathway-specific, whereas genes with the highest expression variation were common to all pathways. Finally, we showed that the level of cell-to-cell variation is increased in the most immature compartment of hematopoiesis in myelodysplastic syndromes. These data suggest that differentiation could be better conceptualized as a dynamical stochastic process with a transient stage of cellular indetermination.

cell biology↗

bioRxiv2021

A de novo paradigm for male infertility

IntroductionDe novo mutations (DNMs) are known to play a prominent role in sporadic disorders with reduced fitness1. We hypothesize that DNMs play an important role in male infertility and explain a significant fraction of the genetic causes of this understudied disorder. To test this hypothesis, we performed trio-based exome-sequencing in a unique cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare protein altering DNMs were classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of Loss-of-Function (LoF) DNMs in LoF-intolerant genes (p-value=1.00x10-5) as well as predicted pathogenic missense DNMs in missense-intolerant genes (p-value=5.01x10-4). One DNM gene identified, RBM5, is an essential regulator of male germ cell pre-mRNA splicing2. In a follow-up study, 5 rare pathogenic missense mutations affecting this gene were observed in a cohort of 2,279 infertile patients, with no such mutations found in a cohort of 5,784 fertile men (p-value=0.009). Our results provide the first evidence for the role of DNMs in severe male infertility and point to many new candidate genes affecting fertility.

genetics↗

bioRxiv2021

TRIM71 deficiency causes germ cell loss during mouse embryogenesis and promotes human male infertility

Mutations affecting the germline can result in infertility or the generation of germ cell tumors (GCT), highlighting the need to identify and characterize the genes controlling the complex molecular network orchestrating germ cell development. TRIM71 is a stem cell-specific factor essential for embryogenesis, and its expression has been reported in GCT and adult mouse testes. To investigate the role of TRIM71 in mammalian germ cell embryonic development, we generated a germline-specific conditional Trim71 knockout mouse (cKO) using the early primordial germ cell (PGC) marker Nanos3 as a Cre-recombinase driver. cKO mice are infertile, with male mice displaying a Sertoli cell-only (SCO) phenotype, which in humans is defined as a specific subtype of non-obstructive azoospermia characterized by the absence of developing germ cells in the testes seminiferous tubules. Infertility originates during embryogenesis, as the SCO phenotype was already apparent in neonatal mice. The in vitro differentiation of mouse embryonic stem cells (ESCs) into PGC-like cells (PGCLCs) revealed reduced numbers of PGCLCs in Trim71-deficient cells. Furthermore, in vitro growth competition assays with wild type and CRISPR/Cas9-generated TRIM71 mutant NCCIT cells, a human GCT-derived cell line which we used as a surrogate model for proliferating PGCs, showed that TRIM71 promotes NCCIT cell proliferation and survival. Our data collectively suggest that germ cell loss in cKO mice results from combined defects during the specification and maintenance of PGCs prior to their sex determination in the genital ridges. Last, via exome sequencing analysis, we identified several TRIM71 variants in a cohort of infertile men, including a loss-of-function variant in a patient with SCO phenotype. Our work reveals for the first time an association of TRIM71 variants with human male infertility, and uncovers further developmental roles for TRIM71 in the generation and maintenance of germ cells during mouse embryogenesis.

developmental biology↗

bioRxiv2019

Biallelic mutations in M1AP are a frequent cause of meiotic arrest leading to male infertility

Male infertility affects [~]7% of men in Western societies, but its causes remain poorly understood. The most clinically severe form of male infertility is non-obstructive azoospermia (NOA), which is, in part, caused by an arrest at meiosis, but so far only few genes have been reported to cause germ cell arrest in males. To address this gap, whole exome sequencing was performed in 60 German men with complete meiotic arrest, and we identified in three unrelated men the same homozygous frameshift variant c.676dup (p.Trp226LeufsTer4) in M1AP, encoding meiosis 1 arresting protein. Then, with collaborators from the International Male Infertility Genomics Consortium (IMIGC), we screened a Dutch cohort comprising 99 infertile men and detected the same homozygous variant c.676dup in a man with hypospermatogenesis predominantly displaying meiotic arrest. We also identified two Portuguese men with NOA carrying likely biallelic loss-of-function (LoF) and missense variants in M1AP among men screened by the Genetics of Male Infertility Initiative (GEMINI). Moreover, we discovered a homozygous missense variant p.(Pro389Leu) in M1AP in a consanguineous Turkish family comprising five infertile men. M1AP is predominantly expressed in human and mouse spermatogonia up to secondary spermatocytes and previous studies have shown that knockout male mice are infertile due to meiotic arrest. Collectively, these findings demonstrate that both LoF and missense M1AP variants that impair its protein cause autosomal-recessive meiotic arrest, non-obstructive azoospermia and male infertility. In view of the evidence from several independent groups and populations, M1AP should be included in the growing list of validated NOA genes.

genetics↗

bioRxiv2019

SYCP2 translocation-mediated dysregulation and frameshift variants cause human male infertility

Infertility is one of the most common disorders for men of reproductive age. To identify novel genetic etiologies, we studied a male with severe oligozoospermia and 46, XY,t(20;22)(q13.3;q11.2). We identified exclusive overexpression of SYCP2 from the der(20) allele that is hypothesized to result from enhancer adoption. Modeling the dysregulation in budding yeast resulted in disruption of the synaptonemal complex, a common cause of defective spermatogenesis in mammals. Exome sequencing of infertile males revealed three novel heterozygous SYCP2 frameshift variants in additional subjects with cryptozoospermia and azoospermia. This study provides the first evidence of SYCP2-mediated male infertility in humans.

genetics↗