The thymocyte-specific RNA-binding protein Arpp21 provides TCR repertoire diversity by binding to the 3'-UTR and promoting Rag1 mRNA expression.

The regulation of thymocyte development by RNA-binding proteins (RBPs) is largely unexplored. We identify 642 RBPs in the thymus and focus on Arpp21, which shows selective and dynamic expression in early thymocytes. Arpp21 is downregulated in response to T cell receptor (TCR) and Ca2+ signals. Downregulation requires Stim1/Stim2 and CaMK4 expression and involves Arpp21 protein phosphorylation, polyubiquitination and proteasomal degradation. Arpp21 directly binds RNA through its R3H domain, with a preference for uridine-rich motifs, promoting the expression of target mRNAs. Analysis of the Arpp21-bound transcriptome reveals strong interactions with the Rag1 3'-UTR. Arpp21-deficient thymocytes show reduced Rag1 expression, delayed TCR rearrangement and a less diverse TCR repertoire. This phenotype is recapitulated in Rag1 3'-UTR mutant mice harboring a deletion of the Arpp21 response region. These findings show how thymocyte-specific Arpp21 promotes Rag1 expression to enable TCR repertoire diversity until signals from the TCR terminate Arpp21 and Rag1 activities.

Plant MDL proteins synergize with the cytokine MIF at CXCR2 and CXCR4 receptors in human cells.

Mammalian macrophage migration inhibitory factor (MIF) and its paralog, D-dopachrome tautomerase, are multifunctional inflammatory cytokines. Plants have orthologous MIF and D-dopachrome tautomerase-like (MDL) proteins that mimic some of the effects of MIF on immune cells in vitro. We explored the structural and functional similarities between the three Arabidopsis thaliana MDLs and MIF. X-ray crystallography of the MDLs revealed high structural similarity between MDL and MIF homotrimers and suggested a potential explanation for the lack of tautomerase activity in the MDLs. MDL1 and MDL2 interacted with each other and with MIF in vitro, in yeast, and in plant leaves and formed hetero-oligomeric complexes with MIF in vitro. The MDLs stimulated signaling through the MIF receptors CXCR2 or CXCR4 and enhanced the responses to MIF in a yeast reporter system, in human neutrophils, and in human lung epithelial cells. Pharmacological inhibitors that disrupted MIF activity or prevented the formation of MIF-MDL hetero-oligomers blocked the observed synergism. These findings demonstrate that MDLs can enhance cellular responses to MIF, which may have functional implications in tissues exposed to MDLs from the diet or environment.

Unrestrained cleavage of Roquin-1 by MALT1 induces spontaneous T cell activation and the development of autoimmunity.

Constitutive activation of the MALT1 paracaspase in conventional T cells of Malt1TBM/TBM (TRAF6 Binding Mutant = TBM) mice causes fatal inflammation and autoimmunity, but the involved targets and underlying molecular mechanisms are unknown. We genetically rendered a single MALT1 substrate, the RNA-binding protein (RBP) Roquin-1, insensitive to MALT1 cleavage. These Rc3h1Mins/Mins mice showed normal immune homeostasis. Combining Rc3h1Mins/Mins alleles with those encoding for constitutively active MALT1 (TBM) prevented spontaneous T cell activation and restored viability of Malt1TBM/TBM mice. Mechanistically, we show how antigen/MHC recognition is translated by MALT1 into Roquin cleavage and derepression of Roquin targets. Increasing T cell receptor (TCR) signals inactivated Roquin more effectively, and only high TCR strength enabled derepression of high-affinity targets to promote Th17 differentiation. Induction of experimental autoimmune encephalomyelitis (EAE) revealed increased cleavage of Roquin-1 in disease-associated Th17 compared to Th1 cells in the CNS. T cells from Rc3h1Mins/Mins mice did not efficiently induce the high-affinity Roquin-1 target IκBNS in response to TCR stimulation, showed reduced Th17 differentiation, and Rc3h1Mins/Mins mice were protected from EAE. These data demonstrate how TCR signaling and MALT1 activation utilize graded cleavage of Roquin to differentially regulate target mRNAs that control T cell activation and differentiation as well as the development of autoimmunity.

Targeting the glycine-rich domain of TDP-43 with antibodies prevents its aggregation in vitro and reduces neurofilament levels in vivo.

Cytoplasmic aggregation and concomitant nuclear clearance of the RNA-binding protein TDP-43 are found in ~ 90% of cases of amyotrophic lateral sclerosis and ~ 45% of patients living with frontotemporal lobar degeneration, but no disease-modifying therapy is available. Antibody therapy targeting other aggregating proteins associated with neurodegenerative disorders has shown beneficial effects in animal models and clinical trials. The most effective epitopes for safe antibody therapy targeting TDP-43 are unknown. Here, we identified safe and effective epitopes in TDP-43 for active and potential future passive immunotherapy. We prescreened 15 peptide antigens covering all regions of TDP-43 to identify the most immunogenic epitopes and to raise novel monoclonal antibodies in wild-type mice. Most peptides induced a considerable antibody response and no antigen triggered obvious side effects. Thus, we immunized mice with rapidly progressing TDP-43 proteinopathy ("rNLS8" model) with the nine most immunogenic peptides in five pools prior to TDP-43ΔNLS transgene induction. Strikingly, combined administration of two N-terminal peptides induced genetic background-specific sudden lethality in several mice and was therefore discontinued. Despite a strong antibody response, no TDP-43 peptide prevented the rapid body weight loss or reduced phospho-TDP-43 levels as well as the profound astrogliosis and microgliosis in rNLS8 mice. However, immunization with a C-terminal peptide containing the disease-associated phospho-serines 409/410 significantly lowered serum neurofilament light chain levels, indicative of reduced neuroaxonal damage. Transcriptomic profiling showed a pronounced neuroinflammatory signature (IL-1ß, TNF-α, NfκB) in rNLS8 mice and suggested modest benefits of immunization targeting the glycine-rich region. Several novel monoclonal antibodies targeting the glycine-rich domain potently reduced phase separation and aggregation of TDP-43 in vitro and prevented cellular uptake of preformed aggregates. Our unbiased screen suggests that targeting the RRM2 domain and the C-terminal region of TDP-43 by active or passive immunization may be beneficial in TDP-43 proteinopathies by inhibiting cardinal processes of disease progression.

Features of Isoforms of Human Soluble TACI.

The BAFF/APRIL-system with the two cytokines BAFF and APRIL and their three receptors, transmembrane activator and CAML interactor (TACI), BAFF receptor, and B-cell maturation Ag, is important for B cell maintenance. The BAFF/APRIL system is a therapeutic target in B cell-derived malignancies and autoimmune diseases. However, unexpected outcomes of clinical trials with atacicept (TACI-Fc) underline our incomplete understanding of this system. Shedding of the three receptors is one important regulatory element. In humans, TACI exists in two isoforms generated through alternative splicing in their extracellular portion: TACI-long (l) has two cysteine-rich domains, whereas TACI-short (s) lacks the first low-affinity one. In this study, we discriminated soluble (s) forms of TACI-l and TACI-s with newly generated mAbs and found that both were spontaneously released from activated human B cells, with a predominance of sTACI-l. Furthermore, sTACI-l was also the dominant isoform in human serum. Vaccination with the mRNA vaccine from BioNTech does not significantly affect the serum levels of sTACI-l. Both TACI-s and TACI-l were shed by a disintegrin and metalloproteinase domain-containing protein 10. TACI-l and TACI-s formed homo- and hetero-oligomers in soluble and membrane-bound forms. Both sTACI-l and sTACI-s acted as decoy receptors for BAFF, but only sTACI-l also efficiently inhibited APRIL. Dimerization of sTACI-l enhanced its decoy functions only slightly. Together, we extend our knowledge of the complexity of the BAFF/APRIL system by identifying and characterizing the two soluble isoforms of TACI.

Translational Studies Using the MALT1 Inhibitor (S)-Mepazine to Induce Treg Fragility and Potentiate Immune Checkpoint Therapy in Cancer.

Introduction: Regulatory T cells (Tregs) play a critical role in the maintenance of immune homeostasis but also protect tumors from immune-mediated growth control or rejection and pose a significant barrier to effective immunotherapy. Inhibition of MALT1 paracaspase activity can selectively reprogram immune-suppressive Tregs in the tumor microenvironment to adopt a proinflammatory fragile state, which offers an opportunity to impede tumor growth and enhance the efficacy of immune checkpoint therapy (ICT). Methods: We performed preclinical studies with the orally available allosteric MALT1 inhibitor (S)-mepazine as a single-agent and in combination with anti-programmed cell death protein 1 (PD-1) ICT to investigate its pharmacokinetic properties and antitumor effects in several murine tumor models as well as patient-derived organotypic tumor spheroids (PDOTS). Results: (S)-mepazine demonstrated significant antitumor effects and was synergistic with anti-PD-1 therapy in vivo and ex vivo but did not affect circulating Treg frequencies in healthy rats at effective doses. Pharmacokinetic profiling revealed favorable drug accumulation in tumors to concentrations that effectively blocked MALT1 activity, potentially explaining preferential effects on tumor-infiltrating over systemic Tregs. Conclusions: The MALT1 inhibitor (S)-mepazine showed single-agent anticancer activity and presents a promising opportunity for combination with PD-1 pathway-targeted ICT. Activity in syngeneic tumor models and human PDOTS was likely mediated by induction of tumor-associated Treg fragility. This translational study supports ongoing clinical investigations (ClinicalTrials.gov Identifier: NCT04859777) of MPT-0118, (S)-mepazine succinate, in patients with advanced or metastatic treatment-refractory solid tumors.

Establishment and Functional Characterization of Murine Monoclonal Antibodies Recognizing Neuritin.

Neuritin represents a neurotrophic factor that is not only important in neuronal development and plasticity but also impacts endothelial angiogenesis, cell migration, tumor growth and the production of antibodies by B cells. We established monoclonal mouse anti-mouse neuritin antibodies by immunizing knock-out mice with two different neuritin-derived peptides. Because neuritin is well conserved between species, these new monoclonal antibodies recognize the neuritin of a wide variety of species, including human. Moreover, they not only recognize specifically surface-bound neuritin expressed by murine follicular regulatory T cells but also the block binding of recombinant neuritin to germinal center B cells. This suggests that these newly generated tools will be of great use in studying neuritin expression and function.

Depletion of the RNA-binding protein PURA triggers changes in posttranscriptional gene regulation and loss of P-bodies.

The RNA-binding protein PURA has been implicated in the rare, monogenetic, neurodevelopmental disorder PURA Syndrome. PURA binds both DNA and RNA and has been associated with various cellular functions. Only little is known about its main cellular roles and the molecular pathways affected upon PURA depletion. Here, we show that PURA is predominantly located in the cytoplasm, where it binds to thousands of mRNAs. Many of these transcripts change abundance in response to PURA depletion. The encoded proteins suggest a role for PURA in immune responses, mitochondrial function, autophagy and processing (P)-body activity. Intriguingly, reduced PURA levels decrease the expression of the integral P-body components LSM14A and DDX6 and strongly affect P-body formation in human cells. Furthermore, PURA knockdown results in stabilization of P-body-enriched transcripts, whereas other mRNAs are not affected. Hence, reduced PURA levels, as reported in patients with PURA Syndrome, influence the formation and composition of this phase-separated RNA processing machinery. Our study proposes PURA Syndrome as a new model to study the tight connection between P-body-associated RNA regulation and neurodevelopmental disorders.

Medin co-aggregates with vascular amyloid-ß in Alzheimer's disease.

Aggregates of medin amyloid (a fragment of the protein MFG-E8, also known as lactadherin) are found in the vasculature of almost all humans over 50 years of age1,2, making it the most common amyloid currently known. We recently reported that medin also aggregates in blood vessels of ageing wild-type mice, causing cerebrovascular dysfunction3. Here we demonstrate in amyloid-ß precursor protein (APP) transgenic mice and in patients with Alzheimer's disease that medin co-localizes with vascular amyloid-ß deposits, and that in mice, medin deficiency reduces vascular amyloid-ß deposition by half. Moreover, in both the mouse and human brain, MFG-E8 is highly enriched in the vasculature and both MFG-E8 and medin levels increase with the severity of vascular amyloid-ß burden. Additionally, analysing data from 566 individuals in the ROSMAP cohort, we find that patients with Alzheimer's disease have higher MFGE8 expression levels, which are attributable to vascular cells and are associated with increased measures of cognitive decline, independent of plaque and tau pathology. Mechanistically, we demonstrate that medin interacts directly with amyloid-ß to promote its aggregation, as medin forms heterologous fibrils with amyloid-ß, affects amyloid-ß fibril structure, and cross-seeds amyloid-ß aggregation both in vitro and in vivo. Thus, medin could be a therapeutic target for prevention of vascular damage and cognitive decline resulting from amyloid-ß deposition in the blood vessels of the brain.

A non-canonical vitamin K cycle is a potent ferroptosis suppressor.

Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation1, has a key role in organ injury, degenerative disease and vulnerability of therapy-resistant cancers2. Although substantial progress has been made in understanding the molecular processes relevant to ferroptosis, additional cell-extrinsic and cell-intrinsic processes that determine cell sensitivity toward ferroptosis remain unknown. Here we show that the fully reduced forms of vitamin K-a group of naphthoquinones that includes menaquinone and phylloquinone3-confer a strong anti-ferroptotic function, in addition to the conventional function linked to blood clotting by acting as a cofactor for γ-glutamyl carboxylase. Ferroptosis suppressor protein 1 (FSP1), a NAD(P)H-ubiquinone reductase and the second mainstay of ferroptosis control after glutathione peroxidase-44,5, was found to efficiently reduce vitamin K to its hydroquinone, a potent radical-trapping antioxidant and inhibitor of (phospho)lipid peroxidation. The FSP1-mediated reduction of vitamin K was also responsible for the antidotal effect of vitamin K against warfarin poisoning. It follows that FSP1 is the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle6. The FSP1-dependent non-canonical vitamin K cycle can act to protect cells against detrimental lipid peroxidation and ferroptosis.

A novel monoclonal IgG1 antibody specific for Galactose-alpha-1,3-galactose questions alpha-Gal epitope expression by bacteria.

The alpha-Gal epitope (α-Gal) with the determining element galactose-α1,3-galactose can lead to clinically relevant allergic reactions and rejections in xenotransplantation. These immune reactions can develop because humans are devoid of this carbohydrate due to evolutionary loss of the enzyme α1,3-galactosyltransferase (GGTA1). In addition, up to 1% of human IgG antibodies are directed against α-Gal, but the stimulus for the induction of anti-α-Gal antibodies is still unclear. Commensal bacteria have been suggested as a causal factor for this induction as α-Gal binding tools such as lectins were found to stain cultivated bacteria isolated from the intestinal tract. Currently available tools for the detection of the definite α-Gal epitope, however, are cross-reactive, or have limited affinity and, hence, offer restricted possibilities for application. In this study, we describe a novel monoclonal IgG1 antibody (27H8) specific for the α-Gal epitope. The 27H8 antibody was generated by immunization of Ggta1 knockout mice and displays a high affinity towards synthetic and naturally occurring α-Gal in various applications. Using this novel tool, we found that intestinal bacteria reported to be α-Gal positive cannot be stained with 27H8 questioning whether commensal bacteria express the native α-Gal epitope at all.

Spatial centrosome proteome of human neural cells uncovers disease-relevant heterogeneity.

The centrosome provides an intracellular anchor for the cytoskeleton, regulating cell division, cell migration, and cilia formation. We used spatial proteomics to elucidate protein interaction networks at the centrosome of human induced pluripotent stem cell-derived neural stem cells (NSCs) and neurons. Centrosome-associated proteins were largely cell type-specific, with protein hubs involved in RNA dynamics. Analysis of neurodevelopmental disease cohorts identified a significant overrepresentation of NSC centrosome proteins with variants in patients with periventricular heterotopia (PH). Expressing the PH-associated mutant pre-mRNA-processing factor 6 (PRPF6) reproduced the periventricular misplacement in the developing mouse brain, highlighting missplicing of transcripts of a microtubule-associated kinase with centrosomal location as essential for the phenotype. Collectively, cell type-specific centrosome interactomes explain how genetic variants in ubiquitous proteins may convey brain-specific phenotypes.

PAT2 regulates vATPase assembly and lysosomal acidification in brown adipocytes.

OBJECTIVE: Brown adipocytes play a key role in maintaining body temperature as well as glucose and lipid homeostasis. However, brown adipocytes need to adapt their thermogenic activity and substrate utilization to changes in nutrient availability. Amongst the multiple factors influencing brown adipocyte activity, autophagy is an important regulatory element of thermogenic capacity and activity. Nevertheless, a specific sensing mechanism of extracellular amino acid availability linking autophagy to nutrient availability in brown adipocytes is unknown. METHODS: To characterize the role of the amino acid transporter PAT2/SLC36A2 in brown adipocytes, loss or gain of function of PAT2 were studied with respect to differentiation, subcellular localization, lysosomal activity and autophagy. Activity of vATPase was evaluated by quenching of EGFP fused to LC3 or FITC-dextran loaded lysosomes in brown adipocytes upon amino acid starvation, whereas the effect of PAT2 on assembly of the vATPase was investigated by Native-PAGE. RESULTS: We show that PAT2 translocates from the plasma membrane to the lysosome in response to amino acid withdrawal. Loss or overexpression of PAT2 impair lysosomal acidification and starvation-induced S6K re-phosphorylation, as PAT2 facilitates the assembly of the lysosomal vATPase, by recruitment of the cytoplasmic V1 subunit to the lysosome. CONCLUSIONS: PAT2 is an important sensor of extracellular amino acids and regulator of lysosomal acidification in brown adipocytes.

Active site geometry stabilization of a presenilin homolog by the lipid bilayer promotes intramembrane proteolysis.

Cleavage of membrane proteins in the lipid bilayer by intramembrane proteases is crucial for health and disease. Although different lipid environments can potently modulate their activity, how this is linked to their structural dynamics is unclear. Here, we show that the carboxy-peptidase-like activity of the archaeal intramembrane protease PSH, a homolog of the Alzheimer's disease-associated presenilin/γ-secretase is impaired in micelles and promoted in a lipid bilayer. Comparative molecular dynamics simulations revealed that important elements for substrate binding such as transmembrane domain 6a of PSH are more labile in micelles and stabilized in the lipid bilayer. Moreover, consistent with an enhanced interaction of PSH with a transition-state analog inhibitor, the bilayer promoted the formation of the enzyme's catalytic active site geometry. Our data indicate that the lipid environment of an intramembrane protease plays a critical role in structural stabilization and active site arrangement of the enzyme-substrate complex thereby promoting intramembrane proteolysis.

Cutting proteins into pieces is a crucial process in the cell, allowing several important processes to take place, including cell differentiation (which allows cells to develop into specific types), cell death, protein quality control, or even where in the cell a protein will end up. However, the specialized proteins that carry out this task, known as proteases, can also be involved in the development of disease. For example, in the brain, a protease called γ-secretase cuts up the amyloid-ß protein precursor, producing toxic forms of amyloid-ß peptides that are widely believed to cause Alzheimer's disease. Proteases like γ-secretase carry out their role in the membrane, the layer of fats (also known as lipids) that forms the outer boundary of the cell. The environment in this area of the cell can influence the activity of proteases, but it is poorly understood how this happens. One way to address this question would be to compare the activity of γ-secretase in the lipid environment of the membrane to its activity when it is entirely surrounded by different molecules, such as detergent molecules. Unfortunately, γ-secretase is not active when it is removed from its lipid environment by a detergent, making it difficult to perform this comparison. To overcome this issue, Feilen et al. chose to study PSH, a protease similar to γ-secretase that produces the same amyloid-ß peptides but remains active in detergent. When Feilen et al. mixed PSH with lipid molecules like those found in the membrane and amyloid-ß precursor protein, PSH produced amyloid-ß peptides including those that are thought to cause Alzheimer's. However, when a detergent was substituted for the lipid molecules this led to longer amyloid-ß peptides than usual, indicating that PSH was not able to cut proteins as effectively. The change in environment appeared to reduce PSH's ability to progressively trim small segments from the peptides. Computer modelling of the protease's structure in lipids versus detergent supported the experimental findings: the model predicted that the areas of PSH important for recognizing and cutting other proteins would be more stable in the membrane compared to the detergent. These results indicate that the cell membrane plays a vital role in the stability of the active regions of proteases that are cleaving in this environment. In the future, this could help to better understand how changes to the lipid molecules in the membrane may contribute to the activity of γ-secretase and its role in Alzheimer's disease.

A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape.

CD73 catalyzes the conversion of ATP to adenosine, which is involved in various physiological and pathological processes, including tumor immune escape. Because CD73 expression and activity are particularly high on cancer cells and contribute to the immunosuppressive properties of the tumor environment, it is considered an attractive target molecule for specific cancer therapies. In line, several studies demonstrated that CD73 inhibition has a significant antitumor effect. However, complete blocking of CD73 activity can evoke autoimmune phenomena and adverse side effects. We developed a CD73-specific antibody, 22E6, that specifically inhibits the enzymatic activity of membrane-tethered CD73 present in high concentrations on cancer cells and cancer cell-derived extracellular vesicles but has no inhibitory effect on soluble CD73. Inhibition of CD73 on tumor cells with 22E6 resulted in multiple effects on tumor cells in vitro, including increased apoptosis and interference with chemoresistance. Intriguingly, in a xenograft mouse model of acute lymphocytic leukemia (ALL), 22E6 treatment resulted in an initial tumor growth delay in some animals, followed by a complete loss of CD73 expression on ALL cells in all 22E6 treated animals, indicating tumor immune escape. Taken together, 22E6 shows great potential for cancer therapy, favorably in combination with other drugs.

FK506-Binding Protein 11 Is a Novel Plasma Cell-Specific Antibody Folding Catalyst with Increased Expression in Idiopathic Pulmonary Fibrosis.

Antibodies are central effectors of the adaptive immune response, widespread used therapeutics, but also potentially disease-causing biomolecules. Antibody folding catalysts in the plasma cell are incompletely defined. Idiopathic pulmonary fibrosis (IPF) is a fatal chronic lung disease with increasingly recognized autoimmune features. We found elevated expression of FK506-binding protein 11 (FKBP11) in IPF lungs where FKBP11 specifically localized to antibody-producing plasma cells. Suggesting a general role in plasma cells, plasma cell-specific FKBP11 expression was equally observed in lymphatic tissues, and in vitro B cell to plasma cell differentiation was accompanied by induction of FKBP11 expression. Recombinant human FKBP11 was able to refold IgG antibody in vitro and inhibited by FK506, strongly supporting a function as antibody peptidyl-prolyl cis-trans isomerase. Induction of ER stress in cell lines demonstrated induction of FKBP11 in the context of the unfolded protein response in an X-box-binding protein 1 (XBP1)-dependent manner. While deficiency of FKBP11 increased susceptibility to ER stress-mediated cell death in an alveolar epithelial cell line, FKBP11 knockdown in an antibody-producing hybridoma cell line neither induced cell death nor decreased expression or secretion of IgG antibody. Similarly, antibody secretion by the same hybridoma cell line was not affected by knockdown of the established antibody peptidyl-prolyl isomerase cyclophilin B. The results are consistent with FKBP11 as a novel XBP1-regulated antibody peptidyl-prolyl cis-trans isomerase and indicate significant redundancy in the ER-resident folding machinery of antibody-producing hybridoma cells.

Soluble TREM2 in CSF and its association with other biomarkers and cognition in autosomal-dominant Alzheimer's disease: a longitudinal observational study.

BACKGROUND: Therapeutic modulation of TREM2-dependent microglial function might provide an additional strategy to slow the progression of Alzheimer's disease. Although studies in animal models suggest that TREM2 is protective against Alzheimer's pathology, its effect on tau pathology and its potential beneficial role in people with Alzheimer's disease is still unclear. Our aim was to study associations between the dynamics of soluble TREM2, as a biomarker of TREM2 signalling, and amyloid ß (Aß) deposition, tau-related pathology, neuroimaging markers, and cognitive decline, during the progression of autosomal dominant Alzheimer's disease. METHODS: We did a longitudinal analysis of data from the Dominantly Inherited Alzheimer Network (DIAN) observational study, which includes families with a history of autosomal dominant Alzheimer's disease. Participants aged over 18 years who were enrolled in DIAN between Jan 1, 2009, and July 31, 2019, were categorised as either carriers of pathogenic variants in PSEN1, PSEN2, and APP genes (n=155) or non-carriers (n=93). We measured amounts of cleaved soluble TREM2 using a novel immunoassay in CSF samples obtained every 2 years from participants who were asymptomatic (Clinical Dementia Rating [CDR]=0) and annually for those who were symptomatic (CDR>0). CSF concentrations of Aß40, Aß42, total tau (t-tau), and tau phosphorylated on threonine 181 (p-tau) were measured by validated immunoassays. Predefined neuroimaging measurements were total cortical uptake of Pittsburgh compound B PET (PiB-PET), cortical thickness in the precuneus ascertained by MRI, and hippocampal volume determined by MRI. Cognition was measured using a validated cognitive composite (including DIAN word list test, logical memory delayed recall, digit symbol coding test [total score], and minimental status examination). We based our statistical analysis on univariate and bivariate linear mixed effects models. FINDINGS: In carriers of pathogenic variants, a high amyloid burden at baseline, represented by low CSF Aß42 (ß=-4·28 × 10-2 [SE 0·013], p=0·0012), but not high cortical uptake in PiB-PET (ß=-5·51 × 10-3 [0·011], p=0·63), was the only predictor of an augmented annual rate of subsequent increase in soluble TREM2. Augmented annual rates of increase in soluble TREM2 were associated with a diminished rate of decrease in amyloid deposition, as measured by Aß42 in CSF (r=0·56 [0·22], p=0·011), in presymptomatic carriers of pathogenic variants, and with diminished annual rate of increase in PiB-PET (r=-0·67 [0·25], p=0·0060) in symptomatic carriers of pathogenic variants. Presymptomatic carriers of pathogenic variants with annual rates of increase in soluble TREM2 lower than the median showed a correlation between enhanced annual rates of increase in p-tau in CSF and augmented annual rates of increase in PiB-PET signal (r=0·45 [0·21], p=0·035), that was not observed in those with rates of increase in soluble TREM2 higher than the median. Furthermore, presymptomatic carriers of pathogenic variants with rates of increase in soluble TREM2 above or below the median had opposite associations between Aß42 in CSF and PiB-PET uptake when assessed longitudinally. Augmented annual rates of increase in soluble TREM2 in presymptomatic carriers of pathogenic variants correlated with decreased cortical shrinkage in the precuneus (r=0·46 [0·22]), p=0·040) and diminished cognitive decline (r=0·67 [0·22], p=0·0020). INTERPRETATION: Our findings in autosomal dominant Alzheimer's disease position the TREM2 response within the amyloid cascade immediately after the first pathological changes in Aß aggregation and further support the role of TREM2 on Aß plaque deposition and compaction. Furthermore, these findings underpin a beneficial effect of TREM2 on Aß deposition, Aß-dependent tau pathology, cortical shrinkage, and cognitive decline. Soluble TREM2 could, therefore, be a key marker for clinical trial design and interpretation. Efforts to develop TREM2-boosting therapies are ongoing. FUNDING: German Research Foundation, US National Institutes of Health.

Sirtuin-1 sensitive lysine-136 acetylation drives phase separation and pathological aggregation of TDP-43.

Trans-activation response DNA-binding protein of 43 kDa (TDP-43) regulates RNA processing and forms neuropathological aggregates in patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Investigating TDP-43 post-translational modifications, we discovered that K84 acetylation reduced nuclear import whereas K136 acetylation impaired RNA binding and splicing capabilities of TDP-43. Such failure of RNA interaction triggered TDP-43 phase separation mediated by the C-terminal low complexity domain, leading to the formation of insoluble aggregates with pathologically phosphorylated and ubiquitinated TDP-43. Introduction of acetyl-lysine at the identified sites via amber suppression confirmed the results from site-directed mutagenesis. K84-acetylated TDP-43 showed cytoplasmic mislocalization, and the aggregation propensity of K136-acetylated TDP-43 was confirmed. We generated antibodies selective for TDP-43 acetylated at these lysines, and found that sirtuin-1 can potently deacetylate K136-acetylated TDP-43 and reduce its aggregation propensity. Thus, distinct lysine acetylations modulate nuclear import, RNA binding and phase separation of TDP-43, suggesting regulatory mechanisms for TDP-43 pathogenesis.

Phosphorylation of serine-893 in CARD11 suppresses the formation and activity of the CARD11-BCL10-MALT1 complex in T and B cells.

CARD11 acts as a gatekeeper for adaptive immune responses after T cell or B cell antigen receptor (TCR/BCR) ligation on lymphocytes. PKCθ/ß-catalyzed phosphorylation of CARD11 promotes the assembly of the CARD11-BCL10-MALT1 (CBM) complex and lymphocyte activation. Here, we demonstrated that PKCθ/ß-dependent CARD11 phosphorylation also suppressed CARD11 functions in T or B cells. Through mass spectrometry-based proteomics analysis, we identified multiple constitutive and inducible CARD11 phosphorylation sites in T cells. We demonstrated that a single TCR- or BCR-inducible phosphorylation on Ser893 in the carboxyl terminus of CARD11 prevented the activation of the transcription factor NF-κB, the kinase JNK, and the protease MALT1. Moreover, CARD11 Ser893 phosphorylation sensitized BCR-addicted lymphoma cells to toxicity induced by Bruton's tyrosine kinase (BTK) inhibitors. Phosphorylation of Ser893 in CARD11 by PKCθ controlled the strength of CARD11 scaffolding by impairing the formation of the CBM complex. Thus, PKCθ simultaneously catalyzes both stimulatory and inhibitory CARD11 phosphorylation events, which shape the strength of CARD11 signaling in lymphocytes.

Normality sensing licenses local T cells for innate-like tissue surveillance.

The increasing implication of lymphocytes in general physiology and immune surveillance outside of infection poses the question of how their antigen receptors might be involved. Here, we show that macromolecular aggregates of intraepidermal γδ T cell antigen receptors (TCRs) in the mouse skin aligned with and depended on Skint1, a butyrophilin-like (BTNL) protein expressed by differentiated keratinocytes (KCs) at steady state. Interruption of TCR-mediated 'normality sensing' had no impact on γδ T cell numbers but altered their signature phenotype, while the epidermal barrier function was compromised. In addition to the regulation of steady-state physiology, normality sensing licensed intraepidermal T cells to respond rapidly to subsequent tissue perturbation by using innate tumor necrosis factor (TNF) superfamily receptors. Thus, interfering with Skint1-dependent interactions between local γδ T cells and KCs at steady state increased the susceptibility to ultraviolet B radiation (UVR)-induced DNA damage and inflammation, two cancer-disposing factors.