Brice Gaudilliere

A Calcium-Regulated MEF2 Sumoylation Switch Controls Postsynaptic Differentiation

Postsynaptic differentiation of dendrites is an essential step in synapse formation. We report here a requirement for the transcription factor myocyte enhancer factor 2A (MEF2A) in the morphogenesis of postsynaptic granule neuron dendritic claws in the cerebellar cortex. A transcriptional repressor form of MEF2A that is sumoylated at lysine-403 promoted dendritic claw differentiation. Activity-dependent calcium signaling induced a calcineurin-mediated dephosphorylation of MEF2A at serine-408 and, thereby, promoted a switch from sumoylation to acetylation at lysine-403, which led to inhibition of dendritic claw differentiation. Our findings define a mechanism underlying postsynaptic differentiation that may modulate activity-dependent synapse development and plasticity in the brain.

A CaMKII-NeuroD Signaling Pathway Specifies Dendritic Morphogenesis

The elaboration of dendrites is fundamental to the establishment of neuronal polarity and connectivity, but the mechanisms that underlie dendritic morphogenesis are poorly understood. We found that the genetic knockdown of the transcription factor NeuroD in primary granule neurons including in organotypic cerebellar slices profoundly impaired the generation and maintenance of dendrites while sparing the development of axons. We also found that NeuroD mediated neuronal activity-dependent dendritogenesis. The activity-induced protein kinase CaMKII catalyzed the phosphorylation of NeuroD at distinct sites, including endogenous NeuroD at Ser336 in primary neurons, and thereby stimulated dendritic growth. These findings uncover an essential function for NeuroD in granule neuron dendritic morphogenesis. Our study also defines the CaMKII-NeuroD signaling pathway as a novel mechanism underlying activity-regulated dendritic growth that may play important roles in the developing and mature brain.

Clinical recovery from surgery correlates with single-cell immune signatures

Single-cell mass cytometry revealed immune correlates of patient-associated variability in surgical recovery. Signaling Surgical Recovery The speed and ease of recovery after surgery differ for every patient, and determining the mechanisms that drive recovery could lead to patient-specific recovery protocols. Gaudilliere et al. used mass cytometry to characterize postsurgical immunological insult at a single-cell level and found a surgical immune signature that correlated with clinical recovery across patients. Specifically, cell signaling responses, but not cell frequency, were linked to recovery. Moreover, the correlated signaling responses occurred most notably in CD14+ monocytes, suggesting that these cells may play a predominant role in surgical recovery. The consistency of this signature across patients suggests a tightly regulated immune response to surgical trauma, which, if validated, may form the basis of a diagnostic guideline for personalized postsurgical care. Delayed recovery from surgery causes personal suffering and substantial societal and economic costs. Whether immune mechanisms determine recovery after surgical trauma remains ill-defined. Single-cell mass cytometry was applied to serial whole-blood samples from 32 patients undergoing hip replacement to comprehensively characterize the phenotypic and functional immune response to surgical trauma. The simultaneous analysis of 14,000 phosphorylation events in precisely phenotyped immune cell subsets revealed uniform signaling responses among patients, demarcating a surgical immune signature. When regressed against clinical parameters of surgical recovery, including functional impairment and pain, strong correlations were found with STAT3 (signal transducer and activator of transcription), CREB (adenosine 3′,5′-monophosphate response element–binding protein), and NF-κB (nuclear factor κB) signaling responses in subsets of CD14+ monocytes (R = 0.7 to 0.8, false discovery rate <0.01). These sentinel results demonstrate the capacity of mass cytometry to survey the human immune system in a relevant clinical context. The mechanistically derived immune correlates point to diagnostic signatures, and potential therapeutic targets, that could postoperatively improve patient recovery.

A FOXO–Pak1 transcriptional pathway controls neuronal polarity

Neuronal polarity is essential for normal brain development and function. However, cell-intrinsic mechanisms that govern the establishment of neuronal polarity remain to be identified. Here, we report that knockdown of endogenous FOXO proteins in hippocampal and cerebellar granule neurons, including in the rat cerebellar cortex in vivo, reveals a requirement for the FOXO transcription factors in the establishment of neuronal polarity. The FOXO transcription factors, including the brain-enriched protein FOXO6, play a critical role in axo-dendritic polarization of undifferentiated neurites, and hence in a switch from unpolarized to polarized neuronal morphology. We also identify the gene encoding the protein kinase Pak1, which acts locally in neuronal processes to induce polarity, as a critical direct target gene of the FOXO transcription factors. Knockdown of endogenous Pak1 phenocopies the effect of FOXO knockdown on neuronal polarity. Importantly, exogenous expression of Pak1 in the background of FOXO knockdown in both primary neurons and postnatal rat pups in vivo restores the polarized morphology of neurons. These findings define the FOXO proteins and Pak1 as components of a cell-intrinsic transcriptional pathway that orchestrates neuronal polarity, thus identifying a novel function for the FOXO transcription factors in a unique aspect of neural development.

PIASx Is a MEF2 SUMO E3 Ligase That Promotes Postsynaptic Dendritic Morphogenesis

Postsynaptic morphogenesis of dendrites is essential for the establishment of neural connectivity in the brain, but the mechanisms that govern postsynaptic dendritic differentiation remain poorly understood. Sumoylation of the transcription factor myocyte enhancer factor 2A (MEF2A) promotes the differentiation of postsynaptic granule neuron dendritic claws in the cerebellar cortex. Here, we identify the protein PIASx as a MEF2 SUMO E3 ligase that represses MEF2-dependent transcription in neurons. Gain-of-function and genetic knockdown experiments in rat cerebellar slices and in the postnatal cerebellum in vivo reveal that PIASx drives the differentiation of granule neuron dendritic claws in the cerebellar cortex. MEF2A knockdown suppresses PIASx-induced dendritic claw differentiation, and expression of sumoylated MEF2A reverses PIASx knockdown-induced loss of dendritic claws. These findings define the PIASx-MEF2 sumoylation signaling link as a key mechanism that orchestrates postsynaptic dendritic claw morphogenesis in the cerebellar cortex and suggest novel functions for SUMO E3 ligases in brain development and plasticity.

Expression of specific inflammasome gene modules stratifies older individuals into two extreme clinical and immunological states

Low-grade, chronic inflammation has been associated with many diseases of aging, but the mechanisms responsible for producing this inflammation remain unclear. Inflammasomes can drive chronic inflammation in the context of an infectious disease or cellular stress, and they trigger the maturation of interleukin-1β (IL-1β). Here we find that the expression of specific inflammasome gene modules stratifies older individuals into two extremes: those with constitutive expression of IL-1β, nucleotide metabolism dysfunction, elevated oxidative stress, high rates of hypertension and arterial stiffness; and those without constitutive expression of IL-1β, who lack these characteristics. Adenine and N4-acetylcytidine, nucleotide-derived metabolites that are detectable in the blood of the former group, prime and activate the NLRC4 inflammasome, induce the production of IL-1β, activate platelets and neutrophils and elevate blood pressure in mice. In individuals over 85 years of age, the elevated expression of inflammasome gene modules was associated with all-cause mortality. Thus, targeting inflammasome components may ameliorate chronic inflammation and various other age-associated conditions.

Transcription factor Sp4 regulates dendritic patterning during cerebellar maturation

Integration of inputs by a neuron depends on dendritic arborization patterns. In mammals, the genetic programs that regulate dynamic remodeling of dendrites during development and in response to activity are incompletely understood. Here we report that knockdown of the transcription factor Sp4 led to an increased number of highly branched dendrites during maturation of cerebellar granule neurons in dissociated cultures and in cerebellar cortex. Time-course analysis revealed that depletion of Sp4 led to persistent generation of dendritic branches and a failure in resorption of transient dendrites. Depolarization induced a reduction in the number of dendrites, and knockdown of Sp4 blocked depolarization-induced remodeling. Furthermore, overexpression of Sp4 wild type, but not a mutant lacking the DNA-binding domain, was sufficient to promote dendritic pruning in nondepolarizing conditions. These findings indicate that the transcription factor Sp4 controls dendritic patterning during cerebellar development by limiting branch formation and promoting activity-dependent pruning.

Transient Partial Permeabilization with Saponin Enables Cellular Barcoding Prior to Surface Marker Staining

Fluorescent cellular barcoding and mass‐tag cellular barcoding are cytometric methods that enable high sample throughput, minimize inter‐sample variation, and reduce reagent consumption. Previously employed barcoding protocols require that barcoding be performed after surface marker staining, complicating combining the technique with measurement of alcohol‐sensitive surface epitopes. This report describes a method of barcoding fixed cells after a transient partial permeabilization with 0.02% saponin that results in efficient and consistent barcode staining with fluorescent or mass‐tagged reagents while preserving surface marker staining. This approach simplifies barcoding protocols and allows direct comparison of surface marker staining of multiple samples without concern for variations in the antibody cocktail volume, antigen‐antibody ratio, or machine sensitivity. Using this protocol, cellular barcoding can be used to reliably detect subtle differences in surface marker expression.

Patient-specific Immune States before Surgery Are Strong Correlates of Surgical Recovery.

Background:Recovery after surgery is highly variable. Risk-stratifying patients based on their predicted recovery profile will afford individualized perioperative management strategies. Recently, application of mass cytometry in patients undergoing hip arthroplasty revealed strong immune correlates of surgical recovery in blood samples collected shortly after surgery. However, the ability to interrogate a patient’s immune state before surgery and predict recovery is highly desirable in perioperative medicine. Methods:To evaluate a patient’s presurgical immune state, cell-type–specific intracellular signaling responses to ex vivo ligands (lipopolysaccharide, interleukin [IL]-6, IL-10, and IL-2/granulocyte macrophage colony-stimulating factor) were quantified by mass cytometry in presurgical blood samples. Selected ligands modulate signaling processes perturbed by surgery. Twenty-three cell surface and 11 intracellular markers were used for the phenotypic and functional characterization of major immune cell subsets. Evoked immune responses were regressed against patient-centered outcomes, contributing to protracted recovery including functional impairment, postoperative pain, and fatigue. Results:Evoked signaling responses varied significantly and defined patient-specific presurgical immune states. Eighteen signaling responses correlated significantly with surgical recovery parameters (|R| = 0.37 to 0.70; false discovery rate < 0.01). Signaling responses downstream of the toll-like receptor 4 in cluster of differentiation (CD) 14+ monocytes were particularly strong correlates, accounting for 50% of observed variance. Immune correlates identified in presurgical blood samples mirrored correlates identified in postsurgical blood samples. Conclusions:Convergent findings in pre- and postsurgical analyses provide validation of reported immune correlates and suggest a critical role of the toll-like receptor 4 signaling pathway in monocytes for the clinical recovery process. The comprehensive assessment of patients’ preoperative immune state is promising for predicting important recovery parameters and may lead to clinical tests using standard flow cytometry.

Implementing Mass Cytometry at the Bedside to Study the Immunological Basis of Human Diseases: Distinctive Immune Features in Patients with a History of Term or Preterm Birth

Single‐cell technologies have immense potential to shed light on molecular and biological processes that drive human diseases. Mass cytometry (or Cytometry by Time Of Flight mass spectrometry, CyTOF) has already been employed in clinical studies to comprehensively survey patients’ circulating immune system. As interest in the “bedside” application of mass cytometry is growing, the delineation of relevant methodological issues is called for. This report uses a newly generated dataset to discuss important methodological considerations when mass cytometry is implemented in a clinical study. Specifically, the use of whole blood samples versus peripheral blood mononuclear cells (PBMCs), design of mass‐tagged antibody panels, technical and analytical implications of sample barcoding, and application of traditional and unsupervised approaches to analyze high‐dimensional mass cytometry datasets are discussed. A mass cytometry assay was implemented in a cross‐sectional study of 19 women with a history of term or preterm birth to determine whether immune traits in peripheral blood differentiate the two groups in the absence of pregnancy. Twenty‐seven phenotypic and 11 intracellular markers were simultaneously analyzed in whole blood samples stimulated with lipopolysaccharide (LPS at 0, 0.1, 1, 10, and 100 ng mL−1) to examine dose‐dependent signaling responses within the toll‐like receptor 4 (TLR4) pathway. Complementary analyses, grounded in traditional or unsupervised gating strategies of immune cell subsets, indicated that the prpS6 and pMAPKAPK2 responses in classical monocytes are accentuated in women with a history of preterm birth (FDR<1%). The results suggest that women predisposed to preterm birth may be prone to mount an exacerbated TLR4 response during the course of pregnancy. This important hypothesis‐generating finding points to the power of single‐cell mass cytometry to detect biologically important differences in a relatively small patient cohort.