Brain pathology (Zurich, Switzerland)
Rossini, L;De Santis, D;Cecchini, E;Cagnoli, C;Maderna, E;Cartelli, D;Morgan, BP;Torvell, M;Spreafico, R;di Giacomo, R;Tassi, L;de Curtis, M;Garbelli, R;
PMID: 36564349 | DOI: 10.1111/bpa.13141
Dendritic spines are the postsynaptic sites for most excitatory glutamatergic synapses. We previously demonstrated a severe spine loss and synaptic reorganization in human neocortices presenting Type II focal cortical dysplasia (FCD), a developmental malformation and frequent cause of drug-resistant focal epilepsy. We extend the findings, investigating the potential role of complement components C1q and C3 in synaptic pruning imbalance. Data from Type II FCD were compared with those obtained in focal epilepsies with different etiologies. Neocortical tissues were collected from 20 subjects, mainly adults with a mean age at surgery of 31 years, admitted to epilepsy surgery with a neuropathological diagnosis of: cryptogenic, temporal lobe epilepsy with hippocampal sclerosis, and Type IIa/b FCD. Dendritic spine density quantitation, evaluated in a previous paper using Golgi impregnation, was available in a subgroup. Immunohistochemistry, in situ hybridization, electron microscopy, and organotypic cultures were utilized to study complement/microglial activation patterns. FCD Type II samples presenting dendritic spine loss were characterized by an activation of the classical complement pathway and microglial reactivity. In the same samples, a close relationship between microglial cells and dendritic segments/synapses was found. These features were consistently observed in Type IIb FCD and in 1 of 3 Type IIa cases. In other patient groups and in perilesional areas outside the dysplasia, not presenting spine loss, these features were not observed. In vitro treatment with complement proteins of organotypic slices of cortical tissue with no sign of FCD induced a reduction in dendritic spine density. These data suggest that dysregulation of the complement system plays a role in microglia-mediated spine loss. This mechanism, known to be involved in the removal of redundant synapses during development, is likely reactivated in Type II FCD, particularly in Type IIb; local treatment with anticomplement drugs could in principle modify the course of disease in these patients.
Brain pathology (Zurich, Switzerland)
Cooze, BJ;Dickerson, M;Loganathan, R;Watkins, LM;Grounds, E;Pearson, BR;Bevan, RJ;Morgan, BP;Magliozzi, R;Reynolds, R;Neal, JW;Howell, OW;
PMID: 35132719 | DOI: 10.1111/bpa.13054
The extent of grey matter demyelination and neurodegeneration in the progressive multiple sclerosis (PMS) brains at post-mortem associates with more severe disease. Regional tissue atrophy, especially affecting the cortical and deep grey matter, including the thalamus, is prognostic for poor outcomes. Microglial and complement activation are important in the pathogenesis and contribute to damaging processes that underlie tissue atrophy in PMS. We investigated the extent of pathology and innate immune activation in the thalamus in comparison to cortical grey and white matter in blocks from 21 cases of PMS and 10 matched controls. Using a digital pathology workflow, we show that the thalamus is invariably affected by demyelination and had a far higher proportion of active inflammatory lesions than forebrain cortical tissue blocks from the same cases. Lesions were larger and more frequent in the medial nuclei near the ventricular margin, whilst neuronal loss was greatest in the lateral thalamic nuclei. The extent of thalamic neuron loss was not associated with thalamic demyelination but correlated with the burden of white matter pathology in other forebrain areas (Spearman r = 0.79, p < 0.0001). Only thalamic neuronal loss, and not that seen in other forebrain cortical areas, correlated with disease duration (Spearman r = -0.58, p = 0.009) and age of death (Spearman r = -0.47, p = 0.045). Immunoreactivity for the complement pattern recognition molecule C1q, and products of complement activation (C4d, Bb and C3b) were elevated in thalamic lesions with an active inflammatory pathology. Complement regulatory protein, C1 inhibitor, was unchanged in expression. We conclude that active inflammatory demyelination, neuronal loss and local complement synthesis and activation in the thalamus, are important to the pathological and clinical disease outcomes of PMS.
Ahrens JM, Jones JD, Nieves NJ, Mitzey AM, DeLuca HF, Clagett-Dame M.
PMID: 29182680 | DOI: 10.1371/journal.pone.0188887
While all 2-methylene-19-nor analogs of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) tested produce an increase in epidermal thickness in the rhino mouse, only a subset reduce utricle size (comedolysis). All-trans retinoic acid (atRA) also causes epidermal thickening and a reduction in utricle size in the rhino mouse. We now report that 2-methylene-19-nor-(20S)-1α-hydroxybishomopregnacalciferol (2MbisP), a comedolytic analog, increases epidermal thickening more rapidly than does atRA, while both reduce utricle area at an equal rate. Whereas unlike atRA, 2MbisP does not alter the epidermal growth factor receptor ligand, heparin-binding epidermal growth factor-like growth factor, it does increase the expression of both amphiregulin and epigen mRNA, even after a single dose. In situ hybridization reveals an increase in these transcripts throughout the closing utricle as well as in the interfollicular epidermis. The mRNAs for other EGFR ligands including betacellulin and transforming growth factor-α, as well as the epidermal growth factor receptor are largely unaffected by 2MbisP. Another analog, 2-methylene-19-nor-(20S)-26,27-dimethylene-1α,25-dihydroxyvitamin D3 (CAGE-3), produces epidermal thickening but fails to reduce utricle size or increase AREG mRNA levels. CAGE-3 modestly increases epigen mRNA levels, but only after 5 days of dosing. Thus, 2-MbisP produces unique changes in epidermal growth factor receptor ligand mRNAs that may be responsible for both epidermal proliferation and a reduction in utricle size.
Vu, R;Jin, S;Sun, P;Haensel, D;Nguyen, QH;Dragan, M;Kessenbrock, K;Nie, Q;Dai, X;
PMID: 35926463 | DOI: 10.1016/j.celrep.2022.111155
Delayed and often impaired wound healing in the elderly presents major medical and socioeconomic challenges. A comprehensive understanding of the cellular/molecular changes that shape complex cell-cell communications in aged skin wounds is lacking. Here, we use single-cell RNA sequencing to define the epithelial, fibroblast, immune cell types, and encompassing heterogeneities in young and aged skin during homeostasis and identify major changes in cell compositions, kinetics, and molecular profiles during wound healing. Our comparative study uncovers a more pronounced inflammatory phenotype in aged skin wounds, featuring neutrophil persistence and higher abundance of an inflammatory/glycolytic Arg1Hi macrophage subset that is more likely to signal to fibroblasts via interleukin (IL)-1 than in young counterparts. We predict systems-level differences in the number, strength, route, and signaling mediators of putative cell-cell communications in young and aged skin wounds. Our study exposes numerous cellular/molecular targets for functional interrogation and provides a hypothesis-generating resource for future wound healing studies.
Hammond TR, Dufort C, Dissing-Olesen L, Giera S, Young A, Wysoker A, Walker AJ, Gergits F, Segel M, Nemesh J, Marsh SE, Saunders A, Macosko E, Ginhoux F, Chen J, Franklin RJM, Piao X, McCarroll SA, Stevens B.
PMID: 30471926 | DOI: 10.1016/j.immuni.2018.11.004
Microglia, the resident immune cells of the brain, rapidly change states in response to their environment, but we lack molecular and functional signatures of different microglial populations. Here, we analyzed the RNA expression patterns of more than 76,000 individual microglia in mice during development, in old age, and after brain injury. Our analysis uncovered at least nine transcriptionally distinct microglial states, which expressed unique sets of genes and were localized in the brain using specific markers. The greatest microglial heterogeneity was found at young ages; however, several states-including chemokine-enriched inflammatory microglia-persisted throughout the lifespan or increased in the aged brain. Multiple reactive microglial subtypes were also found following demyelinating injury in mice, at least one of which was also found in human multiple sclerosis lesions. These distinct microglia signatures can be used to better understand microglia function and to identify and manipulate specific subpopulations in health and disease.
Byeon SJ, Lee HS, Kim MA, Lee BL, Kim WH.
PMID: 28399526 | DOI: 10.1159/000464250
Abstract
OBJECTIVE:
Gastric cancer (GC) is the second most common cancer and the third leading cause of cancer-related death in Korea. Alterations in the ERBB (homology to the erythroblastoma viral gene product, v-erbB) receptor family and ERBB-related signaling pathways are frequently observed in GC. However, the roles of the ERBB receptors and their ligands in GC are not well established.
METHODS:
We evaluated the expression levels of various ERBB receptor ligands (i.e., heparin-binding epidermal growth factor-like growth factor [HBEGF], transforming growth factor-α [TGFA], amphiregulin [AREG], epiregulin [EREG], epidermal growth factor [EGF], and betacellulin [BTC]) and 3 ERBB family receptors (i.e., epidermal growth factor receptor [EGFR], human EGFR2 [HER2], and ERBB3) in 313 cases of GC using immunohistochemistry, fluorescence in situ hybridization, and mRNA in situ hybridization.
RESULTS:
A high expression of EGFR, HER2, and ERBB3 was observed in 30, 32, and 27 cases, respectively. A high expression of HBEGF, TGFA, AREG, EREG, EGF, and BTC was observed in 91, 97, 151, 74, 26, and 37 cases, respectively. A high expression of TGFA was associated with better survival, while a high expression of BTC was associated with worse survival. These results were confirmed using Cox proportional hazards analysis. HBEGF, TGFA, AREG, tumor-node-metastasis classification, Lauren's classification, and ERBB3 were significant survival parameters in multivariate analysis.
CONCLUSION:
Among the ERBB family receptors and ligands examined, 3 ligands (i.e., TGFA, HBEGF, and AREG) and ERBB3 had a prognostic impact.
Melderis, S;Warkotsch, MT;Dang, J;Hagenstein, J;Ehnold, LI;Herrnstadt, GR;Niehus, CB;Feindt, FC;Kylies, D;Puelles, VG;Berasain, C;Avila, MA;Neumann, K;Tiegs, G;Huber, TB;Tharaux, PL;Steinmetz, OM;
PMID: 35468361 | DOI: 10.1016/j.jaut.2022.102829
Systemic lupus erythematosus (SLE) is a common autoimmune disorder with a complex and poorly understood immuno-pathogenesis. Lupus nephritis (LN) is a frequent and difficult to treat complication, which causes high morbidity and mortality. The multifunctional cytokine amphiregulin (AREG) has been implicated in SLE pathogenesis, but its function in LN currently remains unknown. We thus studied the model of pristane-induced LN and found increasing renal and systemic AREG expression during the course of disease. Importantly, renal injury was significantly aggravated in the absence of AREG, revealing a net anti-inflammatory role. Analyses of immune responses showed dual effects. On the one hand, AREG enhanced activation of pro-inflammatory myeloid cells, which however did not play a major role for the course of LN. More importantly, on the other hand, AREG strongly suppressed pathogenic cytokine production by T helper effector cells. This effect was more general in nature and could be reproduced in response to antigen immunization. Since AREG has been postulated to downregulate T cell responses via enhancing Treg suppressive capacity, we followed up on this aspect. Interestingly, however, in vitro studies revealed potential direct and Treg independent effects of AREG on T helper effector cells. In favor of this notion, we found significantly enhanced T cell responses and consecutive aggravation of LN, only if epidermal growth factor receptor (EGFR) signaling was abrogated in total T cells, but not if the EGFR was absent on Tregs alone. Finally, we also found enhanced AREG expression in plasma and renal biopsies of patients with LN, supporting the relevance of our findings for human disease. In summary, our data identify AREG as an anti-inflammatory mediator of LN via broad downregulation of pathogenic T cell immunity. These findings further highlight the AREG/EGFR axis as a potential therapeutic target.
The Journal of investigative dermatology
Basset, J;Marchal, L;Hovnanian, A;
PMID: 36116508 | DOI: 10.1016/j.jid.2022.08.045
Pachyonychia congenita (PC) is a rare keratinizing disorder characterized by painful palmoplantar keratoderma (PPK) for which there is no standard current treatment. PC is caused by dominant mutations in keratin 6A, 6B, 6C, 16, and 17 genes involved in stress, wound healing, and epidermal barrier formation. Mechanisms leading to pain and PPK in PC remain elusive. Here, we show overexpression of EGFR ligands epiregulin and TGF-α as well as HER1-EGFR and HER2 in the upper spinous layers of PC lesions. EGFR activation was confirmed by upregulated MAPK/ERK and mTOR signaling. Abnormal late terminal keratinization was associated with elevated transglutaminase-1 (TG1) activity. Additionally, the Ca2+ permeable channel TRPV3 was significantly increased in PC-lesional skin suggesting a predominant role of the TRPV3/EGFR signaling complex in PC. We hypothesized that this complex contributes to promoting TG1 activity and induces the expression and shedding of EGFR ligands. To counteract this biological cascade, we treated 3 PC patients with oral erlotinib for 6 to 8 months. The treatment was well tolerated and led to an early, drastic, and sustained reduction of neuropathic pain with a major improvement of quality-of-life. Our study provides evidence that targeted pharmacological inhibition of EGFR is an effective strategy in PC.
Proc Natl Acad Sci U S A.
Kalish BT, Cheadle L, Hrvatin S, Nagy MA, Rivera S, Crow M, Gillis J, Kirchner R, Greenberg ME.
PMID: 29343640 | DOI: 10.1073/pnas.1717871115
Coordinated changes in gene expression underlie the early patterning and cell-type specification of the central nervous system. However, much less is known about how such changes contribute to later stages of circuit assembly and refinement. In this study, we employ single-cell RNA sequencing to develop a detailed, whole-transcriptome resource of gene expression across four time points in the developing dorsal lateral geniculate nucleus (LGN), a visual structure in the brain that undergoes a well-characterized program of postnatal circuit development. This approach identifies markers defining the major LGN cell types, including excitatory relay neurons, oligodendrocytes, astrocytes, microglia, and endothelial cells. Most cell types exhibit significant transcriptional changes across development, dynamically expressing genes involved in distinct processes including retinotopic mapping, synaptogenesis, myelination, and synaptic refinement. Our data suggest that genes associated with synapse and circuit development are expressed in a larger proportion of nonneuronal cell types than previously appreciated. Furthermore, we used this single-cell expression atlas to identify the Prkcd-Cre mouse line as a tool for selective manipulation of relay neurons during a late stage of sensory-driven synaptic refinement. This transcriptomic resource provides a cellular map of gene expression across several cell types of the LGN, and offers insight into the molecular mechanisms of circuit development in the postnatal brain.
Matson, KJE;Russ, DE;Kathe, C;Hua, I;Maric, D;Ding, Y;Krynitsky, J;Pursley, R;Sathyamurthy, A;Squair, JW;Levi, BP;Courtine, G;Levine, AJ;
PMID: 36163250 | DOI: 10.1038/s41467-022-33184-1
After spinal cord injury, tissue distal to the lesion contains undamaged cells that could support or augment recovery. Targeting these cells requires a clearer understanding of their injury responses and capacity for repair. Here, we use single nucleus RNA sequencing to profile how each cell type in the lumbar spinal cord changes after a thoracic injury in mice. We present an atlas of these dynamic responses across dozens of cell types in the acute, subacute, and chronically injured spinal cord. Using this resource, we find rare spinal neurons that express a signature of regeneration in response to injury, including a major population that represent spinocerebellar projection neurons. We characterize these cells anatomically and observed axonal sparing, outgrowth, and remodeling in the spinal cord and cerebellum. Together, this work provides a key resource for studying cellular responses to injury and uncovers the spontaneous plasticity of spinocerebellar neurons, uncovering a potential candidate for targeted therapy.
A RIPK1-regulated inflammatory microglial state in amyotrophic lateral sclerosis
Proceedings of the National Academy of Sciences of the United States of America
Mifflin, L;Hu, Z;Dufort, C;Hession, CC;Walker, AJ;Niu, K;Zhu, H;Liu, N;Liu, JS;Levin, JZ;Stevens, B;Yuan, J;Zou, C;
PMID: 33766915 | DOI: 10.1073/pnas.2025102118
Microglial-derived inflammation has been linked to a broad range of neurodegenerative and neuropsychiatric conditions, including amyotrophic lateral sclerosis (ALS). Using single-cell RNA sequencing, a class of Disease-Associated Microglia (DAMs) have been characterized in neurodegeneration. However, the DAM phenotype alone is insufficient to explain the functional complexity of microglia, particularly with regard to regulating inflammation that is a hallmark of many neurodegenerative diseases. Here, we identify a subclass of microglia in mouse models of ALS which we term RIPK1-Regulated Inflammatory Microglia (RRIMs). RRIMs show significant up-regulation of classical proinflammatory pathways, including increased levels of Tnf and Il1b RNA and protein. We find that RRIMs are highly regulated by TNFα signaling and that the prevalence of these microglia can be suppressed by inhibiting receptor-interacting protein kinase 1 (RIPK1) activity downstream of the TNF receptor 1. These findings help to elucidate a mechanism by which RIPK1 kinase inhibition has been shown to provide therapeutic benefit in mouse models of ALS and may provide an additional biomarker for analysis in ongoing phase 2 clinical trials of RIPK1 inhibitors in ALS.
Das, M;Mao, W;Shao, E;Tamhankar, S;Yu, G;Yu, X;Ho, K;Wang, X;Wang, J;Mucke, L;
| DOI: 10.1016/j.isci.2021.103245
Nonconvulsive epileptiform activity and microglial alterations have been detected in people with Alzheimer’s disease (AD) and related mouse models. However, the relationship between these abnormalities remains to be elucidated. We suppressed epileptiform activity by treatment with the antiepileptic drug levetiracetam or by genetic ablation of tau and found that these interventions reversed or prevented aberrant microglial gene expression in brain tissues of aged human amyloid precursor protein transgenic mice, which simulate several key aspects of AD. The most robustly modulated genes included multiple factors previously implicated in AD pathogenesis, including TREM2, the hypofunction of which increases disease risk. Genetic reduction of TREM2 exacerbated epileptiform activity after mice were injected with kainate. We conclude that AD-related epileptiform activity markedly changes the molecular profile of microglia, inducing both maladaptive and adaptive alterations in their activities. Increased expression of TREM2 seems to support microglial activities that counteract this type of network dysfunction.
