Global Excellence in Cellular Neuropathology: Europe

Editorial

25 October 2023

Editorial: Global excellence in cellular neuropathology: Europe

Ryszard Pluta

Aurel Popa-Wagner

and

Dirk M. Hermann

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Editors

Aurel Popa-Wagner

University of Medicine and Pharmacy of Craiova

Ryszard Pluta

Laboratory of Ischemic and Neurodegenerative Brain Research, Mossakowski Medical Research Institute (PAS)

Dirk M. Hermann

University of Duisburg-Essen

Impact

Original Research

30 January 2023

Brain blood vessel autoantibodies in patients with NMDA and GABAA receptor encephalitis: identification of unconventional Myosin-X as target antigen

Lucie Y. Li

, 11 more and

Markus Höltje

Antibody 011-138 colocalizes with commercial anti-Myosin-X antibodies in brain sections and hCMEC/D3 cells and downregulates Myosin-X. (A) Unfixed and unpermeabilized sections from adult mouse brains were incubated with 5 μg/ml of human monoclonal antibody (mAb) 011-138 and a commercial monoclonal mouse antibody directed against unconventional Myosin-X (Myo-X). Shown is the 3-layer cerebellar cortex consisting of the granule cell layer (GCL), Purkinje cell layer (PCL), and outermost molecular layer (ML). Incubation with mAb 011-138 resulted in staining of the Purkinje cell somata and larger blood vessels (upper panel). Staining with commercial anti-Myo-X antibody showed a comparable pattern (lower panel, no larger vessels present). Double staining revealed a high degree of signal overlap between patient 011-138 and commercial MyoX antibodies in Purkinje cells. (B) Likewise, double staining revealed a high degree of signal overlap between patient 011-138 and commercial Myo-X antibodies in mid-size (insets) to larger blood vessels. (C) Double incubation of fixed hCMEC/D3 cells with antibody 011-138 and commercial anti-Myosin-X IgG. Both stainings yielded a similar staining pattern with partially overlapping signals (see insets). (D) Incubation of hCMEC/D3 cells with mAb 011-38 results in the downregulation of Myo-X. Following incubation of hCMEC/D3 cells with 5 μg/ml of mAb 011-138 or mGO53 as control for 48 h cells were homogenized and subjected to Western blotting and quantitative PCR to check for protein expression and gene regulation. Incubation with mAb 011-138 decreased protein expression of Myo-X by 20% (left chart), gene regulation was unaltered (right chart). Data are given as normalized means ± SEM adjusted to loading from two individual experiments analyzed in duplicates (Western blot) and as two individual experiments (qPCR). *p ≤ 0.05.

Introduction: The antibody repertoire from CSF-derived antibody-secreting cells and memory B-cells in patients with encephalitis contains a considerable number of antibodies that do not target the disease-defining autoantigen such as the GABA or NMDA receptors. This study focuses on the functional relevance of autoantibodies to brain blood vessels in patients with GABA_A and NMDA receptor encephalitis.

Methods: We tested 149 human monoclonal IgG antibodies from the cerebrospinal fluid of six patients with different forms of autoimmune encephalitis on murine brain sections for reactivity to blood vessels using immunohistochemistry. Positive candidates were tested for reactivity with purified brain blood vessels, effects on transendothelial electrical resistance (TEER), and expression of tight junction proteins as well as gene regulation using human brain microvascular endothelial hCMEC/D3 cells as in vitro blood-brain barrier model. One blood-vessel reactive antibody was infused intrathecally by pump injection in mice to study in vivo binding and effects on tight junction proteins such as Occludin. Target protein identification was addressed using transfected HEK293 cells.

Results: Six antibodies reacted with brain blood vessels, three were from the same patient with GABA_AR encephalitis, and the other three were from different patients with NMDAR encephalitis. One antibody from an NMDAR encephalitis patient, mAb 011-138, also reacted with cerebellar Purkinje cells. In this case, treatment of hCMEC/D3 cells resulted in decreased TEER, reduced Occludin expression, and mRNA levels. Functional relevance in vivo was confirmed as Occludin downregulation was observed in mAb 011-138-infused animals. Unconventional Myosin-X was identified as a novel autoimmune target for this antibody.

Discussion: We conclude that autoantibodies to blood vessels occur in autoimmune encephalitis patients and might contribute to a disruption of the blood-brain barrier thereby suggesting a potential pathophysiological relevance of these antibodies.

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7 citations

Parvalbumin (PV)-positive interneurons are reduced in the ipsilateral subiculum 21 days after kainate (KA). Representative sections immunostained for PV in control mice (A–C) and 21 days after KA injection (D–I). The localization of the CA1 pyramidal cell layer is indicated. The borders of the subicular pyramidal cell layer toward the alveus (alv) and the molecular layer (m) are delineated as dashed lines according the brain atlas (Franklin and Paxinos, 2008). (A) Dorsal subiculum after NaCl injection. PV+ cells are evenly distributed throughout the pyramidal cell layer of the proximal and distal subiculum and PV+ fibers form a dense plexus. (B,C) Similar pattern at the level of the injection site (B) and further ventral (C). (D) Ipsilateral subiculum 21 days after KA, dorsal position. PV+ somata and PV+ fibers are strongly diminished, mainly in the part of the pyramidal cell layer adjacent to the molecular layer. (E) Similar pattern for the level of the injection site and further ventral (F). (G–I) Contralateral subiculum 21 days after KA. The density of PV+ interneurons is comparable to controls. (J–L) Quantification of PV+ cells in the subiculum of NaCl-injected mice, ipsi- and contralateral subiculum of KA-injected mice 21 days after injection. Individual mice are color-coded, bars show means (cells/mm2) with standard deviation (SD). (J) Reduced density of PV+ cells in the ipsilateral (KAi) but not contralateral dorsal subiculum (KAc) (one-way ANOVA: p = 0.0002, Tukey’s multiple comparison test NaCl–KAi: p = 0.0008; KAi–KAc: p = 0.0004). (K) Reduction of PV+ cell density in the ipsilateral subiculum at the injection site (one-way ANOVA: p = 0.026, Tukey’s multiple comparison test NaCl–KAi: p = 0.025). (L) The effect on KA on PV+ interneurons is weaker in the ventral subiculum (one-way ANOVA: p = 0.051). Scale bars: 250 μm. ***p < 0.001, *p < 0.05. alv, alveus, CA1, cornu ammonis 1, m, molecular layer, PrS presubiculum, sr stratum radiatum.

Original Research

29 March 2023

Differential vulnerability of neuronal subpopulations of the subiculum in a mouse model for mesial temporal lobe epilepsy

Julia Franz

, 4 more and

Ute Häussler

2,727 views

0 citations

Erythromelalgia (EM) patient human induced pluripotent stem cell–derived sensory neurons (hiPSC–SNs) are hypersensitive to mild increases in ambient temperature. (A) Representative raw imaging traces of control hiPSC-SNs (top, orange) and EM patient–SNs (bottom, green) at 37 and 40°C. (B) Comparison of spike frequency filled black dots indicate average values. Error bars indicate SEM. [EM patientdSNs N = 10, control human induced pluripotent stem cell-derived sensory neurons (hiPSCdSNs) N = 6, Wilcoxon rank-sumtest, control: *p = 0.87, EM patient: *p = 0.027].

Original Research

17 January 2023

Bringing to light the physiological and pathological firing patterns of human induced pluripotent stem cell-derived neurons using optical recordings

Therese C. Alich

, 6 more and

Istvan Mody

Human induced pluripotent stem cells (hiPSCs) are a promising approach to study neurological and neuropsychiatric diseases. Most methods to record the activity of these cells have major drawbacks as they are invasive or they do not allow single cell resolution. Genetically encoded voltage indicators (GEVIs) open the path to high throughput visualization of undisturbed neuronal activity. However, conventional GEVIs perturb membrane integrity through inserting multiple copies of transmembrane domains into the plasma membrane. To circumvent large add-ons to the plasma membrane, we used a minimally invasive novel hybrid dark quencher GEVI to record the physiological and pathological firing patterns of hiPSCs-derived sensory neurons from patients with inherited erythromelalgia, a chronic pain condition associated with recurrent attacks of redness and swelling in the distal extremities. We observed considerable differences in action potential firing patterns between patient and control neurons that were previously overlooked with other recording methods. Our system also performed well in hiPSC-derived forebrain neurons where it detected spontaneous synchronous bursting behavior, thus opening the path to future applications in other cell types and disease models including Parkinson’s disease, Alzheimer’s disease, epilepsy, and schizophrenia, conditions associated with disturbances of neuronal activity and synchrony.

6,009 views

6 citations

Brief Research Report

21 July 2022

GABA tonic currents and glial cells are altered during epileptogenesis in a mouse model of Dravet syndrome

Rosa Chiara Goisis

, 6 more and

Gabriele Losi

Dravet Syndrome (DS) is a rare autosomic encephalopathy with epilepsy linked to Na_v1.1 channel mutations and defective GABAergic signaling. Effective therapies for this syndrome are lacking, urging a better comprehension of the mechanisms involved. In a recognized mouse model of DS, we studied GABA tonic current, a form of inhibition largely neglected in DS, in brain slices from developing mice before spontaneous seizures are reported. In neurons from the temporal cortex (TeCx) and CA1 region, GABA tonic current was reduced in DS mice compared to controls, while in the entorhinal cortex (ECx) it was not affected. In this region however allopregnanonole potentiation of GABA tonic current was reduced in DS mice, suggesting altered extrasynaptic GABA_A subunits. Using THIP as a selective agonist, we found reduced δ subunit mediated tonic currents in ECx of DS mice. Unexpectedly in the dentate gyrus (DG), a region with high δ subunit expression, THIP-evoked currents in DS mice were larger than in controls. An immunofluorescence study confirmed that δ subunit expression was reduced in ECx and increased in DG of DS mice. Finally, considering the importance of neuroinflammation in epilepsy and neurodevelopmental disorders, we evaluated classical markers of glia activation. Our results show that DS mice have increased Iba1 reactivity and GFAP expression in both ECx and DG, compared to controls. Altogether we report that before spontaneous seizures, DS mice develop significant alterations of GABA tonic currents and glial cell activation. Understanding all the mechanisms involved in these alterations during disease maturation and progression may unveil new therapeutic targets.

2,883 views

8 citations

(A) GluN variants pathogenicity annotations based on current annotations (in red) and on GRIN structural algorithm-inferred annotations from homologous variants (in yellow). (B) GluN variants functional annotations based on current available annotations (in blue) and GRIN structural algorithm-inferred annotations from homologous variants (in yellow). (C) Summary of GRIN structural algorithm-based computational annotation of non-classified GluN1, GluN2A, and GluN2B subunits missense variants. GRIN predictive power of the algorithm was applied, and showed an expansion of disease-associated (top row) GRIN variants, together with a reduction of GRIN variants number previously classified as “Uncertain significance” (central row) and an increase of functional annotations of GRIN variants affecting the ATD, LBD, and TMD domains (bottom row).

Original Research

23 December 2022

Identification of homologous GluN subunits variants accelerates GRIN variants stratification

Ana Santos-Gómez

, 4 more and

Mireia Olivella

2,101 views

3 citations