Anti-Neuronal IgG4 Autoimmune Diseases and IgG4-Related Diseases May Not Be Part of the Same Spectrum: A Comparative Study.

Background: IgG4 is associated with two emerging groups of rare diseases: 1) IgG4 autoimmune diseases (IgG4-AID) and 2) IgG4-related diseases (IgG4-RLD). Anti-neuronal IgG4-AID include MuSK myasthenia gravis, LGI1- and Caspr2-encephalitis and autoimmune nodo-/paranodopathies (CNTN1/Caspr1 or NF155 antibodies). IgG4-RLD is a multiorgan disease hallmarked by tissue-destructive fibrotic lesions with lymphocyte and IgG4 plasma cell infiltrates and increased serum IgG4 concentrations. It is unclear whether IgG4-AID and IgG4-RLD share relevant clinical and immunopathological features. Methods: We collected and analyzed clinical, serological, and histopathological data in 50 patients with anti-neuronal IgG4-AID and 19 patients with IgG4-RLD. Results: A significantly higher proportion of IgG4-RLD patients had serum IgG4 elevation when compared to IgG4-AID patients (52.63% vs. 16%, p = .004). Moreover, those IgG4-AID patients with elevated IgG4 did not meet the diagnostic criteria of IgG4-RLD, and their autoantibody titers did not correlate with their serum IgG4 concentrations. In addition, patients with IgG4-RLD were negative for anti-neuronal/neuromuscular autoantibodies and among these patients, men showed a significantly higher propensity for IgG4 elevation, when compared to women (p = .005). Last, a kidney biopsy from a patient with autoimmune paranodopathy due to CNTN1/Caspr1-complex IgG4 autoantibodies and concomitant nephrotic syndrome did not show fibrosis or IgG4+ plasma cells, which are diagnostic hallmarks of IgG4-RLD. Conclusion: Our observations suggest that anti-neuronal IgG4-AID and IgG4-RLD are most likely distinct disease entities.

Dexamethasone enhances necrosis-like neuronal death in ischemic rat hippocampus involving µ-calpain activation.

Transient forebrain ischemia (TFI) leads to hippocampal CA1 pyramidal cell death which is aggravated by glucocorticoids (GC). It is unknown how GC affect apoptosis and necrosis in cerebral ischemia. We therefore investigated the co-localization of activated caspase-3 (casp-3) with apoptosis- and necrosis-like cell death morphologies in CA1 of rats treated with dexamethasone prior to TFI (DPTI). In addition, apoptosis- (casp-9, casp-3, casp-3-cleaved PARP and cleaved α-spectrin 145/150 and 120kDa) and necrosis-related (calpain-specific casp-9 cleavage, µ-calpain upregulation and cleaved α-spectrin 145/150kDa) cell death mechanisms were investigated by Western blot analysis. DPTI expedited CA1 neuronal death from day 4 to day 1 and increased the magnitude of CA1 neuronal death from 66.2% to 91.3% at day 7. Furthermore, DPTI decreased the overall (days 1-7) percentage of dying neurons displaying apoptosis-like morphology from 4.7% to 0.3% and, conversely, increased the percentage of neurons with necrosis-like morphology from 95.3% to 99.7%. In animals subjected to TFI without dexamethasone (ischemia-only), 7.4% of all dying CA1 neurons were casp-3-immunoreactive (IR), of which 3.1% co-localized with apoptosis-like and 4.3% with necrosis-like changes. By contrast, DPTI decreased the percentage of dying neurons with casp-3 IR to 1.4%, of which 0.3% co-localized with apoptosis-like changes and 1.1% with necrosis-like changes. Western blot analysis from DPTI animals showed a significant elevation of µ-calpain, a calpain-produced necrosis-related casp-9 fragment (25kDa) and cleavage of α-spectrin into 145/150kDa fragments at day 4, whereas in ischemia-only animals a significant increase of casp-3-cleaved PARP, cleavage of α-spectrin into 145/150 and 120kDa fragments was detected at day 7. We conclude that DPTI, in addition to augmenting and expediting CA1 neuronal death, causes a shift from apoptosis-like cell death to necrosis involving µ-calpain activation.

Anti-apoptotic signaling and failure of apoptosis in the ischemic rat hippocampus.

Several anti-apoptotic proteins are induced in CA1 neurons after transient forebrain ischemia (TFI), but fail to protect the majority of these cells from demise. Correlating cell death morphologies (apoptosis-like and necrosis-like death) with immunohistochemistry (IHC), we investigated whether anti-apoptosis contributes to survival, compromises apoptosis effector functions and/or delays death in CA1 neurons 1-7 days after TFI. As surrogate markers for bioenergetic failure, the IHC of respiratory chain complex (RCC) subunits was investigated. Dentate granule cell (DGC) apoptosis following colchicine injection severed as a reference for classical apoptosis. Heat shock protein 70 (Hsp70), neuronal apoptosis inhibitory protein (NAIP) and manganese superoxide dismutase (MnSOD) were upregulated in the majority of intact CA1 neurons paralleling the occurrence of CA1 neuronal death (days 3-7) as well as in a proportion of apoptosis-(<50%) and necrosis-like (<30%) CA1 neurons. Colchicine did not provoke an anti-apoptotic response in DGC at all. In addition, more than 70% of apoptosis- and necrosis-like CA1 neurons had completely lost their RCC subunits suggesting bioenergetic failure; by contrast, following colchicine injection, 88% of all apoptotic DGC presented RCC subunits. Thus, anti-apoptotic proteins may, in a subset of ischemic CA1 neurons, prevent cell death, while in others, affected by pronounced energy failure, they may cause secondary necrosis.

Expression of glutamic acid decarboxylase and identification of GABAergic cells in the ischemic rat dentate gyrus.

We have investigated the glutamic acid dexcarboxylase (GAD) mRNA and protein isoforms as markers for ischemic loss of GABAergic neurons in the dentate hilus. Stereological counts of these neurons were performed in rats surviving 8 days after 10 min of transient forebrain ischemia, and in control rats (sham-operated and naïve). GAD65 and GAD67 were detected by both in situ hybridization and immunocytochemistry. No differences (three-way ANOVA, P > 0.05) were found between treatments (ischemia, sham-operated or naïve) when cell counts of identical GAD isoforms were compared at the same level (mRNA or protein). However, irrespective of treatments, the number of neurons expressing GAD65 mRNA was significantly higher than the number of neurons expressing GAD65 protein, and the number of neurons expressing GAD67 mRNA was significantly lower than the number of neurons expressing GAD67 protein. In parallel, we investigated the colocalization of the cell death marker Fluorojade B (FJB) with somatostatin- or GAD67-immunoreactivity in hilus of control and ischemic rats. Although the majority of FJB positive cells also contained somatostatin, a small number of GAD67 immunoreactive neurons contained FJB, suggesting that a small number of GABAergic neurons die after ischemia. In conclusion, this study provides direct evidence that a small proportion of GABAergic hilar neurons succumbs to ischemia. Stereological counts of neurons identified from their expression of either GAD isoform of mRNA or protein revealed a high inter-animal variation at any detection level in both naïve, sham-operated and ischemic rats. Therefore, counts of GABAergic neurons should be carefully interpreted in accordance with the marker used for identification.

A role for mixed lineage kinases in granule cell apoptosis induced by cytoskeletal disruption.

Microtubule disruption by colchicine induces apoptosis in selected neuronal populations. However, little is known about the upstream death signalling events mediating the neurotoxicity. We investigated first whether colchicine-induced granule cell apoptosis activates the c-Jun N-terminal kinase (JNK) pathway. Cultured murine cerebellar granule cells were exposed to 1 microm colchicine for 24 h. Activation of the JNK pathway was detected by western blotting as well as immunocytochemistry using antibodies against phospho-c-Jun (p-c-Jun). Next, adult male rats were injected intracerebroventricularly with colchicine (10 microg), and JNK pathway activation in dentate granule cells (DGCs) was detected by antibodies against p-c-Jun. The second part of the study tested the involvement of mixed lineage kinases (MLK) as upstream activators of the JNK pathway in colchicine toxicity, using CEP-1347, a potent MLK inhibitor. In vitro, significant inhibition of the JNK pathway, activated by colchicine, was achieved by 100-300 nm CEP-1347, which blocked both activation of cell death proteases and apoptosis. Moreover, CEP-1347 markedly delayed neurite fragmentation and cell degeneration. In vivo, CEP-1347 (1 mg/kg) significantly prevented p-c-jun increase following injection of colchicine, and enhanced survival of DGCs. We conclude that colchicine-induced neuronal apoptosis involves the JNK/MLK pathway, and that protection of granule cells can be achieved by MLK inhibition.

Ischemia leads to apoptosis--and necrosis-like neuron death in the ischemic rat hippocampus.

Morphological evidence of apoptosis in transient forebrain ischemia is controversial. We therefore investigated the time sequence of apoptosis-related antigens by immunohistochemistry and correlated it with emerging nuclear patterns of cell death in a model of transient forebrain ischemia in CA1 pyramidal cells of the rat hippocampus. The earliest ischemic changes were found on day 2 and 3, reflected by an upregulation of phospho-c-Jun in a proportion of morphologically intact CA1 neurons, which matched the number of neurons that succumbed to ischemia at later time points. At day 3 and later 3 ischemic cell death morphologies became apparent: pyknosis, apoptosis-like cell death and necrosis-like cell death, which were confirmed by electron microscopy. Activated caspase-3 was present in the vast majority of cells with apoptosis-like morphology as well as in a small subset of cells undergoing necrosis; its expression peaked on days 3 to 4. Silver staining for nucleoli, which are a substrate for caspase-3, revealed a profound loss of nucleoli in cells with apoptosis-like morphology, whereas cells with necrosis-like morphology showed intact nucleoli. Overall, cells with apoptosis-like morphology and/or caspase-3 expression represented a minor fraction (<10%) of ischemic neurons, while the vast majority followed a necrosis-like pathway. Our studies suggest that CA1 pyramidal cell death following transient forebrain ischemia may be initiated through c-Jun N-terminal kinase (JNK) pathway activation, which then either follows an apoptosis-like cell death pathway or leads to secondary necrosis.