Dendritic spine loss in epileptogenic Type II focal cortical dysplasia: Role of enhanced classical complement pathway activation.
Brain pathology (Zurich, Switzerland)
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.
10.1111/bpa.13141
Complement system biomarkers in epilepsy.
Kopczynska Maja,Zelek Wioleta M,Vespa Simone,Touchard Samuel,Wardle Mark,Loveless Samantha,Thomas Rhys H,Hamandi Khalid,Morgan B Paul
Seizure
PURPOSE:To explore whether complement dysregulation occurs in a routinely recruited clinical cohort of epilepsy patients, and whether complement biomarkers have potential to be used as markers of disease severity and seizure control. METHODS:Plasma samples from 157 epilepsy cases (106 with focal seizures, 46 generalised seizures, 5 unclassified) and 54 controls were analysed. Concentrations of 10 complement analytes (C1q, C3, C4, factor B [FB], terminal complement complex [TCC], iC3b, factor H [FH], Clusterin [Clu], Properdin, C1 Inhibitor [C1Inh] plus C-reactive protein [CRP]) were measured using enzyme linked immunosorbent assay (ELISA). Univariate and multivariate statistical analysis were used to test whether combinations of complement analytes were predictive of epilepsy diagnoses and seizure occurrence. Correlation between number and type of anti-epileptic drugs (AED) and complement analytes was also performed. RESULTS:We found: CONCLUSION: This study adds to evidence implicating complement in pathogenesis of epilepsy and may allow the development of better therapeutics and prognostic markers in the future. Replication in a larger sample set is needed to validate the findings of the study.
10.1016/j.seizure.2018.05.016
Early treatment with C1 esterase inhibitor improves weight but not memory deficits in a rat model of status epilepticus.
Physiology & behavior
BACKGROUND:Status epilepticus (SE) is a prolonged and continuous seizure that lasts for at least 5 min. An episode of SE in a healthy system can lead to the development of spontaneous seizures and cognitive deficits which may be accompanied by hippocampal injury and microgliosis. Although the direct mechanisms underlying the SE-induced pathophysiology remain unknown, a candidate mechanism is hyperactivation of the classical complement pathway (C1q-C3 signaling). We recently reported that SE triggered an increase in C1q-C3 signaling in the hippocampus that closely paralleled cognitive decline. Thus, we hypothesized that blocking activation of the classical complement pathway immediately after SE may prevent the development of SE-induced hippocampal-dependent learning and memory deficits. METHODS:Because C1 esterase inhibitor (C1-INH) negatively regulates activation of the classical complement pathway, we used this drug to test our hypothesis. Two groups of male rats were subjected to 1 hr of SE with pilocarpine (280-300 mg/kg, i.p.), and treated with either C1-INH (SE+C1-INH, 20 U/kg, s.c.) or vehicle (SE+veh) at 4, 24, and 48 h after SE. Control rats were treated with saline. Body weight was recorded for up to 23 days after SE. At two weeks post SE, recognition and spatial memory were determined using Novel Object Recognition (NOR) and Barnes maze (BM), respectively, as well as locomotion and anxiety-like behaviors using Open Field (OF). Histological and biochemical methods were used to measure hippocampal injury including cell death, microgliosis, and inflammation. RESULTS:One day after SE, both SE groups had a significant loss of body weight compared to controls (p<0.05). By day 14, the weight of SE+C1-INH rats was significantly higher than SE+veh rats (p<0.05), and was not different from controls (p>0.05). At 14 days post-SE, SE+C1-INH rats displayed higher mobility (distance travelled and average speed, p<0.05) and had reduced anxiety-like behaviors (outer duration, p<0.05) than control or SE+veh rats. In NOR, control rats spent significantly more time exploring the novel object vs. the familiar (p<0.05), while rats in both SE groups spent similar amount of time exploring both objects. During days 1-4 of BM training, the escape latency of the control group significantly decreased over time (p<0.05), whereas that of the SE groups did not improve (p>0.05). Compared to vehicle-treated SE rats, SE+C1-INH rats had increased levels of C3 and microglia in the hippocampus, but lower levels of caspase-3 and synaptic markers. CONCLUSIONS:These findings suggest that acute treatment with C1-INH after SE may have some protective, albeit limited, effects on the physiological recovery of rats' weight and some anxiolytic effects, but does not attenuate SE-induced deficits in hippocampal-dependent learning and memory. Reduced levels of caspase-3 suggest that treatment with C1-INH may protect against cell death, perhaps by regulating inflammatory pathways and promoting phagocytic/clearance pathways.
10.1016/j.physbeh.2019.112705
Synaptic Pruning by Microglia in Epilepsy.
Andoh Megumi,Ikegaya Yuji,Koyama Ryuta
Journal of clinical medicine
Structural and functional collapse of the balance between excitatory (E) and inhibitory (I) synapses, i.e., synaptic E/I balance, underlies the pathogeneses of various central nervous system (CNS) disorders. In epilepsy, the synaptic E/I balance tips toward excitation; thus, most of the existing epileptic remedies have focused on how to directly suppress the activity of neurons. However, because as many as 30% of patients with epilepsy are drug resistant, the discovery of new therapeutic targets is strongly desired. Recently, the roles of glial cells in epilepsy have gained attention because glial cells manipulate synaptic structures and functions in addition to supporting neuronal survival and growth. Among glial cells, microglia, which are brain-resident immune cells, have been shown to mediate inflammation, neuronal death and aberrant neurogenesis after epileptic seizures. However, few studies have investigated the involvement of synaptic pruning-one of the most important roles of microglia-in the epileptic brain. In this review, we propose and discuss the hypothesis that synaptic pruning by microglia is enhanced in the epileptic brain, drawing upon the findings of previous studies. We further discuss the possibility that aberrant synaptic pruning by microglia induces synaptic E/I imbalance, promoting the development and aggravation of epilepsy.
10.3390/jcm8122170
Gene expression in the epileptic (EL) mouse hippocampus.
Lee Tih-Shih,Li Alexander Y,Rapuano Amedeo,Mantis John,Eid Tore,Seyfried Thomas N,de Lanerolle Nihal C
Neurobiology of disease
The neuropathology of hippocampal seizure foci in human temporal lobe epilepsy (TLE) and several animal models of epilepsy reveal extensive neuronal loss along with astrocyte and microglial activation. Studies of these models have advanced hypotheses that propose both pathological changes are essential for seizure generation. However, some seizure foci in human TLE show an extreme loss of neurons in all hippocampal fields, giving weight to hypotheses that favor neuroglia as major players. The epileptic (EL) mouse is a seizure model in which there is no observable neuron loss but associated proliferation of microglia and astrocytes and provides a good model to study the role of activated neuroglia in the presence of an apparently normal population of neurons. While many studies have been carried out on the EL mouse, there is a paucity of studies on the molecular changes in the EL mouse hippocampus, which may provide insight on the role of neuroglia in epileptogenesis. In this paper we have applied high throughput gene expression analysis to identify the molecular changes in the hippocampus that may explain the pathological processes. We have observed several classes of genes whose expression levels are changed. It is hypothesized that the upregulation of heat shock proteins (HSP70, HSP72, FOSL2 (HSP40), and their molecular chaperones BAG3 and DNAJB5 along with the down regulated gene MALAT1 may contribute to the neuroprotection observed. The increased expression of BDNF along with immediate early gene expression (FosB, JunB, ERG4, NR4A1, NR4A2, FBXO3) and the down regulation of GABRD, DBP and MALAT1 it is hypothesized may contribute to the hyperexcitability of the hippocampal neurons in this model. Activated astrocytes and microglia may also contribute to excitability pathomechanisms. Activated astrocytes in the ELS mouse are deficient in glutamine synthetase and thus reduce the clearance of extracellular glutamate. Activated microglia which may be associated with C1Q and MHC class I molecules we propose may mediate a process of selective removal of defective GABAergic synapses through a process akin to trogocytosis that may reduce neuronal inhibition and favor hyperexcitability.
10.1016/j.nbd.2020.105152
Complement factor C1q mediates sleep spindle loss and epileptic spikes after mild brain injury.
Science (New York, N.Y.)
Although traumatic brain injury (TBI) acutely disrupts the cortex, most TBI-related disabilities reflect secondary injuries that accrue over time. The thalamus is a likely site of secondary damage because of its reciprocal connections with the cortex. Using a mouse model of mild TBI (mTBI), we found a chronic increase in C1q expression specifically in the corticothalamic system. Increased C1q expression colocalized with neuron loss and chronic inflammation and correlated with disruption in sleep spindles and emergence of epileptic activities. Blocking C1q counteracted these outcomes, suggesting that C1q is a disease modifier in mTBI. Single-nucleus RNA sequencing demonstrated that microglia are a source of thalamic C1q. The corticothalamic circuit could thus be a new target for treating TBI-related disabilities.
10.1126/science.abj2685
Plasma Small Extracellular Vesicles with Complement Alterations in / and Sporadic Frontotemporal Lobar Degeneration.
Cells
Cutting-edge research suggests endosomal/immune dysregulation in /-associated frontotemporal lobar degeneration (FTLD). In this retrospective study, we investigated plasma small extracellular vesicles (sEVs) and complement proteins in 172 subjects (40 Sporadic FTLD, 40 Intermediate/Pathological expansion carriers, and 49 Heterozygous/Homozygous mutation carriers, 43 controls). Plasma sEVs (concentration, size) were analyzed by nanoparticle tracking analysis; plasma and sEVs C1q, C4, C3 proteins were quantified by multiplex assay. We demonstrated that genetic/sporadic FTLD share lower sEV concentrations and higher sEV sizes. The diagnostic performance of the two most predictive variables (sEV concentration/size ratio) was high (AUC = 0.91, sensitivity 85.3%, specificity 81.4%). C1q, C4, and C3 cargo per sEV is increased in genetic and sporadic FTLD. C4 (cargo per sEV, total sEV concentration) is increased in Sporadic FTLD and reduced in + Homozygous, suggesting its specific unbalance compared with Heterozygous cases. C3 plasma level was increased in genetic vs. sporadic FTLD. Looking at complement protein compartmentalization, in control subjects, the C3 and C4 sEV concentrations were roughly half that in respect to those measured in plasma; interestingly, this compartmentalization was altered in different ways in patients. These results suggest sEVs and complement proteins as potential therapeutic targets to mitigate neurodegeneration in FTLD.
10.3390/cells11030488
Enhanced synaptic connectivity and epilepsy in C1q knockout mice.
Chu Yunxiang,Jin Xiaoming,Parada Isabel,Pesic Alexei,Stevens Beth,Barres Ben,Prince David A
Proceedings of the National Academy of Sciences of the United States of America
Excessive CNS synapses are eliminated during development to establish mature patterns of neuronal connectivity. A complement cascade protein, C1q, is involved in this process. Mice deficient in C1q fail to refine retinogeniculate connections resulting in excessive retinal innervation of lateral geniculate neurons. We hypothesized that C1q knockout (KO) mice would exhibit defects in neocortical synapse elimination resulting in enhanced excitatory synaptic connectivity and epileptiform activity. We recorded spontaneous and evoked field potential activity in neocortical slices and obtained video-EEG recordings from implanted C1q KO and wild-type (WT) mice. We also used laser scanning photostimulation of caged glutamate and whole cell recordings to map excitatory and inhibitory synaptic connectivity. Spontaneous and evoked epileptiform field potentials occurred at multiple sites in neocortical slices from C1q KO, but not WT mice. Laser mapping experiments in C1q KO slices showed that the proportion of glutamate uncaging sites from which excitatory postsynaptic currents (EPSCs) could be evoked ("hotspot ratio") increased significantly in layer IV and layer V, although EPSC amplitudes were unaltered. Density of axonal boutons was significantly increased in layer V pyramidal neurons of C1q KO mice. Implanted KO mice had frequent behavioral seizures consisting of behavioral arrest associated with bihemispheric spikes and slow wave activity lasting from 5 to 30 s. Results indicate that epileptogenesis in C1q KO mice is related to a genetically determined failure to prune excessive excitatory synapses during development.
10.1073/pnas.0913449107
Selective expression of clusterin (SGP-2) and complement C1qB and C4 during responses to neurotoxins in vivo and in vitro.
Rozovsky I,Morgan T E,Willoughby D A,Dugichi-Djordjevich M M,Pasinetti G M,Johnson S A,Finch C E
Neuroscience
This study concerns expression of the genes encoding three multifunctional proteins: clusterin and two complement cascade components, C1q and C4. Previous work from this and other laboratories has established that clusterin, Clq and C4 messenger RNAs are elevated during Alzheimer's disease, and in response to deafferenting and excitotoxic brain lesion. This study addresses hippocampal clusterin, ClqB and C4 expression in response to neurotoxins that caused selective neuron death. Kainate, which preferentially kills hippocampal CA3 pyramidal neurons but not dentate gyrus granule neurons induced clusterin immunoreactivity in CA1 and CA3 pyramidal neurons and adjacent astrocytes, but not in dentate gyrus granule neurons. In contrast, colchicine, which preferentially kills the dentate gyrus granule neurons, induced clusterin immunoreactivity in the local neuropil as punctate deposits, but not in the surviving or degenerating dentate gyrus granule neurons. Clusterin messenger RNA was increased in astrocytes. ClqB and C4 messenger RNAs increased within 48 h after kainate injections, particularly in the CA3 pyramidal layer, less in the dentate gyrus-CA4, and less in CA1. Clq immunoreactivity was detected in CA1 pyramidal neurons and also as small punctate deposits in the CA1 region at eight and 14 days after kainate. The increase of both clusterin and ClqB messenger RNAs after kainate injections was blocked by barbiturates that prevented seizures and neurodegeneration. In primary hippocampal neuronal cultures treated with glutamate, a subpopulation of cultured neurons that survived glutamate toxicity also had parallel elevations of clusterin and ClqB messenger RNA. In conclusion, cytotoxins that target selective hippocampal neurons increase the expression of both clusterin and ClqB in vivo and in vitro. These results show that elevations of clusterin messenger RNA or protein can be dissociated from each other and from cell death. These increased messenger RNAs were associated with immunoreactive deposits that differed by cell type and intra- versus extracellular locations. These results suggest that the complement system is involved in brain responses to injury.
10.1016/0306-4522(94)90473-1