Extracellular aggregates of amyloid β (Aβ) peptides, which are characteristic of Alzheimer's disease (AD), act as an essential trigger for glial cell activation and the release of ATP, leading to the ...stimulation of purinergic receptors, especially the P2X7 receptor (P2X7R). However, the involvement of P2X7R in the development of AD is still ill-defined regarding the dual properties of this receptor. Particularly, P2X7R activates the NLRP3 inflammasome leading to the release of the pro-inflammatory cytokine, IL-1β; however, P2X7R also induces cleavage of the amyloid precursor protein generating Aβ peptides or the neuroprotective fragment sAPPα. We thus explored in detail the functions of P2X7R in AD transgenic mice. Here, we show that P2X7R deficiency reduced Aβ lesions, rescued cognitive deficits and improved synaptic plasticity in AD mice. However, the lack of P2X7R did not significantly affect the release of IL-1β or the levels of non-amyloidogenic fragment, sAPPα, in AD mice. Instead, our results show that P2X7R plays a critical role in Aβ peptide-mediated release of chemokines, particularly CCL3, which is associated with pathogenic CD8
T cell recruitment. In conclusion, our study highlights a novel detrimental function of P2X7R in chemokine release and supports the notion that P2X7R may be a promising therapeutic target for AD.
In the CA3 region of the hippocampus, pyramidal cells excite other pyramidal cells and interneurons. The axons of CA3 pyramidal cells spread throughout most of the region to form an associative ...network. These connections were first drawn by Cajal and Lorente de No. Their physiological properties were explored to understand epileptiform discharges generated in the region. Synapses between pairs of pyramidal cells involve one or few release sites and are weaker than connections made by mossy fibers on CA3 pyramidal cells. Synapses with interneurons are rather effective, as needed to control unchecked excitation. We examine contributions of recurrent synapses to epileptiform synchrony, to the genesis of sharp waves in the CA3 region and to population oscillations at theta and gamma frequencies. Recurrent connections in CA3, as other associative cortices, have a lower connectivity spread over a larger area than in primary sensory cortices. This sparse, but wide-ranging connectivity serves the functions of an associative network, including acquisition of neuronal representations as activity in groups of CA3 cells and completion involving the recall from partial cues of these ensemble firing patterns.
Using transcriptomics, anatomical studies, imaging and ELISA, Morin-Brureau et al. examine microglia in patients with temporal lobe epilepsies. In highly sclerotic regions such as CA1, the ...anti-inflammatory cytokine IL-10 regulates microglial phenotype. Seizures induce a transient microglial phenotype associated with secretion of inflammatory cytokines including human CXCL8.
Abstract
Microglia, the immune cells of the brain, are highly plastic and possess multiple functional phenotypes. Differences in phenotype in different regions and different states of epileptic human brain have been little studied. Here we use transcriptomics, anatomy, imaging of living cells and ELISA measurements of cytokine release to examine microglia from patients with temporal lobe epilepsies. Two distinct microglial phenotypes were explored. First we asked how microglial phenotype differs between regions of high and low neuronal loss in the same brain. Second, we asked how microglial phenotype is changed by a recent seizure. In sclerotic areas with few neurons, microglia have an amoeboid rather than ramified shape, express activation markers and respond faster to purinergic stimuli. The repairing interleukin, IL-10, regulates the basal phenotype of microglia in the CA1 and CA3 regions with neuronal loss and gliosis. To understand changes in phenotype induced by a seizure, we estimated the delay from the last seizure until tissue collection from changes in reads for immediate early gene transcripts. Pseudotime ordering of these data was validated by comparison with results from kainate-treated mice. It revealed a local and transient phenotype in which microglia secrete the human interleukin CXCL8, IL-1B and other cytokines. This secretory response is mediated in part via the NRLP3 inflammasome.
Intracerebral injections of tracers or viral constructs in rodents are now commonly used in the neurosciences and must be executed perfectly. The purpose of this article is to update existing ...protocols for intracerebral injections in adult and neonatal mice. Our procedure for stereotaxic injections in adult mice allows the investigator to improve the effectiveness and safety, and save time. Furthermore, for the first time, we describe a two-handed procedure for intracerebral injections in neonatal mice that can be performed by a single operator in a very short time. Our technique using the stereotaxic arm allows a higher precision than freehand techniques previously described. Stereotaxic injections in adult mice can be performed in 20 min and have 〉90% efficacy in targeting the injection site. Injections in neonatal mice can be performed in 5 min. Efficacy depends on the difficulty of precisely localizing the injection sites, due to the small size of the animal. We describe an innovative, effortless, and reproducible surgical protocol for intracerebral injections in adult and neonatal mice.
Lipid homeostasis is dysregulated in some neurodegenerative diseases and after brain injuries due to excess glutamate or lack of oxygen. However the kinetics and cell specificity of dysregulation in ...different groups of lipids during excitotoxic neuronal death are not clear. Here we examined the changes during excitotoxic neuronal death induced by injecting kainic acid (KA) into the CA1 region of mouse hippocampus. We compared neuronal loss and glial cell proliferation with changes in lipid‐related transcripts and markers for different lipid groups, over 12 days after KA‐treatment. As neurons showed initial signs of damage, transcripts and proteins linked to fatty acid oxidation were up‐regulated. Cholesterol biosynthesis induced by transcripts controlled by the transcription factor Srebp2 seems to be responsible for a transient increase in neuronal free cholesterol at 1 to 2 days. In microglia, but not in neurons, Perilipin‐2 associated lipid droplets were induced and properties of Nile red emissions suggest lipid contents change over time. After microglial expression of phagocytotic markers at 2 days, some neutral lipid deposits co‐localized with lysosome markers of microglia and were detected within putative phagocytotic cups. These data delineate distinct lipid signals in neurons and glial cells during excitotoxic processes from initial neuronal damage to engagement of the lysosome–phagosome system.
We used lipid markers and transcriptomics of lipid‐related molecules to examine changes during 12 days of excitotoxic neuronal death after hippocampal kainic acid injection. Immunohistochemistry, live cell imaging and electron microscopy reveal distinct changes in hippocampal neurons and glia. Neurons transiently accumulate cholesterol while microglia express Nile red positive droplets of neutral lipid (Figure) during excitotoxic processes from inflammation to the phagocytosis of dead neurons.
Intra-hippocampal kainate injection induces the emergence of recurrent seizures after a delay of 3â4 weeks. We examined the
cellular and synaptic basis of this activity in vitro using extracellular ...and intracellular records from longitudinal hippocampal slices. These slices permitted recordings from
the dentate gyrus, the CA3 and CA1 regions and the subiculum of both the injected and the contralateral non-injected hippocampus.
A sclerotic zone was evident in dorsal regions of slices from the injected hippocampus, while ventral regions and tissue from
the contralateral hippocampus were not sclerotic. Interictal field potentials of duration 50â200 ms were generated spontaneously
in both ipsilateral and contralateral hippocampal slices, but not in the sclerotic region, at 3â12 months after injection.
They were initiated in the CA1 and CA3 regions and the subiculum. They were blocked by antagonists at glutamatergic receptors
and were transformed into prolonged epileptiform events by GABAergic receptor antagonists. The membrane potential and the
reversal potential of GABAergic synaptic events were more depolarized in CA1 pyramidal cells from kainate-treated animals
than in control animals. Ictal-like events of duration 8â80 s were induced by tetanic stimulation (50 Hz, 0.2â1 s) preferentially
in dorsal contralateral and ventral ipsilateral slices. Similar events were initiated by focal application of a combination
of high K + and GABA. These data show that both interictal and ictal-like activities can be induced in slices of both ipsilateral and
contralateral hippocampus from kainate-treated animals and suggest that changes in cellular excitability and inhibitory synaptic
signalling may contribute to their generation.
The GGGGCC intronic repeat expansion within
is the most common genetic cause of ALS and FTD. This mutation results in toxic gain of function through accumulation of expanded RNA foci and aggregation ...of abnormally translated dipeptide repeat proteins, as well as loss of function due to impaired transcription of
. A number of
and
models of gain and loss of function effects have suggested that both mechanisms synergize to cause the disease. However, the contribution of the loss of function mechanism remains poorly understood. We have generated C9ORF72 knockdown mice to mimic C9-FTD/ALS patients haploinsufficiency and investigate the role of this loss of function in the pathogenesis. We found that decreasing C9ORF72 leads to anomalies of the autophagy/lysosomal pathway, cytoplasmic accumulation of TDP-43 and decreased synaptic density in the cortex. Knockdown mice also developed FTD-like behavioral deficits and mild motor phenotypes at a later stage. These findings show that C9ORF72 partial loss of function contributes to the damaging events leading to C9-FTD/ALS.
Focal epilepsies of the mesial temporal lobe are often associated with a hippocampal sclerosis. Changes in Cl homeostasis and GABAergic signalling in downstream regions, may contribute to the human ...pathology. A review of changes in cellular and synaptic function and connectivity after de-afferentation suggests this epileptic syndrome may involve a pathological replay of developmental mechanisms.
Microglia exhibit multiple, phenotype-dependent motility patterns often triggered by purinergic stimuli. However, little data exist on motility of human microglia in pathological situations. Here we ...examine motility of microglia stained with a fluorescent lectin in tissue slices from female and male epileptic patients diagnosed with mesial temporal lobe epilepsy or cortical glioma (peritumoral cortex). Microglial shape varied from ramified to amoeboid cells predominantly in regions of high neuronal loss or closer to a tumor. Live imaging revealed unstimulated or purine-induced microglial motilities, including surveillance movements, membrane ruffling, and process extension or retraction. At different concentrations, ADP triggered opposing motilities. Low doses triggered process extension. It was suppressed by P2Y12 receptor antagonists, which also reduced process length and surveillance movements. Higher purine doses caused process retraction and membrane ruffling, which were blocked by joint application of P2Y1 and P2Y13 receptor antagonists. Purinergic effects on motility were similar for all microglia tested. Both amoeboid and ramified cells from mesial temporal lobe epilepsy or peritumoral cortex tissue expressed P2Y12 receptors. A minority of microglia expressed the adenosine A2A receptor, which has been linked with process withdrawal of rodent cells. Laser-mediated tissue damage let us test the functional significance of these effects. Moderate damage induced microglial process extension, which was blocked by P2Y12 receptor antagonists. Overall, the purine-induced motility of human microglia in epileptic tissue is similar to that of rodent microglia in that the P2Y12 receptor initiates process extension. It differs in that retraction is triggered by joint activation of P2Y1/P2Y13 receptors.
Microglial cells are brain-resident immune cells with multiple functions in healthy or diseased brains. These diverse functions are associated with distinct phenotypes, including different microglial shapes. In the rodent, purinergic signaling is associated with changes in cell shape, such as process extension toward tissue damage. However, there are little data on living human microglia, especially in diseased states. We developed a reliable technique to stain microglia from epileptic and glioma patients to examine responses to purines. Low-intensity purinergic stimuli induced process extension, as in rodents. In contrast, high-intensity stimuli triggered a process withdrawal mediated by both P2Y1 and P2Y13 receptors. P2Y1/P2Y13 receptor activation has not previously been linked to microglial morphological changes.
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