Francesca Cerbai

The Neuron-Astrocyte-Microglia Triad in Normal Brain Ageing and in a Model of Neuroinflammation in the Rat Hippocampus

Ageing is accompanied by a decline in cognitive functions; along with a variety of neurobiological changes. The association between inflammation and ageing is based on complex molecular and cellular changes that we are only just beginning to understand. The hippocampus is one of the structures more closely related to electrophysiological, structural and morphological changes during ageing. In the present study we examined the effect of normal ageing and LPS-induced inflammation on astroglia-neuron interaction in the rat hippocampus of adult, normal aged and LPS-treated adult rats. Astrocytes were smaller, with thicker and shorter branches and less numerous in CA1 Str. radiatum of aged rats in comparison to adult and LPS-treated rats. Astrocyte branches infiltrated apoptotic neurons of aged and LPS-treated rats. Cellular debris, which were more numerous in CA1 of aged and LPS-treated rats, could be found apposed to astrocytes processes and were phagocytated by reactive microglia. Reactive microglia were present in the CA1 Str. Radiatum, often in association with apoptotic cells. Significant differences were found in the fraction of reactive microglia which was 40% of total in adult, 33% in aged and 50% in LPS-treated rats. Fractalkine (CX3CL1) increased significantly in hippocampus homogenates of aged and LPS-treated rats. The number of CA1 neurons decreased in aged rats. In the hippocampus of aged and LPS-treated rats astrocytes and microglia may help clearing apoptotic cellular debris possibly through CX3CL1 signalling. Our results indicate that astrocytes and microglia in the hippocampus of aged and LPS-infused rats possibly participate in the clearance of cellular debris associated with programmed cell death. The actions of astrocytes may represent either protective mechanisms to control inflammatory processes and the spread of further cellular damage to neighboring tissue, or they may contribute to neuronal damage in pathological conditions.

Cannabinoid receptor stimulation is anti-inflammatory and improves memory in old rats

The number of activated microglia increase during normal aging. Stimulation of endocannabinoid receptors can reduce the number of activated microglia, particularly in the hippocampus, of young rats infused chronically with lipopolysaccharide (LPS). In the current study we demonstrate that endocannabinoid receptor stimulation by administration of WIN-55212-2 (2mg/kg day) can reduce the number of activated microglia in hippocampus of aged rats and attenuate the spatial memory impairment in the water pool task. Our results suggest that the action of WIN-55212-2 does not depend upon a direct effect upon microglia or astrocytes but is dependent upon stimulation of neuronal cannabinoid receptors. Aging significantly reduced cannabinoid type 1 receptor binding but had no effect on cannabinoid receptor protein levels. Stimulation of cannabinoid receptors may provide clinical benefits in age-related diseases that are associated with brain inflammation, such as Alzheimers disease.

Differential activation of mitogen-activated protein kinase signalling pathways in the hippocampus of CRND8 transgenic mouse, a model of Alzheimer's disease

Transgenic Centre for Research in Neurodegenerative Diseases 8 (TgCRND8) mice expressing a double mutant form of human amyloid precursor protein represent a good model of Alzheimers disease, and can be useful to clarify the involvement of mitogen-activated protein kinases (MAPK) dysregulation in the pathophysiology of this neurodegenerative disorder. Activation of extracellular regulated kinase (ERK) 1/2, jun kinase (JNK) and p38MAPK was studied in the hippocampus of 7-month-old TgCRND8 mice by immunohistochemistry and Western blot analysis using antibodies selective for the phosphorylated, and thus active, forms of the enzymes. We demonstrated that the three main MAPK pathways were differentially activated in cells of the hippocampus of TgCRND8 mice in comparison to wild type (Wt) littermates, p38MAPK and JNK being more activated, while ERK less activated. p38MAPK was significantly activated in microglia, astrocytes and neurons, around and distant from the plaques. JNK was highly activated in cells closely surrounding the plaques. No difference was observed in the activation of the two major bands of JNK, at a molecular weight of 46 kDa and 54 kDa. These data indicate the possible involvement of p38MAPK and JNK pathways dysregulation in the pathogenesis of Alzheimers disease. The ERK2 isoform of the ERK pathway was less activated in the hippocampal dentate gyrus of Tg mice in basal conditions. Furthermore activation of the ERK pathway by ex vivo cholinergic stimulation with carbachol caused significantly higher activation of ERK in the hippocampus of Wt mice than in Tg mice. These findings may pose a molecular basis for the memory disruption of Alzheimers disease, since proper functioning of the basal forebrain cholinergic neurons and of ERK2 is critical for memory formation.

Inhibition of acetylcholine-induced activation of extracellular regulated protein kinase prevents the encoding of an inhibitory avoidance response in the rat

It has been demonstrated that the forebrain cholinergic system and the extracellular regulated kinase signal transduction pathway are involved in the mechanisms of learning, encoding, and storage of information. We investigated the involvement of the cholinergic and glutamatergic systems projecting to the medial prefrontal cortex and ventral hippocampus and of the extracellular regulated kinase signal transduction pathway in the acquisition and recall of the step-down inhibitory avoidance response in the rat, a relatively simple behavioral test acquired in a one-trial session. To this aim we studied by microdialysis the release of acetylcholine and glutamate, and by immunohistochemistry the activation of extracellular regulated kinase during acquisition, encoding and recall of the behavior. Cholinergic, but not glutamatergic, neurons projecting to the medial prefrontal cortex and ventral hippocampus were activated during acquisition of the task, as shown by increase in cortical and hippocampal acetylcholine release. Released acetylcholine in turn activated extracellular regulated kinase in neurons located in the target structures, since the muscarinic receptor antagonist scopolamine blocked extracellular regulated kinase activation. Both increased acetylcholine release and extracellular regulated kinase activation were necessary for memory formation, as administration of scopolamine and of extracellular regulated kinase inhibitors was followed by blockade of extracellular regulated kinase activation and amnesia. Our data indicate that a critical function of the learning-associated increase in acetylcholine release is to promote the activation of the extracellular regulated kinase signal transduction pathway and help understanding the role of these systems in the encoding of an inhibitory avoidance memory.

P2 receptor antagonists prevent synaptic failure and extracellular signal-regulated kinase 1/2 activation induced by oxygen and glucose deprivation in rat CA1 hippocampus in vitro.

To investigate the role of purinergic P2 receptors under ischemia, we studied the effect of P2 receptor antagonists on synaptic transmission and mitogen‐activated protein kinase (MAPK) activation under oxygen and glucose deprivation (OGD) in rat hippocampal slices. The effect of the P2 antagonists pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonate (PPADS, unselective, 30 μm), N 6‐methyl‐2′‐deoxyadenosine‐3′,5′‐bisphosphate (MRS2179, selective for P2Y1 receptor, 10 μm), Brilliant Blue G (BBG, selective for P2X7 receptor, 1 μm), and 5‐[[[(3‐phenoxyphenyl)methyl][(1S)‐1,2,3,4‐tetrahydro‐1‐naphthalenyl]amino]carbonyl]‐1,2,4‐benzenetricarboxylic acid (A‐317491, selective for P2X3 receptor, 10 μm), and of the newly synthesized P2X3 receptor antagonists 2‐amino‐9‐(5‐iodo‐2‐isopropyl‐4‐methoxybenzyl)adenine (PX21, 1 μm) and 2‐amino‐9‐(5‐iodo‐2‐isopropyl‐4‐methoxybenzyl)‐N 6‐methyladenine (PX24, 1 μm), on the depression of field excitatory postsynaptic potentials (fEPSPs) and anoxic depolarization (AD) elicited by 7 min of OGD were evaluated. All antagonists significantly prevented these effects. The extent of CA1 cell injury was assessed 3 h after the end of 7 min of OGD by propidium iodide staining. Substantial CA1 pyramidal neuronal damage, detected in untreated slices exposed to OGD injury, was significantly prevented by PPADS (30 μm), MRS2179 (10 μm), and BBG (1 μm). Western blot analysis showed that, 10 min after the end of the 7 min of OGD, extracellular signal‐regulated kinase (ERK)1/2 MAPK activation was significantly increased. MRS2179, BBG, PPADS and A‐317491 significantly counteracted ERK1/2 activation. Hippocampal slices incubated with the ERK1/2 inhibitors 1,4‐diamino‐2,3‐dicyano‐1,4‐bis(2‐aminophenylthio)butadiene (U0126, 10 μm) and α‐[amino[(4‐aminophenyl)thio]methylene]‐2‐(trifluoromethyl) benzeneacetonitrile (SL327, 10 μm) showed significant fEPSP recovery after OGD and delayed AD, supporting the involvement of ERK1/2 in neuronal damage induced by OGD. These results indicate that subtypes of hippocampal P2 purinergic receptors have a harmful effect on neurotransmission in the CA1 hippocampus by participating in AD appearance and activation of ERK1/2.

Hippocampal long term memory: Effect of the cholinergic system on local protein synthesis

The present study was aimed at establishing a link between the cholinergic system and the pathway of mTOR and its downstream effector p70S6K, likely actors in long term memory encoding. We performed in vivo behavioral experiments using the step down inhibitory avoidance test (IA) in adult Wistar rats to evaluate memory formation under different conditions, and immunohistochemistry on hippocampal slices to evaluate the level and the time-course of mTOR and p70S6K activation. We also examined the effect of RAPA, inhibitor of mTORC1 formation, and of the acetylcholine (ACh) muscarinic receptor antagonist scopolamine (SCOP) or ACh nicotinic receptor antagonist mecamylamine (MECA) on short and long term memory formation and on the functionality of the mTOR pathway. Acquisition test was performed 30 min after i.c.v. injection of RAPA, a time sufficient for the drug to diffuse to CA1 pyramidal neurons, as demonstrated by MALDI-TOF-TOF imaging. Recall test was performed 1 h, 4 h or 24 h after acquisition. To confirm our results we performed in vitro experiments on live hippocampal slices: we evaluated whether stimulation of the cholinergic system with the cholinergic receptor agonist carbachol (CCh) activated the mTOR pathway and whether the administration of the above-mentioned antagonists together with CCh could revert this activation. We found that (1) mTOR and p70S6K activation in the hippocampus were involved in long term memory formation; (2) RAPA administration caused inhibition of mTOR activation at 1 h and 4 h and of p70S6K activation at 4 h, and long term memory impairment at 24 h after acquisition; (3) scopolamine treatment caused short but not long term memory impairment with an early increase of mTOR/p70S6K activation at 1 h followed by stabilization at longer times; (4) mecamylamine plus scopolamine treatment caused short term memory impairment at 1 h and 4 h and reduced the scopolamine-induced increase of mTOR/p70S6K activation at 1 h and 4 h; (5) mecamylamine plus scopolamine treatment did not impair long term memory formation; (6) in vitro treatment with carbachol activated mTOR and p70S6K and this effect was blocked by scopolamine and mecamylamine. Taken together our data reinforce the idea that distinct molecular mechanisms are at the basis of the two different forms of memory and are in accordance with data presented by other groups that there exist molecular mechanisms that underlie short term memory, others that underlie long term memories, but some mechanisms are involved in both.

Clasmatodendrosis and β-amyloidosis in Aging Hippocampus

Alterations of the tightly interwoven neuron/astrocyte interactions are frequent traits of aging, but also favor neurodegenerative diseases, such as Alzheimer disease (AD). These alterations reflect impairments of the innate responses to inflammation‐related processes, such as β‐amyloid (Ab) burdening. Multidisciplinary studies, spanning from the tissue to the molecular level, are needed to assess how neuron/astrocyte interactions are influenced by aging. Our study addressed this requirement by joining fluorescence‐lifetime imaging microscopy/phasor multiphoton analysis with confocal microscopy, implemented with a novel method to separate spectrally overlapped immunofluorescence and Aβ autofluorescence. By comparing data from young control rats, chronically inflamed rats, and old rats, we identified age‐specific alterations of neuron/astrocyte interactions in the hippocampus. We found a correlation between Aβ aggregation (+300 and +800% of aggregated Aβ peptide in chronically inflamed and old vs. control rats, respectively) and fragmentation (clasmatodendrosis) of astrocyte projections (APJs) (+250 and +1300% of APJ fragments in chronically inflamed and old vs. control rats, respectively). Clasmatodendrosis, in aged rats, associates with impairment of astrocyte‐mediated Aβ clearance (245% of Aβ deposits on APJs, and +33% of Aβ deposits on neurons in old vs. chronically inflamed rats). Furthermore, APJ fragments colocalize with Aβ deposits and are involved in novel Aβ‐mediated adhesions between neurons. These data define the effects of Aβ deposition on astrocyte/neuron interactions as a key topic in AD biology.—Mercatelli, R., Lana, D., Bucciantini, M., Giovannini, M. G., Cerbai, F., Quercioli, F., Zecchi‐Orlandini, S., Delfino, G., Wenk, G. L., Nos, D. Clasmatodendrosis and β‐amyloidosis in aging hippocampus. FASEB J. 30, 1480–1491 (2016). www.fasebj.org

Changes in glia-neuron interactions in rat hippocampus induced by aging and inflammation

Aging is accompanied by a decline in cognitive functions, along with a variety of neurobiological changes. Recently, the term “inflammaging” has been coined to characterize a widely accepted paradigm that aging is accompanied by a low-grade chronic up-regulation of certain proinflammatory responses. The association between inflammation, aging, and Alzheimer Disease, is based on complex molecular and cellular changes that we are only just beginning to understand. The hippocampus plays a critical role in memory formation and is one of the structures more closely related to electrophysiological, structural and morphological changes during aging. In the present study, we examined the effect of normal aging and LPS-induced inflammation on glia/neuron interaction in the CA1 region by confocal immunofluorescence. The density of GFAP positive astrocytes was significantly lower in CA1 region of aged rats, with a mean hippocampal density of 522±8/mm2in the young (n=12) and of 420±16/mm2 in the aged rat (n=15; -20% vs young rats, P < 0.0001) but it did not change in LPS-treated rats (548.5±38/ mm2, n=6, n.s.). Confocal microscopy indicated that in the hippocampus of aged and LPS-treated rats astrocytes were smaller, with thicker and shorter branchings than in young rats and with morphological signs of clasmatodendrosis. In aged and LPS-treated rats apoptotic neurons (as evidenced by AIF or CytC immunohistochemistry) were surrounded by astrocyte branchings, apparently in the process of clearing up the cellular debris. Apoptotic cell debris, scattered throughout the CA1 region, were significantly higher in aged (496±29/mm2, n=10) and LPS-treated rats (106±25/mm2, n=6) than in young rats (22.7±5/mm2, n=12, P < 0.001 vs both other groups) and were all juxtaposed to astrocytes cell bodies and branchings. Scarse activated microglia (OX-6 immunopositive) were present in CA1 of young rats (2.5±1, n=10) while substantial infiltration of activated microglia were present in CA1 of aged and LPS treated rats. These cells were hypertrophic, ranging from densely arborized cells to cells with a bushy appearance with swollen cell bodies and intensely stained short processes, often in close association with apoptotic cells. These data show that senescence-induced modifications of astrocytes and microglia in the hippocampus of aged and LPS treated rats may help clearing the cellular debris derived from apoptotic mechanisms. This might be a protective mechanism that possibly controls inflammatory processes and spread of further cellular damage to neighboring tissues.