BackgroundAlzheimer’s disease (AD) is the most prevalent form of age-related dementia, and its effect on society increases exponentially as the population ages. Accumulating evidence suggests that neuroinflammation, mediated by the brain’s innate immune system, contributes to AD neuropathology and exacerbates the course of the disease. However, there is no experimental evidence for a causal link between systemic inflammation or neuroinflammation and the onset of the disease.MethodsThe viral mimic, polyriboinosinic-polyribocytidilic acid (PolyI:C) was used to stimulate the immune system of experimental animals. Wild-type (WT) and transgenic mice were exposed to this cytokine inducer prenatally (gestation day (GD)17) and/or in adulthood. Behavioral, immunological, immunohistochemical, and biochemical analyses of AD-associated neuropathologic changes were performed during aging.ResultsWe found that a systemic immune challenge during late gestation predisposes WT mice to develop AD-like neuropathology during the course of aging. They display chronic elevation of inflammatory cytokines, an increase in the levels of hippocampal amyloid precursor protein (APP) and its proteolytic fragments, altered Tau phosphorylation, and mis-sorting to somatodendritic compartments, and significant impairments in working memory in old age. If this prenatal infection is followed by a second immune challenge in adulthood, the phenotype is strongly exacerbated, and mimics AD-like neuropathologic changes. These include deposition of APP and its proteolytic fragments, along with Tau aggregation, microglia activation and reactive gliosis. Whereas Aβ peptides were not significantly enriched in extracellular deposits of double immune-challenged WT mice at 15 months, they dramatically increased in age-matched immune-challenged transgenic AD mice, precisely around the inflammation-induced accumulations of APP and its proteolytic fragments, in striking similarity to the post-mortem findings in human patients with AD.ConclusionChronic inflammatory conditions induce age-associated development of an AD-like phenotype in WT mice, including the induction of APP accumulations, which represent a seed for deposition of aggregation-prone peptides. The PolyI:C mouse model therefore provides a unique tool to investigate the molecular mechanisms underlying the earliest pathophysiological changes preceding fibrillary Aβ plaque deposition and neurofibrillary tangle formations in a physiological context of aging. Based on the similarity between the changes in immune-challenged mice and the development of AD in humans, we suggest that systemic infections represent a major risk factor for the development of AD.

In addition to the fundamental role of the extracellular glycoprotein Reelin in neuronal development and adult synaptic plasticity, alterations in Reelin-mediated signaling have been suggested to contribute to neuronal dysfunction associated with Alzheimer's disease (AD). In vitro data revealed a biochemical link between Reelin-mediated signaling, Tau phosphorylation, and amyloid precursor protein (APP) processing. To directly address the role of Reelin in amyloid-␤ plaque and Tau pathology in vivo, we crossed heterozygous Reelin knock-out mice (reeler) with transgenic AD mice to investigate the temporal and spatial AD-like neuropathology. We demonstrate that a reduction in Reelin expression results in enhanced amyloidogenic APP processing, as indicated by the precocious production of amyloid-␤ peptides, the significant increase in number and size of amyloid-␤ plaques, as well as age-related aggravation of plaque pathology in double mutant compared with single AD mutant mice of both sexes. Numerous amyloid-␤ plaques accumulate in the hippocampal formation and neocortex of double mutants, precisely in layers with strongest Reelin expression and highest accumulation of Reelin plaques in aged wild-type mice. Moreover, concentric accumulations of phosphorylated Tau-positive neurons around amyloid-␤ plaques were evident in 15-month-old double versus single mutant mice. Silver stainings indicated the presence of neurofibrillary tangles, selectively associated with amyloid-␤ plaques and dystrophic neurites in the entorhinal cortex and hippocampus. Our findings suggest that age-related Reelin aggregation and concomitant reduction in Reelin-mediated signaling play a proximal role in synaptic dysfunction associated with amyloid-␤ deposition, sufficient to enhance Tau phosphorylation and tangle formation in the hippocampal formation in aged Reelin-deficient transgenic AD mice.

Cajal-Retzius cells play a crucial role during ontogeny in regulating cortical lamination via release of reelin. In adult brain, they comprise small calretinin-positive interneurons located in the marginal zone of the cerebral cortex and in the hippocampal fissure. Alterations of reelin signaling or expression have been involved in major neurological disorders, and they underlie granule cell dispersion (GCD) in mesial temporal lobe epilepsy (TLE). Here, we investigated in a mouse model of TLE the contribution of Cajal-Retzius cells to reelin production in epileptic hippocampus and the molecular mechanisms underlying GCD. Following unilateral intrahippocampal Kainic acid injection in adult mice to induce an epileptic focus, we observed that Cajal-Retzius cells gradually became strongly immunopositive for reelin, due to intracellular accumulation. This phenotype resembled the morphology of Cajal-Retzius cells in reeler Orleans (reln (orl/orl) ) mice, which express a secretion-deficient 310-kDa reelin fragment. The possibility that GCD might result from abnormal reelin processing in Cajal-Retzius cells, leading to a lack of reelin secretion, was confirmed by KA injection in reln (orl/+) mice, which induced severe GCD. Furthermore, Western blot analysis in KA-treated wildtype mice revealed increased production of ∼300-kDa reelin fragments, confirming abnormal proteolytic processing. This effect was not seen upon treatment with Botulinum neurotoxin E (BoNT/E), which prevents GCD in KA-lesioned hippocampus by chronic blockade of synaptic transmission. Furthermore, BoNT/E blocked upregulation of TrkB in Cajal-Retzius cells, suggesting that production of truncated reelin in KA-treated hippocampus is activity-dependent and regulated by BDNF. Altogether, these data reveal that GCD results from abnormal reelin processing in Cajal-Retzius cells under the control of BDNF. Our findings highlight the critical role played by Cajal-Retzius cells for hippocampal neuronal reorganization in TLE.

Reelin is a large extracellular glycoprotein required for proper neuronal positioning during development. In the adult brain, Reelin plays a crucial modulatory role in the induction of synaptic plasticity and successful formation of long-term memory. Recently, alterations in Reelin-mediated signaling have been suggested to contribute to neuronal dysfunction associated with Alzheimer's disease (AD). We previously reported that aging in several species is characterized by a decline in Reelin-expressing interneurons and concomitant accumulation in amyloid-like plaques in the hippocampal formation, significantly correlating with cognitive impairments. In transgenic AD mice, we detected Reelin in oligomeric amyloid-beta aggregates and in tight association with fibrillary plaques. Here, we used immunohistochemistry at the light and electron microscopy level to characterize further the morphology, temporal and spatial progression, as well as the potential of Reelin-positive plaques to sequester murine amyloid-beta peptides in wild-type mice. We developed a new immunohistochemical protocol involving a stringent protease pretreatment which markedly enhanced Reelin-immunoreactivity and allowed specific detection of variable shapes of murine anti-amyloid-beta protein precursor-immunoreactivity in plaques in the hippocampus, likely representing N-terminal fragments and amyloid-beta species. Ultrastructural investigations confirmed the presence of Reelin in extracellular space, somata of interneurons in young and aged wild-type mice. In aged mice, Reelin- and amyloid-beta-immunoreactivity was detected in extracellular, spherical deposits, likely representing small intermediates or fragments of amyloid fibrils. Our results suggest that Reelin itself aggregates into abnormal oligomeric or protofibrillary deposits during aging, potentially creating a precursor condition for fibrillary amyloid-beta plaque formation.

Besides its critical role during neurodevelopment, the extracellular glycoprotein reelin is also a pivotal regulator of adult synaptic function and plasticity, and altered reelin-mediated signalling has been suggested to contribute to neuronal dysfunction associated with Alzheimer's disease. We have recently discovered, in aged rodents and non-human primates, a pronounced decline in reelin-positive interneurons and concomitant accumulation of reelin in extracellular amyloid-like deposits, both being associated with episodic-like memory impairments. Here, we report that these age-related neuropathological changes in hippocampus, entorhinal and piriform cortices of aged wild-type mice are accompanied by abnormal axonal varicosities and altered expression profiles of calcium-binding proteins in plaque-dense areas, as well as a significant reduction in the number of parvalbumin-positive gamma-aminobutyric acid (GABA)ergic projection neurons in basal forebrain areas, including medial septum (MS), ventral and horizontal diagonal Band of Broca (VDB/HDB) and substantia innominata (SI), compared with young subjects. In addition, a significant reduction in the number of choline acetyltransferase-positive cholinergic projection neurons was evident in the HDB/SI area but not in the MS of aged compared with young wild-type mice. No reelin-deposits were found in these basal forebrain regions. Our findings suggest that the elevated reelin plaque load in the projection areas of afferent subcortical GABAergic and cholinergic neurons including hippocampus, entorhinal and piriform cortices affects the axonal integrity and survival of these neurons, potentially contributing to the cognitive impairments observed in aged wild-type mice.