In line with this hypothesis and the critical role of Reelin in synaptic transmission and learning and memory, several recent findings suggest that alterations in Reelin expression and abnormal Reelin signaling may contribute to neuronal dysfunction associated with AD. In addition, several studies have investigated the molecular mechanism by which Reelin, its receptors and downstream signaling proteins may contribute to the pathophysiology of this progressive neurodegenerative disease. The purpose of this section is to summarize the current knowledge and recent findings related to the molecular link between Reelin dysfunction and AD-related neuropathology.
AD is a complex neurodegenerative disease that afflicts an increasing fraction of our aging population. It is characterized by progressive cognitive decline and severe neurodegeneration [81
]. Neuropathological hallmarks comprise neurofibrillary tangles (NFTs), consisting of hyperphosphorylated Tau [82
] and the formation of senile plaques, primarily composed of amyloid-β (Aβ) peptides [83
]; the amyloidogenic cleavage product of APP that results from sequential cleavage by β- and γ-secretases [84
]. Abnormal amyloidogenic Aβ processing and formation of amyloid-β plaques have been suggested to lead to synaptic dysfunction, synapse loss, and ultimately to neuronal death. Non-amyloidogenic processing by cleavage through α- and γ-secretase results in the production and release of a large N-terminal extracellular fragment and smaller, membrane-bound C-terminal fragments with putative neuroprotective and transcriptional functions [86
]. Recently, the team of Tessier-Lavigne discovered an additional disease-modifying, N-terminal fragment of APP that acts as ligand of the death receptor 6 (DR6, a member of the tumor necrosis factor receptor superfamily). This fragment is produced upon growth factor withdrawal by β-secretase plus an additional, as yet unidentified, protease and has been shown to induce Caspase-6-mediated axonal degeneration [89
], a process involved in axonal pruning during development. These recent results suggest that abnormal neurodegeneration associated with AD might be due to inappropriate activation or reactivation of a developmental pathway in the aged brain.
Recent insights into the pathophysiology of Aβ peptides suggested a potent negative impact of Aβ oligomers on synaptic functions that may underlie impairments in long-term synaptic plasticity [90
]. Incubation of hippocampal neurons with Aβ oligomers lead to intracellular trapping or functional impairment of AMPA and NMDA receptors [93
], thereby decreasing LTP [90
]. As already mentioned, several studies showed an increase in LTP following Reelin application to hippocampal slices [28
], demonstrating that Reelin has an opposite effect on synaptic function compared to Aβ oligomers. A recent study conducted in Joachim Herz’ lab demonstrated that Reelin could indeed antagonize the suppressive effects of Aβ oligomers on synaptic NMDA receptor-mediated neurotransmission [100
]. They demonstrated that Reelin signaling in excitatory synapses could restore oligomeric Aβ–induced impairments in synaptic plasticity to normal levels. Activation of SFKs by Reelin through ApoER2 and VLDLR binding was necessary for neutralizing the Aβ-mediated suppression, supporting again the crucial role of Reelin-mediated signaling for normal synaptic function.
The group of Saez-Valero has recently addressed the putative link between abnormal Reelin levels and AD pathogenesis. They analyzed the expression and glycosylation pattern of Reelin in CSF and cortical tissue extracts obtained from AD patients and non-demented controls [101
]. They reported abnormal glycosylation of Reelin as well as a preferential up-regulation of the N-terminal 180-kDa Reelin fragment in the CSF and frontal cortex of AD patients. Full-length and other proteolytic fragments of Reelin remained unaltered in control and AD subjects [101
], suggesting alterations in Reelin processing and signaling in AD. Another recent study showed that the N-terminal fragment of Reelin can be generated within the endosomes after internalization of the full-length form [104
], pointing to the possibility that in AD patients the endosomal recycling and re-secretion of this fragment into the extracellular space could also be significantly affected.
Our own studies provide an alternative explanation for the selective increase in the N-terminal Reelin fragment. We have recently observed that Reelin accumulates in oligomeric amyloid-like plaques in the hippocampal formation of several aged species [39
], indicating that the N-terminal fragments may preferentially oligomerize and aggregate in the extracellular matrix during aging (). Indeed, preliminary biochemical investigations confirmed that these Reelin deposits contain besides the full-length form also significant amounts of N-terminal Reelin fragments (JD, IK unpublished data). Our observations further revealed that Reelin was selectively associated but not completely co-localized with fibrillary Aβ species. Oligomeric Aβ deposits on the other hand showed a very high degree of co-localization with Reelin [105
], confirming and extending previous findings involving transgenic AD mouse models [106
]. To address the putative functional implications, we have investigated whether early accumulations of extracellular protein deposits in the projection areas of subcortical neurons can act retrogradly to induce degeneration of cholinergic neurons in the basal forebrain [108
], known to be affected early in AD pathogenesis and significantly contributing to the progressive hippocampus-dependent memory impairments [109
]. Considering the highly consistent finding of aging-related Reelin plaque deposition in target areas of cholinergic neurons, such as the hippocampus, entorhinal and piriform cortices, we reasoned that these plaques could impair the integrity of axonal terminals, potentially resulting in or contributing to the degeneration of basal forebrain projection neurons. We found that the age-related neuropathological changes in the target areas of these neurons were indeed accompanied by abnormal axonal varicosities and altered expression profiles of calcium-binding proteins in plaque dense areas. Moreover, we reported a significant reduction in the number of parvalbumin-positive GABAergic as well as choline acetyltransferase-positive cholinergic projection neurons in several basal forebrain areas. No Reelin deposits were found in these regions, suggesting that the loss of projection neurons was not due to adverse effects of local protein deposition and plaque formation. Altogether, our findings suggest that the elevated Reelin plaque load in the projection areas of afferent subcortical GABAergic and cholinergic neurons affects the axonal integrity and survival of these neurons, potentially also contributing to the cognitive impairments observed in aged wild-type mice.
Figure 3: Reelin accumulates in extracellular deposits in aged individuals. A-B) Anti-Reelin (G10 antibody) immunoreactivity (IR) in the hippocampus of 3 months (A) and 12 months old wild-type mice (B). Note the decrease in intraneuronal IR and concomitant accumulation (more ...)
Our findings so far provided important information regarding putative temporal processes underlying the transition from normal to pathological aging. (1) We found that the reduced numerical density of Reelin-expressing interneurons as well as the survival rate of basal forebrain GABAergic neurons appears to be a consistent feature of normal aging. (2) The observation of axonal varicosities selectively in the vicinity of Reelin plaques indicates that the degeneration of GABA- and cholinergic projection neurons is potentially a consequence of the abnormal protein deposition in their target areas. (3) The immunohistochemical findings suggested the presence of several proteins within or associated with Reelin-positive deposits [39
In order to identify the putative neurotoxic factors within these extracellular Reelin aggregates, we recently initiated a biochemical and proteomic approach to investigate their composition as well as temporal and spatial progression. We developed a new immunohistochemical protocol involving a stringent protease pretreatment to enhance Reelin-immunoreactivity. This procedure allowed a significant increase in Reelin-immunoreactivity within protease-resistant plaques and a parallel reduction in the putative soluble pool of Reelin proteins. Unexpectedly, it also allowed the specific detection of several murine proteolytic APP fragments within the Reelin plaques in the aged hippocampus (, [105
]). The same treatment in APP knockout mice did not reveal any comparable staining pattern, confirming the specificity of the immunoreactivity. Moreover, our investigations using this adapted immunohistochemical protocol allowed the detection of fibrillary Aβ deposits as seen in human, indicating that similar aging-related pathophysiological changes occur in aged rodents. 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-β-immunoreactivity was detected in extracellular, spherical deposits; potentially representing small intermediates or fragments of amyloid fibrils (), confirming our immunohistochemical data and pointing to the usefulness of non-transgenic animals to investigate early molecular mechanisms that underlie the shift from normal to pathological forms of aging. These results confirmed that Reelin itself aggregates into abnormal oligomeric or protofibrillary deposits during aging, potentially creating a precursor condition for senile amyloid-β plaque formation in sporadic AD.
Figure 4: Reelin-positive plaques are associated with astrocytes as well as proteolytic APP fragments. A–C) Representative confocal images of hippocampal brain sections processed for triple-immunofluorescence stainings using Cy3-GFAP (red), anti-Aβ (more ...)
To directly test the role of Reelin in AD pathophysiology, we crossed heterozygous reeler
mice into a transgenic AD background [110
] to investigate the effect of reduced Reelin-mediated signaling on amyloid-β plaque and neurofibrillary tangle formation, as well as neurodegenerative processes. We provided first biochemical evidence of enhanced amyloidogenic APP processing and accelerated amyloid-β plaque formation in transgenic AD mice with genetically reduced Reelin levels [111
], complementing recent in vitro
]. Furthermore, we observed that the amyloid-β plaque pathology strongly aggravated in Reelin-deficient AD mice during aging. This was accompanied by significant micro- and astrogliosis and a striking concentric accumulation of phospho-Tau-positive neurons and neurofibrillary tangles (NFTs) around amyloid-β plaques in the aged hippocampal formation. Futhermore, this was associated with a significant ventricular enlargement and cortical shrinkage, again predominantly affecting the entorhinal cortex and hippocampus. Our findings thereby provide the first in vivo
support that dysfunctional Reelin-mediated signaling is a critical upstream modulator of amyloidogenic APP processing and Tau hyperphosphorylation, both likely contributing to progressive neurodegeneration observed in Reelin-deficient AD mice. Altogether, these observations add to our understanding of the putative molecular mechanisms that underlie the pathogenesis of AD. Reduced Reelin-dependent signaling during aging appears as crucial driving force able to shift APP processing from non-amyloidogenic to amyloidogenic forms in vivo