MicroRNA-132and Egr-1in Nucleus Basalis Of Meynert During The Course Of Alzheimer’ Disease

Aims:The Nucleus Basalis of Meynert (NBM) is the productive area of cholinergic system in the brain. It is of crucial importance for memory processes and is strongly affected in late Alzheimer’s disease (AD). Early activation of NBM in the course of AD (Braak stage II-III) has been observed by our and other’s previous postmortem studies and confirmed by an in vivo study, while neurodegeneration were observed in late AD. In addition we found also neuronal activation taking place in the prefrontal cortex (PFC) in early AD (Braak stages II-III), with many genes involved in synaptic activity and plasticity showing increased expression in early AD while decreased expression in late AD. All these studies indicate early activation is a more general phenomenon in the early AD brain. While the activation may cast a protective function on neurons, compensating for the fuction impairment caused by the pathological changes. Therefore, revealing the mechanism of ’up-down’ pattern in AD brain, especially the early activaion, plays an important role in finding a therapy strategy and drug target for AD.In the present study, we investigated the mechanism in two candidates, microRNA-132(miR132) and the transcription factor Early Growth Response-1(Egr-1) which not only stimulate synaptic activity and plasticity, but are also involved in AD pathology and might affect cholinergic function. We investigated the expression of miR-132and Egr-1in the NBM in the course of AD (From Braak0and VI), in relation to the typical AD neuropathology, i.e. hyperphosphorylated tau and intra-and extracellular Amyloid beta (Aβ), and to the alterations in choline acetyltransferase (ChAT) at the protein level, and to the apolipoprotein E (APOE) s4carriers and parameters of neurodegeneration, i.e. neuron atrophy and neuronal density. We want to figure out whether miR-132and/or Egr-1may play a role in the up-down pattern of functional changes in the cholinergic NBM.Materials and Methods:Forty-nine formalin-fixed block containing the hypothalamus and NBM were obtained from the Netherlands Brain Bank (NBB), ranging from non-demented controls (Braak stage=0) to6Braak stages of AD (7patients in each group). The7stages were well matched for age, sex, postmortem delay (PMD), cerebrospinal fluid (CSF) pH, APOE genotype, clock time of death, fixation time, and storage time. In addition,3pooled groups were distinguished on the basis of their main clinical phenotypes as found before, i.e. Braak0-II (mainly’no symptom-stage’, called NS group in the present study), Braak III-IV (mainly’MCI stage’, called MCI group in the present study), and Braak stage V-VI (mainly’AD stage’, called AD group in the present study). The sections at the level of the fornix, and/or the anterior commissure, i.e. Ch4-am or Ch4-al areas were mounted for further study. The NBM sections were stained by thionin (for orientation, neuron numbers, and neuron atrophy), and immunocytochemically by AT8(hyperphosphorylated tau),4G8(intra-and extra-cellular Aβ), Chat (for cholinergic neurons in NBM), Egr-1(for Egr-1expression) and Gallys silver staining for argyrophilic neurofibrillary tangles (NFT) and by in situ hybridization for miR-132. Quantification of the integrated optical density (IOD) of ICC or ISH signals were done with image analysis software for comparison among groups.Results:miR-132and Egr-1in the NBM did not decrease until late AD (S-N-K, P=0.055and P<0.05). They both showed positive correlations with ChAT expression (r=0.49, P<0.001and r=0.61, P<0.001). Neuron density was preserved (ANOVA, P=0.101) while neuron profile size only decreased during the late Braak stages (K-W, compared to NS, P=0.00001and compared to MCI, P=0.007). The number of ChAT-ir neuron seems to be preserved until late AD (S-N-K, P<0.05). The characteristic AD neuropathological changes (Neurofibrillary tangles, NFT and amyloid beta, Aβ) appeared to rise dramatically between Braak stage Ⅱ and Ⅲ, followed by either further increase (NFTs) or staying stable (AP) untill Braak Ⅵ (S-N-K, P<0.05). The alterations in hyperphoshorylated tau (AT8) dominated in the earlier (Braak Ⅱ-Ⅳ), and the silver stained NFT and neuropil threads (NT) in later phases of the AD process (Braak Ⅴ-Ⅵ). While ChAT immunoreactivity (-ir) showed a significant negative correlation with AT8-ir, no such correlation was found between ChAT-ir and4G8-ir, underlining the idea that the tau pathology is predominant in the AD process in the NBM. A striking finding was that practically all Aβ staining in the NBM was extracellular in all Braak stages. A significant lower expression of ChAT was observed in ApoEε4carriers in MCI (S-N-K, P<0.05) while more4G8-ir in NS in ApoEε4carriers were found when compared to the non-carriers (S-N-K, P<0.05).Conclusion:From the functional changes of miR-132and Egr-1along the course of Alzheimer’s disease we conclude i) that these two molecules may play a role in keeping the cholinergic function intact in early Alzheimer’s disease stages and ii) that these molecules may contribute to the late neurodegeneration of this cholinergic nucleus.