Preferential accumulation of amyloid-beta in presynaptic glutamatergic terminals (VGluT1 and VGluT2) in Alzheimer's disease cortex.

Amyloid-beta (Aß) is thought to play a central role in synaptic dysfunction (e.g. neurotransmitter release) and synapse loss. Glutamatergic dysfunction is involved in the pathology of Alzheimer's disease (AD) and perhaps plays a central role in age-related cognitive impairment. Yet, it is largely unknown whether Aß accumulates in excitatory boutons. To assess the possibility that glutamatergic terminals are lost in AD patients, control and AD synaptosomes were immunolabeled for the most abundant vesicular glutamate transporters (VGluT1 and VGluT2) and quantified by flow cytometry and immunoblot methods. In post-mortem parietal cortex from aged control subjects, glutamatergic boutons are fairly abundant as approximately 40% were immunoreactive for VGluT1 (37%) and VGluT2 (39%). However, the levels of these specific markers of glutamatergic synapses were not significantly different among control and AD cases. To test the hypothesis that Aß is associated with excitatory terminals, AD synaptosomes were double-labeled for Aß and for VGluT1 and VGluT2, and analyzed by flow cytometry and confocal microscopy. Our study demonstrated that Aß immunoreactivity (IR) was present in glutamatergic terminals of AD patients. Quantification of Aß and VGluT1 in a large population of glutamatergic nerve terminals was performed by flow cytometry, showing that 42% of VGluT1 synaptosomes were immunoreactive for Aß compared to 9% of VGluT1 synaptosomes lacking Aß-IR. Percentage of VGluT2 synaptosomes immunoreactive for Aß (21%) was significantly higher than VGluT2 synaptosomes lacking Aß-IR (9%). Moreover, Aß preferentially affects VGluT1 (42% positive) compared to VGluT2 terminals (21%). These data represent the first evidence of high levels of Aß in excitatory boutons in AD cortex and support the hypothesis that Aß may play a role in modulating glutamate transmission in AD terminals.

High levels of synaptosomal Na(+)-Ca(2+) exchangers (NCX1, NCX2, NCX3) co-localized with amyloid-beta in human cerebral cortex affected by Alzheimer's disease.

Synaptosomal expression of NCX1, NCX2, and NCX3, the three variants of the Na(+)-Ca(2+) exchanger (NCX), was investigated in Alzheimer's disease parietal cortex. Flow cytometry and immunoblotting techniques were used to analyze synaptosomes prepared from cryopreserved brain of cognitively normal aged controls and late stage Alzheimer's disease patients. Major findings that emerged from this study are: (1) NCX1 was the most abundant NCX isoform in nerve terminals of cognitively normal patients; (2) NCX2 and NCX3 protein levels were modulated in parietal cortex of late stage Alzheimer's disease: NCX2 positive terminals were increased in the Alzheimer's disease cohort while counts of NCX3 positive terminals were reduced; (3) NCX1, NCX2 and NCX3 isoforms co-localized with amyloid-beta in synaptic terminals and all three variants are up-regulated in nerve terminals containing amyloid-beta. Taken together, these data indicate that NCX isoforms are selectively regulated in pathological terminals, suggesting different roles of each NCX isoform in Alzheimer's disease terminals.