Analysis of microdissected human neurons by a sensitive ELISA reveals a correlation between elevated intracellular concentrations of Abeta42 and Alzheimer's disease neuropathology.
ABSTRACT In Alzheimer's disease (AD), Purkinje neurons in the cerebellum are spared, while, for instance, pyramidal neurons in the hippocampus are neuropathologically affected. Several lines of evidence suggest that the pathogenesis could be induced by the concentration-dependent polymerization of the amyloid beta-peptide (Abeta) into extracellular oligomers. The role of intracellular Abeta is not fully investigated, but recent data indicate that also this pool could be of importance. Here, we use laser capture microdissection microscopy for isolation of Purkinje neurons from AD cases and controls, and quantify the low levels of intracellular Abeta using a novel and highly sensitive ELISA. Similar to Cornu Ammonis 1 pyramidal neurons, the intracellular levels of the most toxic variant, Abeta42, as well as the Abeta42/Abeta40 ratio, were increased in Purkinje neurons from sporadic AD cases as compared to controls. However, the levels of Abeta42 as well as Abeta40 were clearly lower in Purkinje neurons than in pyramidal neurons. Based on the volume of the captured Purkinje neurons, the intraneuronal concentrations of Abeta42 were calculated to be 200 nM in sporadic AD cases and 90 nM in controls. The corresponding concentrations in pyramidal neurons from hippocampus were 3 muM and 660 nM, respectively. The Abeta40 concentration was not significantly altered in AD cases compared to controls. However, we found ten times higher concentration of Abeta40 in pyramidal neurons (10 muM) compared to Purkinje neurons (1 muM). Finally, we suggest that high concentration of intracellular Abeta42 correlates with vulnerability to AD neuropathology.
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ABSTRACT: The enzyme complex γ-secretase generates amyloid β-peptide (Aβ), a 37–43-residue peptide associated with Alzheimer disease (AD). Mutations in presenilin 1 (PS1), the catalytical subunit of γ-secretase, result in familial AD (FAD). A unifying theme among FAD mutations is an alteration in the ratio Aβ species produced (the Aβ42/Aβ40 ratio), but the molecular mechanisms responsible remain elusive. In this report we have studied the impact of several different PS1 FAD mutations on the integration of selected PS1 transmembrane domains and on PS1 active site conformation, and whether any effects translate to a particular amyloid precursor protein (APP) processing phenotype. Most mutations studied caused an increase in the Aβ42/Aβ40 ratio, but via different mechanisms. The mutations that caused a particular large increase in the Aβ42/Aβ40 ratio did also display an impaired APP intracellular domain (AICD) formation and a lower total Aβ production. Interestingly, seven mutations close to the catalytic site caused a severely impaired integration of proximal transmembrane/hydrophobic sequences into the membrane. This structural defect did not correlate to a particular APP processing phenotype. Six selected FAD mutations, all of which exhibited different APP processing profiles and impact on PS1 transmembrane domain integration, were found to display an altered active site conformation. Combined, our data suggest that FAD mutations affect the PS1 structure and active site differently, resulting in several complex APP processing phenotypes, where the most aggressive mutations in terms of increased Aβ42/Aβ40 ratio are associated with a decrease in total γ-secretase activity.01/2014; 4. DOI:10.1016/j.fob.2014.04.006
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ABSTRACT: Intracellular accumulation of amyloid-β (Aβ) protein has been proposed as an early event in AD pathogenesis. In patients with mild cognitive impairment, intraneuronal Aβ immunoreactivity was found especially in brain regions critically involved in the cognitive deficits of AD. Although a large body of evidence demonstrates that Aβ42 accumulates intraneuronally (inAβ), the action and the role of Aβ42 buildup on synaptic function have been poorly investigated. Here, we demonstrate that basal synaptic transmission and LTP were markedly depressed following Aβ42 injection into the neuron through the patch pipette. Control experiments performed with the reverse peptide (Aβ42-1) allowed us to exclude that the effects of inAβ depended on changes in oncotic pressure. To further investigate inAβ synaptotoxicity we used an Aβ variant harboring oxidized methionine in position 35 that does not cross the neuronal plasma membrane and is not uploaded from the extracellular space. This Aβ42 variant had no effects on synaptic transmission and plasticity when applied extracellularly, but induced synaptic depression and LTP inhibition after patch-pipette dialysis. Finally, the injection of an antibody raised against human Aβ42 (6E10) in CA1 pyramidal neurons of mouse hippocampal brain slices and autaptic microcultures did not, per se, significantly affect LTP and basal synaptic transmission, but it protected against the toxic effects of extracellular Aβ42. Collectively, these findings suggest that Aβ42-induced impairment of glutamatergic synaptic function depends on its internalization and intracellular accumulation thus paving the way to a systemic proteomic analysis of intracellular targets/partners of Aβ42.The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 09/2014; 34(38):12893-903. DOI:10.1523/JNEUROSCI.1201-14.2014 · 6.75 Impact Factor
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ABSTRACT: Numerous studies have implicated the abnormal accumulation of intraneuronal amyloid-beta (Abeta) as an important contributor to Alzheimer's disease (AD) pathology, capable of triggering neuroinflammation, tau hyperphosphorylation and cognitive deficits. However, the occurrence and pathological relevance of intracellular Abeta remain a matter of controversial debate. In this study, we have used a multidimensional approach including high-magnification and super-resolution microscopy, cerebro-spinal fluid (CSF) mass spectrometry analysis and ELISA to investigate the Abeta pathology and its associated cognitive impairments, in a novel transgenic rat model overexpressing human APP. Our microscopy studies with quantitative co-localization analysis revealed the presence of intraneuronal Abeta in transgenic rats, with an immunological signal that was clearly distinguished from that of the amyloid precursor protein (APP) and its C-terminal fragments (CTFs). The early intraneuronal pathology was accompanied by a significant elevation of soluble Abeta42 peptides that paralleled the presence and progression of early cognitive deficits, several months prior to amyloid plaque deposition. Abeta38, Abeta39, Abeta40 and Abeta42 peptides were detected in the rat CSF by MALDI-MS analysis even at the plaque-free stages; suggesting that a combination of intracellular and soluble extracellular Abeta may be responsible for impairing cognition at early time points. Taken together, our results demonstrate that the intraneuronal development of AD-like amyloid pathology includes a mixture of molecular species (Abeta, APP and CTFs) of which a considerable component is Abeta; and that the early presence of these species within neurons has deleterious effects in the CNS, even before the development of full-blown AD-like pathology.06/2014; 2(1):61. DOI:10.1186/2051-5960-2-61