Department of Neurobiology, Care Sciences and Society (NVS), Karolinska Institutet and Dainippon Sumitomo Pharma Alzheimer Center (KASPAC), KI-Alzheimer's Disease Research Center, Karolinska Institutet, Novum plan 5, Huddinge, Sweden.
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.
[Show abstract][Hide abstract] ABSTRACT: Synaptic degeneration is one of the earliest hallmarks of Alzheimer disease. The molecular mechanism underlying this degeneration is not fully elucidated but one key player appears to be the synaptotoxic amyloid β-peptide (Aβ). The exact localization of the production of Aβ and the mechanisms whereby Aβ is released remain elusive. We have earlier shown that Aβ can be produced in crude synaptic vesicles and it has been reported that increased synaptic activity results in increased secreted but decreased intracellular Aβ levels. Therefore, we considered whether Aβ could be produced in synaptic vesicles and/or released through the same mechanisms as neurotransmitters in synaptic vesicle exocytosis. Small amounts of Aβ were found to be produced in pure synaptic vesicle preparations. We also studied the release of glutamate and Aβ from rat cortical nerve terminals (synaptosomes). We found that large amounts of Aβ were secreted from non-stimulated synaptosomes, from which glutamate was not released. On the contrary, we could not detect any differences in Aβ release between non-stimulated synaptosomes and synaptosomes stimulated with KCl or 4-aminopyridine, whereas glutamate release was readily inducible in this system. To conclude, our results indicate that the major release mechanism of Aβ from isolated nerve terminals differs from the synaptic release of glutamate and that the activity-dependent increase of secreted Aβ, reported by several groups using intact cells, is likely dependent on post-synaptic events, trafficking and/or protein synthesis mechanisms.
"Increasing attention is currently being paid to intraneuronal Aβ, generated via the endosomal system and accumulated in endosomes/lysosomes, mitochondria and autophagosomes, as an early player in the pathogenesis of AD . In fact, intraneuronal Aβ accumulation occurs well before the increase of extracellular Aβ , it is detectable in plaque-free animals , and converging reports indicate a key pathogenetic role of intraneuronal β-amyloid in neurodegeneration and AD pathology [44,45]. For example, the intracellular concentration of Aβ42, the most toxic Aβ variant, in pyramidal (CA1) human neurons has been estimated to be 3 μM in the case of sporadic AD patients, as compared to 660 nM in the case of neurons from control, unaffected subjects . "
[Show abstract][Hide abstract] ABSTRACT: Background
Alzheimer disease is a multifactorial disorder characterized by the progressive deterioration of neuronal networks. The pathological hallmarks includes extracellular amyloid plaques and intraneuronal neurofibrillary tangles, but the primary cause is only partially understood. Thus, there is growing interest in developing agents that might target multiple mechanisms leading to neuronal degeneration. CHF5074 is a nonsteroidal anti-inflammatory derivative that has been shown to behave as a γ-secretase modulator in vitro and to inhibit plaque deposition and to reverse memory deficit in vivo in transgenic mouse models of Alzheimer’s disease (AD). In the present study, the effects of a long-term (13-month) treatment with CHF5074 on indicators of brain functionality and neurodegeneration in transgenic AD mice (Tg2576) have been assessed and compared with those induced by a prototypical γ-secretase inhibitor (DAPT).
To this end, plaque-free, 6-month-old Tg2576 mice and wild-type littermates were fed with a diet containing CHF5074 (125 and 375 ppm/day), DAPT (375 ppm/day) or vehicle for 13 months. The measured indicators included object recognition memory, amyloid burden, brain oligomeric and plasma Aβ levels, intraneuronal Aβ, dendritic spine density/morphology, neuronal cyclin A positivity and activated microglia. Tg2576 mice fed with standard diet displayed an impairment of recognition memory. This deficit was completely reverted by the higher dose of CHF5074, while no effects were observed in DAPT-treated mice. Similarly, amyloid plaque burden, microglia activation and aberrant cell cycle events were significantly affected by CHF5074, but not DAPT, treatment. Both CHF5074 and DAPT reduced intraneuronal Aβ content, also increasing Aβ40 and Aβ42 plasma levels.
This comparative analysis revealed a profoundly diverse range of clinically relevant effects differentiating the multifunctional anti-inflammatory derivative CHF5074 from the γ-secretase inhibitor DAPT and highlighted unique mechanisms and potential targets that may be crucial for neuroprotection in mouse models of AD.
"Actually, neurodegeneration in AD patients does not affect only the cerebrum but also the cerebellum, although generally in later stages of the disease. Many lines of research have shown that, in AD patients, the cerebellum is reduced in volume in a similar fashion as cerebral hemispheres –, the Purkinje cell (PC) number is decreased  and the levels of Aβ1–42 are increased more than twofold relative to controls . "
[Show abstract][Hide abstract] ABSTRACT: In Alzheimer's disease (AD), the severity of cognitive symptoms is better correlated with the levels of soluble amyloid-beta (Aβ) rather than with the deposition of fibrillar Aβ in amyloid plaques. In APP/PS1 mice, a murine model of AD, at 8 months of age the cerebellum is devoid of fibrillar Aβ, but dosage of soluble Aβ(1-42), the form which is more prone to aggregation, showed higher levels in this structure than in the forebrain. Aim of this study was to investigate the alterations of intrinsic membrane properties and of synaptic inputs in Purkinje cells (PCs) of the cerebellum, where only soluble Aβ is present. PCs were recorded by whole-cell patch-clamp in cerebellar slices from wild-type and APP/PS1 mice. In APP/PS1 PCs, evoked action potential discharge showed enhanced frequency adaptation and larger afterhyperpolarizations, indicating a reduction of the intrinsic membrane excitability. In the miniature GABAergic postsynaptic currents, the largest events were absent in APP/PS1 mice and the interspike intervals distribution was shifted to the left, but the mean amplitude and frequency were normal. The ryanodine-sensitive multivescicular release was not altered and the postsynaptic responsiveness to a GABA(A) agonist was intact. Climbing fiber postsynaptic currents were normal but their short-term plasticity was reduced in a time window of 100-800 ms. Parallel fiber postsynaptic currents and their short-term plasticity were normal. These results indicate that, in the cerebellar cortex, chronically elevated levels of soluble Aβ(1-42) are associated with alterations of the intrinsic excitability of PCs and with alterations of the release of GABA from interneurons and of glutamate from climbing fibers, while the release of glutamate from parallel fibers and all postsynaptic mechanisms are preserved. Thus, soluble Aβ(1-42) causes, in PCs, multiple functional alterations, including an impairment of intrinsic membrane properties and synapse-specific deficits, with differential consequences even in different subtypes of glutamatergic synapses.
PLoS ONE 04/2012; 7(4):e34726. DOI:10.1371/journal.pone.0034726 · 3.23 Impact Factor
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