Amyloid Beta annular protofibrils in cell processes and synapses accumulate with aging and Alzheimer-associated genetic modification.

Amyloid beta (Abeta) annular protofibrils (APFs) have been described where the structure is related to that of beta barrel pore-forming bacterial toxins and exhibits cellular toxicity. To investigate the relationship of Abeta APFs to disease and their ultrastructural localization in brain tissue, we conducted a pre-embedding immunoelectron microscopic study using anti-annular protofibril antiserum. We examined brain tissues of young- and old-aged amyloid precursor protein transgenic mice (APP23), neprilysin knockout APP23 mice, and nontransgenic littermates. alphaAPF-immunoreactions tended to be found (1) on plasma membranes and vesicles inside of cell processes, but not on amyloid fibrils, (2) with higher density due to aging, APP transgene, and neprilysin deficiency, and (3) with higher positive rate at synaptic compartments in aged APP23, especially in neprilysin knockout APP23 mice. These findings imply that APFs are distinct from amyloid fibrils, interact with biological membranes, and might be related to synaptic dysfunction in Alzheimer model mouse brains.

Neprilysin-sensitive synapse-associated amyloid-beta peptide oligomers impair neuronal plasticity and cognitive function.

A subtle but chronic alteration in metabolic balance between amyloid-beta peptide (Abeta) anabolic and catabolic activities is thought to cause Abeta accumulation, leading to a decade-long pathological cascade of Alzheimer disease. However, it is still unclear whether a reduction of the catabolic activity of Abeta in the brain causes neuronal dysfunction in vivo. In the present study, to clarify a possible connection between a reduction in neprilysin activity and impairment of synaptic and cognitive functions, we cross-bred amyloid precursor protein (APP) transgenic mice (APP23) with neprilysin-deficient mice and biochemically and immunoelectron-microscopically analyzed Abeta accumulation in the brain. We also examined hippocampal synaptic plasticity using an in vivo recording technique and cognitive function using a battery of learning and memory behavior tests, including Y-maze, novel-object recognition, Morris water maze, and contextual fear conditioning tests at the age of 13-16 weeks. We present direct experimental evidence that reduced activity of neprilysin, the major Abeta-degrading enzyme, in the brain elevates oligomeric forms of Abeta at the synapses and leads to impaired hippocampal synaptic plasticity and cognitive function before the appearance of amyloid plaque load. Thus, reduced neprilysin activity appears to be a causative event that is at least partly responsible for the memory-associated symptoms of Alzheimer disease. This supports the idea that a strategy to reduce Abeta oligomers in the brain by up-regulating neprilysin activity would contribute to alleviation of these symptoms.

Oligomeric proteins ultrastructurally localize to cell processes, especially to axon terminals with higher density, but not to lipid rafts in Tg2576 mouse brain.

We examined the ultrastructural localization of oligomeric proteins, Abeta42, and flotillin-1 in Tg2576 mouse brains by triple immunoelectron microscopy. Oligomer-specific immunoreactions localized to cell processes, especially to axon terminals with higher density in Tg than in nonTg mouse brains. The oligomer was less frequently colocalized to flotillin-1-immunoreactive rafts than Abeta42, suggesting that rafts are one of the sites of polymeric Abeta deposition, but not of oligomeric proteins including Abeta.

Soluble Abeta oligomers ultrastructurally localize to cell processes and might be related to synaptic dysfunction in Alzheimer's disease brain.

Soluble Abeta oligomers have recently been considered to be responsible for cognitive dysfunction prior to senile plaque (SP) formation in Alzheimer's disease (AD) brain. To investigate the ultrastructural localization of soluble Abeta oligomers, we conducted the post-embedding immunoelectron microscopic (IEM) study using an antibody against a molecular mimic of oligomeric Abeta. We examined autopsied brains from AD patients and nondemented subjects. Oligomer-specific immunoreactions detected by IEM tended to be found with higher density (1) in AD than in nondemented brains and (2) at the axon and axon terminal in AD than in nondemented brains. These findings imply that soluble Abeta oligomers might be related to synaptic dysfunction in AD brain.

Part of membrane-bound Abeta exists in rafts within senile plaques in Tg2576 mouse brain.

To clarify whether rafts are the site of abnormal amyloid beta protein (Abeta) deposition, we examined the ultrastructural localization of both flotillin-1 (pre-embedding) and Abeta (post-embedding) in Tg2576 mouse brains. After observing the exact areas of senile plaques by reflection contrast microscopy, we observed these same plaques under an electron microscope. Membrane-bound Abeta was predominantly observed on plasma membranes of small processes in diffuse plaques. Non-fibrillar and fibrillar Abeta was increased in primitive plaques, and the fibrillar form was predominant in mature plaques. The number of flotillin-1-positive rafts per field in mature plaques was prominently less than those outside of the plaques, in diffuse plaques and in primitive plaques. The colocalization of flotillin-1 with Abeta42 appeared approximately 10% of flotillin-1-positive rafts within senile plaques, while there was no colocalization found outside of the plaques. This study ultrastructurally demonstrated that part of membrane-bound Abeta exists in lipid rafts within senile plaques, and suggests that rafts could be one of the sites for initial Abeta deposition.

Ultrastructural localization of flotillin-1 to cholesterol-rich membrane microdomains, rafts, in rat brain tissue.

There is much interest in research on cholesterol-rich membrane microdomains, rafts, in the field of neurobiology. However, no one has shown the ultrastructure of rafts in tissues. We examined the ultrastructure of rafts in rat brain tissue by pre-embedding immunoelectron microscopy using flotillin-1 antibody, which is a biochemical marker of lipid rafts, and BCtheta, which is nicked and biotinylated theta-toxin, and binds to membrane cholesterol of rafts. Flotillin-1- and BCtheta-labeled areas were patchy and prominent on the plasma membranes of small processes and synapses in the neuropil. The size of flotillin-1 labeling was 40-200 nm. In addition, the membrane of lysosome and Golgi apparatus were frequently labeled for flotillin-1 with a patchy pattern. Flotillin-1 and BCtheta were mostly colocalized in double immunolabeling on a part of the plasma membranes of small processes and secondary lysosome membranes. We first indicate that flotillin-1 localizes to BCtheta-positive cholesterol-rich membrane microdomains in vivo, and that flotillin-1 and BCtheta could be ultrastructural raft markers in neural tissue.