AB5074P Sigma-AldrichAnti-Amyloid Antibody, β 1-40

In Alzheimer's disease (AD), β-amyloid (Aβ) plaques are tightly enveloped by microglia processes, but the significance of this phenomenon is unknown. Here we show that microglia constitute a barrier with profound impact on plaque composition and toxicity. Using high-resolution confocal and in vivo two-photon imaging in AD mouse models, we demonstrate that this barrier prevents outward plaque expansion and leads to compact plaque microregions with low Aβ42 affinity. Areas uncovered by microglia are less compact but have high Aβ42 affinity, leading to the formation of protofibrillar Aβ42 hotspots that are associated with more severe axonal dystrophy. In ageing, microglia coverage is reduced leading to enlarged protofibrillar Aβ42 hotspots and more severe neuritic dystrophy. CX3CR1 gene deletion or anti-Aβ immunotherapy causes expansion of microglia coverage and reduced neuritic dystrophy. Failure of the microglia barrier and the accumulation of neurotoxic protofibrillar Aβ hotspots may constitute novel therapeutic and clinical imaging targets for AD.

Therapeutic approaches for prevention or reduction of amyloidosis are currently a main objective in basic and clinical research on Alzheimer's disease. Among the agents explored in clinical trials are anti-Aβ peptide antibodies and secretase inhibitors. Most anti-Aβ antibodies are considered to act via inhibition of amyloidosis and enhanced clearance of existing amyloid, although secretase inhibitors reduce the de novo production of Aβ. Limited information is currently available on the efficacy and potential advantages of combinatorial antiamyloid treatment. We performed a chronic study in APPLondon transgenic mice that received treatment with anti-Aβ antibody gantenerumab and BACE inhibitor RO5508887, either as mono- or combination treatment. Treatment aimed to evaluate efficacy on amyloid progression, similar to preexisting amyloidosis as present in Alzheimer's disease patients. Mono-treatments with either compound caused a dose-dependent reduction of total brain Aβ and amyloid burden. Combination treatment with both compounds significantly enhanced the antiamyloid effect. The observed combination effect was most pronounced for lowering of amyloid plaque load and plaque number, which suggests effective inhibition of de novo plaque formation. Moreover, significantly enhanced clearance of pre-existing amyloid plaques was observed when gantenerumab was coadministered with RO5508887. BACE inhibition led to a significant time- and dose-dependent decrease in CSF Aβ, which was not observed for gantenerumab treatment. Our results demonstrate that combining these two antiamyloid agents enhances overall efficacy and suggests that combination treatments may be of clinical relevance.

Dendritic spines are the site of most excitatory synapses, the loss of which correlates with cognitive impairment in patients with Alzheimer disease. Substantial evidence indicates that amyloid-β (Aβ) peptide, either insoluble fibrillar Aβ deposited into plaques or soluble nonfibrillar Aβ species, can cause spine loss but the concurrent contributions of fibrillar Aβ and nonfibrillar Aβ to spine loss has not been previously assessed. We used multiple-label immunohistochemistry to measure spine density, size, and F-actin content surrounding plaques in the cerebral cortex in the PSAPP mouse model of Aβ deposition. Our approach allowed us to measure fibrillar Aβ plaque content and an index of nonfibrillar Aβ species concurrently. We found that spine density was reduced within 6 μm of the plaque perimeter, remaining spines were more compact, and F-actin content per spine was increased. Measures of fibrillar Aβ plaque content were associated with reduced spine density near plaques, whereas measures of nonfibrillar Aβ species were associated with reduced spine density and size but not altered F-actin content. These findings suggest that strategies to preserve dendritic spines in AD patients may need to address both nonfibrillar and fibrillar forms of Aβ and that nonfibrillar Aβ may exert spine toxicity through pathways not mediated by depolymerization of F-actin.

The occurrence and progression of cerebral β-amyloid angiopathy and β-amyloid plaques in sporadic Alzheimer's disease may be attributed to aging-related deficiencies in β-amyloid drainage along cerebral perivascular pathways. To elucidate high-definition characteristics of cerebral β-amyloid deposition, we performed immunogold silver staining for β-amyloid-40 and β-amyloid-42 on semithin LR White-embedded tissue sections from 7 Alzheimer's disease/severe cerebral β-amyloid angiopathy, 9 Alzheimer's disease/mild cerebral β-amyloid angiopathy, 5 old control, and 4 young control autopsy brains. In vessel walls, β-amyloid-40 and β-amyloid-42 deposits were unevenly distributed along the adventitia and among the medial smooth muscle cells. β-Amyloid-40 immunoreactivity appeared greater than that of β-amyloid-42 in vessel walls, with β-amyloid-42 being preferentially located on their abluminal regions. In capillary walls, either β-amyloid-40 or β-amyloid-42 deposits or both were present in 6 of 7 severe cerebral β-amyloid angiopathy and in 1 of 9 mild cerebral β-amyloid angiopathy cases, with a marked variation in thickness and focally abluminal excrescences. In 5 of 7 severe cerebral β-amyloid angiopathy cases, a subset of β-amyloid-laden capillaries revealed either β-amyloid-40 or β-amyloid-42 deposits or both radiating from their walls into the surrounding neuropil ("pericapillary deposits"). No vascular β-amyloid-40 or β-amyloid-42 deposits were observed in any of the controls. In conclusion, the patterns of β-amyloid-42 and β-amyloid-40 immunoreactivity in vessel walls suggest that β-amyloid deposits occur in the vascular basement membranes along cerebral perivascular drainage pathways, extending from cortical capillaries to leptomeningeal arteries. The presence of pericapillary β-amyloid deposits suggests that a subset of β-amyloid plaques originate from β-amyloid-laden capillaries, particularly in Alzheimer's disease brains that exhibit preferential capillary involvement by cerebral β-amyloid angiopathy.

The amyloid-beta (Abeta) precursor protein (APP), a transmembrane protein that undergoes proteolytic cleavage into defined fragments, has been implicated in axonal transport. The proposed role of APP as a vesicle receptor for the microtubule motor kinesin-1 has relevance for the pathogenesis of Alzheimer's disease. Nevertheless, this function, which relies on the transport to the cell periphery of full-length APP rather than its cleavage fragments, remains controversial. Other proposed functions of APP, such as regulating transcription, neurogenesis, cell movement, or neurite growth also rely on APP's presence as a full-length protein at the cell surface, implying that APP cleavage occurs after its transport to the cell periphery. To test this hypothesis, we mapped the localization of various APP epitopes in neurons in culture and in the mouse brain. Surprisingly, epitopes from the N-terminal, C-terminal, and central (Abeta) domains of APP each showed a distinct distribution throughout the cell and rarely colocalized. Within neurites, these epitopes were localized to distinct transport vesicles that associated with different sets of microtubules and, occasionally, actin filaments. C-terminal APP fragments were preferentially transported into neurites as phosphorylated forms, entered the lamellipodium and filopodia of growth cones, and concentrated in regions of growth cone turning and advancement (unlike the N-terminal and Abeta fragments). We conclude that, under normal conditions, the proteolytic cleavage of APP primarily occurs before its sorting into axonal transport vesicles and the cleaved fragments segregate into separate vesicle populations that reach different destinations, and thus have different functions.

We have investigated the possibility that soluble, blood-borne amyloid beta (Abeta) peptides can cross a defective blood-brain barrier (BBB) and interact with neurons in the brain. Immunohistochemical analyses revealed extravasated plasma components, including Abeta42 in 19 of 21 AD brains, but in only 3 of 13 age-matched control brains, suggesting that a defective BBB is common in AD. To more directly test whether blood-borne Abeta peptides can cross a defective BBB, we tracked the fate of fluorescein isothiocyanate (FITC)-labeled Abeta42 and Abeta40 introduced via tail vein injection into mice with a BBB rendered permeable by treatment with pertussis toxin. Both Abeta40 and Abeta42 readily crossed the permeabilized BBB and bound selectively to certain neuronal subtypes, but not glial cells. By 48 h post-injection, Abeta42-positive neurons were widespread in the brain. In the cerebral cortex, small fluorescent, Abeta42-positive granules were found in the perinuclear cytoplasm of pyramidal neurons, suggesting that these cells can internalize exogenous Abeta42. An intact BBB (saline-injected controls) blocked entry of blood-borne Abeta peptides into the brain. The neuronal subtype selectivity of Abeta42 and Abeta40 was most evident in mouse brains subjected to direct intracranial stereotaxic injection into the hippocampal region, thereby bypassing the BBB. Abeta40 was found to preferentially bind to a distinct subset of neurons positioned at the inner face of the dentate gyrus, whereas Abeta42 bound selectively to the population of large neurons in the hilus region of the dentate gyrus. Our results suggest that the blood may serve as a major, chronic source of soluble, exogenous Abeta peptides that can bind selectively to certain subtypes of neurons and accumulate within these cells.

Our goal is to understand the pathogenesis of amyloid-beta (Abeta) deposition in the Alzheimer's disease (AD) brain. We established a cell culture system where central nervous system-derived neuronal cells (CAD cells) produce and accumulate within their processes large amounts of Abeta peptide, similar to what is believed to occur in brain neurons, in the initial phases of AD. Using this system, we show that accumulation of Abeta begins within neurites, prior to any detectable signs of neurodegeneration or abnormal vesicular transport. Neuritic accumulation of Abeta is restricted to a small population of neighboring cells that express normal levels of amyloid-beta precursor protein (APP) but show redistribution of BACE1 to the processes, where it colocalizes with Abeta and markers of late endosomes. Consistently, cells that accumulate Abeta appear in isolated islets, suggesting their clonal origin from a few cells that show a propensity to accumulate Abeta. These results suggest that Abeta accumulation is initiated in a small number of neurons by intracellular determinants that alter APP metabolism and lead to Abeta deposition and neurodegeneration. CAD cells appear to recapitulate the biochemical processes leading to Abeta deposition, thus providing an experimental in vitro system for studying the molecular pathobiology of AD.

Proteolytic processing of amyloid precursor protein (APP) generates amyloid-beta peptide and has been implicated in the pathogenesis of Alzheimer's disease. However, the normal function of APP, whether this function is related to the proteolytic processing of APP, and where this processing takes place in neurons in vivo remain unknown. We have previously shown that the axonal transport of APP in neurons is mediated by the direct binding of APP to the kinesin light chain subunit of kinesin-I, a microtubule motor protein. Here we identify an axonal membrane compartment that contains APP, beta-secretase and presenilin-1. The fast anterograde axonal transport of this compartment is mediated by APP and kinesin-I. Proteolytic processing of APP can occur in the compartment in vitro and in vivo in axons. This proteolysis generates amyloid-beta and a carboxy-terminal fragment of APP, and liberates kinesin-I from the membrane. These results suggest that APP functions as a kinesin-I membrane receptor, mediating the axonal transport of beta-secretase and presenilin-1, and that processing of APP to amyloid-beta by secretases can occur in an axonal membrane compartment transported by kinesin-I.