beta-amyloid modulation of synaptic transmission and plasticity

The sequencing of β amyloid protein (Aβ) in 1984 led to the formulation of the “amyloid hypothesis” of Alzheimer's disease (AD) (Glenner and Wong, 1984). The hypothesis proposed that accumulation of Aβ is responsible for AD-related pathology, including Aβ deposits, neurofibrillary tangles, and eventual neuronal cell death (Tanzi and Bertram, 2005). Within a few years, four groups cloned the amyloid precursor protein (APP) gene from which Aβ is processed (Goldgaber et al., 1987; Kang et al., 1987; Robakis et al., 1987; Tanzi et al., 1987). Linkage analysis mapped the gene to chromosome 21, and mutations in APP were found that led to the inappropriate processing of APP into the Aβ1–42 peptide (Goate et al., 1991; Mullan et al., 1992) (for review, see Tanzi and Bertram, 2005). However, these mutations are responsible for only a small fraction of the early-onset familial AD, and the search began for other genes that might also influence the processing of Aβ. Several novel mutations were identified in the presenilins (Levy-Lahad et al., 1995; Rogaev et al., 1995; Sherrington et al., 1995), and apolipoprotein E4 was identified as a major risk factor for the most frequent form of AD (Strittmatter et al., 1993; Mahley et al., 2006)

Conditional inactivation of presenilin 1 prevents amyloid accumulation and temporarily rescues contextual and spatial working memory impairments in amyloid precursor protein transgenic mice.

Accumulation of -amyloid (A ) peptides in the cerebral cortex is considered a key event in the pathogenesis of Alzheimers disease (AD). Presenilin 1 (PS1) plays an essential role in the -secretase cleavage of the amyloid precursor protein (APP) and the generation of A peptides. Reduction of A generation via the inhibition of -secretase activity, therefore, has been proposed as a therapeutic approach for AD. In this study, we examined whether genetic inactivation of PS1 in postnatal forebrain-restricted conditional knock-out (PS1 cKO) mice can prevent the accumulation ofA peptides and ameliorate cognitive deficits exhibited by an amyloid mouse model that overexpresses human mutant APP. We found that conditional inactivation of PS1 in APP transgenic mice (PS1 cKO;APP Tg) effectively prevented the accumulation of A peptides and formation of amyloid plaques and inflammatory responses, although it also caused an age-related accumulation of C-terminal fragments of APP. Short-term PS1 inactivation in young PS1 cKO;APP Tg mice rescued deficits in contextual fear conditioning and serial spatial reversal learning in a water maze, which were associated with APP Tg mice. Longer-term PS1 inactivation in older PS1 cKO;APP Tg mice, however, failed to rescue the contextual memory and hippocampal synaptic deficits and had a decreasing ameliorative effect on the spatial memory impairment. These results reveal that in vivo reduction of A via the inactivation of PS1 effectively prevents amyloid-associated neuropathological changes and can, but only temporarily, improve cognitive impairments in APP transgenic mice.This work was supported by National Institute of Neurological Disorders and Stroke Grant R01NS041783 (J.S.), the Alzheimers Association (C.A.S., J.S.), the Medical Research Council, and the Alzheimers Research Trust (R.G.M.M). We thank L. Mucke for the APP transgenic mice, D. Selkoe for the C7 and A antibodies, M. Shoji for the Saeko antiserum, and W. Xia and J. Zheng for ELISA. We are grateful to V. Beglopoulos, W. Cheng, M. Goldberg, and C. Lemere for assistance

The morphology of human rod ERGs obtained by silent substitution stimulation

YesPurpose To record transient ERGs from the lightadapted human retina using silent substitution stimuli which selectively reflect the activity of rod photoreceptors. We aim to describe the morphology of these waveforms and examine how they are affected by the use of less selective stimuli and by retinal pathology. Methods Rod-isolating stimuli with square-wave temporal profiles (250/250 ms onset/offset) were presented using a 4 primary LED ganzfeld stimulator. Experiment 1: ERGs were recorded using a rodisolating stimulus (63 ph Td, rod contrast, Crod = 0.25) from a group (n = 20) of normal trichromatic observers. Experiment 2: Rod ERGs were recorded from a group (n = 5) using a rodisolating stimulus (Crod = 0.25) which varied in retinal illuminance from 40 to 10,000 ph Td. Experiment 3: ERGs were elicited using 2 kinds of nonisolating stimuli; (1) broadband and (2) rod-isolating stimuli which contained varying degrees of L- and M-cone excitation. Experiment 4: Rod ERGs were recorded from two patient groups with rod monochromacy (n = 3) and CSNB (type 1; n = 2). Results The rod-isolated ERGs elicited from normal subjects had a waveform with a positive onset component followed by a negative offset. Response amplitude was maximal at retinal illuminances\100 ph Td and was virtually abolished at 400 ph Td. The use of non-selective stimuli altered the ERG waveform eliciting more photopic-like ERG responses. Rod ERGs recorded from rod monochromats had similar features to those recorded from normal trichromats, in contrast to those recorded from participants with CSNB which had an electronegative appearance. Conclusions Our results demonstrate that ERGs elicited by silent substitution stimuli can selectively reflect the operation of rod photoreceptors in the normal, light-adapted human retina.Deutsche Forschungsgemeinschaft (DFG) (KR1317/13-1) and Bundesministerium für Bildung und Forschung (BMBF) (01DN14009) provided financial support for JK

Accumulation of intraneuronal Aβ correlates with ApoE4 genotype

In contrast to extracellular plaque and intracellular tangle pathology, the presence and relevance of intraneuronal Aβ in Alzheimer’s disease (AD) is still a matter of debate. Human brain tissue offers technical challenges such as post-mortem delay and uneven or prolonged tissue fixation that might affect immunohistochemical staining. In addition, previous studies on intracellular Aβ accumulation in human brain often used antibodies targeting the C-terminus of Aβ and differed strongly in the pretreatments used. To overcome these inconsistencies, we performed extensive parametrical testing using a highly specific N-terminal Aβ antibody detecting the aspartate at position 1, before developing an optimal staining protocol for intraneuronal Aβ detection in paraffin-embedded sections from AD patients. To rule out that this antibody also detects the β-cleaved APP C-terminal fragment (β-CTF, C99) bearing the same epitope, paraffin-sections of transgenic mice overexpressing the C99-fragment were stained without any evidence for cross-reactivity in our staining protocol. The staining intensity of intraneuronal Aβ in cortex and hippocampal tissue of 10 controls and 20 sporadic AD cases was then correlated to patient data including sex, Braak stage, plaque load, and apolipoprotein E (ApoE) genotype. In particular, the presence of one or two ApoE4 alleles strongly correlated with an increased accumulation of intraneuronal Aβ peptides. Given that ApoE4 is a major genetic risk factor for AD and is involved in neuronal cholesterol transport, it is tempting to speculate that perturbed intracellular trafficking is involved in the increased intraneuronal Aβ aggregation in AD

Preparation of Pre-Confluent Retinal Cells Increases Graft Viability In Vitro and In Vivo: A Mouse Model

PURPOSE: Graft failure remains an obstacle to experimental subretinal cell transplantation. A key step is preparing a viable graft, as high levels of necrosis and apoptosis increase the risk of graft failure. Retinal grafts are commonly harvested from cell cultures. We termed the graft preparation procedure "transplant conditions" (TC). We hypothesized that culture conditions influenced graft viability, and investigated whether viability decreased following TC using a mouse retinal pigment epithelial (RPE) cell line, DH01. METHODS: Cell viability was assessed by trypan blue exclusion. Levels of apoptosis and necrosis in vitro were determined by flow cytometry for annexin V and propidium iodide and Western blot analysis for the pro- and cleaved forms of caspases 3 and 7. Graft viability in vivo was established by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase 3 immunolabeling of subretinal allografts. RESULTS: Pre-confluent cultures had significantly less nonviable cells than post-confluent cultures (6.6%±0.8% vs. 13.1%±0.9%, p<0.01). Cell viability in either group was not altered significantly following TC. Caspases 3 and 7 were not altered by levels of confluence or following TC. Pre-confluent cultures had low levels of apoptosis/necrosis (5.6%±1.1%) that did not increase following TC (4.8%±0.5%). However, culturing beyond confluence led to progressively increasing levels of apoptosis and necrosis (up to 16.5%±0.9%). Allografts prepared from post-confluent cultures had significantly more TUNEL-positive cells 3 hours post-operatively than grafts of pre-confluent cells (12.7%±3.1% vs. 4.5%±1.4%, p<0.001). Subretinal grafts of post-confluent cells also had significantly higher rates of cleaved caspase 3 than pre-confluent grafts (20.2%±4.3% vs. 7.8%±1.8%, p<0.001). CONCLUSION: Pre-confluent cells should be used to maximize graft cell viability

Low-Density Lipoprotein Receptor-Related Protein 1 (LRP1) Mediates Neuronal Aβ42 Uptake and Lysosomal Trafficking

Alzheimer's disease (AD) is characterized by the presence of early intraneuronal deposits of amyloid-beta 42 (Abeta42) that precede extracellular amyloid deposition in vulnerable brain regions. It has been hypothesized that endosomal/lysosomal dysfunction might be associated with the pathological accumulation of intracellular Abeta42 in the brain. Our previous findings suggest that the LDL receptor-related protein 1 (LRP1), a major receptor for apolipoprotein E, facilitates intraneuronal Abeta42 accumulation in mouse brain. However, direct evidence of neuronal endocytosis of Abeta42 through LRP1 is lacking.Here we show that LRP1 endocytic function is required for neuronal Abeta42 uptake. Overexpression of a functional LRP1 minireceptor, mLRP4, increases Abeta42 uptake and accumulation in neuronal lysosomes. Conversely, knockdown of LRP1 expression significantly decreases neuronal Abeta42 uptake. Disruptions of LRP1 endocytic function by either clathrin knockdown or by removal of its cytoplasmic tail decreased both uptake and accumulation of Abeta42 in neurons. Finally, we show that LRP1-mediated neuronal accumulation of Abeta42 is associated with increased cellular toxicity.These results demonstrate that LRP1 endocytic function plays an important role in the uptake and accumulation of Abeta42 in neuronal lysosomes. These findings emphasize the central function of LRP1 in neuronal Abeta metabolism

Abeta42-Induced Neurodegeneration via an Age-Dependent Autophagic-Lysosomal Injury in Drosophila

The mechanism of widespread neuronal death occurring in Alzheimer's disease (AD) remains enigmatic even after extensive investigation during the last two decades. Amyloid beta 42 peptide (Aβ1–42) is believed to play a causative role in the development of AD. Here we expressed human Aβ1–42 and amyloid beta 40 (Aβ1–40) in Drosophila neurons. Aβ1–42 but not Aβ1–40 causes an extensive accumulation of autophagic vesicles that become increasingly dysfunctional with age. Aβ1–42-induced impairment of the degradative function, as well as the structural integrity, of post-lysosomal autophagic vesicles triggers a neurodegenerative cascade that can be enhanced by autophagy activation or partially rescued by autophagy inhibition. Compromise and leakage from post-lysosomal vesicles result in cytosolic acidification, additional damage to membranes and organelles, and erosive destruction of cytoplasm leading to eventual neuron death. Neuronal autophagy initially appears to play a pro-survival role that changes in an age-dependent way to a pro-death role in the context of Aβ1–42 expression. Our in vivo observations provide a mechanistic understanding for the differential neurotoxicity of Aβ1–42 and Aβ1–40, and reveal an Aβ1–42-induced death execution pathway mediated by an age-dependent autophagic-lysosomal injury

Mechanism of Neuronal versus Endothelial Cell Uptake of Alzheimer's Disease Amyloid β Protein

Alzheimer's disease (AD) is characterized by significant neurodegeneration in the cortex and hippocampus; intraneuronal tangles of hyperphosphorylated tau protein; and accumulation of β-amyloid (Aβ) proteins 40 and 42 in the brain parenchyma as well as in the cerebral vasculature. The current understanding that AD is initiated by the neuronal accumulation of Aβ proteins due to their inefficient clearance at the blood-brain-barrier (BBB), places the neurovascular unit at the epicenter of AD pathophysiology. The objective of this study is to investigate cellular mechanisms mediating the internalization of Aβ proteins in the principle constituents of the neurovascular unit, neurons and BBB endothelial cells. Laser confocal micrographs of wild type (WT) mouse brain slices treated with fluorescein labeled Aβ40 (F-Aβ40) demonstrated selective accumulation of the protein in a subpopulation of cortical and hippocampal neurons via nonsaturable, energy independent, and nonendocytotic pathways. This groundbreaking finding, which challenges the conventional belief that Aβ proteins are internalized by neurons via receptor mediated endocytosis, was verified in differentiated PC12 cells and rat primary hippocampal (RPH) neurons through laser confocal microscopy and flow cytometry studies. Microscopy studies have demonstrated that a significant proportion of F-Aβ40 or F-Aβ42 internalized by differentiated PC12 cells or RPH neurons is located outside of the endosomal or lysosomal compartments, which may accumulate without degradation. In contrast, BBME cells exhibit energy dependent uptake of F-Aβ40, and accumulate the protein in acidic cell organelle, indicative of endocytotic uptake. Such a phenomenal difference in the internalization of Aβ40 between neurons and BBB endothelial cells may provide essential clues to understanding how various cells can differentially regulate Aβ proteins and help explain the vulnerability of cortical and hippocampal neurons to Aβ toxicity

Tg2576 Cortical Neurons That Express Human Ab Are Susceptible to Extracellular Aβ-Induced, K+ Efflux Dependent Neurodegeneration

Background: One of the key pathological features of AD is the formation of insoluble amyloid plaques. The major constituent of these extracellular plaques is the beta-amyloid peptide (Aβ), although Aβ is also found to accumulate intraneuronally in AD. Due to the slowly progressive nature of the disease, it is likely that neurons are exposed to sublethal concentrations of both intracellular and extracellular Aβ for extended periods of time. Results: In this study, we report that daily exposure to a sublethal concentration of Aβ1-40 (1 μM) for six days induces substantial apoptosis of cortical neurons cultured from Tg2576 mice (which express substantial but sublethal levels of intracellular Aβ). Notably, untreated Tg2576 neurons of similar age did not display any signs of apoptosis, indicating that the level of intracellular Aβ present in these neurons was not the cause of toxicity. Furthermore, wildtype neurons did not become apoptotic under the same chronic Aβ1-40 treatment. We found that this apoptosis was linked to Tg2576 neurons being unable to maintain K⁺ homeostasis following Aβ treatment. Furthermore, blocking K⁺ efflux protected Tg2576 neurons from Aβ-induced neurotoxicity. Interestingly, chronic exposure to 1 μM Aβ1-40 caused the generation of axonal swellings in Tg2576 neurons that contained dense concentrations of hyperphosphorylated tau. These were not observed in wildtype neurons under the same treatment conditions. Conclusions: Our data suggest that when neurons are chronically exposed to sublethal levels of both intra- and extra-cellular Aβ, this causes a K⁺-dependent neurodegeneration that has pathological characteristics similar to AD.9 page(s

Protective Effects of Positive Lysosomal Modulation in Alzheimer's Disease Transgenic Mouse Models

Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid β peptide (Aβ) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aβ1–42. Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK) was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of Aβ pathology. Systemic PADK injections in APPSwInd and APPswe/PS1ΔE9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both Aβ immunostaining as well as Aβx-42 sandwich ELISA measures in APPSwInd mice of 10–11 months. More extensive Aβ deposition in 20-22-month APPswe/PS1ΔE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aβ1–38 occurs as Aβ1–42 levels decrease in the mouse models, indicating that PADK treatment leads to Aβ truncation. Associated with Aβ clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces Aβ1–42 accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of Aβ pathology and provides proof-of-principle for small molecule therapeutic development for AD and possibly other protein accumulation disorders