Alzheimer's amyloid-ß and tau protein accumulation is associated with decreased expression of the LDL receptor-associated protein in human brain tissue.

INTRODUCTION: One of the major neuropathological features of Alzheimer's disease (AD) is the accumulation of amyloid-ß (Aß) protein in the brain. Evidence suggests that the low-density lipoprotein receptor-associated protein (RAP) binds strongly to Aß and enhances its cellular uptake and that decreased RAP expression correlates with increased Aß production in animal models of AD. METHODS: The current study examined whether RAP levels change in AD human brain tissue and whether they are related to the amount of AD pathology. RAP and NeuN levels were determined by Western blot, while low-density lipoprotein receptor-related protein 1 (LRP1), tau and Aß levels were determined by ELISA in the temporal cortex of 17 AD and 16 control cases. RESULTS: An increase in total Aß and insoluble and soluble tau protein was observed in AD brain tissue. In contrast, RAP levels were significantly decreased in AD brain tissue compared to controls. Correlation analysis revealed that levels of RAP correlated with both total Aß and soluble and insoluble tau levels. Neither LRP1 nor NeuN levels were significantly altered in AD brain tissue homogenates and did not correlate with Aß or tau protein levels. CONCLUSION: Reduction in RAP may contribute to the accumulation and aggregation of Aß in the AD brain.

Knockout of Amyloid ß Protein Precursor (APP) Expression Alters Synaptogenesis, Neurite Branching and Axonal Morphology of Hippocampal Neurons.

The function of the ß-A4 amyloid protein precursor (APP) of Alzheimer's disease (AD) remains unclear. APP has a number of putative roles in neuronal differentiation, survival, synaptogenesis and cell adhesion. In this study, we examined the development of axons, dendrites and synapses in cultures of hippocampus neutrons derived from APP knockout (KO) mice. We report that loss of APP function reduces the branching of cultured hippocampal neurons, resulting in reduced synapse formation. Using a compartmentalised culture approach, we found reduced axonal outgrowth in cultured hippocampal neurons and we also identified abnormal growth characteristics of isolated hippocampal neuron axons. Although APP has previously been suggested to play an important role in promoting cell adhesion, we surprisingly found that APPKO hippocampal neurons adhered more strongly to a poly-L-lysine substrate and their neurites displayed an increased density of focal adhesion puncta. The findings suggest that the function of APP has an important role in both dendritic and axonal growth and that endogenous APP may regulate substrate adhesion of hippocampal neurons. The results may explain neuronal and synaptic morphological abnormalities in APPKO mice and the presence of abnormal APP expression in dystrophic neurites around amyloid deposits in AD.

Peripheral treatment with enoxaparin exacerbates amyloid plaque pathology in Tg2576 mice.

Alzheimer's disease (AD) is a complex, progressive neurological disorder characterized by the formation of extracellular amyloid plaques composed of ß-amyloid protein (Aß), the key component in pathogenesis of AD. Peripheral administration of enoxaparin (ENO) reportedly reduces the level of Aß and the amyloid plaques in the cortex of amyloid precursor protein (APP) transgenic mice. However, the exact mechanism of these effects is unclear. Our previous studies indicated that ENO can inhibit APP processing to Aß in primary cortical cells from Tg2576 mice by downregulating BACE1 levels. This study examines whether ENO-induced reduction of amyloid load is due to the decreased APP processing to Aß in Tg2576 mice. Surprisingly, our results indicated that ENO significantly increases the Aß42/Aß40 ratio in cortex and enhances the amyloid plaque load in both cortex and hippocampus, although overall APP processing was not influenced by ENO. Moreover, ENO stimulated the aggregation of both Aß40 and Aß42 in vitro. Although ENO has been reported to improve cognition in vivo and has potential as a therapeutic agent for AD, the results from our study suggest that ENO can exacerbate the amyloid pathology, and the strategy of using ENO for the treatment of AD may require further assessment. © 2016 Wiley Periodicals, Inc.

N-terminal domain of Bothrops asper Myotoxin II Enhances the Activity of Endothelin Converting Enzyme-1 and Neprilysin.

Neprilysin (NEP) and endothelin converting enzyme-1 (ECE-1) are two enzymes that degrade amyloid beta in the brain. Currently there are no molecules to stimulate the activity of these enzymes. Here we report, the discovery and characterisation of a peptide referred to as K49-P1-20, from the venom of Bothrops asper which directly enhances the activity of both ECE-1 and NEP. This is evidenced by a 2- and 5-fold increase in the Vmax of ECE-1 and NEP respectively. The K49-P1-20 concentration required to achieve 50% of maximal stimulation (AC50) of ECE-1 and NEP was 1.92 ± 0.07 and 1.33 ± 0.12 µM respectively. Using BLITZ biolayer interferometry we have shown that K49-P1-20 interacts directly with each enzyme. Intrinsic fluorescence of the enzymes change in the presence of K49-P1-20 suggesting a change in conformation. ECE-1 mediated reduction in the level of endogenous soluble amyloid beta 42 in cerebrospinal fluid is significantly higher in the presence of K49-P1-20 (31 ± 4% of initial) compared with enzyme alone (11 ± 5% of initial; N = 8, P = 0.005, unpaired t-test). K49-P1-20 could be an excellent research tool to study mechanism(s) of enzyme stimulation, and a potential novel drug lead in the fight against Alzheimer's disease.

Do Microglia Default on Network Maintenance in Alzheimer's Disease?

Although the cause of Alzheimer's disease (AD) remains unknown, a number of new findings suggest that the immune system may play a critical role in the early stages of the disease. Genome-wide association studies have identified a wide array of risk-associated genes for AD, many of which are associated with abnormal functioning of immune cells. Microglia are the brain's immune cells. They play an important role in maintaining the brain's extracellular environment, including clearance of aggregated proteins such as amyloid-ß (Aß). Recent studies suggest that microglia play a more active role in the brain than initially considered. Specifically, microglia provide trophic support to neurons and also regulate synapses. Microglial regulation of neuronal activity may have important consequences for AD. In this article we review the function of microglia in AD and examine the possible relationship between microglial dysfunction and network abnormalities, which occur very early in disease pathogenesis.

Neurogenin 2 mediates amyloid-ß precursor protein-stimulated neurogenesis.

Amyloid-ß precursor protein (APP) is well studied for its role in Alzheimer disease, although its normal function remains uncertain. It has been reported that APP stimulates the proliferation and neuronal differentiation of neural stem/progenitor cells (NSPCs). In this study we examined the role of APP in NSPC differentiation. To identify proteins that may mediate the effect of APP on NSPC differentiation, we used a gene array approach to find genes whose expression correlated with APP-induced neurogenesis. We found that the expression of neurogenin 2 (Ngn2), a basic helix-loop-helix transcription factor, was significantly down-regulated in NSPCs from APP knock-out mice (APPKO) and increased in APP transgenic (Tg2576) mice. Ngn2 overexpression in APPKO NSPCs promoted neuronal differentiation, whereas siRNA knockdown of Ngn2 expression in wild-type NSPCs decreased neuronal differentiation. The results demonstrate that APP-stimulated neuronal differentiation of NSPCs is mediated by Ngn2.

The N-terminal fragment of the ß-amyloid precursor protein of Alzheimer's disease (N-APP) binds to phosphoinositide-rich domains on the surface of hippocampal neurons.

The function of the ß-amyloid precursor protein (APP) of Alzheimer's disease is poorly understood. The secreted ectodomain fragment of APP (sAPPα) can be readily cleaved to produce a small N-terminal fragment (N-APP) that contains heparin-binding and metal-binding domains and that has been found to have biological activity. In the present study, we examined whether N-APP can bind to lipids. We found that N-APP binds selectively to phosphoinositides (PIPs) but poorly to most other lipids. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 )-rich microdomains were identified on the extracellular surface of neurons and glia in primary hippocampal cultures. N-APP bound to neurons and colocalized with PIPs on the cell surface. Furthermore, the binding of N-APP to neurons increased the level of cell-surface PI(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate. However, PIPs were not the principal cell-surface binding site for N-APP, because N-APP binding to neurons was not inhibited by a short-acyl-chain PIP analogue, and N-APP did not bind to glial cells which also possessed PI(4,5)P2 on the cell surface. The data are explained by a model in which N-APP binds to two distinct components on neurons, one of which is an unidentified receptor and the second of which is a PIP lipid, which binds more weakly to a distinct site within N-APP. Our data provide further support for the idea that N-APP may be an important mediator of APP's biological activity.

Insights into the physiological function of the ß-amyloid precursor protein: beyond Alzheimer's disease.

The ß-amyloid precursor protein (APP) has been extensively studied for its role as the precursor of the ß-amyloid protein (Aß) of Alzheimer's disease. However, the normal function of APP remains largely unknown. This article reviews studies on the structure, expression and post-translational processing of APP, as well as studies on the effects of APP in vitro and in vivo. We conclude that the published data provide strong evidence that APP has a trophic function. APP is likely to be involved in neural stem cell development, neuronal survival, neurite outgrowth and neurorepair. However, the mechanisms by which APP exerts its actions remain to be elucidated. The available evidence suggests that APP interacts both intracellularly and extracellularly to regulate various signal transduction mechanisms. This article reviews studies on the structure, expression and post-translational processing of ß-amyloid precursor protein (APP), as well as studies on the effects of APP in vitro and in vivo. We conclude that the published data provide strong evidence that APP has a trophic function. APP is likely to be involved in neural stem cell development, neuronal survival, neurite outgrowth and neurorepair. However, the mechanisms by which APP exerts its actions remain to be elucidated. The available evidence suggests that APP interacts both intracellularly and extracellularly to regulate various signal transduction mechanisms.

ß-Amyloid precursor protein: function in stem cell development and Alzheimer's disease brain.

Stem cell therapy may be a suitable approach for the treatment of many neurodegenerative diseases. However, one major impediment to the development of successful cell-based therapies is our limited understanding of the mechanisms that instruct neural stem cell behaviour, such as proliferation and cell fate specification. The ß-amyloid precursor protein (APP) of Alzheimer's disease (AD) may play an important role in neural stem cell proliferation and differentiation. Our recent work shows that in vitro, APP stimulates neural stem or progenitor cell proliferation and neuronal differentiation. The effect on proliferation is mediated by an autocrine factor that we have identified as cystatin C. As cystatin C expression is also reported to inhibit the development of amyloid pathology in APP transgenic mice, our finding has implications for the possible use of cystatin C for the therapy of AD.

Role of cystatin C in amyloid precursor protein-induced proliferation of neural stem/progenitor cells.

The amyloid precursor protein (APP) is well studied for its role in Alzheimer disease. However, little is known about its normal function. In this study, we examined the role of APP in neural stem/progenitor cell (NSPC) proliferation. NSPCs derived from APP-overexpressing Tg2576 transgenic mice proliferated more rapidly than NSPCs from the corresponding background strain (C57Bl/6xSJL) wild-type mice. In contrast, NSPCs from APP knock-out (APP-KO) mice had reduced proliferation rates when compared with NSPCs from the corresponding background strain (C57Bl/6). A secreted factor, identified as cystatin C, was found to be responsible for this effect. Levels of cystatin C were higher in the Tg2576 conditioned medium and lower in the APP-KO conditioned medium. Furthermore, immunodepletion of cystatin C from the conditioned medium completely removed the ability of the conditioned medium to increase NSPC proliferation. The results demonstrate that APP expression stimulates NSPC proliferation and that this effect is mediated via an increase in cystatin C secretion.

Proteoglycans in the central nervous system: role in development, neural repair, and Alzheimer's disease.

Proteoglycans (PGs) are major components of the cell surface and extracellular matrix and play critical roles in development and maintenance of the central nervous system (CNS). PGs are a family of proteins, all of which contain a core protein to which glycosaminoglycan side chains are covalently attached. PGs possess diverse physiological roles, particularly in neural development, and are also implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD). The main functions of PGs in the CNS are reviewed as are the roles of PGs in brain injury and in the development or treatment of AD.

Mechanisms of transthyretin aggregation and toxicity.

Amyloidoses are characterised by the deposition of insoluble protein that occurs in the extracellular compartment of various tissues. One form of amyloidosis is caused by transthyretin (TTR) misfolding and deposition in target tissues. It is clear that many amyloidoses share common features of fibrillogenesis and toxicity. This chapter examines the mechanisms of TTR aggregation with a view to understanding the possible therapeutic interventions in amyloid disease.

Size and sulfation are critical for the effect of heparin on APP processing and Aß production.

Alzheimer's disease is associated with abnormal accumulation of Aß, which is produced from the ß-amyloid precursor protein (APP) by the ß-site APP-cleaving enzyme (BACE1) and γ-secretase. Our previous studies showed that heparin can decrease APP processing by decreasing the levels of BACE1 and ADAM10. In this study, we examined the effects of glycosaminoglycans (GAGs) on APP processing and Aß production with the aim of understanding the specificity of the effects. Various GAG analogs were incubated with primary cortical cells derived from APP (SW)Tg2576 mice and the level of APP, proteolytic products of APP and APP-cleavage enzymes were measured. The effect of GAGs on APP processing was both size- and sulfation-dependent. 6-O-Sulfation was important for the effect on APP processing as heparin lacking 6-O sulfate were less potent than native heparin. However, deletion of carboxyl groups on heparin had no significant effect on APP processing. Our studies suggest that there is structural specificity to the effect of GAGs on APP processing and that certain GAGs have a greater effect on Aß production than others. This suggests that it might be possible to alter the structure of GAGs to achieve more specific inhibitors of APP processing that can cross the blood-brain barrier.

STIM1 is necessary for store-operated calcium entry in turning growth cones.

Coordinated calcium signalling is vital for neuronal growth cone function and axon pathfinding. Although store-operated calcium entry (SOCE) has been suggested to be an important source of calcium in growth cone navigation, the mechanisms that regulate calcium signalling, particularly the regulation of internal calcium stores within growth cones, are yet to be fully determined. Stromal Interaction Molecule 1 (STIM1) is a calcium-sensing protein localized in the endoplasmic reticulum membrane that interacts with Orai proteins in the plasma membrane to initiate SOCE and refilling of intracellular calcium stores. We hypothesize that STIM1- and Orai1/2-mediated SOCE are necessary for growth cone turning responses to extracellular guidance cues. We show that STIM1 and Orai reorganize into puncta upon store depletion and during growth cone turning with STIM1 localization biased towards the turning side (high calcium side) of the growth cone. Importantly, STIM1 knock-down perturbed growth cone turning responses to the guidance cues brain-derived neurotrophic factor and semaphorin-3a (Sema-3a), as well as abolishing Sema-3a-induced growth cone collapse. Furthermore, STIM1 knock-down abolished SOCE induced by brain-derived neurotrophic factor, but not Sema-3a. Our data suggest that STIM1 is essential for correct growth cone navigation, playing multiple roles in growth cone motility, including the activation of SOCE.

Dysregulation of Ca2+ homeostasis in Alzheimer's disease: role in acetylcholinesterase production and AMPA receptor internalization.

Amyloid-ß (Aß)-induced Ca(2+) influx into neurons has been well described since it was first reported almost 20 years ago. Ca(2+) influx can disrupt mechanisms of long-term potentiation and long-term depression and increase neuronal susceptibility to excitotoxicity. Our studies show that Aß also causes an increase in acetylcholinesterase (AChE) levels and induces AMPA receptor internalization through Ca(2+)-dependent mechanisms. As Aß-induced Ca(2+) entry may increase neuronal excitability, the increase in AChE and the downregulation of cell surface AMPA receptors may be part of a homeostatic mechanism which maintains normal levels of cholinergic and glutamatergic signaling.

Characterization of early stage intermediates in the nucleation phase of Aß aggregation.

Alzheimer's disease (AD) is a common form of dementia, which is characterized by the presence of extracellular amyloid plaques comprising the amyloid ß peptide (Aß). Although the mechanism underlying AD pathogenesis remains elusive, accumulating evidence suggests that the process of amyloid fibril formation is a surface-mediated event, which plays an important role in AD onset and progression. In this study, the mechanism of Aß aggregation on hydrophobic surfaces was investigated with dual polarization interferometry (DPI), which provides real-time information on early stages of the aggregation process. Aggregation was monitored on a hydrophobic C18 surface and a polar silicon oxynitride surface. The DPI results showed a characteristic Aß aggregation pattern involving a decrease in the density of Aß at the surface followed by an increase in the thickness on the hydrophobic C18 chip. Most importantly, the DPI measurements provided unique information on the early stages of Aß aggregation, which is characterized by the presence of initially slow nucleus formation process followed by exponential fibril elongation. The dimensions of the putative nucleus corresponded to a thickness of ∼5 nm for both Aß40 and Aß42, which may represent about 10-15 molecules. The results thus support the nucleation-dependent polymerization model as indicated by the presence of a nucleation phase followed by an exponential growth phase. These results are the first reported measurements of the real-time changes in Aß molecular structure during the early stages of amyloid formation at the nanometer level.

Altered expression of brain acetylcholinesterase in FTDP-17 human tau transgenic mice.

Pathological hyperphosphorylation and aggregation of the tau protein is associated with dementia and can be the central cause of neurodegeneration. Here, we examined potential alterations in the level of the cholinergic enzyme acetylcholinesterase (AChE) in the brain of transgenic mice (Tg-VLW) expressing human tau mutations. Overexpression of mutant hyperphosphorylated tau (P-tau) led to an increase in the activity of AChE in the brain of Tg-VLW mice, paralleled by an increase in AChE protein and transcripts; whereas the levels of the enzyme choline acetyltransferase remained unaffected. VLW tau overexpression in SH-SY5Y cells also increased AChE activity levels. All major molecular forms of AChE were increased in the Tg-VLW mice, including tetrameric AChE, which is the major species involved in hydrolysis of acetylcholine in the brain. Colocalization of human P-tau and AChE supports the conclusion that P-tau can act to increase AChE. This study is the first direct evidence of a modulatory effect of P-tau on brain AChE expression.

Neurodegeneration in familial amyloidotic polyneuropathy.

Familial amyloid polyneuropathies (FAP) constitute a group of inherited amyloidoses that affect peripheral nerves. One common form of FAP is caused by transthyretin (TTR) misfolding and deposition in the peripheral nervous system, leading to neuronal toxicity and death. The molecular mechanisms responsible for this toxicity are unclear; however, there is good biochemical and histopathological evidence that the toxicity of TTR mutations is correlated to their aggregation state. In addition, neuronal calcium dysregulation is a mechanism that has been suggested to drive the pathogenesis of FAP. Amyloidogenic TTR mutations cause significant calcium influx via L-type calcium channels in neuronal cell lines, while in primary sensory neurons, TTR mediates a calcium influx via a novel mechanism of transient receptor potential melanostatin (TRPM8) and voltage-gated sodium and calcium channel activation. Significantly, calcium dysregulation is a pathological hallmark of other neurodegenerative diseases involving amyloidosis, for example Alzheimer's disease, and this mechanism could explain the molecular events that drive amyloid toxicity in other neurodegenerative diseases.