Advances in the pathogenesis of Alzheimer’s disease: a re-evaluation of amyloid cascade hypothesis

Although APP has been implicated in the pathology of AD, much evidence shows that APP has its own physiological functions, especially in regulation of synaptic function and neuronal activity. Mice lacking APP and APP-like protein 2 show deficits in structure and function in neuromuscular synapses[10]. In cultured hippocampal neurons, lack of APP also affects synapse formation and transmission[11]. On the contrary, mice overexpressing APP exhibit enhanced synaptic plasticity and spatial memory[12]. Kamenetz et al found that APP processing could have a normal negative feed-back function in modulating Aβ levels to maintain proper neuronal activity[13]. In addition, APP processing also regulates cholesterol metabolism. When Aβ is produced, AICD is stabilized by Fe65, localized to the nucleus and binds to transcription factor Tip60. The protein-protein interaction initiates the transcription of the Aβ degradation enzyme, neprilysin, thus reduces the Aβ levels[14]. AICD-Fe65-Tip60 complex has been shown to suppress the transcription of lipoprotein receptor LRP1, which is known to regulate ApoE and cholesterol levels in CNS, suggesting a biological interaction between APP and ApoE/cholesterol metabolisms[15]. Furthermore, APP possesses the biological function in controlling cholesterol biosynthesis and sphingomyelin production via Aβ-dependent modulation of neuronal levels of Hydroxymethylglutaryl-CoA reductase (HMGR) and sphingomyelinases (SMases), indicating a functional basis of APP processing for the link between lipids and AD[16]. Endogenous AICD in primary neurons is temporally up-regulated during neuronal differentiation, and such a physiological function is negatively mediated by neuron-specific c-Jun N-terminal kinase JNK3 via phosphorylation of APP[17]. APP and its mammalian paralogs, the amyloid precursor-like proteins 1 and 2, have been demonstrated to be capable of forming homo- and hetero-complexes that exhibit physiological function in promoting trans-cellular adhesion in vivo[18]. Han et al also characterized a neuroprotective function of APP in preventing tau hyperphosphorylation via suppressing overactivation of Cdk5 (Cyclin-dependent kinase 5)[19].

It is well known that the pathologcial function of APP lies on its amyloidogenic processing. It has been recognized that many APP mutations cause autosomal dominant early-onset AD. Increasing of gene copy number including genomic duplication in the APP locus[20, 21] may also lead to AD dementia in earlier life. Interestingly, a recently identified mutation adjacent to β-site (A673T) of APP gene was shown to result in Aβ reduction and protection against cognitive decline in the elderly without AD[22]. On the other hand, however, overexpression of FAD-linked mutant APP could lead to olfactory sensory neuron apoptosis in the absence of amyloid plaque, which might be the mechanism of deficits in odor detection, one of the earliest AD symptoms[23]. All these indicate that both APP genomic duplication and mutations can lead to changes in APP function and subsequent Aβ metabolism, strongly implicating a central role of not only APP but also its β-cleavage in pathogenesis of AD. To identify the pathological functions of APP, many APP transgenic mice including wild-type human APP and FAD-linked APP mutations have been generated. FAD-linked APP mutation mice show an increase in the amount, length, and fibrillogenic generation of Aβ species and have amyloid deposits at the age of 18 months[24] while, surprisingly, mice overexpressing APP do not develop AD pathologies or memory deficits but instead exhibit enhanced spatial memory, which depends on the function of AICD generated by β-secretase-mediated cleavage[12]. Studies on APP mutation transgenic mice have given us much information of AD pathogenesis, but the molecular mechanisms still need further investigation.

PS1 mutations are the most common genetic cause for early-onset familial AD (FAD). PS genes harbor about 90% of identified FAD mutations. There have been more than 100 PS1 mutations being described. Some of them, such as L85P, P117L, P117S, insF1, and L166P, are associated with very early onset (usually before age of 30 years old) of cognitive decline[39]. Many of the PS1 mutations lead to an increase in relative production of more toxic Aβ42 peptides. The prevailing amyloid hypothesis posits that deposits of Aβ peptides, especially the more hydrophobic and aggregation-prone Aβ42, initiates a pathogenic cascade, leading to neurodegeneration in AD[40]. This has been referred to as the toxic gains-of-function of PS in triggering neurodegeneration in AD. The amyloid cascade hypothesis is supported by the results from FAD-linked mutant transgenic mice. PS1 mutation significantly accelerates the rate of Aβ deposition in mutant APP transgenic mice[24]. Expression of human mutant PS1 in PS1 null mice is sufficient to elevate Aβ1-42, supporting a gain-of-function activity of PS1 mutation[41].

However, the most recent evidence from several independent PS transgenic model-based studies emerged that supports the “PS loss of function” hypothesis as a potential pathogenic mechanism of AD. Firstly, mice lacking both PSs in the forebrain show AD-like progressive neurodegenerative phenotypes including forebrain degeneration, impaired synaptic plasticity and spatial memory without Aβ production[42‐46]. A number of PS1 mutations (L113P, G183V and insR352) have been found in patients with familial forms of frontotemporal dementia (FTD), a common neurodegenerative dementia that lacks amyloidogenesis[47‐49]. These observations suggest that neurodegeneration can take place in the absence of Aβ.

PS genes have been identified to play an important role in many normal physiological activities. These physiological functions can be classified to γ-secretase-dependent and -independent functions. There are many identified γ-secretase substrates. By cleavage of these substrates, PSs mediate their multiple functions in development, calcium homeostasis, cell adhesion, transport, trafficking/localization, and apoptosis[36, 38, 50]. FAD-linked PS mutations might impair the γ-secretase-dependent proteolysis of some of the substrates, such as Notch, N-cadherin and tyrosinase, resulting in loss of the related functions of PS[51, 52]. Meanwhile, FAD-linked PS mutations might also impair some γ-secretase-independent functions, such as the regulation of β-catenin-dependent signaling[53], modulation of phosphatidylinositol 4,5-bisphosphate metabolism[54], endoplasmic reticulum [Ca²⁺ leak function[55], PI3K/Akt signaling pathway-dependent neuroprotective roles[56], synaptic homeostasis[57] and fast axonal transport of APP[58]. In addition to involving in Aβ generation, PS genes participate in the regulation of Aβ degradation mediated by AICD-dependent transcriptional modulation of the degrading enzyme, neprilysin[14]. FAD-linked PS mutations may disrupt the physiological function of PSs in regulating Aβ levels.

Surprisingly, many γ-secretase inhibitors at low concentration enhance Aβ42 production while reducing Aβ40 levels, similar to the effects of FAD-linked PS mutations[59‐61], suggesting that PS mutation could result in a partial loss of its function. Furthermore, PS mutations are scattered throughout the protein’s N-terminus, C-terminus and transmembrane domains, occurring at about 20% of the amino acid residues. As it is impossible for different PS mutations to gain the same toxic function, it is therefore most likely that the loss of normal PS function by ‘random’ changes of amino acid residues is the culprit for triggering AD pathogenesis. However, the “PS loss of function” hypothesis is still unable to explain the exact mechanism for FAD-linked APP mutations that cause AD. In this regard, it has been assumed that Aβ42 might act as an inhibitor of γ-secretase. APP mutations may interfere with the physiological roles of PS and hereby initiate the pathogenic cascades of AD[51]. Further investigations are required to confirm the hypothesis.

NEP is a 90 ~ 110 kDa plasma membrane glycoprotein of the neutral zinc metalloendopeptidase family that degrades enkephalins, endothelins, and Aβ peptides[82]. Particularly, NEP is the major enzyme to degrade soluble extracellular Aβ in the brain. Recent studies demonstrated that NEP levels decline in an age-dependent manner and inversely correlate with levels of insoluble Aβ in the temporal and frontal cortex of AD and normal brain[83, 84]. NEP expression or activities are decreased significantly in AD brain[85, 86]. The finding that possession of ApoE4 was related to obvious reduction in NEP levels[85] suggests that down-regulation of NEP might be implicated in AD pathogenesis.

The transcription of NEP has been demonstrated to be regulated by AICD which is released during the Aβ generation[14]. There is a physiological negative feedback in vivo that keeps Aβ homeostasis, in which Aβ production could lead to translocation of AICD to nucleus and transactivation of NEP. NEP therefore has a role in governing the balance between Aβ production and degradation. If such a balance is disrupted, Aβ would come to be oligomerized and lead to formation of the fibrillar Aβ protein (fAβ). The resulting fAβ can inhibit the proteolytic activities of the proteases by binding to NEP and IDE[87], leading to the formation of a positive feedback that accelerates amyloid deposition. Indeed, studies on transgenic mice of AD have shown that NEP-mediated degradation of Aβ plays a key role in AD neurodegeneration and serves as a novel therapeutic approach to AD[88]. These findings also suggest that AD pathogenesis might result from deficits in Aβ clearance. On the other hand, however, recent observations in drosophila demonstrated that NEP overespression could result in the inhibition of CREB-mediated transcription, age-dependent axon degeneration and shortened lifespan[89]. Studies on crossing hAPP transgenic mice and NEP transgenic mice also showed that, although NEP overexpression inhibits plaque formation, it fails to reduce pathogenic Aβ oligomers and improve the impaired learning and memory function[90].

IDE is an 110 kDa zinc metalloendopeptidase that highly expresses in the liver, testis, muscle and brain[82]. The enzyme has been implicated in the pathogenesis of AD and type II diabetes due to its capabilities in degrading Aβ, AICD[91, 92], amylin, insulin and insulin-like growth factors[82]. IDE gene is located in chromosome 10 that is highly associated with later-onset AD (LOAD)[93]. Some genetic variants of IDE have also been strongly implicated in LOAD[94, 95]. IDE mRNA and protein levels are markedly decreased in hippocampus of AD patients with ApoE ε4 allele, the genotyping known as a high risk factor for LOAD[96]. Membrane-bound IDE levels and its activity are significantly decreased in subjects with mild cognitive impairment (MCI) and appear to decrease continuously during the conversion from MCI to AD[97]. IDE activity is reduced in affected versus unaffected subjects of three chromosome 10-linked AD pedigrees, although no significant difference of IDE expression has been observed[98]. However, recent studies on transgenic AD mouse models showed that cortical IDE mRNA and protein levels are elevated in parallel with Aβ40 and Aβ42 generation[99]. In transgenic tg2576 mice, IDE expression is increased with age and is located around amyloid plaque as a result of Aβ-induced inflammation[100]. This phenomena is similar to the observation that IDE is immunopositive in senile plaques in human AD brain[101]. Studies on triple-transgenic mice (hAPPswe/PS1 M146V/hTau P301L) showed that the expression of IDE was regulated by 17beta-estrodiol via an ERbeta/PI3K pathway[102]. Unlike NEP that hydrolyzes both monomeric and oligomeric Aβ, IDE is found to degrade only soluble monomeric Aβ[103]. A recent study by Llovera et al demonstrated that the catalytic domain of IDE could form a stable complex with Aβ, which might disrupt Aβ clearance and facilitate AD neurodegeneration[104]. There have been also studies showing that IDE can cleave C-terminal domain of human acetylcholinesterase (hAChE) and trigger its conformational conversion from α to β-structure, which acts as the seed of Aβ fibrils and enhances the rate of amyloid elongation[105]. This suggests an important role of IDE digestion of C-terminal domain of hAChE in amyloidogenic pathogenesis of AD.

IDE plays essential role in insulin homeostasis, implicating a close relationship between AD and type II diabetes (DM2). A large body of evidence has indicated that cognitive capacity is often impaired in patients with diabetes[106] while insulin resistance is a high risk factor of AD[107]. IDE knockout mice exhibit hallmarks of both AD and DM2[92]. Diet-induced insulin resistance leads to increased γ-secretase activity and decreased IDE activity, resulting in elevated Aβ40 and Aβ42 levels in the brain of Tg2576 mice[108]. Further exploration of the underlying mechanism has shown that defective insulin receptor signaling may lead to up-regulation of Aβ generation. Insulin resistance induced by intake of sucrose-sweetened water or a safflower oil-enriched diet exacerbates the AD pathology in transgenic AD animal models[71, 109].

Aβ has long been shown to affect excitatory synaptic neurotransmission[13] and hippocampal synaptic plasticity[110, 126, 141]. Aβ oligomers are able to bind specifically to excitatory pyramidal neurons and affect their synaptic structure, composition and density and the membrane expression of NMDA receptor[135]. Similar observations on rat hippocampal slices have also shown that Aβ oligomers induce loss of hippocampal synapses and spines in a NMDA receptor-dependent manner[130]. Aβ exhibits a specific inhibitory role in a presynaptic P/Q calcium current, which is required for synaptic plasticity[128]. Aβ also plays an important role in activity-dependent presynaptic vesicle release[142]. Moreover, Aβ can induce neuronal network dysfunction including abnormal induction of excitatory neuronal activity and compensatory inhibitory circuits[143]. The abnormalities of synapse and neuronal network resulting from Aβ might be the physiological basis of cognitive decline in AD animal models and patients.

Microglia is rapidly recruited around amyloid plaques after its appearance[144]. Aβ can trigger the translocation of microglia from bone marrow to the sites around amyloid plaques[145]. Aβ up-regulates P38 MAPK or p44/42 MAPK signaling, which may lead to microglia activation with release of cytokines including tumor necrosis factor α (TNF-α) and interleukin-1 β (IL1-β)[146]. The microglia around plaques maintains the stability of the plaques[147]. Both pharmacological blockade and genetic knock-out of TNF-α or iNOS down-regulate Aβ-induced cognitive dysfunction in AD mouse model, revealing that TNF-α and iNOS are key mediator of Aβ neurotoxicity[148]. Genetic disruption of transforming growth factor β (TGF-β) signaling mitigates Aβ levels and amyloid plaques, and partially rescues the cognitive abnormality in Tg2576 mice[149]. However, the roles of TGF-β signaling are in a debate. Tesseur I et al showed that deficiency in TGF-β signaling promotes Aβ accumulation and neuronal degeneration[150]. Accumulating evidence also suggests that functional loss of TGF-β signaling may contribute to Aβ-induced neurodegeneration and tau pathology, indicating a neuroprotective role of this pathway[151].