An Integrative View on Intra- and Inter-Cellular Cooperation Mechanisms in Alzheimer’s Disease

Review Article

Austin Alzheimers J Parkinsons Dis. 2014;1(1): 11.

An Integrative View on Intra- and Inter-Cellular Cooperation Mechanisms in Alzheimer’s Disease

Ancuta Augustina Gheorghisan Galateanu1,2 and Ana-Maria Enciu1,3*

1Department of Cellular and Molecular Medicine, Carol Davila University of Medicine and Pharmacy, Romania

2C. I. Parhon National Institute of Endocrinology, Romania

3V. Babes National Institute of Pathology, Romania

*Corresponding author: Enciu AM, Department of Cellular and Molecular Medicine, Carol Davila University of Medicine and Pharmacy, no8 Eroilor Sanitari Blvd, 050474 Bucharest, Romania.

Received: August 02, 2014; Accepted: September 08, 2014; Published: September 09, 2014


Alzheimer’s disease (AD) has been identified as central nervous system pathology more than 100 years ago and for a long time the diagnostic criteria remained the same, based on the anatomopathological findings. Failure of clinical trials in almost every field of AD therapy forced a widening of perspective on molecular pathologies, to the point which, now, AD is considered a multifactorial disease. Recent advancement in AD cell biology uncovered new data regarding amyloid precursor protein and its enzymatic cleavage products, the amyloid beta peptides, such as oligomerization and membrane pore formation. More intracellular deregulated events have been highlighted, e.g. endoplasmic reticulum stress and mitochondrial dysfunction, possibly related through the newly discovered Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM). During the last few years, non-neuronal cell populations came into focus in AD research, such as glial cells, endothelial cells forming the blood-brain barrier or brain non-neuronal stem cells of mesenchymal nature. All these cellular players interact or react to what was considered the central dogma of AD – the amyloid cascade – to the point which this dogma is about to be overthrown.

Keywords: Alzheimer’s disease; Amyloid pores; Endoplasmic reticulum stress; Mitochondrial dysfunction; Blood-brain barrier; Brain mesenchymal stem cells


Alzheimer’s disease (AD) has been identified as central nervous system pathology more than 100 years ago and for a long time the diagnostic criteria remained the same, based on the anatomopathological findings: amyloid plaques and fibrillary tangles. For almost half of century no significant progress has been made for this immutable disease, until electron micrograph studies identified amyloid fibrils in both Alzheimer’s disease and Down syndrome patients [1]. Combined with genetic studies, the Amyloid Precursor Protein (APP) gene and splicing proteins were identified and soon, familial forms of the disease and subsequent mutations were reported [2-4]. Familial studies, as well as molecular studies of late-onset AD led to identification of other risk factors, such as apolipo protein E [5,6]. The new wave of knowledge came from attempts to identify the proteases involved in fibril generation and AD pathology. Observation that mutated presenilins (enzymes involved in Notch signaling) are related to aggressive forms of early onset AD [7] led to a demonstrated relationship between these gamma secretase and APP [8] followed by identification of alpha secretase [9] and beta secretase [10]. Beta secretase has soon become the main point of interest in the development of an efficient, targeted, anti-AD therapy [11], but failed to fulfill the expectations during the next twenty years. Failure of clinical trials in almost every field of AD therapy forced a widening of perspective on molecular pathologies, to the point which now, AD is considered a multifactorial disease. Genome-wide association studies have identified during the last 5 years involvement of cholesterol metabolism, endosomal system and innate immunity in AD, albeit at frequencies below 50% and modest impact on risk [12]. Although most in vitro studies used neuronal cell populations, lately, new cell types (microglia, endothelial cells) were shown to contribute to, or be influenced by molecular pathogenesis of AD. Parallel advancement in other types of dementias, such as vascular dementia, raised the possibility that pathologic and diagnostic limits may not be so clearcut. Far from been exhaustive, the present review addresses recent advances in several fields of neuroresearch related one way or the other to AD, from intracellular, compartment-specific processing of AD related proteins, to types of cells of importance in the progression of the disease.

Intracellular Pathogenic Links

Amyloid cascade and tau hyperphosphorylation- a 5 years update

Amyloid cascade: Amyloid cascade is a two-step enzymatic cleavage, initiated normally by a class of proteases (ADAM), also called a secretases, that shed the extracellular domain of APP and generate a C-terminal fragment, further cleaved by a gamma-secretase complex, with presenilins as main enzymatic component. In AD, a β secretase cleaves the extracellular domain of APP, generating the C99 fragment and creating the premises to yield Aβ peptides 40 and 42, with the latter exhibiting high propensity to aggregate in the extracellular space and create amyloid plaques. Besides these two main Aβ species, several other truncated species have been identified Cerebrospinal Fluid (CSF), plasma or interstitial fluid [13]. Very recently, through nuclear magnetic resonance and electron paramagnetic resonance spectroscopy, it was shown that C99 has a flexible transmembrane domain with a binding site for cholesterol [14]. It is now known that amyloid peptides are detrimental to synaptic activity even inoligomeric forms [15], or intracellular deposits [16]. Up to this moment, all three classes of secretase are fairly well described along with the secretory pathway, alternative enzymatic cleaving sites and subsequent peptide products. Cleavage occurs with optimum efficiency in the endosomal compartment, characterized by a lower pH than the rest of cellular compartments.

Amyloid precursor protein and cholesterol metabolism: Although the relationship between amyloid plaque and cholesterol has been long reported, only recently, functional cholesterol-binding domains in several amyloidogenic proteins have been identified. For APP, although cholesterol binding domains are found in both C99 and Aβ peptides, they are only partially overlapping, leading to greater affinity of cholesterol for Aβ. Furthermore, cholesterol binding enables Aβ peptides to form membrane pores by oligomerization. This data offered the perspective of an original therapeutic strategy using cholesterol competing-drugs, such as bexarotene for the treatment of Alzheimer’s and other neurodegenerative diseases that involve cholesterol-dependent toxic oligomers [17].

But not only cholesterol and apolipo proteins are shown to influence Aβ deposition and cognition, recently other players involved in cholesterol metabolisms were studied and reported to exert varia influences on AD pathology.

Hemizygosity of ATP-binding cassette transporter A1 (ABCA1) transporter (that regulates cholesterol efflux and formation of High- Density Lipoprotein (HDL)) increases Aβ deposition, but only when associated with ApoE4 phenotype [18].

In turn, overexpression of human apoA-I in the circulation prevents learning and memory deficits in APP/PS1 mice, but apparently without changing the burden of Aβ deposition in the brain. The protective effect was supposedly due partly to anti neuroinflammatory effect and diminished cerebral amyloid angiopathy [19].

Hormonal signaling: AD has become today the “diabetes of the brain”, or type 3 diabetes, due to its well characterized resistance to insulin signaling, accompanied by IGF-1 resistance and closely associated with IRS-1 dysfunction, potentially triggered by Aβ oligomers [20].“Resistance to insulin” in AD brain has grossomodo the same significance as in diabetes – defective downstream insulin signaling, in spite of sufficient hormone levels. Mechanisms responsible for brain insulin resistance, reported so far are: i) increased IGF-1R levels with aberrant distribution in AD cortex [21]; ii) phosphorylation of insulin receptor substrate 1 and 2 (IRS 1/2) [22], acting a negative feedback exerted by various kinases (ERK2, glycogensynthase kinase–3 (GSK-3), mammalian target of rapamycin/ S6K1 (mTOR/S6K1)and certain isoforms of Protein Kinase C (PKC)) and from feed-forward inhibition exerted by I?β kinase β (IKKβ) and JNK1/2 [20]; iii)reduced cytosolic and/or membranous levels of PI3K [23]; iv) reduced GLUT-1 expression at blood-brain barrier in AD patients [24].

In turn, early hyperinsulinemia is enough to exacerbate AD pathology observed in APP/PS1 mice [25]. Intranasal administration of insulin seems to improve cognition in both healthy and MCI patients [26].

Circulating leptin was associated with a reduced incidence of dementia and AD [27], but excess Aβ can potentially lead to a pathologically low leptin state, early in the disease process, that progressively worsens as the amyloid burden increases [28].

Anti-pathogen activity of Aβ: Possible roles played by pathogens in AD were repeatedly reported, from viral [29,30] to bacterial [31] and fungal [32] infections, as well as in other neurodegenerative diseases, such as Parkinson’s Disease and amyotrophic lateral sclerosis [33]. Most notably, recent evidence indicate the herpes simplex virus-1 to be a strong presence in the pathogenesis of AD, leading to generation of multiple APP fragments with neurotoxic potentials [34], induces accumulation of intracellular Ca(2+) and downstream signaling to Ca(2+)-dependent APP phosphorylation and intracellular accumulation of Aβ42 [35] and abnormally hyperphosphorylated tau [36].

In 2010, Soscia et al proposed Aβ as an antimicrobial peptide and proved it to have antimicrobial effect in vitro, comparable with LL-37, an archetypical human antimicrobial peptide. The authors also showed that brain homogenates of AD patients have higher antimicrobial activity than aged matched non-AD samples and that antimicrobial action correlates with tissue Abeta levels [37]. Further on, other peptides generated by enzymatic c leavage from the extracellular domain of APP exert antimicrobial effects in vitro against Gram-negative and Gram-positive bacteria, putatively via a membrane permeabilising activity [38]. Aβ42 was also shown to reduce uptake of IAV by epithelial cells and appeared to possess direct antiviral effect, possibly by promoting viral aggregation and further helping of phagocytic viral clearance [39].

Tau hyperphosphorylation: The second main pathogenic link involved in AD is hyperphosphorylation of tau – a microtubule associated protein, involved in cytoskeleton stability. Unlike APP, able to induce by itself a complete AD phenotype (both amyloid plaques and tau hyperphosphorylation) when overexpressed in animal models, abnormal tau phosphorylation leads to tautopathies. In AD tau pathology is most likely secondary to amyloid cascade, the main link between the two being glycogen synthase kinase 3, activated by the Aβ peptides via insulin and Wnt signaling pathways [40].

Intraneuronal interaction between Aβ peptides and tau has been proposed more than 5 years ago to occur on multiple tau peptides, especially those in exons 7 and 9, enhancing tau phosphorylation by GSK3beta [41] and reconfirmed lately in postmortem brains from AD patients at different stages of disease progression and animal models [42]. Furthermore, recent evidence suggests that tau protein may mediate amyloid-β peptide (Aβ) toxicity by modulating the tyrosine kinase Fyn [43]. Animal models with a heterozygous Fyn phenotype showed increased soluble Aβ accumulation and worsened spatial learning in the absence of changes in tau phosphorylation [44]. Tau-Fyn interaction has been recently investigated in order to map Fyn binding site on Tau and target interaction inhibitors by highthroughput screening [45]. Phosphorylation of tau is also influenced by Cdk5, a cyclin kinase which, unlike its other family members is not involved in cell cycle progression but in phosphorylation of cytoskeletal proteins and synatpic formation. In AD was reported as one of the major players causing aberrant hyperphosphorylation of tau through phosphorylation of specific repeats in neurofilaments of heavy and medium molecular weight [46]. Silencing of CDK5 reduces the phosphorylation of tau in primary neuronal cultures and in the brain of wild-type mice [47]. Recent data report, however, that there is a possibility that a therapeutic effect would be attainted only by dual kinase inhibition [48].

Another recent input on tau biology is the relationship between the Unfolded Protein Response (UPR) and early tau phosphorylation [49] as well as tau involvement in mitochondrial dysfunction [50]. Truncated tau induced significant mitochondrial fragmentation in neurons and when combined with Aβ at sublethal concentrations, increased the levels of oxidative stress. It also enhanced Aβ-induced mitochondrial potential loss in primary neurons [51].

Tau phosphorylation seems to be modulated also by neurotransmitters input, as specific serotonergic denervation increased tau phosphorylation in denervated cortex, without affecting amyloid-beta (Aβ) pathology [52].

Posttranslational control of main players in AD by microRNAs

MicroRNAs are small non-coding RNAs, than can pair, perfectly or imperfectly, with the 3’ untranslated region (UTR) of different mRNAs. A perfect complementarity between the mRNA and miRNA will lead to mRNA degradation, while an imperfect match will stop the protein translation. Either way, microRNAs act as downregulators of protein synthesis, for one, several or, in some cases, tens of mRNAs. It is also possible for one mRNA to be targeted by several different microRNAs that regulate its expression at different time points in the life of the cell, or under different environmental conditions.

As this posttranslational regulatory pathway has been identified no more than two decades ago, the bulk of data regarding the main actors in AD pathology (APP, beta and gamma- secretases) regulated by microRNA is no older than 5-10 years. The research in the field of microRNA involvement in AD pathology is expanding, along with discovery of new microRNA sequences. Up-to-date, in one microRNA data base ( are listed over 1800 sequences of identified microRNAs in humans, out of which many species or clusters are expressed in the central nervous system in a both timedependent manner [53] and cell-specific manner [54]. Many of micro RNAs expressed in the human brain are not conserved between different families of primates, suggesting that, phylogenetically, they are a recent acquisition [55].

From the full range of miAR Nuri identified in the brain, so far only a few have been associated with neuro degenerative processes: miR-133b [56], miR433 [57] in Parkinson’s disease, miR-9 in Huntington’s disease [58,59], miR-132, miR-124a, miR-125b, miR- 107, miR-219 andmiR-128 in Alzheimer’s disease [60-62].

One of the first reports of a miRNA profile modification in patients with AD belongs to Walter Lukiw, studying human hippocampus from fetal and adult patients with AD. The results indicate the abnormal expression ofmiR-9, miR-125b andmiR-128 in the hippocampus of AD brains [61]. Another study by Wang W X et al. On AD patients’ brains, compared with control subjects without cognitive impairment, showed a marked decrease of miARN-107, even in the early stages. Wang et al. demonstrated that the 3’-UTR sequence of BACE1 mRNA is a possible site of attachment of them iARN-107, regulating the expression of the enzyme. In addition to the decrease observed in the cortex of the temporal, the same trend was observed in the motor cortex. These changes indicate a global change miR-107 levels in the whole brain, even in area sun affected by AD pathology [63] . The 3 ‘UTR of mRNA of BACE1 is also targeted by miRNA-29a/b-1cluster, as decreased expression of these microRNAs was demonstrated in patients withsporadicAD. Consistent with the literature [60,64-68] an increase of BACE1 expression was demonstrated through quantitative RT-PCR. Decreased expression miR-29a/b-1 and increased BACE1 is not specific to a particular cortical area, as demonstrated by cerebellum assessment [69]. Cogswell metal, Quantified the expression of over 300 miRNA sites in the hippocampus, medial front algyrus and cerebellum taken from different stages of AD compared with agematched controls. These data show modifications of certain species of miRNA – out of which miR-9 and miR-132 were repressed in the hippocampus and frontal gyrus, correlating with the progress and location of pathological lesions. In addition they report detecting miRNAs in the CSF, with different levels from controls [70]. Over expression of miR-125b causes tau hyperphosphorylation and targets the phosphatases DUSP6 and PPP1CA and the anti-apoptotic factor Bcl-W [71], whereas miR-922 increased the levels of phosphorylated tau by regulating ubiquitin carboxy-terminal hydrolase L1 [72].

Recently, other species of miRNAs are found dysregulated in animal models of AD, related not to amyloid cascade but to other pathogenic links such as synaptic plasticity (upregulation of miR-181 [73]), or immune-related (miR-155, upregulated simultaneously with an increase of microglia and astrocyte activation [74]).

Mitochondrial dysfunction

Alteration of mitochondrial metabolism in AD patients has been well documented in the literature and confirmed in in vitro studies showing that Aβ affects mitochondrial DNA and proteins, leading to impairments of the Electronic Transport Chain (ETC) and ultimately mitochondrial dysfunction [75]. In a triple transgenic mouse AD model, mitochondrial dysfunction was detectable from embryonic stage, continues throughout the reproductive period and is exacerbated during reproductive senescence, unlike control wild type littermates, in which oxidative stress and a significant decline in mitochondrial function was demonstrated only with reproductive senescence [76]. A significant decrease in mitochondrial membrane potential was also noted in two cell models of AD, also accompanied by a decrease in ATP synthesis [77]. Interesting results have also been reported in selected nerve cell lines [78], along with already consecrated in vitro neuronal models PC-12 [79] and SHSY-5Y [80] cell lines

The relationship between mitochondrial dysfunctions and other pathogenic links in AD was under intense scrutiny, most efforts concentrated on relationship between this organelle and aberrant APP processing. Correlating with the newly demonstrated ability of Aβ to oligomerize at cell membrane site, increasing evidence suggested that both APP and Aβ peptides accumulate also in mitochondrial membranes, forming pores that increase permeability and further promote the excess accumulation of Ca(2+) [81]. Both full length APP and C99 were shown to target to the mitoplast (inner membrane and matrix compartments) in brains of an AD transgenic mice, which seemed to be almost completely dependent on BACE 1 activity [82]. As for Aβ peptides, they were found at mitoplast level in both monomeric and oligomeric forms, even as early as 2 months of age in transgenic AD mice, generating free radicals, ultimately leading to oxidative damage [75]. Mass spectrometry studies identified heat shock protein 60 (HSP60) to be responsible for Swedish mutated APP (KM670/671NL) translocation to mitochondrial matrix, along with critical components of gamma-secretase complex [83].

Studies on the integrity of the inner mitochondrial membranes and functional status of electron transfer complexes reported that APP treatment negatively influences the complex IV activity, while the activities of complexes I and II did not change. Furthermore, activity of complex III was significantly enhanced in APP expressing cells, as compensatory response, in order to balance the defect of complex IV, compensatory mechanisms that was, however, unable to prevent the strong impairment of total mitochondrial electron transport chain [84].

Another pathogenic interaction proposed recently to explain the mitochondrial dysfunction under AD burden was upregulation of voltage-dependent anion channel 1 protein (VDAC1) levels, found in the cortical tissues from the brains of patients with AD, relative to controls, as well as in transgenic laboratory mice [85].

The association between mutant APP and mitochondrial dysfunction in not cell-type specific, as it has been very recently reported to also occur in the striated muscle fibres of a transgenic animal used as an AD model [86].

Last, but not least, mitochondrial dynamics have also been reported to be altered in AD, with a shift of balance towards organelle fission and decreased mitochondrial anterograde movement, to accompany the defective functions [87]. Furthermore, downregulation of Akt signaling, also possibly related to insulin resistance in AD, was shown to diminish mitochondrial biogenesis, with subsequent memory impairment in laboratory animals [88]. Another pathologic link relating mitochondria and AD is Aβ-binding alcohol dehydrogenase (ABAD) - a direct molecular link from Aβ to mitochondrial toxicity. Aβ was shown to interact with ABAD in the mitochondria of AD patients and transgenic mice overexpressing ABAD, with the latter manifesting exaggerated neuronal oxidative stress and impaired memory in an Aβ-rich environment [89].

Endoplasmic reticulum stress in AD

Endoplasmic Reticulum (ER) stress is manifested by several functional dysregulations such as calcium release or accumulation of unfolded proteins in the lumen of the organelle. This loss of homeostasis is sensed by specific transmembrane proteins that trigger the Unfolded Protein Response (UPR) – three different signaling pathway that regulate gene transcription to increase chaperone levels into assisting ER to properly fold proteins, or even to downregulate production of mRNA of unfolded proteins [90] (Figure 1).

Citation: Galateanu AAG and Ana-Maria Enciu. An Integrative View on Intra- and Inter-Cellular Cooperation Mechanisms in Alzheimer’s Disease. Austin Alzheimers J Parkinsons Dis. 2014;1(1): 11. ISSN: 2377-357X