Potential Novel Approaches to Understand the Pathogenesis and Treat Alzheimer’s Disease

INTRODUCTION

Alzheimer’s disease (AD) is a complex, multi-factorial disease, which is unique to humans. AD is defined neuropathologically by the accumulation of amyloid-β (Aβ) into extracellular plaques in the brain parenchyma and in the vasculature (known as congophilic amyloid angiopathy [CAA]), and abnormally phosphorylated tau that accumulates intraneuronally forming neurofibrillary tangles (NFTs) [1–4]. Pathological aggregation of phosphorylated tau and Aβ occurs in a sequential process. Monomers first aggregate into oligomers intraneuronally, which then continue to aggregate into the fibrils observed in amyloid plaques and NFTs, with this pathology then spreading in a characteristic brain topography that is distinct for NFTs and plaques [1, 5–7]. Much evidence indicates that oligomers are the most neurotoxic species in AD as levels of these species correlate much better with cognitive decline compared to the burden of plaques or NFTs [5, 9]. Amyloid plaques primarily consist of aggregated Aβ, with the most abundant forms of Aβ being Aβ_1 - 40 and Aβ_1 - 42. However, amyloid deposits also contain Aβ species with heterogeneity at both the amino and carboxyl termini and with post-translation modifications such as pyroglutamate modifications at residues 3 or 11 (Aβ_N3pE or Aβ_N11pE), as well as phosphorylation at serine residues 8 and 26 (pSer8Aβ and pSer26Aβ) [5, 10]. The presence and amount of these different Aβ species is important since some species are particularly prone to aggregation and are more toxic than others, with the presence of species such as pSer8Aβ having been linked to a “biochemical staging” of amyloid plaques [10, 11]. Evidence indicates this process initially occurs predominately in synapses [8, 12]. All species of Aβ are derived from cleavage products of the amyloid-β protein precursor (AβPP), a type 1 transmembrane protein present in all cells including neurons. In the amyloidogenic pathway, AβPP is initially cleaved by BACE1 and then cleaved by γ-secretase (a protease composed of presenilin-1, presenilin enhancer 2, nicastrin and APH-1), to release monomeric soluble Aβ (sAβ) [13], which has normal physiological functions with neurotrophic properties [14–16]. In AD, either increased production of sAβ and/or production of more aggregation prone species of sAβ (in the case of familial AD) or impaired clearance of sAβ (in the case of sporadic AD [sAD]) results in Aβ accumulation in the brain [5]. There are many environmental and genetic factors that increase the risk for AD; however, understanding the interplay between these risk factors and their individual contribution to the pathogenesis of AD, as well as in different subtypes of AD is a process in evolution. AD is characterized as either familial early-onset (EOAD; <5% of all AD patients, with onset at <65 years) or sporadic late-onset (sAD; onset >65 years). Autosomal dominant mutations in presenilin 1 (PSEN1), presenilin 2 (PSEN2), or the amyloid precursor protein (APP) gene account for ∼10% of all EOAD cases (∼1% of all AD cases), leaving the cause of the majority of EOAD unexplained [17–20]. sAD afflicts >95% of patients with AD and is related to both genetic and environmental factors [18, 21–23]. A combination of genome-wide association studies, linkage, and whole genome/exome sequencing have identified over 30 loci that confer increased risk for sAD, including genes involved in innate immunity, cholesterol metabolism, and synaptic/neuronal membrane function, suggesting that the pathogenesis of sAD has considerable heterogeneity [18, 25]. The strongest identified genetic risk factor for sAD is the inheritance of the apolipoprotein (apo) E4 allele, the protein product of which influences both the aggregation and clearance of brain Aβ [26–28]. Much more rare variants of another gene that encodes the triggering receptor expressed on myeloid cells 2 (TREM2) have been reported as a significant risk factor for sAD, with an odds ratio similar to apoE4 [29, 30]. This genetic diversity that drives AD pathogenesis suggests that AD is a syndrome with a final common pathway that involves the accumulation of Aβ and tau oligomers. Our understanding of these complex pathways has greatly increased in recent years; however, despite this expanding knowledge base there has been a very high failure rate of ∼99.6% with AD targeting clinical trials. There are many reasons for this high failure rate; however, an important factor has been the frequent premature translation of successful pathology reduction in transgenic (Tg) mouse models, which have pathology driven by overexpression of very rare EOAD mutations, to humans with sAD in whom the pathology is driven by substantially different pathways, which may vary in importance from patient to patient [3, 31–33]. In addition, studies in these animal models of AD ignore the very significant age associated neuronal loss that occurs in many brain regions, without correlation to NFT or Aβ pathology, that underlies an individual’s “neuronal reserve” [34]. To overcome these limitations, one possible direction is greater research use of human tissue. AD pathogenesis heterogeneity could be better examined using omics approaches that allow genome- or proteome-wide screening for altered networks during disease, focusing on particular subset samples of AD [3, 35]. The use of various unbiased omics approaches is being increasingly recognized as essential for the future of effective AD research [36]. AD therapeutic approaches need to better reflect the diversity of disordered pathways that can drive AD pathology and be less amyloidocentric. In this review, we outline our recent attempts to use proteomic approaches to better understanding the heterogeneity of AD pathogenesis and our preclinical studies using a number of different, potentially synergistic, therapeutic approaches that we hope will have relevance for sAD.

PROTEOMIC STUDIES USING HUMAN POSTMORTEM TISSUE

There is a plethora of molecular alterations in the AD brain that occur in addition to the accumulation of pathologic Aβ and tau species. Proteomics offers an unbiased comprehensive way to explore how these molecular changes are interrelated and how they contribute to AD pathophysiology. The hypothesis-free, exploratory nature of unbiased proteomics enables the simultaneous examination of thousands of proteins, which will ultimately provide a much broader overview of AD pathophysiology [37, 38]. The majority of proteomics studies using AD tissue have analyzed homogenates of large regions of tissue [39–50]. This approach has allowed the identification of proteins with large, region-wide differences between AD and controls. Many of these studies are limited by small numbers of subjects included and by the lack of sub-fractionation of tissue homogenates, meaning that detected protein changes are limited to abundant proteins only. However, these limitations are being addressed in more recent studies which include larger numbers of subjects across a range of disease states (ranging from preclinical to advanced AD), therefore providing an invaluable resource comprehensively detailing the proteome of the frontal cortex in these subjects [47, 51]. Other studies have used subcellular fractionation to examine protein alterations in AD in more targeted biochemically extracted fractions such as insoluble proteins [51–53], synaptic fractions [54, 55], phosphopeptides [56], and membrane associated proteins [57]. This type of approach allows more specific examination of proteomic differences in AD tissue in each of these fractions, but it still does not allow for direct analysis of proteomic differences in specific cell types or neuropathological features. Therefore, we developed our localized proteomics technique, which combines laser capture microdissection and label-free quantitative LC-MS to allow proteomics of specific regions, neuropathological features, or cells of interest [58–60]. Other groups have also used localized proteomics on frozen postmortem AD tissue specimens, analyzing the proteome of microdissected neurons [61], NFTs [62–64], plaques [65], CAA [66, 67], and specific hippocampal subregions [68]. However, a particular advantage of our localized proteomics technique is that it was deliberately optimized to allow the use of formalin-fixed paraffin embedded (FFPE) tissue. This is because the vast majority of human tissue specimens are FFPE blocks collected at autopsy, which are an underutilized, but exceptionally valuable resource for medical research.

We recently used localized proteomics to show that the protein composition of amyloid plaques was significantly different in patients separated into two subtypes of AD based on the rate of disease progression: those with rapidly progressive AD (rpAD) and those with typical sporadic AD (sAD) [35, 69]. Patients with rpAD have a particularly aggressive form of AD where median survival time is limited to 7–10 months after diagnosis in comparison to a survival time of ≥10 years in sAD [70, 71]. Little is known about the pathological changes that underlie the rapid disease progression, and there are currently no gross neuropathological differences or differences in AD CSF biomarkers that can be used for either diagnosis or to explain the rapid progression of AD in these patients in comparison to sAD [70, 71]. However, it is important to note that recent studies have shown that Aβ₄₂ oligomers in rpAD have distinct properties from those in sAD, which may promote the faster spread of Aβ pathology [70, 73]. The aim of our study was to compare the plaque proteome in rpAD and sAD patients (n = 22/group). We found that rpAD plaques had a significantly different protein composition in comparison to sAD; 141 proteins had significantly different levels in rpAD plaques [35, 69]. Many of the proteins with altered expression are known to have a role in the development and maintenance of amyloid plaques (e.g., Aβ, gelsolin, GFAP, and α-synuclein), suggesting that these proteins may have a particularly important role in rpAD pathogenesis. Interestingly, rpAD plaques were found to contain significantly higher levels of neuronal proteins and significantly lower levels of astrocyte proteins. Immunohistochemistry validated and extended the proteomic data to show that the decreased levels of astrocyte proteins in rpAD plaques was due to fewer plaque-associated astrocytes in rpAD in comparison to sAD. Comparison of our plaque proteomic data with previous proteomic datasets generated using AD tissue showed that proteins with higher expression in rpAD plaques typically have either lower expression in sAD (39% of proteins upregulated in rpAD plaques are typically downregulated in sAD) or have no known involvement in sAD (46% upregulated proteins). In sum, this suggests that rpAD is unlikely to be simply a more extreme version of sAD, but instead a separate subtype of AD that is mediated by different pathological mechanisms.

One of the advantages of using unbiased proteomics to characterize protein differences is that it allows the detection and quantification of novel proteins linked to AD pathogenesis. One example of such a novel protein that we characterized in further studies is secernin-1. Very little is known about the general function of secernin-1, and its role in AD has never been examined. Our proteomics data showed that secernin-1 had a consistently high expression in plaques in both rpAD and sAD [35]. Consequent immunohistochemistry showed that particularly high levels of secernin-1 were observed in plaque-associated dystrophic neurites and in NFTs. The consistently high degree of colocalization with phosphorylated tau could imply an important relationship between secernin-1 and the generation of NFTs. Further studies characterizing the distribution and function of secernin-1 in AD are currently underway. The detection of novel proteins involved in AD pathogenesis (such as secernin-1) highlights the powerful nature of unbiased ‘omics studies. Studies such as these have great potential to increase our understanding of the broad molecular mechanisms that underlie AD and the large amount of data generated in these studies can be used as the basis for future targeted studies to specifically examine the role of each of these proteins in the development of AD [36, 69]. The simultaneous analysis of thousands of proteins at once provides a much more complete overview of the molecular changes that occur in the AD brain and will ultimately help with the identification of the most promising drug targets beyond Aβ and tau.

APOLIPOPROTEIN E TARGETING THERAPEUTIC APPROACHES FOR AD

The apolipoprotein E (apoE)/Aβ interaction plays a major part in the conformational transformation of soluble Aβ and Aβ deposits in typical sAD (an exception is the subtype of rpAD, as discussed above) [26, 75]. ApoE has a number of important functions in the brain, including being the major CNS cholesterol and other lipid carrier. It is also involved in synaptic plasticity, glucose metabolism, mitochondrial function, and vascular integrity [27, 28]. ApoE affects both the clearance and aggregation state of Aβ in an isotype specific manner in AD [26, 75–77]. For example, apoE has been shown to enhance aggregation of Aβ with the order of apoE4 >apoE3 >apoE2 [78–81]; also, effects have been shown on the stabilization of Aβ oligomers, where apoE4 is found to have the greatest impact [82, 83]. Under physiological conditions, it has been determined that relatively little normal, sAβ binds to apoE [84] and apoJ is the major CNS Aβ binding protein [85, 86]. In AD, however, as the aggregate state of Aβ shifts, there is a greater interaction with apoE [77, 88]. Research has also established that apoE4 is less effective at clearance of Aβ than apoE3 [89]. It is, therefore, possible to suggest that blocking the binding between apoE and Aβ could promote Aβ deposition, as it would inhibit clearance. However, pivotal in vivo studies show that this does not occur. Eliminating apoE reduces fibrillar amyloid deposition significantly [90], with apoE4 expressing AD Tg mice having greater amyloid deposition compared to apoE3 or E2 expressing mice [91, 92]. Further, other Aβ binding proteins, including apoJ or α2-macroglobulin, are associated with pathways that have greater effectiveness at Aβ clearance in contrast to apoE mediated Aβ clearance [26, 93]. It can be concluded, therefore, that the net effect of blocking the Aβ/apoE interaction is to inhibit deposition and enhance clearance. This strategy also has the advantage of not interfering with the many normal and beneficial functions of apoE. We have shown in several past studies, that treatment with Aβ 12-28P—a peptide homologous to the specific apoE binding domain of Aβ—in two AD Tg mouse models with primarily amyloid plaque deposition and in one AD model with primarily CAA, all produced a major reduction of Aβ burden, both in brain parenchyma and in brain vasculature when compared to age-matched vehicle-treated Tg mice [94–96]. Our studies additionally showed that blocking the apoE/Aβ interaction with Aβ12-28P in triple transgenic mice reduces AD-related Aβ and tau pathology [97]. In other results, Aβ12-28P treatment in an amyloid mouse model with apoE2-targeted replacement (TR) or apoE4-TR mouse backgrounds produced a reduction in Aβ oligomer and plaque load, also alleviating neuritic degeneration, which indicates that inhibition of Aβ/apoE interactions appears to materially block aggregation and deposition of Aβ, irrespective of apoE isoform [98]. In recent work, we sought to sharpen our approach toward possible clinical application. For this purpose, we undertook to design 9 pairs of related linear and cyclic peptoid compounds derived from the Aβ12-28P sequence to screen for new apoE/Aβ binding inhibitors, looking to demonstrate higher efficacy and safety [99]. The lead peptoid screened by surface plasmon resonance (SPR), CPO_Aβ17-21P decreased the apoE4/Aβ₄₂ binding at a 2:1 molar ratio (peptoid:apoE4) and virtually blocked all binding at a 8:1 molar ratio (peptoid:apoE4). The half-maximal inhibition (IC₅₀) derived from a one-site competition, nonlinear, regression equation of CPO_Aβ17-21P was 1.02 nM, which is much improved compared to 36.7 nM for the parent peptide Aβ12-28P [95]. Other earlier studies showed that there is a critical region for Aβ binding to apoE in the residue range 17–21, with the lysine at residue 16 being special of importance [100, 101]. It can be expected that a peptoid conforming to this sequence would be a most effective inhibitor of the Aβ/apoE interaction. APP/PS1 AD mice treated with CPO_ Aβ17-21P had a major cognitive improvement, including reduction of soluble and insoluble Aβ peptide/oligomer levels in brain and lower total amyloid burden in cortex and hippocampus [99]. It is important to note that CPO_ Aβ17–21P treatment reduces Aβ related pathology and cognitive deficit using a 7.5 fold reduced dose (0.2 mg per mouse, twice per week) in contrast to the Aβ12-28P treatment dose used previously on 3xTg-mice (1 mg per mice, three times per week) [97, 99]. It suggested that the new peptoid inhibitor CPO_Aβ17–21P, with a very low molecular weight (<1 kDa) and inherent protease resistance, has improved bioavailability/biostability over Aβ12-28P.

There is a potential risk in targeting Aβ deposition in that increasing the pool of soluble Aβ may facilitate formation of the toxic oligomer species. That has been demonstrated by some other immunotherapeutic approaches [102, 103]. Although apoE has a dual role in Aβ deposition and clearance, CPO_Aβ17-21P inhibition of apoE4/Aβ₄₂ interaction in APP/PS1 AD mice did not affect the soluble Aβ pool. Another potential risk is brain inflammation when targeting Aβ deposition. Our work has shown, that Iba1 and CD11b (both markers for microgliosis), and GFAP (a biomarker for astrogliosis) immunoreactivity is reduced or unchanged in the CPO_Aβ17-21P treated Tg mice [99]. Our novel therapy of blocking apoE/Aβ interaction has ameliorated all AD pathological features tested, including: improved memory deficits, reduction of amyloid burden and tau pathology and reduction of vascular amyloid deposition [94–96, 99]. A research project utilizing Aβ12-28P to block apoE/Aβ interaction in an amyloid mouse model with apoE2-TR or apoE4-TR mouse background produced a reduction in Aβ plaque load and oligomer and ameliorated neuritic degeneration [98]. Therefore, it can be stated that this therapy is not apoE isoform restrictive. This approach does not preclude the simultaneous therapies discussed below, as they may have a synergistic effect that procures a more effective treatment.

STIMULATION OF INNATE IMMUNITY AS A THERAPEUTIC APPROACH FOR AD

Genome-wide association and other genetic studies have shown the linkage of a number of innate immunity related genes in late-onset AD, in particular TREM2 [29, 104]. These studies are suggestive of the importance of microglia in AD pathogenesis, by identifying several AD associated genes that are expressed primarily in microglial cells. Microglia are critical regulators of innate immune responses in the brain. However, depending on the circumstances, their activation can have opposing effects [30, 106]. Stimulation of innate immunity via Toll-like receptor (TLR) signaling pathways has been shown to be beneficial in modulating AD pathology in a number of studies [107–110]. On the other hand, manipulation of TLRs can also produce adverse effects in AD models [110–113]. Discrepancies between studies may be the consequence of variations in the types and doses of TLR ligands used, as well as administration frequencies. It appears that therapeutic immune activation should follow the “Goldilocks Principle”: it needs to be just right. In addition, disease stage and underlying brain’s immune status should be considered in designing future applications. We have focused on ameliorating immunosenescence and its associated AD pathology via TLR9 stimulation. TLR9 recognizes the unmethylated CpG sequences present at high frequency in bacterial and viral DNA and at low frequency in human DNA. Oligodeoxynucleotides (ODNs) containing these unmethylated CpG sequences trigger cells that express TLR9 (including cells of the monocytes/macrophage lineage, plasmacytoid dendritic cells and B cells) to mount an innate immune response. Several CpG ODNs have shown excellent safety profiles with >600 preclinical studies investigating the treatment or prevention of cancers, infections, and allergies, and >100 human clinical trials having been completed or are ongoing using CpG ODNs [114–117]. Immunotherapy has emerged as an attractive approach for disease intervention in AD; yet significant associated adverse events are the occurrence of amyloid related imaging abnormalities (ARIA) and cerebral hemorrhages, which are linked with the rapid clearance of CAA, with resulted blood-brain barrier break down, and excessive neuroinflammation [103, 118]. Our earlier studies using the Tg2576 and 3xTg-AD mouse models document that stimulation of innate immunity via TLR9 ligand class B CpG ODN has the advantage of concurrently ameliorating Aβ and tau pathologies, in association with behavioral improvements [119, 120]. However, several studies suggest that inflammation and altered microglial activation may exacerbate tau deposition [121–123]. Our research findings clearly demonstrate CpG ODN reduces both tau and plaque pathology in 3xTg-mice [120]; however, in this study we could not exclude the possibility that the tau pathology reduction was secondary to the decreased amyloid burden. To resolve whether CpG ODN directly reduces pathological tau, we are currently conducting studies in Tg4510 AD model mice which have only tau related pathology.

The experimental mouse models utilized in our initial studies have minimal vascular amyloid. Hence, more recently we evaluated the therapeutic profile of CpG ODN in TgSwDI mice, which are an AD model with very extensive vascular amyloid [124, 125], testing the hypothesis that CpG ODN can harness innate immunity to reduce the age-dependent accumulation of CAA pathology in both young mice (prior to the onset of pathology) and in aged mice (with established pathology) [126, 127]. Our data documents that peripheral administration of CpG ODN negated short term memory deficits assessed by novel object recognition test as well as, being effective at improving spatial and working memory evaluated using a radial arm maze in both young and old age cohorts of TgSwDI mice [126, 127]. Detailed neuropathological evaluation accompanied by quantitative image analyses demonstrated significant reductions in total amyloid burden in CpG ODN-treated Tg animals compared to Tg controls. Even though fibrillar deposits are less amenable to clearance, quantification of Thioflavine-S stained sections confirmed a significant reduction in fibrillar vascular amyloid burden without associated microhemorrhages in CpG ODN groups. Importantly, we did not detect any microhemorrhages in wild-type animals after CpG ODN administration thus providing additional evidence of the safety of our approach. These favorable histological findings were corroborated by measurements of Aβ levels in the brain homogenates, which revealed a significant decrease in the levels of total and soluble Aβ_40/42 fractions and Aβ oligomer levels in CpG ODN-treated TgSwDI mice. Peripheral administration of TLR9 agonist, class B CpG ODN, successfully triggered a targeted immune response polarizing macrophages/microglia toward beneficial states of activation with improved phagocytic function, resulting in restriction of AD pathology in the absence of apparent toxicity. Therefore, our recent findings together with prior studies, validate this novel concept of immunomodulation as a safe method to successfully prevent and ameliorate AD related pathologies, supporting the potential clinical applicability of CpG ODN [119, 127]. Studies are currently ongoing using a non-human primate model of AD, squirrel monkeys, which naturally develop extensive CAA as well as ARIA, making them a particularly appropriate AD model to test an immunomodulatory therapeutic approach [128–130].

THERAPEUTIC IMMUNOMODULATION TARGETING Aβ AND TAU OLIGOMER TOXICITY CONCURRENTLY

Soluble oligomeric forms of Aβ and tau, which could spread via a “prion-like” mechanism, are thought to be the key mediators of neuronal toxicity in AD [5, 131–136]. The change in conformation to oligomeric misfolded conformers presents the possibility of specific immunological recognition using either active or passive approaches [103, 138]. Initial trials of active vaccination in AD failed as a result of autoimmune toxicity from the use of self-immunogens, such as aggregated Aβ [103, 139]. Clinical trials of passive immunization have also produced disappointing results related to the targeting of both physiological and pathological forms of Aβ, without specific targeting of the most toxic oligomeric species [103, 141]. It is now recognized that the soluble toxic oligomeric forms of pathologic proteins or peptides might be more efficient immunologic targets for both active and passive immunization approaches. This realization has led to the production of a limited number of anti-conformation monoclonal antibodies and new formulation vaccines, as was previously reviewed [103, 142]. Two significant problems need to be addressed for therapeutic success in targeting toxic oligomeric structures. The first is the widespread use of primary structure self-antigens to determine the tertiary structure of the oligomeric immunogens used for active immunization and the production/selection of possible anti-conformation monoclonal antibodies, with the remaining possibility of cross-reactive autoimmune toxicity due to incomplete selectivity for the pathological conformation. The second is the restrictive specificity of the immunogen to a single or limited number of pathological conformers [141]. To overcome these problems, we recently developed a methodology to produce anti-β-sheet secondary structure conformational monoclonal antibodies [137]. Our prior work using three different AD Tg mouse models, has shown that active immunization based on this approach produces a therapeutic polyclonal response which reduces all key neuropathological features of AD, including amyloid plaques, CAA, and tau-related pathology, in association with significant cognitive improvements [103, 143–145]. Amyloid plaques and CAA were shown to be reduced in APP/PS1 (amyloid plaque model) and TgSwDI (CAA Tg model) model mice, respectively, while in 3xTg-mice (amyloid plaque and tau pathology model) p13Bri immunization led to reductions of both tau and Aβ fibrillar and oligomeric pathology [103, 143–145]. Inoculation of p13Bri with Alum as an adjuvant in these three AD Tg models produced a systemic polyclonal response to pathologic/oligomeric forms of both Aβ and tau, as well as demonstrating cross-specificity to AD, prion disease, and Lewy body disease human brain tissue (and not control human tissue). These promising results led us to the production of hybridomas from which we could select monoclonal antibodies (mAbs) with potential diagnostic or therapeutic value, by their specific reactivity to β-sheet secondary structures found in unrelated primary sequences of pathologic conformers of diverse neurodegenerative disorders [137]. The β-sheet secondary structure of proteins can be derived from very diverse and unrelated primary sequences, but generally is dominant in the production of any pathologic misfolded proteins or peptides. For an immunogen we used a small 13 amino acids peptide of the carboxyl terminus of the very rare British amyloidosis (ABri), which is derived from an intronic DNA sequence expressed by a missense mutation and has no sequence homology to any other mammalian protein (including all other known amyloid proteins) [118, 147]. The peptide was subject to controlled polymerization by an extensive glutaraldehyde reaction to form immunogenic, covalently bound 10–100 kDa soluble and stable oligomers with high β-sheet secondary structure content (p13Bri) [143, 144]. p13Bri inoculation, with a suitable adjuvant, produced an array of antibodies to the non-self motif and the β-sheet secondary structure. Stable hybridomas were obtained, with cloned mAbs selected by the novel approach of specifically using as selector compounds, oligomeric conformers from different neurodegenerative disorders with the only commonality being the shared β-sheet secondary structure. Due to the novel method by which we generated our anti-β-sheet conformational mAbs and their poly-reactivity to toxic conformers found in most common neurodegenerative disorders, we believe our approach to be innovative and more likely to have therapeutic success in humans, compared to other existing oligomer targeting mAbs [138]. The potential advantages include: 1) a reduced risk of inducing auto-immune complications since the immunogen used has no primary structure homology to any human peptide/protein (with the exception of ABri found in the very rare patients with British amyloidosis); 2) selective targeting of the β-sheet secondary structure found in toxic oligomers; hence, avoiding interference with the multiple beneficial and physiological functions of soluble Aβ, tau, α-synuclein, and PrP; 3) diminished risk of producing ARIA like complications, related to the direct clearance of fibrillar Aβ vascular deposits, since mainly oligomeric forms of Aβ and tau are being targeted; 4) simultaneous targeting of Aβ, tau, and α-syn related pathologic conformers (that have the potential to cross seed each other), addressing the mixed pathologies found in the majority of neurodegenerative disease patients [148–152]; 5) negligible risk of increasing toxic oligomer species by the mobilization of fibrillary Aβ and tau species as has been shown to occur with some vaccination methods [102]; 6) potential therapeutic activity for prion diseases by interfering with the spread of PrP^Res. No other reported methodology for producing mAbs to oligomeric conformations, published so far, has this unique combination of properties. Therefore, we believe that our technological approach has the potential to develop tools for the detection, monitoring and treatment of multiple neurodegenerative disorders [137].

Another somewhat related therapeutic approach is the potential blocking of both Aβ and tau oligomer mediated toxicity by inhibiting/competing with their binding to the normal PrP^C, which acts as an oligomer receptor on the surface of neurons. The Strittmatter group has demonstrated that extracellular oligomeric Aβ binds PrP^C with high affinity, activating an intracellular signaling cascade coupled to the protein tyrosine kinase Fyn [153]. The ability of oligomeric Aβ to activate Fyn is dependent on the presence of PrP^C and requires mGluR5, suggesting that in AD, oligomeric Aβ triggers neuronal signal transduction from PrP^C to mGluR5 to Fyn kinase [154–156]. Fyn activation, in turn, hyperphosphorylates and mislocalizes tau in the dendritic spines, leading to destabilized microtubules, and the production of NFTs which results in the cognitive impairment characteristic of AD patients [156, 157]. We have previously shown that anti-PrP mAbs such as 6D11 can ameliorate cognitive deficits in an AD mouse model with advanced disease by blocking oligomer mediated synaptic toxicity via inhibiting binding to PrP^C, without affecting the amyloid burden or altering Aβ oligomer levels [158]. This same anti-PrP mAb is an effective therapeutic agent in prion disease models, preventing PrP^Sc replication [142, 160]. More recently, we have also shown that PrP^C expression is critical in mediating tau-related pathology and neuronal toxicity in the setting of traumatic brain injury (TBI) [161]. TBI and its associated chronic traumatic encephalopathy (CTE) is now recognized to be a tauopathy related to tau oligomer mediated toxicity [161–163]. Hence, we hypothesize that PrP^C is a receptor for Aβ and tau oligomers, as well as, PrP^Sc. Therefore, blocking the Aβ, PrP^Sc, and tau oligomer interactions with PrP^C may reduce the pathology and cognitive deficits associated with AD, prion disease, frontotemporal dementia, and CTE.

CONCLUSIONS

Our understanding of the pathogenesis of AD has increased exponentially in recent years. Despite this growing knowledge base, translating preclinical therapeutic successes from animal models to the clinic remains an elusive goal. We believe that future therapeutic approaches need to better reflect the diversity of disordered pathways that can drive AD pathology. These approaches need to be tested using human tissue and a diversity of animal models that better reflect the wide spectrum of AD pathogenesis. Multiple different therapeutic approaches must be developed targeting the numerous distinct pathways that can ultimately lead to Aβ oligomer accumulation and the triggering of tau pathology. These therapeutic approaches such as the blocking of Aβ/apoE interaction, stimulation of innate immunity, and the simultaneous immune interference of Aβ/tau oligomer toxicity can be individually tailored to each patient depending on what is the primary driver of their AD pathology and/or their stage in the disease. In addition, it may be necessary to combine therapeutic approaches and/or to develop multi-target-directed ligands [164], to better reflect the complexity of the abnormal pathways triggered in AD pathogenesis.