NEURAL REGENERATION RESEARCH www.nrronline.org Review Amyloid-beta peptide and tau protein crosstalk in Alzheimer’s disease https://doi.org/10.4103/1673-5374.332127 Alejandro R. Roda1, Gabriel Serra-Mir1, Laia Montoliu-Gaya2, Lidia Tiessler1, Date of submission: December 22, 2020 Sandra Villegas1, * Date of decision: February 19, 2021 Abstract Date of acceptance: June 3, 2021 Alzheimer’s disease is a neurodegenerative disease that accounts for most of the 50-million dementia cases worldwide in 2018. A large amount of evidence supports Date of web publication: January 7, 2022 the amyloid cascade hypothesis, which states that amyloid-beta accumulation triggers tau hyperphosphorylation and aggregation in form of neurofibrillary tangles, and these aggregates lead to inflammation, synaptic impairment, neuronal loss, and thus to cognitive From the Contents decline and behavioral abnormalities. The poor correlation found between cognitive Introduction 1666 decline and amyloid plaques, have led the scientific community to question whether amyloid-beta accumulation is actually triggering neurodegeneration in Alzheimer’s disease. Search Strategy and Selection Criteria 1666 The occurrence of tau neurofibrillary tangles better correlates to neuronal loss and clinical Amyloid-beta Peptide as the Alzheimer’s Disease symptoms and, although amyloid-beta may initiate the cascade of events, tau impairment Trigger: the Amyloid Cascade Hypothesis 1666 is likely the effector molecule of neurodegeneration. Recently, it has been shown that amyloid-beta and tau cooperatively work to impair transcription of genes involved in Tau and Its Role in Neurodegeneration 1667 synaptic function and, more importantly, that downregulation of tau partially reverses Amyloid-beta Peptide and Tau Protein Interplay: transcriptional perturbations. Despite mounting evidence points to an interplay between More Than Casual Partners 1669 amyloid-beta and tau, some factors could independently affect both pathologies. Thus, the dual pathway hypothesis, which states that there are common upstream triggers causing Combined Therapies as a New Hope: Targeting both amyloid-beta and tau abnormalities has been proposed. Among others, the immune Amyloid-beta and Tau 1670 system seems to be strongly involved in amyloid-beta and tau pathologies. Other factors, as the apolipoprotein E ε4 isoform has been suggested to act as a link between amyloid-beta and tau hyperphosphorylation. Interestingly, amyloid-beta-immunotherapy reduces not only amyloid-beta but also tau levels in animal models and in clinical trials. Likewise, it has been shown that tau-immunotherapy also reduces amyloid-beta levels. Thus, even though amyloid-beta immunotherapy is more advanced than tau-immunotherapy, combined amyloid-beta and tau-directed therapies at early stages of the disease have recently been proposed as a strategy to stop the progression of Alzheimer’s disease. Key Words: aggregation; Alzheimer; amyloid-beta; dementia; immunotherapy; inflammation; neurodegeneration; tau Introduction nature of AD (Karran and De Strooper, 2016; Selkoe and Hardy, 2016). The aim of this review is to discuss new findings on the interplay Most of the 50 million people living with dementia worldwide in between Aβ and tau in the context of an updated view of the ACH 2018 suffered from Alzheimer’s disease (AD). AD is a major cause and to summarize the state of the art in AD clinical trials that are of disability and dependency among elderly people, so economic the basis for finding subsequent anti-Aβ and anti-tau combined impact is huge, with an estimate of US$ 1,000,000 million just therapies. for the year 2018 (Alzheimer’s Disease International, 2018). AD is a neurodegenerative disease characterized by memory loss, cognitive impairment, and behavioral and psychological symptoms Search Strategy and Selection Criteria of dementia. The main histopathological hallmarks of AD include Literature search was performed on PubMed until November the extracellular accumulation of the amyloid-beta (Aβ) peptide 2020. Only papers published in English were considered. The key in senile plaques and formation of neurofibrillary tangles (NFTs) words/terms were aggregation, Alzheimer, amyloid-beta, cascade by hyperphosphorylated tau protein. Mounting evidence supports hypothesis, dementia, crosstalk, interaction, immunotherapy, the assumption that cerebral Aβ deposition begins decades before inflammation, neurodegeneration, and tau. AD clinical onset and prior to cortical spreading of tau pathology. However, a complete understanding of the role of Aβ, from loss of Amyloid-beta Peptide as the Alzheimer’s Disease proteostasis to synaptic toxicity is still lacking. Similarly, it is accepted that tau hyperphosphorylation plays a critical role in neuron death; Trigger: the Amyloid Cascade Hypothesis however, whether it is cause or consequence of disease development Amyloid precursor protein (APP) is an ubiquitous single-pass remains unknown. Although controversial, the amyloid cascade transmembrane protein that contains an extracellular domain, a hypothesis (ACH) is the only hypothesis covering the multifactorial hydrophobic transmembrane domain, and an intracellular domain 1 Protein Design and Immunotherapy Group, Departament de Bioquímica i Biologia Molecular, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain; 2Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy, University of Gothenburg, Sweden *Correspondence to: Sandra Villegas, PhD, Sandra.villegas@uab.cat. https://orcid.org/0000-0001-8644-4304 (Sandra Villegas) Funding: The work was supported by grant SAF2017-89613-R from the Ministerio de Economía y Competitividad (MINECO) / FEDER (Madrid, Spain) (to SV). How to cite this article: Roda AR, Serra-Mir G, Montoliu-Gaya L, Tiessler L, Villegas S (2022) Amyloid-beta peptide and tau protein crosstalk in Alzheimer’s disease. Neural Regen Res 17(8):1666-1674. 1666 ｜NEURAL REGENERATION RESEARCH｜Vol 17｜No. 8｜August 2022 Review NEURAL REGENERATION RESEARCH www.nrronline.org (Kang et al., 1987). Under physiological conditions, soluble APP (sAPP) apolipoprotein E, and the risk specifically occurs when the APOEε4 is involved in neurite outgrowth, synaptogenesis, synaptic plasticity, allele is present (Roda et al., 2019; Andrews et al., 2020). and cell survival through N-methyl-D-aspartate and gamma- aminobutyric acid receptors modulation, which maintain intracellular The fact that several clinical trials for Aβ-immunotherapy-based calcium homeostasis (Müller et al., 2017; Rice et al., 2019). drugs have failed make made some researchers to question whether the ACH explains the physiopathology of AD by itself (Herrup, 2015). APP can be cleaved by a combination of different secretases However, the decision on the accelerated approval of aducanumab complexes, following two pathways (Figure 1A). In the non- (Aduhelm, Biogen) was recently make (https://www.fda.gov/drugs/ amyloidogenic APP processing, the combined action of α- and news-events-human-drugs/fdas-decision-approve-new-treatment- γ-secretases releases sAPPα, P3 peptide (which corresponds to the alzheimers-disease). An accelerated decision means that the drug is Aβ17–40/42 sequence) and the APP intracellular domain (De Strooper shown to have an effect on a surrogate endpoint that is reasonably et al., 1998). The amyloidogenic processing of APP is initiated by likely to predict a clinical benefit to patients and there remains some β-secretase (or β-site APP-cleaving enzyme 1 (BACE1)), instead of uncertainty about the drug’s clinical benefit. α-secretase so sAPPα is released; next, γ-secretase generates Aβ peptides with different C-terminal residues and APP intracellular Different aggregation states of Aβ coexist in AD brains, monomers, domain. This cleavage, at what is known as the β-site, can be also oligomers, fibrils and amyloid plaques (Figure 3A). Experimental performed by BACE2 (59% identity to BACE1 and similar structural evidence suggests that plaques maintain a dynamic equilibrium with organization), although BACE2 tends to cleavage at the β’-site, toxic oligomer species (Benilova et al., 2012). Monomeric Aβ is an rendering an N-truncated, non-amyloidogenic β-peptide (Kimura intrinsically disordered protein; hence,, Aβ does not adopt a single et al., 2016; Wang et al., 2019). Therefore, cleavage by γ-secretase folded shape in aqueous environment but acquires an ensemble is the one generating heterogeneity at the C-terminus end of Aβ of conformations which are prone to aggregation in the form of peptide, producing mainly Aβ1–40 and, to a lesser extent, the most Aβ oligomers (Ball et al., 2011). Aβ oligomers are the transient amyloidogenic Aβ1–42 form (Figure 1B). Aβ1–40 tends to accumulate in intermediate states between monomer and fibril formation. Both the vasculature whereas Aβ1–42 constitutes the predominant form in Aβ oligomers and fibrils are heterogeneous in structure and size amyloid plaques. and they are known to be the most toxic Aβ species (Benilova et al., Three decades ago, mutations in APP (Citron et al., 1992) (21q11.2– 2012), particularly oligomers. Although solving the structure of the q21) (Figure 1C), as well as in the catalytic units of γ-secretase Aβ peptide from human samples has been quite challenging, the genes, presenilin 1 (Sherrington et al., 1995) (PSEN1, 14q24.3) and 3D-structure of both, Aβ1–40 and Aβ1–42 fibrils has been solved in the presenilin 2 (Levy-Lahad et al., 1995) (PSEN2, 1q42.2), were reported last decade (Lu et al., 2013; Qiang et al., 2017). Aβ1–40 fibrils from to cause familial AD (FAD) (Esquerda-Canals et al., 2017b). These one single patient shows that Aβ1–40 peptide monomers aggregate mutations result in Aβ accumulation because of amyloidogenic APP into multiple of three oligomers (trimers, hexamers, nonamers, and processing enhancing. One of the most remarkable FAD mutations dodecamers), where N-termini (DAEFR, residues 1–5) are exposed to is the Swedish mutation, a double mutation at the β-site (K670/ the solvent and hydrophobic C-termini are buried in the trimer core N, M671/L) that enhances APP processing by β-secretase and so (Figure 3B; pdb 2M4J) (Lu et al., 2013). Because the concentration generates increased levels of Aβ compared with control individuals. of Aβ1–40 is much higher than Aβ1–42, such multiple of three oligomers In contrast, Icelandic mutation (A673T), located at the β-site as well, are found in most studies with animal models for AD, i.e., in the 3xTg- lessens amyloidogenic APP processing and so protects against AD AD model (Giménez-Llort et al., 2013). The structure of Aβ1–42 fibrils onset (Jonsson et al., 2012). Some other relevant mutations on APP shows a quite different structure, with two twisted protofilaments gene are located on the N-terminal part of the Aβ peptide or close to stacked in parallel and thus built of “LS”-shaped dimers, with the the γ-secretase site (Esquerda-Canals et al., 2017b). N-terminus slightly solvent-exposed and the C-terminus completely buried (Figure 3C; pdb 5OQV) (Gremer et al., 2017). How oligomers The variety of mutations on PSEN genes encompasses missense promote cell toxicity has not been completely unraveled. However, mutations, small deletions and insertions, as well as genomic exon several molecular mechanisms as activation of metabotropic deletions, being PSEN1 mutations the most usual in FAD (Park et glutamate receptor 5, Caspase-3 activation, N-methyl-D-aspartate al., 2017; Bagyinszky et al., 2018; Giau et al., 2018a, b). Although receptor interaction, and α7 nicotinic acetylcholine receptor hundreds of mutations on APP, PSEN1, and PSEN2 have been described, they only account for a small part of early-onset AD cases, upregulation have been proposed (Bezprozvanny, 2009). attributable when AD onset occurs before 65 years of age. Non- Although amyloid deposits do not correlate with either disease FAD early-onset AD cases are clustered with late-onset AD into what progression or cognitive decline (Hanseeuw et al., 2019), their spatial is called the sporadic form of the disease (SAD). Several years ago, and temporal emergence are well described (Braak and Braak, 1991). it was observed that the serum of non-AD individuals shows auto- In a first stage, amyloid deposits occur in the frontal, temporal, and antibodies against Aβ, which indicates that some amyloidogenic occipital lobes of the isocortex. In a second stage, they spread to all processing also occurs in normal conditions. In addition, there is a the isocortical association areas, except on primary sensory areas differential physiological role of BACE1 (which generates Aβ peptide, and motor field. Disperse amyloid deposits also occur in the frontal as mentioned) and BACE2 (which generates both Aβ peptide and and parietal lobes. In this stage, the hippocampus is slightly affected. an N-truncated form, as mentioned). BACE1 is indispensable for The final stage is characterized by depositions in primary isocortical proper astrogenesis, axonal growth and migration, myelination and areas and progressive spreading to the striatum, thalamus and remyelination, neuronal excitation, and synaptic plasticity; whereas hypothalamus. BACE2 is involved in different processes, as glucose homeostasis and pigmentation (Yan, 2017). Studies using physiological Aβ concentrations (pM) show a role in normal synaptic physiology, Tau and Its Role in Neurodegeneration enhancing long term potentiation, promoting survival of neurons, As mentioned before, the lack of success in approving any anti-Aβ and promoting a neurogenic effect on neural progenitor cells. In therapy up to now, as well as the poor correlation found between addition, Aβ is involved in the repair of leaks in the blood-brain cognitive decline and the occurrence of amyloid plaques, have led barrier, promotes recovery from injury, and acts as an antimicrobial the scientific community to question whether Aβ accumulation is and tumor suppressor peptide (Kent et al., 2020). actually triggering neurodegeneration in AD. The occurrence of tau In 1992, taking into consideration FAD cases, together with the fact NFTs better relates to neuronal loss and clinical symptoms (Hanseeuw that location of APP gene on chromosome 21 leads Down syndrome et al., 2019) and, although Aβ may initiate a cascade of events, tau individuals to suffer from AD (Macleod et al., 2015) by the time they impairment is likely the effector molecule of neurodegeneration. reach middle to late age, the ACH was postulated (Hardy and Higgins, Tau, apart from being a stabilizer of microtubules and being 1992) (Figure 2). ACH states that Aβ peptide deposition triggers tau involved in their dynamics, plays a role in several physiological hyperphosphorylation and aggregation in the form of NFTs, and these processes including myelination, axonal transport, neurogenesis, aggregates lead to inflammation, synaptic impairment, neuronal loss, motor function, learning and memory, neuronal excitability, glucose and thus to cognitive decline and behavioral abnormalities. However, metabolism, iron homeostasis, and DNA protection (Kent et al., Aβ overproduction is not the only cause of AD since the reduction 2020). It is important to mention that while Aβ accumulation in Aβ auto-antibodies featuring SAD points to an impairment in Aβ is characteristic of AD, tau pathology also exists in a group of clearance. In consonance, the most well-known genetic risk factor for neurodegenerative diseases known as tauopathies, which are developing SAD is the apolipoprotein E (APOE) gene, which encodes different to AD. NEURAL REGENERATION RESEARCH｜Vol 17｜No. 8｜August 2022｜1667 NEURAL REGENERATION RESEARCH www.nrronline.org Review A C Figure 1 ｜ Amyloid precursor protein (APP) processing. (A) α- and γ-secretase act in the non- amyloidogenic pathway, whereas the activity of β-secretase (or BACE) instead of α-secretase defines the amyloidogenic pathway generating the Aβ peptide. BACE1 hydrolyses the β-site (M671-D672 bond) and BACE2 cleaves the β’-site (681Y-682E bond), rendering an N-t truncated form of the precursor (E11-Xx), although also cleaves the β-site. Thus, β′- site cleavage is amyloidolytic rather than amyloidogenic. Finally, γ-secretase releases the Aβ peptide, or its N-t truncated form, after preferentially cleaving the V711-D712 and A713-T714 bonds rendering Aβ1–40 and Aβ1–42, respectively. (B) Target proteolytic B sites in the Aβ peptide. (C) Some of the main FAD-related mutations in human APP. ACID: APP intracellular domain; Aβ: amyloid-beta peptide; BACE: beta-site APP cleaving enzyme; CTF: carboxyterminal fragments; FAD: familial Alzheimer’s disease. Figure 2 ｜ The amyloid cascade hypothesis (ACH). Whether by an increase in production (FAD) or an impairment in clearance (SAD), Aβ peptide accumulates through an aggregation pathway populated by different species, i.e. monomers, oligomers, fibrils, and amyloid plaques. Aβ oligomers are the most toxic species, and to a lesser extent fibrils, causing calcium disruption, mitochondrial failure, inflammatory response, oxidative stress, transcriptional impairment, synaptic loss and, finally, neuronal death. In addition, Aβ oligomers induce the hyperphosphorylation of tau, which aggregates into oligomers that evolve to paired helical filaments and finally to NFTs. Tau oligomers are also toxic for neurons, with a strong effect on synapses, and, in turn, induce the formation of Aβ oligomers. In addition to this positive feedback between Aβ and tau aggregation, Aβ and tau cooperatively work to lead to neuronal death. Although still controversial, the ACH covers the multifactorial nature of AD. Aβ: Amyloid- beta peptide; FAD: familial Alzheimer’s disease; NFTs: neurofilament tangles; PHF: paired helical filaments; SAD: sporadic Alzheimer’s disease. A Figure 3 ｜ Amyloid fibril formation and three- dimensional structure. (A) Aggregation of Aβ from the formation of different kind of oligomers to amyloid fibrils. (B) Aβ1–40 amyloid fibrils are composed by multiple ordered trimers. The N-termini are completely solvent-exposed, whereas B C the C-termini are buried in the hydrophobic core (PDB 2M4J) (Lu et al., 2013). Each color represents a monomer. (C) Aβ1–42 amyloid fibrils are made of dimers. The N-termini are slightly solvent-exposed, whereas the C-termini are completely buried in the interface between monomers (PDB 5OQV) (Gremer et al., 2017). Each monomer in the dimer shows higher β-content than in the trimer. Each color represents a monomer. Images were generated with the Swiss- PdbViewer software (Guex and Peitsch, 1997). Aβ: Amyloid-beta peptide; PDB: protein data bank. 1668 ｜NEURAL REGENERATION RESEARCH｜Vol 17｜No. 8｜August 2022 Review NEURAL REGENERATION RESEARCH www.nrronline.org The link between tau dysfunction and neurodegeneration was set et al., 2011), and it is thought to be secreted under physiological through human genetics. Mutations in MAPT, the microtubule- conditions. Microglial cells may propagate tau through exosome- associated protein Tau gene, give rise to an inherited form of dependent mechanisms (Asai et al., 2015). Spread of tau aggregated Frontotemporal Dementia (FTD) and parkinsonism with abundant assemblies may seed and promote aggregation (Falcon et al., 2015) filamentous tau deposits in the brain, in the absence of Aβ deposits along different brain regions. In AD, deposition of tau aggregates (Goedert, 2015). Six different isoforms of tau are expressed in the follows a highly stereotyped pattern, beginning in the entorhinal adult human central nervous system via alternative splicing of the cortex and hippocampus before propagation to other regions. Their MAPT gene, which comprises 16 exons and is found on chromosome stereotypical appearance in nerve cells underlies Braak´s stages 17q21.3 (Figure 4A). Regulated expression of exons 2 and 3 gives rise of AD (Jellinger, 1998), according to which inclusions form first in to tau isoforms with 0, 1, or 2 N-terminal inserts, whereas exclusion subcortical regions, transentorhinal cortex, and entorhinal cortex or inclusion of exon 10 leads to expression of tau isoforms with three (stages I and II). They then appear in the hippocampal formation and (3R) or four (4R) microtubule-binding repeats (Connell et al., 2005). some parts of the neocortex (stages III and IV), followed by most 3R or 4R isoforms are predominant in tau inclusions depending on of the neocortex (stages V and VI). Individuals at stages I and II are the neurodegenerative disease. Progressive supranuclear palsy (PSP), asymptomatic, some individuals at stages III and IV show signs of corticobasal degeneration, argyrophylic grain disease, among others, memory impairment, and those at stages V and VI suffer AD (Schöll exhibit overexpression of 4R isoforms; whereas Pick’s disease, and et al., 2016). familial FTD and parkinsonism (MAPT mutations such as G272V and Q336R) are mainly characterized by tau inclusions rich in 3R tau isoforms (Irwin, 2016). In other diseases such as AD, Down syndrome Amyloid-beta Peptide and Tau Protein Interplay: or Chronic traumatic encephalopathy, both 3R and 4R isoforms are More Than Casual Partners found in tau tangles. The N-terminal phosphatase-activating domain Currently it is becoming widely accepted that Aβ is the “trigger” in tau, which triggers a signaling pathway that disrupts axonal and tau the “bullet” driving AD (Bloom, 2014). In addition, and transport, is exposed in all the isoforms. The extent of phosphatase- although tau load better relates to cognitive decline than Aβ load, as activating domain exposure and oligomerization is larger for tau mentioned, it has recently been reported that the presence of both aggregates composed of 4R isoforms than 3R isoforms. However, Aβ and tau is necessary for memory decline in the preclinical stages aggregates of all isoforms exhibit enough phosphatase-activating of AD (Sperling et al., 2019). However, the mechanisms behind Aβ domain exposure to impair axonal transport (Cox et al., 2016). and tau interplay in AD remain elusive. Tau is predominantly expressed in the central and peripheral nervous Recent data supports the hypothesis that there is a bidirectional system, where it is most abundant in nerve cell axons. In the brain, interplay between Aβ and tau. Few years ago, it was demonstrated tau is subject to several posttranslational modifications, including that addition of Aβ to human cells induces tau aggregation in the phosphorylation, acetylation, methylation, glycation, isomerization, form of paired helical filaments-like filaments (Ferrari et al., 2003). O-GlcNAcetylation, nitration, sumoylation, ubiquitination, and Interestingly, cellular models have shown that over-expression of APP truncation (Morris et al., 2015), but the role all these modifications and PSEN1 increases not only Aβ, but also tau aggregation (Choi et play in tau function remains unknown. The most studied tau al., 2014). A relation between Aβ and tau was also found in animal posttranslational modification is phosphorylation, as despite the models, as in APPPS1 transgenic mice (APP KM670/671NL (Swedish), significant heterogeneity that exists between and within the different PSEN1 L166P) overexpressing Aβ also show an increase in CSF- tauopathies, the deposited tau in pathological lesions is invariably tau levels. Furthermore, tau is able to facilitate Aβ aggregation, as highly phosphorylated (Noble et al., 2013). Phosphorylation of tau tau deletion in APPPS1 transgenic mice ameliorated Aβ deposition negatively regulates its ability to interact with microtubules, but (Peters et al., 2019); whereas the addition of human tau increased whether phosphorylation is a trigger for aggregation remains to be plaque size, supporting the hypothesis that there is a bidirectional proved. Although aggregated tau is heavily phosphorylated in the interplay between Aβ and tau. However, the APPPS1 mouse model of human brain, not all phosphorylated tau is aggregated (Goedert AD does not develop NFTs, neither do other APP and/or PS1 models et al., 2017). Full-length tau assembles into filaments through its (Esquerda-Canals et al., 2017b). The only mouse model developing repeats, with the N-terminal half and the C-terminus forming the NFTs is the triple transgenic mouse model (3xTg-AD; PS1M146V, APPSwe, fuzzy coat (Figure 4B; pdb 6HRE) (Falcon et al., 2018). The filament tau P301L) and this is so because one of the three transgenes is a core contains the repeat sequences that are required to the binding mutated MAPT (tauP301L), found in familial FTD (Oddo et al., 2003). of soluble tau to microtubules, suggesting that physiological function In addition, this mouse model is the only one where an evident and pathological assembly are mutually exclusive (loss of function). neuron loss has been quantified even at the early stages of the Brains from different individuals with the same tauopathy show disease. Interestingly, cellular density showed a strong correlation the same kind of tau filaments (Falcon et al., 2018), whereas each with intracellular Aβ burden in glutamatergic neuronal populations disease is characterized by its own unique tau fold (Scheres et al., (Esquerda-Canals et al., 2017a). At the most advanced stage of the 2020). Cross-seeding experiments have shown that Aβ oligomers disease, it has been demonstrated that Aβ-immunotherapy not only induce tau oligomerization in vitro (Lasagna-Reeves et al., 2010), decreases Aβ-burden but also affects tau load in this mouse model indicating that early abnormal Aβ oligomerization precedes tau (Roda et al., 2020). Similarly, active DNA Aβ42 immunization in 3xTg- aggregation. Interestingly, it has been reported that structure- AD mice reduced not only Aβ deposition but also tau pathology based inhibitors of Aβ are able to avoid tau aggregation, suggesting (Rosenberg et al., 2018). that there is a common aggregation interface in both tau and Aβ (Griner et al., 2019). Although tau is mainly an intracellular protein, The challenging modeling of AD in rats has been achieved with extracellular tau is present in the brain and interstitial fluid (Yamada the line TgF344-AD expressing APPSwe and PS1ΔE9 genes (Cohen et Figure 4 ｜ Three-dimensional structure of tau NFTs and tau isoforms scheme. A B (A) Six different isoforms of tau are expressed in the adult human central nervous system via alternative splicing of the MAPT gene. Regulated expression of exons 2 and 3 gives rise to tau isoforms with 0, 1, or 2 N-terminal inserts whereas exclusion or inclusion of exon 10 leads to expression of tau isoforms with three (3R) or four (4R) microtubule-binding repeats. (B) Paired helical filaments from AD brain. The structure shows the fuzzy core formed by aggregated repeats (residues G304-E380) (PDB 6HRE)(Falcon et al., 2018). Each color represents a monomer. Images were generated with the Swiss- PdbViewer software (Guex and Peitsch, 1997). AD: Alzheimer’s disease; MAPT: microtubule associated protein tau; PDB: protein data bank. NEURAL REGENERATION RESEARCH｜Vol 17｜No. 8｜August 2022｜1669 NEURAL REGENERATION RESEARCH www.nrronline.org Review al., 2013). This rat model shows Aβ oligomers, Aβ plaques, tau in neurons. Interestingly, GSK-3 is also involved in other dementias, pathology, behavioral impairment and neuron loss. Abundant NFTs as in FTD (Schaffer et al., 2008), and its inhibition reduces tau were detected in close proximity to β-amyloid plaques in aged phosphorylation in vitro (Singh et al., 1995; Wagner et al., 1996), transgenic rats that were reminiscent of NFTs found in AD patients as well as in animal models (Uno et al., 2009). Thus, apoE or GSK-3 brains. Taken into account that rats are 4–5 million years closer independent-Aβ alterations could enhance tau phosphorylation and to humans than mice in evolution and that they express all six tau aggregation, supporting the dual pathway hypothesis. Furthermore, isoforms found in humans, this rat model supports the validity of the it has been proposed that GSK-3 could be a linker between Aβ and ACH in humans. Several studies show that Aβ and tau co-localize in tau hyperphosphorylation (Medina and Avila, 2012). Regarding Aβ, neurons, as well as in astrocytes (Pickett et al., 2019). Aβ peptide is it has been widely reported that apoE ε4 enhances Aβ production mainly accumulated in the synapses near to senile plaques, whereas and aggregation as well as impairs its clearance (Roda et al., 2019). tau synaptic localization is no dependent on senile plaques vicinity. Recently, an interaction effect between apoE ε4 and Aβ, rather than Moreover, only 0.02% of the synapses were positive for Aβ and tau, the sum of their independent effects, has been related to increased indicating that direct Aβ-tau interaction might not be the major tau load in humans (Therriault et al., 2020). However, not only apoE link between both pathologies. Interestingly, different crossings of ε4 is a common trigger of Aβ and tau aggregation. Several metabolic MAPTnull + APP/PS1 + rTg21221 AD mouse lines (APP/PS1 + Tau), diseases have been suggested to be related with neurodegeneration. homozygous for deletion of mouse tau and heterozygous for the In fact, diet-induced insulin resistance in animal models stimulates human wild-type tau inducible transgene, have shown that Aβ and Aβ and tau aggregation, as well as neurodegeneration (Bharadwaj tau cooperatively work to impair transcription of genes involved in et al., 2017). Which are the exact mechanisms behind such a synaptic function. In addition, accumulation of tau was observed in stimulation remains to be elucidated, but the occurrence of amylin (or synapses of this mouse model and in human postmortem tissues. islet amyloid polypeptide) deposits in senile plaques suggests that a Downregulation of tau in these mice after behavioral deficits link between this pancreatic amyloid and AD could exist. Moreover, emerged, restored behavioral deficits, reduced synaptic tau levels, immune system dysregulation and oxidative stress, both present in and partially reversed transcriptional perturbations (Pickett et al., type-2 diabetes, are involved in AD progression. 2019). Therefore, this is another evidence of tau being the effector Thus, a large amount of evidence supports the assumption that molecule in AD. Aβ peptide and tau protein are not casual partners. There is a link Another proposed link between Aβ and tau alterations is the between both pathologies that cooperate to produce synaptic C-terminus of hsp70-interacting protein, as it has been reported dysfunction, neuronal loss, and the eventual cognitive decline that Aβ accumulation decreases C-terminus of hsp70-interacting characterizing AD. protein expression and increases tau levels. In addition, and Aβ- induced effects on tau are rescued by restoring C-terminus of hsp70- Combined Therapies as a New Hope: Targeting interacting protein levels (Oddo et al., 2008; Lyon and Milligan, 2019). Amyloid-beta and Tau A common hallmark among neurodegenerative diseases is Even though there is a bidirectional interaction and some factors neuroinflammation. The innate immune system could be another could independently affect both molecules, Aβ oligomerization link between amyloid and tau pathologies. Interestingly, both Aβ and seems to precede tau hyperphosphorylation. The fact that Aβ- tau induce a neuroinflammatory response (Yoshiyama et al., 2007; immunotherapy still raises some concerns is in agreement with Alawieyah Syed Mortadza et al., 2018) that leads to neuronal death, the new idea of Aβ acting just as a switch-on. Once Aβ triggers the cascade of events that leads to neuronal death and dementia, plasmatic leakage, and brain atrophy. On the one hand, microglia and it is not that important whether its levels can be reduced or not complement factors seem to be involved in the early synaptic loss because the effector molecule driving to synaptic impairment and induced by oligomeric species of Aβ (Hong et al., 2016). On the other neuronal death is tau. This would explain why previous clinical trials hand, some studies suggest that microglia could play a pivotal role in for Aβ-immunotherapy enrolling mild to moderate AD patients, did tau spreading by making synapses more vulnerable to tau-induced not reach the end-points. Nowadays, active clinical trials for Aβ- synaptic impairment. Inhibition of exosome secretion by microglia immunotherapy enroll patients in early, even prodromal stages of the suppressed tau propagation to the dentate gyrus in a mouse model, disease (see below). recovering non-pathological excitability in this brain region. In contrast, extracellular soluble, but not intracellular, Aβ has been In the last years, it has been reported that Aβ-immunotherapy related with increased exosome-dependent tau spreading in both reduces not only Aβ but also tau levels in animal models (Roda et al., animal models and human (Gomes et al., 2019). These observations 2020) and clinical trials (Blennow et al., 2012; Sevigny et al., 2016). support the important role microglia, and particularly microglial Likewise, it has been shown that tau-immunotherapy also reduces exosomes, play in prion-like spreading of tau throughout the brain Aβ levels (Dai et al., 2017). Since the interplay between Aβ and tau (Asai et al., 2015). It is worth noting that the immune system is becomes evident, several researchers claim that a combined therapy not only a mediator between Aβ or tau and neuronal death. Aβ would be a better strategy to treat AD at early stages of the disease oligomers could activate the immune system, prior to senile plaque (Busche and Hyman, 2020; Tolar et al., 2020). formation, triggering tau hyperphosphorylation and, subsequently, the formation of NFTs. Aβ oligomers are able to activate the Currently, there are three principal therapeutic approaches focused inflammasome (Nod-like receptor family pyrin domain containing 3, on Aβ: avoiding Aβ aggregation, reducing Aβ production, and NLRP3) that, in turn, regulates kinases, as glycogen synthase kinase 3 enhancing Aβ clearance (Table 1). Only one drug intended for (GSK-3) and phosphatases, as protein phosphatase 2A, inducing tau avoiding Aβ aggregation, Tramiprosate (reformulated as ALZ-801), abnormal phosphorylation (Ising et al., 2019) and evidencing a clear has reached phase III but beneficial effect was only observed in link among Aβ, the immune system, and tau pathology. APOE ε4/4 carriers (Abushakra et al., 2017; NIH, 2021). Other Aβ aggregation inhibitors, such as ELND005, did not reach primary Despite the evidence that the bidirectional interplay between outcomes in phase II (Rafii et al., 2017; NIH, 2021). Most secretases Aβ and tau better explains the ACH, it is also true that there are inhibitors/activators have reached phase III but safety concerns factors that could independently affect and trigger both Aβ and tau occurred in most of them (Vellas et al., 2012; Penninkilampi et al., abnormalities (Small and Duff, 2008). This idea has been formulated 2016; Rombouts et al., 2021). The first active immunotherapy clinical as “The dual pathway hypothesis”, which states that there are trial for AD tested the AN1792 vaccine, composed of fibrillar Aβ (1– common upstream triggers causing both Aβ and tau abnormalities 42) and QS21 adjuvant. Although phase II was discontinued because (Small and Duff, 2008). Supporting this hypothesis, it has been shown 6% of immunized patients suffered meningoencephalitis, clearance of that Aβ is necessary but not enough to induce tau pathology. Aβ plaques and reduction in cognitive decline were demonstrated (Vellas plaque-bearing mouse models that do not overexpress tau need et al., 2009). Next generation of Aβ active vaccines have consisted of injection of misfolded AD-tau seeds to develop the three major types N-terminal Aβ sequences (i.e., 1–6, 1–14) encapsulated in different of AD-relevant tau pathologies: dystrophic neurites surrounding delivery systems (Hull et al., 2017; Lopez Lopez et al., 2019), The Aβ plaques (NP tau), neuropil threads and NFTs. These distinct rationale behind the use of the N-terminal sequence is found in the tau pathologies show different temporal onsets and functional 3D-structures of trimers (1–40) and dimers (1–42), both solvent consequences on neural activity and behavior (He et al., 2018). exposing the N-termini, as mentioned. This rationale was also used Among other factors, APOE, specifically the apoE ε4 isoform, would for obtaining some of the monoclonal antibodies (mAbs) for passive have an important role in this new scenario. ApoE ε4 increases the Aβ-immunotherapy (La Porte et al., 2012; Salloway et al., 2014; activity of one of the major tau kinases, GSK-3 (Liang et al., 2019), Logovinsky et al., 2016; The Lancet Neurology, 2017; Manolopoulos 1670 ｜NEURAL REGENERATION RESEARCH｜Vol 17｜No. 8｜August 2022 Review NEURAL REGENERATION RESEARCH www.nrronline.org Table 1 ｜ Aβ therapeutics in clinical trials Therapeutic approach Mechanism of action Drugs (companies) Clinical trials Identifier Aβ aggregation β-Breakers Tramiprosate (3APS, Alzhemed, Phase III did not reach primary outcomes, but had NCT00314912 inhibitors homotaurine, ALZ-801) (Bellus beneficial effect on APOE e4/4 patients with mild Health Inc.) AD ELND005 (Scyllo-inositol) (Ellan) Phase II did not reach primary outcomes NCT00934050 Secretases inhibitors / β-Secretase (BACE) MK-8931 (Merk Sharp & Dohme Safety concerns and no effects after Phase III NCT01739348 modulators/activators inhibitors Corp.), JNJ-54861911 (Janssen), studies NCT02569398 E2609 (Eisai Inc.), AZD3293 NCT02956486 (LY3314814) (Eli Lilly and Co. & NCT02783573 AstraZeneca) γ-Secretase inhibidor or Semagacestat (LY-450139) (Eli Lilly) Dangerous effects because their relation with NCT00594568 modulator and Flurizan (Tarenflurbil, MPC- cancer (+ for T-cell leukemia) NCT00105547 7869) (Myriad Genetics’) α-Secretase activator Etazolate (ETH 0202) (Exonhit) No effect in Phase III NCT00880412 Active immunotherapy Fibrillar Aβ (1–42), QS21 AN1792 (Elan and Wyeth) Phase II discontinued (2002). 6% of patients with NCT00021723 adjuvant meningoencephalitis. Clearance of plaques, decrease in cognitive decline. N-t Aβ (1–6) in viral CAD106 (Novartis) Phase II/III ongoing with cognitively healthy APOE NCT02565511 particles e4/4 N-t Aβ (1–14) + T-cell UB-311 (United Neuroscience Ltd.) Phase II recently terminated NCT03531710 epitope, delivery system Passive immunotherapy Humanized mAb, IgG1 Bapineuzumab (AAB-001) (Janssen Phase III terminated (2012), mild to moderate AD. NCT00574132 & Pfizer) Fibrillar Aβ clearance (PET), CSF-p-tau reduction Anti-N-t (1–5) No cognitive improvement. 5% ARIA-E (APOE e4 carriers) Binds monomeric, oligomeric and fibrillar Humanized mAb, IgG2 Ponezumab (PF- Phase II repurposed for CAA (1–40) NCT01821118 04360365RN1219) Anti-C-t (30–40) (Pfizer) Hippocampal amyloid burden reduction. Binds plasmatic Aβ No cognitive improvement (peripheral sink) Humanized mAb, IgG1 Solanezumab (LY206243) (Eli Lilly No benefit in Phase III, mild AD NCT01760005 and Co.& Hoffmann–La Roche) Anti-mid-region (16–24) NCT02008357 Binds monomers Phase III (DIAN-TU study, FAD asymptomatic) (peripheral sink) Phase III (A4 study, asymptomatic) Fully human mAb, IgG1 Gantenerumab (RO4909832, Eight Phase III studies, prodromal and/or mild AD NCT03443973 RG1450) (Hoffmann-La Roche) on going (i.e.) Anti-N-t (1–10) and -mid- NCT04339413 region (23–25) Binds fibrils NCT03444870 Phase II (DIAN-TU study, FAD asymptomatic) NCT01760005 Humanized mAb, IgG4 Crenezumab (MABT5102A) Phase III terminated (2019), mild to moderate AD NCT03977584 (Genentech) Anti-mid-region (12–23) CSF Aβ-oligomers’ decrease, no ARIA-E Binds protofibrils No cognitive improvement API study (pre-symptomatic PS1E280A carriers) Fully human mAb, IgG1 Aducanumab (BIIB037) (Biogen Accelerated approval by the FDA, June 7, 2021 NCT02477800 Idec) Anti-N-t (3–6) and mid- region Binds all aggregated forms E22G “arctic” mutation BAN2401 (Eisai Inc.) Phase III just initiated, early AD NCT03887455 (protofibrils) Clears amyloid plaques, Cognitive improvement NCT04468659 10% ARIA-E (APOE e4 carriers) Intravenous IgGIV Gammagard (Baxalta US Inc.); All just completed Phase III, mild to moderate AD NCT00818662 Octagam (Octapharma); Flebogamma (Grifols Biol. Inc) IgG fraction, polyclonal Reduction in amyloid, cognitive improvement Binds different linear and Low statistical potential, under analysis conformational epitopes ClinicalTrials.gov accessed February 27, 2021. AD: Alzheimer's disease; APOE: apolipoprotein E; ARIA-E: amyloid-related imaging anomalies-E; Aβ: amyloid-beta; BACE: beta-site APP cleaving enzyme; CAA: cerebral amyloid angiopathy; CSF: cerebro-spinal fluid; FAD: familiaol Alzheimer's disease; IgG: immunoglobulin-G; mAb: monoclonal antibody; PET: positron emission tomography. et al., 2019; Servick, 2020; Yoshida et al., 2020). Bapineuzumab, an APOE ε4 carriers, and no effect on cognitive decline was achieved humanized mAb, was the first reaching phase III clinical trials, but (Salloway et al., 2014). Apart from the accelerated approval for amyloid related imaging abnormalities were observed, mainly in aducanumab mentioned above, gantenerumab and BAN2401 have NEURAL REGENERATION RESEARCH｜Vol 17｜No. 8｜August 2022｜1671 NEURAL REGENERATION RESEARCH www.nrronline.org Review potential for near term approval (Tolar et al., 2020). Aducanumab this sense, a phase I clinical trials with the oligonucleotide NIO752 and gantenerumab are fully human mAbs that target epitopes (Novartis) aimed to treat PSP is being initiated (NCT04539041) (NIH, composed of the N-terminus and a central region of the Aβ peptide, 2021). In the specific case of the treatment of AD, IONIS MAPTRx whereas BAN2401 is a humanized mAb directed to an epitope on the oligonucleotide is being tested in phase I/II for decreasing tau mRNA fibrils made by the E22G “arctic” mutation. (NIH, 2021). Another approach is the inhibition of tau aggregation that is being tested in a phase III clinical trial with methylene blue Similarly, treatments to reduce tau aggregation or decrease tau levels (Rember), already used for malaria. Concerning the enhancing of tau are under development (Table 2), but not as advanced as for Aβ. A clearance, it must be taken into account that tau oligomers can exist new therapeutic approach for treating AD or other taupathologies in a variety of states including hyperphosphorylated forms, which can is the use of antisense RNAs interfering the expression of MAPT be both soluble and insoluble. In addition, the fact that they are six mRNA (Evers et al., 2015). Morpholinos are synthetic RNA analogs isoforms complicates the election of the best target. It remains to be that are stable and specifically bind to the designed target mRNA. In determined (a) which of these oligomeric species of tau are causally neuroblastoma cell cultures, reductions in tau levels up to 80% were involved in neurodegeneration, and (b) which trigger the beginning achieved by exon skipping inducing an out-of-frame deletion in tau of the formation paired helical filaments filaments. Meanwhile, mRNA (Sud et al., 2014). As mentioned before, six tau isoforms are phase II trial for active immunotherapy with a fragment of tau found in the human brain (Figure 4A) and the ration between R3 (not (294–305) (AADvac1 (Axon Neuroscience SE) and several phase II including exon 10) and R4 isoforms (including exon 10) is impaired in trials for passive immunotherapy with gosuranemab, tilavonemab, several diseases such as PSP, Corticobasal Degeneration, Argyrophylic semorinemab zagotenemab are ongoing (Alzforum, 2020). All these grain disease, Pick’s disease, and FTD-P, among others. Although humanized mAbs have been obtained on an IgG4 scaffold and this is not the case in AD, it is worth mentioning the capability of recognize the N-terminus of tau and hence extracellular tau, with antisense oligonucleotides for controlling the skipping of exon 10 the exception of zagotenemab that also recognizes a mid-region and therefore their therapeutic potential (Peacey et al., 2012). In characterizing aggregated tau. Table 2 ｜ Tau therapeutics in clinical trials Therapeutic approach Mechanism of action Drugs (companies) Clinical trials Identifier Antisense RNA MAPT mRNA splicing and/or NIO752 (Novartis) Phase I registered for the NCT04539041 silencing treatment of PSP IONIS MAPTRx (BIIB080, ISIS 814907) (Ionis Phase I/II ongoing NCT03186989 Pharma, Inc.) Tau aggregation Several mechanism (i.e. for malaria, TRx0237/LMT-X/Methylene Blue (Rember) Phase III ongoing NCT03446001 inhibitors others) Active immunotherapy Tau (294–305) + keyhole limpet AADvac1 (Axon Neuroscience SE) Phase II slowed NCT02579252 hemocyanin, Al(OH)3 adjuvant. neurodegeneration biomarkers 66 copies of a synthetic, ACI-35 (AC Immune) Elicited a weak immune NCT04445831 S396&S404-phosphorylated tau into response, and needed to be liposomes. MPLA adjuvant redesigned. Phase II ongoing. Passive Humanized mAb, IgG4 Gosuranemab (BIIB092, IPN002) (Biogen) Phase II just initiated, early AD NCT03352557 immunotherapy Anti-Nt (15–24) Binds e-tau Humanized mAb, IgG4 Tilavonemab (ABBV-8E12, C2N-8E12) (AbbVie) Phase II just initiated, early AD NCT03712787 Anti-Nt (22–34), human NCT02880956 Binds e-tau Humanized mAb, IgG4 Semorinemab (RO7105705, Genentech, Inc.) Phase II just initiated, prodromal NCT03289143 to mild and moderate AD Anti-Nt (2–24), human NCT03828747 Binds e-tau Humanized mAb, IgG4 Zagotenemab (LY3303560) (Eli Lilly Co) Phase II just initiated, early AD NCT03518073 Anti-Nt (7–9) and mid (312–322) Binds aggregated-tau ClinicalTrials.gov accessed February 27, 2021. AD: Alzheimer's disease; e-tau: extracellular tau; IgG: immunoglobulin; mAb: monoclonal antibody; MAPT: microtubule associated protein tau; mRNA: messenger ribonucleic acid; PSP: progressive supranuclear palsy. In conclusion, the evidence of a bidirectional interplay between Aβ appropriate credit is given and the new creations are licensed under the and tau better explains the ACH, which is currently complemented identical terms. by the dual pathway hypothesis. Combined anti-Aβ and anti- ©Article author(s) (unless otherwise stated in the text of the article) 2022. tau therapies will be tested in the near future because Aβ- All rights reserved. No commercial use is permitted unless otherwise immunotherapy is quite advanced, with a recent accelerated expressly granted. approval for Aduhelm, and tau-immunotherapy is on the path. In addition, the determination of the three-dimensional structures of the fibrils made by Aβ1–40, Aβ1–42, and tau may be helpful in the design References of new therapeutic molecules. Therefore, it is quite likely we are on Abushakra S, Porsteinsson A, Scheltens P, Sadowsky C, Vellas B, Cummings J, Gauthier the path to find a cure for this devastating disease. S, Hey JA, Power A, Wang P, Shen L, Tolar M (2017) Clinical effects of tramiprosate in APOE4/4 homozygous patients with mild Alzheimer’s disease suggest disease Author contributions: ARR designed the manuscript; ARR, GSM, LMG and modification potential. J Prev Alzheimers Dis 4:149-156. SV collaborated in data search and manuscript writing; LT participated in Alawieyah Syed Mortadza S, Sim JA, Neubrand VE, Jiang LH (2018) A critical role of manuscript revision; SV performed final manuscript revision. All authors TRPM2 channel in Aβ42-induced microglial activation and generation of tumor approved the final version of the manuscript. necrosis factor-α. Glia 66:562-575. Conflicts of interest: The authors declare no conflicts of interest. 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Yoshiyama Y, Higuchi M, Zhang B, Huang SM, Iwata N, Saido TCC, Maeda J, Suhara T, Rosenberg RN, Fu M, Lambracht-Washington D (2018) Active full-length DNA Aβ42 Trojanowski JQ, Lee VMY (2007) Synapse loss and microglial activation precede immunization in 3xTg-AD mice reduces not only amyloid deposition but also tau tangles in a P301S tauopathy mouse model. Neuron 53:337-351. pathology. Alzheimers Res Ther 10:115. C-Editors: Zhao M, Liu WJ, Qiu Y; T-Editor: Jia Y 1674 ｜NEURAL REGENERATION RESEARCH｜Vol 17｜No. 8｜August 2022