Pathways to Alzheimer s disease

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1 Key Symposium Click here for more articles from the symposium Watch Guest Editor Professor Mila Kivipelto talk about the 9th Key Symposium: Updating Alzheimer s Disease Diagnosis here. Pathways to Alzheimer s disease doi: /joim J. Hardy 1, N. Bogdanovic 2, B. Winblad 3, E. Portelius 4, N. Andreasen 3, A. Cedazo-Minguez 3 & H. Zetterberg 1,4 From the 1 Department of Molecular Neuroscience, Reta Lila Weston Research Laboratories, UCL Institute of Neurology, London, UK; 2 Section of Clinical Geriatrics, Karolinska Institutet; 3 KI-Alzheimer Disease Research Center, Karolinska Institutet, NVS, Stockholm; and 4 Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden Abstract. Hardy J, Bogdanovic N, Winblad B, Portelius E, Andreasen N, Cedazo-Minguez A, Zetterberg H (UCL Institute of Neurology, London, UK; Karolinska Institutet, Stockholm; Karolinska Institutet, NVS; and The Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden). Pathways to Alzheimer s disease. (Key Symposium). J Intern Med 2014; 275: Recent trials of anti-amyloid agents have not produced convincing improvements in clinical outcome in Alzheimer s disease; however, the reason for these poor or inconclusive results remains unclear. Recent genetic data continue to support the amyloid hypothesis of Alzheimer s disease with protective variants being found in the amyloid gene and both common low-risk and rare high-risk variants for disease being discovered in genes that are part of the amyloid response pathways. These data support the view that genetic variability in how the brain responds to amyloid deposition is a potential therapeutic target for the disease, and are consistent with the notion that anti-amyloid therapies should be initiated early in the disease process. Keywords: Alzheimer s disease, amyloid, cell biology, genetics, tau. Introduction There is no doubt that for the last 20 years, the amyloid cascade hypothesis has dominated opinion about the aetiology and pathogenesis of Alzheimer s disease (AD), as well as guided the efforts to find treatments. However, in the last 2 years, several clinical trials of agents targeting amyloid-b (Ab), including two c-secretase inhibitors (semagacestat and avagacestat) and two anti-ab antibodies (bapineuzumab and solanezumab), have failed to reach their primary clinical endpoints. These trials have produced ambivalent results, with positive findings only in ad hoc secondary end-points, and have led to divergent opinions on whether it is worthwhile persevering with Ab therapies or whether this approach should be abandoned. It is not the primary purpose of this review to discuss these issues although we will offer our opinion. Rather, as the initial evidence supporting the amyloid cascade hypothesis came from genetic analysis of individuals with Down syndrome and autosomal dominant AD, our primary purpose is to assess more recent genetic data from genome-wide association studies and exome sequencing to consider the potential additional drug targets highlighted by these analyses and discuss how they relate to the amyloid pathway. Deciphering the results of clinical trials of semagacestat, bapineuzumab and solanezumab The amyloid cascade hypothesis of AD, first postulated in the early 1990s, incorporates histopathological, genetic and biochemical information. It posits that the deposition of Ab in the brain parenchyma, due to an imbalance between the production and clearance of Ab initiates a sequence of events that ultimately lead to AD dementia [1 5]. The accumulation of Ab in the brain is thus the primary force driving the AD pathogenesis. The hypothesis is not simply an academic construct and will only have been useful if it leads to effective clinical management of the disease. Against this background, it is essential to reach a conclusion as soon as possible as to whether the results of clinical trials reported to date support or refute this hypothesis, or are inconclusive. A number of anti-ab strategies are discussed below. Semagacestat and avagacestat Semagacestat and avagacestat are c-secretase inhibitors. c-secretase has more than a hundred substrates including Notch, and we now know that 296 ª 2014 The Association for the Publication of the Journal of Internal Medicine

2 there is almost no selectivity of these drugs for different substrates. Therefore, the fact that cognitive decline was worse in the treatment arm, compared with placebo, in these trials is difficult to interpret at present [6 8]. Bapineuzumab and solanezumab The antibodies bapineuzumab and solanezumab target different epitopes at Ab. Bapineuzumab is a humanized monoclonal IgG1 antibody against the Ab N-terminus (Ab1 5), based on the murine antibody 3D6, which was intended to promote Ab clearance from the brain [9]. Solanezumab, on the other hand, is a humanized version of the mouse monoclonal antibody m266, raised against Ab13 28 [10, 11]. It differs from bapineuzumab in several ways. First, it recognizes various N-terminal truncated species (such as Ab4 42) that are known to exist alongside full-length Ab1 42 in AD plaques [12]. Secondly, whereas bapineuzumab binds amyloid plaques more strongly than soluble Ab, solanezumab selectively binds to soluble Ab with little or no affinity for the fibrillar form [13]. Thirdly, in small Phase I and Phase II studies [11], there was no clinical, cerebrospinal fluid (CSF) biomarker or imaging evidence of meningoencephalitis or vasogenic oedema, the latter of which has been a problem with bapineuzumab. Bapineuzumab had no clinical efficacy [9]. However, it was associated with reduced amyloidbinding as evidenced by Pittsburgh Compound B and decreased CSF tau levels suggesting that there was some reduction in neuronal damage, which was not clinically useful, at least in the Phase III trial that was recently stopped. However, it is important to note that 38% of enrolled patients did not fulfil criteria for AD. Likewise, in the overall analysis, solanezumab did not have an effect on clinical outcome. However, in the group of patients with mild AD, there was a small reduction in clinical worsening but this was not associated with a reduction in CSF tau levels. Taken together, the two antibody studies produced inconclusive results, which makes it difficult to draw any firm conclusions on whether or not the approach will be effective. Other issues concerning these and future trials As others have noted, all these trials illustrate the difficulties in testing the amyloid hypothesis [7, 14]. A consensus is emerging that drug candidates need to be administered early in the disease process in drug-na ıve patients with AD, and possibly in carriers of amyloid precursor protein (APP) and PSEN1 and PSEN2 mutations. However, achieving the goals set out by this consensus is extremely challenging from an organizational and ethical perspective. APP and PSEN1 and PSEN2 mutation carriers are rare, and apart from the extended presenilin 1, pedigree in Colombia are under the care of a large number of clinical centres each of which has few patients. Many mutation carriers do not want to know their mutation status, complicating their involvement in clinical trials. Furthermore, these trials require extended clinical follow-up and sufficient patient numbers with high enough conversion rates. Despite these difficulties, randomized controlled trials on AD prevention (e.g. ADCS-A4, API and DIAN) are already underway, as discussed in other articles of this themed issue. Recent genetic data and the implications for pathways to AD A summary of all the currently known genetic loci for AD is shown in Fig. 1. APP and presenilin variability The role of APP and presenilin mutations in causing early AD has long been recognized. However, genetic analysis of late-onset AD has surprisingly revealed that these mutations are also pathogenic factors in some cases [15, 16]. Indeed, given the prevalence of late-onset disease, it is likely that there are more cases of disease with ages over 60 years who have APP and PSEN1 and PSEN2 Causes Alzheimer s disease Risk of Alzheimer s disease High Medium Low PSEN1 PSEN2 APP Very rare TREM2 APOE4 APOE4 Frequency in the population MS4A CR1 PICALM BIN1 CD2AP EPHA1 CLU ABCA7 CD33 Very common Fig. 1 Risk loci for Alzheimer s disease. Adapted from Manolio et al. [20]. ª 2014 The Association for the Publication of the Journal of Internal Medicine 297

3 mutations than early-onset disease, although they still account for <1% of cases. The Swedish APP (N/L670/671K/M) mutation causes AD because it is a better substrate for b-secretase and thus more APP is metabolized along the amyloidogenic pathway [5]. Recently, Jonsson and colleagues [17] noted that a specific mutation in APP, which was previously found to be located close to the beta-secretase site [16, 18], made it a poorer substrate for BACE1, and accordingly, it was associated with a lower risk of developing AD. This is a remarkable observation which, at least anecdotally, has been replicated [19], implying that a lifelong rather modest reduction in flux through the amyloidogenic pathway is associated with a reduction in the risk of developing AD. Clearly, this is seen as prima facie support for BACE1 inhibition as a valid target for AD therapy and is strong evidence for the amyloid hypothesis. Genome-wide association studies Genome-wide association studies identify common loci (typically frequencies of 10 50%), which have modest effects on risk (typically with odds ratios in the range of ). Over the last 5 years, this approach has begun to yield large numbers of risk loci, and this harvest continues as study results are pooled and as more samples are collected [20 23]. The utility of these studies in terms of predicting who will develop disease is modest but real. However, of importance, they identify pathways and processes in which genetic variability affects disease risk. In broad terms, these studies have identified three such pathways: (i) endosomal vesicle recycling, (ii) cholesterol metabolism and (iii) the innate immune system [24]. It is not yet possible to definitively relate these pathways directly either to each other or to APP but some suggestions are emerging, and they are all potential targets for therapy. Endosomal vesicle recycling Several genes apparently involved in endosomal vesicle recycling have been identified in genomewide association studies. These include bridging integrator 1 (BIN1), phosphatidylinositol-binding clathrin assembly protein (PICALM) and sortilinrelated receptor 1 (SORL1). SORL1 was identified, at least in part, through its role in APP metabolism [25], some of which takes place in the endosomal pathway. Thus, it seems plausible that at least some of the genetic variability in this pathway contributes through variability in APP metabolism. It is notable that these loci, in contrast to the others, do not encode genes whose expression is altered in APP transgenic mice [26]. This suggests that genetic variability at these loci causes actions upstream of amyloid deposition, in contrast to the other types of pathways. Cholesterol metabolism Considerable evidence from genetic, epidemiological or basic research studies suggests a pathogenic link between the dysregulation of cholesterol homeostasis and AD. The strongest known risk factor influencing the incidence of sporadic AD is the presence of the E4 allele of the apolipoprotein E (ApoE) gene. ApoE is the major carrier of cholesterol in the central nervous system. Individuals with one or two copies of the apoe4 allele have a higher risk of developing AD, compared with carriers of other isoforms [27]. ApoE has an important role in Ab metabolism, aggregation and deposition. Increased plaque deposition has been observed in apoe4 carriers and in animal models of brain amyloidosis carrying the human apoe4 allele [28 30]. ApoE isoforms have different abilities to transport cholesterol, with apoe4 being the least potent. Moreover, reduced levels of total ApoE have been seen in the brain and CSF of apoe4 carriers and in apoe4 transgenic animals [31]. The detrimental effect of apoe4 has being shown to be enhanced by environmental risk factors for AD, both in humans [32] and in animal models [33]. A reduced capacity of apoe4 for neuronal delivery of cholesterol is believed to affect synaptogenesis and repair mechanisms, and has been proposed to directly contribute to disease progression. The interplay between brain cholesterol metabolism and Ab formation has long been recognized. In vitro studies have shown that intracellular cholesterol levels can modulate Ab generation [34] and also that Ab may directly regulate cholesterol metabolism in the brain [35]. ApoE also seems to regulate Ab aggregation and deposition, although this regulation remains poorly understood. Studies in microglia have shown that ApoE3 cells are more efficient in degrading Ab compared with ApoE4 cells [36]. Moreover, ApoE4 binds Ab with lower affinity than the E3 allele, suggesting that ApoE4 might be less efficient in mediating Ab clearance 298 ª 2014 The Association for the Publication of the Journal of Internal Medicine

4 Table 1 Currently known loci for Alzheimer s disease from genome-wide association studies Gene Odds ratio Function Apolipoprotein E >3.0 Lipid metabolism Clusterin 1.4 Lipid metabolism, innate immunity PICALM 1.3 Endosomal vesicle recycling ABCA7 1.3 Lipid metabolism CR1 1.3 Innate immunity BIN1 1.2 Endosomal vesicle recycling MS4A 1.2 Innate immunity TREM2 3.0 Innate immunity All the above loci were found by genome-wide association studies, except TREM2 that was found by exome sequencing. [37]. Finally, the findings of recent studies suggest that ApoE may also affect AD risk by immunomodulation, which may make individuals more or less vulnerable to build-up of amyloid in the brain [38, 39]. Recent APP transgenic mouse data have clearly shown that experimental manipulation of brain cholesterol metabolism has therapeutic potential [40]. Recently, a unique aspect in the relationship between APP and cholesterol metabolism has emerged. Cholesterol forms a complex with the Ab domain of APP, suggesting that strategies aimed at preventing the binding of cholesterol to APP may have therapeutic potential in AD [41]. Moreover, a novel role of APP in neurons as been identified in controlling cholesterol biosynthesis and turnover, indicating for the importance of synaptic function in damage repair [42] Table 1. Indeed, the findings of genetic studies have implicated several genes related to cholesterol synthesis, transport, uptake or metabolism (i.e. APOE, ABCA7, ABCA1, CLU and CYP46A1) in AD [24, 43, 44]. This evidence suggests that altered cholesterol levels, transport and cellular distribution are intimately linked to amyloid accumulation and AD pathogenesis. However, a complete understanding of the exact underlying mechanisms is still lacking. The links between cholesterol and AD pathogenesis are summarized in Fig. 2. APP Chl synthesis Chl turnover CLU, SORL1, LRP1, ABCA1, ABCA7, Cyp46 Chl transport Chl metabolism Unbalanced neuronal Chl Decreased synaptogenesis and repair ApoE4 Decreased Chl transport Decreased ApoE levels Decreased Aβ clearance Enhanced Aβ levels Fig. 2 Cholesterol-related pathways to Alzheimer disease (AD). Evidence supporting that altered cholesterol levels, transport and cellular distribution contribute to amyloid accumulation and AD pathogenesis: (i) The interplay between brain cholesterol homeostasis and Ab formation. (ii) Novel findings from GWAS on genes related to cholesterol metabolism (APOE, ABCA7, ABCA1, CLU, CYP46A1) in AD. (iii) The lack of function of apoe4 in transporting cholesterol to neurons and in clearing Ab from the brain. An unbalanced neuronal cholesterol homeostasis would lead to decreased synaptogenesis and repair mechanisms. The innate immune system Neuropathological analysis of AD brains has long indicated a role of the complement cascade in the pathogenesis of AD [45] and indeed Ab has been shown to activate the complement cascade [46]. Morphological characteristics of an inflammatory response include a clustering of activated microglia and complement factors within amyloid plaques. Fibrillary amyloid can bind complement factor C1 and activate the classical complement pathway without involvement of antibodies [47, 48]. Inflammatory proteins contribute to pathological processes in the AD brain, including amyloid homeostasis, gliosis and phosphorylation of tau. Inflammation may have both beneficial effects, such as phagocytosis, and detrimental effects, such as off-target actions on neighbouring cells. Inflammation is a relatively early pathogenic event, which was revealed by neuroradiological and neuropathological observations in patients with mild cognitive impairment [49, 50]. Astroglia and microglia both play a crucial role in innate immunity and use Toll receptors to recognize fibrillar amyloid. Both types of receptors, Toll-like and CD14, are innate receptors that mediate activation of transcription factors and production of pro-inflammatory cytokines. Stimulation of the innate immune system reduces parenchymal and vascular amyloid. The innate ª 2014 The Association for the Publication of the Journal of Internal Medicine 299

5 immune system can be activated by interaction of amyloid and LDL in both AD and atherosclerosis; the latter is a major risk factor for late-onset AD. Thus, activated microglia can be, at the same time, in two functionally different activated states either as pro-inflammatory (detrimental) or involved in repair of the brain. Increased CSF levels of complement factors and microglial activation markers have been described in AD [51 53] but longitudinal data to determine the sequence of these alterations in relation to the onset of amyloid deposition and neurotoxicity markers are lacking. On the other hand, genetic analysis has now clearly shown that variability in innate immunity is an important determinant of risk of AD with several loci, for example the complement receptor 1 (CR1), clusterin and TREM2, mapping to this system [24]. Exome sequencing Exome sequencing has the potential to identify high-risk loci for disease. The expense and analytical burdens that these large-scale sequencing projects entail have only recently begun to be overcome and the first data from what will undoubtedly be a large number of projects are starting to be reported. Rare, heterozygous loss of function mutations in the TREM2 gene have very recently been found to increase the risk of AD by up to fivefold [26, 54]. TREM2 has being shown to suppress the inflammatory response by inhibiting cytokine production and secretion in microglia [55]. TREM2 is also involved in the regulation of phagocytic pathways that are responsible for the removal of cell debris and possibly of amyloid. Thus, a lack of function of TREM2 could result in increased systemic inflammation, damage of neurons and accumulation of amyloid. The findings of these studies, and of those mentioned above, point to the microglial activation state as a key determinant of the pathogenesis of AD. Recently, Pottier and colleagues [56] found a high frequency of potentially pathogenic SORL1 mutations in autosomal dominant early-onset AD. SORL1 is an important mediator of APP localization and its access to secretases, and was previously linked to the disease; in addition, SORL1 was found to be a risk gene in genome-wide association studies. Moreover, a mutation in NOTCH3 (p.r1231c), previously found to cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy was observed in a Turkish family with AD [57], indicating that this method could contribute to the differential diagnosis. These new findings indicate the presence of genetic heterogeneity in AD that involves other genes besides the Mendelian forms of APP and presenilin genes. Exome sequencing will allow the identification of unexpected genetic causes, clinical phenotypes and therefore of unknown pathways contributing to the disease pathogenesis. An attempt at synthesizing the genetic data into a model for disease It is clearly too early to attempt to construct a scheme that explains all the genetic loci for AD, but there is certainly some predictability in the identified genes. We suggest that Ab deposition remains central to the disease process. Endosomal processing of APP is a determinant of this deposition process. Ab deposition is modulated by ApoE by mechanisms that remain unclear. Microglia respond to Ab deposition, at first by phagocytosis of amyloid deposits, but eventually microglial activation switches to an inflammatory phenotype with abundant cytokine production, which is both neurotoxic and leads to progression of the clinical disease process (Fig. 3). Tangle pathology is downstream of this process as illustrated by the fact that tau mutations cause tangle pathology in the absence of amyloid pathology in frontotemporal dementia as well as by the finding that APP transgenes potentiate tangle pathology in MAPT transgenic mice (reviewed in [5]) Of note, it has proven very difficult to find a direct biochemical link between Ab and tau pathology, and these recent findings implicating the innate immune system may suggest that the link is not direct and cell autonomous but instead is mediated through an inflammatory process. It is also clear that the recent data showing that Ab and tangle pathology can spread independently of each other, by what we presume is a template mechanism, is an important consideration with respect to the relationship between these pathologies [58]. A testable, detailed prediction of this model is that biomarkers of imbalanced endosomal processing of APP would be the first to indicate increased risk of disease, followed by a drop in CSF Ab42 levels and an increase in amyloid positron emission 300 ª 2014 The Association for the Publication of the Journal of Internal Medicine

6 Phagocytic response Aβ Complement activation CR1 CLU Acts as a lock on microglial inflammatory responses, promoting protective functions TREM2 Promotes protective functions CR1 CLU TREM1 Low cytokine and neurotrophin production Phagocytosis Inflammatory activation Neuron Survival and repair Aβ deposition, plaque formation and toxicity Apoptotic bodies and aids repair Microglia Inflammatory Response Aβ Complement activation CR1 CLU TREM2 Promotes protective functions CR1 CLU TREM1 Cytokine production e.g. TNFa secreation Phagocytosis Inflammatory activation Fig. 3 Effects of Ab on microglia activation and the effects of genetic variability in CLU, CR1 and TREM2. Neuron Neurodegeneration Aβ build-up, plaque formation and toxicity Apoptotic cell bodies and hinders repair Microglia tomography (PET) signal in individuals who are truly progressing towards AD. This latter change may be linked to ApoE or other cholesterol-related factors. The importance of cholesterol for vesicle formation and Ab formation is clear. Moreover, new data showing that APP controls cholesterol synthesis and turnover further strengthen a molecular link between AD and cholesterol. Although some controversies remain, all results indicate that there are clear relationships between dysregulation of cholesterol metabolism or transport and both cognitive dysfunction and AD pathology. Brain amyloid pathology is initially relatively nontoxic, as evidenced by normal CSF tau levels, absence of brain atrophy on imaging and normal cognition but, eventually, through unknown mechanisms, Ab aggregates over-activate microglia, which mediate neurotoxicity through cytokine production, release of proteases and activation of complement proteins. Microglial over-activation (the inflammatory M1 phenotype) should be detectable using PET ligands and/or microglial activation markers in CSF, and would according to this model result in neurotoxicity, indicated by ª 2014 The Association for the Publication of the Journal of Internal Medicine 301

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