Imipramine and olanzapine block apoE4-catalyzed polymerization of Aβ and show evidence of improving Alzheimer’s disease cognition

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Imipramine and olanzapine block apoE4-catalyzed polymerization of Aβ and show evidence of improving Alzheimer’s disease cognition

• Noah R. Johnson,
• Athena C.-J. Wang,
• Christina Coughlan,
• Stefan Sillau,
• Esteban Lucero,
• Lisa Viltz,
• Neil Markham,
• Cody Allen,
• A. Ranjitha Dhanasekaran,
• Heidi J. Chial &
• Huntington Potter 

Alzheimer's Research & Therapy volume 14, Article number: 88 (2022) Cite this article

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Abstract

Background

The apolipoprotein E (APOE) ε4 allele confers the strongest risk for late-onset Alzheimer’s disease (AD) besides age itself, but the mechanisms underlying this risk are debated. One hypothesis supported by evidence from multiple labs is that apoE4 binds to the amyloid-β (Aβ) peptide and catalyzes its polymerization into neurotoxic oligomers and fibrils. Inhibiting this early step in the amyloid cascade may thereby reduce or prevent neurodegeneration and AD.

Methods

Using a design of experiments (DOE) approach, we developed a high-throughput assay to identify inhibitors of apoE4-catalyzed polymerization of Aβ into oligomers and fibrils. We used it to screen the NIH Clinical Collection of small molecule drugs tested previously in human clinical trials. We then evaluated the efficacy and cytotoxicity of the hit compounds in primary neuron models of apoE4-induced Aβ and phosphorylated tau aggregation. Finally, we performed retrospective analyses of the National Alzheimer’s Coordinating Center (NACC) clinical dataset, using Cox regression and Cox proportional hazards models to determine if the use of two FDA-approved hit compounds was associated with better cognitive scores (Mini-Mental State Exam), or improved AD clinical diagnosis, when compared with other medications of the same clinical indication.

Results

Our high-throughput screen identified eight blood-brain barrier (BBB)-permeable hit compounds that reduced apoE4-catalyzed Aβ oligomer and fibril formation in a dose-dependent manner. Five hit compounds were non-toxic toward cultured neurons and also reduced apoE4-promoted Aβ and tau neuropathology in a dose-dependent manner. Three of the five compounds were determined to be specific inhibitors of apoE4, whereas the other two compounds were Aβ or tau aggregation inhibitors. When prescribed to AD patients for their normal clinical indications, two of the apoE4 inhibitors, imipramine and olanzapine, but not other (non-hit) antipsychotic or antidepressant medications, were associated with improvements in cognition and clinical diagnosis, especially among APOE4 carriers.

Conclusions

The critical test of any proposed AD mechanism is whether it leads to effective treatments. Our high-throughput screen identified two promising FDA-approved drugs, imipramine and olanzapine, which have no structural, functional, or clinical similarities other than their shared ability to inhibit apoE4-catalyzed Aβ polymerization, thus identifying this mechanism as an essential contribution of apoE4 to AD.

Background

Genetic factors can increase the risk for developing AD, in particular in individuals who carry the ε4 allele of the APOE gene [1]. The three common apoE isoforms, apoE2, apoE3, and apoE4, differ by single amino acid substitutions at positions 112 and 158. Of the three common allelic variants of APOE, ε3 is most prevalent, accounting for 70–80% of the total alleles in the human population, followed by ε4, which accounts for 10–15%, and then ε2, which accounts for 5–10% [2]. Carrying one copy of APOE4 more than triples the risk for AD, whereas being homozygous for APOE4 increases the risk by greater than 12-fold [1]. Indeed, despite its low allelic frequency in the general population, approximately 60–65% of individuals with AD carry at least one copy of APOE4 [3]. The onset of AD symptoms occurs earlier in APOE4 carriers than in non-carriers and is accompanied by more severe plaque deposition, intraneuronal Aβ accumulation, cerebral amyloid angiopathy, and BBB dysfunction [1, 4, 5].

Multiple potential mechanisms by which apoE4 increases the risk for AD have been proposed and investigated. For example, apoE, and especially apoE4, binds to Aβ with high affinity and acts as a catalyst to accelerate the rate of Aβ oligomer and fibril formation [6,7,8,9], increase their stability [10, 11], and promote their neurotoxicity [12,13,14]. Consistent with this premise, human apoE4 expressed in mice seeded Aβ aggregation [15], and conversely, knockout of the mouse Apoe gene in transgenic mice expressing human amyloid precursor protein (APP) abolished amyloid fibril and plaque formation and cognitive decline [16, 17]. Furthermore, careful longitudinal evaluation in prodromal AD has revealed that APOE genotype plays the greatest role during the initial seeding stages of Aβ deposition and that APOE4 genotype is strongly associated with increased Aβ oligomer levels in the brain [18,19,20]. Additional contributors to the increased genetic risk of APOE4 in AD may include impaired Aβ clearance, exacerbated oxidative stress and neuroinflammation (reviewed in [21,22,23]), and loss of critical apoE functions. Notably, apoE is found co-deposited in amyloid plaques in the AD brain, suggesting a direct interaction with Aβ [24]. Rare mutations in the Aβ binding domain of apoE markedly reduce the risk for AD in humans [25, 26]. Taken together, substantial evidence supports a role for apoE as an essential molecular chaperone for Aβ aggregation in the brain and suggests that inhibiting this process is a promising therapeutic approach to preventing AD.

ApoE-targeted therapeutics for AD have focused predominantly on modulating the overall levels of apoE or the degree of its lipidation. ApoE depletion in AD mouse models has been accomplished using antisense oligonucleotides, immunotherapies, or tamoxifen-inducible APOE repression, each of which was found to reduce amyloid pathology [27,28,29]. However, given that apoE is expressed throughout the body where it carries out many critical functions, a reduction in total apoE levels is expected to have many undesirable side effects [30]. Thus, focusing on the interaction between apoE and Aβ may yield a more precise therapeutic benefit for AD without interfering with the many beneficial functions of apoE. Small molecule “structure correctors” or gene editing have been used to block the formation of the pathological conformation of apoE4 [31, 32]. Additionally, synthetic peptides or peptoids designed to block the apoE-binding site on Aβ were also found to reduce Aβ aggregation in vitro and in AD mouse models [12, 33, 34]. Although the clinical translatability of these therapies remains to be determined, together, they validate the inhibition of the apoE4-Aβ interaction as a tractable therapeutic approach for AD.

Here, we describe the identification of a set of small molecule drugs that can block the interaction between apoE4 and Aβ. We developed an apoE4-catalyzed Aβ fibrillization assay and employed it for high-throughput screening (HTS) of the National Institutes of Health (NIH) Clinical Collection (NCC) library of small molecules with a history of use in clinical trials, many of which are Food and Drug Administration (FDA)-approved drugs. Repurposing known drugs has numerous benefits, such as the availability of safety and dosing information that allows for faster and more cost-effective clinical testing. Through a series of HTS assays, we identified eight hit compounds that reduced apoE4-catalyzed Aβ fibrillization in a dose-dependent manner. We present evidence that two of those hit compounds — imipramine and olanzapine — reduced Aβ and phosphorylated tau (pTau) neuropathology in cell culture models and, when taken by AD patients for their other normal clinical indications, were associated with improved cognition and greater incidence of receiving an improved clinical diagnosis. Because imipramine and olanzapine are completely different drugs with regard to their structures, designed mechanisms of action, and current approved clinical indications, and their only common link is our discovery of their shared ability to block the apoE4-catalyzed polymerization of Aβ into neurotoxic fibrils, these findings validate this mechanism as an essential contribution of apoE4 to AD.

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