A Pathway Towards Alzheimer’s Disease Treatments: Understanding the Role of ApoE in Human Neuron Physiology

Huang YWA, Zhou B, Wernig M, Sudhof TC (2017). “ApoE2, ApoE3, and ApoE4 Differentially Stimulate APP Transcription and Aβ Secretion.” Cell 168(3): 427-441.

http://www.cell.com/cell/fulltext/S0092-8674(16)31760-3

 More than five million individuals are affected by Alzheimer’s Disease (AD) in the United States. This dementia is the sixth leading cause of death nationwide, and one of every three seniors dies from AD or a related dementia. AD patients progressively worsen in their memory loss, difficulty in performing familiar tasks, disorientation to time or place, and inability to make decisions or solve problems. By 2050, it is estimated that the number of cases could reach 16 million, yet there is no treatment for this condition.

Figure 1: Amyloid-beta plaques in brain tissue from Alzheimer’s Disease patients.

Adapted from: http://bigthink.com/articles/the-brain-plaques-and-tangles-that-cause-alzheimers-disease

Finding a treatment for AD has proven to be very challenging due to its multifactorial nature. Age is the greatest risk factor, combined with important genetic contributions and environmental factors. Research has primarily sought to understand the biology underlying early-onset familial Alzheimer’s, using genes with well-defined autosomal dominant mutations. However, familial early-onset AD occurs in less than ten percent of Alzheimer’s Disease cases. The majority of cases are sporadic, late-onset, and associated with “risk genes”, particularly APOE. APOE encodes for Apolipoprotein E, a protein that regulates cholesterol metabolism. In the brain, it is thought to perform cholesterol-independent functions necessary for neuronal physiology that were previously poorly understood. Humans carry three allelic variants that result in three protein isoforms: ApoE2 is carried by eight percent of the population, and thought to be protective; ApoE3 is found in 78 percent of the population and confers neutral risk; and, 14 percent of the population carries ApoE4, which is thought to promote AD. Among AD patients, 50 to 65 percent carry one copy of ApoE4. Two copies of ApoE4 leads to 20 times the risk of developing AD. Elucidating the underlying mechanism of ApoE signaling in human neurons is critical to understanding AD pathophysiology and identifying possible treatments.

Huang et al. (2017) seek to understand how ApoE regulates neuronal physiology and how it contributes to AD pathogenesis. Traditionally, AD has been studied in a variety of mouse models; however, mice lack the three isoforms of ApoE seen in humans. To be able to compare ApoE2, ApoE3, and ApoE4, the authors utilize induced neurons (iN), which are human neurons trans-differentiated from H1 human embryonic stem cells by inducible expression of the Ngn2 transcription factor (Figure 1A; Zhang et al., 2013). The resulting glutamatergic iN were cultured on murine embryonic fibroblasts (MEFs), a feeder cell layer to support long-term growth, along with ApoE2, ApoE3, or ApoE4 recombinant proteins at a physiological CSF concentration (Figure 1C, 1D; Huang et al., 2017).

A key process in AD is the production of amyloid-beta protein aggregates from the cleavage of amyloid precursor protein (APP). How ApoE isoforms may differentially contribute to the production of APP and of amyloid-beta is currently unknown. The authors first ask whether the three isoforms of ApoE differentially regulate amyloid-beta production. While all three isoforms enhance amyloid-beta production relative to controls, ApoE4 resulted in the highest amount of amyloid-beta while ApoE2 resulted in the lowest amount, both relative to the neutral ApoE3 (Figure 1E). Overall, this correlation supports the idea that ApoE4 may confer the strongest risk to developing AD by increasing the amount of insoluble amyloid-beta protein that aggregates into plaques and fibrils.

How might ApoE actually increase amyloid-beta production? Huang et al. next investigate the effects of ApoE on mRNA and protein levels of APP, the precursor to amyloid-beta  (Figure 4A, 4C). Addition of recombinant ApoE protein isoforms to iN cultures resulted in APP RNA and protein that was greatest in ApoE4-treated cells, followed by ApoE3, and lastly ApoE2. The authors show that adding RAP, a competitive inhibitor of ApoE receptors (ApoER), prevents APP production and suggests that APP expression is dependent on ApoE binding the ApoER (Figure 4A).

By which mechanism might ApoE stimulate APP expression? Huang et al. analyze candidate signaling pathways, and find that ApoE stimulates phosphorylation of the ERK/MAPK pathway, an intracellular molecular signaling pathway. The steps of this pathway are shown in Figure 1 (right). Ultimately, this pathway culminates in APP transcription, driven (in decreasing order) by ApoE4, ApoE3, and ApoE2. The transcription of APP is mediated by the phosphorylation of c-Fos, which activates the AP-1 transcription factor. Overall, these findings provide a mechanistic understanding of downstream ApoE signaling, and how this process is differentially regulated by three ApoE isoforms.

Huang et al. sought to elucidate ApoE signaling in neuronal physiology and how this process might be differentially regulated by ApoE isoforms that confer different levels of risk to developing AD. The authors demonstrate that ApoE acts as a signaling molecule via binding of ApoER, which in turn activates DLK, the gate-keeper of the downstream cascade. In the cytosol, ERK and MAPK are activated and this results in phosphorylation of c-Fos, which stimulates the transcription factor AP-1 to induce expression of APP. This cascade is most activated (in decreasing order) by ApoE4, ApoE3, and ApoE2. Thus, the allele of ApoE that one receives may in fact be a direct driver of AD. While the iN cultures are glia-free, a shortcoming is that the feeder cell layer of MEFs is not from human cells; ideally, the iN would be cultured long-term without the need of a feeder cell layer. The present study is significant as it provides the first ApoE signaling pathway that may explain AD in human neurons, and provides novel pharmacological targets for potential treatments.

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“Alzheimer’s Disease.” Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention, 12 Sept. 2017, www.cdc.gov/chronicdisease/resources/publications/aag/alzheimers.htm.

“Alzheimer’s Disease Genetics Fact Sheet.” National Institute on Aging, 2015, https://www.nia.nih.gov/health/alzheimers-disease-genetics-fact-sheet.

Huang YWA, Zhou B, Wernig M, Sudhof TC (2017). “ApoE2, ApoE3, and ApoE4 Differentially Stimulate APP Transcription and Aβ Secretion.” Cell 168(3): 427-441.

Liu CC, Kanekiyo T, Xu H, Bu G (2013). “Apolipoprotein E and Alzheimer disease: risk, mechanisms, and therapy.” Nat Rev Neurol 9(2): 106-118.