The amyloid-B precursor protein (APP) plays a key role in the development of the amyloid plaques that are the hallmark of Alzheimer’s disease. Now, researchers claim to have identified thousands of genetic variants of the APP gene that codes for the protein in the brains of patients with the most common form of Alzheimer’s disease, known as late-onset or sporadic AD (SAD). The study, reported in the journal Nature in November 2018 (doi.org/10.1038/s41586-018-0718-6), also reveals for the first time how this genetic variation occurs – by a mechanism involving the enzyme reverse transcriptase, the same type of enzyme used by HIV to infect cells.
‘Our findings provide a scientific rationale for immediate clinical evaluations of HIV antiretroviral therapies in people with AD,’ says Jerold Chun, senior VP of Neuroscience Drug Discovery at Sanford Burnham Prebys Medical Discovery Unit (SBP), an idea that the researchers say is supported by the relative absence of proven AD in ageing HIV patients on antiretroviral medication.
The APP gene variants were created by reverse transcription, the researchers note, when RNA acts as a template to form complementary DNA sequences that are then reinserted back into the original genome.
This process of gene recombination – which occurs each time cells divide to make new ones – has not previously been reported in nerve cells (neurons) in the brain but could also help to explain the complexity and diverse functions of our brain cells.
‘Gene recombination was discovered as both a normal process for the brain and one that goes wrong in AD,’ Chun says. Using cutting edge analysis techniques, Chun and his team found that 100% of the AD brain samples contained a disproportionately high number of APP gene variants, compared with healthy brains. Eleven of the observed single-nucleotide changes were already known mutations in a very rare form of early onset or familial Alzheimer’s disease (FAD).
One high accuracy sequencing method, single molecule real time (SMRT) circular consensus sequencing (CCS), identified 6299 unique sequences in the APP gene neurons of SAD brains. ‘The difference with our sequencing approach is that it sequences one gene molecule multiple times, sequencing both strands forward and reverse,’ explains Jonas Korlach, CSO of CA-based Pacific Biosciences. ‘Sequencing single molecules means SMRT-CCS is very sensitive to these diverse products.’
Chun and the team, also involving researchers at the University of California, San Diego, point out that reverse transcriptase is an ‘error-prone’ enzyme that makes lots of mistakes. This explains both the diversity of our brain neurons, they believe, and why copies of the APP gene in AD are ‘not accurate’ – and possibly why many promising drugs targeted at betaamyloid have disappointed in clinical trials.
‘The thousands of APP gene variations in AD provide a possible explanation for the failures of more than 400 clinical trials targeting single forms of beta-amyloid or involved enzymes,’ says Chun. ‘APP gene DRUG DISCOVERY HIV therapy may help AD recombination in AD may be producing many other genotoxic changes as well as disease-related proteins that were therapeutically missed in prior clinical trials. The functions of APP and betaamyloid that are central to the amyloid hypothesis can now be re-evaluated in light of our gene recombination discovery.’
Nearly 6m people in the US have AD, with the number expected to reach 14m by 2060. Genetics is a major factor, with individuals carrying the ApoE4 isoform of human apolipoprotein at greatest risk. ApoE3 is neutral and ApoE2 is protective. Although ApoE4 was previously known to affect the accumulation and clearance of beta-amyloid, new research reported in 2017 by researchers at Stanford University also revealed how it may affect beta-amyloid synthesis. In a paper in Cell (doi: 10.1016/j. cell.2016.12.044), the team demonstrated how ApoE activates a pathway that enhances APP transcription and beta-amyloid synthesis in mice. Activation followed the potency rank order ApoE4>ApoE3>ApoE2, which over a lifetime could add up to a big cumulative risk.
The new research highlights the value of high accuracy sequencing methods, says Korlach. Methods such as SMRT-CCS are more expensive than other ca $1000 whole genome sequencing methods by firms such as Illumina.
‘But while you may save a few thousand dollars on upfront sequencing, it could end up costing millions more in development costs if the drug ends up failing clinical trials,’ he says.