PHAGO

Over 46 million people live with dementia worldwide and this number is estimated to increase to more than 100 million by 2050. It’s known that Alzheimer’s disease – the major cause of dementia – is associated with a progressive loss of neurons and their connectivity in the brain, triggered by the formation of amyloid plaques. The immune cells of the brain, known as microglia, attack the amyloid plaques – but over time, the microglia themselves start to behave abnormally and can attack healthy cells.

There are several genes known to be associated with Alzheimer’s disease – two of which are the TREM2 and CD33/SIGLEC33 genes. Before the PHAGO project launched in 2016, little was known about how these genes contribute to the progression of Alzheimer’s disease. PHAGO set out to close this knowledge gap, investigating how these genes function in the brain and contribute to the progression of Alzheimer’s, as well as developing a series of tools that will support future therapy development.

Unlocking the complexity of the brain

PHAGO unveiled a slew of new discoveries relating to TREM2 and CD33/SIGLEC33, but overall what they found was that the TREM2 and CD33/SIGLEC33 interactions were far more complex than anticipated, meaning that we are further away from developing a therapy targeting either TREM2 or CD33/SIGLEC33 than previously thought.

The TREM2 gene codes for the TREM2 protein, which activates immune cells like microglia in the brain after sensing potential threats. The CD33/SIGLEC33 gene codes for a protein that counter-regulates TREM2 signalling, suppressing immune cells like microglia when the conditions are right.

One key finding was that the signalling of TREM2 to activate microglia is necessary to combat disease-causing pathogens in patients with Alzheimer’s disease. This means that a functioning TREM2 receptor is essential because it contributes to the removal of amyloid plaques. However, over time activated microglia may begin to attack healthy synapses and neurons, leading to further deterioration.

People with Alzheimer’s disease and a faulty TREM2 gene are less likely to be able to remove plaques, causing the disease to progress more rapidly. If the TREM2 protein is not activated in the correct way, the microglia may continue to attack synapses and neurons even within a healthy person.

Another important finding was that the extracellular part of the TREM2 protein is cleaved and shed. This shedding impacts the microglia's capacity to remove plaques. It was also found that the shed TREM2 blocks the aggregation of beta-amyloid, the protein which clumps together to form amyloid plaques, which lead to Alzheimer’s disease. This may be another possible beneficial function. Deutsches Zentrum fur Neurodegenerative Erkrankungen EV, a German public research organization involved in the project, now holds a patent to target the TREM2 cleavage site, which might be used in future drug development.

The TREM2 shedding mechanism discovery was utilised to generate a highly sensitive assay using the shed soluble TREM2 as a novel biomarker. As Alzheimer’s disease develops, TREM2 sheds in order to attack amyloid plaques. This leads to an increase in TREM2 in the cerebrospinal fluid, which can be detected and is a very early indicator of the presence of an inflammatory disease in the brain, such as Alzheimer’s disease.

Antibodies to TREM2 and/or CD33 were established and analysed and continue to be investigated by the industrial partners. On a more basic level, in a small drug library screen with a TREM2 reporter cell line, the chemical amphotericin B was identified as an activator of TREM2 receptor or signalling pathway.

A toolbox to deepen understanding of signalling pathways in the brain

The project successfully developed a series of tools – including animal models, in vitro assays, induced pluripotent stem cell (iPSC) lines and AI-based simulations – which could be used by industry and academia alike to identify possible treatments for Alzheimer’s disease.

By inserting human TREM2 and CD33/SIGLE33 gene variants into mice, the project developed several new mouse models for further analysis of these signalling pathways. Although there are increasing attempts to reduce animal use in science, transgenic mouse models remain an important tool and are particularly needed for therapy development, since they yield relevant translatable results.

Before the PHAGO project, it was very difficult to get high yield, high quality microglial cell cultures derived from induced pluripotent stem cells (iPSC). Led by LIFE & BRAIN GmbH, a German-based SME, the project developed a new, effective method for deriving high yields of microglial cells from iPSCs. The company patented this methodology.

More than 40 induced pluripotent stem cell lines exhibiting different variants of the TREM2 and CD33/SIGLEC33 genes were developed by the project. Cell lines are stable populations of cells that can be maintained in culture for an extended period of time, making them perfect tools for carrying out reproducible in vitro studies. In this case, the researchers developed cell lines of microglial cells that enabled them to examine the processes underlying the function and activity of the TREM2 and CD33/SIGLEC33 genes.

Bioinformaticians within the PHAGO consortium used RNA sequencing data to exploit the genetic profiles that are related to neurodegenerative diseases. They then developed algorithms to make predictions about how certain drugs might affect these genetic profiles and pathways. These types of studies can be used as a first test for how we might expect drugs to work, prior to carrying out additional in vitro and animal studies. In this way, these computational models can help the pharmaceutical industry to find potential drug candidates at a reduced cost.

Paving the way towards a therapy for Alzheimer’s disease

Although the PHAGO project discovered that the TREM2 and CD33/SIGLE33 signalling pathways were far more complex than previously imagined, the strides that have been made by the project have significantly increased the knowledge of these two genes and their role as well as the role of their signalling pathways in Alzheimer’s disease. Moreover, the tools developed by the project will help researchers to deepen our understanding of these mechanisms. More than forty peer-reviewed publications were produced by the project with upwards of 3000 citations. Two patents were also secured. The TREM2 and CD33 variant iPSC lines generated by the PHAGO consortium have been deposited in the stem cell bank of EBISC, to allow easy availability of these lines to the scientific community.