Microglia are the resident immune cells in the brain – they maintain law and order when trouble kicks off. And when things get really bad, these cells change shape, become “activated”, and start to absorb toxins, debris and anything else that they feel should not be there – via a process called phagocytosis.
And they are ruthless in this task.
When we are young, these cells function very well at maintaining a general sense of ‘homeostasis‘ (or stable equilibrium). But as we age,… well, let’s just say things start to slip a little.
Recently a group of researchers at Stanford University have discovered by inhibiting a single protein, called CD22, they can restore microglial homeostasis in the ageing brain, and this had beneficial effects in a model of Parkinson’s.
In today’s post, we will look at what microglia are, what phagocytosis is, and what these new CD22 results could mean for Parkinson’s.
My father often says: Ageing is not for sissies.
And as the birthdays have started to mount up, I’ve come to better understand what he means.
There are days when I feel like an old man trapped in a 27 year old’s body. For the record, I’m 27. And for the record, I’m going to be 27 until I die (27 was a great year!).
An amazing journey. Source: Topsimages
While some are able (and foolishly gleeful) to avoid taxes, until recently no one has been able to escape the rentless march of ageing. Until recently, the vast majority of us have been resigned to our fates. And until recently, the fountain of youth has only existed in the realm of the Hollywood movies.
The force is strong with this one. Source: Reddit
Recently there has been an enormous amount of research focused on stopping ageing and preventing death (both of which are being viewed as “curable diseases” – click here to read more about this). Now to be honest, much of this is still quackery.
But there does seem to be progress being made in the biology of extending ‘healthspan’ (as opposed to lifespan).
And some of that research could have implications for Parkinson’s.
Well, for example, this research report was recently published:
Title: CD22 blockade restores homeostatic microglial phagocytosis in ageing brains.
Authors: Pluvinage JV, Haney MS, Smith BAH, Sun J, Iram T, Bonanno L, Li L, Lee DP, Morgens DW, Yang AC, Shuken SR, Gate D, Scott M, Khatri P, Luo J, Bertozzi CR, Bassik MC, Wyss-Coray T.
Journal: Nature. 2019 Apr;568(7751):187-192.
In this study, the researchers were interested in the maintanence of homeostasis (or stable equilibrium) in the brain by a group of cells called microglia.
What are microglia?
Microglia are some of the helper cells in the brain. Specifically, they act as the resident immune cells. When infection or damage occurs, the microglia become ‘activated’ and start cleaning up the area.
Different types of cells in the brain. Source: Dreamstime
When they become activated, microglia do generally do three things:
1. They change shape – microglia usually have outstretched branches when they are in their ‘resting’ state. These branches are constantly monitoring the surrounding environment for any signs of trouble. But when trouble appears, microglia will become activated and retract their branches, giving them a more sperical appearance.
2. They release cytotoxic proteins – these toxic proteins encourage a wounded or sick cell to die, helping the microglia to determine which cells are too sick/damage to survive and need to be removed.
3. They start to be very active with regards to phagocytosis.
What is phagocytosis?
Phagocytosis comes from Ancient Greek φαγεῖν (phagein) , meaning ‘to eat’, and κύτος, (kytos) , meaning ‘cell’. It is used in biology to refer to the process of engulfing or consuming objects.
A schematic of a macrophage. Source: Meducator
Microglial phagocytosis is a process by which dying cells/debris/rubbish can be vaccumed up, broken down and disposed of (Click here to read a good review of microglia-based phagocytosis in the context of Parkinson’s). By doing this amazing clean up job, microglia are able to help maintain ‘homeostasis’ in the brain.
When we are young, microglia manage this task very well.
But as we get older, microglia become less robust in their ability to phagocytose. And as a result, homeostasis becomes impaired. And many researchers believe that this reduced ability to maintain homeostasis could be influential in neurodegenerative conditions, like Parkinson’s.
Interesting. So what did the researchers report in their new study?
The researchers were interested in determining the mechanisms by which microglial homeostatic function becomes impaired over time.
So they conducted a CRISPR-based screening experiment.
What is a CRISPR-based screen?
There is a revolution going on in biological research at the moment.
Everyone is adopting a new technique called CRISPR to help identify proteins involved in particular cellular pathways.
DNA editing using CRISPR technology. Source: EIC
I have previously written a long post explaining the science behind CRISPR (Click here to read that post).
But briefly, Clustered Regularly Interspaced Short Palindromic Repeats (or CRISPR) are a series of repetitive sequences (exact repeats) of DNA that were found in particular bacteria, which form a frontline defensive mechanism against infection from nasty viruses.
During the infection of a bacteria, a phage (a virus that infects bacteria) will inject its DNA into the cell. This DNA will be recognised as foreign DNA by two proteins (these are called Cas1 and Cas2), and they will chop it up into small pieces which will then be inserted into the bacterial DNA (a region referred to as a CRISPR locus). And once it is embedded in the DNA, this CRISPR locus can be passed on to any further cells, if that bacteria decides to divide.
Now these CRISPR loci are important as they will be used to defend the bacteria (and its prodgny) against future viral infections.
CRISPR regions of the DNA are regularly being transcribed (that is the process of producing RNA). The RNA from CRISPR loci are called crRNA (CRISPR-RNA), and they are basically small RNA molecules whose sequence matches a region of phage DNA. This crRNA then attaches to another piece of RNA to become what is called a tracrRNA.
This tracrRNA then joins the Cas9 protein and together they start wandering around the bacteria looking for sections of viral DNA which match that particular crRNA. Like a guard wandering around on patrol duty, looking to see if a particular inmate is on the loose at a prison.
How CRISPR works in bacteria. Source: Sciencedirect
Great but what does this have to do with humans?
Well, researchers have adapted this bacterial defense mechanism for use in mammalian cells. By designing crRNAs, scientists are using CRISPR-Cas9 to target sections of mammalian DNA for genetic editing.
Hang on a second. What is Cas9?
Cas9 (or CRISPR associated protein 9) is the important piece of the puzzle. It is the protein that does the magic.
It is an endonuclease.
What is an endonuclease?
Replication of DNA is never 100% perfect and there are occasionally errors. Millions of years of evolution has given rise to a very sophisticated, but extremely efficient system of DNA monitoring within cells. Many different proteins are involved in this process, and endonucleases play a key role.
An endonucleases is an enzyme that directly binds to DNA and cuts it.
So Cas9 is an enzyme that can cut DNA?
Exactly. And it does this in a very targeted manner, being guided by pieces of RNA (the crRNA mentioned above) that are produced from the CRISPR regions of bacterial DNA. The crRNA is now often referred to as the ‘guide RNA’ (or gRNA – there are too many different types of RNA now, I know).
Cas9 protein interacting with DNA. Source: Stackexchange
Using the CRISPR-Cas9 technology, researchers can now cut out sections of DNA and replace them with new section – opening up the potential for a new field of genetic medicine.
They can also use this approach in research. For example, by mutating specific genes, researchers can investigate what function that region of DNA has.
And this research application can be taken one step further: by randomly mutating one gene per cell, scientists can conduct ‘CRISPR-based screening’ experiments to identify lists of genes involved in a particular function (such as phagocytosis).
Sounds interesting, but how was this applied to microglia and phagocytosis?
Well, the researchers conducted a ‘CRISPR-based screening’ experiment to identify a list of genes involved in phagocytosis.
They inserted the Cas9 protein into the microglia they were growing in cell culture. Then they infected the cells with a large pool of viruses – the viruses were identical except for one critical difference: each virus had a single guideRNA. The investigators then tested the cells for their ability to phagocytose. By sequencing the DNA of the cells after this experiment, they were able to reveal the genes that regulate phagocytosis.
By randomly mutating an individual gene in each microglial cell, the researchers were able to identify 286 genes that were strong negative or positive regulators of phagocytosis.
Next they looked to see which of these genes become more active in aged microglia cells.
And this was how they found CD22.
What is CD22?
Cluster of differentiation-22 (or CD22) is a transmembrane protein.
What does that mean?
Transmembrane proteins are proteins that spans the entire wall of a cell (or the membrane of the cell). They are permanently attached to the membrane, and can come in different formations.
Ok, and what does CD22 do?
CD22 acts as an inhibitor of receptor for B-cell receptor (BCR) signaling.
B-cells are bone marrow-derived cells which produce antibodies that are used to attack invading pathogens (such as viruses). The ‘B’ actually comes from the name of the place they were discovered, the Bursa of Fabricus. The Bursa is an organ only found in birds.
B-cells do not actually kill pathogens, they just spend their short life producing antibodies which trap and neutralise them.
The BCR controls the activation of B-cell. CD22 reduces this activation, by inhibiting BCR signalling.
I see. So what did the microglia researchers find?
The researchers found that as microglia cells get older, CD22 becomes more active. Given that their CRISPR screen inidicated that CD22 is a negative regulator of phagocytosis, the investigators asked themselves if perhaps CD22 has a role in age-related impairment of homeostasis.
To test this idea, the researchers injected mice with some brain matter waste (myelin debris) and then either a CD22 blocking antibody or a control antibody.
What is a blocking antibody?
Antibodies are Y-shaped proteins that the immune system naturally and continuously produces to identify anything in the body that is ‘not self’ (that is, not a normally occurring part of you – think of viruses, bacteria, etc).
Monoclonal antibodies. Source: Astrazeneca
Antibodies act like alert flags for the immune system.
When antibodies bind to something, they alert the immune system to investigate and potentially remove. Each antibody targets a very specific structure (called an antigen), while ignoring everything else. An antigen is defined as any substance or molecule that is capable of causing an immune response in an organism.
When a pathogen (an agent that causes disease or damage) is detected in your body, it will quickly be determined to be not ‘self’. This judgement will be made by the identification of antigens on the surface of the pathogen. If an molecule on the surface of the pathogen is not familiar to the immune system, it will be considered an antigen and an immune response will be initiated to have the pathogen removed.
In this fashion, antibodies are a very powerful method of removing items from the body that are causing trouble or not wanted.
Antibodies binding to a virus. Source: Biology-questions-and-answers
Ok, but what is meant by a “blocking” antibody?
Given that antibodies are so specific for their targets, scientists have designed antibodies that can bind to a protein and block the function of that protein without alerting the immune system. Antbiodies can block function in two general ways:
- By binding to the ligand (the protein that activates the receptor)
- By binding to the receptor
Ok, so what happened when the researchers injected a CD22 blocking antibody into the mice?
To test if CD22 has a role in age-related impairment of homeostasis, the researchers injected mice with some brain matter waste (myelin debris) and then either a CD22 blocking antibody or a control antibody.
After 48 hours, the mice injected with the CD22 blocking antibody had less debris compared to the control mice. This result suggested to the researchers that blocking CD22 on microglia increased their ability to phagocytose. Note: the investigators also reported that microglia appear to be the only cells in the brain that produce CD22 protein, so the researchers concluded that this effect was specific to microglia.
Next, they tested the CD22 blocking antibody on mouse models of Parkinson’s and Alzheimer’s. The researchers found that the antibody treatment resulted in significantly more clearance of the accumulation of extracellular aggregated proteins (alpha synuclein and beta amyloid) than in control treatment animals after 48 hours.
The CD22 blocking antibody was really good at helping microglia to remove the troublesome Parkinson’s associated protein, alpha synuclein.
48 hours? That is a rather short term experiment. What happens in a long term treatment of CD22 blocking antibody scenario?
The researchers tested this next. They treated mice for one month continuously with the CD24 blocking antibody and they found that the microglia in the brains of aged treated mice appeared to be younger and healthier (based on the genes that were active in them) than untreated aged mice.
And as one final experiment, the investigators generated some mice without the CD22 gene, and they found these mice to be better in various cognitive tests.
Wow! Are there any clinically available CD22 antibodies?
Not that I am aware of.
There was, however, a CD22 antibody called Epratuzumab which was being developed by a biotech firm called Immunomedics.
Not the past tense of the sentence above.
Epratuzumab was being developed for autoimmune conditions. It was a humanised monoclonal antibody that targeted CD22, which in the peripheral immune system is found on white blood cells (called B-lymphocytes).
In 2015, two critical Phase III clinical trials (EMBODY1/2) of Epratuzumab in systemic lupus erythematosus (an autoimmune condition) did not meet their primary endpoints. Soon after this, a partnership with pharma company UCB was terminated, slowing any further development of the drug (Source), and it no longer appears on the Immunomedics website (Click here to see their pipeline).
Now it is not clear that Epratuzumab can access the brain (getting past the blood brain barrier), but I am assuming that the researchers behind today’s reviewed report will be exploring methods of blocking CD22 on microglia in the brain.
So what does it all mean?
Researchers have recently published a study suggesting that blocking a protein called CD22 can help to restore impaired homeostatic functions in microglia in the aging brain. The treatment literally returned microglia to a more youthful state. Some readers may be excitedly trying to download DIY instructions for making CD22 blocking antibodies in their kitchen sink with the goal of living forever, but it will be important to see these new results independently replicated before we get too carried away.
If even if the results are varified, translation of this research may be problematic. It will firstly be important to determine whether the results found in mice are the same in humans – there are large differences between the two immune systems (Click here to read more about this).
Plus, given that B-lymphocytes in the blood also have CD22, getting enough of the blocking antibodies into the brain will be tricky as the bulk will be bound in the blood. We can be sure that the researchers behind today’s report will be working on this issue, however, and it will be interesting to see what kind of a solution they (or others) propose.
The banner for today’s post was sourced from Diacomp