Could controlling the brain’s own cleaning team help?

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Microglia are believed to be the primary driver of many neurological diseases, including Alzheimer’s disease. Roxana Wegner/Getty Images
  • Evidence suggests that microglia – the brain’s primary immune cells – may directly contribute to the development of neurodegenerative diseases such as Alzheimer’s disease (AD).
  • Due to technical challenges, scientists have not been able to decipher the molecular mechanisms underlying microglia activity or function in healthy and diseased brains.
  • Scientists have now developed a new method based on the CRISPR gene-editing tool to identify genes that modulate microglia function.
  • By identifying genes involved in pathogenic states of microglial activityscientists were able to switch genes on and offpaving the way for the development of new therapies for AD.

In a recent study published inNatural neurosciencescientists have revealed a new screening platform for characterizing genes that regulate microglial functions that may contribute to Alzheimer’s disease (AD).

Characterizing the regulatory genes that switch microglia from a healthy to a diseased state, such as in the brains of people with AD and other neurodegenerative diseases, could help develop therapies that target these genes or the proteins encoded by these genes.

“Given that microglia are the gatekeepers of brain homeostasis, identifying the specific drivers that drive neuronal toxicity is important for therapeutic intervention. Our new CRISPR screening platform […] allows us to identify these drivers in a fast and scalable way. We have already discovered drug targets that control microglia states, and the next steps would be to test them in relevant preclinical models.
– Dr. Li Gan, study co-author and neuroscientist at Weill Cornell Medical College, speaking to Medical News Today

AD is the most common form of dementia, accounting for 60-80% of all dementia cases. Despite advances in the understanding of AD, there is a lack of effective treatments for this neurodegenerative disease.

The accumulation of evil bends beta-amyloid protein in clumps or plaques is one of the hallmarks of AD. A considerable amount of research has focused on mutations that lead to the abnormal processing of amyloid-beta protein and, subsequently, its accumulation.

However, treatments targeting the pathways involved in beta-amyloid processing have not been successful.

Additionally, researchers have found that people with Alzheimer’s disease do not show it mutations in genes associated with the accumulation of amyloid protein. In contrast, recent evidence suggests that people with AD often have deficits in the permission or removal of misfolded beta-amyloid.

This may be due to malfunctioning microglia, which are the main immune cells in the brain. One of the functions of microglia includes phagocytosis, a process involving the ingestion of dead cells, pathogens, and misfolded proteins to facilitate their removal.

There is growing evidence that the aptitude microglia to remove beta-amyloid protein may be impaired in AD. Microglia may also contribute to the development of AD by secreting inflammatory proteins and causing excessive neuron shedding and synapsesthe links between neurons that allow them to “communicate”.

In addition to AD, there is evidence to suggest that microglia can also to contribute to the development of other neurodegenerative disorders.

However, the molecular mechanisms underlying the wide range of functions performed by microglia in normal conditions and diseases such as AD are not well understood.

Functional genetic screening is a tool used to identify genes involved in a specific cellular function. Such screens involve inhibiting or activating a specific gene in a cell to assess whether changing expression levels of that gene impacts some function of interest, such as cell proliferation.

In recent years, researchers have adapted the gene-editing tool known as CRISPR-Cas9 to identify genes implicated in various diseases, including cancer. The advantages of the CRISPR screening platform include its higher sensitivity and greater reproducibility than previously used screening methods.

CRISPR-Cas9 is made up of a small piece of RNA called the guide sequence and the Cas9 enzyme. Guide RNA binds to the DNA region of interest, allowing Cas9 to bind and cleave DNA at the targeted site.

In the current study, the researchers used a modified CRISPR-Cas9 system involving a disabled Cas9 (dCas9) enzyme that does not cleave DNA. Besides the deactivated Cas9 enzyme, the modified CRISPR-dCas9 platform also consists of proteins that can up-regulate or down-regulate the gene of interest, or in other words, turn them on and off.

Such CRISPR screens involve the delivery of guide RNA to the cell using a genetically modified virus – a viral vector. However, using viruses to deliver guide RNA to mature microglia has been difficult.

To circumvent these difficulties, the researchers used induced pluripotent stem cells (iPSC). IPSCs are derived by reprogramming adult cells from tissues such as skin, hair, or blood, into an embryonic state.

Similar to stem cells in the embryo, these iPSCs can mature into any desired cell type, including neurons or microglia. The advantage of using iPSC-derived cells is that they resemble human cells more than conventional cell lines.

Additionally, mouse and human microglia differ in the molecules released during an immune response. Thus, microglia derived from human iPSCs represent a better model to understand how genes regulate microglial functions.

In the current study, the researchers used induced pluripotent stem cell lines, which were modified to express genes encoding the CRISPR-dCas9 machinery. The CRISPR machinery in iPSCs, however, was inactive and could only be activated in the presence of the antibiotic trimethoprim.

The researchers then used viral vectors to deliver guide RNAs to the iPSCs. The iPSCs used by the researchers were genetically engineered to rapidly differentiate or mature into microglia-like cells upon exposure to a specialized culture medium.

When differentiating iPSCs into microglial cells, the researchers activated the CRISPR machinery by adding trimethoprim to the cell culture medium. This means that although the scientists introduced the guide RNAs into the iPSCs, the genes targeted by the guide RNAs were not activated or inhibited until after the iPSCs had differentiated into microglia-like cells.

If the expression of these targeted genes is disrupted, this could have a negative impact on the development of microglia. This could make it difficult to distinguish whether the change in expression of targeted genes impacted microglia development or adult microglia function.

This new CRISPR platform thus enables scientists to assess the function of genes in adult microglia.

After validating the modified CRISPR screens, the researchers were able to identify microglia genes involved in cellular processes such as proliferation, survival, activation of an immune response and phagocytosis.

For example, they identified genes that modulate phagocytosis – the cellular process of eliminating potentially toxic particles such as NFP1 and INPP5Dthat have been implicated in neurodegenerative disorders.

Microglia respond adaptively to their local environment and exist in a wide range of context-specific states. Each microglial state, such as a diseased state, a healthy state, or the state producing an immune response, is characterized by a specific gene expression profile.

Getting cells back on track

The researchers used RNA sequencing at the single-cell level to characterize different microglial states.

Based on the differences in gene expression profiles, the researchers were able to characterize nine distinct microglial states.

For example, one of the functional states was characterized by increased expression of SPP1 gene that is upregulated in microglia in AD and other neurodegenerative conditions.

In addition, by inhibiting the expression of genes using the CRISPR platform, the researchers were able to identify genes regulating the adoption of these functional states.

For example, researchers have found that downregulation of the colony stimulating factor 1 receptor (CSF1R) using the CRISPR platform reduced the number of cells expressing high levels of the SPP1 embarrassed.

Scientists observed a similar reduction in the number of microglia in the SPP1 disease state when using a drug that inhibits the CSF1R protein. So, by targeting the genes — or the proteins encoded by those genes — that regulate the disease state, scientists could return microglia to a healthy state.

These findings show that this CRISPR-based platform could be used to identify genes that regulate microglial states associated with neurodegenerative conditions. This could then help scientists develop treatments targeting these genes or the gene products.

“CRISPR screens in human microglia have the potential to uncover therapeutic targets that can ‘reprogram’ microglia to enhance their beneficial functions and block their toxicity in disease,” explained the study’s lead author, Dr. Martin Kampmann, professor at the University of California. , SF.


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