A Novel Approach to Chronic Traumatic Encephalopathy (CTE) - Pattern Recognition Receptors

Introduction

Chronic Traumatic Encephalopathy (CTE) is a neurodegenerative disorder that is associated mainly with contact sports such as boxing and football. This brain disorder results in the death or degeneration of nerve cells in the brain. Although there is no treatment available currently for this condition, it is important for athletes to be aware of the risk and to do what can be done to prevent the condition. The purpose of this research is to find advancements on a treatment for this condition as it affects individuals throughout their lives and will eventually lead to dementia and possibly death. (Mayo Clinic 2024)

What is CTE?

CTE is a rare condition, and the cause is unclear, but it has been linked to multiple head injuries rather than a single injury to the head. It is believed that CTE develops mainly when a person/athlete receives a head injury prior to the complete resolution of a previous head injury. Due to this discovery, this disease is linked to athletes in boxing, American football, and other contact sports where players receive multiple hits to the head. It has also been linked to soldiers in the military and physical abuse victims. However, CTE can only be diagnosed after the patient’s death by autopsy, and not everyone will develop CTE even if they partake in boxing for 20 years, which makes it such a difficult disease to cure. (ELS, L.C. 2024)

Causes

CTE is caused by repeated head injuries that damage the blood vessels and cells in the brain. Typically, when an individual receives another head injury before the prior injury has fully resolved, it will heighten the risk of developing CTE. Repeated head injuries cause the neurons in the brain to slowly die or decrease in function by abnormal bleeding and in turn abnormal protein deposits on the brain cells, as seen in figure 1 below. CTE can be caused even when the head injury does not result in concussion. (ELS, L.C. 2024)

Figure 1: Figure shows a head injury to a patient and the damage caused to the internal tissue and blood vessels.

Motor symptoms

• Issues with walking and balance

• Parkinsonism causes shaking, slow movements and trouble with speech

• Motor neuron disease which destroys cells that control walking, speaking, swallowing, and breathing. (Mayo Clinic 2024)

Current Treatment

Currently, there is no cure for CTE, so many of the treatments focus on enhancing the quality of life of the patients and improving the functioning of the individual. Behavioural therapy focus on addressing the psychological challenges of patients with CTE, it helps to cope with the mood swings, depression and the potential aggression that comes in the later stages of CTE (Concussion Legacy Foundation 2024).

Non-pharmacological treatment avenues:

1. Sleep management: disordered sleep is a common symptom of patients with CTE, so it is a common avenue for doctors to look at. Sleep hygiene education and cognitive behaviour therapy can be prescribed for insomnia.

2. Exercise: exercise has been shown to improve the quality of life of CTE patients by improving sleep and mood, lower anxiety, increasing memory and decreasing pain and lower dementia risk.

3. Diet: the mediterranean diet is often prescribed as it is known for its reduced dementia risk and age-related cognitive decline (Campbell et al. 2024).

Nutraceuticals can also be prescribed to offset some of the symptoms of CTE. Vitamins such as vitamin D has been found to be neuroprotective in neurons and the adult brain, (low levels of 1,25-hydroxyvitamin D₃ (1,25(OH)₂D₃) has been found to be linked with neurodegenerative diseases like Alzheimer’s and Parkinson’s disease) (Cui and Eyles 2022). Vitamin B2 has been found to improve multi domain cognitive function in middle-aged and older populations (Ji et al.2024).

Coenzyme Q has been found to play a role in the process of cellular energy supply via oxidative phosphorylation in mitochondria, shuttling electrons from complex I and II to complex III of the mitochondrial respiratory chain. Levels of coenzyme Q have been found to be lower in patients with neurological diseases such as Parkinson’s, motor neuron disease, stroke and multiple system atrophy (Mantle et al. 2021).

Magnesium is involved in the maintenance of homeostasis of all tissues, including the brain. It harmonises nerve signals transmissions and preserves blood-brain barrier integrity (Maier et al. 2022). Melatonin is found to have an antioxidant activity and also has neuroprotective effects (Lee et al. 2019). It is also often prescribed for insomnia as it has been found to help patients fall asleep faster (Johns Hopkins Medicine 2024)

There are some pharmacological treatments that can help manage some of the symptoms of CTE. These treatments will not cure or slow the disease progression, thus it shows the need for a treatment option that will target the disease itself. Medications such as Donepezil, galantamine and rivastigmine are often prescribed to patients with CTE as they can  ‘turn back the clock’ on memory loss in patients with dementia 6-12 months. This can improve a patient's cognition, function and irritability. Selective serotonin reuptake inhibitors (SSRI) are prescribed for a patient suffering with depression and anxiety (e.g  sertraline and escitalopram). Antipsychotics, such as Brexpiprazole, are also prescribed if severe aggression, Bipolar disorder or psychosis are present (Campbell et al. 2024).

Currently, there are also many clinical trials ongoing that are looking at potential treatment strategies for CTE. These trials range from questionnaires for athletes who have played contact sports for a certain number of years (e.g the Head Impact and Trauma Surveillance Study (HITSS), to studies that are investigating methods to diagnose CTE in life rather than death (e.g., Tau Agent for the NeurodeGenerative Lesion of CTE (T.A.N.G.L.E.).

Also, studies that are seeking participants to have their blood drawn in order to investigate biomarkers for CTE  and make a brain donation pledge so they can study the brain after death and gain further insight into how CTE works (Blood Analysis with Neuropathological Knowledge of CTE (Bank CTE) (Boston University 2024).

Prevention strategies

The only way to prevent CTE is to avoid repeated head injuries. According to the HSE, you can reduce the risk of developing CTE by taking steps to ensure your head is protected while engaging in contact sports or activities that have a higher risk of head injury. It is recommended to wear protective equipment such as a padded helmet during sports such as boxing, martial arts, rugby, football or other sports where a knock to the head is common. (HSE.ie 2024).

It is recommended to seek medical advice following a head injury and to adhere to doctors’ advice for returning to sport following a concussion as returning prematurely could have adverse effects. Concussions have been divided into 3 categories based on their severity. The three deciding factors that determine the severity are: Loss of consciousness (LOC), Post-traumatic amnesia (PTA) and Post-concussion signs and symptoms (PCSS).

Table 1: The grading features of concussions.

Table 2: Grade of concussion, the number of times the patient has been concussed and how long they should wait before returning to play (RTP).

A recent study by (Rydzik et al. 2023)  titled “Comparison of head strike incidence under K1 rules of kickboxing with and without helmet protection- a pilot study” found that the rate of head strikes actually increased when the kickboxer was wearing a helmet. The helmet gives the athletes a false sense of security when a helmet is worn and they are more likely to go for headshots, compared to when a helmet is not worn and the athletes are more strategic in their hits and the placement of them. Head protection is an important feature in protecting against TBI, but other factors to consider are:

-       proper training in techniques and skills in the sport,

-       proper teaching and spreading information among athletes about the dangers of head injuries and the importance of taking concussions serious and

-       potentially changing rules in sports to ban hits to the head, such as in sports like MMA, kickboxing, taekwondo. 

Novel Approach Summary:

Our novel approach to treat Chronic Traumatic Encephalopathy (CTE) starts with using stem cells from CTE patients, which avoids the risk of immune rejection, and reprogramming them into pluripotent stem cells (iPSCs) by means of Klf4 plasmid transfection. The aim is to engineer the stem cells to only target misfolded proteins by having them express pattern recognition receptors (PRR), TLR2. Enabling the use of a lentiviral vector design which incorporates these specific pattern recognition receptors (PRR) allows for stable expression in the host genome. Electroporation of the cells will transfect the cells and allow for the isolation of iPSCs colonies that successfully express the PRRs. Further characterisation methods and proteomic activities, evaluate the cell's functionality and characteristics.

Transplantation of the cells into the CTE patients involves the use of a biodegradable scaffold application directly implanted in the brain region, allowing the cells to integrate with the surrounding tissue. Post transplant care requires close monitoring of the patient to assess the integration of the stem cells, their functionality and any potential immune responses. Immunosuppressive therapy may need to be completed to prevent rejection of the transplanted cells.

What Are Pattern Recognition Receptors?

Pattern Recognition Receptors (PRRs) refer to a group of receptors that recognise pathogens – induced infections through proteins and other molecular motifs. PRRs are primarily expressed in immune cells such as dendritic cells and macrophages but can also be found in other immune cells. These receptors play an important role in the body’s innate immune system by recognising pathogens – associated molecular patterns (PAMPS). PAMPS are specific and highly conserved molecular structures which are shared by microorganisms, including lipids, proteins and nucleic acids.

In addition, PRRs can recognise damage – associated molecular patterns (DAMPs), which are unique molecules expressed on stressed, injured, or infected human cells, indicating a harmful microorganism.

In neurodegenerative diseases like Chronic Traumatic Encephalopathy (CTE), misfolded proteins such as tau tend to accumulate in the brain, leading to cellular stress and neuroinflammation. PRRs are known to detect some of these stress-related signals, such as oxidative stress markers and other DAMPs released by affected cells. When activated by these DAMPs, PRRs can initiate pathways that help the brain’s immune cells and attempt to clear cellular debris, including protein aggregates. However, if this activation persists, it can contribute to chronic inflammation and further neuronal damage.

By combining PRRs with stem cell therapy there is a potential to modulate the immune response in a specific and targeted approach. Detecting cellular stress signals related to misfolded proteins while clearing DAMPs is essential. If there is a high level of DAMPs, damaged neurons may further amplify neuroinflammation and aggravate the disease.

To ensure our novel approach is realistic we must:

• Create a stem cell expressing pattern recognition receptors capable of recognising misfolded proteins via DAMPs, oxidative and cellular stress

• Implement strategies capable of modulating PRR activity preventing overactivation.

Choosing The Correct Pattern Recognition Receptor:

There are several pattern recognition receptors which play a key role in innate immunity. Among these receptors are Toll – Like Receptors (TLR), NOD – Like Receptors (NLRs), C- type lectin receptors (CLR) and RIG - 1 like receptors (RLR). It is important that the correct PRRs are chosen for our novel therapy to contribute to protein clearance while minimising adverse side effects such as inflammation and tissue damage.

TLR2 is a PRR belonging to the toll – like receptor family. They are divided into six major groups based on their function, location and signalling pathways. TLR2 is most associated with binding to lipoproteins which are expressed on a type of bacteria, known as gram – positive bacteria. TLR2 is associated with several DAMPs across many conditions including High – Mobility Group Box 1 (HMGB1), Heat Shock proteins (HSPs) and Bigylcan, all of which can be observed in a person who has suffered a brain injury.

Mechanism of PRRs:

TLR2 signalling pathways are complex and selective. Since we are dealing with misfolded proteins, we need to fuse our TLR receptors with a domain that is capable of recognising motifs in misfolded proteins. The domain we have chosen is a single – chain variable fragment which will specifically bind to aggregated tau, a protein which is abundant in CTE. This Anti – Tau scFv, derived from Tau5 antibodies, will be fused to the TLR receptor to act as a chimeric receptor leading to downstream signalling pathways. In normal TLR2 signalling, LPS, a PAMP in bacteria, binds to LBP, a binding protein, which initiates this signalling pathway. Our therapy looks to bypass this step as in the context of CTE, LPS and LBP are irrelevant, as our DAMPs are misfolded proteins.

In the case of TLR2, when Tau5 scFv binds to misfolded proteins, two separate TLR2 receptors join which begins the recruitment of an adaptor protein called MyD88, a protein which connects a protein that receives signals outside the cell to a protein relaying signals inside the cell. This in turn activates IRAK (interleukin – 1 receptor – associated kinase), another receptor which regulates TLRs, and TRAF6 (tumour necrosis factor receptor), which is another adaptor protein involved in TLR signalling.  TRAF6 activates TAK1, TGF- β- activated kinase 1, through a process called polyubiquitination, a process in which two different types of proteins join. TAK1 activates MAPKs (mitogen – activated protein kinases) which phosphorylate and activate NF-κB. NF-κB, a transcription factor involved in cytokine production then enters the nucleus promoting the expression of IL-6, IL-1 Beta and TNF- alpha, all pro – inflammatory cytokines, which help recruit immune cells to the site of build up of aggregated Tau.

Figure 2: Toll - Like Receptor (TLR) cascade reaction.

Control of inflammation:

In this therapeutic strategy we aim to implement co-expression of an anti-inflammatory cytokine such as interleukin-10 (IL-10), alongside the engineered TLR-scFv receptor to balance immune activation and prevent excessive inflammation. IL-10 is an anti-inflammatory cytokine that limits the immune response by inhibiting the production of pro-inflammatory cytokines like TNF-α and IL-6. By expressing IL-10 in our engineered stem cells we aim to create a passive “brake” on inflammation. This ensures that the immune response does not exceed therapeutic levels. When the TLR-scFv receptor binds to misfolded tau proteins and initiates immune signalling. IL-10 is simultaneously present to modulate this response. It then promotes the clearance of toxic aggregates without risking damage to healthy tissue. This approach provides an effective way to control inflammation in the brain’s sensitive environment while reducing potential side effects and preserving the therapeutic effects of the TLR pathway. Co-expression of IL-10 is used in cellular therapies and to manage inflammation which makes it an ideal candidate to add to our chimeric receptor system.

Genetically engineering stem cells:

Genetically modifying induced pluripotent stem cells (iPSCs) involves transforming somatic cells back into a pluripotent state, which enables them to develop into different cell types. This process usually employs the use of a specific factor such as Klf4 which is delivered into the somatic cells through viral vectors.

By genetically engineering these pluripotent stem cells, it will then be modified to express the pattern recognition receptor TLR2 and target misfolded proteins.

The approach involves engineering a patient's own iPSCs to avoid immune rejection.

The steps to genetically engineer induced pluripotent stem cells (iPSCs) and to express pattern recognition receptors (PRR) will involve several key phases;

1. Cell Sourcing: The first step of the process is to obtain somatic cells, which can be either skin or blood cells, taken from a CTE patient. Our novel approach will involve cell sourcing using a CTE patient's blood cells as it’s a relatively non-invasive procedure compared to other methods, and can provide a rich diversity of cell types. Blood collection will be taken through standard venipuncture procedure, the blood sample is then processed to isolate mononuclear cells through density gradient centrifugation, allowing the mononuclear cells to be separated from other blood components. T-cells or fibroblasts are then reprogrammed into iPSCs.

2. Reprogramming: Introducing the reprogramming factor Klf4 into the somatic cell using the viral vector method, plasmid transfection. The decision to use Klf4 over other reprogramming factors is due to Klf4 playing a significant role in regulating cell proliferation and differentiation, it also helps inducing pluripotency.
In plasmid transfection, a plasmid containing the Klf4 gene is introduced into somatic cells using an electroporation transfection agent. The plasmid can replicate independently within the cell, and once inside it can be transcribed and translated to proceed with the Klf4 protein. This method is in general less efficient than other viral vectors, however it will allow for easier manipulation and control over the expression levels of Klf4.

3. Autologous iPSCs: As the iPSCs are derived from the patient's own cells, they are autologous. This significantly reduces the risk of immune rejection.

4. Vector Design for PRR Expression: Next, an expression vector contains the gene for the desired pattern recognition receptor TLR2, along with appropriate regulatory elements to ensure expression in the iPSCs. The aim is to engineer the stem cells which only target misfolded proteins by having them express the pattern recognition receptor (PRR) TLR2. Lentiviral vectors are particularly effective for the stable integration into the host genome, making them a good choice for the long term expression in iPSCs. This vector will also enable our novel approach to carry the gene interest along with strong promoters and regulatory elements.

5. Transfection: Introduce the PRR expression vector into the patients iPSCs using electroporation or viral transduction. The iPSCs are cultured under optimal conditions until reaching desired confluence. Our approach will aim for 70-80% for successful electroporation.

Next, preparation of the PRR expression vector through linearisation and purifying the plasmid DNA using a purification kit. Trypsinizing the iPSCs to detach from the cell culture, centrifuge to a pellet and then re-suspending in an electroporation buffer such as PBS. The addition of the purified PRR expression vector to the cell suspension allows for the cell DNA mixture transfer to an electroporation cuvette. After electroporation, the procedure is to recover the cells through pre-warmed culture medium and incubation. After a recovery period of 48 hours, selection of successful transfected cells using fluorescent sorting, then characterisation steps follow.

6. Selection of Transfected Cells: Using fluorescent sorting to isolate the iPSC colonies that successfully express the PRR.

7. Characterisation: Confirmation that the expression of the PRR using qPCR and evaluating its functionality through appropriate assays. qPCR allows for quantification of DNA by measuring the fluorescence emitted during the amplification process. It is a common tool used for gene expression analysis and will allow for real time monitoring, providing a more accurate quantification than endpoint analysis.

The patients iPSCs cells have now been successfully genetically engineered to express pattern recognition receptors (PRR) whilst avoiding immune rejection.

8. Transplantation: Finally, transplanting the engineered cells back into the patient, minimising the risk of immune rejection since they are derived from their own tissue. The means of transplantation into the patients involves the use of a matrix application. A biodegradable scaffold that can support the engineered stem cells and provide a conductive environment for tissue regeneration. The scaffold can be integrated with the brain region, allowing the cells to integrate with the surrounding area.

9. Post transplant care: This is essential, requiring close monitoring of the patient to assess the integration of the stem cells, their functionality and any potential immune responses. Immunosuppressive therapy may need to be completed to prevent rejection of the transplanted cells.

Figure 3: Schematic representing the steps taken to create iPSCs transfected with Pattern Recognition Receptors.

Proteomic activities: 

Our novel strategy to genetically engineer iPCSs stem cells to express specific pattern recognition receptor (PRR) TLR2 to target misfolded proteins, will require carrying out certain proteomic activities to enable the analysis of protein expression and interactions, providing insights into the signalling pathways actuated by the PRR engagement. This approach can enhance our understanding of immune mechanisms and potentially lead to further advancements in therapies for diseases involving immune dysfunction.

Protein profiling of the engineered stem cells using techniques such as mass spectroscopy will allow for investigation into how the proteome changes in response to PRR activation or the presence of misfolded proteins.

Conduction of interaction studies such as affinity purification to identify interacting partners of the PRRs along with revealing downstream signalling pathways and potential targets for therapeutic intervention.

Investigation into the proteomes of the engineered stem cells expressing pattern recognition receptors with stem cells, allows for the comparison between the novel stem cell treatment for CTE to that of control stem cells.

Post translational modifications (PTMs) of protein such as phosphorylation or glycosylation which can be crucial for protein function and stability, especially in the context of misfolded proteins. Biomarker discovery using functional studies and clinical utility assessments will be imperative for identifying the potential biomarkers for CTE association with misfolded proteins by analysing proteomic data. Advancements in this area of research could potentially lead to the development of diagnostic tools or therapeutic agents.

Conclusion

Chronic Traumatic Encephalopathy is a very dangerous disease and not much is known about its cause. As CTE is only able to be diagnosed by autopsy, it is difficult to understand the mechanism of CTE. Throughout this study we had aimed to delve deeper and to further understand CTE. We came up with a novel approach using stem cells that will target misfolded proteins by having them express pattern recognition receptors (PRR), TLR2. The recognition receptor will be bound to a single – chain variable fragment that specifically binds to aggregated tau, thus making a chimeric antibody that is specific to tau proteins (a protein that is found to be misfolded in CTE and thought to be a key player in the disease). The cells the antibody will be transfected into will be iPSC, taken directly from the patient, altered and re-injected back into the patient, lowering the immune rejection rate. CTE is a serious but yet relatively unknown disease. More needs to be done to find a cure for this disease but we hope our novel treatment may potentially be a step forward.