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Towards Identifying Pharmacodynamic Blood Biomarkers of MAP3K12 Inhibition

Jirström, Elisabeth LU (2017) KBK820 20171
Pure and Applied Biochemistry
Abstract
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons in the brain and spinal cord, for which there is no cure. Activation of the stress-induced c-Jun N-terminal kinase (JNK) signaling pathway, a critical mediator of neuronal apoptosis and axon degeneration, is evident in ALS and is therefore considered a potential target to prevent neurodegeneration. Pharmacological inhibition of an upstream activator of the JNK pathway, the mitogen-activated protein kinase kinase kinase (MAP3K12), has demonstrated neuroprotection in vitro as well as suppression of pathway activation in vivo. These observations, along with the largely neuronal-specific expression of... (More)
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons in the brain and spinal cord, for which there is no cure. Activation of the stress-induced c-Jun N-terminal kinase (JNK) signaling pathway, a critical mediator of neuronal apoptosis and axon degeneration, is evident in ALS and is therefore considered a potential target to prevent neurodegeneration. Pharmacological inhibition of an upstream activator of the JNK pathway, the mitogen-activated protein kinase kinase kinase (MAP3K12), has demonstrated neuroprotection in vitro as well as suppression of pathway activation in vivo. These observations, along with the largely neuronal-specific expression of MAP3K12, makes inhibition of this kinase a promising therapeutic target for ALS. Furthermore, a pharmacodynamic (PD) blood biomarker would be beneficial to help assess efficacy of MAP3K12 inhibition. A crucial step to interpreting the PD effects of MAP3K12 inhibition using blood biomarkers is to acquire evidence of MAP3K12 protein expression in blood, which was the aim of this study. First, we found Map3k12 transcript levels to be highly reduced in blood from Map3k12 conditional knockout (cKO) mice, demonstrating that these are excellent negative controls for studying MAP3K12 expression in blood. Next, three antibodies were identified that recognized mouse and human MAP3K12. Utilizing these antibodies, an intracellular flow cytometry method was developed that successfully detected MAP3K12 protein in human peripheral blood mononuclear cells (PBMCs). Collectively, our data provide evidence of MAP3K12 expression in human blood and thus pave the way for identifying PD blood biomarkers of MAP3K12 inhibition. (Less)
Popular Abstract
Blood Biomarkers – a Window into the Brain?

Little is known about the underlying cause of neurodegenerative diseases such amyotrophic lateral sclerosis (ALS). The protein MAP3K12 was recently identified as an important player in neurodegeneration and a potential therapeutic target for ALS. Our study found evidence of MAP3K12 in blood, which brings us closer towards identifying blood biomarkers to evaluate the biological effects of ALS drugs.

ALS is a rare but terrifying neurodegenerative disease that kills half of the patients within 3 years from onset. The disease attacks the cells that are in charge of muscle movement, so called motor neurons. As the disease worsens, ALS patients lose their ability to move, speak, eat and... (More)
Blood Biomarkers – a Window into the Brain?

Little is known about the underlying cause of neurodegenerative diseases such amyotrophic lateral sclerosis (ALS). The protein MAP3K12 was recently identified as an important player in neurodegeneration and a potential therapeutic target for ALS. Our study found evidence of MAP3K12 in blood, which brings us closer towards identifying blood biomarkers to evaluate the biological effects of ALS drugs.

ALS is a rare but terrifying neurodegenerative disease that kills half of the patients within 3 years from onset. The disease attacks the cells that are in charge of muscle movement, so called motor neurons. As the disease worsens, ALS patients lose their ability to move, speak, eat and ultimately breathe.

After decades of research, there is still no cure to ALS. The cause of motor neuron death still puzzles scientists, but more clues are being revealed about the underlying disease mechanisms. Recent studies have uncovered a molecular pathway leading to degeneration of motor neurons in ALS. With increased attention to this pathway, a protein called MAP3K12 was found to be one of its key components. This new piece to the puzzle makes MAP3K12 a promising therapeutic target to prevent the death of motor neurons in people with ALS.

These findings have led to development of drugs, so called inhibitors that block the activation of MAP3K12. However, the impossibility of collecting brain samples makes it difficult to measure the effect of these inhibitors within the brain. A way to overcome this obstacle is to use biomarkers.

Biomarkers are measurable indicators that provide a window into health and disease. A classic example of a biomarker is the cholesterol levels in blood that serve as a risk indicator for heart disease. Another use of biomarkers is to assess the biological effects produced by a drug. Finding such biomarkers of MAP3K12 inhibitors would open up ways to monitor their effectiveness to help find the right dose for ALS patients. Ultimately this could speed up drug trials for ALS, which is of great importance for a disease of such urgent unmet medical need.

Although biomarkers can be found throughout the body, we set out to find a biomarker in blood. Why, you might ask? Well, just imagine being able to predict the inhibitors effects in the brain with only a few drops of blood. The main challenge with finding such biomarkers is that MAP3K12 protein mainly exists in the brain and has not previously been found in blood. Without the protein, the drug would have little to no effect in blood, which would make the use of a blood biomarker pointless. Promisingly, the gene encoding the protein is indeed present in blood, which motivated us to search for protein expression in the blood.

First off, we used mice in which the gene encoding MAP3K12 had been genetically removed, so called knockout mice, as well as cells that we genetically engineered to express the protein. Using these two sources where we clearly knew the presence or absence of the protein, we tested the ability of different antibodies to recognize MAP3K12 and found several good candidates to use for protein detection in blood. By using our newfound antibodies in flow cytometry, a laser-based technique useful for studying protein expression, we developed a novel method for detecting the protein in human blood cells.

Armed with laser and antibodies, we found evidence of MAP3K12 protein expression in human blood, which brings us one step closer to finding a blood biomarker of this protein. In the future, a quick blood sample from an ALS patient could allow us to measure levels of MAP3K12 activity in the blood and predict the effects of MAP3K12 inhibition in the brain. (Less)
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author
Jirström, Elisabeth LU
supervisor
organization
course
KBK820 20171
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Mitogen-activated protein kinase kinase kinase, MAP3K12, Amyotrophic lateral sclerosis, DLK, ALS, Pharmacodynamic, Biomarker, applied biochemistry, tillämpad biokemi
language
English
id
8923833
date added to LUP
2017-09-18 13:58:39
date last changed
2017-09-18 13:58:39
@misc{8923833,
  abstract     = {Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons in the brain and spinal cord, for which there is no cure. Activation of the stress-induced c-Jun N-terminal kinase (JNK) signaling pathway, a critical mediator of neuronal apoptosis and axon degeneration, is evident in ALS and is therefore considered a potential target to prevent neurodegeneration. Pharmacological inhibition of an upstream activator of the JNK pathway, the mitogen-activated protein kinase kinase kinase (MAP3K12), has demonstrated neuroprotection in vitro as well as suppression of pathway activation in vivo. These observations, along with the largely neuronal-specific expression of MAP3K12, makes inhibition of this kinase a promising therapeutic target for ALS. Furthermore, a pharmacodynamic (PD) blood biomarker would be beneficial to help assess efficacy of MAP3K12 inhibition. A crucial step to interpreting the PD effects of MAP3K12 inhibition using blood biomarkers is to acquire evidence of MAP3K12 protein expression in blood, which was the aim of this study. First, we found Map3k12 transcript levels to be highly reduced in blood from Map3k12 conditional knockout (cKO) mice, demonstrating that these are excellent negative controls for studying MAP3K12 expression in blood. Next, three antibodies were identified that recognized mouse and human MAP3K12. Utilizing these antibodies, an intracellular flow cytometry method was developed that successfully detected MAP3K12 protein in human peripheral blood mononuclear cells (PBMCs). Collectively, our data provide evidence of MAP3K12 expression in human blood and thus pave the way for identifying PD blood biomarkers of MAP3K12 inhibition.},
  author       = {Jirström, Elisabeth},
  keyword      = {Mitogen-activated protein kinase kinase kinase,MAP3K12,Amyotrophic lateral sclerosis,DLK,ALS,Pharmacodynamic,Biomarker,applied biochemistry,tillämpad biokemi},
  language     = {eng},
  note         = {Student Paper},
  title        = {Towards Identifying Pharmacodynamic Blood Biomarkers of MAP3K12 Inhibition},
  year         = {2017},
}