The Discovery of ATP Synthase as a Drug Target and its Effects on Aging and Dementia
Since 2004 only one Alzheimer’s disease therapeutic has been approved. Alzheimer’s disease drugs are a result of target-based screens, and with the lack of reliable drug targets the search for working medicine has been limited. Age has a huge role in the development of Alzheimer’s disease, although it is still uncertain how closely the two are at the molecular level. Because age is so closely related to developing the disease, scientist have developed a specific screening designed to mimic some of the most common age-associated central nervous system toxicities. They discovered a compound called J147, which is said to enhance the memory, restore cognition, and help aid the rapid deterioration caused by aging in mouse subjects used in the experiment. Through this single drug target J147, scientists are now able to lightly confirm that metabolic regulation, aging, and dementia, are all connected. Three different approaches were used to identify the molecular target of J147 and then later used to confirm the molecular target. First, they used the drug affinity responsive target stability also known as (DARTS), this is an unbiased and small target identification technique used to identify binding partners. Next, they used affinity precipitation pull-downs, which confirmed that ATP5A was the target of J147. ATP5A was the most highly enriched protein within both experiments. Just after these two approaches, J147 was now known to bind to and inhibit the activity of the mitochondrial ATP synthase. Later, they used biochemical and localization experiments to support the idea that ATP5A was a target of J147.
Disease progression in the elderly and patients who suffer from Alzheimer’s is linked to mitochondrial dysfunction that reduces levels of ATP. Scientist tested J147’s effect on the mitochondrial membrane. After a significant dose, within the next sixty minutes an increase in the mitochondrial membrane’s potential was noticed. This affect is very closely related with the regulation of ATP synthase activity. By inhibiting ATP activity scientists were able to discover that this protects from neurotoxic insults, and that J147 blocks intracellular amyloid-induced cell death. Along with ATP5A knockdown which also prevents cell death. With these findings we find it true that the new drug candidate J147 contains compounds that promotes cell survival and reduces changes associated with aging.
The J147 drug, which is identified through phenotypic screenings, has recently shown benefits in animals that have Alzheimer’s disease, AD. This drug acts through the mitochondrial protein called ATP5A to encourage cell survival and reduce certain changes that come with aging. Having the correct metabolic control is important for having a successful response to the horrific stresses that affect the aging brain and they may also provide other ways to the amyloid pathway for AD-targeted therapeutic interventions. The proteins VDAC, Slc25a, and IP3R3 as well as ATP synthase have all been used in the configuration of the mitochondria permeability transition (mPT) pore that is responsible for implementing cell death during fatal conditions of stress. That is what led to the idea of using the mPT pore as possible neurodegeneration therapy. There was a specific in vivo role for ATP synthase inhibition in protection of brain neurons that was used against excitotoxic damage. It was demonstrated through generation of a qualified mouse model that expressed the human form of mutant ATPase inhibitory factor 1, hIF1.The results show an increase in IF1 mediated protection of the toxicity analysis validate the neuroprotective effect that was seen with ATP5A and it supports that ATP synthase inhibition is neuroprotective. It was observed that J147 increased cytosolic calcium levels most likely explains the initiation of CamKK2. That led to the investigation of the role CamKK2 plays in AMPK phosphorylation. Interference of the CamKK2 mixed with STO609 eliminated the effects of the J147 on AMPK activation. It is important that AMPK is used because it was needed to facilitate J147 protection, which indicates a direct role for AMPK in J147’s mechanical pathway. AMPK is considered an energy sensor, which interferes with anabolic process, which consume energy, and endorses catabolic processes, which create energy. The question was asked if the pathways were affected in vitro or in vivo by the J147 treatment. In cells, J147 stimulated AMPK/mTOR signaling through increased phosphorylation of ACC1. The variation of ATP synthase activity through siRNA specified destruction of ATP5A phenocopied J147’s effect on AMPK/mTOR targets. The western blot analysis on hippocampal lysates from J147 treated mice showed that AMPK is activated in vivo. What all this data shows is that the J147 facilitated neuroprotection produced by targeting ATP synthase could possibly regulate both metabolism and aging. The J147 drug increased the lifespan in drosophila by 12.5%. The hippocampal gene expression was examined and targeted metabolic data of J147 treated and untreated mice. The drug treatment stabilized the hippocampal transcriptome in a young age as well as the plasma metabolome against age associated increases in drift variance which point to a biologically younger transcriptome and metabolome. Since the J147 was recognized based on its capability of protecting cells from old age-associated neurotoxicity’s in vitro, the results suggest that aging and age-associated dementia are more closely related that previously thought and could possibly share common drug targets. If the close relationship between averting aging and dementia shown for J147 are true for other genetic targets, the pathways would offer a new source of AD drugs that are very much needed.
J147 was put through a couple of assessments before given to humans. First, J147 was applied to an approach called drug affinity responsive target stability; which is an unbiased small molecule target identification approach to detect putative binding partners. Lysates, taken from HT22 hippocampal nerve cell line, were incubated for 15 minutes before treated with pronase to take down unbound protein complexes. Results showed ATP5A, which is a catalytic subunit of the mitochondrial ATP synthase complex, was the most enriched presumed target related to controls. ATP5A is responsible for the synthesis of ATP. To ensure ATP5A was the most highly enriched protein, scientist tested whether the activity of ATP synthase complex was affected by J147. The experiment was isolated in a bovine heart mitochondria. J147 was added to the heart and an incubation period of 1 hour was required before analyzing the results. Scientist found that J147 binds to and partially restricts the active of the mitochondrial ATP synthase. Next scientist concluded whether the intracellular localization of J147 in HT22 cells stayed the same with a mitochondrial target by confocal fluorescent microscopy. An imaging time course exhibited J147 rapidly colocalized with the mitochondrial marker cytochrome C oxidase. Therefor, both biochemical and localization experiments support ATP5A as a target of J147.
The phenomenal discovery of J147 and its target is groundbreaking evidence of the potential for an actual cure for Alzheimer’s disease, which will positively impact the lives of hundreds of thousands of people. The diversified studies and approaches taken to prove that the drug J147 targets the ATP synthase are broad and targeting at the same time. My critique, due to scientists’ efforts of amplifying their research and not limiting their investigation to just one resource, is a positive one. Any given hypothesis that is tested with several experiments helps with its accuracy, if all the experiments give precise and accurate results, which at the end give the overall results a low standard deviation value. The lower the standard deviation value is, the higher the accuracy and trustworthiness of the theory will be.
From the experiments performed which include: DARTS, cell viability and acute toxicity, transcriptome/metabolome drift analysis, cell lines, immunofluorescent staining, J147 pull downs, complex V activity assay, western blot, ROS and membrane potential measurements, cytosolic calcium measurements, ATP, transfections, and drosophila stocks, I would like to expand and point out the importance of using hippocampal HT22 cells found in the mice and MC65 neuronal cells found in humans. (Goldberg, 2018). Since it is believed as we age that plaques and neurofibrillary tangles are found in places like the hippocampus (associated with memory processing), using these cells to test is an excellent approach to reverse memory loss by rejuvenating the neuron cells. (Martini, 2006, p. 542). Not only would it help minimize memory loss by reversing the aging of the cell, but it could be beneficial to all other systems of the human body affected by aging.
If I could add any procedures to the existing experimental procedures, it would be to include standard deviation statistics. Having a value that defines how accurate and precise your results have been from the beginning, middle, and end speaks relatively loud with confidence that all the testing wasn’t in vain. Having a standard deviation value to back up all your findings would just reinforce the discovery of this new elixir of life, “the J147 drug.” (Salk News, 2018)
The Discovery of ATP Synthase as a Drug Target and its Effects on Aging and Dementia