Research Findings

According to the rules outlined in each grant awarded by The CART Fund, researchers are required to submit regular progress reports on their work. We are pleased to provide the final updates from previous CART Fund grant recipients outlining the results of their research. Please note out of respect for their intellectual and proprietary material, we will only publish final reports rather than the progress reports that have been submitted. If you have an interest in a particular ongoing research project, please do not hesitate to contact us for additional information.

We are encouraged by these researchers’ continued efforts to provide cutting-edge research data at world-class facilities in the United States thanks to grants from The CART Fund.

Dr. Keith Vossel (University of Minnesota) was the recipient of a $112,500 grant from The CART Fund in 2018. (Posted Feb 17, 2021)

Keith VosselPeople with Alzheimer’s disease often experience seizures and silent seizure activity in the brain, accelerating the memory loss associated with the disease. We know that the tau protein in brain cells is linked to seizures and memory loss in patients with Alzheimer’s, but we do not know exactly how tau contributes to these seizures and ultimately causes cell death in the brain.

With support from The CART Fund, Dr. Vossel’s team has discovered new mechanisms by which tau contributes to epilepsy and memory loss in Alzheimer’s disease. Dr. Vossel’s team has found that by creating modifications in tau’s structure, they can prevent seizures and cell death that occur in Alzheimer’s disease models. They have used advanced molecular imaging to determine that the modifications prevent tau from attaching to molecules that are implicated in both epilepsy and Alzheimer’s disease.

They are testing the effects of these tau modifications on cellular signaling regulated by these molecules, and in the process, are discovering new molecules that are involved. With a better understanding of how tau’s binding to such molecules contributes to seizures in Alzheimer’s disease, Vossel’s team can design drugs to block these interactions and potentially slow or halt the progression of the disease. This contribution is expected to be significant because it will have broad translational importance in the prevention and treatment of seizures and associated memory loss in Alzheimer’s disease.

Dr. Chihiro Sato (Washington University School of Medicine) was the recipient of a $125,000 grant from The CART Fund in 2016. (Posted July 31, 2020)
Thanks to the generous support from CART, Drs. Sato and Barthelemy published multiple papers describing distinct tau profiles in the human brain, cerebrospinal fluid, and neurons in a dish. Dr. Barthelemy also found that the phosphorylation ratio at position T217 on the tau protein serves as a more sensitive AD biomarker compared to the commonly used T181.

In their research supported by CART, they developed methods to measure tau, phosphorylated tau (p-tau) and specific isoforms that result from alternative splicing and truncations in the human brain, CSF and cell culture models. They used tau antibodies to capture and concentrate tau proteins in the sample, then used mass spectrometry methods to describe and measure which types of tau (splicing variants, truncations, chemical modifications such as phosphorylation) are present in the brain, CSF and neurons in cell culture.

They found that tau is full-length in the brain, but CSF tau is truncated at the middle of the protein. Neurons derived from human skin fibroblasts using induced pluripotent stem cell (iPSC) technologies were also analyzed. Tau is full-length inside the cells but when it is released in the media, it is truncated, recapitulating tau profiles in the brain and CSF, respectively. This supports that neurons in a dish can serve as a translational model for studying tau in the human central nervous system (Sato, Barthelemy et al., Neuron, 2018).

They further analyzed p-tau profiles in the individuals with AD in the CSF (Barthelemy et al., Front Aging Neurosci, 2019) and brain. In AD CSF and brain, p-tau at specific sites were increased. When brain samples were biochemically analyzed for local correlation between amyloid and p-tau, soluble p-tau was more associated with the amount of local amyloid compared to insoluble p-tau. This suggests a direct link between local amyloid and p-tau in AD brains (Horie, Barthelemy, Sato et al., submitted).

Furthermore, they found that specific peptides in the Microtubule Binding Region (MTBR) of tau that is closer to the end of the protein increase in AD (Horie, Barthelemy, Sato et al., under revision), potentially serving as another CSF biomarker of AD.

Second, they tested whether other CSF p-tau species can serve as more sensitive AD biomarkers compared to the most commonly used phosphorylation site at amino acid Threonine 181. They found that the ratio of phosphorylation at Threonine 217 (pT217) to T217 (phosphorylation occupancy at T217) in the middle of the protein associates with amyloid more strongly than the ratio of pT181 to T181 in late onset sporadic AD (Barthelemy, Sato et al., Alz Res Ther 2020). This led to another study in individuals with familial AD mutations in the Dominantly Inherited Alzheimer’s Network (DIAN) cohort at Washington University. Dr. Barthelemy and the team found that the ratio of pT217 to T217 increases almost 20 years before onset of AD symptoms, right at the time amyloid aggregation starts in the brain as detected by positron emission tomography (PET) imaging (Barthelemy, McDade et al., Nat Med 2020).

Dr. Nicola Allen (Salk Institute) was the recipient of a $112,500 grant from The CART Fund in 2018. (Posted July 19, 2020)

In their research, the Allen lab has shown that the star-shaped support cells called astrocytes regulate neuronal function throughout life. In the young brain, astrocytes initiate and maintain connections between neurons, while in the aging brain astrocytes may contribute to the loss of neuronal connections and function. Given this close relationship between the two types of brain cells, astrocytes are likely central to development of Alzheimer’s disease (AD).

AD is characterized by the loss of neuronal connections, known as synapses, which leads to cognitive decline. The Allen lab aims to better understand the role of astrocytes in AD and test whether targeting astrocytes can be used as a therapy to slow or reverse the loss of synapses. In their CART project, the researchers have used AD mouse models to analyze gene expression in astrocytes to understand the specific changes to astrocytes that accompany synapse loss. They identified multiple astrocyte genes as being altered in aging, and have now found that some of these genes are also altered in expression at the earliest stages of AD. They are in the process of asking if blocking these gene expression changes in mouse models of AD is protective to synapses.

In a parallel approach the lab is asking if re-expressing signals that astrocytes use to regulate synapses in the young brain, is also protective of synapses in AD. Their findings provide insight into how certain genes could become dysregulated, contributing to synapse loss in AD.

Dr. Tong Li (Johns Hopkins University) was the recipient of a $75,000 grant from The CART Fund in 2017. (Posted March 9, 2020)

Age is the most important risk factor for development of Alzheimer’s disease (AD). How aging increase the chance for people to develop AD is still unclear. To address this critical question, we proposed to use a newly developed mouse model of AD to test whether aging could facilitate the development of a protein aggregation called the tau tangle, which is a key hallmark in the brain of AD patients. We found that when mice were allowed to start accumulation of tau aggregation at 6 month or 12 month of age (mimicking young adult and middle age in humans), the onset and development of tau pathology were not changed. However, when tau accumulation in mice were started at 16 month of age (mimicking advanced age in humans), much more severe tau aggregation, neuron cell loss and brain atrophy were observed. These results demonstrated that aging accelerated development of tau aggregations. In addition, we also found that age also play an essential role in the sex differences in AD. Our studies demonstrate that the age could increase the risk of AD by accelerating the development of tau aggregation. Understanding the aging related mechanism in AD will provide new strategies for designing effective treatments for AD.

Dr. Tsuneya Izeku (Boston University) was the recipient of $100,000 grant from The CART Fund in 2017. (Posted February 27, 2020)

We have completed the assessment of selected drugs for targeting an enzyme tau-tubulin kinase 1 (TTBK1) by biochemistry, toxicity to cells, toxicity to animals and efficacy of inhibiting the toxic modification of tau protein in animal brain.  After assessment of these data, it was determined that none of the selected drugs are good enough for next step. We performed second screening of modified drugs from the original best candidate drug, but none of them show inhibition activation against target enzyme. New drug candidates are found based on the screening software for simulation of drug-enzyme interaction. We plan to test those new candidates for its inhibition activity against target enzyme as a backup strategy in the future.

Dr. Michael Karin (University of California San Diego School of Medicine) was the recipient of $150,000 grant from The CART Fund in 2017.  (Posted August 21, 2019)

The foundation of our work is the proposal that chronic neuronal inflammation (i.e neuroinflammation) plays a key role in the progression of Alzheimer’s Disease and other types of late onset neurodegenerative diseases. While inflammation is usually associated with pain, fever and swelling, there is evidence that it also plays an important role in neurodegeneration. Accordingly, we hypothesize that damage to the brain’s neurons due to aging and/or trauma results in the release of normal cellular components, such as the energy storing molecule ATP, that can evoke an inflammatory response when encountered by specialized immune cells that reside in the brain called microglia.

When microglia are stimulated by such damage-released molecules, they secrete inflammatory signals called cytokines, which are small proteins through which microglia can affect neuronal functions. IL-1 and IL-18 are two of these cytokines produced by activated microglia and they can amplify the initial damage to neuronal cells and thereby worsen the neuronal loss that eventually gives rise to the dementia and impaired cognitive ability associated with Alzheimer’s disease.

IL-1 and IL-18 are made by a large intracellular complex of proteins called the NLRP3 inflammasome. We have proposed that the most effective way to halt the production of IL-1 and IL-18 by activated microglia, and thereby protect early Alzheimer’s disease patients from progressive neuronal loss, is to inhibit, or shut down, the NLRP3 inflammasome factory. We therefore investigated how the NLRP3 inflammasome factory is powered and identified an important role for the cellular “power stations” called mitochondria.

Mitochondria are organelles (sub-cellular organs) that produce energy for cells. Just like us, mitochondria have their own DNA. We found that the NLRP3 inflammasome and the production of mitochondrial DNA are stimulated at the same time. However, the conditions that stimulate the NLRP3 inflammasome also damage the newly produced mitochondrial DNA. To get rid of their bad DNA and repair the damage, the mitochondria spit out fragments of bad DNA to the cytosol, where they bind to the NLRP3 inflammasome and start the production of IL-1 and IL-18.

Although short-term production of IL-1 and IL-18 is important for tissue repair, their chronic production worsens neuronal damage and accelerates the progression of Alzheimer’s disease. Based on these findings, we suggest that a highly effective way to shut down the NLRP3 inflammasome factory is to prevent the generation of bad mitochondrial DNA. To this end, we identified a critical enzyme that helps produce mitochondrial DNA in microglia and have shown that stopping this enzyme, called CMPK2, prevents the generation of bad mitochondrial DNA, thereby blocking NLRP3 inflammasome activation and production of IL-1 and IL-18.

So, to put all of this in an easier to understand context, the NLRP3 inflammasome can be considered a factory, IL-1 and IL-18 are the products made by the factory and bad mitochondrial DNA and CMPK2 are the control boards that control how fast the factory spits out its products.

Since the initiation of our CART-sponsored project, we have confirmed that exposing microglia to inflammatory signals begins the production of CMPK2. Furthermore, we recently produced mice that are deficient in CMPK2 and are now expanding this line of mice so that we will be able to crossbreed them with a mouse model of Alzheimer’s disease. This will enable us to combine our discoveries and determine if shutting down CMPK2 will be useful for halting the progression of the neuronal degeneration and dementia that characterize Alzheimer’s disease.

Dr. Philip Copenhaver (Oregon Health & Science University) was the recipient of a $75,000 grant from The CART Fund in 2017. (Posted August 12, 2019)

“A Novel Modulator of membrane estrogen receptors for treating AD”

During Alzheimer’s Disease, toxic amyloid proteins accumulated in the brain of patients, but clinical trials designed to reduce amyloid have failed.  As an alternative strategy, we have tested a promising compound called STX that might protect patients by supporting the overall health of brain cells.  STX was originally developed as a replacement for estrogen (the female sex hormone).

Like estrogen, STX can prevent the loss of brain cells in an animal model of stroke.  However, STX does not cause the side effects of estrogen that can harm AD patients, suggesting that it can be used to treat both men and women. In early studies, we discovered that STX protected isolated nerve cells against toxic amyloid, in part by improving the production of cellular energy in the brain. 

With support from The CART Fund, we have explored the protective mechanisms of STX. We found that STX activates an important signaling cascade inside nerve cells (called “PI3K” signaling), which stimulates protective responses in other diseases. In particular, we found activation of PI3K signaling by STX improves mitochondrial function, which generate cellular energy needed to keep nerve cells healthy. 

Using CART funds, we also showed that STX can be safely fed to mice for many months (with no side effects). More importantly, treatment with STX improved behavioral responses and memory in a mouse model of AD. The results of our CART project will provide the foundation for a more extensive analysis of STX, with the long-term goal of advancing STX to clinical trials.

Dr. Christelle Anaclet (University of Massachusetts Medical School) was the recipient of a $250,000 grant from The CART Fund in 2017. (Posted July 10, 2019)

The goal of our work is to develop new mouse models to test the role of sleep in memory, both in healthy individuals and in Alzheimer’s Disease models. Alzheimer’s Disease is associated with both sleep impairment and memory deficit. Two major sleep stages are distinguished, slow-wave-sleep (SWS) and rapid eye movement sleep (REMS).

Historically, loss-of-sleep experiments have suggested a beneficial role of SWS in declarative memory and a beneficial role of REMS in non-declarative memory, but these experiments have been unable to describe what it is about sleep that might enhance these functions.

We are testing the hypothesis that sleep enhancement can improve memory function and reverse the deficits associated with Alzheimer’s Disease, using – for the first time – gain-of-sleep experiments. We have previously validated a mouse model of SWS enhancement.

The CART grant allowed us to validate a mouse model of REM sleep enhancement, establish cognitive testing in our lab, and produce and validate two triple transgenic mouse models to enhance sleep in Alzheimer’s Disease mice.


Dr. Wenjie Luo (Cornell University) was the recipient of a $100,000 grant from The CART Fund in 2015. (Posted December 17, 2018)

Investigating the role of microglia in tau clearance in Alzheimer’s Disease

During these three years supported by the CART Fund, we have performed the experiments as proposed in the original plan and obtained extensive knowledge about how microglia degrade pathological tau based on the following scientific results. In our original proposal, we planned to investigate the mechanism how brain phagocytic cell microglia play a role in the brain clearance of tau, a pathologic protein that can spread from neuron to neuron and form deadly neurofibrillary tangles in Alzheimer’s Disease (AD). The ultimate goal of this research project is to search for therapeutic agents that can enhance the tau-degrading activity of microglia, thus reducing tau pathology and preventing cognitive decline in AD patients. During these funded years, we have made scientific discoveries about the mechanism how pathologic tau is internalized and degraded by microglia. We also identified the activation conditions as well as genetic factors that regulate this tau degradation process in microglia. Finally, we have collected data showing that brain microglia approach pathologic tau injected into the mouse brain (see figure to right).

We deeply appreciate the tremendous help provided by The CART fund to help us initiate and extend this project. The scientific observation and conclusion are significant for the AD basic research and also impact greatly in AD therapeutic study.

Dr. Thomas Anastasio (University of Illinois) was the recipient of a $50,000 grant from The CART Fund in 2016.  (Posted December 3, 2018)

CART-funded Computational Research Identifies Potentially Effective Drug Combinations for Alzheimer Disease Treatment:

Alzheimer Disease (AD) remains the leading neurological killer, and “the amyloid hypothesis” remains the leading theory of AD. “Amyloid-beta” is the abnormal protein fragment that accumulates in AD brains and that composes the “amyloid plaques” that are found in AD brains.  The amyloid hypothesis says that amyloid-beta causes AD – but it’s wrong! Treatment strategies aimed at reducing amyloid-beta don’t work!

These and other observations show that amyloid-beta is not the only factor that underlies AD. Instead, research shows that AD is “multifactorial” in that it is caused by many different factors, not just amyloid-beta. And because AD is multifactorial, it probably should be treated with combination therapies that target multiple factors. But this poses a problem because the design of combination therapies to treat multifactorial diseases is highly complex. One project funded by the CART foundation addressed this problem.

This computational project proceeded in three stages. The first was to create a computer model of the interactions between many of the key factors that are known to underlie AD. The second was to analyze an extensive database containing data on real AD patients, which also included lists of all the drugs they took. Fortunately, the patients in the database took commonly prescribed drugs in many different combinations, so the third stage was to see if the benefits to the patients of specific combinations of drugs could be correlated with predictions of the efficacies of those same drug combinations from the computer model.

Statistical analysis showed that the database benefits and the predicted model efficacies were significantly correlated! That meant that the computer model was picking up something real about the complex, multifactorial, pathological processes that underlie AD. The main payoff was that the agreement between the database and the model could be used to identify new drug combinations that might actually be effective in treating AD, and they involve commonly used drugs that elderly people already take.

The approach identified many potentially effective combinations, but combinations of certain drugs rose to the top. They included the NSAIDs ibuprofen (and similar drugs) and aspirin, along with drugs that are commonly used to treat high blood pressure. This CART funded project suggests that these combinations should be explored experimentally and/or clinically to see if they really can be effective weapons in the fight against AD.

Dr. Frank Sharp (University of California at Davis) was the recipient of a $125,000 grant from The CART Fund in 2016. (Posted October 23, 2018)

We were the first to propose that lipopolysaccharide (LPS, found in the wall of all Gram-negative bacteria) could play a role in causing sporadic Alzheimer’s disease (AD). This is based in part upon recent studies showing that: Gram-negative E. coli bacteria can form extracellular amyloid; bacterial-encoded 16S rRNA is present in all human brains with over 70% being Gram-negative bacteria; ultrastructural analyses have shown microbes in erythrocytes of AD patients; blood LPS levels in AD patients are 3-fold the levels in control; LPS combined with focal cerebral ischemia and hypoxia produced amyloid-like plaques and myelin injury in adult rat cortex. Moreover, Gram-negative bacterial LPS was found in aging control and AD brains, though LPS levels were much higher in AD brains. In addition, LPS co-localized with amyloid plaques, peri-vascular amyloid, neurons, and oligodendrocytes in AD brains. Based upon the postulate LPS caused oligodendrocyte injury, degraded Myelin Basic Protein (dMBP) levels were found to be much higher in AD compared to control brains. Immunofluorescence showed that the dMBP co-localized with β amyloid (Aβ) and LPS in amyloid plaques in AD brain, and dMBP and other myelin molecules were found in the walls of vesicles in periventricular White Matter (WM). These data led to the hypothesis that LPS acts on leukocyte and microglial TLR4-CD14/TLR2 receptors to produce NFkB mediated increases of cytokines which increase Aβ levels, damage oligodendrocytes and produce myelin injury found in AD brain. Since Aβ1-42 is also an agonist for TLR4 receptors, this could produce a vicious cycle that accounts for the relentless progression of AD. Thus, LPS, the TLR4 receptor complex, and Gram-negative bacteria might be treatment or prevention targets for sporadic AD.

Dr. Yueming Li (Memorial Sloan Kettering Cancer Center, NYC) was the recipient of a $250,000 grant from The CART Fund in 2016.  (Posted October 15, 2018)

Alzheimer’s disease (AD) is characterized by an aggregation of toxic proteins that build up in the brain. Normally, a process called autophagy allows cells to rid themselves of any “garbage” they hold. We define that garbage as proteins that are unnecessary or dysfunctional components.

When people have AD, the autophagy process fails with two specific proteins, toxic amyloid beta plaques and tau tangles. The cells cannot rid themselves of these proteins. In fact, we can see the cumulative toxicity when we examine the brain of a patient with AD who has passed away.

AD is caused by progressive brain cell death over time. We believe that AD, as well as other neurodegenerative disorders, can be treated by addressing breakdown in the cells’ autophagic pathways. If the pathways can return to functioning properly, the cells may able to engage in their own housekeeping and free themselves of the damaging detritus.

My team is looking at ways to clear the autophagic pathways in these cells. Because of advanced technologies, we can use chemical libraries to screen for and identify existing drug molecules that promote the proper function of these cleaning pathways.

With this generous grant, we have identified such molecules. We have also synthesized the molecules because in doing so we can improve their drug properties.

As we move forward, we’re continuing to develop this class of molecules as drug candidates to further understand, and possibly treat, AD. Our progress has brought us closer to our goal of bringing relief to individuals and families living with this difficult and progressive neurological disease.