Brain Entry and Exit Consortium

The brain is a remarkable but fragile organ with limited ability for self-renewal following injury; it also has a very high metabolism, using approximately 20% of all energy consumed by the body and producing significant waste. Consequently, it has evolved a complex system of barriers to control the entry and exit of materials and maintain its delicate healthy balance. The CureAlz Brain Entry & Exit Consortium is investigating how each component of the brain’s entry and exit structures, along with the cerebrospinal fluid that flows through the brain, must function together to maintain health. Restoration of a single disrupted pathway may be sufficient to rescue detrimental aspects of numerous routes, offering an attractive therapeutic intervention to prevent widespread impairment.

The consortium’s members bring cutting-edge expertise to the study of these interconnected structures. They are examining how the blood-brain barrier must be selective, rather than simply exclusive, and how clearance of debris through the meningeal lymphatics system is vital to immune communication between the brain and periphery. They also are assessing how cerebrospinal fluid flow dynamics affect both exit and entry, and how it may be altered in AD as amyloid beta pathology builds up. The only drug currently FDA approved for its impact on AD pathology is delivered from the blood across the blood-brain barrier; it then tags amyloid beta for clearance from the brain. The work of the Brain Entry & Exit Consortium offers a powerful opportunity to identify new therapeutic possibilities and improve those already in development.

BRAIN ENTRY & EXIT CONSORTIUM: FUNDED RESEARCHERS

• Helene Benveniste, M.D., Ph.D., Yale University School of Medicine
• Se Hoon Choi, Ph.D., Massachusetts General Hospital
• Richard Daneman, Ph.D., University of California, San Diego
• Ali Ertürk, Ph.D., Helmholtz Munich
• Fanny Herisson, M.D., Ph.D., Massachusetts General Hospital
• Roger Kamm, Ph.D., Massachusetts Institute of Technology
• Jony Kipnis, Ph.D., Washington University School of Medicine in St. Louis, Consortium Chair
• Fernanda Marques, Ph.D., Minho University School of Medicine
• Laura Santambrogio, M.D., Ph.D., Weill Cornell Medicine
• Allen Tannenbaum, Ph.D., Stony Brook University

DEVELOPMENT OF SMALL MOLECULE INHIBITORS OF CHOLESTEROL 25-HYDROXYLASE

Identifying novel therapeutic targets for Alzheimer’s disease is a high priority. The two drugs most recently approved for use in treating Alzheimer’s disease share the same target the same target — beta amyloid aggregates — and provide only limited therapeutic benefit to a relatively small percentage of the patient population. Molecules and brain cells involved in the innate immune response — the body’s first line of defense against harmful substances — are of particular interest given the strong evidence implicating neuroinflammation in Alzheimer’s disease. Indeed, many known genetic risk factors for late-onset Alzheimer’s are primarily expressed by brain immune cells (astrocytes and microglia). Two of these genes, APOE and TREM2, are involved in the transition of microglia from a homeostatic sentry state to a DAM (disease-associated microglia) state in response to beta amyloid and cell debris. Both also play key roles in cholesterol and lipid handling. As strategies targeting APOE and TREM2 make their way through the clinical drug discovery and trial pipeline, the field continues to search for other promising candidates that can positively intervene in this complicated immune response.

The team is pursuing a drug discovery effort that leverages cutting-edge computational (in silico) modeling to address the lack of structural information about the target. Molecules affect a protein’s activity by physically interacting with them, so knowing the structure of a protein and how it changes its conformation in its biological environment is vital to predicting what molecules might be able to access and bind to different positions on the protein. Historically, the techniques to determine protein structure were expensive and difficult, and very few proteins had been well defined. Scientists had to repeatedly combine a protein of interest with as many of its potential binding partners as the lab could afford to test and observe the outcomes to find pairs that interacted, a low-potential and tedious approach given the immense number of potential partners. In silico modeling provides an alternative approach to studying these proteins with limited structural information.

In their first year, the Cashikar and Elgendy team, in collaboration with Dr. Lamees Hegazy’s lab, used Alphafold—an AI-driven tool to predict protein structures that won its developers the 2024 Nobel Prize in Chemistry—to establish a well-predicted experimental physical structure for CH25H. After the refinement of the structure, they performed virtual (in silico) modeling of the structure and its molecular dynamics in relevant biological conditions (e.g., embedded in bi- and monolayer cellular membranes). They then performed in silico screening of over one million candidate molecules for likely binding affinity to CH25H’s predicted structure and available binding sites. Thanks to these computational methods, what would have taken years with old methods (if even possible) was completed in just the first year of this project. In parallel preparation for the eventual biological validation of the in silico hits, the team also developed assays to quantify both the amount of CH25H and the enzymatic activity of CH25H in primary mouse microglia. These assays will be critical to assessing the efficacy of candidate compounds to inhibit CH25H and will be used during the remaining screening and validation experiments.

The team is now synthesizing and experimentally validating the binding activity of the lead candidates from the screening in cell culture, including measuring which ones best inhibit CH25H activity. They will also be evaluated for target specificity, drug-like properties, and likely brain penetrance. Building on their earlier in silico and in vitro work, they will then advance to in vivo studies that will bring the work closer to human biology. These studies will test whether their top candidate can delay the onset of neuroinflammation and neurodegeneration in a tau mouse model. The team will measure several outcomes in the mouse brain, including markers of inflammation in microglia and astrocytes, tissue loss, and pathological forms of tau.

Drs. Cashikar and Elgendy ultimately seek to leverage these data to apply for NIH funding to support further small molecule drug discovery efforts, the filing of an Investigational New Drug application, and phase I clinical testing.

DOES A GENETIC MUTATION PREVENT ALZHEIMER’S DISEASE?

In a study exploring the interaction between genetics and Alzheimer’s disease (AD) pathology, researchers discovered how the APOE3 Christchurch (APOE3ch) mutation protects against AD. Through support from Cure Alzheimer’s Fund, an APOE3ch mouse model was created, allowing researchers to investigate the influence of the Christchurch mutation on amyloid and tau pathology. The findings showcase how APOE3ch revs up the efficiency of microglia surrounding amyloid plaques to remove aggregated tau. Although APOE3ch reduced the levels of amyloid beta, its real power was the ability to prevent the spread of tau, which precedes neuronal death and dementia. These findings reinforce the potential of APOE3ch as a genetic shield against AD, and present a novel perspective on the genetic mechanisms leading to AD resilience.

In 2019, a Nature Medicine article described the intriguing case of a woman who, despite inheriting a gene mutation responsible for a rare, early-onset form of AD, escaped developing AD dementia. In fact, she remained cognitively normal into her 70s and only then developed mild cognitive impairment, which lasted until her death from cancer at age 78. Postmortem analysis of her brain showed unusually high levels of amyloid beta but limited levels of tau and neurodegeneration. The key to her resilience appeared to rest with the fact she also had a specific mutation in both copies of the APOE3 gene. This mutation is known as the APOE3 Christchurch (APOE3ch) mutation.

However, because this unique combination of mutations only was found in one person, scientists could not prove that APOE3ch prevented the development of AD dementia. Now, with the development of an APOE3ch mouse model, researchers have been able to study the influence of APOE3ch on amyloid and tau pathology and provide evidence of how APOE3ch safeguards against dementia.

The woman highlighted in the Nature Medicine article was part of a large, extended Colombian family in South America with a high prevalence of early-onset AD due to a mutation in the Presenilin I (PSEN1) gene. This mutation causes the overproduction of amyloid beta. Members of this kindred who inherit the PSEN1 mutation develop symptoms of AD when they enter their 40s, much earlier than the more common late-onset form of AD, which typically affects people in their mid-60s and beyond. Although AD can be characterized as early- or late-onset depending on when clinical symptoms appear, they all share the same classic pathological hallmarks of amyloid plaques and neurofibrillary (tau) tangles.

A significant genetic factor for late-onset AD is the APOE gene, which has three forms:

• APOE2, known to be protective.
• APOE4, which increases risk.
• APOE3, which typically does not influence risk.

In the case of the Colombian woman, a mutation in APOE3 appears to confer remarkable resilience against AD. To study how this was possible, researchers—with support from a grant provided by Cure Alzheimer’s Fund—created a mouse model containing two copies of APOE3ch and modified it to overproduce amyloid beta. Since mice don’t naturally replicate the tau pathology seen in humans, a small amount of human tau was introduced into the brains of the mice which, in the presence of amyloid beta, would pathologically spread through the brain.

However, this is not what researchers saw in the APOE3ch model. Like the Colombian woman, these mice had extensive amyloid beta throughout their brains but very little tau. The Christchurch mutation energized the brain’s natural immune cells, the microglia, to engulf and degrade tau more efficiently. They were so efficient that tau was prevented from spreading.

This discovery is important because it showcased how APOE3ch blocked the transition between amyloid beta buildup and tau spread. In AD, amyloid beta accumulates in the brain for decades before symptoms appear. The authors of this study, along with many other scientists in the Alzheimer’s disease research community, theorize that the buildup of amyloid beta eventually triggers tau to tangle and spread throughout the brain. As a result, the brain becomes inflamed, neurons die, and memory and cognition suffer. The question of why amyloid beta triggers tauopathy is not clearly understood. However, the spread of tau is a greater indicator of cognitive impairment than the accumulation of amyloid beta. By impacting the activity of microglia, APOE3ch blocked the destructive cascade that leads to AD dementia.

While APOE3ch modestly decreased the amount of amyloid beta present compared with mice that didn’t have the Christchurch mutation, its effect on microglia holds the most promise for disease prevention. Developing a therapy to mimic APOE3ch’s impact on microglia efficiency could harness its power to render amyloid beta accumulation harmless and prevent AD-associated dementia.

Published in Cell:

APOE3ch Alters Microglial Response and Suppresses Ab-Induced Tau Seeding and Spread

Marco Colonna, M.D., Washington University in St. Louis

Jason D. Ulrich, Ph.D., Washington University in St. Louis

David Holtzman, M.D., Washington University in St. Louis

THE FIRST BIOMARKER FOR TAU TANGLES

Researchers have identified a promising biomarker, MTBR-tau243, that could transform the early detection of Alzheimer’s disease (AD). This biomarker, measured in the cerebrospinal fluid, detects the type of tau found in tangles that accumulate in the brain. Unlike the currently available phosphorylated tau markers, which are more sensitive to rising amyloid beta levels, MTBR-tau243 directly mirrors tau tangle pathology, offering a clearer picture of disease progression. The strong association of MTBR-tau243 with tau tangles and cognitive decline positions it as a prime candidate for diagnosing AD, tracking its advancement and evaluating tau-targeted therapies.

As Alzheimer’s disease (AD) unfolds, it is the spread of tau tangles, an event that happens years to decades after amyloid plaques accumulate in the brain, that more immediately precedes cognitive decline. Consequently, tau has become a focal point for emerging therapies aimed at halting AD’s advance. This focus requires tools that can track tau’s harmful spread and monitor the impact of drug interventions. While positron emission tomography (PET) imaging accurately detects tau tangles in the brain, its high cost and the need for specialized facilities limit its widespread, routine use. Fluid biomarkers are a more feasible option, but pinpointing those that accurately reflect tau levels in tangles has been difficult. However, after an extensive search, scientists have identified MTBR-tau243 as the first fluid biomarker that reliably tracks tau accumulation in the brain.

Tau proteins are a normal part of the internal support structure of brain cells. With AD, tau is chemically changed in a process called phosphorylation. While some phosphorylation is normal, in AD, tau becomes overly phosphorylated, causing it to forgo its structural responsibilities and, instead, stick to itself and form insoluble aggregates known as tau tangles.

Years before tau tangles form and AD symptoms manifest, amyloid beta begins to accumulate in the brain. Usually, amyloid beta is cleared out of the brain, where it is detectable in the blood and cerebrospinal fluid (CSF). However, when amyloid beta forms sticky plaques during AD, less of it is flushed out, and the amyloid beta concentration in biofluids decreases. Intriguingly, as more and more plaques form in the brain, the amount of phosphorylated tau (or p-tau) in biofluids increases. The level of p-tau continues to rise in parallel with amyloid plaque formation until the moment tau tangles form in the brain. At that point, p-tau levels in biofluids plateau and do not respond to the rising accumulation and spread of tau tangles. Thus, although seemingly counterintuitive, p-tau biomarkers provide information about amyloid plaques, not tau tangles. While amyloid beta and p-tau biomarkers continue to be instrumental in the early diagnosis of AD, there is a pressing need for a reliable fluid biomarker for tau tangles.

To pinpoint biomarkers that indicate the emergence and proliferation of tau tangles, researchers turned to the extensive patient data found in two AD cohorts. The cohorts included individuals representing a full range of disease stages and, with corresponding amyloid and tau PET imaging, providing a rich dataset for analysis. Within these groups, the biomarker MTBR-tau243 stood out after analyzing the data as it reliably mirrored the presence of tau tangles seen in PET scans while showing no association with amyloid beta. This finding is particularly significant considering the strong link between tau accumulation and cognitive deterioration.

While MTBR-tau243 alone was a significant marker for tau pathology and cognitive decline, researchers found that its predictive power was enhanced when combined with another biomarker, p-tau205, in CSF. This combination approached the diagnostic accuracy of the more expensive PET scans.

These findings have the potential to advance diagnostic standards for AD. Currently, tau pathology is assessed through costly PET scans or by evaluating p-tau levels in CSF. The specificity of MTBR-tau243 for the presence and progression of tau tangles makes it a strong candidate for measuring tau pathology while being less expensive and more accessible than a PET scan. Discovering a tau biomarker that precisely traces tau pathology and predicts cognitive decline marks a significant advancement in AD diagnostic and monitoring tools.

Published in Nature Medicine: CSF MTBR-tau243 is a specific biomarker of tau tangle pathology in Alzheimer’s disease
David M. Holtzman, M.D., Washington University School of Medicine in St. Louis
John C. Morris, M.D., Washington University School of Medicine in St. Louis
Rik Ossenkoppele, Ph.D., Amsterdam University Medical Centers, The Netherlands; Lund University, Sweden
Oskar Hansson, M.D., Ph.D., Lund University, Sweden
Randall J. Bateman, M.D., Washington University School of Medicine in St. Louis

Mitochondrial Alzheimer’s Risk Factors Control APOE Expression and Secretion

In this project, funded by CureAlz, the Faundez lab is exploring a hypothesis that would upend the field’s current thinking about the role of mitochondrial dysfunction in Alzheimer’s disease onset and progression.

Mitochondria are the powerhouses of the cell, producing energy needed, in a process called Electron Transport Chain (ETC) for all essential functions. With AD, mitochondria become dysfunctional. This means less energy, more stress, and more damage in cells, especially brain cells.

While many researchers believe the risk gene APOE4 causes classic Alzheimer’s features (like amyloid beta and tau buildup), which in turn lead to mitochondrial failure, the Faundez team’s data suggest the reverse may be true.

The team disrupted the mitochondrial function in brain cells. And in mouse models, APOE levels rose after the increase in amyloid and APP — hinting that mitochondrial dysfunction might actually drive APOE increases, not the other way around. 

APOE is made by different cell types and influences other various pathways. Of its three variants, APOE4 is associated with the strongest risk of AD, while APOE3 is the strongest protective genetic risk factor; APOE2 is considered neutral risk. While the functions of APOE in disease are not fully understood, Faundez’s finding suggests that rising APOE levels could actually be a protective cellular response to mitochondrial stress, which then might later contribute to the disease.

To explore this further, the team is examining how different electron transport chain components influence the APOE expression in the three major APOE-producing brain cells: astrocytes, microganglia and neurons. So far, their finding show that astrocytes, which are normally the highest producing APOE cells, are also the most sensitive to mitochondrial dysfunction.

During the project’s first year, the lab created a set of gene-edited astrocyte stem cells lacking key Electron Transport Chain (ETC) and APOE genes.

If APOE is truly protective, they’d expect cells lacking ETC and APOE to struggle the most. But if cells in mitochondrial distress do better without APOE, that suggests the APOE response may not be so helpful afterall. So far, they’ve found that the astrocyte cells that have the APOE gene deleted actually do better, and those cells with APOE-3 were more resilient than those with APOE4.

The results indicate that while APOE increase may be a protective response at first, it is harmful in the long run; and that not all APOE is the same. APOE3 seems more neutral or mildly helpful; APOE4, the risky type for AD, may make things worse.

Their ongoing work aims to clarify how mitochondrial stress affects APOE—and whether protecting mitochondrial function could limit APOE4’s harmful effects and slow Alzheimer’s progression.

Insights from the Aging Mouse Brain

Aging is the biggest risk factor for developing sporadic Alzheimer’s disease. But the changes that take place in the human brain as we age, leading to increased risk, remain largely unknown. A new investigation set out to identify the molecular underpinnings that occur in the aging brain. As a result, it was discovered that previously unsuspected regions of the brain are more vulnerable to aging, and two anti-aging treatments can rejuvenate the brain in unexpected ways.

The use of magnetic resonance imaging (MRI) has revealed that the human brain does not age uniformly. While these imaging studies provide a broad view of structural changes in the brain, they do not reveal the molecular underpinning of the aging process. Instead, understanding how gene activity changes—which genes are turned on or off—may be a better indicator of how the brain ages.

To gain systematic insight, in a recent study funded by CureAlz, researchers developed a workflow to assess gene activity changes across the whole brain of male and female mice, resulting in the most comprehensive atlas of the aging brain. Fifteen regions in both brain hemispheres were studied in mice, whose ages were equivalent to 20–80 human years. A set of 82 genes whose activity changed over time across all regions was identified, and this common aging signature was compared across different regions as the mice aged. The set included an increase in activity of genes regulating inflammation, and a drop in that of genes regulating protein quality control.

Comparing the common aging signature across brain regions revealed that certain regions were more vulnerable to aging, while others remained relatively unchanged. The earliest and strongest gene activity changes occurred in regions rich in white matter, such as the corpus callosum and cerebellum, two areas often overlooked in the research of aging. These changes were observed in mice whose age was equivalent to a person in their 50s. Roughly half of the brain is composed of white matter, which plays a crucial role in transmitting signals across the brain and primarily is made up of myelinated nerve fibers. White matter vulnerability to aging is a new discovery, challenging previous focus on grey matter-rich regions, like the cortex or hippocampus, whose aging was slower in comparison (grey matter contains the brain’s neuronal cell bodies).

The study also revealed that aging impacts cells in the brain differently based on their location. Glial cells, which play a role in inflammation, had the strongest changes of the common aging signature throughout the brain, with white matter glia aging more rapidly than their cortical counterparts. This highlights the possibility that white matter glial cells may be driving overall brain aging. Meanwhile, neurons showed region-specific aging signatures, reflective of the neurons and regions’ functional roles.

Designed to understand how aging may be reversed at the molecular level, the study also included the testing of two interventions. The first was a dietary intervention of caloric reduction over a four-week period. The second was the injection of plasma from young mice into older mice. While both interventions have been proposed to redress aging, they had very different effects on gene activity across various brain regions, suggesting that multiple potential pathways to redress aging could be in play. Results showed that the dietary intervention activated genes linked to circadian rhythms across the whole brain, an observation that is in line with prior research suggesting that the effects of caloric reduction on longevity may depend on the timing of food intake. In contrast, the plasma intervention had a direct effect on the common aging signature, and reversed it in several different brain regions.

The study also examined whether genes linked to disease also changed in region-specific ways with aging. The results revealed that the activity of the APOE gene (whose APOE4 variant is the strongest genetic risk factors for sporadic AD) changed most profoundly with age regions not unusually associated with AD. “The region-specific differential regulation of such genes could be an additional factor modulating disease risk,” the authors wrote.

In summary, the findings of the research suggest that regions of the brain age differently on the molecular level. This provides insights into why certain regions of the brain are more susceptible to disease, and provides a valuable resource for future discovery of targets for novel Alzheimer’s therapeutics. An outcome of the study is available to the entire science community and includes an online interactive data visualization resource of the results to facilitate future scientific advancements.

Published in Cell: Atlas of the Aging Mouse Brain Reveals White Matter as Vulnerable Foci

Tony Wyss-Coray, Ph.D., Stanford University

EXERCISE AND ALZHEIMER’S DISEASE: THE PROTECTIVE POWER OF IRISIN

Engaging in physical exercise may bolster overall health and act as a safeguard for our brain, particularly with respect to Alzheimer’s disease (AD). Using a 3D cell culture model designed to mimic AD pathology, scientists discovered that the hormone irisin, which is released from the muscles during exercise, increases the production of neprilysin in specific brain cells known as astrocytes. Upon its release from astrocytes, neprilysin breaks down amyloid beta, the protein that forms harmful plaques in AD. Consequently, the levels of amyloid beta were significantly reduced. These results cast exercise and irisin in a new light as potential deterrents against the development of Alzheimer’s disease, and highlight a new therapeutic angle to prevent amyloid beta accumulation.

Recent research has revealed a fascinating connection between physical exercise and Alzheimer’s disease (AD). It centers on a small protein called irisin. Produced during physical activity, irisin plays a pivotal role in brain health by potentially mitigating one of AD’s hallmark features—the accumulation of amyloid beta plaques.

Discovered in 2012, irisin gets its name from Iris, the Greek goddess who carried good news from the gods to humans. Echoing the role of its namesake, irisin also acts as a messenger, but in this case, as a chemical that ferries the beneficial effects of exercise throughout the body. Muscles release irisin into the bloodstream during physical exertion, and from there, irisin travels the body, carrying out its effects, such as converting white fat into energy-burning brown fat, strengthening bones and controlling sugar levels. Its reach even extends to the brain, where it is thought to sharpen cognitive abilities and play a role in AD prevention.

Irisin is present in both human and mouse brains, and its levels are lower in patients with AD and mouse models of the disease. Deleting the irisin gene in mice removes the cognitive benefits usually associated with exercise, partly because it interferes with the birth of new neurons in the hippocampus, a region of the brain important for learning and memory. On the other hand, in the same mouse models, increasing irisin levels in the bloodstream enhances cognition and reduces brain inflammation.

In addition to its ability to boost cognition in mouse models, physical exercise also reduces amyloid beta levels and inflammation in the brain. However, the connection between exercise, irisin and reduced amyloid beta levels is unclear.

To determine whether there is a connection, scientists turned to a 3D cell culture model of AD known as Alzheimer’s in a Dish™, created initially through a grant provided by Cure Alzheimer’s Fund. This model can be considered a mini-brain that fully replicates Alzheimer’s pathology, including amyloid plaques, tau tangles and neuroinflammation.

Introducing irisin to the model reduced amyloid beta levels by targeting brain cells known as astrocytes to produce more neprilysin, an enzyme that digests amyloid beta. Irisin binds to a specific receptor on astrocytes, which triggers a cascade of signaling events within the cell, culminating in the increased production of neprilysin. Astrocytes release neprilysin into their environment, allowing the enzyme to find and digest amyloid beta.

These findings suggest that increasing irisin levels in humans could combat the buildup of amyloid beta, which, according to the amyloid cascade theory of AD, eventually would prevent the trigger of tau tangles and cell death. The potential of irisin as a therapeutic agent is underscored by its ability to cross the blood-brain barrier. However, in the 3D cell models used in this study, irisin only decreased the amyloid burden in the earliest stages of the disease. When added to 3D cell models where the disease was more advanced, the levels of amyloid beta did not change. Therefore, treating patients with irisin in the early stages of the disease—or before symptoms appear—would be optimal.

This research reinforces the value of physical exercise in maintaining cognitive health and opens new pathways for developing treatments that leverage the body’s natural response to exercise. As scientists continue to unravel the complexities of AD and the potential of proteins like irisin, the hope is that such discoveries will lead to effective interventions for this debilitating disease.

Published in Neuron:
Irisin Reduces Amyloid-b by Inducing the Release of Neprilysin from Astrocytes Following Downregulation of ERK-STAT3 Signaling

Joseph Park, Ph.D., Massachusetts General Hospital

Luisa Quinti, Ph.D., Massachusetts General Hospital

Doo Yeon Kim, Ph.D., Massachusetts General Hospital/Harvard Medical School

Christiane Wrann, D.V.M, Ph.D., Massachusetts General Hospital

Rudolph Tanzi, Ph.D., Massachusetts General Hospital/Harvard Medical School

Se Hoon Choi, Ph.D., Massachusetts General Hospital/Harvard Medical School