Blood-Brain Barrier Transport in Aging and Alzheimer’s Disease

Session I: Tools and Models to Study BBB Transport and Permeability in Aging and AD/ADRD

Chair: Peter Searson, Ph.D., The Johns Hopkins University

Generation of HiPSC-derived Brain Microvascular Endothelial Cells Using a Combination of Directed Differentiation and Transcriptional Reprogramming Strategies
Dritan Agalliu, Ph.D., and Andrew Sproul, Ph.D., Columbia University Irving Medical Center

The brain is highly vascularized, closely associating between blood vessels, neurons and glial cells. Each neuron is an average of 10 μM away from a blood vessel. In healthy brains, this blood remains confined to the vasculature; in disease, the BBB becomes dysfunctional, and molecules can leak into the brain. While researchers have created many tools for studying the interactions between endothelial cells, neurons, and astrocytes in mice, they are just beginning to develop such tools for use in humans. At the forefront of this nascent technology is the use of iPSCs to generate different cell types and create a vascularized, three-dimensional (3D) model of the BBB with the ultimate goals of identifying therapeutic drugs and developing tools for personalized medicine.

To study the neurovascular unit (NVU), composed of endothelial cells, neurons, and glia that regulate blood flow, the researchers relied on transcriptomics to ensure they were generating cells with high-molecular fidelity and good barrier function. Drs. Agalliu and Sproul transformed iPSC-derived BBB-primed endothelial cells (bpECs) into reprogrammed brain microvascular endothelial cells (rBMECs) through overexpression of BBB-critical transcription factors FOXF2/ZIC3, which showed elevated expression of tight junction-related proteins and responded to pro-inflammatory and disease-relevant stimuli such as oligomeric Aβ42 via the upregulation of certain cytokines and leukocyte adhesion molecules (LAMs). The rBMECs also showed decreased leakage of biocytin and 70-kilodalton dextran, functional P-glycoprotein activity, and reduced transcytosis compared to primary human BMECs. The researchers also generated human iPSC (hiPSC)-derived pericytes (PCs) and astrocytes (ACs). They showed that the rBMECs could form a tubule in a 3D microfluidic system and could generate a 3D NVU when co-cultured with induced PCs and ACs.

Gaps include the low efficiency and scalability (although still useable) of generating rBMECs, the need to improve the reproducibility of the 3D NVU system, the immaturity of iPSC-derived NVU cells compared to the human brain, the long-term functioning of the 3D NVU system, and the unknown congruence of the iPSC-based system to the in vivo human NVU. Opportunities include the ability to test genetic vs. environmental risk factors for disease, assess the role of the immune system, blood, and sera on the NVU, model a large number of neurologic and psychiatric diseases, screen drugs for precision medicine on a moderate scale, and integrate other cell types into the NVU model.

Blood-Brain Barrier Transport in Tissue-Engineered Models
Peter C. Searson, Ph.D., Johns Hopkins University

Recent advances in stem cell technology and biomaterials have led to the rapid development of a new generation of tissue-engineered BBB models. The phenotype and function of the brain microvascular endothelial cell varies along the arterial and venous axis leading to the concept of a neurovascular complex (NVC) instead of a universal neurovascular unit (NVU) and highlighting the importance of supporting cells and the local microenvironment. An important gap in the development of BBB models is that innovation has outpaced efforts to ensure accuracy and validate results. However, the observed characteristics in humans to be used as benchmarks are limited or inconsistent. Therefore, there is an urgent need to establish a consensus database of human benchmarks to assess model accuracy.

Dr. Searson identified how scientists can use these models to create a very reductive approach to disease modeling and screening therapeutics. Many diseases of the brain are heterogeneous, with a wide range of etiologies derived from multiple risk factors, creating many possible combinations of stressors or perturbations on the BBB. To understand the mechanism of disease progression, researchers need to understand how these perturbations contribute to BBB dysfunction. One of the simplest examples is Huntington’s disease (HD), for which genetics is the major risk factor. Microvessel models with induced BMECs (iBMECs) that carry the HTT mutation show functional differences in cell turnover, immune cell adhesion, and angiogenesis compared to wild type microvessels. In a more complex example, in familial AD (FAD) models, iBMEC microvessels showed no changes in permeability when perfused with sera from young individuals. However, perfusion with serum from aged individuals resulted in a significant increase in permeability. This approach highlights the opportunities afforded by a reductive approach to determining the impact of perturbations associated with disease risk factors on BBB function. The next step, according to Dr. Searson, is to develop models for the risk factors for cerebrovascular and neurodegenerative diseases to assess the mechanisms of BBB dysfunction and to screen for new therapies.

Quantification of Blood-Brain Barrier Permeability In Vivo and In Vitro
Bingmei M. Fu, Ph.D., The City College of New York

In the brain, tiny capillary and post-capillary venules are responsible for material exchange between the vasculature and neural cells. The glycocalyx, a dense, gel-like layer made of carbohydrates, covers the luminal surface and tight junctions of the vascular wall, creating a major barrier for large molecules by acting as a molecular sieve while tight junctions are the major barriers for small molecules and ions. Dr. Fu showed that immunostaining can be used to identify and quantify the glycocalyx and junction proteins in two in vitro BBB models: human cerebral microvascular endothelial cells and mouse brain microvascular endothelial cells with and without astrocytes. The structure of the glycocalyx and junction proteins determines their transport functions. The transport coefficients for passive transport across membranes or paracellular transport across the BBB can be calculated using Starling’s law for water filtration and the Kedem-Katchalsky equation for solute flux, respectively.

Dr. Fu identified several methods to quantify BBB permeability in vivo, including multiphoton microscopy, which measures both BBB permeability to a solute and the solute effective diffusion coefficient in brain tissue. The permeability of pial microvessels (the vasculature servicing the inner layer of the meninges) can also be quantified in vivo using epi-fluorescence inverted microscopy. For BBB samples grown on a Transwell filter, Dr. Fu used a special Ohm meter to measure the trans-endothelial electrical resistance (TEER). Calculating the inverse of TEER indicates the BBB permeability to ions. The BBB permeability to a solute can also be determined by measuring the flux of the fluorescently labeled solutes across the BBB grown on a Transwell filter

Dr. Fu said that it will be important to increase BBB permeability to drug delivery using non-invasive approaches, such as transcranial electric current, magnetic stimulation, and FUS stimulation. She says it will also be important to determine BBB permeability in AD and aging animal models and identify drugs that can reverse the dysfunctional BBB dynamics. Mathematical modeling will help identify the limits of BBB transport and the parts of the brain most likely to be compromised by AD and/or aging. Improvements to high-resolution optical microscopy will allow deeper penetration of the brain.

Pharmacokinetic Analysis of BBB Integrity and Permeability in Animal Models
Ulrich Bickel, M.D., Texas Tech University

Scientists have been working to measure BBB permeability for decades. The transport can be expressed in terms of an uptake clearance. Techniques in animal models include carotid artery perfusion and intravenous (IV) injection techniques. Brain uptake is either analyzed at a single sampling time, by the multiple time point graphical method, or by compartmental analysis. Multiple time graphic analysis (Patlak method) is used in particular with non-invasive in vivo techniques like Positron Emission Tomography (PET) scans and dynamic contrast enhanced MRI. Unidirectional brain uptake of permeability markers is assumed to hold during short experimental durations and in the absence of active efflux mechanisms.

Dr. Bickel spoke of how both low-molecular-weight, hydrophilic substances (such as radiolabeled or stable-isotope labeled sucrose, mannitol, or sodium fluorescein) and high-molecular-weight markers (including Evans blue, inulin, and dextrans) are being used as markers for BBB integrity. Evans Blue is problematic because of its tight, yet non-covalent binding to albumin and other proteins, leaving a small free fraction, which makes quantification interpretation of the uptake mechanism unreliable. Sodium fluorescein is widely used as a paracellular marker due to its simple and sensitive quantification by fluorescence detection. Because its protein-bound fraction in plasma can vary, the free fraction must be measured for accurate analysis of BBB permeability. Radiolabeled sucrose has been used since the 1960s for its metabolic stability and absence of protein binding. Undefined, lipophilic contaminants and radioisotope licenses can limit its utility. Stabile isotope-labeled sucrose can be quantified with ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) with high sensitivity and specificity. Dr. Bickel’s work is approaching the true BBB permeability values of sucrose, although accurate experimental measurement of brain uptake clearance (Kin) is challenging. Scientists need to correct for brain intravascular content to prevent the overestimation of Kin. With multiple time point analysis of BBB permeability, the determination of the proper experimental duration is critical. Dr. Bickel has also been working to detect differences in BBB permeability between awake animals and those under different types of anesthesia. Future questions to answer include how to detect age-dependent changes in BBB permeability, whether transgenic AD models lead to increased sucrose permeability, and whether these methods can be adapted to measure permeability in small regions of the brain.

Session I Discussion

BBB Perturbation as an AD Risk Factor

A workshop participant asked whether genes influencing BBB dysfunction are linked to AD. Dr. Searson said that work using transcriptomic changes and functional measurements will be helpful in answering this question. Our understanding is still in its early days, and researchers are still in the process of figuring out how risk factors might translate to BBB perturbations and lead to brain dysfunction. Since neurodegenerative diseases are multifactorial, it is difficult to develop a one-size-fits-all drug. Characterizing the perturbations that contribute to disease phenotype may create an integrated approach.
Dr. Thorne said that it will be important to test some novel perturbations and measure the various functional attributes that are changed. Doing this in vivo will help provide correlations with both behavior change and in vitro measurements. These models will have tremendous value for pharma and biotech.

Need for a Consensus Database

The field needs a consensus database of human data with which to compare data from animal models. There is so little data available, and this is a huge limitation to developments in the field. Dr. Searson urged the FDA and NIH to invest effort in building that database, including funding specific experiments to create some needed data.

Vascular Space Correction

Without a direct measurement of the vascular contribution to the uptake of therapeutics, it can be difficult to make the needed measurement corrections. Especially if uptake is low, vascular correction is very important. But vascular space is also being altered by disease and various experimental variables, which further complicates correction calculation. Some researchers are using laser microdissection to learn more.

Extracellular Space Volume

Neonatal mice have twice the volume of extracellular space as adults, and a participant asked whether modeling approaches accounted for this. Dr. Bickel said that uptake clearance from the blood in newborn animals is not expected to be higher, because the BBB is intact well before birth. A different extracellular space fraction will have a proportionate effect on the efflux rate constant from brain tissue.

Influence of Anesthetics

The mice used in some of the microscopy studies were anesthetized during some of the experiments, which could have a transient or persistent effect on BBB. From the drug delivery point of view, the BBB impacts of these anesthetics have gone under the radar in terms of interest and concern about mechanistic details.

Cell Morphology and Drug Delivery

Some modeling studies assume cells are cuboidal, but they often are not. Dr. Sproul said that when his lab makes tubules, they will need to measure cell diameter. The other point about tubules is that the microfluidic channel used by both Dr. Sproul and Dr. Searson for tubule formation is 200 microns in size, which is larger than capillaries (arteriolar size) and could affect drug delivery. Experiments in Dr. Searson’s lab that used laser ablation to blast out a small number of cells and track microvessel recovery indicated that neighboring cells might stretch to heal the injury. This implies interesting dynamics in endothelial cell communication.

BBB Models and Neurovascular Interactions

While the various models used to study the BBB are extremely useful to detect permeability changes, their utility in addressing neurovascular interactions (e.g., the link between endothelial cells and astrocytes) is unclear. Evidence is accumulating that neurovascular interactions are important in AD. Dr. Searson pointed out that the field’s understanding of these interactions is still in its infancy, and there are some basic morphological questions that need to be resolved to begin creating these models.

Session II: Basic BBB Biology and Transport Mechanisms and How They are Affected in Aging and AD/ADRD

Chair: Emilie Reas, Ph.D., University of California, San Diego

Extracellular Vesicles (EVs) at the Brain Barriers and Implications for Neuroinflammation and Alzheimer's Disease
Roosmarijn Vandenbroucke Ph.D., Vlaams Instituut voor Biotechnologie (VIB)

The choroid plexus (ChP) consists of epithelial cells surrounding a core of highly vascularized, fenestrated capillaries, located in the cerebrospinal fluid (CSF)-filled brain ventricles. This blood-CSF barrier is a large surface area between blood and brain. The Aβ oligomers associated with AD have been associated with increased blood-CSF barrier permeability and with morphological changes to ChP epithelial cells. Extracellular vesicles (EVs), which contain luminal contents of the cell, such as proteins, metabolites, and nucleic acids, enclosed in a lipid bilayer, can form at the blood-CSF interface. Initially, EVs were seen as part of the cell’s garbage disposal system, but it has become increasingly clear that EVs are very important in cell-cell signaling. Early experiments by the Vandenbroucke lab showed that systemic inflammation and Aβ oligomers induce increased EV production at the blood-CSF barrier, which itself induces neuroinflammation.

Dr. Vandenbroucke observed high vesicular activity at the basolateral side of ChP epithelial cells, and experiments in her lab showed that ChP epithelial cell-derived EVs show brain-homing capacity upon IV injection, traveling from the CSF to the brain and delivering their cargo to brain cells. Peripheral EVs can also have an impact on the brain via the gut-microbiota-brain axis. Outer membrane vesicles (OMVs) from Helicobacter pylori can be loaded with Cre recombinase. Cargo from OMVs can reach the brain and be taken up by astrocytes. In an AD mouse model, OMVs were shown to aggravate plaque load.

One major gap in ChP research is that most data come from animal models, so not much is known about the mechanisms of EV production and blood-CSF barrier crossing in humans. It is also unclear what proportion of EVs comes from ChP epithelial cells, other local cells, or the blood itself. EV distribution throughout the brain is another murky area. One more major question is how much of EV signaling is from intracellular cargo release vs. ligand-receptor signaling. However, in vitro models of human and mouse blood-CSF barriers are available, including well-characterized ChP organoids, which provide a promising avenue for future research. The potential anti-inflammatory effects of EVs also add another layer of complexity to their role in neuroinflammation.

Human Neuroimaging of Blood-Brain Barrier Breakdown in Early Alzheimer’s Disease: Associations with APOE and Brain Microstructure
Emilie Reas Ph.D., University of California, San Diego

Human BBB research is still in its infancy compared to animal and in vitro studies. Measuring BBB permeability in human subjects is challenging but can be done using biochemical techniques (common biofluid markers of pericyte injury and blood-derived proteins) as well as neuroimaging methods, such as dynamic contrast-enhanced magnetic resonance imaging (MRI) and water exchange MRI.

Prior studies with dynamic contrast-enhanced MRI show evidence of APOE4-dependent BBB breakdown in the hippocampus and parahippocampal gyrus independent of Aβ, tau, and brain atrophy. A decade of human studies has led to inconsistent and inconclusive findings. Evidence supports BBB breakdown in MCI, but associations with Aβ are weak and evidence for an association with APOE4 is limited. Dr. Reas’ lab investigated when BBB dysfunction emerges in the early AD cascade and whether early BBB breakdown is localized to medial temporal regions or globally distributed. In a sample enriched for AD risk and pathology, BBB permeability was unrelated to age or cognitive impairment. Cortical gray matter BBB permeability was elevated in APOE4 carriers, even in the absence of Aβ pathology or cognitive decline. BBB permeability was related to entorhinal and white matter microstructural abnormalities, particularly for APOE4 carriers. The work showed a widespread, APOE4-dependent increase in BBB permeability early in the course of AD, which may be related to neurodegeneration in the entorhinal cortex.

Dr. Reas says that researchers need to develop multimodal integration models to isolate structural and functional properties of the BBB. Scientists also need to know more about both genetic and environmental factors for BBB permeability differences. It is also important to clarify the temporal dynamics over disease course and day-to-day variability and to integrate fluid and imaging biomarkers. This will help elucidate APOE4-dependent avenues and causal pathways towards neurodegeneration and cognitive decline.

Adaptations and Dysfunctions of the Blood-Brain Barrier Transporters in Aging and Alzheimer’s Disease
William A. Banks, M.D., University of Washington

The BBB is more accurately thought of as an interface rather than a barrier. It plays a role in nutrition (delivering glucose, amino acids, free fatty acids, and vitamins), homeostasis, and communication (transferring signals, peptides, and regulatory proteins). BBB transport is highly modifiable via a variety of strategies and pharmaceutical approaches. Over the lifespan, the BBB changes. This is expected and appropriate, as the BBB responds to the needs of the brain. Changes in BBB functions unrelated to changing brain needs, however, can cause disease. In AD, brain endothelial cell functions change, and in vitro studies show that Aβ peptides themselves can alter brain endothelial cell function. Dr. Banks’ work from the 1980s found that old, healthy rats show a decreased brain-to-blood efflux of enkephalins by peptide transport system-1. BBB changes associated with normal aging show a proportionate decrease in molecule transport and may reflect altered central nervous system (CNS) demands or susceptibility to CNS disease. These transporters are often altered in AD.

The Banks lab has worked on brain-to-blood efflux of Aβ peptides for several decades. In mouse models of AD, Aβ efflux was already impaired in young mice and worsened with age. Dr. Banks found that decreasing expression of the low-density lipoprotein (LDL) receptor-related protein (LRP) at the BBB with antisense would subsequently decrease Aβ efflux, increase Aβ levels in the brain and impair cognition. This work supported the idea that LRP could contribute to Aβ accumulation in AD. Measurements in the hippocampus of AD patients showed a non-statistical decrease in LRP levels, but a significant increase in oxidized LRP. The delivery of amyloid precursor protein antisense oligonucleotides restored Aβ efflux in an AD mouse model. Newer work from the Banks lab has identified brain insulin resistance as a factor in impaired BBB transport.

The field’s understanding of BBB transporters in aging and AD includes several major gaps, such as which transporters are altered and why, how the NVU and peripheral tissues communicate with the BBB, and how maladaptations arise. BBB transporters also provide various opportunities, including the ability to restore Aβ efflux in aging and AD with antioxidants and/or antisense oligonucleotides. Researchers can also learn more about the strategies needed to restore physiological function to altered BBBs in aging and AD.

Signaling and Trafficking Disruptions at the Blood-Brain Barrier in Alzheimer’s Disease and Metabolic Syndrome
Karunya Kandimalla Ph.D., University of Minnesota College of Pharmacy

Cerebrovascular pathology is one of the pathophysiological hallmarks of AD, and cerebrovascular disease commonly co-occurs with AD. In healthy brains, neuronal activity and cerebral blood flow are closely coupled like a well-coordinated ballet, which is regulated by the neurovascular unit (NVU) that consists of BBB endothelium, pericytes, neurons, and astrocytes. The BBB endothelium itself has a distinct functional duality: It exists as a formidable barrier and an efficient transport portal to move nutrients and metabolites in and out of the brain. In AD, a traffic jam forms at the BBB that disrupts the transport of endogenous molecules like insulin into the brain and reduces the clearance of Aβ from the brain. PET scans show that impaired Aβ clearance likely leads to the deposition of amyloid plaques in the brain. An increase in CSF Aβ42 is one of the earliest pathological changes in the Alzheimer’s brain.

Dr. Kandimalla pointed out that some of the factors that increase AD risk, such as inflammation, cerebrovascular dysfunction, oxidative stress, and autophagic dysfunction, are also implicated in Type 2 diabetes mellitus. Work in mouse models showed that the injection of insulin into the internal carotid artery led to reduced Aβ42 accumulation but increased Aβ40. Insulin administration also promoted the brain clearance of Aβ42 but not Aβ40. This suggests that insulin resistance increases the influx of Aβ42 and enhances its accumulation in the brain. PET scans show that brain uptake of the radioactive tracer fluorodeoxyglucose (FDG) is lower in AD mice than in healthy controls, and that brain influx clearance of FDG is lower after exposure to Aβ. Further experiments revealed that Aβ peptides disrupt the expression of glucose transporter 1 and insulin signaling. A downregulation of receptors expressed in the NVU constituent cells that regulate extracellular matrix (ECM) turnover also occurs in the AD brain.

Dr. Kandimalla noted that current brain atlases have major gaps in cataloging NVU vascular components, particularly endothelial cells and pericytes. Deep learning methods could enable the deconvolution of specific NVU gene expression profiles from bulk tissue data to create an NVU cell-type-specific atlas of AD patients and controls.

Age-Related Dynamics of the Blood-Brain Barrier and Neurovascular Unit: The Role of TGF-β Signaling in Maintaining Brain Health
Daniela Kaufer Ph.D., University of California at Berkeley

BBB dysfunction is common in neurological and psychiatric diseases, and symptoms may be correlated with the extent and location of the BBB dysfunction. This dysfunction also increases with age in healthy individuals as the BBB structure and function changes during the aging process. Dr. Kaufer hypothesized that vascular damage triggers neurological disease via BBB dysfunction. Her work showed that albumin accumulates in astrocytes and activates transforming growth factor beta (TGF-β) receptor signaling and provided evidence of increased astrocytic TGF-β signaling in aged mouse and human brains.

To answer the question as to whether TGF-β is correlated with or causes BBB dysfunction, experiments from the Kaufer lab revealed that albumin induces astrocytic activation, which led to the upregulation of inflammatory cytokines including TGF-β. Over time, this activation induced astrocytic senescence. Ectopic brain exposure to serum albumin recapitulated aging-related pathology as measured by gene expression signature, synaptic transmission, inflammation, cell senescence, neural dysfunction, and cognitive dysfunction. These results show that albumin activates a positive feedback loop of TGF-β receptor signaling. Use of a small molecule TGF-β receptor kinase inhibitor restored vascular integrity, reversed inflammation, blocked astrocytic senescence, restored astrocytic buffering, and blocked synaptogenesis. It restored BBB and neural function and reduced inflammation in the aging brain.

Major gaps and opportunities in understanding BBB dysfunction include underlying mechanisms and molecular signatures of BBB dysfunction in different clinical scenarios. Researchers also need dependable, higher throughput assays to quantify BBB-specific dysfunction. Reversing BBB dysfunction may also be a useful therapeutic target, although questions remain as to whether this restoration can be too much, and whether there is a role for dynamic BBB function.

Session II Discussion

BBB Dysfunction Detection and AD Diagnosis

Dr. Reas noted that BBB dysfunction appears early in the AD cascade and precedes the accumulation of Aβ. She asked how this emerging evidence might alter prevention, screening, and treatment approaches. Are researchers focusing on the right disease stages? Should there be more of a focus on repairing vascular dysfunction, or would addressing some of the downstream pathology be sufficient? Dr. Thorne noted that AD develops over decades, and it is still not clear what the first event in the AD cascade is.

Dr. Bickel pointed out the gap between the accumulation of Aβ and the beginning of MCI. Scientists know the NVU is changing, but it has been difficult to investigate how. Even if researchers could magically remove the accumulated Aβ plaque, some of the neurovascular damage may not be repairable since it has persisted for several decades. Dr. Kaufer said that animal models are limited in their ability to answer this question because they do not recapitulate the aging process. What’s more, neural activity can modulate BBB function, adding an additional layer of complexity.

Interpreting BBB Dysfunction

Dr. Thorne asked whether researchers might be misinterpreting dysfunction and dysregulation in the BBB for other types of pathological changes. He mentioned the blood-CSF barrier work at the ChP and the role that the CSF plays in AD. Dr. Kaufer said that the role of CSF will depend on the permeability changes to the BBB. She said that multiple events, including peripheral inflammation, likely contribute to the final result. Measuring changes to the BBB and blood-CSF barrier will require an oligomer-specific approach.

Blood proteins could gain increased access to the brain by a variety of mechanisms, and different researchers have different “pet” mechanisms. By the end, there is increasingly less selectivity in what accumulates in the brain and what cells are involved. Even minor and transient accumulation of blood proteins in the brain could lead to significant changes over time. Opening the BBB does not turn brain access into a free-for-all. There is definitely dysfunction, but details on how that increases brain penetration of biomolecules is less clear.

APOE4 and BBB Dysfunction

The APOE4 risk allele is only one amino acid different from the wild-type version, so how does this make the BBB leaky? Groups investigating this phenomenon have found that inflammation plays an important role including the Cyclophilin A-matrix metalloproteinase-9. Parasite-mediated pathways also appear to be important.

Leakiness Threshold

Changes to BBB function occur on a sliding scale, which led Dr. Searson to ask about the pathological threshold for BBB function or leakiness. Some of this will likely depend on the microenvironment in the brain, but it comes down to an understanding of what changes are part of the normal aging process and how the body tolerates these changes. Understanding what thresholds exist for normal activities of daily life would be important for both in vitro and in vivo studies.

Gold Standards for Animal Models

Given the discovery that Evans Blue may not be the best marker of BBB function, the question naturally arises as to what should be the gold standard measure? Some of the critiques of Evans Blue also include details on the benefits of other existing markers. Some of the best sensitivity and specificity has come from radioactive markers.

Session III: Current BBB Penetrating Drug Transport Strategies and Drug Studies for Alzheimer’s Disease

Chair: Robert Thorne, Ph.D., Denali Therapeutics

Introduction and Overview: Leveraging Physiology and Engineering for Drug Delivery Across the Blood-Brain Barrier: Taking Antibodies, Enzymes and Other Proteins to the Final Frontier
Robert Thorne, Ph.D., Denali Therapeutics

The BBB is not just a barrier but rather a highly selective interface and regulated border between the periphery of the body and CNS tissue and fluids. These borders obviously pose a problem for delivering biologics to the brain. Currently, the FDA has approved seven biotherapeutics as CNS drugs. The first approvals were for direct CSF infusions or injections, with the systemic application of monoclonal antibodies for AD appearing later. Key challenges for the field have included uncertainties around the precise biodistribution mechanisms and trafficking pathways for different approaches, and the need to develop new methods for increasing CNS drug exposure and translating them to the clinic for AD and other diseases.

Dr. Thorne divided currently considered technologies for delivering drugs to the brain as either invasive or non-invasive. Convection-enhanced delivery is an invasive method that uses pressure to force a flow of liquid to overcome diffusion limitations in brain tissue during intraparenchymal delivery. For the CSF administration approaches, it has proven challenging to deliver therapies such as large molecules and gene therapy vectors (e.g., AAVs) deeper into the brain, leading to an expectation of limited distribution with sharp gradients at brain and spinal cord pial surfaces. A less-invasive strategy that has been examined carefully over the last decade uses focused ultrasound (FUS) with intravenously injected microbubbles to transiently open the BBB for drug delivery. While researchers have learned a lot about how this process happens, there remain major questions about paracellular vs. transcellular permeability, the potential for sterile inflammatory processes, and translatability from animal models to humans. Other non-invasive methods include CNS delivery targeting endogenous BBB systems such as transferrin receptor 1 (TfR1) and CD98 heavy chain (CD98hc). Engineered transport vehicles (TVs) targeting TfR1 and CD98hc have distinct exposure properties. Experiments in mice have shown that TVs targeted to TfR1 localize throughout the brain parenchyma, targeting primarily neurons. Similarly, studies in mice have shown that TVs targeted to CD98hc also biodistribute to brain in a diffuse pattern, but with more preferential localization to glial cells. Dr. Thorne says the work performed to date has shown that engineering these TVs and other similar architectures could potentially open up a new strategy for treating AD across multiple platforms, e.g. by enabling better brain exposures for antibodies, other proteins, and oligonucleotides.

To move the various approaches further forward, researchers need to develop a more detailed knowledge of the biodistribution complexities associated with different CNS delivery approaches, as well as better understand trafficking and uptake dynamics for approaches aimed at crossing the BBB. Scientists also need to better understand how aging and diseases like AD may affect delivery and biodistribution, although there is historical and emerging evidence that AD does not make the BBB significantly more permeable to antibody therapeutics, further emphasizing the need for new delivery approaches. Dr. Thorne also said that the field needs more communication between researchers devoted to studying the CNS barriers and the AD and aging community, because there exists unique specialist knowledge in each of these research areas that is needed across the different disciplines. Nevertheless, there is much reason for optimism in the coming years that CNS delivery approaches will mature, and new therapies will be approved.

Focused Ultrasound Blood-Brain Barrier (BBB) Opening in Alzheimer's Disease
Ali Rezai, M.D., West Virginia University

Focused ultrasound (FUS) induces a transient opening of the BBB and can be varied in terms of power, duration, energy, dose of microbubbles, and location of treatment. The goal is to achieve stable expansion and contraction needed to open the BBB. Too much energy can create microbleeds, but too little energy won’t cause an opening. Dr. Rezai mentioned the Insightec system, in which a shaped transducer delivers sound waves to any brain target with no incisions. The BBB can be opened with circulating microbubbles where the ultrasound energy converges. Animal studies show this method is safe and reversible, increasing the penetrance of endogenous antibodies, reducing Aβ and tau, and improving behavior and cognition.

Dr. Rezai shared statistics on 300+ patients who have undergone more than 500 BBB—opening sessions. Human data show immediate BBB opening and closure within 24-48 hours. This patient data showed some reduction of Aβ plaque after a two-hour outpatient procedure. Multiple brain regions with high Aβ load can be treated in one session. Dr. Rezai says that FUS can specifically target areas implicated in AD. In the rapidly changing and evolving area of AD therapeutics, the use of monoclonal Aβ antibodies offers promise, but with significant side effects. Dr. Rezai’s preliminary tests combining Aβ antibody infusions with FUS BBB opening demonstrates five-fold higher antibody penetration in the brain than infusion alone. The Rezai lab is currently conducting a first-in-human safety and feasibility study in three patients with mild AD to compare infusion alone with infusion + FUS. Preliminary data indicates that the side of the brain that received FUS showed greater reduction of Aβ plaques.

Dr. Rezai says that scientists need to explore other systems, conduct long-term studies of results and safety, and scale up the number of patients and treatment volumes to demonstrate meaningful clinical responses. FUS BBB opening should also be studied with other antibodies to determine whether it increases drug penetration and availability. Researchers also need to measure drug biodistribution with BBB opening, investigate the potential of FUS BBB opening for reducing drug dose, frequency, duration, and side effects, and develop therapies that take advantage of FUS BBB opening.

Low Intensity Focused Ultrasound Brain Delivery of AAV Gene Therapy for Alzheimer's Disease
Rachel Bailey, Ph.D., University of Texas Southwestern Medical Center

Gene therapy using AAV shows promise for treating a variety of diseases, including AD. The challenge is getting the therapeutics into the appropriate cells at meaningful doses. AAVs have long been used for gene therapy for their sustained transgene expression and good safety profile. The tropism of the virus is determined by the capsid: AAV9 and AAVrh10 can cross the BBB to transduce both neurons and glia. AAVs can be delivered via IV, but the strategy requires a very high dose for adults, limiting their efficacy. Direct brain injection uses a lower dosage and avoids neutralization antibodies, but it is highly invasive. CSF injection of AAVs has a medium level of invasiveness and can lead to an expression gradient from the site of expression and potentially cause systemic escape. Low intensity focused ultrasound (LIFU) may enhance AAV delivery to the CNS.

The Bailey lab has been working with AAVs targeting tau and tauopathies. Decreasing tau is a potential therapeutic approach for several neurodegenerative diseases. In an AD mouse model, injections of an AAV9 vector carrying artificial tau-specific microRNA transgene (AAV9/hTaui) decreased brain tau mRNA and were effective in reducing brain pathology even when used later in disease. However, these results show that better delivery methods were needed. Adding LIFU to temporarily open the BBB improved uptake of intrathecally injected AAV9/hTau, said Dr. Bailey.

Clathrin Nanoparticles for Delivery of Biologics Across the Blood-Brain Barrier and Treating Alzheimer’s Disease
Franco Vitaliano and Gordana Vitaliano M.D., ExQor Technologies Inc.

ExQor has developed a clathrin nanoplatform to improve the CNS delivery of biologics and macromolecules across the BBB. Clathrin transports macromolecules across the BBB, and ExQor has been working to deliver brain-derived neurotrophic factor (BDNF), a key molecule involved in learning and memory, using the clathrin nanoplatform to treat AD. In an in vivo preclinical study of human immunodeficiency virus (HIV) dementia, the ExQor clathrin transporter had up to 400 times higher hippocampal concentration of BDNF compared to control. ExQor trials show their clathrin transporter can successfully lead to in vivo BBB passing and CNS delivery of neurotrophic factors, antibodies, dopamine transporter ligands, gadolinium contrast agents, and superparamagnetic iron oxide contrast agents. Dr. Vitaliano says that the clathrin transporter also carries structural advantages. Coating liposomes with clathrin improves their stability and rigidity as the clathrin lattice is 100 times stiffer than typical liposomal membranes. ExQor can create a clathrin transporter in three different configurations: decorated clathrin cages, clathrin-coated vesicles, and clathrin triskelia, all of which cross the BBB via clathrin-mediated endocytosis (CME).

Previous studies have shown that CME plays a critical role in AD pathogenesis, especially in synaptic dysfunction and Aβ production. CME is crucial for synaptic vesicle recycling and neurotransmitter release at presynaptic terminals. The dysregulation of CME appears to be an early event in AD and can lead to synaptic dysfunction and cognitive deficits. Preclinical data from ExQor showed that clathrin can penetrate cells and deliver transferrin via endocytosis. In rat studies, clathrin nanoplatforms administered via intranasal or intraperitoneal methods crossed the BBB. Clathrin could also penetrate the nasal mucosa via transcytosis and penetrate the brain. Clathrin transporters carrying BDNF were shown to target tyrosine protein kinase B (TrkB) and increase TrkB signaling in rats. In a 3xTg-AD mouse model, BDNF clathrin transporters increased neurogenesis, synaptogenesis, dendritic integrity and hippocampal gray matter volume; they also decreased apoptosis and reversed oxidative stress. The Vitalianos envision a future of precision CNS medicine via neuro-theranostics for early screening, diagnosis, and treatment of neurodegenerative disorders.

High-Affinity Transferrin Receptor Antibody for Brain Drug Delivery in Alzheimer’s Disease Mouse Models
Rachita Sumbria, Ph.D., Chapman University

Several decades ago, researchers identified transferrin receptors (TfRs) at the BBB and showed they could transport molecules across the BBB. Studies in mice showed that TfR monoclonal antibodies (TfRMAbs) could deliver biologics to the brain, and a version of TfRMAb targeting amyloid-beta is currently in Phase II trials. Dr. Sumbria said that TfRMAb could deliver neurotrophins such as erythropoietin (Epo), decoy tumor necrosis factor alpha (TNF-α) receptors, as well as therapeutic antibodies. She highlighted Epo, since both Epo and the Epo receptor are expressed in the brain, citing a growing body of literature showing the neuroprotective effects of Epo. However, Epo has limited BBB penetration, requiring high systemic doses that result in hematopoietic side effects.

The Sumbria lab tethered Epo to a TfRMAb and showed high-affinity binding to both TfR and the Epo receptor. In an amyloidosis mouse model of AD, Dr. Sumbria showed that TfRMAb-Epo could reduce Aβ load and neuroinflammation in WT mice, while also increasing synaptophysin. Further, in a tau mouse model, TfRMAb-Epo led to a reduction in phosphorylated tau and improvements in behavior outcomes in mice. TfRMAb-Epo showed better therapeutic effects and safety indices than Epo alone, which Dr. Sumbria attributed to the TfR-mediated BBB penetration and faster peripheral clearance of TfRMAb-Epo. In a separate mouse study, the Sumbria lab demonstrated the safety of high-affinity bivalent TfRMAbs and a reversal of reticulocyte suppression with no change in brain TfR or iron levels. Dr. Sumbria says that researchers still need to study TfR expression in different mouse models and ages and to understand the impact of chronic dosing on plasma bioavailability. Also needed are measurements of how the fusion partner impacts TfRMAb clearance.

Neurotropic Gectosomes as a CNS Gene Delivery System
Gan Zhang, Ph.D., Vesicle Therapeutics, Inc. and Xuedong Liu, Ph.D., University of Colorado-Boulder

Gectosomes are engineered, cargo-specific, and fusogenic EVs that can be used to deliver therapeutics. They can be engineered with 293T producer cells using split green fluorescent protein (GFP) complementation to load the cargo into the gectosome that leads to endosome escape, fusion with target cells, and the release of cargo in target cells. GFP complementation also provides a fluorescent signal to measure and verify cargo loading. Rodent studies showed that gectosomes could deliver gene editing constructs targeting proprotein convertase subtilisin/kexin type 9 and reducing low density lipoprotein (LDL) cholesterol levels.

The cellular tropism of the gectosome depends on the viral glycoprotein used in its construction. The human Chandipura virus is a neurotropic pathogen, and overexpression of the Chandipura viral glycoprotein (CNV-G) induces the robust production of CNV-G gectosomes equivalent to gectosomes produced via the expression of vesicular stomatitis virus G (VSV-G). The CNV-G gectosomes were cell-type selective in the delivery of Cre recombinase to primary neuronal cells and led to better brain exposure with the same IV injection dose compared to VSV-G. Dr. Zhang says that researchers still need to develop a delivery system for crossing the BBB, optimizing neurotropic viral glycoproteins, and identifying appropriate disease models and target validation.

Functionalized Nanoparticles to Deliver Nucleic Acid Therapeutics to Brain for Neurodegenerative Diseases
Jagdish Singh Ph.D., North Dakota State University

The BBB is a tight junction between two endothelial cells that restricts the passage of water-soluble agents. Molecules can cross the BBB via the paracellular aqueous pathway, transcellular lipophilic pathway, transport proteins, receptor-mediated transcytosis, and adsorptive transcytosis. Nanotechnology-based gene and drug therapies can use liposomal nanoparticles as a brain-targeted gene carrier. Specific ligands can target receptors on target cells, and conjugating polyethylene glycol (PEG) chains to proteins via PEGylation can prevent immune system recognition. These liposomal nanoparticles can pass through the BBB via transcytosis and deliver plasmid DNA into brain cells. Dr. Singh’s work showed that modifying the surface of PEGylated liposomal nanoparticles with transferrin and the cell-penetrating peptide penetratin (Pen) can improve transfection efficiency and facilitate endosomal escape. Brain-specific liposomal nanoparticles loaded with therapeutics are synthesized using thin film hydration techniques followed by the insertion of coupled micelles using a post-insertion technique.

Studies in C57BL/6J mice using near-infrared imaging showed that liposomal nanoparticles injected into the tail vein preferentially reached the brain. Immunofluorescence analysis of the cortex and hippocampus showed that the liposomes crossed the BBB and transfected mouse brain cells. The specificity of PenTf-liposomes carrying plasmid DNA coding for nerve growth factor (NGF) in transfecting neurons was demonstrated by GFP that was mainly expressed in cells co-localized with the neuronal marker NeuN. The liposomes showed a dose-dependent effect on NGF expression in the mouse brain and stimulation of new cell formation. The Singh lab also developed a novel combination therapy for AD using liposomal nanoparticles encapsulating cannabidiol (CBD) and BDNF that were functionalized with specific targeting ligands. In an immortalized microglia cell line, the CBD liposomes showed anti-inflammatory properties following cell stimulation with lipopolysaccharide and Aβ oligomers. In vivo studies in mice showed a reduction of cortical Aβ and phosphorylated tau following CBD liposome treatment. These mice showed subsequent improvements in cognitive function. This demonstrates the potential for a safe and efficient non-viral gene delivery vector that specifically targets brain cells.

In addition, the researchers also discussed a non-invasive intranasal route to deliver VGF in wild-type mice using VGF polyplexed with the cell-penetrating peptide TAT and mannose-functionalized oleic acid grafted chitosan nanomicelles. VGF expression was significantly higher (p<0.05) after intranasal vs. intravenous administration.

Session III Discussion

Need for BBB Delivery Approaches

Dr. Thorne asked whether, given the known BBB dysfunction in AD, biologics like aducanemab and lecanemab needed specific delivery approaches. Dr. Rezai said that is an important question, and these drugs might not need an extra delivery effort. But it would also need to be studied in various AD subtypes, including APOE4, to get a better understanding of the kinetics and dosing. Dr. Thorne said that the “gorilla in the room” is whether antibodies sufficiently cross the BBB in AD and whether there is a size-dependent nature to permeability changes. Is the BBB damage sufficient to allow a vesicle or antibody to cross? And is the therapeutic in question using an influx ligand?

Dr. Bailey added that the BBB dysfunction is not necessarily in the parts of the brain that need therapeutics. The field needs better studies to look at FUS targeting and benefit, and localized changes in molecule uptake and BBB changes. Scientists also need to better understand whether these therapeutics need to be delivered to broad areas of the brain (such as those with tau pathology) or more targeted regions. Evidence is showing a need for some sort of delivery system, because passive transport is not sufficient.

Neural Damage from FUS

Dr. Rezai says that his studies do indicate some microdamage in the brain with FUS. When they use a higher ultrasound dose, it can create an initial cavitation that causes larger hemorrhages because blood vessels are mechanically impacted. It is still not clear whether there is damage, but they have not documented any long-term clinical impact on AD.
Dr. Banks pointed out that how the BBB is opened will be critical, as is the size of the molecule, vesicle, or virus that is being transported. Larger cargo may need the vesicular system. Toxicity with BBB opening is important and can lead to the release of prostaglandins and cytokines, necessitating a close focus on neuroinflammatory effects. There also needs to be a better understanding of regional differences in BBB dysfunction.

Workshop Discussion: Identification of Gaps and Opportunities
Chairs: Peter Searson, Ph.D., Emilie Reas, Ph.D., and Robert Thorne, Ph.D.

BBB Changes and Aging

Dr. Reas kicked off the discussion by asking how the changes that occur with aging inform the translational strategies that the field is thinking about. What parts of the basic science does the field still need to learn? And how will that impact the development of therapeutics? Dr. Banks pointed out that scientists still really do not know what normal aging is. For a long time, cognitive decline and dementia were seen as just a normal part of the human condition. But just because something is common does not mean it is normal. If the BBB becomes dysfunctional, that can set the stage for disease, as the brain is no longer receiving and removing the molecules in the needed proportions. Then, researchers also need to figure out the implications of BBB changes. Dr. Reas says she also does work on in utero enzyme replacement therapy, so understanding the biology of the BBB across the lifespan is important for a variety of conditions. The field has the tools to study it; researchers just need the right people asking the right questions.

Understanding BBB changes also has implications for AAVs and gene therapy, according to Dr. Bailey. Often, AAV vectors are tested in neonatal mice, and then scientists are frustrated when the approach does not work in aged adults. She points out that therapies need to be tested at relevant ages as well as in appropriate disease models.

Promise of AI on BBB Research

Based in Silicon Valley, Dr. Thorne commented that the field of aging, with its huge datasets, is ripe for an AI approach. Several workshop participants said they already are using AI or planning to do so soon. AI is especially useful for the analysis of large-scale, longitudinal imaging studies, such as one in which Dr. Rezai is following several thousand patients in Appalachia. Other workshop participants noted the potential for AI in spatial phenotyping, although more progress needs to be made in deconvolution approaches.

Animal Models for Behavioral Changes

Most of the animal models used in AD are focused on the molecular pathology of disease. But these models may also be useful in understanding the behavioral changes that result from BBB dysfunction. It is hard to capture big changes in BBB permeability, which necessitates different models to understand different aspects of BBB dysfunction. By creating different cell types, researchers can see interactions, change genetics, alter environmental stressors and decode cross-talk.

Behavioral changes due to phenotype are still very meaningful to identify and study, but scientists need to figure whether they are studying vascular changes or neurological changes. Researchers understand in a reductive way how perturbations drive particular modes of dysfunction, but they need to explore possible therapies to repair BBB function. Specifically, researchers need to spend more time on understanding endothelial cell function.

Survey: One Area of BBB Research to be Developed

Representatives from the NIA asked the researchers to each share their perspective on one area that needs to be investigated sooner rather than later.

• Dr. Fu: Studies have shown marked effects on drug delivery with the use of FUS and transcranial direct current stimulation, but the mechanism is not clear. Dr. Fu says that more and better animal models are needed to understand the mechanism.
• Dr. Kandimalla: The field does not have much molecular level data on endothelial cells in vasculature in humans, so it is not clear how transcriptomics and proteomics change with disease. Without that information, it is hard to see what changes to target and identify novel targets. The advent of AI and other sequencing technologies will really open this area up to new and useful investigations. Dr. Kandimalla says that what happened in the cancer field would be a good roadmap for the cerebrovascular field.
• Dr. Kaufer: Funding of team science approaches is critical. She noted that in the workshop, each scientist specialized in their own specific cell, molecule, and/or signaling pathway, and it was eye-opening to get a full view of the complexity. By funding interdisciplinary teams, researchers will be able to get a more complete grasp of the biology of AD.
• Dr. Franco Vitaliano: Most researchers are sitting on a ton of data, and they are often filtering their data to drive forward certain hypotheses. Perhaps it is time to use these datasets to investigate other hypothesis and treatment approaches that are complementary or supplementary to a plaque-driven AD approach.
• Dr. Rezai: The field needs more preclinical studies and animal research to learn about clinical mechanisms. With such a high failure rate in developing medications to bring to the clinic, improving the likelihood of success by gaining a fuller basic science picture will be important.
• Dr. Thorne: Omics technologies must be brought to the study of neurovasculature. Proteomics, transcriptomics, glycomics, and metabolomics approaches have not yet been applied. Cross-species studies will also be helpful to identify factors that alter normal BBB function. He also spoke of the need to evaluate biomarker-based approaches and begin discussions with regulatory bodies and stakeholders. The FDA has shown a willingness to approve drugs based on biomarkers, and AI is showing tremendous power in identifying and using biomarkers.
• Dr. Banks: Researchers typically frame the BBB as binary, either on or off, either disrupted or intact. But the BBB is cellular-based and so has complex functions. The BBB is not only a barrier; it actively participates in brain health. Because it is polarized, it does a lot of things that most cells can’t do. Additionally, the field needs to move away from an Aβ-centric view of AD to open up possibilities for therapeutics. Dr. Banks fears over-reliance on plaque-directed therapeutics illustrates Kahneman’s hypothesis-induced blindness.
• Dr. Zhang: The field needs to begin to select an animal model to use in this field. Dr. Zhang also spoke of the need for better data sharing among scientists while also respecting intellectual property concerns.
• Dr. Thorne: Platform-based approaches are needed to monitor disease and track response to therapy more cheaply and easily. Especially as trials get incredibly complex, these methods are going to be very useful.
• Dr. Vandenbroucke: More attention must be paid to the role of immune cells in BBB function and dysfunction. Whether via in silico studies or animal models, scientists need to understand more about how immune cells are interacting with brain cells, microvasculature, and neurovasculature.

After the question-and-answer session, the NIA conveners thanked the researchers for their participation and concluded the workshop.

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Acronyms

3D: Three-dimensional

Aβ: Amyloid beta

AAV: Adeno-associated virus

AAV9/hTaui: AAV9 vector carrying artificial tau-specific microRNA transgene

AC: Astrocyte

AD: Alzheimer’s disease

ADRD: Alzheimer’s disease and related dementias

AI: Artificial intelligence

APOE4: Apolipoprotein E4

BBB: Blood-brain barrier

BDNF: Brain-derived neurotrophic factor

bpECs: BBB-primed endothelial cells

CBD: Cannabidiol

CD98hc: CD98 heavy chain

ChP: Choroid plexus

CME: Clathrin-mediated endocytosis

CNS: Central nervous system

CNV-G: Chandipura viral glycoprotein

CSF: Cerebrospinal fluid

ECM: Extracellular matrix

Epo: Erythropoietin

EV: Extracellular vesicle

FAD: Familial Alzheimer’s disease

FDA: Food and Drug Administration

FDG: Fluorodeoxyglucose

FUS: Focused ultrasound

GFP: Green fluorescent protein

HD: Huntington’s disease

hiPSCs: Human induced pluripotent stem cells

HIV: Human immunodeficiency virus

iBMECs: Induced brain microvascular endothelial cells

iPSCs: Induced pluripotent stem cells

IV: Intravenous

Kin: Brain uptake clearance

LDL: Low-density lipoprotein

LIFU: Low-intensity focused ultrasound

LRP: LDL receptor–related protein

MCI: Mild cognitive impairment

MRI: Magnetic resonance imaging

MS: Mass spectrometry

NGF: Nerve growth factor

NIA: National Institute on Aging

NIH: National Institutes of Health

NINDS: National Institute of Neurologic Disease and Stroke

NVC: Neurovascular complex

NVU: Neurovascular unit

OMV: Outer membrane vesicle

PC: Pericyte

PEG: Polyethylene glycol

Pen: Penetratin

PET: Positron emission tomography

rBMECs: Reprogrammed brain microvascular endothelial cells

TEER: Trans-endothelial electrical resistance

TfR: Transferrin receptor

TfR1: Transferrin receptor 1

TfRMAb: TfR monoclonal antibodies

TGF-β: Transforming growth factor-β

TNF-α: Tumor necrosis factor alpha

TrkB: Tyrosine protein kinase B

TV: Transport vehicle

UPLC-MS/MS: Ultra-high performance liquid chromatography tandem mass spectrometry

VSV-G: Vesicular stomatitis virus glycoprotein