Investigating Patterns of Degeneration in Alzheimer’s Disease

Alzheimer’s disease (AD) is known to cause memory loss and cognitive decline, but other functions of the brain can remain intact. The reasons cells in some brain regions degenerate while others are protected is largely unknown. In a paper to be published in Stem Cell Reports, researchers from Brigham and Women’s Hospital have found that factors encoded in the DNA of brain cells contribute to the patterns of degeneration, or vulnerability, in AD.

AD is characterized by plaques composed of amyloid β-protein (Aβ) and tangles composed of Tau protein; accumulation of Aβ protein leads to disruption of Tau and, eventually, neurodegeneration which affects brain regions in a variety of ways. The front, rostral, portion of the brain is generally more damaged by plaque build-up while the back, caudal, portion is generally spared.

Though there are several mechanisms that could cause these differences, the team focused on the potential contributions of cell-autonomous factors among neuronal subtypes that could affect both the generation of and the responses to Aβ. In a novel application of human induced-pluripotent stem cell (iPSC) technology, the team generated powerful culture systems that represent different areas of the brain. The systems were developed by taking skin cells from patients with a familial Alzheimer’s disease mutation and turning these skin cells into stem cells. Stem cells divide to make more stem cells, providing an unlimited supply of cells. Stem cells also can be turned into any type of cell in the body, including brain cells. In this study, the authors showed that vulnerable brain cells made more toxic Aβ protein compared to brain cells from more protected regions of the brain.

In addition, the researchers found that brain cells in the protected, caudal portion of the brain have a less toxic response to Aβ than their rostral counterparts. Though early-onset, familial Alzheimer’s disease (fAD) accounts for a small number of AD cases, the study of fAD patients, or samples in this case, can reveal important aspects of the cell and molecular mechanisms underlying all types of AD. The team is currently using this information to investigate exactly why caudal neurons are protected and what differences in cell type cause neurons to be protected from AD.

“These findings illuminate our understanding of why some neurons are spared and why others are not spared in AD,” said Christina Muratore, PhD, of the Department of Neurology. “If we can find out more information about why these subtypes of cells are protected, we may be able to use this information to tailor therapies to protect the vulnerable cells.”

This work is part of the AD Deep Phenotyping program supported by a gift from Rick and Nancy Moskovitz, and is supported by the Bright Focus Foundation, Brigham Research Institute, and NIH.

Paper Cited: Muratore, C et al. “Cell-type dependent Alzheimer’s disease phenotypes: probing the biology of selective neuronal vulnerability” Stem Cell Reports DOI: 10.1016/j.stemcr.2017.10.015

A Delicate Crossing: Controller Developed to Open the Blood-Brain Barrier with Precision

Representation of the drug delivery system. Serving as the acoustic indicator of drug delivery dosage, the microbubble emission signal y(t) is used to find the expected controller output u(f) which is fed back to help control ultrasound transducer transmission.

The blood-brain barrier – the semi-permeable membrane that surrounds the brain – offers important protection for a delicate organ, but in some cases, clinicians need to get past the barrier to deliver vital drugs to treat the brain. Researchers at Brigham and Women’s Hospital are investigating a way to temporarily loosen the blood-brain barrier to deliver drugs with the assistance of microbubbles. In a new advancement, they have developed a system in preclinical models that offers a finer degree of control – and, therefore, safety – in opening the barrier. Their findings are published this week in The Proceedings of the National Academy of Sciences.

“We want to be able to monitor our ability to open the blood-brain barrier in real-time by listening to echoes – this could give us immediate information on the stability of the microbubbles oscillations and give us fast, real-time control and analysis,” said lead author Tao Sun, a PhD candidate in the labs of co-authors Nathan McDannold, PhD, in the Focused Ultrasound Laboratory in the Department of Radiology at BWH, and Eric Miller, PhD, chair and professor in the Department of Electrical and Computer Engineering at Tufts.

McDannold and his colleagues have been working for years on using focused ultrasound and microbubbles to disrupt the blood-brain barrier and deliver drugs to the brain. However, a major challenge for translating research advancements in this area into clinical impact has been a lack of a reliable way to get instantaneous feedback on how well microbubbles are vibrating inside the brain. Microbubbles can help temporarily open the blood-brain barrier without incision or radiation, but if these bubbles destabilize and collapse, they can damage the critical vasculature in the brain.

In the lab, the research team used a rat model to develop a closed-loop controller – a device that can give them a metaphorical window into the brain. By placing sensors on the outside of the brain that act like secondary microphones, the research team could listen to ultrasound echoes bouncing off the microbubbles to determine how stable the bubbles were. They could then tune and adjust their ultrasound input instantly to stabilize the bubbles, excite them to open the barrier, and deliver a drug of a predefined dose, while maintaining safe ultrasound exposure. The team tested the approach in healthy rats as well as an animal model of glioma brain cancer.

Further research will be needed to adapt the technique for humans, but the approach could offer improved safety and efficacy control for human clinical trials, which are now underway in Canada.

This work was supported by National Institutes of Health P01 CA174645.

Paper cited: Sun T et al. “Closed-loop control of targeted ultrasound drug delivery across the blood-brain/tumor barriers in a rat glioma model” Proceedings of the National Academy of Sciences doi:10.1073/pnas.1713328114

Insulin Found to Control Cardiac Function Through Reactive Oxygen Species

More than 30 million Americans have diabetes, according to the American Diabetes Association. A BWH research team has now shown that insulin may also impact the body in a unique way.

The study, led by Thomas Michel, MD, PhD, of BWH’s Cardiovascular Division, was recently published in Free Radical Biology and Medicine, a journal of the Society for Redox Biology and Medicine (SfRBM).

This new discovery unveiled an entirely unexpected mechanism whereby insulin-dependent generation of hydrogen peroxide (H₂O₂) in cardiac myocytes controls heart contractility and critical cell signaling pathways.  These research findings have direct implications for our understanding of diabetic cardiomyopathy, which is a major cause of morbidity and mortality for patients with diabetes,” said Michel.

Michel added, “We usually think of H₂O₂ as a harmful molecule that can cause pathological oxidative stress in diabetes and other diseases. Our recent discoveries help to establish that H₂O₂ actually plays a central role in modulating insulin action in the normal heart.  We now need to more fully understand the factors that govern the transition from the physiological roles of H₂O₂ in normal cells to the pathological roles of H₂O₂ and related  molecules in disease states.”

Michel’s team first observed that insulin treatment attenuated the usual response seen when adrenaline-like drugs are used to stimulate the contraction of cardiac myocytes isolated from mice. Benjamin Steinhorn, a Harvard MD-PhD student, along with postdoctoral fellow Juliano Sartoretto, used novel biosensor methods and genetic approaches to demonstrate that the attenuated contractile response was due to cellular generation of H₂O₂ by two distinct but closely-related enzymes, NOX2 and NOX4, following insulin treatment. However, these effects of insulin were absent in cardiac myocytes isolated from diabetic mice. These findings suggest that insulin resistance in the diabetic heart may lead to deleterious potentiation of contractile responses and promote the development of heart failure.

Paper cited: Steinhorn B et al. “Insulin-dependent metabolic and inotropic responses in the heart are modulated by hydrogen peroxide from NADPH-oxidase isoforms NOX2 and NOX4.” Free Radical Biology and Medicine DOI: 10.1016/j.freeradbiomed.2017.09.006

Uncovering a Reversible Master Switch for Development

In a paper published in Genes & Development, BWH principal investigator Mitzi Kuroda, PhD, and her team identified a reversible “master switch” on most developmental genes. The team unearthed this biological insight through studies in the fruit fly —a powerful model organism for studying how human genes are organized and function.

The human genome contains billions of DNA “letters,” that can only be read as words, phrases and sentences with the help of proteins that, metaphorically, mark the DNA with punctuation. Together, the DNA-protein combinations form chromatin which provides the essential annotation for gene transcription. However, it is still not understood how the annotation and readout of a single genome differs across cell types. The differences are crucial for normal development and are mutated in cancer. Currently, it is thought that different combinations of proteins act at each of the thousands of genes, and deciphering the numerous complex patterns is a difficult task.

In Kang et al., the Kuroda lab identifies a reversible “master switch” that sits on potentially all developmental genes in a model organism, the fruit fly.  Their bivalent master switch model provides a conceptually simple explanation for how each developmental step is made along the path to different cell types, dependent on cell type-specific proteins, but acting through this common module.

In this case the fly model is likely to extend and synergize with seminal work by Harvard Medical School professor Brad Bernstein, MD, PhD, and colleagues on the regulation of key developmental genes in mammalian embryos.

This work was supported by NIH grant GM101958 to M.I.K. K.A.M. was supported by the Joint Training Program in Molecules, Cells, and Organisms (T32GM007598 from the NIH). B.M.Z. was supported by a fellowship from the Jane Coffin Childs Memorial Fund.

Paper Cited: Kang, H et al. “Bivalent complexes of PRC1 with orthologs of BRD4 and MOZ/MORF target developmental genes in Drosophila” Genes & Development DOI 10.1101/gad.305987.117

Using Networks to Understand Tissue-Specific Gene Regulation

Researchers at Brigham and Women’s Hospital have discerned that different tissue functions arise from a core biological machinery that is largely shared across tissues, rather than from their own individual regulators. In a paper published in Cell Reports, Kimberly Glass, PhD, of the Channing Division of Network Medicine, and her team explain how they have used PANDA (Passing Attributes between Networks for Data Assimilation) to create network models of the interactions between transcription factors and genes, finding that the presence of different tissue functions is the result of subtle, tissue-specific shifts in a regulatory network. For each of these tissue-specific functions, the network has the same core components, but they’re combined in different ways with added genetic and environmental information. The team analyzed data from the Genotype-Tissue Expression (GTEx) consortium, among other regulatory information sources, to reconstruct and characterize regulatory networks for 38 tissues.

PANDA, a model created by Glass and her team in 2013, was uniquely qualified for this investigation because it can more accurately model interactions between transcription factors – which help control where, when and to what extent genes get activated – and their targets. Summarizing the complex interactions between transcription factors and genes is an important step in understanding patterns in the network that inform how gene regulation gives rise to a variety of specific tissue functions.

The authors also observed that the regulation of specific tissue function is largely independent of transcription factor expression. They note that there are approximately 30,000 genes in the human genome, but fewer than 2,000 of them encode transcription factors.

“A large number of processes must be carried out for a tissue to function properly,” said Glass. “Rather than activating particular transcription factors to carry out these various processes, we find that that the networks connecting these regulators to their target genes is reconfigured to more effectively coordinate the activation of those tissue functions.”

The team notes that their work highlights the importance of considering the context of specific tissues when developing drug therapies. Given that shifted regulatory networks govern different functions, this will be important in order to understand the potential side effects of drugs outside of the target tissue.

This work was supported by grants from the US National Institutes of Health, including grants from the National Heart, Lung, and Blood Institute (5P01HL105339, 5R01HL111759, 5P01HL114501, K25HL133599), the National Cancer Institute (5P50CA127003, 1R35CA197449, 1U01CA190234, 5P30CA006516, P50CA165962), the National Institute of Allergy and Infectious Disease (5R01AI099204), and the Charles A. King Trust Postdoctoral Research Fellowship Program, Sara Elizabeth O’Brien Trust, Bank of America, N.A., Co-Trustees. Additional funding was provided through a grant from the NVIDIA foundation. This work was conducted under dbGaP approved protocol #9112 (accession phs000424.v6.p1).

Paper Cited: Sonawane AR et al. “Understanding Tissue-Specific Gene Regulation.” Cell Reports DOI: 10.1016/j.celrep.2017.10.001

T-cell Receptors (gold, upper) on the surface of T-cells selectively bind the lipid antigen presenting molecule on dendritic cells, CD1b (lower), presenting a rare cellular phospholipid (gold), over common phospholipids found in membranes (pink, cyan and green), which in turn induces an autoimmune response.

The Skinny on Lipid Immunology

Phospholipids – fat molecules that form the membranes found around cells – make up almost half of the dry weight of cells, but when it comes to autoimmune diseases, their role has largely been overlooked. Recent research has pointed to a role for them in numerous diseases, including psoriasis, contact hypersensitivities and allergies. In a new study published in Science Immunology, researchers from Brigham and Women’s Hospital and Monash University in Australia reveal new insights into the basis for T cell receptor (TCR) autoreactivity to self-phospholipids, with implications for autoimmune diseases.

“Lipids have been under appreciated in immunology,” said co-corresponding author D. Branch Moody, MD, a principal investigator in the Division of Rheumatology, Immunology and Allergy. “We’ve been interested in autoimmune diseases for decades, and it’s thought that in certain autoimmune diseases like psoriasis, multiple sclerosis and type 1 diabetes are driven by particular tissues. The search for the particular molecules, known as antigens, that trigger autoimmune diseases has focused on proteins and peptides, but we should also be thinking about lipids as candidate antigens for autoimmune disease.”

For 30 years, researchers have known that T cells play an important role in autoimmune disorders, but it was thought that T cells could only respond to proteins. Previous studies conducted by investigators at the Brigham provided the first hint that a T cell could also respond to lipids. The newly published study suggests that many T cells can respond lipids, and illuminates the physical structures that make this recognition of lipids possible.

T cells are activated when another key part of the immune system, dendritic cells, present them with an antigen. Moody and his colleagues, Ildiko Van Rhijn and Tan-yun Cheng, set out to detect what molecules were being captured and presented, stimulating a T cell response. Using structural biology, Jamie Rossjohn and Adam Shahine of the Australian Research Council Centre of Excellence in Advanced Molecular Imaging at Monash University in Australia showed how a protein on the surface of dendritic cells – known as CD1b – binds to lipids. This complex of CD1b and a lipid then binds to a T cell receptor, activating an immune response.

“The advanced imaging facilities of the Australian Synchrotron have allowed us to generate three-dimensional models of T-cell receptor interaction against CD1b and lipid antigens,” said Shahine. “These results highlight the role of CD1b in a phospholipid-mediated immune response, and grant us a deeper understanding of the mechanisms of lipid-based autoimmune disease.”

The work may have implications for specific forms of autoimmune disease, including systemic lupus erythematosus. Previous studies have found that patients with lupus have antibodies that bind to phospholipids, which cause clotting and strokes. The new study shows that T cells also recognize phospholipids, opening up new perspectives on T cell and antibody cooperation in this disease.

“We now have these beautiful, three-dimensional images of how three different molecules can interact, which explains some detail about which part of the lipid matters. Knowing the precise structure of the complexes involved in this process could be useful for designing new kinds of lipids that could turn on or off the immune response,” said Moody.

Other researchers who contributed to this work include Sarah Iwany and Stephanie Gras.

Funding for this work was provided by the Australian Research Council and National Health and Medical Research Council, the National Institutes of Health (AR048632 and AI049313). J.R. is supported by an ARC Laureate Fellowship.

Paper cited: Shahine A et al. “A molecular basis of human T cell receptor autoreactivity toward self-phospholipids” Science Immunology DOI: 10.1126/sciimmunol.aao1384

Investigating the Most Common Genetic Contributor to Parkinson’s Disease

LRRK2 gene mutations are the most common genetic cause of Parkinson’s disease (PD), but the normal physiological role of this gene in the brain remains unclear. In a paper published in Neuron, Brigham and Women’s Hospital principal investigator, Jie Shen, PhD, of the Department of Neurology, and her team describe an essential role of LRRK in the brain during aging that may help to shed light on the causes of PD in human patients. Their results appear this week in Neuron.

The team generated LRRK-deficient mice where both the LRRK2 gene and the LRRK1 gene were inactivated using a genetic technique called gene knock out. These mice showed signs of age-dependent dopaminergic (DA) neuron degeneration. Surprisingly, this double knock out, where two genes are simultaneously rendered inoperative, caused mice to exhibit earlier mortality and body-weight loss but largely normal brain-weight. Mice with only one gene knocked out did not develop the age-dependent DA neuron degeneration that the double knock out mice experienced.

Previous investigations by Shen’s team connected the LRRK2 gene to the autophagy-lysosomal pathway. This pathway is an important mechanism for the cell to remove excess and abnormal proteins, and impairment of this pathway is increasingly recognized as a factor in neurodegenerative disorders like PD. The PD-like phenotypes of LRRK2 knockout mice, however, are only present in the aged kidney but not in the brain.

“A logical explanation for the lack of phenotypes in LRRK2 knockout mice was that the LRRK1 gene is still there to carry out normal LRRK function and compensate for the loss of LRRK2,” said Shen. “So we generated the double knock out used in this study. With both LRRK genes removed, the double knockout mice lost the LRRK1 protection of the brain and developed age dependent degeneration.”

In the study published in Neuron, the research team reports age-dependent, significant DA neuron degeneration in double knock out mice accompanied by several other complications including impaired autophagy-lysosomal pathways. Further analysis revealed increased programmed cell death, or apoptosis, and higher levels of alpha-synuclein, a hallmark of PD.

“These findings revealed an essential role of LRRK in the survival of DA neurons and in the regulation of the autophagy-lysosomal pathway in the aging brain,” said Shen.

This work was supported by grants from the NINDS (R01NS071251 and P50NS094733 to J.S.).

Paper Cited: Giaime E et al. “Age-dependent dopaminergic neurodegeneration and impairment of the autophagy-lysosomal pathway in LRRK-deficient mice.”  Neuron DOI: 10.1016/j.neuron.2017.09.036