molecular sensor finds earliest signs of leaky blood brain barrier in MS -- consistent with methanol-formaldehyde paradigm -- Katerina Akassoglou, Dimitrios Davalos, Gladstone Center for In Vivo Imaging Research CIVIR, UCSF, Annals of Neurology: Rich Murray 2013.12.03
- molecular sensor finds earliest signs of leaky blood brain barrier in MS -- consistent with methanol-formaldehyde paradigm -- Katerina Akassoglou, Dimitrios Davalos, Gladstone Center for In Vivo Imaging Research CIVIR, UCSF, Annals of Neurology: Rich Murray 2013.12.03Prof. WC Monte gives free archive of 782 full text medical research references for new paradigm of methanol toxicity -- becomes formaldehyde inside cells of 20 human tissues with high levels of ADH1 enzyme: Rich Murray 2013.12.03Methanol (wood alcohol) from cigarettes and aspartame circulates with blood half-life 3 hours, entering every cell -- made into uncontrolled formaldehyde inside cells with high ADH1 enzyme levels -- WC Monte paradigm: Rich Murray 2013.11.06Only humans cells lack a functioning biochemical defense against the ADH1 enzyme, in high levels in 20 tissues, rapidly making methanol into uncontrolled, free floating highly reactive acidic hydrated formaldehyde right inside the cytosol. Thus, humans are ten to a hundred times more vulnerable than any other creature.similar macular harm in multiple sclerosis as from formaldehyde made by ADH enzyme inside retina capillary walls from methanol, Prof. Woodrow C. Monte text "While Science Sleeps" 2012 Jan -- some quotes re retina harm: Rich Murray 2012.05.10
Molecular sensor detects early signs of multiple sclerosisUsing advanced detection and imaging techniques, Gladstone scientists were able to track thrombin activity in mice modified to mimic MS (left) compared to healthy controls. Credit: Dimitrios Davalos/Kim Baeten
For some, the disease multiple sclerosis (MS) attacks its victims slowly and progressively over a period of many years. For others, it strikes without warning in fits and starts. But all patients share one thing in common: the disease had long been present in their nervous systems, hiding under the radar from even the most sophisticated detection methods. But now, scientists at the Gladstone Institutes have devised a new molecular sensor that can detect MS at its earliest stages—even before the onset of physical signs.
In a new study from the laboratory of Gladstone Investigator Katerina Akassoglou, PhD, scientists reveal in animal models that the heightened activity of a protein called thrombin in the brain could serve as an early indicator of MS. By developing a fluorescently labeled probe specifically designed to track thrombin, the team found that active thrombin could be detected at the earliest phases of MS—and that this active thrombin correlates with disease severity. These findings, reported online in Annals of Neurology, could spur the development of a much-needed early-detection method for this devastating disease.
MS, which afflicts millions of people worldwide, develops when the body's immune system attacks the protective myelin sheath that surrounds nerve cells. This attack damages the nerve cells, leading to a host of symptoms that include numbness, fatigue, difficulty walking, paralysis and loss of vision. While some drugs can delay these symptoms, they do not treat the disease's underlying causes—causes that researchers are only just beginning to understand.
Last year, Dr. Akassoglou and her team found that a key step in the progression of MS is the disruption of the blood brain barrier (BBB). This barrier physically separates the brain from the blood circulation and if it breaks down, a blood protein called fibrinogen seeps into the brain. When this happens, thrombin responds by converting fibrinogen into fibrin—a protein that should normally not be present in the brain. As fibrin builds up in the brain, it triggers an immune response that leads to the degradation of the nerve cells' myelin sheath, over time contributing to the progression of MS.
"We already knew that the buildup of fibrin appears early in the development of MS—both in animal models and in human patients, so we wondered whether thrombin activity could in turn serve as an early marker of disease." said Dr. Akassoglou, who directs the Gladstone Center for In Vivo Imaging Research (CIVIR). She is also a professor of neurology at the University of California, San Francisco, with which Gladstone is affiliated. "In fact, we were able to detect thrombin activity even in our animal models—before they exhibited any of the disease's neurological signs."
In laboratory experiments on mice modified to mimic the signs of MS, the team employed an Activatable Cell-Penetrating Peptide (ACPP), a special type of molecular probe that delivers fluorescent agents to a region of interest. For this study, they developed a thrombin-specific ACPP that could track thrombin activity in mice as the disease progressed. They then carefully analyzed where—and at what stage of disease—thrombin activity was found.
"We detected heightened thrombin activity at specific disease 'hot-spots,' regions where neuronal damage developed over time," said Gladstone Staff Research Scientist Dimitrios Davalos, PhD, associate director of the CIVIR and one of the paper's lead authors. "And when we compared those results to those of a separate, healthy control group of mice, we saw that thrombin activity in the control group was wholly absent."
"Our results are proof of principle that a thrombin-specific molecular probe could be used as an early-detection method," added former Gladstone Postdoctoral Researcher Kim Baeten, PhD, the paper's other lead author.
The team's results offered significant support for the notion that thrombin activity is directly tied to the degradation of nerve cell's myelin sheath—and the subsequent destruction of nerve cells—that characterizes MS. But they also shed light on what has been a long-standing mystery: the underlying molecular processes that kick-start the progression of MS.
"In the future," said Dr. Akassoglou, "this thrombin-specific ACPP could be developed to one day allow for early patient diagnosis and therapeutic intervention—including a way to effectively monitor how patients are responding to the latest treatments."
Journal reference: Annals of Neurology
Provided by Gladstone Institutes
Research reveals new understanding, warning signs, and potential treatments for multiple sclerosis
Scientists are gaining a new level of understanding of multiple sclerosis (MS) that may lead to new treatments and approaches to controlling the chronic disease, according to new research released today at Neuroscience 2013, the annual meeting of the Society for Neuroscience and the world's largest source of emerging news about brain science and health.
MS is a severe, often crippling, autoimmune disease caused by the body's immune system attacking the nervous system. Today, more than two million people worldwide suffer from MS and other neuroinflammatory diseases. MS usually strikes in early adulthood and manifests with symptoms including vision loss, paralysis, numbness, and fatigue. The disease can be intermittent or progressive and currently has no cure.
Today's new findings show that:
- Scientists are one step closer to understanding how antibodies in the blood stream break past the brain's protective barrier to attack the optic nerves, spinal cord, and brain, causing the symptoms of neuromyelitis optica, a rare disease similar to MS. Understanding how the antibodies bypass the protective blood-brain barrier could provide new approaches to treating the disease (Yukio Takeshita, MD, PhD, abstract 404.09, see attached summary).
- A protein involved in blood clotting mightserve as an early detection method for MS before symptoms occur. Early detection of the disease could lead to more effective early treatments (Katerina Akassoglou, PhD, abstract 404.11, see attached summary).
- Low levels of a cholesterol protein correlate with the severity of a patient's MS in both human patients and mouse models. The finding suggests the protein, known to protect against inflammation, may protect against developing MS, and possibly even aid in the regeneration of damaged neurons. This research opens the door to cholesterol drugs as a possible new avenue for MS treatment (Lidia Gardner, PhD, abstract 404.01, see attached summary).
Other recent findings discussed show that:
- A type of immune system cell has been found to directly target and damage nerve cell axons, a hallmark of MS. This may reveal a target for new therapies (Brian Sauer, PhD, presentation 404.06, see attached speaker summary).
- While no treatments to rebuild cells damaged by MS currently exist, scientists have found that when exosomes—tiny, naturally occurring "nanovesicles"—are produced by dendritic cells and applied to the brain, they can deliver a mixture of proteins and RNAs that promote regeneration of protective myelin sheaths and guard against MS symptoms (Richard Kraig, MD, PhD, presentation 812.02, see attached speaker summary).
"The findings shown today represent real promise for the millions suffering from MS," said press conference moderator Jeffrey Rothstein of Johns Hopkins University and an expert in neurodegenerative diseases. "These studies are breakthroughs in understanding and treating a disease that remains uncured, difficult to diagnose, and for which it is very difficult to prevent progression."
Explore further: MS research could help repair damage affecting nerves
Provided by Society for NeuroscienceNat Commun. 2012;3:1227. doi: 10.1038/ncomms2230.
Fibrinogen-induced perivascular microglial clustering is required for the development of axonal damage in neuroinflammation.Davalos D, Ryu JK, Merlini M, Baeten KM, Le Moan N, Petersen MA, Deerinck TJ, Smirnoff DS, Bedard C, Hakozaki H, Gonias Murray S, Ling JB, Lassmann H,Degen JL, Ellisman MH, Akassoglou K.
Gladstone Institute of Neurological Disease, University of California, San Francisco, 1650 Owens Street, San Francisco, CA 94158, USA.
Blood-brain barrier disruption, microglial activation and neurodegeneration are hallmarks of multiple sclerosis.
However, the initial triggers that activate innate immune responses and their role in axonal damage remain unknown.
Here we show that the blood protein fibrinogen induces rapid microglial responses toward the vasculature and is required for axonal damage in neuroinflammation.
Using in vivo two-photon microscopy, we demonstrate that microglia form perivascular clusters before myelin loss or paralysis onset and that, of the plasma proteins, fibrinogen specifically induces rapid and sustained microglial responses in vivo.
Fibrinogen leakage correlates with areas of axonal damage and induces reactive oxygen species release in microglia.
Blocking fibrin formation with anticoagulant treatment or genetically eliminating the fibrinogen binding motif recognized by the microglial integrin receptor CD11b/CD18 inhibits perivascular microglial clustering and axonal damage.
Thus, early and progressive perivascular microglial clustering triggered by fibrinogen leakage upon blood-brain barrier disruption contributes to axonal damage in neuroinflammatory disease.
- [PubMed - indexed for MEDLINE]
free full text
By in vivo imaging of BBB disruption, microglia and axons, we identified fibrinogen as a novel molecular signal that triggers rapid perivascular microglial responses, and contributes to axonal damage in neuroinflammatory disease. Intriguingly, microglia exhibit cell motility patterns directed specifically toward the vasculature that precede the onset of neurological signs and lesion formation. Therefore, our data suggest that BBB disruption is necessary for the mobilization of the resident innate immune cells around the CNS vasculature, which might represent one of the earliest events in MS pathology that can trigger and amplify inflammatory demyelination and axonal damage (Fig. 8b).
Our study demonstrates a novel pattern of microglial motility toward the vasculature not previously observed in other injury or disease models. In contrast to Alzheimer’s disease14 and laser-induced injury11,13, in neuroinflammation, microglia primarily respond to the vasculature by a combination of process extension and cell body motility. Microglial responses toward the vasculature occur already at the pre-onset stage of EAE. As demyelination and axonal damage are prominent at the peak of EAE as shown in our study and previously31, microglial clusters might demarcate the areas of new lesion formation. Extensive axonal deformities appear in synchrony with rapid microglial process extension and retraction, and such events were recorded only perivascularly, at sites of BBB leakage with fibrin deposition and not in other areas in the parenchyma. Although this might be a real-time in vivo representation of the classic role of microglia as phagocytic cells that remove cellular debris, microglia might have a primary role in axonal destruction. Indeed, as we recorded multiple microglial interactions with abnormal axonal formations and fragments still expressing CFP, and confirmed with confocal microscopy engulfment of axonal fragments by microglia, we postulate that microglia might attack and phagocytose living axonal fragments at areas of BBB leakage. This agrees with the observations that microglia and macrophages are close to degenerating axons with intact myelin sheaths40, and phagocytosis of axonal fragments by microglia occurs in MS lesions36. Therefore, our three-color in vivo imaging studies identify the vasculature as a niche for activation of innate immune responses and axonal damage in neuroinflammation. Furthermore, they support neuropathological studies in MS plaques that have suggested a primary role for microglia and their inflammatory activity in axonal damage41,42,43.
Our study reveals a novel function for fibrinogen as a signal that induces rapid, yet sustained microglial responses in vivo in the healthy brain. In vivo real-time imaging previously identified rapid microglial process extension upon injection of ATP11. Fibrinogen is a novel molecular signal that can mediate a similar response. ATP mediates microglial responses through the P2Y12 receptor44. As rapid microglial process extension is associated with dramatic cytoskeletal rearrangements, presumably involving activation of the Rho signaling pathway, cross-talk of P2Y12 with integrin receptors might mediate the rapid microglial response induced by fibrinogen. Indeed, integrins are primarily responsible for cytoskeletal rearrangements, and fibrinogen induces Rho activation in microglia via CD11b/CD18 (ref. 27). ATP induces integrin-β1 expression in microglia through P2Y12 receptor and integrin-β1 activation is involved in the directional process extension by microglia in brain tissue45. Moreover, ATP increases CD11b expression in microglia46and eosinophils47. Future studies will determine the potential cross-talk between P2Y12 and microglial integrin receptors upon BBB disruption.
The perivascular responses we observe are not restricted to microglia, but also include the resident pial macrophages. Meningeal and parenchymal blood vessels have different architecture, as leptomeningeal blood vessels are not surrounded by the glia limitans perivascularis. Interestingly, we observe fibrin deposition and perivascular clustering around both meningeal and parenchymal blood vessels. The concomitant responses of microglia and resident pial macrophages to BBB leakage imply an aspect of functional convergence of the two CNS-resident monocytic populations in perivascular clustering during neuroinflammatory disease. Fibrin/CD11b signaling appears to mediate both responses of microglia and pial macrophages, since anticoagulant treatment or studies in the Fibγ390-396A mice ameliorate responses of both cell populations (our study) and clinical EAE severity25,27. Although our study demonstrated the interplay between BBB disruption with the Cx3cr1GFP/+ CNS-resident microglia and pial macrophages, fibrin/CD11b signaling might also be responsible for peripheral macrophage recruitment in neuroinflammation. Infiltration of peripheral monocytes in the CNS parenchyma is crucial for disease progression and depends on CCR2 expression8,17. Cx3cr1GFP/+ mice are primarily reporters for resident monocytes17. In the Cx3cr1GFP/+ mice there are only a few peripherally circulating GFP-positive cells, which are not expressing CCR217, suggesting that these cells do not infiltrate in the CNS. Future studies with differentially labeled resident and peripheral monocyte populations will dissect the role of fibrinogen in macrophage recruitment in CNS disease. Thus, molecular events at the BBB that trigger early perivascular plasma leakage and immune cell clustering may regulate peripheral inflammatory responses and infiltration of the CNS.
A fundamental question in the development of inflammatory demyelination has been the identity of the early triggers and the amplifiers of disease pathogenesis. It is considered axiomatic that large-scale transmigration across the BBB is associated with loss of barrier function. Studies from neuropathology in early MS lesions that showed fibrin deposition and microglial activation in the absence of parenchymal T cells or demyelination suggested that there are other mechanisms in play that might open the BBB and activate microglia2,3,4,5. However, standard histopathology techniques cannot decipher which comes first (that is, microglial activation or BBB disruption). Our study shows that fibrinogen that enters into the CNS after BBB leakage is sufficient and specific among blood proteins to trigger rapid microglial responses even in the healthy CNS in the absence of a pre-existing lesion. Thus, it is possible that in genetically susceptible individuals, early events at the gliovascular interface, such as fibrinogen leakage in the CNS, might function as triggers that are sufficient to promote autoimmune responses resulting in further BBB disruption, demyelination and plaque formation. Indeed, CD11b-positive monocytes modulate autoimmune responses48 and perivascular microglia and pial macrophages have properties of antigen-presenting cells49,50,51. Therefore, early cluster formation mediated by fibrin/CD11b signaling may contribute to the development of autoimmunity. These mechanisms might also be in play in active MS plaques, where release of proinflammatory cytokines by infiltrating macrophages and T cells may further open the BBB and enhance fibrin deposition in the CNS. Similar to microglia, astrocyte activation also occurs early in EAE before significant T-cell entry, and is associated with axonal damage in the optic nerve52. As fibrinogen induces astrocyte activation53, it is possible that it might also contribute to the early activation of astrocytes in neuroinflammation. Identifying fibrinogen as a trigger for perivascular microglial activation in early disease stages is crucial for the understanding of microglial functions and their contribution to CNS autoimmune disease as well as for the development of therapies targeting early stages in MS pathogenesis. Future in vivo imaging studies using differentially labeled lymphocytes, peripheral macrophages, microglia, astrocytes and fibrinogen will establish the temporal and spatial relationships that govern adaptive and innate immune responses during the course of EAE.
Microglia and macrophages are considered main effector cells of the innate immune response in the CNS that might be involved in axonal damage32. In MS lesions, a major mechanism of axonal damage is related to the effects of ROS, which may lead to axonal damage via mitochondrial impairment54. In accordance, neutralization of ROS in EAE reduced axonal damage40. We show that fibrinogen induces ROS release in microglia and that its signaling via the microglial receptor CD11b is required for development of axonal damage in inflammatory disease. Fibrin deposition correlates with acute axonal damage and microglial activation in early pre-demyelinating MS lesions before the infiltration of lymphocytes2. Moreover, fibrin accumulates on demyelinated axons in chronic-progressive MS21,35. Axonal damage is considered a critical cause of disability in MS and is associated with activation of the innate immune system, suggesting that suppressing innate immune responses could be an important therapeutic strategy for progressive MS42. Therefore, targeting the interactions of fibrinogen with the CD11b/CD18 integrin receptor might be a potent new therapeutic approach to suppress the activation of innate immune responses and prevent axonal damage in neuroinflammatory disease.
Biochemical Journal Poster Prize winner
Kim Baeten obtained her Masters degree in Pharmaceutical Sciences at the KU-Leuven in Belgium. She followed this up with a Ph.D. at the University of Aberdeen (U.K.) in the laboratory of Professor N.A. Booth. She studied how platelets can increase the breakdown of blood clots, and found that platelet-associated plasminogen caused localized activation of single-chain urokinase-type plasminogen activator, thereby enhancing fibrinolysis.
She is now a post-doctoral Research Fellow in the laboratory of Professor K. Akassoglou at the Gladstone Institute of Neurological Diseases in San Francisco, CA, U.S.A., investigating the role of the blood protein fibrinogen in neurological disease.
More about Dr. Davalos
Dr. Dimitrios Davalos studies microglia, the resident immune cells of the brain and spinal cord, and seeks to better understand their functions under physiological conditions and in different animal models of neurological disease. Microglia are the first responders to any pathological insult, whether injury or disease, in the brain or the spinal cord.
During his graduate years Dr. Davalos performed the first in vivo imaging study of microglia, taking advantage of powerful microscopy technologies that allow researchers to follow the behavior of individual cells inside the intact living brain, in real time. This work redefined our understanding of the role of microglia in the brain and, with over 650 citations to date, is considered one of the classic studies in microglial biology.
Dr. Davalos is currently investigating mechanisms of blood brain barrier disruption, a pathological phenomenon that is very common among neurological diseases, such as multiple sclerosis, Alzheimer’s disease, and stroke. Using cutting-edge imaging techniques, he studies the interactions between blood vessels, neurons, and glia, and seeks to understand how their relationships change between health and disease. Specifically, Dr. Davalos investigates how microglia become activated when the brain vasculature is compromised, and how microglial responses relate to the neuronal deficits observed in neurological diseases. In doing so, his ultimate goal is to identify new targets for therapeutic intervention.
As the associate director of the Center for In Vivo Imaging Research (CIVIR), Dr. Davalos oversees the day-to-day operations of the Center, designs experiments, and performs and trains collaborating scientists in surgical and in vivo imaging procedures. He has significant experience in addressing the technical challenges that are inherent with in vivo imaging experiments in different tissues and has developed and published new methods to make such experiments possible. The Center is equipped with two cutting-edge two-photon microscopes especially customized for multicolor, simultaneous, deep tissue imaging of multiple fluorescently labeled cells in vivo.
Dr. Davalos earned a BS in biology from the University of Athens in Greece, and a Master’s and a PhD in physiology and neuroscience from New York University. He then joined the laboratory of Dr. Katerina Akassoglou for his postdoctoral training at the University of California, San Diego (UCSD), and moved with her to the Gladstone Institute of Neurological Disease in 2008. In 2010, Drs. Akassoglou and Davalos established the CIVIR, which collaborates with researchers from all over the world.
Before joining Gladstone, Dr. Davalos helped to establish in vivo imaging at the National Center for Microscopy and Imaging Research (NCMIR) at UCSD, where he is still a visiting scientist. He serves as a member of the pilot grant review committee for the National Multiple Sclerosis Society (NMSS) and received postdoctoral and young investigator awards from the NMSS, the American Heart Association and the Nancy Davis Foundation for Multiple Sclerosis.
Prof. WC Monte gives free archive of 782 full text medical research references for new paradigm of methanol toxicity -- becomes formaldehyde inside cells of 20 human tissues with high levels of ADH1 enzyme: Rich Murray 2013.12.03
Methanol (wood alcohol) from cigarettes and aspartame circulates with blood half-life 3 hours, entering every cell -- made into uncontrolled formaldehyde inside cells with high ADH1 enzyme levels -- WC Monte paradigm: Rich Murray 2013.11.06
Only humans cells lack a functioning biochemical defense against the ADH1 enzyme, in high levels in 20 tissues, rapidly making methanol into uncontrolled, free floating highly reactive acidic hydrated formaldehyde right inside the cytosol. Thus, humans are ten to a hundred times more vulnerable than any other creature.
similar macular harm in multiple sclerosis as from formaldehyde made by ADH enzyme inside retina capillary walls from methanol, Prof. Woodrow C. Monte text "While Science Sleeps" 2012 Jan -- some quotes re retina harm: Rich Murray 2012.05.10
Evidence exists that autism results from exposure to pregnant women in the fourth week, since ADH1 levels are high in the Purkinje cells of the vermis in the cerebellum, while other plausible birth defects include spina bifida, premature birth, and Fetal Alcohol Spectrum Disorder.
The leading methanol sources are cigarette smoke and aspartame (E951).
WC Monte gives 782 free full text medical research references at WhileScienceSleeps.com .
California OEHHA sets methanol ingestion level 23 mg daily, same as from 1 can aspartame diet soda, 10 cigarettes, 3 tomatoes, or 4 cans green beans: Rich Murray 2013.07.03
"However, the anticipated exposure to methanol from consumption of aspartame would not be considered an exposure within the meaning of Proposition 65 because aspartame is not listed under Proposition 65."
[ Rich Murray: Many pregnant women drink one 12-oz can aspartame diet drink daily, with 200 mg aspartame that gives 11% methanol, 22 mg, which is just under the OEHHA limit of 23 mg daily.
The smoke from 10 cigarettes gives 20 mg methanol, the same as from 3 full size fresh tomatoes, or 4 cans of unfresh green beans. ]
smoke from a pack cigarettes gives 40 mg methanol (wood alcohol), same as from 2 aspartame diet drinks -- becomes formaldehyde inside brain and retina cells via ADH1 enzyme -- WC Monte paradigm: Rich Murray 2013.08.30
11% of aspartame is methanol, which becomes free floating formaldehyde inside human cells -- methanol also in cigarettes and canned fruits and vegetables: Rich Murray 2013.08.30
Human epidemiological studies so far fail to control for additional common methanol sources: cigarettes and wood and peat smoke, smoked foods, fresh
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