BrightFocus-Funded Alzheimer’s Research at Neuroscience 2015
This research was supported by BrightFocus
The tweets start arriving a week before it starts: “Our lab is excited to be heading to Chicago for SfN.” “Proud to be presenting at SfN.” “Any tips for surviving my first SfN?”
If there’s a nervous undertone, it’s because the annual conference of the Society for Neuroscience (SfN) can be overwhelming to beginners. Sometimes even for seasoned scientists.
It stands to reason that when 30,000 neuroscientists gather in one place from close to half of the world’s countries (85-plus, at last count), you’re in for a heady experience. Public radio described this year’s SfN, also known as Neuroscience 2015, as “science’s hottest hangout.
Hundreds of data-packed presentations take place each day. To have an abstract accepted at SfN, and be a presenter there, is an honor among peers. Either way, SfN provides the chance to collaborate with others who also are looking for answers to today’s most perplexing diseases.
Finding Ways to Boost the Brain’s Immunity Against Alzheimer’s
Recent discoveries about AD pathology have hammered home the point that it’s important to diagnose and treat AD at its earliest stages, before memory loss and other clinical symptoms become noticeable. That’s because during this preclinical stage of Alzheimer’s, which can last for decades, a critical conversion takes place. “Normal” amyloid beta (Aβ) and tau protein undergo biochemical changes that ultimately cause them to become misshapen and collect in neurotoxic plaques and tangles.
Growing evidence shows that, at Alzheimer’s earliest stages, an army of immune cells are responsible for clearing neurotoxic Aβ protein from the brain. The host defense forces include inflammatory cells, brain-residing microglia (eg, the brain’s indigenous immune cells), and infiltrating monocyte-derived macrophage cells, which are a reserve force of cells in the peripheral bloodstream that provide reinforcement for the brain’s own immune cells, as needed.
Even so, over the course the decades-long battle, as brain tissue succumbs to chronic, AD-associated inflammation, the host defenses get worn down. They become less and less efficient at removing Aβ, and ultimately are believed to become pro-inflammatory themselves, thus making them cytotoxic to neurons.
At Cedars-Sinai Medical Center in Los Angeles, 2013-16 BrightFocus grantee Maya Koronyo-Hamaoui, PhD is showing how it may be possible to “recruit” fresh immune cells to modify or invigorate the brain’s existing immune cells that have become inefficient and dysfunctional .
In mice, Koronyo-Hamaoui and her team have experimented with different methods of invigorating the brain’s host defense against synaptotoxic Aβ species. These include weekly immunization with an approved drug, glatiramer acetate (GA), which was shown to enhance natural recruitment of blood-borne monocytes to diseased parts of the brain; and injections of therapeutic bone-marrow derived immune cells (monocytes) into the peripheral bloodstream. They also tried both therapies together.
Results were successful, and showed that in 13-month-old mice exhibiting AD symptoms, these interventions helped rescue and preserve synapses, ie, the ends of neurons through which nerve signals pass, and helped to maintain cognitive function. In addition, treatment with GA helped direct macrophages to clear pathological forms of Aβ, as well as amyloid plaque sites, thus reducing overall amyloid burden and neuroinflammation (eg, astrogliosis) (M. Koronyo-Hamaoui, Abstract No. 195.01A. A. Rentsendorj, Abstract No. 395.13).
Koronyo-Hamaoui and her team published earlier results from this BrightFocus-funded project in major scientific journals. Last spring, in the Journal of Clinical Investigation, they described their experiments using genetically modified mice to examine the effect of targeted angiotensin-converting enzyme (ACE) overexpression on AD; the results showed that both plaque burden and astrogliosis were drastically reduced in their AD-positive, ACE-over-expressing mouse model (Bernstein KE et al, 2015).
Then last summer, the group’s article in Brain highlighted their method of grafting green fluorescent protein -labeled monocytes into the blood, thus enabling them to track the same cells in the brain. In this paper they demonstrated, for the first time, that it is possible to change disease progression in mice using their immunomodulation techniques. Their results pointed to reduced Aβ levels and reduced local inflammation contributing to preservation of synapses and cognition. The suppressed inflammation is something they attributed to GA’s induction of regulatory interleukin 10 (IL-10) in the brain. IL-10 is an immune cell circulating throughout the body that targets innate immune and other glial cells in the brain and may aid in terminating detrimental neuroinflammation.
Also discussed in that paper were the team’s in vitro studies using primary cultures of bone marrow-derived macrophage cells. Results showed that the increased clearance of toxic Aβ species were mediated by increased scavenger receptor expression on the membrane of these macrophages and also by increased levels matrix metallopeptidase 9 (MMP-9), an enzyme that is capable of degrading Aβ (Koronyo Y et al, Brain, 2015).
How Memories Are Made—and Lost—in Alzheimer’s
It’s often assumed that memory dysfunction in Alzheimer’s is a consequence of dying neurons. However, the BrightFocus-funded work of Carlos Saura, PhD, of Universitat Autònoma de Barcelona, has challenged that assumption.
In 2005, Saura published a paper showing that, in a mouse model of AD, memory impairment occurred prior to the accumulation of amyloid plaques. This suggested that something besides plaques causes synaptic dysfunction and cognition loss early in the disease process.
From that time forward, Saura, a 2014-17 BrightFocus grantee, devoted the better part of a decade to investigating how genes encode memories in the brain, and how that breaks down in Alzheimer’s. Basically, genes tell our cells what to do by signaling through specialized proteins known as “transcriptional factors.” In the hippocampus, transcriptional factors cause neurons to activate the circuits for memory processing; however, the process is poorly understood.
Saura has focused on the CREB-regulated transcription coactivator 1 (CRTC1) gene, and in a series of mouse experiments, has shown it plays a critical role in regulating the hippocampus’ ability to encode memory. He and Arnaldo Parra-Damos, PhD, of his team, shared their latest results in a poster and at a symposium, where Saura spoke.
Their experiments have shown that in normal adult mice, spatial and associative learning cause CRTC1 activation leading to gene expression in memory circuits. In AD-mutant mice with increased levels of toxic Aβ, this activity of CRTC1 was reduced, and associated with memory deficits. [Saura et al, Abstract #370.04; Parra-Damas et al, Abstract #131.17]
Apart from mice, they also looked at changes in CRTC1 expression in postmortem brain samples from 68 adults who suffered from varying stages of AD. They found that dysregulation of CRTC1-dependent transcription is associated with decreased CRTC1 levels in the human brain in the middle stages of Alzheimer’s (Braak III–IV pathological stages).
Together, the evidence suggests that early Aβ-induced dysregulation of CRTC1 may underlie memory deficits early in AD. Importantly, when AD mice were treated to overexpress CRTC1 in the hippocampus, spatial learning and memory improved.
Saura thinks that enhancing CRTC1 function might provide therapeutic benefits that help overcome the breakdown in gene transcription, and assist in the preservation of memory deficits in the early stages of AD. Up ahead, he is hoping that these alterations in gene regulation and transcription may serve as novel biomarkers and therapeutic targets to restore brain function in humans with early Alzheimer’s.
Already his BrightFocus-funded work has been published in two journals. The first article is a “proof of concept” milestone; the team’s mice data are some of the first evidence that targeting brain transcriptome reverses memory loss in AD (Parra-Damas et al, J Neurosci 2014). The second, a review article, puts the work into broader perspective and articulates the team’s vision and goals for translating their work into human therapies (Saura et al, Front Cell Neurosci. 2015).
Immune Cells React Differently to Alzheimer’s, Based on Their Genetic Make-up
With a 2013-16 BrightFocus grant, Paramita Chakrabarty, PhD, of the University of Florida, has been investigating the interplay between innate immunity and genetic risk in Alzheimer’s disease (AD). In particular, she’s looking at differences in microglial activation and inflammatory response in various mouse models with different risk factors for AD.
As noted before, Alzheimer's disease invokes an immune response and causes massive inflammation, a natural reaction of the body to toxins, bacteria, and foreign particles. Cytokines are protein molecules that act as messengers between d cells to regulate the inflammatory response.
For Chakrabarty and her mentor and coauthor, Todd Golde, PhD, a driving question has been to determine how an anti-inflammatory response might alter disease onset. In general, an immune response that increases anti-inflammatory proteins is thought to be beneficial. However, in their previous study published earlier this year, these researchers showed that gene therapy to induce a cytokine known as Interleukin 10 (IL-10) actually increased the APOE protein expression and led to greater plaque burden in mice (Chakrabarty et al, Neuron, 2015.
Those results have been described as “counterintuitive” and “novel,” but in some ways they make sense to Chakrabarty. “The interactions between Alzheimer pathology and functioning of the immune system is still enigmatic, with seemingly disparate data emerging from various laboratories,” she notes.
Thus, her ongoing quest is to use modern techniques to identify the exact transcriptomic profile, or “genetic signature” of microglia cells in animal models of different AD risk categories, in response to toxic Aβ and other Alzheimer’s-related pathogens. This is important given that many laboratories are now focused on developing disease-modifying therapies targeted to a specific immune signature. And “as you may be aware, we do not have the ‘perfect’ model of Alzheimer’s disease that fully recapitulates all the facets of Alzheimer’s pathology,” Chakrabarty says.
Her latest results, presented at SfN, are “discordant.” It appears that microglial cells in various mouse models of AD have a unique genetic signature and response differently to pathologic threats, including Aβ, based on both their genes and environment. “Such discordant immunophenotyping data calls for cautious interpretation of studies conducted exclusively in primary microglia and their relevance to testing therapies in culture,” she says.
Chakrabarty’s research, a merger of genetics and cell biology, is like a microcosm of “personalized medicine,” that new field where therapies for cancer and other diseases are “customized” based on a person’s genetic make-up. Only in this case, her “patient” is an immune cell in a mouse that’s been genetically altered to develop AD, and she’s testing how it responds to an array of pathologic proteins under different conditions. Like a forensic scientist, she’s helping others avoid wrong conclusions that could lead them down a blind alley way in their search for effective treatments and cures.
How Well Do We Know Amyloid Beta?
David Brody, MD, PhD, is another researcher whose BrightFocus-funded discoveries are helping to keep the field of Alzheimer’s research on target. A board-certified neurologist, Brody spends most time in research at the Washington University School of Medicine in St. Louis, MO (WUSTL)
With a 2014-17 grant from BrightFocus, he is studying the toxic forms of Aβ that build up in the brain and are thought to trigger Alzheimer’s pathology. In fact, he may be studying Alzheimer’s-related forms of Aβ more closely than anyone has before.
That’s because Brody has developed specialized methods and equipment to purify Aβ strains, or “species,” in human brain tissue from donors who have died of Alzheimer’s disease. It’s a painstaking process. After isolating each purified strain of AB, the team is using state-of-the-art chemical analysis, called mass spectrometry, to characterize its structure.
Why is this important? Amyloid proteins, including AB, are among the most prevalent proteins in the human body. Aβ can be present in the body, and even in the brain, for a lifetime without causing harm. However, in the decades-long lead up to Alzheimer’s, something causes AB to “oligomerize,” or break up into smaller fragments known as “oligomers.” And whereas in its longer conformation, Aβ is mostly benign, the shorter AB fragments are prone to aggregation, which means they clump together. This can lead to AB plaque formation and to cerebral amyloid angiopathy, where amyloid deposits collect on the walls of the brain’s blood vessels—another condition associated with AD.
Brody reasons that to only by understanding these toxic forms of amyloid-beta will it be possible to efficiently design effective treatments to prevent them from forming, block their toxicity, or destroy them in the brain. And here’s the kicker: whereas Alzheimer’s research has so far only focused on two toxic Aβ species, Aβ40 and Aβ42, Brody and his team have by now identified more than a dozen additional Aβ strains.
After so much Alzheimer’s research has focused on the buildup of AD-related plaques, and attempts to connect their formation to upstream and downstream pathologies, Brody is calling for the field to “rethink” the basic premise that plaques themselves kill neurons, leading to memory loss. Even though it’s true that plaques are a hallmark sign of Alzheimer’s pathology, plaque deposition only moderately correlates with the progression to dementia. In contrast, AB oligomers (Aβ 40 and Aβ 42) have demonstrated a stronger correlation with dementia status and exhibit significantly greater toxicity.
He believes that the driving force beyond synaptic dysfunction and loss in AD might instead be modification to Aβ structure, and that this, rather than plaques per say, may accelerate memory loss and cognitive decline. Having discovered and authenticated so many native Aβ oligomers from human brains, his goal now is to test that hypothesis.
Watching His Grandmother, He Was Inspired to Study Weight Loss in Alzheimer’s
As a clinician, Mac Ishii, MD, PhD, is trained to notice people. And one of the things he started noticing about people with Alzheimer’s is that they almost invariably lose weight as the disease takes hold and progresses.
Ishii first recognized this in his own grandmother. “My grandmother was never a thin woman, but within a few years of her diagnosis she had lost a significant amount of weight, despite eating what appeared to be her normal amount of food. ‘What causes this unintended weight loss? Is it part of the disease?,’” he wondered at the time.
Ishii is an assistant faculty member in neurology and neuroscience at Weill-Cornell Medical College in New York City. After talking to fellow clinicians there, and reviewing the literature, it became clear to him that weight loss and the associated metabolic deficits are a central part of Alzheimer’s, and that weight loss can occur early, before mental decline.
This launched Ishii’s entry into Alzheimer’s research. Years earlier, while pursuing his PhD, Ishii had investigated pathways in the brain that control appetite and body weight. Now, as a 2015-17 BrightFocus grantee, he’s looking at how toxic AB affects the hypothalamus, a major brain center that controls appetite, by disrupting proteins and hormones produced by fat cells (ie, adipose tissue), that are essential for regulating metabolism and body weight.
He’s discovered that in mice models of AD, as AB accumulates, the animals express low levels of the hormone leptin proportional to their fat stores. Normally, low leptin signals that fat stores are depleted and causes animals to eat more or use less energy to maintain a healthy body weight. Since the mice did neither, there seemed to be a problem with the signaling process.
Ishii and colleagues next found that a key type of brain cells that signal the hypothalamus to stimulate appetite were not responding to leptin and other important metabolic signals. This kink in the signaling system could resulted in a spiral of weight loss.
This early work was published in the Journal of Neuroscience last year (Ishii et al, 2014) At his SfN talk, Ishii gave the results of more recent experiments where they tested the hypothesis that dysfunction in key hypothalamic areas promotes catabolism, an energy drain. Catabolism is a biochemical reaction that breaks down molecules and disperses their energy prior to their disposal from the body. Ishii and colleagues have gathered evidence that in AD mice, catabolism results in a decrease in the size of fat molecules and increased “browning” of white adipose tissue, and that this outcome appears to be a sympathetic reaction to aberrant hypothalamic signaling.
[Correction: An earlier version of this study erroneously reported that the above work had been published at the time of posting.]
Ishii’s next step with his BrightFocus-funded project is to see whether this same metabolic dysfunction occurs in humans, and whether it might potentially lead to biomarkers for detecting AD risk. A sizable drop in leptin concentration—in conjunction with other tools—could help furnish a more accurate early diagnosis of AD which, in early stages, can be hard to differentiate from other forms of cognitive decline. It thus might be a tool to help identify high-risk patients and place them in clinical trials for emerging medications.
In addition, Ishii and colleagues are hopeful that there are ways to reverse the dysfunctional signaling and restore healthy body weight to help already-diagnosed Alzheimer's patients. Otherwise, weight loss is associated with worsening disease progression and even increased risk of death.
These are lessons Ishii took to heart as a neurology resident, when he watched his own grandmother and other elderly patients lose weight before and during their struggle with Alzheimer’s.
"When you see this in your family you say, ‘We have to do something.’ My grandmother was a big impetus for me personally,” he said.
“As a grandson, I feel at times helpless as my grandmother’s dementia continues to worsen. As a clinician-scientist, I see the potential advances we can make by exploring clinical observations that can help solve the complexities of Alzheimer’s disease. It began as a very personal inquiry, but hopefully now will make a broader contribution.”
Differences Seen in Effects of Human vs. Mouse ApoE
At Washington University in St. Louis (WUSTL), Fan Liao, PhD, and her mentor, David Holtzman, MD, have been working to develop immunotherapy against apolipoprotein E (apoE), a protein which affects Aβ clearance and aggregation.
Having the ApoE4 genotype, meaning the allele 4 mutation on the APOE gene, is one of the few known genetic risk factors for late-onset AD. ApoE4 is associated with higher Aβ plaque load, as well as a higher volume and prevalence of cerebral amyloid angiopathy (CAA), a condition associated with AD. In CAA, Aβ deposits collect on the walls of the brain’s blood vessels. The vast majority of patients diagnosed with AD also have CAA.
With a 2013-15 BrightFocus grant, Liao has assessed the effects of ApoE immunization on cognitive behavior, intrinsic brain network function, and amyloid plaque load in mice; as well as monitored for potential side-effects.
In addition, she investigated the mechanisms behind how apoE antibodies work, including their site of action. To do that, Liao monitored the distribution of apoE and apoE antibodies in the brain and blood following short-term immunization; and she also applied apoE antibodies directly into the brain and monitored removal of existing plaques using two-photon microscopy (a fluorescence imaging technique) in live mice.
At SfN, Liao focused on these latter findings from her BrightFocus-funded work. A key theme was t differences between human and animal apoE when administered to mouse models.
Of 299 amino acids, human apoE shares only 70% homology with mouse apoE. That may explain why the two affect plaque formation and CAA differently. Indeed, Liao found that when mouse and human apoE are expressed at the same level, they co-aggregate differently and have different effects on plaque deposition versus CAA in the mouse brain.
TREM2 and Tau: Basic Investigation Into Another Alzheimer’s Risk
The immune molecule known as TREM2 is a relative newcomer to the Alzheimer’s field. It’s name stands for “triggering receptor expressed on myeloid cells 2,” and the TREM2 genetic variant has been associated with an increased risk of developing late-onset AD, as well as elevated risk for other neurodegenerative diseases, including frontotemporal dementia, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS).
Since 2012, it’s been known that mutations in the microglial gene TREM2 increase Alzheimer’s risk by about as much the ApoE4 allele does. Almost nothing is known about the role of TREM2 in tauopathy, even though tau is a pathology shared among many of those same degenerative disorders.
Bruce Lamb, PhD, of the Cleveland Clinical Lerner Research Institute, has a 2015-18 BrightFocus grant to investigate the interaction between TREM2 and tau in a mouse model developed in his own lab. Pathological hyperphosphorylation of microtubule-associated protein tau (MAPT) also is a feature of AD as well as other neurodegenerative disorders collectively known as tauopathies.
They are exploring the role of TREM2 in modulating pathological outcomes, including inflammation, neurodegeneration, and behavior. A strong body of literature exists linking inflammation and innate immunity to AD pathogenesis through the use of genomic approaches and animal models.
In addition, the group is investigating the impact of TREM2 deficiency on gene expression patterns within immune cells of the brain. There are different immune cell types in the brain, some of which are normally present, and some of which are recruited into the brain when it is diseased, and better understanding of the interactions of these cells will help to identify specific and unique treatment targets.
At SfN, Scott Bemiller, PhD, a graduate student in Lamb’s lab, presented findings from Lamb’s BrightFocus-funded work. In previous experiments the group showed that TREM2-deficient mice had complete elimination of plaque-associated macrophages, decreased pro-inflammatory cytokine production, and a modest reduction in amyloid burden. In other words, TREM2’s absence was protective against Aβ.
TREM2 signaling and expression in a mouse model of AD tauopathy sets up a different pattern, however. Strikingly, these researchers showed that TREM2 signaling results in opposing effects between amyloid and tau pathologies, exacerbating amyloid pathology while suppressing tau pathology. From their results, TREM2 deficiency results in earlier and increased phosphorylation of several MAPT epitopes, earlier MAPT aggregation, altered microglial activation, and cognitive dysfunction. When they analyzed the underlying mechanisms linking TREM2 deficiency to MAPT phosphorylation, the search revealed dysregulation of multiple MAPT kinases.
They are still investigating the reasons for TREM2’s opposite effects on Aβ vs. tau pathology. So far, Bemiller speculates it may be due to a reduction in critical neuronal-microglial cross-talk that results in a lack of suppression signaling, which detrimentally alters inflammatory signaling. Another explanation could be that microglia lacking TREM2 are unable to effectively clear extracellular MAPT.
Up ahead, this ongoing study will add critical knowledge to the function of TREM2 and innate immune pathways in AD and other neurodegenerative tauopathies, and will help uncover novel therapeutic targets that can be translated from mice studies to human patients.