Penn Medicine Researchers Pinpoint Potential New Drug Target for Protection against Certain Neurodegenerative Diseases
Findings Could Pave Way for Precision Medicine Approach to Treatment of Neurological Diseases
PHILADELPHIA - Penn Medicine researchers have discovered that hypermethylation - the epigenetic ability to turn down or turn off a bad gene implicated in 10 to 30 percent of patients with Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Degeneration (FTD) - serves as a protective barrier inhibiting the development of these diseases. Their work, published this month in Neurology, may suggest a neuroprotective target for drug discovery efforts.
"This is the first epigenetic modification of a gene that seems to be protective against neuronal disease," says lead author Corey McMillan, PhD, research assistant professor of Neurology in the Frontotemporal Degeneration Center in the Perelman School of Medicine at the University of Pennsylvania.
Expansions in the offending gene, C9orf72, have been linked with TAR DNA binding protein (TDP-43) which is the pathological source that causes ALS and FTD. "Understanding the role of C9orf72 has the possibility to be truly translational and improve the lives of patients suffering from these devastating diseases," says senior author, Edward Lee, PhD, assistant professor of Neuropathology in Pathology and Laboratory Medicine at Penn.
McMillan and team evaluated 20 patients recruited from both the FTD Center and the ALS Center at the University of Pennsylvania who screened positive for a mutation in the C9orf72 gene and were clinically diagnosed with FTD or ALS. All patients completed a neuroimaging study, a blood test to evaluate C9orf72 methylation levels, and a brief neuropsychological screening assessment. The study also included 25 healthy controls with no history of neurological or psychiatric diseases.
MRI revealed reduced grey matter in several regions that were affected in patients compared to controls. Grey matter is needed for the proper function of the brain in regions involved with muscle control, memory, emotions, speech, and decision-making. Critically, patients with hypermethylation of C9orf72showed more dense grey matter in the hippocampus, frontal cortex, and thalamus, regions of the brain important for the above described tasks and affected in ALS and FTD, suggesting that hypermethylation is neuroprotective in these regions.
To validate these findings, the Penn team also looked at autopsies of 35 patients with C9orf72 expansions and found that their pathology also suggested that increased methylation was associated with reduced neuronal loss in both the frontal cortex and hippocampus.
Longitudinal analysis was performed in 11 of the study patients to evaluate the neuroprotective effects of hypermethylation in individuals over their disease course. This showed reduced changes in grey matter of the hippocampus, thalamus, and frontal cortex, associated with hypermethylation suggesting that disease progresses more slowly over time in individuals with C9orf72 hypermethylation. Longitudinal neuropsychological assessments also showed a correlation between protected memory decline and hypermethylation.
These findings are consistent with a growing number of studies which have suggested the neuroprotective effects of the hypermethylation of C9orf72. "We believe that this work provides additional data supporting the notion that C9orf72 methylation is neuroprotective and therefore opens up the exciting possibility of a new avenue for precision medicine treatments and targets for drug development in neurodegenerative disease," says McMillan.
Additional Penn authors include Jenny Russ, PhD; Elisabeth M. Wood, MSc; David J. Irwin, MD; Murray Grossman, MD, EdD; Leo McCluskey, MD; Lauren Elman, MD; and Vivianna Van Deerlin, MD, PhD.
This research was funded by the National Institutes of Health (AG043503, AG017586, AG039510, AG10124, AG032953) and the Wyncote Foundation. Dr. Lee is supported by the Doris Duke Charitable Foundation Clinical Scientist Development Award.
Penn Study Describes New Models for Testing Parkinson's Disease Immune-based Drugs
Understanding how disease spreads from neuron to neuron is key to finding treatments
PHILADELPHIA – Using powerful, newly developed cell culture and mouse models of sporadic Parkinson's disease (PD), a team of researchers from the Perelman School of Medicine at the University of Pennsylvania, has demonstrated that immunotherapy with specifically targeted antibodies may block the development and spread of PD pathology in the brain. By intercepting the distorted and misfolded alpha-synuclein (a-syn) proteins that enter and propagate in neurons, creating aggregates, the researchers prevented the development of pathology and also reversed some of the effects of already-existing disease. The a-sync clumps, called Lewy bodies, eventually kill affected neurons, which leads to clinical PD. Their work appears in Cell Reports.
Earlier studies by senior author Virginia M.-Y. Lee, PhD, and her colleagues at Penn's Center for Neurodegenerative Disease Research (CNDR) had demonstrated a novel pathology of PD in which misfolded a-syn fibrils initiate and propagate Lewy bodies via cell-to-cell transmission. This was accomplished using synthetically created a-syn fibrils that allowed them to observe how Parkinson's pathology developed and spread in a mouse and in neurons in a dish. The present study is a proof-of-concept of how these models might be used to develop new PD therapies.
"Once we created these models, the first thing that came to mind is immunotherapy," says Lee, CNDR director and professor of Pathology and Laboratory Medicine. "If you can develop antibodies that would stop the spreading, you may have a way to at least retard the progression of PD." The current work, she explains, uses antibodies that were generated and characterized at CNDR previously to see if they would reduce the pathology both in cell culture and in animal models.
Lee's team focused on anti0a0syn monoclonal antibodies(MAbs). "In animal models," Lee explains, "the question we want to ask is, can we reduce the pathology and also rescue cell loss to improve the behavioral deficits?"
Using their previously established sporadic PD mouse model, the researchers conducted both prevention and intervention preclinical studies. For preventional studies, they injected mouse a-syn synthetic preformed fibrils into wild-type, normal mice, as a control, and then immediately treated the mice with Syn303, one of the MAbs used (or lgG, another type of common antibody, for the control mice).
The control group without MAb administration showed PD pathology in multiple brain areas over time, while the mice treated with Syn303 showed significantly reduced pathology in the same areas. For intervention studies, they treated PD mice with Syn303 several days after fibril injections when Lewy bodies were already present. They found that the progression of pathology was markedly reduced in the Syn303-treated mice versus mice that did not receive Syn303.
"But there are some limitations to experiments in live mice since it is difficult to directly study the mechanism of how it works," Lee says. "To do that, we went back to the cell culture model to ask whether or not the antibody basically prevents the uptake of misfolded a-syn." The cell culture experiments showed that MAbs prevented the uptake of misfolded a-syn fibrils by neurons and sharply reduced the recruitment of natural a-syn into new Lewy body aggregates.
Next steps for the team will be to refine the immunotherapeutic approach. "We need to make better antibodies that have high affinity for pathology and not the normal protein," says Lee.
The team's models also open up new opportunities for studying and treating PD. "The system really allows us to identify new targets for treating PD," Lee says. "The cell model could be a platform to look for small molecular drugs that would inhibit pathology." Their approach could also serve as a foundation for genetically based studies to identify specific genes involved in PD pathology.
"Hopefully more people will use the model to look for new targets or screen for treatments for PD. That would be terrific," concludes Lee.
The work was supported by an National Institute on Aging training grant (T32-AG000235), the National Institute of Neurological Disorders and Stroke Morris K. Udall Parkinson's Disease Center of Excellence (P50 NS053488), the Michael J. Fox Foundation, the Keefer family, and the Parkinson Council.
Coauthors, all from Penn are Hien T. Tran, Charlotte Hiu-Yan Chung, Michiyo Iba, Bin Zhang, John Q. Trojanowski, and Kelvin C. Luk.
Role of Calcium in Familial Alzheimer's Disease Clarified in Penn Study, Pointing to New Therapeutic Options
PHILADELPHIA – In 2008, researchers at the Perelman School of Medicine at the University of Pennsylvania showed that mutations in two proteins associated with familial Alzheimer's disease (FAD) disrupt the flow of calcium ions within neurons. The two proteins interact with a calcium release channel in an intracellular compartment. Mutant forms of these proteins that cause FAD, but not the normal proteins, result in exaggerated calcium signaling in the cell.
Now, the same team, led by J. Kevin Foskett, PhD, chair of Physiology, and a graduate student, Dustin Shilling, has found that suppressing the hyperactivity of the calcium channels alleviate FAD-like symptoms in mice models of the disease. Their findings appear in the Journal of Neuroscience.
Current therapies for Alzheimer's include drugs that treat the symptoms of cognitive loss and dementia, and drugs that address the pathology of Alzheimer's are experimental. These new observations suggest that approaches based on modulating calcium signaling could be explored, says Foskett.
The two proteins, called PS1 and PS2 (presenilin 1 and 2), interact with a calcium release channel, the inositol trisphosphate receptor (IP3R), in the endoplasmic reticulum. Mutant PS1 and PS2 increase the activity of the IP3R, in turn increasing calcium levels in the cell. "We set out to answer the question: Is increased calcium signaling, as a result of the presenilin-IP3R interaction, involved in the development of familial Alzheimer's disease symptoms, including dementia and cognitive deficits?" says Foskett. "And looking at the findings of these experiments, the answer is a resounding 'yes.'"
Exaggerated intracellular calcium signaling is a robust phenomenon seen in cells expressing FAD-causing mutant presenilins, in both human cells in culture and in mice. The team used two FAD mouse models to look for these connections. Specifically, they found that reducing the expression of IP3R1, the dominant form of this receptor in the brain, by 50 percent, normalized the exaggerated calcium signaling observed in neurons of the cortex and hippocampus in both mouse models.
In addition, using 3xTg mice - animals that contain presenilin 1 with an FAD mutation, as well as expressed mutant human tau protein and APP genes — the team observed that the reduced expression of IP3R1 profoundly decreased amyloid plaque accumulation in brain tissue and the hyperphosphorylation of tau protein, a biochemical hallmark of advanced Alzheimer's disease. Reduced expression of IP3R1 also rescued defective electrical signaling in the hippocampus, as well and memory deficits in the 3xTg mice, as measured by behavioral tests.
"Our results indicate that exaggerated calcium signaling, which is associated with presenilin mutations in familial Alzheimer's disease, is mediated by the IP3R and contributes to disease symptoms in animals," says Foskett. "Knowing this now, the IP3 signaling pathway could be considered a potential therapeutic target for patients harboring mutations in presenilins linked to AD."
The 'Calcium Dysregulation' Hypothesis
"The 'calcium dysregulation' hypothesis for inherited, early-onset familial Alzheimer's disease has been suggested by previous research findings in the Foskett lab. Alzheimer's disease affects as many as 5 million Americans, 5 perfect of whom have the familial form. The hallmark of the disease is the accumulation of tangles and plaques of amyloid beta protein in the brain.
The 'amyloid hypothesis' that postulates that the primary defect is an accumulation of toxic amyloid in the brain has long been used to explain the cause of Alzheimer's," says Foskett. In his lab's 2008 Neuron study, cells that carried the disease-causing mutated form of PS1 showed increased processing of amyloid beta that depended on the interaction of the PS proteins with the IP3R. This observation links dysregulation of calcium inside cells with the production of amyloid, a characteristic feature in the brains of people with Alzheimer's disease.
Clinical trials for AD have largely been directed at reducing the amyloid burden in the brain. So far, says Foskett, these trials have failed to demonstrate therapeutic benefits. One idea is that the interventions started too late in the disease process. Accordingly, anti-amyloid clinical trials are now underway using asymptomatic FAD patients because it is known that they will eventually develop the disease, whereas predicting who will develop the common form of AD is much less certain.
"There has been an assumption that FAD is simply AD with an earlier, more aggressive onset," says Foskett. "However, we don't know if the etiology of FAD pathology is the same as that for common AD. So the relevance of our findings for understanding common AD is not clear. What's important, in my opinion, is to recognize that AD could be a spectrum of diseases that result in common end-stage pathologies. FAD might therefore be considered an orphan-disease, and it's important to find effective treatments, specifically for these patients - ones that target the IP3R and calcium signaling."
Coauthors are Dustin Shilling, Marioly Muller, Hajime Takano, Don-On Daniel Mak, Ted Abel, Douglas A. Coulter, all from Penn.
This work was supported by the National Institute of Mental Health (MH059937) and a National Research Service Award Grant (AG038240)
Shape-shifting Disease Proteins May Explain Variable Appearance of Neurodegenerative Diseases, Penn Study Finds
Targeting Distinct Alpha-synuclein Strains a Potential Treatment Approach
PHILADELPHIA – Neurodegenerative diseases are not all alike. Two individuals suffering from the same disease may experience very different age of onset, symptoms, severity, and constellation of impairments, as well as different rates of disease progression. Researchers in the Perelman School of Medicine at the University of Pennsylvania have shown one disease protein can morph into different strains and promote misfolding of other disease proteins commonly found in Alzheimer's, Parkinson's and other related neurodegenerative diseases.
Virginia M.Y. Lee, PhD, MBA, professor of Pathology and Laboratory Medicine and director of the Center for Neurodegenerative Disease Research, with co-director, John Q. Trojanowski MD, PhD, postdoctoral fellow Jing L. Guo, PhD, and colleagues, discovered that alpha-synuclein, a protein that forms sticky clumps in the neurons of Parkinson's disease patients, can exist in at least two different structural shapes, or "strains," when it clumps into fibrils, despite having precisely the same chemical composition.
These two strains differ in their ability to promote fibril formation of normal alpha-synuclein, as well as the protein tau, which forms neurofibrillary tangles in individuals with Alzheimer's disease.
Importantly, these alpha-synuclein strains are not static; they somehow evolve, such that fibrils that initially cannot promote tau tangles acquire that ability after multiple rounds of "seeded" fibril formation in test tubes.
The findings appear in the July 3rd issue of Cell.
Morphed Misfolding Proteins Found In Overlapping Neurodegenerative Diseases
Tau and alpha-synuclein protein clumps are hallmarks of separate diseases – Alzheimer's and Parkinson's, respectively. Yet these two proteins are often found entangled in diseased brains of patients who may manifest symptoms of both disorders.
One possible explanation for this convergence of Alzheimer's and Parkinson's disease pathology in the same patient is a global disruption in protein folding. But, Guo and Lee showed that one strain of alpha-synuclein fibrils which cannot promote tau fibrillization actually evolved into another strain that could efficiently cause tau to fibrillize in cultured neurons, although both strains are identical at the amino acid sequence level. Guo and Lee called the starting conformation "Strain A," and the evolved conformation, "Strain B."
To figure out how A and B differ, Guo showed that the two strains folded into different shapes, as indicated by their differential reactivity to antibodies and sensitivity to protein-degrading enzymes. The two strains also differed in their ability to promote tau fibrillization and pathology in mouse brains, mimicking the results from cultured cells. When analyzing post-mortem brains of Parkinson's patients, the team found at least two distinct forms of pathological alpha-synuclein.
Lee and her team speculate that in humans, alpha-synuclein aggregates may shift their shapes as they pass from cell to cell (much like a cube of silly putty being re-shaped to form a sphere), possibly developing the ability to entangle other proteins such as tau along the way. That process, in turn, could theoretically yield distinct types of alpha-synuclein pathologies that are observed in different brain regions of Parkinson's disease patients.
While further research is needed to confirm and extend these findings, they have potentially significant implications for patients afflicted with Parkinson's and other neurodegenerative diseases. For example, Lee explains, they could account for some of the heterogeneity observed in Parkinson's disease. Different strains of pathological alpha-synuclein may promote formation of distinct types of alpha-synuclein aggregates that may or may not induce tau pathology in different brain regions and in different patients. That, in turn, could explain why some Parkinson's patients, for example, experience only motor impairments while others ultimately develop cognitive impairments.
The findings also have potential therapeutic implications, Lee says. By recognizing that pathological alpha-synuclein can exist in different forms that are linked with different impairments, researchers can now selectively target one or the other, or both, for instance with strain-selective antibodies.
"What we've found opens up new areas for developing therapies, and particularly immunotherapies, for Parkinson's and other neurodegenerative diseases," Lee says.
Other study authors are Dustin J. Covell, Joshua P. Daniels, Michiyo Iba, Anna Stieber, Bin Zhang, Dawn M. Riddle, Linda K. Kwong, Yan Xu, all from Penn.
Research funding was provided the National Institute on Aging and the National Institute of Neurological Disease and Stroke (AG017586, NS053488), the Marian S. Ware Alzheimer Program, the Parkinson's Council, the Dr. Arthur Peck Fund, and the Jeff and Anne Keefer Fund.