The incidence of Alzheimer's Disease is increasing each year and is the most common cause of dementia.

Scientists and engineers at Tufts University and Massachusetts Institute of Technology (MIT) have used a 3D human tissue culture model of the brain to demonstrate a possible causal relationship between sporadic Alzheimer’s disease (AD) and infection with herpes simplex virus 1 (HSV-1), the virus that causes cold sores.

The researchers say their bioengineered system will make it possible to look more closely at the pathogenic mechanisms of sporadic AD, and could ultimately lead to the development of better therapeutic strategies for treating the neurodegenerative disorder. “Our brain tissue model allowed us to take a closer look at the potential causal relationship between herpes and Alzheimer’s disease, and the results were intriguing,” said David Kaplan, PhD, Stern Family professor of engineering and chair of the department of biomedical engineering at Tufts’ School of Engineering. “After just three days of herpes infection, we saw large and dense plaque formations of beta amyloid protein, as well as increased expression of some of the enzymes responsible for generating the plaques. We observed neuron loss, neuroinflammation, and depressed signaling between neurons— everything we observe in patients. Never before have so many facets of the disease been replicated in vitro.”

Kaplan and colleagues reported their findings in Science Advances, in a paper titled, “A 3D human brain-like tissue model of herpes-induced Alzheimer’s disease.”

AD is a progressive neurodegenerative disorder that causes severe cognitive decline as well as short-term memory loss, impaired speech, and ultimately an inability to function in day-to-day activities. There are nearly 5.8 million AD patients in the United States, where the condition is the sixth leading cause of death, the authors noted. In the absence of medical advances to cure or at least halt disease progression, the number of AD patients is also expected to double as the population ages. Hallmark features of AD include the formation of neurofibrillary tangles (NFTs) and senile plaques that develop in the brain. NFTs are aggregates of the microtubule-associated protein Tau, which result from aberrant hyperphosphorylation and misfolding. Senile plaques consist of extracellular accumulation of β-amyloid (Aβ) peptide, which are formed from amyloid precursor protein (APP), an integral transmembrane protein found in neurons.

Early onset AD (EOAD) is a form of AD that develops in people aged 60 years or younger, and represents about 1–6% of all AD cases. Most of these cases are familial, with multiple relatives also having developed the disorder, the team noted. Some cases of EOAD have been linked with specific gene mutations. In contrast with EOAD, about 95% of AD cases are sporadic, or late-onset, and these cases have a “complex, multifactorial etiology that, despite years of research, still remains quite elusive,” the authors noted. Patients with sporadic, late-onset AD start with presumably normal brain tissue, albeit with some likely age-related risk factors.

Environmental conditions are suspected of playing a potentially significant role in triggering the cascade of molecular reactions that lead to the onset of AD, and there is growing evidence to suggest that pathogens may be involved in the development of sporadic AD, the team continued. The AD pathogen hypothesis suggests that different pathogens may trigger accumulation of the Aβ1–42 isoform the β-amyloid (Aβ) peptide that aggregates into fibrils that contribute to formation of the telltale senile plaques seen in AD.

Herpes simplex virus type I (HSV-1) is one of the pathogens that is gaining increasing attention as a potential causative agent in sporadic AD pathogenesis, the investigators pointed out. Studies to date have found HSV-1 DNA in the brain tissue of patients with AD, while recent epidemiological studies have reported HSV-1 infection as a significant risk factor for later onset AD, the investigators noted. “While there is no published correlative evidence of active herpes infections in patients with AD, these clinical findings suggest that active infection may not be a requirement for AD, but rather that prior history of herpes infection is sufficient to initiate AD pathogenesis.”

HSV-1 typically resides in the peripheral nervous system in a latent state, and while virus reactivation may cause no symptoms, it can manifest as cold sores, or, in rare cases, result in herpes simplex encephalitis (HSE), which indicates that HSV-1 can penetrate the blood-brain barrier. “Given the mounting evidence that HSV-1 has potential causality in the development of AD, we aimed to generate a three-dimensional (3D) human brain model of herpes-induced AD,” the scientists explained.

In vitro human models of AD typically use cells derived from patients with familial EOAD, which carry mutations in the key known AD-related genes, amyloid precursor protein (APP), or presenilin 1 or presenilin 2 (PSEN1 or PSEN2). Such models allow scientists to study pathogenesis in these inherited forms of AD, but often provide little mechanistic insight into the causes, and potential environmental risk factors that might be involved in cases of sporadic AD, for which there is no known genetic link, the researchers pointed out. And while multiple studies have linked HSV infection with sporadic AD, to date, “no direct causality of AD by herpes virus in a 3D human brain model has been demonstrated.”

The 3D human brain tissue model developed by the Tufts and MIT group uses genetically normal human-induced neural stem cells (hiNSCs), which allows for a clearer investigation of environmental factors that might potentially lead to disease. The 3D construct consists of a 6 mm wide donut-shaped, sponge-like material made up of silk protein and collagen and populated with normal neural stem cells that are then guided to differentiate into neurons.

This “donut ring” fosters the growth of neurons with the cell bodies situated in the silk-collagen donut, while extending axons into the “donut hole,” making neural connections and mimicking both the grey matter and white matter of the brain, respectively. Using this model, researchers can observe physical and biological changes in the neurons in real-time using imaging methods, and can also monitor electrical activity and integrity of the communication networks between the cells. “The aim of this study was to examine the effects of HSV-1 infection in our established 3D human brain–like tissue model to assay for correlations to AD pathology.”

Fluorescent immunostaining in which the red shows amyloid beta fibrils, the green indicates HSV-1-infected cells, and the blue indicates dead cells stained with DAPI. [Dana Cairns, Tufts University]
When the investigators infected neurons in the bioengineered brain model with HSV-1, they observed the formation of amyloid plaques, together with neuronal loss, neuroinflammation, and diminished neural network functionality, which are all typical features of Alzheimer’s disease. “… we demonstrate that relatively high levels of HSV-1 infection in our hiNSC model resulted in cell death, similar to what has been previously reported using other cell types, while low levels of the virus induced a remarkable AD-like phenotype not previously reported in vitro,” they wrote.

“Our tissue model achieves this robust AD phenotype in a short time relative to other 3D model systems of AD and using unmodified wild-type cells completely in the absence of any exogenous factors specifically known to regulate or induce AD.”

The researchers found that that dozens of Alzheimer’s-associated genes were over-expressed in the HSV-1 infected brain tissue constructs compared with non-infected tissue. “To further understand this AD-like pathology, we performed a qPCR array of known AD mediators … to compare expression levels between mock- and HSV-1–infected 3D human brain–like tissue constructs. Forty genes in this AD-specific array were significantly up-regulated in HSV-1-infected samples,” they wrote. Prominent among the differentially regulated gene were those coding for the enzymes cathepsin G and BACE2, which are both known to be associated with Alzheimer’s disease and involved in the production of β-amyloid peptides found in plaques. Some of the overexpressed gene products observed in the tissue model could feasibly represent targets for future drug candidates.

The team says the study is the first to report a human model of AD that displays so many physiologically relevant features of human disease in one system, “… including the generation of large, multicellular, dense Aβ+ fibrillar PFLs, neuronal loss, reactive gliosis, neuroinflammation, and diminished neural network functionality.”

Encouragingly, treating the HSV-1-infected brain tissue models using the antiviral drug valacyclovir led to reduced plaque formation and mitigated other Alzheimer’s disease-like features. Valacyclovir is an FDA-approved antiviral drug that is used to treat herpes infections. Drug treatment was also associated with the reduction to pre-infection levels of several molecules and enzymes associated with Alzheimer’s disease. “We also demonstrate efficacy in reducing herpes-induced AD phenotypes through the use of clinically relevant antiviral medication, suggesting that this type of pharmaceutical intervention might be useful for preventing and/or treating AD in patients,” the investigators stated. “This robust bioengineered system will provide the opportunity to further elucidate mechanisms of sporadic AD pathogenesis and to ultimately develop more efficacious strategies for treating this complex and devastating disease.”

“This is a model of Alzheimer’s disease which is very different from what other studies have used,” said Dana Cairns, postdoctoral research associate in Kaplan’s lab and first author of the study. “Most other studies relied on using genetic mutations in the neurons to induce Alzheimer’s disease-like phenotypes and ours does not, which is what really sets it apart. Our model using normal neurons allows us to show that herpes virus alone is sufficient to induce Alzheimer’s disease phenotypes.”

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