BME SENIOR CAPSTONE PROJECT

The Role of Traumatic Brain Injury in HSV-Induced Alzheimer's Disease

Yassi Khorsandian, Brooke Smiley, Jordan SmileyFaculty Advisors: David Kaplan, PhD; Dana Cairns, PhD

Kaplan Lab, Tufts University

AbstractIn the world, neurological diseases and damages can be some of the most debilitating problems

that affect individuals. These include a variety of diseases that affect people of all ages, includingtraumatic brain injury (TBI) and Alzheimer's disease (AD). TBI can lead to many negative long-termneurological impacts, such as neuroinflammation and increased rates of mental illness.1 AD also impactsneurological function, leading to memory loss, cognitive impairment, and loss of bodily functions. AD isthe sixth leading cause of death in the US.2 While TBI has a clear cause in many instances, such as animpact injury to the head, the cause of sporadic AD is still unknown. Herpes simplex virus, or HSV, islatent in the entire population and has been shown to have a connection to increasing the incidence of ADwhen activated due to an external factor.1 Previous studies have shown that activation of HSV due to atrigger of shingles can lead to AD.3 Because of the extreme nature of TBI damage, and therefore theupregulation of inflammatory markers post-TBI, we believe that TBI damage could activate and triggerHSV infection leading to AD onset. Therefore, this project plans to analyze the role of traumatic braininjury (TBI) in triggering herpes simplex virus (HSV)-induced AD. As a proof of concept, 2D woundmodels with hiNSCs will be used in various tests to analyze the upregulation of a variety of markers andfactors related to AD due to neural damage. Initially, immunostaining for TUJ1, GFAP, A and HSV willbe completed. In the second stage of this project, 3D silk scaffold donut models seeded with hiNSCsexpressing a latent HSV infection will be subjected to a lab-simulated TBI event to analyze post-damageupregulation of TUJ1, GFAP, A , and HSV seen through immunostaining; changes in neural activitythrough Fluo-4 calcium imaging; and qPCR analysis.

Keywords: Herpes Simplex Virus Type 1 (HSV-1), Alzheimer’s Disease (AD), Traumatic Brain Injury(TBI)

Elements of Engineering DesignThere are two objectives of this project: first, to analyze the upregulation of markers indicative of

AD within a 2D wound model to provide proof-of-concept data, and second, to analyze 3D brain modelsexpressing a latent HSV-infection that are subjected to a lab-simulated TBI event for the same markers.The results from the second objective will extend the proof-of-concept model to a 3D in vitro model thatbetter represents TBI to analyze its relationship to AD. All of these markers (see Appendix A for moreinformation) are selected based on prior evidence of their presence in AD patients, and have been shownto be relevant markers that are upregulated or downregulated due to HSV-induced AD in a previousreport.2

The entire process for testing this experiment needs to be designed. In order to do this, theprocedure seen in the Kaplan Lab’s previous study on Herpes-induced AD will be adapted to fit thisproject's goals.2 The same brain model, along with cells infected by the same HSV virus, will be used, butwith a different process that combines the TBI procedure that is completed through a weight-drop model.5

The same markers seen to be important in the previous HSV/AD study will be used to evaluate the results.From here, more tests may be designed based upon time and the data that has been collected.

In general, this project will rely heavily on the concepts of cell culture, scaffold production andseeding, immunostaining, qPCR, ELISA, and other tests. Therefore, mathematical and statistical conceptsrelated to quantitative data analysis will be important.

The main constraint that needs to be considered for this project is safety. All IBC protocolprocedures must be followed regarding the HSV-1 work, in addition to handling the human-inducedneural stem cells (hiNSCs) and other materials properly. Cost is not considered an issue as the materialsused will be readily available in the lab or easily ordered. Our project aims to make potential insights thatcould provide new information regarding a possible relationship between AD in relation to the commoninjury of TBI.

However, if TBI does not upregulate markers related to AD, other factors will be considered thatcould trigger latent HSV-induced AD. One possible stimulus could include lipopolysaccharide (LPS),which would model a bacterial infection. The same tests of the 3D brain models would be completedwhile replacing the lab-simulated TBI event with this alternative stimulus. If this also does not work, analternative option would involve introducing inflammation directly instead of causing inflammationthrough a stimulus to examine its potential to trigger HSV and cause AD.

The planned tests include analyzing a simulated 2D wound model through immunostaining forthe upregulation of GFAP, vimentin, Lipocalin2 (LCN2), Serpina3 (SERP3), TUJ1, and TNFα. Whenanalyzing the 3D models, immunostaining for the same markers will be completed in addition to cellactivity measurements using assays for interleukin-1β (IL1β), IL-6, interferon γ (IFNγ), APP, BACE1,PSEN1, and PSEN2, and qPCR measures of HSV-UL29, GFAP, and AD mediators (see Appendix A formore information). The quantitative milestones will mirror all of the quantitative data given in theprevious HSV-induced AD paper.2 For each test listed above, results that mirror or are closely related tothose in this paper will provide us with results that show statistical significance of correlation betweenTBI and HSV-induced AD. Despite other research teams working on discovering other triggers (such asthe shingles virus) for AD,3 this project is unique because no studies have directly examined a potentialcorrelation between TBI and HSV-induced AD.

Design Flow Chart

Figure 1. Project design flow chart.

This design flow chart describes our research project from a design perspective. Thisencompasses all steps of the engineering design process, from defining a problem and formulating ideasfor a solution to running experiments, recording results, and presenting the final data. Feedback loops areincluded as indicators of a place to reevaluate the results and modify the proposed solution as needed. Theoverall outcome of this project will include a set of data analyzing the effect of TBI or other alternativestimuli on triggering the upregulation of markers and indicators HSV-induced AD in vitro.

Introduction/BackgroundAlzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive

impairment and memory loss alongside a variety of neuropsychiatric symptoms such as the restriction ofdaily activities.6 Important hallmarks of AD are the formation of beta-amyloid plaques and neurofibrillarytangles.6 AD is commonly divided into two separate disease types according to the causes: familial ADhas been attributed to mutations in 3 specific genes [amyloid precursor protein (APP) gene, presenilin1(PSEN1) gene and presenilin 2 (PSEN2) gene], whereas sporadic AD, which accounts for over 90% ofAD cases, has no known cause.6 Even though a number of environmental and genetic factors have beeninvestigated as potential causes of sporadic AD, genome-wide association studies have only been able tofind weak links between the Apolipoprotein E gene and the rate of sporadic AD occurrence. 6 Moreover,few medications are available that can slow down or stop the progression of AD, but no definitive

treatment or cures are available.7 Furthermore, despite extensive research into many potential genetic andenvironmental factors, there is currently no concrete evidence explaining the cause of sporadic AD.6

Therefore, more robust studies and further investigation is required to establish the root cause of sporadicAD and develop more effective treatments for this condition.

Despite the limited knowledge surrounding the onset of sporadic AD, herpes simplex virus, orHSV, is latent in the entire population and has been shown in a previous study to have a connection toincreasing the incidence of AD when activated due to an external factor.2 HSV-1, or herpes simplex virustype 1, is a double-stranded DNA virus.2,8 This virus infects an individual's epithelial cells, but remainslatent in the body's neurons post-recovery of the initial infection.8 In most individuals this virus is latent inthe peripheral nervous system, leading to no symptoms or apparent problems. However, reactivation ofHSV-1 can result in a variety of symptoms with a range of severity from no symptoms at all or cold soresto herpes simplex encephalitis (HSE).2 One particular study has shown that activation of HSV due to atrigger of shingles can lead to AD3. This raises a question of what other potential triggers could lead toreactivation of HSV that ultimately causes signs of AD.

TBI is one of the most detrimental diseases to neurological function that can impact anyindividual regardless of medical status. Sports players, vehicle drivers, or anyone who falls could end upwith TBI. In fact, physical and mental injuries are mostly caused as a result of TBI, and TBI is also themain cause of death for young people below 45 years old. Any sort of impact to the head can causedisrupted brain morphology, which is the ultimate sign of a confirmed TBI. These morphology changescan be accompanied by concussions, damage to white and gray matter, hemorrhaging, and more.1 Due tothe extreme nature of TBI damage, and therefore the upregulation of inflammatory markers post-TBI, it ispossible that TBI damage could activate and trigger HSV infection leading to indicators of AD onset. Thisproject therefore aims to find a possible trigger of this disease by analyzing the effects of traumatic braininjury in upregulating markers seen in AD.

This study will utilize an in vitro model system used to model HSV-induced AD described in aprevious study completed in the Kaplan lab published in 2020 by Cairns et al. In this study, silk spongescaffolds were seeded with hiNSCs infected with HSV-1. Besides an upregulation of AD markers whenthe HSV-1 was present, the study showed that treatment with valacyclovir, an antiviral drug that is oftenused to treat HSV-1, decreased the signs of AD. Therefore, these models suggest that a latent HSVinfection could be triggered to create the multicellular amyloid plaques, inflammation, and gliosis seen inAD.1 The model created in this recent study provides a framework for future studies analyzing the triggersof AD related to pathogenesis.2 Our planned study will be able to utilize this model by creating a latentHSV-1 infection in the cells by using a low multiple of infection (MOI). We will then attempt to retriggerthis HSV-1 infection with a lab-induced TBI stimulus in order to analyze the relationship between TBIand HSV-induced AD.

The 3D in vitro silk scaffold-based brain models used in this study and many others have beenshown to be the optimal brain disease model as they are able to demonstrate both extensive neuronalgrowth as well as the formation of 3D neural networks that are structurally and functionally connected.9These silk-collagen protein composite scaffolds have already been shown in previous studies to beeffective and useful models of the brain and brain-related disease because they are able to be culturedlong-term, allowing for neural network maturation; furthermore, the porosity of these scaffolds allows forhigh cell viability through optimal nutrient/waste diffusion, and treatment options allow for maximum celladhesion.9 More specifically, the modular 3D brain-like cortical tissue produced using such silk scaffoldshas proven an effective model for simulating TBI through monitoring of electrophysiological andneurochemical changes.5

While this study will examine a relationship based upon a novel hypothesis relating TBI, HSV-1,and AD, other studies present competition to our goal through examining other factors that could triggerHSV-induced AD. Some studies have previously looked at the long-term neurocognitive changes thatresult from triggering HSV with chickenpox (varicella zoster virus, VZV) that had been reactivated asshingles (herpes zoster virus, HZV); these studies found that a remarkably high risk of developingdementia and phenotypes consistent with AD were observed in those with Herpes Zoster Ophthalmicus

(HZO, shingles).4 Other factors similar to HZO that could also trigger HSV-induced AD could beconsidered competitive research to what this project aims to achieve. Despite this competition, all of thesestudies in addition to the one proposed here could all work together to provide a more cohesiveunderstanding of the factors that could lead to AD. Such studies could help develop bettertreatments/preventative measures for AD, which has been shown to be the case for the use of antivirals inpatients with HZO, whose risk of dementia dramatically decreased when treated with anti-herpesantivirals.4 Therefore, examining the specific potential relationship between TBI and HSV-induced AD isa worthwhile endeavor that could eventually make an impact not only within our understanding ofsporadic AD, but also improving patient outcomes in the clinic.

Unifying Figure of entire project:

Figure 2. Schematic project overview.

Specific Aims:Specific Aim 1: Confirm the upregulation of AD markers in 2D wound models. The goal of this aimis to establish a connection between neuronal injury and HSV-induced AD to provide a proof-of-conceptfoundation prior to the completion of a more rigorous 3D study.Experimental Plans:Since this is the pilot study, slightly fewer markers will be tested than in the large 3D study for sake oftime, materials, and resources. For the 2D models, immunostaining will be completed to look for theupregulation of HSV, Aβ, TUJ1, and GFAP (markers listed in Appendix A). 2 These markers were used toindicate upregulation of Alzheimer’s related factors in a previous study showing HSV-inducedAlzheimer’s disease,2 making them ideal factors to analyze for our proof-of-concept experiment. Inaddition, we will measure the change in space in between migratory fronts from the scratch in the cellmonolayer to analyze neurite extension from the damaged hiNSCs.Expected Outcomes:In the wound injury models, we expect to see upregulation of both HSV and Aβ in the conditionsexpressing both injury and the latent HSV-1 infection. This is because HSV-1 has been proven to triggermarkers representative of AD,10 so both markers should be expressed if inflammation is a sufficienttrigger to reactivate HSV-1 and induce the development of qualities seen in neurons due to Alzheimer's

disease. If promising results are obtained, the study will move forward to the 3D models with TBI as astressor to induce damage and inflammation.Alternative Approaches:If no promising upregulation of AD markers are seen, we will move onto using other possible stimuli fortriggering HSV-induced Alzheimer’s. One such option is lipopolysaccharide (LPS), which has also beenshown to lead to neuroinflammation.11

Specific Aim 2: Confirm the upregulation of AD markers in 3D brain models expressing a latentHSV-infection that are subjected to a lab-simulated TBI event. The goal of this aim is to confirm theresults of the 2D proof-of-concept study in more physiologically-relevant, 3D silk donut brain modelsafter TBI-induced neuronal injury.Experimental plans:3D silk donut scaffolds will be prepared to model the brain in vitro, as previous studies in the Kaplan Labhave established this model for similar experiments.1,2,5 After obtaining well-established cultures ofhiNSCs in these scaffolds through at least four weeks of culture, a latent HSV infection will be inducedthrough previously established methods.2 Following this infection the scaffolds will be subjected to alab-simulated weight-drop TBI event as previously described.1 The same markers (HSV, Aβ, TUJ1, andGFAP) will be analyzed through immunostaining as completed in the 2D experiments to prove the resultshold in the more complex 3D models. Furthermore, information regarding neuronal activity will beobtained through Fluo-4 calcium imaging methods. Calcium imaging has been shown to be an effectivemethod of analyzing Alzheimer's disease in vivo,13 making it an important measure to analyze in in vitromodels as well. Last, completing a qPCR array of known AD mediators as completed in a previous studyof HSV-induced AD2 will provide more quantitative results to establish a complete analysis of theupregulation of AD-related markers.Expected Outcomes:As in the 2D proof-of-concept study, we expect to see the upregulation of HSV and Aβ in the conditionsexpressing both injury and the latent HSV-1 infection. This will prove that the 3D neuronal damageinduced by a traumatic event simulated in vitro was capable of reactivating HSV-1 and inducingAD-related cellular markers to be expressed. Similarly, we expect to see differing levels of neuronalactivity indicated by Fluo-4 imaging due to the induced nerve damage and potential development ofAD-like conditions. In this case, we expect to see a downregulation of activity in the injury models; forthe non-HSV models we expect to see a downregulation that may begin to be less pronounced as timegoes on, but for the HSV-injury models we expect to see a decrease that continuously gets worse. Last, weexpect to see the upregulation of known AD mediators as seen in a previous study of HSV-induced AD. 2

Alternative approaches:If promising results are not seen, we can also do protein analysis through the completion of a WesternBlot. If this still does not indicate the upregulation of AD, we can start over in the 2D models with theanalysis of a new stimulus.

Methods2D Models:Human-Induced Neural Stem Cell (hiNSC) Culture:

Mitotically inactivated Mouse Embryonic Fibroblasts (MEF) feeder plates were preparedaccording to the established protocol.12 Briefly, MEFs were grown under normal conditions in 150 mm2

plates, and 0.5 ml of Mitomycin C aliquot (2 mg in 5 ml sterile 1X PBS) were added, allowed to incubateat 37°C for 2 hours, and gelatin was added followed by a 20 minute incubation at room temperature; theplates were then washed three times with sterile 1X PBS, the cells were trypsinized, spun down to apellet, and resuspended in complete medium (10 ml per 100 mm2 plate), and seeded at 4-6 x 104

cells/mm2.To thaw and culture human induced neural stem cells (hiNSCs), the same protocol was

followed.12 Briefly, the frozen cell vial was thawed in a gloved hand, 1 ml hiNSC complete medium was

added, the cells were centrifuged, resuspended in 10 ml of fresh complete medium, and added to thefeeder plates. Media was changed every 1-2 days.

The hiNSCs were differentiated in the 24 well plates used in this experiment according to thesame protocol.12 Briefly, the cells plates were rinsed with 1X PBS, 3-4 ml TrypLE of was added, andincubated at 37°C for 1 min; 6 ml complete medium was added, a serological pipet was used to dislodgethe colonies, and resuspended in hiNSC complete medium without bFGF.

2D Cell Culture for Wound Models:hiNSCs at passage 16 were seeded into two laminin-coated 24-well plates at a cell density of 200,000cells per well. These cells were cultured in KO media without FGF for six days until confluentmonolayers were observed. For the rest of the 2D study, the treatment conditions listed in Appendix Bwere analyzed.

HSV-1 InfectionHSV-1 was added to KO media without FGF or neurobasal (NB) media, and 0.5mL of one was added toeach well that required HSV treatment to achieve a multiple of infection (MOI) of about 0.001. 0.5 mL ofmedia without HSV-1 was added to the wells that did not require HSV treatment. Cells were cultured for24 hours to allow for proper infection.

Valacyclovir Treatment24 hours after infection, valacyclovir was added into KO media or NB media. 0.5mL of media was thenadded to respective infected wells to inactivate HSV-1. This was left for 3 days until injury of the hiNSCcultures. 0.5mL of additional media was added to all of the wells after the first 24 hours.

Injury of 2D hiNSC CulturesA total of 16 wells were subjected to injury treatment, including 8 wells for uninfected conditions and 8wells for HSV-infected conditions. All of the injured wells had been treated with valacyclovir. Four wellswith cultures from each media type were injured. A P1000 pipette tip was scratched through the injurywells in the plates of hiNSC 2D cultures. The cells were incubated for 4 days before fixation in 4% PFA.

ImmunostainingThe cells were first fixed by removing the media, washing with 1X PBS, adding 4% paraformaldehyde tocover the bottom of the dish, shaking for 15 minutes, and washing in PBS after removing theparaformaldehyde. They were then washed 3 times with PBST (100 μl Tween-20 and 99.9 ml 1X PBS)and blocked (blocking reagent: 15 ml goat serum, 250 μl TritonX-100, 5 ml 5% NaAzide, and filled to250 ml with 1X PBS.) for 30 minutes. Next, the primary antibody was added for an hour, washed withPBST 3 times, and the secondary antibody Alexa 488 anti-mouse was added for 30 minutes. The primaryantibodies used for this round of immunostaining consisted of Recombinant Anti-Amyloid Fibril antibody[mOC87] - Conformation-Specific (ab201062) by Abcam, Monoclonal Anti-HSV1 + HSV2 gB antibodyproduced in mouse by Sigma-Aldrich®, Invitrogen GFAP Polyclonal Antibody by Thermo FisherScientific, and Monoclonal Anti-β-Tubulin III (neuronal) antibody produced in mouse bySigma-Aldrich®. After another 10 minute PBST wash, DAPI was added, and the cells were washed twicemore with PBST. Lastly, the cells were imaged under the Keyence BZX-700 series fluorescentmicroscope.

Wound MigrationInjury wells were imaged at day 0 post-injury treatment, and again following fixation. All imaging wasusing the Keyence BZX-700 series microscope under brightfield view. These day 0 and day 4 imageswere compared; the distance between migratory fronts was measured using ImageJ software and thecomputational comparisons were completed in Excel.

3D Models:Silk Processing/Scaffold Production:To begin, 3D in vitro brain donut silk scaffolds were prepared according to an established protocol.8Briefly, Bombyx mori cocoons were cut into small pieces, boiled with Na2CO3 for 30 minutes, washed,dried, and dissolved in a LiBr solution. Porous scaffolds were then prepared by pouring the silk solutioninto a mold, adding NaCl, allowing it to dry, and cutting the scaffolds to the desired size (6mm outerdiameter, 2mm inner diameter) before autoclaving.

3D Cell Culture:The 3D model experiments will be very similar but an expanded version of the 2D study. The hiNSCs willbe seeded at a density of 1 million cells per scaffold. After culturing the cell-laden scaffolds for 4 weeks,infection with HSV-1 at an MOI of about 0.001 will be completed for 2-3 days followed by 2-3 days ofValacyclovir/placebo treatment. The 3D models will first be assessed for the upregulation of HSV, Aβ,TUJ1, and GFAP as completed in the 2D proof-of-concept study. Fluo-4 calcium imaging will also becompleted to analyze differences in live cell activity, while qPCR analysis will be performed to compareexpression levels of neuronal and AD-related markers among the different cultures.

Results:2D Experiments:Figures 3 and 4 depict the results from the immunostaining performed on the 2D wound models. The 6conditions listed consist of 3 MOCK (no infection) and 3 HSV (HSV-infected) conditions; VEH refers tothe lack of valacyclovir and VCV refers to the conditions with valacyclovir added. The INJURY imagesare the wells through which a pipette tip was scratched to produce wound models. Dotted lines indicatethe edge of the wound for reference in the fluorescent images.

Figure 3. Immunostaining results from 2D experiments (TUJ1, GFAP, and DAPI stains).

Figure 4. Immunostaining results from 2D experiments (HSV, Aβ, and DAPI stains).

Figure 5 depicts a sample image of the wound migration well comparison between day 0 and day 4. Agraphical representation of the average change in migratory front distance across condition types isincluded as well.

Figure 5. (A) Sample of a representative wound migration brightfield image. The dotted lines indicate thelocation of the initial injury from the top image. (B) Graphical representation of the change in migratory

front distance.3D Experiments:We made 200 scaffolds that have been autoclaved and are ready to be seeded. Appendix D shows theprocess of making the scaffolds from silk sponges, and our final scaffolds as stored after autoclaving.These scaffolds will be seeded this week for culture over winter break, allowing for the HSV-infectionprocess to begin immediately after break in January.

Discussion:

As seen in Figure 3, strong DAPI (cell nuclei) and TUJ1 (neuronal marker) signals are present inall conditions but the HSV condition without valacyclovir treatment. Similarly, the GFAP signalindicating glial cells and gliosis is not present in this condition. This is expected, as untreated HSV causedthe neurons in culture to become unhealthy and die. Meanwhile, the GFAP signal is present in all of theother conditions, indicating the presence of glial cells, but upregulated in both of the injury conditions.This also is expected, as gliosis occurs due to nerve cell injury.

In particular, Figure 4 shows results that indicate success of the 2d proof-of-concept study. Thegreen stain seen on the top row of the figure is for an active HSV infection, which as expected, onlyproduced a signal for the HSV-infected conditions. The signal for the HSV+VEH condition is faintbecause as shown by the DAPI stain, many of the cells died as a result of the long-term untreated HSVinfection. This faint signal is reduced even further in the HSV+VCV condition because the antiviral drugvalacyclovir treats the HSV infection and results in a chronic version of the disease. Lastly, the strongestsignal can be attributed to the HSV+VCV+INJURY condition, suggesting that the injury was able totrigger HSV to become an active infection.

The red stain seen in the middle row of Figure 4 is for the presence of Aβ, a marker of AD. Theonly condition that has any significant signal with this stain is the HSV+VCV+INJURY condition,suggesting that the combination of the chronic HSV infection and injury can be correlated with theupregulation of Aβ. Overall, the results of the immunostaining from the 2D experiments in Figure 4provide a proof-of-concept that injury can potentially trigger HSV induced-AD, ultimately setting thestage for the 3D studies to follow.

Figure 5 also shows promise for our 3D studies. When looking at the wound migration data, allconditions (across both media types and HSV positive or negative cells) showed approximately a 30%reduction in wound distance. When neurons are injured, they do not proliferate but instead extend theirneurites into the damaged space to attempt to reestablish connections with neighboring cells. Even thoughwe do not see reduced migratory distance in the HSV-infected wound models, the fact that we do not seesignificant differences between treatment types proves that the cells still function in a way that does notcompromise the other data we see in figures 3 and 4.

Future Work:If the link between TBI and HSV-induced AD is established in this study, then future experiments couldtest the use of other antiviral drugs post-TBI to determine their efficacy in preventing HSV-induced AD.On the other hand, if TBI cannot be shown to trigger HSV-induced AD, then other triggers such as LPSand inflammatory factors could be tested to determine if they can lead to HSV-induced AD.

ConclusionsThe goal of this study is to show that brain injury, specifically that of TBI, upregulates the presence of ADmarkers. All of the data that we have obtained in the 2D studies set up a proof-of-concept for our future3D models. The 2D injury models effectively upregulated HSV infection and corresponding beta amyloidplaques, a hallmark of AD. Therefore, the results of the 2D wound models tested this semester shouldcorrelate with results obtained next semester with the 3D TBI models. This sets up a promising study forus to investigate.

Individual Contributions:*When more than one name is listed, we contributed about equally in the writing of that sectionAbstract: Brooke Smiley, Jordan SmileyElements of Engineering Design: Yassi Khorsandian, Brooke Smiley, Jordan SmileyDesign Flow Chart: Jordan SmileyIntroduction/Background: Yassi Khorsandian, Brooke Smiley, Jordan SmileyUnifying Figure: Brooke SmileySpecific Aims:

Specific Aim 1: Yassi Khorsandian, Brooke Smiley, Jordan Smiley

Specific Aim 2: Jordan SmileyMethods:

2D Models:hiNSC Culture: Yassi Khorsandian2D Cell Culture for Wound Models: Jordan SmileyHSV-1 Infection: Jordan SmileyValacyclovir Treatment: Brooke Smiley, Jordan SmileyInjury of 2D hiNSC Cultures: Brooke SmileyImmunostaining: Yassi KhorsandianWound Migration: Brooke Smiley

3D Models:Silk Processing/Scaffold Production: Yassi Khorsandian, Brooke Smiley3D Cell Culture: Brooke Smiley, Jordan Smiley

Results: Yassi Khorsandian, Brooke Smiley, Jordan SmileyDiscussion:

Figure 3: Jordan SmileyFigure 4: Yassi KhorsandianFigure 5: Brooke Smiley

Future Work: Yassi KhorsandianConclusions: Brooke SmileyEditing: Yassi Khorsandian, Brooke Smiley, Jordan Smiley

Acknowledgements:We would especially like to thank Dr. Cairns for her help during this project and for being an amazingmentor during this process. We would also like to thank Dr. Kaplan and Dr. Brown for their ongoingsupport throughout the semester, and Sunny Shaidani for her wonderful feedback and advice.

References:[1] Liaudanskaya, V.; Chung, J. Y.; Mizzoni, C.; Rouleau, N.; Berk, A. N.; Wu, L.; Turner, J. A.;

Georgakoudi, I.; Whalen, M. J.; Nieland, T. J. F.; Kaplan, D. L. Modeling Controlled CorticalImpact Injury in 3D Brain-Like Tissue Cultures. Adv Healthc Mater 2020, 9 (12), e2000122.https://doi.org/10.1002/adhm.202000122.

[2] Cairns, D. M.; Rouleau, N.; Parker, R. N.; Walsh, K. G.; Gehrke, L.; Kaplan, D. L. A 3D HumanBrain-like Tissue Model of Herpes-Induced Alzheimer’s Disease. Sci Adv 2020, 6 (19), eaay8828.https://doi.org/10.1126/sciadv.aay8828.

[3] Cairns, D. M and Itzhaki, R. F. Unpublished Study on Shingles’ Role in Reactivating HSV.[4] Itzhaki, R. F. Corroboration of a Major Role for Herpes Simplex Virus Type 1 in Alzheimer’s

Disease. Front Aging Neurosci 2018, 10, 324. https://doi.org/10.3389/fnagi.2018.00324.[5] Tang-Schomer, M. D.; White, J. D.; Tien, L. W.; Schmitt, L. I.; Valentin, T. M.; Graziano, D. J.;

Hopkins, A. M.; Omenetto, F. G.; Haydon, P. G.; Kaplan, D. L. Bioengineered FunctionalBrain-like Cortical Tissue. Proc Natl Acad Sci U S A 2014, 111 (38), 13811–13816.https://doi.org/10.1073/pnas.1324214111.

[6] Piaceri, I.; Nacmias, B.; Sorbi, S. Genetics of Familial and Sporadic Alzheimer’s Disease. FrontBiosci (Elite Ed) 2013, 5, 167–177. https://doi.org/10.2741/e605.

[7] Mayo Clinic. Alzheimer’s disease - Symptoms and causeshttps://www.mayoclinic.org/diseases-conditions/alzheimers-disease/symptoms-causes/syc-20350447 (accessed 2021 -10 -21).

[8] Saleh, D.; Yarrarapu, S. N. S.; Sharma, S. Herpes Simplex Type 1. In StatPearls; StatPearlsPublishing: Treasure Island (FL), 2021.

[9] Chwalek, K.; Tang-Schomer, M. D.; Omenetto, F. G.; Kaplan, D. L. In Vitro Bioengineered Model ofCortical Brain Tissue. Nat Protoc 2015, 10 (9), 1362–1373.https://doi.org/10.1038/nprot.2015.091.

[10] Marcocci, M. E.; Napoletani, G.; Protto, V.; Kolesova, O.; Piacentini, R.; Li Puma, D. D.; Lomonte,P.; Grassi, C.; Palamara, A. T.; De Chiara, G. Herpes Simplex Virus-1 in the Brain: The Dark Sideof a Sneaky Infection. Trends in Microbiology 2020, 28 (10), 808–820.https://doi.org/10.1016/j.tim.2020.03.003.

[11] Batista, C. R. A., Gomes, G. F., Candelario-Jalil, E., Fiebich, B. L., & De Oliveira, A. C. P. (2019).Lipopolysaccharide-induced neuroinflammation as a bridge to understand neurodegeneration.International journal of molecular sciences, 20(9), 2293.

[12] Cairns, D. M.; Chwalek, K.; Moore, Y. E.; Kelley, M. R.; Abbott, R. D.; Moss, S.; Kaplan, D. L.Expandable and Rapidly Differentiating Human Induced Neural Stem Cell Lines for MultipleTissue Engineering Applications. Stem Cell Reports 2016, 7 (3), 557–570.https://doi.org/10.1016/j.stemcr.2016.07.017.

[13] Rochefort, N. L.; Jia, H.; Konnerth, A. Calcium imaging in the living brain: prospects for molecularmedicine. Trends in Molecular Medicine 2008, 14(9), 389-399.https://doi.org/10.1016/j.molmed.2008.07.005.

Appendix A

Marker or factor What these markers mean or show

GFAP Glial fibrillary acidic protein → astroglial injury/gliosis

TUJ1 Reacts with beta-tubulin III to stain neurons

Aβ Amyloid-Beta plaques characteristic of AD

HSV-1 (gB) Major DNA-binding protein in HSV (glycoprotein B)

Table 1. Markers and their significance.2

Appendix B

Treatments

MOCK + veh

MOCK + VCV (Valacyclovir)

MOCK + VCV + injury

+HSV + veh

+HSV + VCV

+HSV + VCV + injury

Table 2. Treatment descriptions for the 2D wound model study.

Appendix C

Figure 6. Proposed timeline for the project.

Appendix D

Figure 7. (A) Silk donut scaffold production process. (B) Our completed silk donut scaffolds.