Autosomal Dominant Alzheimer’s Disease (ADAD) can result from a pathogenic variant in the PSEN2 gene, which encodes an integral membrane protein called Presenilin 2. This variant has been shown to result in a harmful change in the balance of amyloid-β types in neurons, which has been hypothesized to increase risk of dementia. The diverse capabilities of Presenilin give reason to hypothesize that there may be other effects of the variant protein that are connected to ADAD pathogenesis. Reduced spine density, a feature of ADAD pathology, may be caused by overactivity of synaptic pruning. This activity is mediated by resident innate immune cells in the brain called microglia. We sought to explore the effects of PSEN2 variants on microglia-neuronal interactions via the assessment of synaptic pruning in a human induced pluripotent stem cell (hiPSC) derived in vitro model. We differentiated CVIA2 isogenic and PSEN2 N141I variant hiPSCs into both microglia and neurons. Utilizing a coculture of both wild-type neurons and microglia with the PSEN2 N141I variant, we performed immunocytochemistry for synaptic proteins Synapsin 1 and PSD95a. We then imaged the microglia and neurons using confocal microscopy. We assessed differences in synaptic pruning by quantifying immunofluorescent signal of Synapsin in microglia. Specifically, we looked at the colocalization of synaptic protein expression with signal from microglia-specific protein Iba1. We hypothesized that the variant microglia would contain a significantly different amount of signal for Synapsin compared to that of a control sample of wild-type microglia and neurons, implying a change to synaptic pruning function. If altered microglial synaptic pruning activity is shown to play a role in ADAD pathogenesis, targeting microglia could become a possible therapeutic treatment for patients.

Duchenne Muscular Dystrophy (DMD) is a severe neuromuscular disease caused by mutations in the dystrophin gene and characterized by progressive muscle wasting. Dystrophin protein is essential for the stabilization of muscle fibers; without it, continuous muscle damage eventually has devastating consequences in the skeletal, cardiac, and respiratory systems. While prior AAV-mediated editing strategies have been effective in targeting these mutations, there are significant immunological concerns from the uninterrupted expression of bacterial gene editing components. This project utilizes the mdx mouse model of DMD to address these concerns by developing methods to turn gene editing on and off using novel drug-responsive pA regulator vector constructs. I have been part of this project from the start, contributing to the assembly of the expression constructs using bacterial cultures and standard cloning techniques relying on restriction enzymes and high-fidelity cloning kits to piece together our editing constructs. Initial data was acquired from cell culture tests where I helped perform quantitative dual-luciferase reporter assay analyses. Subsequent in vivo experiments were performed by delivering adeno-associated viral vectors, carrying our constructs, into mdx mice via retro orbital injection. Activation of editing activity was achieved via three intraperitoneal injections of Tetracycline.  During analyses, I played a significant role in processing muscle tissues to extract proteins, DNA, and RNA for quantitative assays. Ultimately, our cell culture tests identified two lead pA regulator sequences exhibiting favorable activation levels at therapeutically relevant Tetracycline doses. Initial in vivo tests are promising, showing drug responsive editing and dystrophin expression in mice. We anticipate that our follow up tests will restore sufficient dystrophin expression and improve muscle function, without persistent editing activity. Overall, the outcomes of these studies could have significant implications for a multitude of genetic conditions amenable to genome editing and may help accelerate translation of effective methods towards clinical trials.

Duchenne Muscular Dystrophy (DMD) is the most common lethal genetic muscular disorder of children and is caused by mutations in the 2.2 MB DMD gene. Absence of the dystrophin protein from the dystrophin-glycoprotein complex, leads to myofibers being highly susceptible to contraction injury, leading to progressive rounds of degeneration and regeneration. There is no cure, and most DMD patients eventually experience cardiorespiratory failure. Utrophin (Utr) is a dystrophin paralog that has long been suggested to be a potential therapeutic for DMD. Here we evaluated 4 novel micro-utrophins (µUtr), with the original micro-utrophin (µUtr1) and micro-dystrophin (µDys5) as controls. Two-week-old mdx^4cv mice were intravenously administered vector genomes of recombinant adeno-associated viral vector (rAAV2/9myo1) with human codon-optimized micro-transgenes, driven by the creatine kinase regulatory cassette (CK8e). At ~8 months of age, we assayed lower limb muscles for contractile performance. After functional testing, I processed heart and gastrocnemius muscle tissues to enable purification of proteins and nucleic acids. I then conducted molecular assays, followed by metabolomics via mass spectroscopy to compare wildtype, mdx^4cv, and treated mdx^4cv mice. These results revealed a leading 6-R µUtr variant (µUtr2) that showed physiological improvements in resistance to contraction-induced injury. We also performed univariate and pathway analysis of ~200 targeted metabolites, revealing fold-changes in tricarboxylic acid (TCA) cycle intermediates along with several genes controlling glucose metabolism. The µUtr2 variant resulted in metabolic and physiological improvements towards alleviating symptoms associated with disease progression in the mdx^4cv mouse model, and may hold promise as a treatment for DMD. Gene delivery of functional utrophin-centric proteins could avoid the adverse immune response events that recently occurred in human clinical trials against dystrophin sequences, potentially providing an alternative therapy for some patients.

Alzheimer’s disease (AD) is a neurodegenerative disease that impacts millions of people and costs billions of dollars annually, with both estimates increasing as our aging population grows. Women are diagnosed with AD at a 2:1 higher rate than men, although the biological drivers of this difference remain elusive. Previous studies have demonstrated that changes to the function of microglia – the brain’s immune cells – observed during AD may be driving disease progression. Furthermore, microglia morphology is related to its function. Thus, we seek to characterize differences in microglia morphology between men and women with and without AD. We hypothesize that microglia from women have, on average, a more disease-associated morphology than those of men, and that differences are exacerbated in individuals with AD. We obtained tissue from the dorsolateral prefrontal cortex of 48 individuals who donated their brains to AD research at UW. We conducted immunohistochemistry (IHC) to stain for microglia markers (IBA1) and two markers of AD pathology (AT8 to stain for phosphorylated Tau and a pan-amyloid β stain). I imaged the samples on a Leica SP8 confocal microscope at multiple depths, which allowed us to compose a 3D rendering of the tissue through an image analysis software called IMARIS. Using IMARIS, I quantitatively measured key aspects of each microglia, such as volume and branching details. Using the data from 12-20 microglia per person, we used multiple regression to test for differences between men and women in both healthy and AD cohorts. We anticipate there are differences in the various measurements of microglial morphology between men and women with AD, which may partially explain the discrepancy in AD rates between sexes. This research is important to better understand the role of sex in AD pathology and help contextualize molecular differences observed in the larger project to which it belongs.

Within the complex landscape of the human genome, even a single mutation can trigger devastating neurological consequences. The reality is exemplified by a single missense mutation p.G192R in the RAB39B gene causing X-linked dominant Parkinson’s disease (PD) with reduced penetrance in females. Previously, loss of function mutations in the gene were associated with X-linked intellectual disability and autism spectrum disorder. RAB39B is a member of the human Rab GTPase family which plays a role in early autophagosome formation and is implicated in intracellular vesicular trafficking. This project investigates how defects in endolysosomal trafficking caused by the p.G192R mutation in RAB39B leads to pathogenic protein aggregates and subsequently, parkinsonism and neurodegeneration. To investigate this, we developed a RAB39 G192R Drosophila model which is characterized by neurodegeneration and protein aggregation using western blot, locomotor deficiency, and lifespans. Complementary to the Drosophila model, we developed a human neuronal model by generating induced pluripotent stem cells (iPSCs) from an affected male and similar age unaffected male from a kindred with X-linked PD due to the p.G192R mutation. Neurons differentiated from the iPSCs are analyzed for endolysosomal trafficking alterations by immunocytochemistry, and western blots for evaluating insoluble ubiquitinated protein aggregates and oligomerized forms of alpha-synuclein. Our preliminary results show increased ubiquitinated protein aggregation when a constitutively active RAB39 transgene was expressed in neuronal tissue. The G196R RAB39 adult flies appear morphologically normal, and the G192R mutation does not seem to affect dRAB39 protein expression in Drosophila. RAB39B G196R neurons also do not have altered expression of RAB39B, but have reduced cellular compartment size of p62-stained autophagolysosomes, and Plin2-stained lipid droplets. Understanding mechanisms underlying the pathogenesis of X-linked PD could reveal novel therapies to slow the rate of progression of neurodegeneration and development of PD.

Mutations in glucosidase, beta acid 1 (GBA) are the strongest genetic risk factor for Parkinson’s Disease (PD) and are associated with faster progression of cognitive and motor symptoms. We hypothesize that GBA mutations disrupt the endolysosomal pathway, altering extracellular vesicle (EV) biogenesis and impairing autophagy, leading to faster spread of Lewy pathology from cell to cell in the brain and consequently accelerated disease progression. To study this connection, we utilize a Drosophila model of GBA deficiency that exhibits increased protein aggregation and neurodegeneration. We found that expression of WT GBA in the muscle of GBA mutant flies reduces protein aggregation in the brain, and EVs isolated from these flies have normalized levels of EV-intrinsic proteins that were elevated in GBA mutant flies. These findings suggest that GBA deficiency mediates PD pathogenesis by accelerating the propagation of protein aggregation to distant tissues. To complement this fly model, we differentiate induced pluripotent stem cells (iPSCs) from a PD patient carrying a null GBA IVS2+1 mutation (GBA IVS PD), isogenic wildtype iPSCs generated by CRISPR repair of the IVS2+1 mutation (GBA WT PD), and iPSCs from an unrelated healthy age and sex match control into neurons. To further investigate how GBA influences EVs, I extract EVs from the GBA IVS PD, isogenic GBA WT PD, and sex-age-match control iPSC-neurons to determine if there is a difference in protein cargo in EVs from GBA deficient neurons. I hypothesize that higher levels of aggregated alpha-synuclein will be present in EVs from GBA deficient neurons. I utilize size exclusion chromatography to isolate EVs from neuronal conditioned media. I then conduct western blots to determine protein within EVs. Understanding how GBA mutations influence EV dysregulation and whether EVs act as a vehicle for spread of Lewy pathology could help us uncover new therapeutic targets to slow neurodegeneration.

Huntington’s disease (HD) is a progressive autosomal-dominant neurodegenerative disease due to the expansion of a CAG-repeat in the huntingtin (HTT) gene. CAG-repeat lengths less than 36 are not associated with disease phenotype, however HD with CAG-repeats greater than 39 causes full disease penetrance, characterized by motor, cognitive, and behavioral symptoms. The onset age of HD symptoms and severity of the disease correlates with the length of the CAG repeat, although there is compelling variability in length and age of onset. Somatic instability (SI) in HD is the occurrence of faulty DNA repair that causes CAG-repeat expansion and continues to lengthen with age. There has been significant research on treating HD but not a treatment addressing both SI and lowering mutant HTT protein. Our lab utilizes multiple therapeutic methods to investigate the relationship between SI and mutant HTT protein with mouse genetic models. More specifically, we devise a CRISPR/Cas9-mediated approach to excise the proximal promoter region in Htt^Q111/+ mice, which have their mutant HTT protein knocked down. My roles in this project include measuring the amount of transcription of the HTT gene from these mutant HTT CRISPR-treated mice to determine whether the amount of HTT gene transcription affects somatic instability. We devised a specially designed qPCR assay to measure the pre-mRNA of the HTT gene. Since these CRISPR-treated mice have been found to have lower somatic instability, we expect the amount of transcription of the HTT gene will directly affect the amount of somatic instability.

Some older individuals exhibit the pathological hallmarks (i.e., amyloid-beta plaques and tau-containing neurofibrillary tangles) of Alzheimer’s disease (AD) yet remain cognitively intact, a phenomenon known as resilience. Microglia, the primary immune cells of the central nervous system are important for clearance of debris and responding to injury in the brain. When exposed to aggregated proteins, they can release inflammatory molecules toxic to neurons.  Because neuroinflammation has been implicated in neurodegeneration, understanding how microglia interact with Aβ could provide insight into immune mechanisms that support cognitive preservation despite AD pathology. In patients with AD who have dementia, it is known that their microglia cluster around amyloid-beta (Aβ) plaques which possibly contribute to damaging inflammation.  Whether microglia in resilient individuals share the same relationship to plaque is unknown. This study investigated whether microglia in resilient individuals differ in their spatial relationship to amyloid plaques compared to non-resilient individuals in the dorsolateral prefrontal cortex. Using confocal montage images from postmortem human brain tissue where immunofluorescence stained for Iba1+ microglia and PanAβ+ Aβ plaques, I quantified the proportion of microglia clustering around Aβ in three groups: 1) individuals with symptomatic AD, 2) cognitively intact individuals with AD pathology (resilient), and 3) cognitively intact individuals with no/low AD pathology (resistant). By generating 2D surface reconstructions, I measured microglia-Aβ overlap and proximity to assess colocalization patterns. I identified differences in microglia-Aβ colocalization between these three groups. This approach can help understand how microglial interactions with Aβ may contribute to resilience mechanisms and could inform novel therapeutic strategies for AD.

Amyotrophic lateral sclerosis (ALS) is a progressive disease affecting 5000 people currently in the United States that is due to the degeneration of motor neurons, leading to muscle weakness, paralysis, respiratory failure, and ultimately death. To date, there has been extensive research investigating the underlying cause of the neurodegeneration that occurs in ALS, as well as attempts at targeted therapeutic interventions. CNM-Au8 is an investigational drug employing active gold (Au) nanocrystals designed to support neuronal survival by enhancing cellular energy production and reducing oxidative stress. The results of two randomized controlled phase 2 studies, the Healey Multiplatform Study and RESCUE-ALS, have suggested possible benefits from this therapy in both delaying disease progression, stabilizing respiratory function, and improving survival. The University of Washington (UW) is also a site for the Second Intermediate Expanded Access Protocol (EAP), allowing patients ineligible for the trials to receive the medication. The EAP follows an open-label, multi-center design, with all participants receiving daily oral doses of CNM-Au8. Participants undergo regular assessments every 12 weeks in person or via remote telehealth visits, allowing flexibility based on disease progression and external factors such as COVID-19 infection. The study tracks disease progression using measures such as the ALS Functional Rating Scale-Revised (ALSFRS-R) and slow vital capacity (SVC). ALSFRS-R is a questionnaire that evaluates a patient’s ability to perform daily activities, including speech, swallowing, mobility, and breathing. SVC is a measure of respiratory function crucial in monitoring ALS progression. 

The Pools Lab is investigating whether reducing tau expression can decrease seizure frequency in temporal lobe epilepsy (TLE). Tau is a microtubule-associated protein that stabilizes neuronal cytoskeletons, but its dysregulation has been implicated in neurodegenerative diseases and epilepsy. Tau dysregulation has been observed in epileptic brain tissue, and previous studies in genetic seizure models in mice suggest that reducing tau expression may decrease seizure susceptibility. However, this hypothesis has not been evaluated in the context of chronic TLE, which our study aims to explore using the pilocarpine rat model of TLE, which mimics chronic seizures in humans. To test this, we administered a CRISPR/Cas9 construct (AAV5-saCas9-sgTau) targeted at tau, injected unilaterally into the left hippocampus for tau knockdown. To assess knockdown efficiency, I performed western blot analysis on hippocampal tissue, comparing tau expression between the CRISPR/Cas9-tau knockdown and contralateral (control) hippocampus. This method allows for quantitative assessment of protein expression using tau-specific antibodies to detect site-specific changes. I then conducted densitometric analysis to quantify band intensities as a measure of tau levels and performed statistical comparisons, including a two-tailed t-test, to determine significant differences. Tissue collection of the CRISPR/Cas9 treated hippocampus versus the contralateral (control) hippocampus at 4 weeks post-injection showed a modest decrease in tau levels. Given tau’s estimated half-life of 23 days, we extended the timeline to 8 weeks to allow for further degradation of pre-existing tau. We predict that reducing tau expression will correlate with decreased seizure frequency, providing insight into tau’s role in epileptogenesis and seizure propagation. Given the heightened risk of neurological and cognitive impairments in epilepsy patients, this research has important implications for understanding tau’s contribution to disease progression and identifying potential therapeutic targets for chronic epilepsy.

The primary objective of this study is to evaluate the safety and effectiveness of the study drug Mevidalen, in alleviating symptoms in individuals with mild to moderate Alzheimer's disease dementia. Mevidalen is a selective positive allosteric modulator of the dopamine D1 receptor. The efficacy of this drug is being assessed by examining the patient's cognitive function, daily activities, sleep patterns, Alzheimer disease progression, physical activity levels, and overall stress. I am conducting patient appointments to collect relevant data for the statistical analysis of the study drugs efficacy and safety. Patients are between the ages of 60-80 years old, and are experiencing mild to moderate memory loss. Cognitive function tests including MMSE to gauge the patients working memory, and C-SSRS to monitor mental health throughout the course of this trial. Vital signs and ECG's are measured multiple times during each appointment to track the patient's overall health. Patients are either assigned and titrated to a placebo, low dose study drug, or moderate dose study drug. This is a double blind study, so both the researchers and the patients are blinded to the drug assignment. Over the course of 14 weeks, the patient is monitored by a neurologist at periodic visits, and via an Ax6 wristwatch device that measures sleep patterns. The hope is that this drug is effective, and will soon become a FDA approved therapy for Alzheimer disease dementia, to alleviate memory loss symptoms from patients around the world.