Neurons, the fundamental cellular units of the human brain and nervous system, are crucial to transmitting signals throughout the human body. In human anatomy, anything that obstructs the communication between neurons could lead to neurodegeneration, decline in function, disorders, and diseases. Alzheimer’s disease (AD), which affects more than 6 million people across the nation as of 2023, is one such disorder. An extensive array of research has been done investigating the underlying cause of the neurodegeneration that occurs in AD. One such theory of neuronal dysfunction, the amyloid hypothesis, points to an accumulation of a protein called beta-amyloid that is present in the brain but in some people accumulates in excess and disrupts neuronal signaling. This ultimately leads to neurodegeneration and cognitive decline. I take part in conducting the AHEAD study, a Phase 2 clinical trial, which is currently underway investigating this theory and a new drug called lecanemab. The drug is designed to remove beta-amyloid from the brain and prevent further neurodegeneration in individuals who have accumulations of amyloid and are at risk of developing AD. This is one of the first preventive clinical trials for Alzheimer's. In this review, I have explored findings from Phase 1 of the AHEAD study and described the screening process for participants for the Phase 2 trial. I have also explored the science behind beta-amyloid, Alzheimer’s disease, and treatment with lecanemab. I expected a large population to pass the screening process, but the statistics show otherwise. I dive into why this is in this project.

Parkinson’s disease (PD), the second most common neurodegenerative disorder, is characterized by Lewy bodies, pathogenic protein aggregates that include alpha-synuclein oligomers. The missense mutation p.G192R in the RAB39B gene was recently found to cause X-linked dominant PD. Loss of function mutations in RAB39B are 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 gene leads to parkinsonism and neurodegeneration. Because RAB39B is highly conserved, we developed a Drosophila model as human RAB39B and Drosophila RAB39 share 75% similarity in amino acid sequence, including 100% identity at p.G192 and flanking amino acids. Using CRISPR/Cas9 genome editing, we created a RAB39G196R Drosophila model that we are currently characterizing for possible neurodegenerative phenotypes. We are examining locomotor deficits and lifespan in RAB39G196R mutant flies compared to isogenic controls, as well as protein aggregation by Western blot. Complementary to the Drosophila model, we developed a human neuronal model by generating induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMC) of an affected male and similar age unaffected male family member kindred with X-linked PD due to the p.G192R mutation. We are investigating endolysosomal trafficking defects in neurons differentiated from iPSCs using antibodies specific for early and late endosomes and lysosomes. We are also examining whether insoluble ubiquitinated protein aggregates and oligomerizes alpha-synuclein are present in RAB39BG192R neurons compared to control neurons. Understanding mechanisms underlying the pathogenesis of X-linked Parkinson’s disease will elucidate the development of PD and potential novel therapeutic targets.

Parkinson's Disease (PD) is a progressive neurodegenerative disease characterized by slowness or stiffness of movement and cognitive impairment. PD is characterized neuropathologically by Lewy Body (LB) aggregates that include lipids, proteins and oligomerized alpha-synuclein. Mutations in the gene glucosidase, beta acid 1 (GBA), are not only the most common genetic risk factor for PD but also accelerate the progression of the disease. We hypothesize that mutations in GBA may mediate faster spread of pathogenic protein aggregation from neuron to neuron. Our previous work has implicated GBA in extracellular vesicle (EV) regulation, suggesting a non-cell autonomous mechanism for GBA accelerating propagation of LBs. To test this hypothesis, we are first exploring how GBA influences EV biogenesis in neurons and astrocytes by examining endolysosomal trafficking in GBA mutated neurons and astrocytes, as well as controls, differentiated from human induced pluripotent stem cells (iPSCs). Our initial results indicate that neurons heterozygous for a GBA null mutation have impaired endolysosomal trafficking with enlarged early endosomes and lysosomes, while astrocytes heterozygous for GBA null do not have impaired early trafficking. These results suggest that GBA mutations differently affect different cell types in the brain and improve our understanding of how GBA influences the spread of LB pathology. I image the iPSC derived neurons and astrocytes using a confocal microscope, for endolysosomal markers and distributions. The goal of this work is to identify novel therapeutic targets for slowing PD progression. 

Alzheimer's Disease (AD) impacts over 6 million people in the U.S, but there are currently no fully effective treatments. Ageing is the biggest risk factor for AD and is associated with cellular changes called senescence. Cellular senescence describes a natural process in cells, leading to cell cycle arrest and metabolic changes due to insults from aging/disease processes. Factors contributing to senescence include DNA damage, and others. A risk factor in neurodegeneration is the ageing of microglia- our brain's immune cells that maintain a healthy brain. Senescent microglia express a senescence associated secretory phenotype- a combination of inflammatory proteins released into their environment- that enhances neurodegenerative processes. My project investigates the relationship between microglial senescence and AD by comparing the levels of senescence markers in AD brains, healthy young brains, and aged brains. I hypothesized that AD brains will contain the greatest amount of senescence markers, followed by aged brains, then healthy young brains. I performed immunohistochemistry for p16Ink4a and gammaH2AX (two robust senescence markers) on 10 human individuals (5 male/5 female per cohort) who donated their brain post-mortem. p16Ink4a is involved in cell cycle regulation and gammaH2AX signals DNA damage. The brain samples were also stained with Iba-1 to identify microglia. A confocal microscope imaged the samples and data was analyzed using the IMARIS software and ImageJ. Senescence markers were quantified in each cohort and localized in microglia or non-microglia cells. I expect to see the greatest amount of p16Ink4a and gammaH2AX in AD brains (specifically AD microglia), with the least amount in healthy young brains. I also expect co-localization of gammaH2AX and p16Ink4a in my samples. Understanding the relationship between microglial senescence and AD pathology could aid in finding methods to target cellular senescence. Slowing down this process could be a usefull tool in decreasing the progression of AD.

Sporadic inclusion body myositis (sIBM) is an acquired progressive inflammatory muscle disease. It is most commonly seen in individuals over 50 years old and affects more men than women. Symptom onset is generally gradual and characterized by progressive muscle weakness and atrophy. Weakness often starts in the quadriceps and finger flexors but can affect other muscles in the arms and legs as the disease progresses. Difficulty swallowing also can be present. The disease remains challenging to diagnose due to its non-uniform presentation. There is currently no cure or standard treatment for sIBM as it is unresponsive to corticosteroids and immunosuppressive drugs. In this study, we investigated the prevalence of and associations between different features considered in the diagnosis of sIBM. We reviewed the electronic medical records of adult patients diagnosed with sIBM using ICD-10 codes at the University of Washington Medical Center from 2003 to 2023. Data was collected including creatine kinase (CK) levels, presence of the anti-cytosolic 5′-nucleotidase 1A (NT5c1A) antibody, pulmonary function testing, presence of dysphagia, muscle strength testing, muscle biopsy findings, electromyography (EMG)/nerve conduction studies, and magnetic resonance imaging (MRI). Statistical analyses were performed to identify the presence of sIBM phenotypes and correlations between them. This study confirms the heterogenous presentation of sIBM and highlights the associated diagnostic challenges this presents. Understanding both typical and atypical presentations is key to preventing delayed diagnosis and misdiagnosis commonly seen in this patient population. Timely diagnosis allows for more tailored management of disease-related symptoms and can help to eliminate the unnecessary administration of ineffective medication and invasive testing. In addition, further characterization of sIBM phenotypes may lead to improvements in both current diagnostic criteria and considerations for clinical trial outcome measures.

The Poolos Lab has made significant progress in elucidating the regulation of tau phosphorylation in the brain and its relationship to both Alzheimer’s disease (AD) and temporal lobe epilepsy (TLE). However, conflicting data exists in the field regarding whether patients with epilepsy exhibit increased or decreased tau phosphorylation and expression compared to health controls. From our preliminary findings using mass spectrometry, we hypothesize that tau undergoes dephosphorylation at several amino acid sites in TLE, as opposed to the hyperphosphorylation observed in AD. To validate this hypothesis, I conducted western blots to separate sample proteins based on their molecular weight via gel electrophoresis. Western blotting has increased sensitivity compared to mass spectrometry in measuring protein phosphorylation levels. I assessed changes in tau expression and phosphorylation using phospho specific antibodies that quantify site specific tau phosphorylation levels. These samples are derived from hippocampal tissues obtained from a chemo-convulsant rat model of TLE, which mimics the chronic seizures experienced by human patients, and compared to age-matched naive controls. Densitometry is employed to quantify the relative amount of phosphorylated tau, and a two-tailed t-test statistical analysis confirms significant changes in tau phosphorylation and expression between tissues from chronically epileptic animals and control subjects. Additionally, I plan to conduct western blots on human tissue from TLE patients to generalize our findings from animal models, deepening our understanding of tau dysregulation in epilepsy. Given the increased risk of premature death and adverse effects on physical and mental health experienced by epilepsy patients, our research holds significant implications for the well-being of epileptic patients and their loved ones. By identifying alterations in tau phosphorylation, we aim to develop a biomarker of epilepsy in cerebral spinal fluid and the blood, thereby advancing diagnostic tests and potential treatments for epilepsy.

Seriously ill hospitalized patients and their families experience elevated stress, anxiety, and depression, resulting from factors like the complexity of serious illness, fragmented medical care, and transitions across settings. They struggle to navigate treatment options, make difficult decisions, and express their values and goals to their clinicians. In addition to communication challenges between patients and clinicians, poor communication among healthcare team members exacerbates the stress associated with serious illness. The aim of this study was to continue work from previous Facilitating Communication Studies to gain knowledge from bedside nurses' experiences with collaborative communication (CC) between patients and families and their healthcare team, and to elicit ideas from nurses about what might improve CC within the hospital system. We defined CC as patients and families being active participants with their healthcare team working towards shared goals of care. We recruited and interviewed 15 bedside nurses from ICU and acute care units at Harborview Medical Center. Following each interview, we input each participant's audio-recorded content into a matrix table, created summaries of each transcript, and synthesized content across transcripts to distill major themes. Nurses reported that breakdowns in CC most often occur when multiple clinicians are involved, during transitions of care, and during the discharge process, resulting in confusion and distress for the patient, family, and bedside nurse. Suggested solutions to overcome these barriers include (1) promoting in-person interactions by rounding as an interprofessional team, (2) inviting bedside nurses to goals of care meetings, (3) implementing a CC training program for clinicians to standardize the CC approach among team members, and (4) empowering patients and families to initiate CC by educating them about their role in their healthcare. These suggestions should be implemented in a future Facilitating Communication Study to reduce distress for seriously ill patients, their families, and their healthcare team. 

The hallmark neuropathological finding of Parkinson’s Disease (PD) is the presence of intraneuronal protein aggregates, consisting of aggregated proteins and misfolded forms of alpha-synuclein. These intraneuronal protein aggregates, known as Lewy bodies, are implicated in many neurodegenerative diseases. Lewy pathology spread in a PD brain correlates with clinical disease progression. Glucosidase, beta, acid (GBA) gene mutations, the strongest genetic risk factor for PD, is also associated with accelerated disease progression and altered extracellular vesicles (EVs). EVs play a crucial role in intercellular communication and delivery of bioactive cargos throughout the central nervous system (CNS). I use a human neuronal cell culture model derived from induced pluripotent stem cells (iPSCs) to examine how GBA mutations alter EV composition, and investigate whether EVs truly act as a vehicle for the seeding of Lewy pathology in other cells, potentially accelerating the propagation of Lewy pathology throughout the CNS. To isolate and purify EVs from the conditioned media of neurons, I use centrifugation and size exclusion chromatography. I visualize and quantify the EV’s size and concentration using a ZetaView nanoparticle analyzer. I perform Western Blot Analysis for candidate cargo proteins within EVs, including alpha-synuclein, ubiquitinated proteins, and EV intrinsic proteins (CD-63 & CD-81). I isolate EVs from the media of GBA PD or WT control neurons expressing alpha-synuclein-GFP fusion protein and apply these EVs to GBA PD or WT neurons. I anticipate that EVs secreted by GBA versus control neurons will contain increased alpha-synuclein protein levels and that increased cell death, endolysosomal trafficking defects, and aggregation of endogenous alpha-synuclein will be associated with the uptake of GBA EVs by recipient neurons. This work will provide evidence supporting the role of GBA in influencing Lewy pathology propagation via EVs, which could elucidate a novel therapeutic mechanism that could be targeted to slow the progression of neurodegeneration.

Rodents are a common model for ischemic stroke research; however, their brains are mostly grey matter while approximately half of tissue affected by stroke in humans is white matter. To study stroke in white matter, we model ischemia in the mouse optic nerve (MON), a pure white matter tract. We observe impaired axonal function and conductance in the MON after ischemia that is improved by ischemic preconditioning (IPC), a phenomenon in which a brief ischemic stimulus protects against subsequent prolonged ischemia. Our prior work demonstrates microglia are required for IPC-mediated axonal protection. Several models of injury and disease report elongation of the nodes of Ranvier (NoR) leading to reduced axonal conductance, but the role of microglia in protecting axons at the NoR is unknown. Here we investigate how NoR are affected by ischemia and microglial depletion. Based on our previous work, we hypothesize that IPC will preserve NoR lengths after exposure to ischemia and this protection will be lost when microglia are absent. Microglia were depleted with PLX5622, a colony stimulating factor 1 receptor antagonist. After treatment, a subset of animals were collected to assess baseline average NoR lengths after microglial depletion alone. Another cohort (N=5) received an in vivo IPC stimulus (15-minute transient common carotid artery occlusion) and 72 hours later experienced ex vivo oxygen-glucose deprivation (ischemic stroke) for 45 minutes. MONs were fixed overnight in paraformaldehyde and prepared for immunohistochemistry using fluorescent antibodies against Nav1.6 (nodes) and Caspr (paranodes) to identify NoRs with confocal microscopy. Nodes are measured using FIJI and the distance between Caspr+ paranodes flanking a Nav1.6+ node is calculated using MATLAB. Microglial depletion alone was found to be associated with increased NoR lengths. Our ongoing work is focusing on the impact of ischemia on NoR lengths and how this may be modulated after IPC.