Autophagy is an essential quality control process in which proteins and organelles are degraded. In this work, we first extended our understanding of autophagic degradation in disease by investigating the use of acidic nanoparticles to restore autophagic flux in a neurotoxic model of Parkinson Disease (PD). Normal autophagic degradation follows two key steps. First, material is engulfed to form a double-membraned autophagosome. Next, autophagosomes fuse with an acidic lysosome to degrade the inner membrane contents. Insufficient lysosomal acidity results in autophagic flux arrest, and in PC-12 cells, we characterized the use of polymeric nanoparticles as a tool to restore lysosomal acidity and rescue autophagic flux in PC-12 cells. Specifically, in an MPP+ model of neurotoxicity, we demonstrated that formulations of poly(lactic-co-glycolic acid) nanoparticles (PLGA) improved lysosomal acidity, autophagic flux, and cell health significantly, but is likely limited in efficacy by polymer degradation rate. To improve upon this, we developed a new acidic nanoparticle formulated with a novel polymer backbone (termed acNPs), engineered to degrade within lysosomes and release tetrafluorosuccinic acid, a highly potent acid (pKa ~1.6). On the benchtop, these engineered nanoparticles demonstrated both colloidal instability and acid release within a weakly acidic environment (pH 6.0) similar to a diseased lysosome but not at a neutral pH of 7.4. In cells, acNPs effectively decreased lysosomal pH within disease lysosomes, thereby restoring autophagic flux and mitochondrial activity in PC-12 cells. Encouragingly, we also were able to show efficacy of acNPs in 2D and 3D models of the human midbrain. acNPs readily trafficked within the lysosomes of cells in 2D midbrain cultures and 3D midbrain organoids. Similar to PC-12 cells, when we challenged these cells in a model of neurotoxicity, we observed restoration of viability in human organoids following acNP treatment. Next, we addressed some current challenges regarding the quantification of autophagy within cells. We repurposed measures of economic income inequality to quantify the spatial dispersion of LC3 signal intensity in a starvation model of autophagy, and then compared these measures to other image-based measurements based on their ability to represent LC3-II levels, a robust protein marker of the autophagosome. Our analysis showed these indices outperformed all other generated measurements, including the current standard of autophagy research, LC3 puncta counting. Additionally, we also explored the linear decomposition properties of the generalized entropy index and found it a facile way to evaluate autophagic flux within 3D imaging datasets of multicellular systems. Specifically, we revealed a differential response to nutrient depravation between neurons and astrocytes. Finally, we translated this paradigm to a high throughput cell assay where we demonstrated EC50 and IC50 curves, produced from datasets acquired through both confocal and automated widefield fluorescence microscopy. Our results agree with standard cell assays.

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No current imaging technology can directly and without significant distortion visualize the defining microscopic features of the human brain. Ex vivo histological techniques yield exquisite planar images, but the cutting, mounting and staining they require induce slice-specific distortions, introducing cross-slice differences that prohibit true 3D analysis. Clearing techniques have proven difficult to apply to large blocks of human tissue and cause dramatic distortions as well. Thus, we have only a poor understanding of human brain structures that occur at a scale of 1–100 μm, in which neurons are organized into functional cohorts. To date, mesoscopic features which are critical components of this spatial context, have only been quantified in studies of 2D histologic images acquired in a small number of subjects and/or over a small region of the brain, typically in the coronal orientation, implying that features that are oblique or orthogonal to the coronal plane are difficult to properly analyze. A serial sectioning optical coherence tomography (OCT) imaging infrastructure will be developed and utilized to obtain images of cyto- and myelo-architectural features and microvasculature network of post-mortem human brain tissue. Our imaging infrastructure integrates vibratome with imaging head along with pre and post processing algorithms to construct volumetric OCT images of cubic centimeters of brain tissue blocks. Imaging is performed on tissue block-face prior to sectioning, which preserves the 3D information. Serial sections cut from the block can be subsequently treated with multiplexed histological staining of multiple molecular markers that will facilitate cellular classification or imaged with high-resolution transmission birefringence microscope. The successful completion of this imaging infrastructure enables the automated reconstruction of undistorted volume of human tissue brain blocks and permits studying the pathological alternations arising from diseases. Specifically, the mesoscopic and microscopic pathological alternations, as well as the optical properties and cortical morphological alternations of the dorsolateral prefrontal cortical region of two difference neurodegeneration diseases, Chronic Traumatic Encephalopathy (CTE) and Alzheimer’s Disease (AD), were evaluated using this imaging infrastructure.

Parkinson’s Disease (PD), the second most common neurodegenerative disease worldwide, pathologically presents with the inclusion of Lewy bodies and dopaminergic cell loss in the brain. Lewy bodies are composed of aggregated a-synuclein protein, and although essential to our understanding of PD, not much is known about the native, pre-synaptic state of a-synuclein (a-syn). Due to its mostly synaptic local, immunostaining results in diffuse signal, ultimately providing little insight into the types of a-syn-resident cells. As a result, insight into a-syn expression driven cellular vulnerability has been difficult to ascertain. Using a knockin mouse model that localizes a-syn to the nucleus of cells by insertion of a nuclear localization signal into the a-syn gene locus (SncaNLS), we overcome visualization issues and map out the topography and cells-of-origin of a-syn in mice. I performed immunohistochemistry on SncaNLS mouse tissue to map out the endogenous distribution of a-synuclein in the brain. Using ilastik machine learning analysis, I determined regions with high a-syn expression, which were subsequently co- stained with cell-type specific markers to gain further topographical granularity. a-syn showed high expression in the olfactory bulb, hippocampus, cerebral cortex, substantia nigra and cerebellum. Within these structures, there was a high level of expression of a-syn in granule cells, pyramidal cells, mitral cells, and dopaminergic neurons. Taken together, the SncaNLS mouse serves as a tool to define an atlas of a-syn topography, potentially providing insight into cellular vulnerability in PD.

Sandhoff disease (SD), a fatal lysosomal storage disease, results from a deficiency of the β-subunit of the β-hexosaminidase A and B enzymes. This deficiency leads to severe accumulation of GM2 gangliosides in lysosomes within the central nervous system (CNS) resulting in mass neuronal apoptosis. The mouse model of SD shows progressive neurodegeneration that closely resembles Sandhoff and Tay Sachs disease (TSD) in humans. SD and TSD consist of infantile, juvenile, and late-onset forms. These diseases can present with a multiplicity of symptoms including cognitive and speech impairments, ataxia, and lower motor neuron disease. Late-onset SD and TSD show motor neuron disease in over 40% of patients. In this study, we explore the role of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in the spinal cord during the development and progression of disease in Sandhoff mice. Using immunocytochemistry and western blotting, we analyzed the expression level and localization of several ER stress and cellular apoptosis markers within the cervical, thoracic, and lumbar regions of the spinal cord of Sandhoff mice. Our results revealed significant upregulation of several ER stress markers in motor neurons that appeared to coincide with significant lysosomal accumulations. In addition, we observed sequential and age-dependent expression changes in ATF6 and CHOP and their prominent nuclear localization within anterior horn motor neurons. Markers of apoptosis, caspases and PARP also appeared to be activated in the spinal cords of Sandhoff mice starting as early as 60 days. Interestingly, we noted more than 50% reduction in neuronal numbers in all regions of the spinal cord of Sandhoff mice between ages 80 and 120 days. Overall, this study provides strong evidence for the role of chronic ER stress and UPR activation in the spine pathophysiology of SD. / Thesis / Master of Science (MSc) / Lysosomal storage diseases are a rare group of inherited neurological disorders that are often fatal at a young age. Two diseases that fall within this category, Sandhoff and Tay Sachs disease, are similar in their cause and symptoms. Current research lacks a complete understanding of the mechanism behind these disorders making the development of new therapeutics challenging. This research highlights a group of cells in the spine that are vulnerable in these diseases. These cells show physical and functional changes in their structure as the diseases progress. We provide evidence of a new stress pathway which appears to be strongly implicated in the development and progression of these diseases. We also show an association between this pathway and the death of these vulnerable cells leading to the symptoms exhibited by patients. These findings expand our current knowledge of these disorders and open new avenues for therapeutic interventions.

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Myelin is a protein complex which plays an integral role in developing and maintaining proper brain function. Due to the plasticity of the brain, and the dynamic nature of myelin, it is critical to develop methods that allow for the investigation of changes in myelin in vivo, to further our understanding of the brain. A substantial amount of myelin is found in the grey matter (GM) of the cerebral cortex – the outermost structure of the brain that supports higher order functions including cognition and more fundamental functions, such as sensation and motor control. While in vivo investigations have traditionally used imaging to focus on myelin in the deep white matter (WM) tracts in the brain, advances in magnetic resonance imaging (MRI) are now allowing investigations of intracortical myelin (ICM). The research in this thesis presents methodology for investigating intracortical myelin levels using magnetic resonance imaging (MRI) in humans, with the aim of developing a better understanding of how myelin contributes to healthy cortical function, and how it may be disrupted in disease. To characterize intracortical myelin, a novel MRI analysis technique was developed early in this work to report the thickness of the heavily myelinated and lightly myelinated layers of the cortex. This measure of myelinated cortical thickness uses a clustering algorithm to separate the layers of the cortex based on voxel intensity in a T1- weighted (T1W) MRI with strong intracortical contrast. The resulting myelinated thickness maps match known myelin profiles of the brain, with cortical regions such as the primary visual and motor cortices displaying proportionally thicker, heavily myelinated layers. The utility of the myelinated cortical thickness for answering clinical questions was tested in bipolar disorder, where a preferential loss of the more myelinated layers in the dorsal lateral prefrontal cortex was found. This study provided the first in vivo evidence of ICM disruptions in bipolar disorder. Later in the thesis work, after surface-based analysis techniques became available, an alternative approach to investigate intracortical myelin was developed that sampled the T1W image intensity at a calculated depth of the cortex as a measure of myelin content. This methodology was used for studying the association of ICM with age in healthy adults ranging from late adolescence to middle-adulthood. It was found that three cortical depths followed a similar trajectory through this age-span, reaching their peak between 35 and 40 years of age. This study contributes to a picture of ICM amounts increasing well into middle age in healthy adults and provides a baseline for studies investigating how this may be disrupted in disease Up to this point, the analysis in the thesis used a specialized T1W MRI that had been optimized to provide strong intracortical contrast, but a question remained of how useful the technique would be if more commonly collected clinical MRIs were used as inputs. This analysis was thus applied to standard T1W and T2-weighted (T2W) anatomical MRIs to test its clinical applicability. 360 participants were investigated from the TRACK-HD dataset to test if intracortical signal analysis could follow the progression of Huntington’s disease. A significant increase in intracortical T1W/T2W signal was found in the most advanced disease group in several cortical regions. This increase in intracortical signal is likely tracking a known increase in iron and/or myelin levels in the Huntington’s disease brain. However, this work suggests that ICM studies would best be conducted with optimized imaging to better be able to characterize the subtle ICM variations within the GM. Overall, the work in this thesis presents two techniques for whole-brain mapping of the distribution of intracortical myelin using MRI. The clinical applicability of the techniques was demonstrated in examples of mental and neurodegenerative disorders. The future directions of this work include developing imaging specific to either myelin or iron as well as revisiting these problems while imaging at greater resolution to better characterize the laminar profile across the cortex. / Thesis / Doctor of Philosophy (PhD)

Neurodegenerative diseases are characterized by selective neuronal vulnerability and subsequent degeneration in specific areas of the brain. Huntington’s Disease (HD) is inherited as an autosomal dominant mutation that primarily affects the cells of the striatum and the cerebral cortex, leading to a triad of symptoms that include the progressive loss of motor function, defects in cognitive ability and psychiatric manifestations. HD is caused by a CAG repeat expansion that exceeds 37 repeats in Exon1 of the ​HTT​ gene, manifesting as a pathogenic polyglutamine (polyQ) amino acid tract expansion in the huntingtin protein. HD is a late onset disorder, with disease onset around 40-50 years of age and symptoms that worsen over 10-20 years. Only a few symptomatic treatments are available and there is currently no cure for the disease. Therapeutics to target the huntingtin gene itself have only been in clinical trial in the past 2 years. The length of the expansion has an inverse relationship with the age of disease onset. Most patients that have repeats between 40-45 CAG, however, have varying age of disease onset. Recent genome-wide association studies (GWAS) have implicated DNA handling and repair pathways as modifiers of age of disease onset up to 6 years. Therapeutic approaches to modify and delay onset indefinitely through other genetic targets will require identification of pathological mechanisms that precede disease onset. Several hallmark phenotypes have been identified in cell and animal models, including pathogenic aggregate formation. These models are not reflective of human biology, using excessively large CAG repeats (>100) associated with the more aggressive, juvenile HD, overlooking the importance of GWAS results and the progression of disease with lower pathogenic CAG repeats (40-50 CAG). We have therefore generated novel, clinically-relevant human patient fibroblast cell lines and have characterized several disease phenotypes. My thesis presents a culmination of several projects that focus on disease modelling, primarily outlining phenotypic differences between wildtype and HD cells that will benefit our understanding of disease pathogenesis. / Thesis / Doctor of Philosophy (PhD)