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Thesis
Peer Reviewed

G3BP1-positive stress granule pathology in brains of Huntington’s disease mice and human patients and a potential role for extracellular vesicle-derived miRNA regulation

Sanchez, Isabella Inez
Advisor(s): Thompson, Leslie M

UC Irvine Electronic Theses and Dissertations (2020)

Stress granules (SGs) are membraneless organelles that form in response to cellular stress. Chronic cellular stress associated with neurodegenerative disease results in the persistence of SG structures. Chronic expression of mutant huntingtin generates various forms of cellular stress, including activation of the unfolded protein response and oxidative stress. However, it has yet to be determined whether SGs are a feature of Huntington’s disease (HD) neuropathology. For my dissertation, I investigated localization and levels of the SG nucleating protein G3BP1 and found a significant increase in the density of G3BP1-positive SGs in the cortex and hippocampus of R6/2 transgenic mice, as well as in the superior frontal cortex of HD patient brains. I also examined the miRNA composition of extracellular vesicles (EVs) present in the cerebrospinal fluid (CSF) of HD patients and show that a subset of their target mRNAs is differentially expressed in the prefrontal cortex of HD patients. Of these targets, there is an enrichment of SG components, including G3BP1. These findings suggest that SG dynamics might play a role in the pathophysiology of HD. Lastly, because the majority of the CSF is produced by choroid plexus epithelial cells, and a significant portion of CSF EVs likely originate from CPECs, I initiated the generation induced pluripotent stem cell (iPSC)-derived CPECs from HD and control lines that will allow for future mechanistic studies of EV secretion. Together, my work provides evidence of a potential role for CSF EV miRNAs in the regulation of SGs, identifies specific miRNAs that modulate the SG component G3BP1, and demonstrates feasibility for the generation of an iPSC-derived CPEC model.

Cover page: G3BP1-positive stress granule pathology in brains of Huntington’s disease mice and human patients and a potential role for extracellular vesicle-derived miRNA regulation

Creative Commons 'BY-NC-ND' version 4.0 license

Article
Peer Reviewed

Dermatomyositis with Eosinophils.

UC Irvine Previously Published Works (2023)

Dermatomyositis is an idiopathic inflammatory myopathy that often presents with symmetric proximal skeletal muscle weakness and characteristic skin findings. Typical skin biopsy findings include vacuolar changes of the basal layer, increased dermal mucin, and a predominantly lymphocytic infiltrate. We report a case of dermatomyositis presenting as intensely pruritic papules and plaques, with initial histopathology being atypical of dermatomyositis due to the presence of eosinophils. The initial biopsy demonstrated a superficial dermatitis with eosinophils, initially thought to represent a drug eruption. A second biopsy of the same cutaneous manifestation was performed at a later time given high clinical suspicion for dermatomyositis and demonstrated a more classic vacuolar interface dermatitis with increased mucin and an absence of eosinophils. Notably, increased pruritus was specifically associated with the lesion that demonstrated tissue eosinophilia. The case illustrates the importance of considering tissue eosinophilia in the histologic presentation of dermatomyositis.

Cover page: Dermatomyositis with Eosinophils.

Article
Peer Reviewed

Longitudinal Biochemical Assay Analysis of Mutant Huntingtin Exon 1 Protein in R6/2 Mice.

UC Irvine Previously Published Works (2018)

Background

Biochemical analysis of mutant huntingtin (mHTT) aggregation species in HD mice is a common measure to track disease. A longitudinal and systematic study of how tissue processing affects detection of conformers has not yet been reported. Understanding the homeostatic flux of mHTT over time and under different processing conditions would aid in interpretation of pre-clinical assessments of disease interventions.

Objective

Provide a systematic evaluation of tissue lysis methods and molecular and biochemical assays in parallel with behavioral readouts in R6/2 mice to establish a baseline for HTT exon1 protein accumulation.

Methods

Established biochemical methods were used to process tissue from R6/2 mice of specific ages following behavior tasks. Aggregation states and accumulation of mHTT exon 1 protein were evaluated using multiple break and assay methods to determine potential conformational flux assay specificity in detection of mHTT species, and tissue specificity of conformers.

Results

Detection of mHTT exon 1 protein species varied based on biochemical processing and analysis providing a baseline for subsequent studies in R6/2 mice. Insoluble, high molecular weight species of mHTT exon 1 protein increased and tracked with onset of behavioral impairments in R6/2 mice using multiple assay methods.

Conclusions

Conformational flux from soluble monomer to high molecular weight, insoluble species of mHTT exon 1 protein was generally consistent for multiple assay methods throughout R6/2 disease progression; however, the results support the use of multiple biochemical techniques to detect mHTT exon 1 protein species for preclinical assessments in HD mouse models expressing mHTT exon 1 protein.

Article
Peer Reviewed

Longitudinal Biochemical Assay Analysis of Mutant Huntingtin Exon 1 Protein in R6/2 Mice

UC Irvine Previously Published Works (2018)

Background

Biochemical analysis of mutant huntingtin (mHTT) aggregation species in HD mice is a common measure to track disease. A longitudinal and systematic study of how tissue processing affects detection of conformers has not yet been reported. Understanding the homeostatic flux of mHTT over time and under different processing conditions would aid in interpretation of pre-clinical assessments of disease interventions.

Objective

Provide a systematic evaluation of tissue lysis methods and molecular and biochemical assays in parallel with behavioral readouts in R6/2 mice to establish a baseline for HTT exon1 protein accumulation.

Methods

Established biochemical methods were used to process tissue from R6/2 mice of specific ages following behavior tasks. Aggregation states and accumulation of mHTT exon 1 protein were evaluated using multiple break and assay methods to determine potential conformational flux assay specificity in detection of mHTT species, and tissue specificity of conformers.

Results

Detection of mHTT exon 1 protein species varied based on biochemical processing and analysis providing a baseline for subsequent studies in R6/2 mice. Insoluble, high molecular weight species of mHTT exon 1 protein increased and tracked with onset of behavioral impairments in R6/2 mice using multiple assay methods.

Conclusions

Conformational flux from soluble monomer to high molecular weight, insoluble species of mHTT exon 1 protein was generally consistent for multiple assay methods throughout R6/2 disease progression; however, the results support the use of multiple biochemical techniques to detect mHTT exon 1 protein species for preclinical assessments in HD mouse models expressing mHTT exon 1 protein.

Article
Peer Reviewed

Huntington’s disease mice and human brain tissue exhibit increased G3BP1 granules and TDP43 mislocalization

UC Irvine Previously Published Works (2021)

Chronic cellular stress associated with neurodegenerative disease can result in the persistence of stress granule (SG) structures, membraneless organelles that form in response to cellular stress. In Huntington's disease (HD), chronic expression of mutant huntingtin generates various forms of cellular stress, including activation of the unfolded protein response and oxidative stress. However, it has yet to be determined whether SGs are a feature of HD neuropathology. We examined the miRNA composition of extracellular vesicles (EVs) present in the cerebrospinal fluid (CSF) of patients with HD and show that a subset of their target mRNAs were differentially expressed in the prefrontal cortex. Of these targets, SG components were enriched, including the SG-nucleating Ras GTPase-activating protein-binding protein 1 (G3BP1). We investigated localization and levels of G3BP1 and found a significant increase in the density of G3BP1-positive granules in the cortex and hippocampus of R6/2 transgenic mice and in the superior frontal cortex of the brains of patients with HD. Intriguingly, we also observed that the SG-associated TAR DNA-binding protein 43 (TDP43), a nuclear RNA/DNA binding protein, was mislocalized to the cytoplasm of G3BP1 granule-positive HD cortical neurons. These findings suggest that G3BP1 SG dynamics may play a role in the pathophysiology of HD.

Cover page: Huntington’s disease mice and human brain tissue exhibit increased G3BP1 granules and TDP43 mislocalization