Biology subjects

Geldenhuys, W. J.

Publications and source records attributed to Geldenhuys, W. J..

6 recordsLinked to original sources

bioRxiv2024

Blocking the ability of huntingtin to bind membranes: a therapeutic strategy for Huntingtons disease

The ordered aggregation of proteins into amyloid fibrils is a hallmark of numerous neurodegenerative diseases. A common strategy in developing therapeutics for amyloid-based diseases relies on preventing or manipulating the aggregation process. However, many amyloid-forming proteins and their aggregates bind and damage organelle and cellular membranes. As such, blocking the ability of these proteins from directly interacting with membranes represents a unique therapeutic strategy. Using a mutant huntingtin (htt) protein associated with Huntingtons disease (HD) as a model system, the viability of this strategy was evaluated. Screening over 1200 compounds for their ability to block htt binding to lipid vesicles, two compounds, Ro90-7501 (Ro) and Benzamil (Ben), were identified and validated. Despite directly interacting with htt, neither compound prevented fibril formation. Molecular dynamics simulations suggested each compound has a unique mechanism of action, consistent with experimental data. Importantly, both compounds ameliorated phenotype in a C. elegans model of HD.

biophysics↗

bioRxiv2024

Preclinical Multi-Omic Assessment of Pioglitazone in Skeletal Muscles of Mice Implanted with Human HER2/neu Overexpressing Breast Cancer Xenografts

Breast cancer (BC) is the most prevalent cancer worldwide and is accompanied by fatigue during both active disease and remission in the majority of cases. Our lab has measured fatigue in isolated muscles from treatment-naive BC patient-derived orthotopic xenograft (BC-PDOX) mice. Here, we conducted a preclinical trial of pioglitazone in BC-PDOX mice to determine its efficacy in ameliorating BC-induced muscle fatigue, as well as its effects on transcriptomic, metabolomic, and lipidomic profiles in skeletal muscle. MethodsThe pioglitazone and vehicle groups were treated orally for 4 weeks upon reaching a tumor volume of 600 mm3. Whole-animal indirect calorimetry was used to evaluate systemic metabolic states. The transcriptome was profiled using short-read bulk RNA sequencing (RNA-seq). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to profile the metabolome and lipidome. Fast and slow skeletal muscle function were evaluated using isolated ex vivo testing. ResultsPioglitazone was associated with a significant overall decrease in metabolic rate, with no changes in substrate utilization. RNA-seq supported the downstream effects of pioglitazone on target genes and displayed considerable upregulation of mitochondrial bioenergetic pathways. Skeletal muscle metabolomic and lipidomic profiles exhibited dysregulation in response to BC, which was partially restored in pioglitazone-treated mice compared to vehicle-treated BC-PDOX mice. Despite molecular support for pioglitazones efficacy, isolated muscle function was not affected by pioglitazone treatment. ConclusionsBC induces multi-omic dysregulation in skeletal muscle, which pioglitazone partially ameliorates. Future research should focus on profiling systemic metabolic dysfunction, identifying molecular biomarkers of fatigue, and testing alternative pioglitazone treatment regimens. Statement of Translational RelevanceBreast cancer-induced fatigue is a prevalent and debilitating symptom that affects a majority of patients, leading to early treatment discontinuation and poorer outcomes. Despite its significant impact on patient quality of life, there are currently no approved therapies for this condition. Our previous work in the clinically relevant breast cancer patient-derived orthotopic xenograft (BC-PDOX) mouse model suggests that disruptions in the PPAR{gamma} signaling pathway may contribute to the development of cancer-related fatigue. Using this model that recapitulates the fatigue phenotype observed in patients, we conducted a preclinical trial evaluating the FDA-approved PPAR{gamma} agonist, pioglitazone, as a treatment for fatigue. Our multi-omic analysis of skeletal muscle from BC-PDOX mice revealed that pioglitazone treatment partially restored dysregulated lipid profiles and mitochondrial bioenergetic transcriptomic alterations. These findings suggest that pioglitazone may have potential as a therapeutic option for managing cancer-related fatigue in breast cancer patients.

cancer biology↗

bioRxiv2023

Oligomerization enhances huntingtin membrane activity but is suppressed by covalent crosslinking

Huntingtin disease (HD) is a neurodegenerative disease caused by expansion of a polyglutamine (polyQ) tract within the huntingtin (htt) protein, leading to aggregation into a variety of species ranging from small oligomers to large fibrils. A consensus concerning which of these aggregate states are primarily responsible for toxicity associated with mutant htt remains elusive. Htt directly binds and damages a variety of membranous surfaces within cells. Here, the ability of different aggregation states of htt to interact with and damage lipid membranes was determined. Oligomers represented the most active lipid binding species, whereas, fibril formation severely limited membrane binding. Thus, strategies to stabilize oligomers were implemented, and conformational flexibility appeared to play a key role in the oligomer/membrane interaction. In particular, stabilizing oligomers with covalent crosslinking with 1,5-difluoro-2,4-dinitrobenzene (DFDNB) effectively eliminated the ability of oligomers to bind lipid membranes and reduced their associated cellular toxicity.

biophysics↗

bioRxiv2022

Interphotoreceptor matrix proteoglycans IMPG1 and IMPG2 proteolyze in the SEA domain and reveal localization mutual dependency

The interphotoreceptor matrix (IPM) is a specialized extracellular mesh of molecules surrounding the inner and outer segments of photoreceptor neurons. Interphotoreceptor matrix proteoglycan 1 and 2 (IMPG1 and IMPG2) are major components of the IPM. Both proteoglycans possess SEA (sperm protein, enterokinase and agrin) domains, which may support proteolysis. Interestingly mutations in the SEA domains of IMPG1 and IMPG2 are associated with vision disease in humans. However, if SEA domains in IMPG molecules undergo proteolysis, and how this contributes to vision pathology is unknown. Therefore, we investigated SEA-mediated proteolysis of IMPG1 and IMPG2 and its significance to IPM physiology. Immunoblot analysis confirmed proteolysis of IMPG1 and IMPG2 in the retinas of wildtype mice. Point mutations mimicking human mutations in the SEA domain of IMPG1 that are associated with vision disease inhibited proteolysis. These findings demonstrate that proteolysis is part of the maturation of IMPG1 and IMPG2, in which deficits are associated with vision diseases. Further, immunohistochemical assays showed that proteolysis of IMPG2 generated two subunits, a membrane-attached peptide and an extracellular peptide. Notably, the extracellular portion of IMPG2 trafficked from the IPM around the inner segment toward the outer segment IPM by an IMPG1-dependent mechanism. This result provides the first evidence of a trafficking system that shuttles IMPG1 and IMPG2 from the inner to outer IPM in a co-dependent manner. In addition, these results suggest an interaction between IMPG1-IMPG2, and propose that mutations affecting one IMPG could affect the localization of the normal IMPG partner contributing to the disease mechanism of vision diseases associated with defective IMPG molecules.

molecular biology↗

bioRxiv2021

Pioglitazone administration restores a PPARG-dependent transcriptional network and ATP levels within skeletal muscles of mice implanted with patient-derived breast tumors

BackgroundFatigue is common in patents with breast cancer (BC), and can occur in patients with early stage disease and in the absence of muscle wasting (i.e. cachexia). We have reported transcriptional and proteomic alterations in muscles from BC patients, which are associated with fatigue. Mice implanted with human BC xenografts recapitulate the muscle molecular composition changes seen in patients, coupled with a greater rate of contraction-induced fatigue. Multiple bioinformatics platforms in both human and mouse muscles have identified peroxisome proliferator activated receptor gamma (PPARG) as central to this phenotype, with multiple PPARG target genes downregulated in response to tumor growth. The current study tested the hypothesis that the PPARG agonist pioglitazone (pio), a commonly prescribed diabetes drug, would rescue the transcriptional alterations observed in muscles of tumor-bearing mice. MethodsSixteen female NSG mice were implanted with breast cancer patient-derived orthotopic xenografts (BC-PDOX) via transplantation of Her2/neu+ human tumor fragments. BC-PDOX mice were randomly assigned to a treatment group that received daily oral pio at 30 mg.kg-1 (n=8), or a control group that received a similar volume of vehicle (n=8). Treatment was initiated when tumors reached a volume of 600mm3, and lasted for 2-weeks. Hindlimb muscles were isolated from BC-PDOX and non-tumor bearing mice for RNA-sequencing, gene expression validation, and ATP quantification. Differentially expressed genes (DEGs) in muscles from BC-PDOX mice relative to non-tumor bearing controls were identified using DESeq2, and multiple bioinformatics platforms were employed to contextualize the DEGs. ResultsWe found that the administration of pio restored the muscle gene expression patterns of BC-PDOX mice to a profile resembling muscles of non-tumor bearing NSG control mice. Validation of skeletal muscle gene expression by qPCR confirmed pio increased the expression of PPARG target genes in skeletal muscles. Isolated mitochondria from muscles of BC-PDOX mice treated with pio contained greater levels of ATP. There were no differences in body weights, muscle weights, or tumor volumes in pio vs. vehicle treated BC-PDOX mice. ConclusionsThese data demonstrate that oral pio supplementation rescues the BC-associated downregulation of PPARG target genes in skeletal muscle. Additionally, muscles from BC-PDOX mice treated with pio had greater levels of ATP, which would be associated a more fatigue-resistant muscle phenotype. Therefore, we propose that the FDA-approved and generic diabetes drug, pio, be considered as a supportive therapy for the treatment of BC-associated muscle fatigue.

cancer biology↗

bioRxiv2019

Targeting the dysfunctional mitochondrial respiration in drug resistant B-cell acute lymphoblastic leukemia

Reprogramming of cellular pathways is a crucial mechanism of drug resistance and survival in refractory acute lymphoblastic leukemia (ALL) cells. In the present study, we performed an unbiased gene expression analysis and identified a dysfunctional mitochondrial respiration program in drug-resistant ALL cells grown in a co-culture system with bone marrow stromal cells (BMSC). Specifically, the activity of the complexes within the electron transport chain was significantly downregulated, correlated with decreased mitochondrial mass and ATP production in drug-resistant ALL cells. To validate mitochondrial respiration as a druggable target, we utilized pyrvinium pamoate (PP), a known inhibitor of mitochondrial respiration and documented its anti-leukemic activity in several ALL cell lines grown alone or in co-culture with BMSC. To increase the bioavailability profile of PP, we successfully encapsulated PP in a nanoparticle drug delivery system and demonstrated that it retained its anti-leukemic activity in a hemosphere assay. PP anti-leukemic activity was decreased by the addition of sodium pyruvate, and furthermore, PP was found to have an additive anti-leukemic effect when used in combination with rotenone, a mitochondrial complex I inhibitor with activity similar to PP on the mitochondrial respiration. Importantly, PPs cell death activity was found to be specific for leukemic cells as primary normal immune cells were resistant to PP-mediated cell death. In conclusion, we have demonstrated that PP is a novel therapeutic lead compound that counteracts the respiratory reprogramming found in refractory ALL cells.

cancer biology↗