Biology subjects

Dirksen, R. T.

Publications and source records attributed to Dirksen, R. T..

10 recordsLinked to original sources

bioRxiv2026

Iron toxicity potentiates cell-type specific amyloid beta proteotoxicity in C. elegans via altered energy homeostasis

Alzheimers disease (AD) is a devastating neurodegenerative disorder characterized by memory loss and a decline in cognitive function. Hallmarks of AD include an age-dependent accumulation of toxic amyloid beta (A{beta}) 42 in the brain, energy dyshomeostasis caused by mitochondrial dysfunction, and iron overload. However, the role of iron overload and mitochondrial dysfunction in AD pathology is unknown and their precise relationship with A{beta} 42 toxicity in AD pathology is unclear. C. elegans provide a powerful model system to untangle and clarify these relationships. In this study, we quantify the temperature-dependence of iron toxicity (16, 20 and 25C) in neurons and muscle of C. elegans that overexpress A{beta} 42. We found that A{beta} 42, regardless of the cell-type expression, caused accelerated paralysis compared to age-matched WT worms with the greatest degree of paralysis observed at an elevated temperature (25C). Moreover, the combination of iron toxicity and A{beta} 42 results in an enhanced paralytic phenotype at 16C. Thus, iron exposure potentiates A{beta} toxicity observed at low temperatures. Iron toxicity stimulated both maximum (State 3) and leak (State 4) respiration in WT and A{beta} 42 worms. A{beta} 42 worms also exhibited increased leak respiration at baseline that was further exacerbated by iron toxicity. Iron burden and sensitivity increased A{beta} 42 peptide toxicity. A{beta} 42 worms exhibited reduced levels of Ca, Zn, Mn, and K. Overall, our results suggest that iron potentiates A{beta} toxicity at low temperature and enhances A{beta} peptide mediated mitochondrial bioenergetic dysfunction in C. elegans. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/714217v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@9eaf46org.highwire.dtl.DTLVardef@542eforg.highwire.dtl.DTLVardef@16d9678org.highwire.dtl.DTLVardef@1b1b16d_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LITemperature stress modulates the synergetic interactions of iron toxicity and A{beta} 42 pathology C_LIO_LIIron sensitivity drives increased cell-type specific A{beta} 42 pathology C_LIO_LIEnergy dyshomeostasis via impaired mitochondrial function and increased proton leak contributes to iron- and A{beta}-induced pathology C_LI

neuroscience↗

bioRxiv2024

DMT1 knockout abolishes ferroptosis induced mitochondrial dysfunction in C. elegans amyloid beta proteotoxicity

Iron is critical for neuronal activity and metabolism, and iron dysregulation alters these functions in age-related neurodegenerative disorders, such as Alzheimers disease (AD). AD is a chronic neurodegenerative disease characterized by progressive neuronal dysfunction, memory loss and decreased cognitive function. AD patients exhibit elevated iron levels in the brain compared to age-matched non-AD individuals. However, the degree to which iron overload contributes to AD pathogenesis is unclear. Here, we evaluated the involvement of ferroptosis, an iron-dependent cell death process, in mediating AD-like pathologies in C. elegans. Results showed that iron accumulation occurred prior to the loss of neuronal function as worms age. In addition, energetic imbalance was an early event in iron-induced loss of neuronal function. Furthermore, the loss of neuronal function was, in part, due to increased mitochondrial reactive oxygen species mediated oxidative damage, ultimately resulting in ferroptotic cell death. The mitochondrial redox environment and ferroptosis were modulated by pharmacologic processes that exacerbate or abolish iron accumulation both in wild-type worms and worms with increased levels of neuronal amyloid beta (A{beta}). However, neuronal A{beta} worms were more sensitive to ferroptosis-mediated neuronal loss, and this increased toxicity was ameliorated by limiting the uptake of ferrous iron through knockout of divalent metal transporter 1 (DMT1). In addition, DMT1 knockout completely suppressed phenotypic measures of A{beta} toxicity with age. Overall, our findings suggest that iron-induced ferroptosis alters the mitochondrial redox environment to drive oxidative damage when neuronal A{beta} is overexpressed. DMT1 knockout abolishes neuronal A{beta}-associated pathologies by reducing neuronal iron uptake. HighlightsO_LIEnergetic imbalance is an early event in iron-induced loss of neuronal function C_LIO_LINeuronal A{beta} increases susceptibility to ferroptosis mediated oxidative damage C_LIO_LIDivalent metal transporter 1 knockout protects against iron-induced oxidative damage and ferroptosis C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/607074v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@927aaorg.highwire.dtl.DTLVardef@10b121forg.highwire.dtl.DTLVardef@1d2d6aforg.highwire.dtl.DTLVardef@10cb797_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2024

Mechano-adaptation: Exercise-driven Piezo1 & Piezo2 augmentation and chondroprotection in articular cartilage

Chondrocytes in adult joints are mechanosensitive post-mitotic quiescent cells with robustly expressed both Piezo1 and Piezo2 ion channels. Here, we examined the mechano-adaptation and Piezo modulations in articular chondrocytes using a mouse exercise model. We first found differential expression patterns of PIEZO1 and PIEZO2 in articular chondrocytes of healthy knee joints; chondrocytes in tibial cartilage (T) exhibit significantly higher PIEZO1 and PIEZO2 than femoral chondrocytes (F). Interestingly, a few weeks of exercise caused both PIEZO1 and PIEZO2 augmentation in F and T compared to the sedentary control group. Despite the increased expression levels of these mechanosensors, chondrocytes in exercised cartilage exhibit significantly reduced mechanical susceptibility against 1mJ impact. PIEZO1 modulation was relatively more rapid than PIEZO2 channels post-exercise. We tested the exercise-induced effect using Piezo1-conditional knockout (Pz1-cKO; Agc1CreERT2;Piezo1fl/fl). Pz1-cKO mice exhibit diminished exercise-driven chondroprotection against 1mJ impact, suggesting essential roles of Piezo1-mediated mechanotransduction for physiologic-induced cartilage matrix homeostasis. In addition, using a mouse OA model, we further found the modulated PIEZO1 in chondrocytes, consistent with reports in Ren et al., but without PIEZO2 modulations over OA progression. In summary, our data reveal the distinctly tuned Piezo1 and Piezo2 channels in chondrocytes post-exercise and post-injury, in turn modulating the mechanical susceptibility of chondrocytes. We postulate that Piezo1 is a tightly-regulated biphasic biomarker; Piezo1 antagonism may increase cellular survival post-injury and Piezo1 (with Piezo2) agonism to promote cartilage ECM restoration.

bioengineering↗

bioRxiv2023

Voluntary wheel running mitigates disease in an Orai1 gain-of-function mouse model of tubular aggregate myopathy

Tubular aggregate myopathy (TAM) is an inherited skeletal muscle disease associated with progressive muscle weakness, cramps, and myalgia. Tubular aggregates (TAs) are regular arrays of highly ordered and densely packed SR straight-tubes in muscle biopsies; the extensive presence of TAs represent a key histopathological hallmark of this disease in TAM patients. TAM is caused by gain-of-function mutations in proteins that coordinate store-operated Ca2+ entry (SOCE): STIM1 Ca2+ sensor proteins in the sarcoplasmic reticulum (SR) and Ca2+-permeable ORAI1 channels in the surface membrane. We have previously shown that voluntary wheel running (VWR) prevents formation of TAs in aging mice. Here, we assessed the therapeutic potential of endurance exercise (in the form of VWR) in mitigating the functional and structural alterations in a knock-in mouse model of TAM (Orai1G100S/+ or GS mice) based on a gain-of-function mutation in the ORAI1 pore. WT and GS mice were singly-housed for six months (from two to eight months of age) with either free-spinning or locked low profile wheels. Six months of VWR exercise significantly increased soleus peak tetanic specific force production, normalized FDB fiber Ca2+ store content, and markedly reduced TAs in EDL muscle from GS mice. Six months of VWR exercise normalized the expression of mitochondrial proteins found to be altered in soleus muscle of sedentary GS mice in conjunction with a signature of increased protein translation and biosynthetic processes. Parallel proteomic analyses of EDL muscles from sedentary WT and GS mice revealed changes in a tight network of pathways involved in formation of supramolecular complexes, which were also normalized following six months of VWR. In summary, sustained voluntary endurance exercise improved slow twitch muscle function, reduced the presence of TAs in fast twitch muscle, and normalized the muscle proteome of GS mice consistent with protective adaptions in proteostasis, mitochondrial structure/function, and formation of supramolecular complexes.

physiology↗

bioRxiv2023

Glutathione supports lipid abundance in vivo

Cells rely on antioxidants to survive. The most abundant antioxidant is glutathione (GSH). The synthesis of GSH is non-redundantly controlled by the glutamate-cysteine ligase catalytic subunit (GCLC). GSH imbalance is implicated in many diseases, but the requirement for GSH in adult tissues is unclear. To interrogate this, we developed a series of in vivo models to induce Gclc deletion in adult animals. We find that GSH is essential to lipid abundance in vivo. GSH levels are reported to be highest in liver tissue, which is also a hub for lipid production. While the loss of GSH did not cause liver failure, it decreased lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we found that GSH promotes lipid abundance by repressing NRF2, a transcription factor induced by oxidative stress. These studies identify GSH as a fulcrum in the livers balance of redox buffering and triglyceride production.

biochemistry↗

bioRxiv2022

Updated Variant Curation Expert Panel Criteria and Pathogenicity Classifications for 251 Variants for RYR1-related Malignant Hyperthermia Susceptibility

The ClinGen malignant hyperthermia susceptibility (MHS) variant curation expert panel specified the ACMG/AMP criteria for RYR1-related MHS and a pilot analysis of 84 variants was published. We have now classified an additional 251 variants for RYR1-related MHS according to current ClinGen standards and updated the criteria where necessary. Criterion PS4 was modified such that individuals with multiple RYR1 variants classified as pathogenic (P), likely pathogenic (LP) or variant of uncertain significance (VUS) were not considered as providing evidence for pathogenicity. Critera PS1 and PM5 were revised to consider LP variants at the same amino acid residue as providing evidence for pathogenicity at reduced strength. Finally, PM1 was revised such that if PS1 or PM5 are used PM1, if applicable, should be downgraded to supporting. Of the 251 RYR1 variants, 42 were classified as P/LP, 16 as B/LB, and 193 as VUS. The primary driver of 176 VUS classifications was insufficient evidence supporting pathogenicity, rather than evidence against pathogenicity. Functional data supporting PS3/BS3 was identified for only 13 variants. Based on the posterior probabilities of pathogenicity and variant frequencies in gnomAD, we estimated the prevalence of individuals with RYR1-related MHS pathogenic variants to be between 1/300 and 1/1,075, considerably higher than current estimates. We have updated ACMG/AMP criteria for RYR1-related MHS and classified 251 variants. We suggest that prioritization of functional studies is needed to resolve the large number of VUS classifications and allow for appropriate risk assessment. RYR1-related MHS pathogenic variants are likely to be more common than currently appreciated.

genetics↗

bioRxiv2021

A novel zebrafish model of SPEG-related centronuclear myopathy (CNM): characterization and comparison with other CNM model zebrafish

Centronuclear myopathy (CNM) is a congenital neuromuscular disorder caused by pathogenic variation in genes associated with membrane trafficking and excitation-contraction coupling (ECC). Bi-allelic autosomal recessive mutations in striated muscle enriched protein kinase (SPEG) account for a subset of CNM patients. Previous research has been limited by the perinatal lethality of Speg knockout mice. Thus, the precise biological role of SPEG in skeletal muscle remains unknown. To address this issue, we generated zebrafish spega, spegb, and spega/spegb (speg-DKO) mutant lines. We demonstrate that speg-DKO zebrafish faithfully recapitulate multiple phenotypes associated with human CNM, including disruption of the ECC protein machinery, dysregulation of calcium homeostasis during ECC, and impairment of muscle performance. Taking advantage of the availability of zebrafish models of multiple CNM genetic subtypes, we compared novel and known disease markers in speg-DKO with mtm1-KO and DNM2-S619L transgenic zebrafish. We observed desmin (DES) accumulation common to all CNM subtypes, and DNM2 upregulation in muscle of both speg-DKO and mtm1-KO zebrafish. In all, we establish a new model of SPEG-related CNM, and identify abnormalities in this model suitable for defining disease pathomechanisms and evaluating potential therapies. Summary StatementWe created a novel zebrafish speg mutant model of centronuclear myopathy that recapitulates key features of the human disorder and provides insight into pathomechanisms of the disease.

genetics↗

bioRxiv2020

Variant Curation Expert Panel Recommendations for RYR1 Pathogenicity Assertions in Malignant Hyperthermia Susceptibility

PurposePrevention of malignant hyperthermia (MH) requires an understanding of RYR1 variant pathogenicity to assess the risk of exposure to triggering agents. Personalized medicine, especially secondary findings and eventually genomic screening, will contribute toward this goal. MethodsWe specified ACMG/AMP criteria for variant interpretation for RYR1 and MH. Proposed rules were piloted on 84 variants. We applied quantitative evidence calibration for several criteria using likelihood ratios based on the Bayesian framework. ResultsSeven ACMG/AMP criteria were adopted without changes, ten were adopted with RYR1-specific modifications, and nine were dropped. The in silico (PP3 and BP4) and hot spot criteria (PM1) were evaluated quantitatively. REVEL gave an OR of 23:1 for PP3 and 16:1 for BP4 using trichotomized cut-offs of >0.85 (pathogenic) and <0.5 (benign). The PM1 hotspot criterion had an OR of 24:1. PP3 and PM1 were implemented at moderate strength. Applying the revised ACMG criteria to 44 recognized MH variants, 30 were assessed as pathogenic, 12 as likely pathogenic, and two as VUS. ConclusionCuration of these variants will facilitate interpretation of RYR1/MH genomic testing results, which is especially important for secondary findings analyses. Our approach to quantitatively calibrating criteria are generalizable to other variant curation expert panels.

genetics↗

bioRxiv2019

Identification of drug modifiers for RYR1 related myopathy using a multi-species discovery pipeline

Ryanodine receptor type I-related myopathies (RYR1-RMs) represent the largest group of non-dystrophic myopathies. RYR1-RMs are associated with severe disabilities and early mortality; despite these facts, there are currently no available treatments. The goal of this study was to identify new therapeutic targets for RYR1-RMs. To accomplish this, we developed a novel discovery pipeline using nematode, zebrafish, and mammalian cell models of the disease. We first performed large-scale drug screens in C. elegans and zebrafish. 74 positive hits were identified in C. elegans, while none were uncovered in the zebrafish. Targeted testing of these hits in zebrafish yielded positive results for two compounds. We examined these compounds using newly created Ryr1 knockout C2C12 cells, and found that p38 inhibition impaired caffeine-induced Ca2+ release. Lastly, we tested one p38 inhibitor in myotubes from Ryr1Y524S/+ (YS) mice, and demonstrated that it blunts the aberrant temperature-dependent increase in resting Ca2+ in these cells. In all, we developed a unique platform for RYR1-RM therapy development that is potentially applicable to a broad range of neuromuscular disorders.

genetics↗

bioRxiv2017

Evidence for, and Metabolic Consequences of, a Cardiac Mitochondrial KNa1.2 Channel

Controversy surrounds the molecular identity of mitochondrial K+ channels that are important for protection against cardiac ischemia-reperfusion injury. While KNa1.2 (Kcnt2 gene) is necessary for cardioprotection by volatile anesthetics, electrophysiologic evidence for a channel of this type in mitochondria is lacking. The endogenous physiologic role of a potential mito-KNa1.2 channel is also unclear. Herein, single channel patch-clamp of 27 independent cardiac mitochondrial inner membrane (mitoplast) preparations from wild type (WT) mice yielded 6 channels matching the known ion-sensitivity, ion-selectivity, pharmacology and conductance properties of KNa1.2 (slope conductance 138{+/-}1 pS). However, similar experiments on 40 preparations from Kcnt2-/- mice yielded zero such channels. The KNa opener bithionol uncoupled respiration in WT but not Kcnt2-/- cardiomyocytes. Furthermore, when oxidizing only fat as substrate, Kcnt2-/- cardiomyocytes and hearts were less responsive to increases in energetic demand. Kcnt2-/- mice also had elevated body fat, but no baseline differences in the cardiac metabolome. These data support the existence of a cardiac mitochondrial KNa1.2 channel, and a role for cardiac KNa1.2 in regulating metabolism under conditions of high energetic demand.

cell biology↗