Biology subjects

Falk, M. J.

Publications and source records attributed to Falk, M. J..

10 recordsLinked to original sources

bioRxiv2026

Targeted modulation of IGFBP5/IGF1, THPO, and P38 MAPK signaling are potent therapeutic strategies generalizable for mitochondrial respiratory chain disease and osteosarcoma

Primary mitochondrial diseases (PMD) have limited disease-modifying therapies, currently applicable to only 3 of over 400 discrete gene disorders. Cycloheximide (CHX) is a global cytosolic translation inhibitor we previously reported to rescue PMD preclinical models, although its toxicity precluded clinical development. To identify specific mediators underlying CHX treatment benefit in PMD, SOMAscan-based proteomics was performed in complex I deficient and genetic disease fibroblast cell line models grown in galactose. Thrombopoietin (THPO) and insulin-like growth factor binding protein 5 (IGFBP5) were the only two differentially regulated proteins, together with ERK/MAPK pathway dysregulation, identified upon CHX treatment in PMD versus healthy control cells. THPO inhibition by siRNA or pharmacologic approaches rescued stress-induced viability loss in patient fibroblasts having diverse PMD gene etiologies, and significantly improved mitochondrial stress, linear growth, and neuromuscular function in a classical ndufs2-/- C. elegans model. IGFBP5 overexpression by lentiviral or mRNA approaches rescued cell viability across distinct PMD gene etiologies, as did IGF1 pharmacologic inhibition across both PMD mutant and C. elegans models. MAPK pharmacologic inhibition rescued multiple distinct complex I disease cells survival, as well as mitochondrial stress in SLC25A46-/- C. elegans. Combination therapies targeting multiple of these glucose signaling pathway proteins, together with glucose and N-acetylcysteine, yielded superior therapeutic benefit in complex I disease cell and C. elegans models. Additionally, single or combined pharmacologic inhibition of THPO or IGF1 significantly enhanced primary and metastatic osteosarcoma cell death. Collectively, targeted small molecule and genetic modulation of THPO, IGF1, or MAPK recapitulated the significant therapeutic benefit of CHX in PMD, while avoiding global translation inhibition. These novel PMD therapies likely confer benefit by attenuating MAPK-driven autophagy and potentially promoting noncanonical glucose uptake, improving cellular energy balance. Overall, these glucose signaling cellular pathway targets hold broad therapeutic promise for PMD patients, warranting further clinical research development.

genetics↗

bioRxiv2026

Dichloroacetate improves animal survival, growth, neuromuscular activity, mitochondrial stress and physiology, and elevated lactate in C. elegans pdha-1 and dld-1 RNAi models of pyruvate dehydrogenase complex deficiency (PDCD)

Pyruvate dehydrogenase complex (PDHc) deficiency (PDCD) is a primary mitochondrial disorder characterized by neurodevelopmental disability, altered intermediary metabolism and early mortality. Dichloroacetate (DCA), a pyruvate analogue, is a well-described PDHc activator that remains under clinical investigation for treatment of PDCD. Here, we studied the in vivo efficacy of a 5-point log concentration range of DCA on animal health and metabolism in C. elegans with feeding RNA interference (RNAi) expression knockdown of either PDHA-1 or DLD-1 homologues at graded degrees to model variable disease severity. These worm models recapitulate phenotypic features of PDCD observed in human patients, including reduced survival, delayed growth, locomotor impairment, and elevated lactate and/or pyruvate tissue levels. DCA treatment appeared well-tolerated, with no gross morphologic toxicity seen at doses up to 25 mM. Significantly improved health, survival, tissue lactate levels, and mitochondrial physiology were observed at 25 mM in pdha-1(RNAi) knockdown animals. DCA treatment in dld-1(RNAi) C. elegans models (undiluted, 1:20 dilution, and 1:100 dilution) showed significant therapeutic benefits on survival, neuromuscular function and metabolic phenotypes primarily in the moderate (1:20) and/or mild (1:100) dld-1(RNAi) deficiency strains, but not in full-dose dld-1(RNAi). Importantly, linear growth, neuromuscular activity, and mitochondrial physiology were significantly improved with DCA treatment even in the most severe dld-1(RNAi) undiluted model. Overall, preclinical modeling provides objective evidence of DCA therapeutic efficacy in C. elegans expression knockdown strains for two well-conserved homologues of PDHA1 and DLD that represent distinct genetic etiologies of PDHc deficiency, with demonstrated beneficial effects on survival, healthspan, tissue lactate, and mitochondrial physiology. These data further confirm that DCAs therapeutic effect correlates with PDHc disease phenotype severity in dld-1(RNAi) animals. SYNOPSIS: Dichloroacetate (DCA) treatment demonstrated significant preclinical beneficial effect on survival, neuromuscular function, linear growth, tissue lactate, and mitochondrial metabolism in two C. elegans models with variable degrees of pyruvate dehydrogenase complex (PDHc) deficiency (PDCD), providing confirmatory evidence to support its therapeutic potential in human PDCD patients.

cell biology↗

bioRxiv2026

Targeting the Mitochondrial Phenotype in Cockayne Syndrome Patient Cells: From Bioenergetic Fragility to Pharmacologic Rescue

BackgroundCockayne syndrome (CS), primarily caused by autosomal recessive pathogenic variants in ERCC6 (CSB) or ERCC8 (CSA), is a transcription-coupled nucleotide excision repair disorder. CS frequently presents with features similar to primary mitochondrial disease (PMD), including leukodystrophy, lactic acidemia, and skeletal muscle mitochondrial DNA (mtDNA) depletion. How this mitochondrial phenotype arises at the cellular level, and whether it can be pharmacologically targeted, is not yet clear. MethodsWe characterized mtDNA content, respiratory chain (RC) protein abundance, mitochondrial biogenesis signaling pathways, and oxidative phosphorylation capacity in primary fibroblasts from two siblings with identical compound heterozygous ERCC6 pathogenic variants (c.1526+1G>T; c.2800C>A, p.Pro934Thr) despite marked intrafamilial phenotypic divergence. A combined metabolic stress exposure (galactose, reduced glutamine, and buthionine sulfoximine, (BSO)) which reduced CS cell survival was used to screen for therapeutic leads among twenty-three candidate mitochondrial disease therapeutic compounds. Lead compounds were mechanistically validated at the level of mitochondrial superoxide, total cellular oxidative stress, glutathione, and autophagic flux. ResultsPatient fibroblasts exhibited several hallmarks of PMD, including reduced mtDNA content, decreased expression of complex I subunit NDUFB8, elevated expression of TOM20 with paradoxically decreased PGC1 suggestive of impaired mitophagic clearance, and decreased mitochondrial respiratory capacity. Under combined metabolic stress, ATP-levels indicative of survival in CS patient fibroblasts selectively collapsed to [~]20% of controls. Five dual-rescue compounds, defined as agents that reproducibly restored ATP-based cell survival in both patient fibroblast lines under stress, were identified, including N-acetylcysteine (NAC), coenzyme Q10 (CoQ10), rapamycin, taurine, and (-)-epicatechin. Mechanistic profiling resolved three functional classes of therapeutic effects in CS cells: (1) upstream mitochondrial reactive oxygen species reduction (NAC, CoQ10); (2) mTORC1 inhibition bypassing defective stress-induced autophagic induction (rapamycin); and (3) extra-mitochondrial improvement in cellular stress resilience ((-)- epicatechin, taurine). ConclusionsERCC6-based CSB deficiency produced a stress-sensitive and physiologically complex mitochondrial phenotype in patient fibroblasts that was pharmacologically treatable by targeting three mechanistically distinct pathways. Oxidative and broader stress buffering, autophagy modulation via mTORC1 inhibition, and enhanced cellular resilience highlight novel therapeutic opportunities to be advanced to clinical trials in CSB patients.

molecular biology↗

bioRxiv2026

In vitro modeling of nutritional and mitochondria-targeted therapies for osteosarcoma

Osteosarcoma is the most common pediatric bone tumor yet has limited treatment options, especially for metastatic cases with a 20% adjusted 5-year survival rate. Current therapies are non-specific, involving primary tumor resection with DNA-damaging chemotherapies like methotrexate, doxorubicin, and cisplatin. Few effective treatment options exist for metastases. Targeting metabolism involving cancers reduced mitochondrial functionality remains underexplored in osteosarcoma. We investigated the therapeutic potential in human osteosarcoma primary and metastatic cell lines of metabolic modulating drugs including metformin, cycloheximide, mitochondrial ETC inhibitors (antimycin A, metformin), dichloroacetate, and imipridones (ONC201, ONC206) on mitochondrial function and cell viability, individually and combined under various nutrient conditions across our lines. Results confirmed osteosarcoma cells are more dependent on glucose than osteoblasts but also require mitochondrial function for survival, highlighting the therapeutic potential of metabolic pathways. Osteosarcoma cell viability was reduced when any metabolic drug treatment was combined with conditions forcing reliance on mitochondrial OXPHOS capacity. Combination metabolic therapies, particularly ONC201/ONC206/metformin in 143B cells, and to a lesser extent DCA and ONC201 with either ONC206 or antimycin A, showed enhanced cytotoxicity compared to single agents, with a good therapeutic index based on minimal toxicity to normal osteoblast cells. The degree of effectiveness varied across cell lines, underscoring the importance of personalized treatment strategies. RNA-Seq transcriptome analysis revealed that effective nutrient and metabolic drug treatments triggered widespread regulatory changes in osteosarcoma cells involving increased translation/splicing with decreased mitochondrial processes such as cholesterol biosynthesis. These results demonstrate the utility of developing combined metabolic and chemotherapeutic treatments for osteosarcoma.

cancer biology↗

bioRxiv2026

Cognate amino acid therapies provide preclinical benefit in 19 C. elegans models of ARS2 deficiency

Mitochondrial aminoacyl-tRNA synthetases (mt-ARS) are essential mitochondrial translation machinery components that catalyze mitochondrial transfer RNAs (tRNAs) charging with their cognate amino acid. Although mt-ARS have a common biochemical function, patients with mt-ARS pathogenic variants commonly develop neurological disorders with varying phenotypes, severity spectrum, and age of onset. Cognate amino acid supplementation has shown reported benefits in select cases of both mt-ARS (ARS2) and cytosolic (ARS1) deficiencies, although the safety and potential benefits of this candidate therapy approach across the full spectrum of mt-ARS disorders remain unclear. Here, C. elegans models were systematically generated for all 19 mitochondrial mt-ARS genes by feeding RNAi knockdown for one or two generations. mt-ARS deficient animals at baseline and upon cognate amino acid treatment were studied at the level of linear growth, neuromuscular activity, lifespan, mitochondrial physiology, and fertility. Results demonstrated that cognate amino acid treatment in a dose-dependent fashion consistently improved worm linear growth and neuromuscular activity, and reduced mitochondrial unfolded protein response stress, in all 19 knockdown models. It further rescued impaired fertility of hars-1 and fars-2 knockdown strains. Collectively, these preclinical studies provide compelling evidence to warrant future cognate amino acid treatment study in rigorous clinical trials spanning all human mt-ARS deficiencies.

physiology↗

bioRxiv2025

Trainable computation in molecular networks

Reports of learning in single cells without genetic change span decades yet remain controversial, in part because there is no accepted general molecular mechanism for training comparable to gradient-based training or Hebbian learning in neural circuits. Here we identify a minimal set of ingredients sufficient to realize non-genetic learning, drawing inspiration from Boltzmann neural networks. First, dense reversible interaction networks provide an expressive substrate in which modulating the concentrations of a small set of mediator species can reprogram function without altering the underlying interaction parameters. Second, a simple rate-sensitive autoregulatory scheme that adjusts these mediator levels provides a local Hebbian-like training rule that can train the same network for diverse tasks, including Pavlovian conditioning, supervised classification, and generative tuning of bet-hedging ratios to match environmental statistics. We show that this autoregulatory training rule is model free and applies to reversible multimerization networks of arbitrary complexity, so training can compensate for unknown or unmodeled interactions present in vivo. These results suggest design principles for trainable synthetic cellular circuits and indicate how molecular systems could learn statistical features of their environments through experience.

systems biology↗

bioRxiv2025

ndufs2-/- zebrafish have impaired survival, neuromuscular activity, morphology, and one-carbon metabolism treatable with folic acid

Mitochondrial complex I (CI) deficiency represents a common biochemical pathophysiology underlying Leigh syndrome spectrum (LSS), manifesting with progressive multi-system dysfunction, lactic acidemia, and early mortality. To facilitate mechanistic studies and rigorous screening of therapeutic candidates for CI deficient LSS, we used CRISPR/Cas9 to generate an ndufs2-/- 16 bp deletion zebrafish strain. ndufs2-/- larvae exhibit markedly reduced survival, severe neuromuscular dysfunction including impaired swimming capacity, multiple morphologic malformations, reduced growth, hepatomegaly, uninflated swim bladder, yolk retention, small intestines, and small eyes and pupils with abnormal retinal ganglion cell layer. Transcriptome profiling of ndufs2-/- larvae revealed dysregulation of the electron transport chain, TCA cycle, fatty acid beta-oxidation, and one-carbon metabolism. Similar transcriptomic profiles were observed in ndufs2-/- missense mutant C. elegans (gas-1(fc21)) and two human CI-disease fibroblast cell lines stressed in galactose media. ndufs2-/- zebrafish had 80% reduced CI enzyme activity. Unbiased metabolomic profiling showed increased lactate, TCA cycle intermediates, and acyl-carnitine species. One-carbon metabolism associated pathway alterations appear to contribute to CI disease pathophysiology, as folic acid treatment rescued the growth defect and hepatomegaly in ndufs2-/-larvae. Overall, ndufs2-/- zebrafish recapitulate severe CI deficiency, complex metabolic pathophysiology, and relevant LSS neuromuscular and survival phenotypes, enabling future translational studies of therapeutic candidates.

pharmacology and toxicology↗

bioRxiv2024

Mitophagy modulation rescues single large-scale mitochondrial DNA deletion (SLSMD) disease symptoms in the C. elegans uaDf5 animal model.

Single large scale mitochondrial DNA (mtDNA) deletions (SLSMD) underlie a range of sporadic or maternally inherited primary mitochondrial diseases having significant morbidity and mortality, including Pearson syndrome, Kearns-Sayre Syndrome, or Chronic Progressive External Ophthalmoplegia. Therapeutic development has been hindered by limited existing knowledge on mtDNA quality control and a lack of SLSMD animal models. To address this challenge, we utilized the C. elegans heteroplasmic SLSMD strain, uaDf5, to objectively screen for potential therapies. As mitophagy modulation has been implicated in mtDNA homeostasis, we screened a library of mitophagy modulating compounds to determine their comparative effects to rescue mitochondrial unfolded protein (UPRmt) stress induction in in uaDf5 SLSMD worms. Interestingly, Thiamine was discovered to be an effective positive control, significantly reducing mitochondrial stress in this model. Two lead therapeutic candidates from the mitophagy library screen were Hemin and Celastrol (Tripterin). Celastrol is a mitophagy activating anti-inflammatory and metabolic modifying natural product derived compound, that rescued multiple fitness outcomes (thrashing, development, survival) and reduced the mitochondrial stress in uaDf5 animals in a mitophagy-dependent fashion. This study highlights the utility of the uaDf5 worm model to enable preclinical identification of therapeutic candidate leads for SLSMD-based heteroplasmic mtDNA diseases and identifies possible therapeutic candidates that serve as mitophagy modulators to improve health and specifically reduce heteroplasmy levels in SLSMD diseases.

cell biology↗

bioRxiv2024

N-Glycosylation of MRS2 balances aerobic and anaerobic energy production by reducing rapid mitochondrial Mg2+ influx in conditions of high glucose or impaired respiratory chain function

N-linked glycoproteins function in numerous biological processes, modulating enzyme activities as well as protein folding, stability, oligomerization, and trafficking. While N-glycosylation of mitochondrial proteins has been detected by untargeted MS-analyses, the physiological existence and roles of mitochondrial protein N-linked glycosylation remain under debate. Here, we report that MRS2, a mitochondrial inner membrane protein that functions as the high flux magnesium transporter, is N-glycosylated to various extents depending on cellular bioenergetic status. Both N-glycosylated and unglycosylated isoforms were consistently detected in mitochondria isolated from mouse liver, rat and mouse liver fibroblast cells (BRL 3A and AFT024, respectively) as well as human skin fibroblast cells. Immunoblotting of MRS2 showed it was bound to, and required stringent elution conditions to remove from, lectin affinity columns with covalently bound concanavalin A or Lens culinaris agglutinin. Following peptide:N-glycosidase F (PNGase F) digestion of the stringently eluted proteins, the higher Mr MRS2 bands gel-shifted to lower Mr and loss of lectin affinity was seen. BRL 3A cells treated with two different N-linked glycosylation inhibitors, tunicamycin or 6-diazo-5-oxo-L-norleucine, resulted in decreased intensity or loss of the higher Mr MRS2 isoform. To investigate the possible functional role of MRS2 N- glycosylation, we measured rapid Mg2+ influx capacity in intact mitochondria isolated from BRL 3A cells in control media or following treatment with tunicamycin or 6-diazo-5-oxo-L-norleucine. Interestingly, rapid Mg2+ influx capacity increased in mitochondria isolated from BRL 3A cells treated with either N-glycosylation inhibitor. Forcing reliance on mitochondrial respiration by treatment with either galactose media or the glycolytic inhibitor 2-deoxyglucose or by minimizing glucose concentration similarly reduced the N-glycosylated isoform of MRS2, with a correlated concomitant increase in rapid Mg2+ influx capacity. Conversely, inhibiting mitochondrial energy production in BRL 3A cells with either rotenone or oligomycin resulted in an increased fraction of N-glycosylated MRS2, with decreased rapid Mg2+ influx capacity. Collectively, these data provide strong evidence that MRS2 N-glycosylation is directly involved in the regulation of mitochondrial matrix Mg2+, dynamically communicating relative cellular nutrient status and bioenergetic capacity by serving as a physiologic brake on the influx of mitochondrial matrix Mg2+ under conditions of glucose excess or mitochondrial bioenergetic impairment.

biochemistry↗

bioRxiv2022

Tissue confinement regulates cell growth and size in epithelia

Cell proliferation is a central process in tissue development, homeostasis and disease. Yet how proliferation is regulated in the tissue context remains poorly understood. Here, we introduce a quantitative framework to elucidate how tissue growth dynamics regulate cell proliferation. We show that tissue growth causes confinement that suppresses cell growth; however, this confinement does not directly affect the cell cycle. This leads to uncoupling between rates of cell growth and division in epithelia and, thereby, reduces cell size. Division becomes arrested at a minimal cell size, which is consistent across diverse epithelia in vivo. Here, the nucleus approaches a volume limit set by the compacted genome. The loss of Cyclin D1-dependent cell size regulation results in an abnormally high nuclear-to-cytoplasmic volume ratio and DNA damage. Overall, we demonstrate how epithelial proliferation is regulated by the interplay between tissue confinement and cell size regulation. Highlights- In epithelia, regulation of cell growth and cycle are uncoupled - Cell growth is regulated by tissue-scale dynamics, which determine confinement - Cell volume in epithelial tissue is described by G1 sizer model with a tunable growth rate - Volume of cells in epithelial tissues is near a minimum set by genome size

biophysics↗