Heart failure is the leading cause of death in individuals with diabetes mellitus (DM), and no effective treatments exist to treat or prevent hyperglycemia-driven cardiomyopathy. Here, we demonstrate that high glucose triggers regulator of G protein signaling 6 (RGS6) up-regulation in both human and murine cardiomyocytes, the hearts of hyperglycemic mice, and cardiac tissue samples from individuals with heart failure and a history of diabetes. Modulation of RGS6 expression in isolated cardiomyocytes resulted in corresponding changes in the expression of the transcription factor Krüppel-like factor 4 (KLF4), a novel RGS6-interacting protein. Further, RGS6-dependent, KLF4-mediated suppression of microRNA 30e (miR-30e) increased expression of the pro-apoptotic miR-30e target Ca2+/calmodulin-dependent kinase II δ isoform (CaMKIIδ). Importantly, inhibition of either KLF4 or CaMKII or overexpression of miR-30e mitigated the deleterious impact of RGS6 overexpression on myocyte viability. Indeed, cardiac-specific RGS6 knockdown provided marked protection against hyperglycemia-driven oxidative stress, mitochondrial dysfunction, and activation of the intrinsic mitochondrial apoptosis pathway in the murine myocardium. Similarly, inhibition of KLF4 decreased cardiotoxicity resulting from viral overexpression of RGS6 in mouse heart. Thus, RGS6 is both necessary and sufficient to drive cardiac damage resulting from chronic elevations in blood glucose. Together, these data point to RGS6/KLF4 as key sources of pathogenic cardiac damage in individuals with DM.

Peptide-based self-assembled nanostructures offer great promise for targeted drug delivery due to their intrinsic biocompatibility, biodegradability, and structural tunability. However, their limited optical properties and lack of functional sites for selective targeting restrict their use in theranostics. Here, we report a fluorophore-integrated, pH-responsive dipeptide nanocarrier engineered from phenylalanine-tryptophan (F-W) conjugated with 4-chloro-7-nitrobenzofurazan (NBD) as a fluorescent probe and vitamin B6 (VitB6) as a pH-sensitive unit. The resulting vitamin B6-modified nanoparticles (PS-Dox) exhibited charge reversal from negative to positive under mildly acidic conditions (pH 5.0), promoting doxorubicin (Dox) release, endosomal escape, and nuclear localization. PS-Dox demonstrated enhanced cytotoxicity, DNA damage, and apoptosis induction in multiple cancer cell lines, while showing negligible toxicity toward non-malignant cardiomyocytes (AC-16 and H9C2). In vivo biodistribution and pharmacokinetic studies revealed increased tumour accumulation and superior tumour growth inhibition compared with Dox. Importantly, PS-mediated encapsulation effectively mitigated Dox-associated cardiotoxicity, a major limitation of conventional chemotherapy. Overall, this study establishes a vitamin B6-mediated, charge-reversible peptide nanocarrier as a biocompatible and efficient platform for targeted anticancer drug delivery, combining tumour-specific therapeutic efficacy with improved cardiac safety.

Cardiotoxicity remains a major limiting factor in the clinical implementation of anthracycline chemotherapy. Though the etiology of doxorubicin-dependent heart damage has yet to be fully elucidated, the ability of doxorubicin to damage DNA and trigger oxidative stress have been heavily implicated in the pathogenesis of chemotherapy-associated cardiomyopathy. Here, we demonstrate that fibronectin type III domain-containing protein 5 (FNDC5), the precursor protein for myokine irisin, is depleted in the hearts of human cancer patients or mice exposed to chemotherapeutics. In cardiomyocytes, restoration of FNDC5 expression was sufficient to mitigate reactive oxygen species (ROS) accumulation and apoptosis following doxorubicin exposure, effects dependent on the irisin encoding domain of FNDC5 as well as signaling via the putative irisin integrin receptor. Intriguingly, we identified two parallel signaling cascades impacted by FNDC5 in cardiomyocytes: the ROS-driven intrinsic mitochondrial apoptosis pathway and the ROS-independent Ataxia Telangiectasia and Rad3-Related Protein (ATR)/Checkpoint Kinase 1 (Chk1) pathway. In fact, FNDC5 forms a co-precipitable complex with Chk1 alluding to possible intracellular actions for this canonically membrane-associated protein. Whereas FNDC5 overexpression in murine heart was cardioprotective, introduction of FNDC5-targeted shRNA into the myocardium was sufficient to trigger Bax up-regulation, ATR/Chk1 activation, oxidative stress, cardiac fibrosis, loss of ventricular function, and compromised animal survival. The detrimental impact of FNDC5 depletion on heart function could be mitigated via treatment with a Chk1 inhibitor identifying Chk1 hyperactivity as a causative factor in cardiac disease. Though our data point to the potential clinical utility of FNDC5/irisin-targeted agents in the treatment of chemotherapy-induced cardiotoxicity, we also found significant down regulation in FNDC5 expression in the hearts of aged mice that attenuated the cardioprotective impacts of FNDC5 overexpression following doxorubicin exposure. Together our data underscore the importance of FNDC5/irisin in maintenance of cardiac health over the lifespan.

Design and development of functionalized and biocompatible drug delivery systems (DDS) for site-specific release of small molecules is emerging as a means to target disease sites while sparing healthy tissue. Cell penetrating short peptides capable of self-assembly and drug encapsulation represent one scaffold with which selective DDSs can be rationally designed due to their chemical diversity, biocompatibility, tunable bioactivity, ease of functionality, and high loading capacity. Herein, we designed and synthesized two tetra peptides, BOC-YWWD (PB1 with Trp-Trp-Sequence) and BOC-WYWD (PB2 without Trp-Trp sequence). Structural rigidification of these two peptides with Zn(II) in the self-assembled state were characterized by the density functional theory (DFT) method and demonstrated to shift self-assembly of their characteristic emission from the ultraviolet to visible range allowing for visualization of cellular entry. PB1-Zn, unlike PB2-Zn, exhibits cell penetrating capabilities and is photo and thermally stable and biocompatible. Self-assembled PB1-Zn effectively encapsulated the chemotherapeutic drug Doxorubicin (Dox) and facilitated intracellular drug delivery. To test the utility of PB1-Zn as a DDS, we chemically modified PB1-Zn with folic acid to target folate receptor α (FLOR1), commonly overexpressed on the surface of cancer cells. In HeLa cervical cancer cells, this chemical conjugation with folic acid significantly improved the ability of Dox to activate the pro-apoptotic DNA damage response and trigger oxidative stress and mitochondrial dysfunction critical for the cancer cell killing actions of the drug. However, PB1-Zn failed to facilitate Dox delivery into the lung cancer epithelial cell line, A549, which does not express high levels of FLOR1. Our results represent an important proof of concept describing the fabrication of fluorescent Zn(II) coordinated, self-assembled short peptides containing the sequential Trp-Trp unit that may be used to develop superior imaging reagents and site-specific DDSs.

The clinical efficacy of psychostimulant drugs, which target monoamine transporters, in treating attention-deficit/hyperactivity disorders (ADHDs) has stimulated interest on the role of transporter proteins like the dopamine (DA) transporter (DAT) in neurotransmission as well as the potential utility of DAT knockout organisms as models for neuropsychiatric disorders. Indeed, the study of DAT-deficient worms, flies, fish, mice, and rats has revealed a conserved role for DAT in the control of motor behavior as well as repetitive behavior, threat aversion, social behavior, and cognition in mammals. However, the disconnect between phenotypes observed in DAT-deficient model organisms and humans, which exhibit an early-onset syndrome characterized by Parkinsonism/dystonia and premature death, challenges the construct validity of DAT knockout models with respect to modeling neurobehavioral disorders. As an alternate approach, several groups have utilized coding variants in the SLC6A3 gene linked to psychiatric conditions, which display divergent molecular phenotypes. This chapter reviews the development and characterization of models of DAT gene deletion and mutation with a particular emphasis on comparing/contrasting the functional impact of DAT deficiency to DAT dysregulation triggered by neuropsychiatric disorder-linked DAT mutants in vivo. Ultimately, the study of DAT knockout and mutant models has revealed novel functions for DA in the mammalian brain, uncovered a dynamic interplay between the monoaminergic systems, highlighted sex differences in the DA system that determine the behavioral trajectory of DAT deregulation, and allowed for the screening of potential leads for therapeutics to treat disorders linked to aberrant dopaminergic neurotransmission.

Women are nearly twice as likely to be diagnosed with Alzheimer’s Disease (AD) over their lifetime. However, historically, preclinical studies utilizing AD rodent models to define new therapeutic targets in AD treatment have neglected to consider the confounding influence of subject sex leading to a lack of mechanistic insight into the biological underpinnings of sex bias in AD. Here, we tracked choice of subject sex over the twenty-year history of the 5xFAD mouse, one of the most frequently cited pre-clinical AD models. We analyzed 1,330 primary research articles indexed on PubMed and recorded information provided regarding subject sex and/or as a rationale for not including datasets separated by sex, if noted. Trends were then plotted as a function of time ending in December 2024. In the last 15 years, the number of published manuscripts on the 5xFAD model omitting information on subject sex has progressively declined. However, the proportion of studies utilizing either males only (29

Aberrant dopamine (DA) signaling is implicated in schizophrenia, bipolar disorder (BPD), autism spectrum disorder (ASD), substance use disorder, and attention-deficit/hyperactivity disorder (ADHD). Treatment of these disorders remains inadequate, as exemplified by the therapeutic use of d-amphetamine and methylphenidate for the treatment of ADHD, agents with high abuse liability. In search for an improved and non-addictive therapeutic approach for the treatment of DA-linked disorders, we utilized a preclinical mouse model expressing the human DA transporter (DAT) coding variant DAT Val559, previously identified in individuals with ADHD, ASD, or BPD. DAT Val559, like several other disease-associated variants of DAT, exhibits anomalous DA efflux (ADE) that can be blocked by d-amphetamine and methylphenidate. Kappa opioid receptors (KORs) are expressed by DA neurons and modulate DA release and clearance, suggesting that targeting KORs might also provide an alternative approach to normalizing DA-signaling disrupted by perturbed DAT function. Here we demonstrate that KOR stimulation leads to enhanced surface trafficking and phosphorylation of Thr53 in wildtype DAT, effects achieved constitutively by the Val559 mutant. Moreover, these effects can be rescued by KOR antagonism of DAT Val559 in ex vivo preparations. Importantly, KOR antagonism also corrected in vivo DA release as well as sex-dependent behavioral abnormalities observed in DAT Val559 mice. Given their low abuse liability, our studies with a construct valid model of human DA associated disorders reinforce considerations of KOR antagonism as a pharmacological strategy to treat DA associated brain disorders.

The nitrogen mustard alkylating agent chlorambucil (CBL) is a critical component of chemotherapeutic regimens used in the treatment of chronic lymphocytic leukemia. The cancer cell-killing actions of CBL are limited by glutathione (GSH) conjugation, a process catalyzed by the GSH transferase hGSTA1-1 that triggers CBL efflux from cells. In the cancer cell microenvironment, intracellular GSH levels are elevated to counterbalance oxidative stress generated due to the high glycolytic demand. As many chemotherapeutic drugs trigger cell death through mechanisms that depend on reactive oxygen species (ROS), antioxidant capacity in cancer cells also represents a barrier to anticancer therapies. Here, we demonstrate that a heightened GSH content in cancer cells can also be exploited for cell-selective drug delivery. We successfully synthesized a malononitrile conjugate terpyridine-based derivative L1, which specifically reacts with GSH in the presence of other biologically relevant amino acids including cysteine (Cys) and homocysteine (Hcy). The significant change in the electronic spectra of L1 in the presence of GSH confirmed GSH detection, which was further corroborated by density functional theory calculations. We next encapsulated CBL into L1-containing, anthracene-functionalized, and 10,12-pentacosadiynoic acid (PCDA)- and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-based liposomes (Lip-CBL-L1). We established successful CBL encapsulation and release from L1-containing liposomes in GSH-enriched cancer cells in vitro. Both Lip-CBL-L1 and the L1-lacking Lip-CBL control displayed cell-killing activity. However, human triple-negative breast cancer cells MDAMB231, human lung cancer cells A549, and murine leukemic WEHI cells were more sensitive to the cytotoxic effects of Lip-CBL-L1 compared to the nonmalignant cells (AC16 and HEK293). Indeed, in these cancer cell lines, Lip-CBL-L1 induced greater ROS generation compared to that of Lip-CBL. Together, our results provide initial evidence of the feasibility of exploiting the unique oxidant environment of cancer cells for optimized drug delivery.

Lip-DT represents an important proof-of-concept exercise demonstrating that functionalized PCDA/DMPC-based liposomes have great promise for the development of new imaging probes and targeted drug-delivery vehicles.

Fabrication of stimuli -responsive superstructure capable of delivering chemotherapeutics directly to the cancer cell by sparing healthy cells is crucial. Herein, we developed redox-responsive hollow spherical assemblies through self -assembly of disulfide -linked cysteine-diphenylalanine (SN). These fluorescent hollow spheres display intrinsic green fluorescence, are proteolytically stable and biocompatible, and allow for real-time monitoring of their intracellular entry. The disulfide bond facilitates selective degradation in the presence of high glutathione (GSH) concentrations, prevalent in cancer cells. We achieved efficient encapsulation (68.72%) of the anticancer drug doxorubicin (Dox) and demonstrated GSH-dependent, redox-responsive drug release within cancerous cells. SN-Dox exhibited a 20 -fold lower effective concentration (2.5 m M) for compromising breast cancer cell viability compared to non-malignant cells (50 m M). The ability of SN-Dox to initiate DNA damage signaling and trigger apoptosis was comparable to that of the unencapsulated drug. Our findings highlight the potential of SN for creating site -specific drug delivery vehicles for sustained therapeutic release.

Background Prior evidence demonstrated that Regulator of G protein Signaling 6 (RGS6) translocates to the nucleolus in response to cytotoxic stress though the functional significance of this phenomenon remains unknown. Methods Utilizing in vivo gene manipulations in mice, primary murine cardiac cells, human cell lines and human patient samples we dissect the participation of a RGS6-nucleolin complex in chemotherapy-dependent cardiotoxicity. Results Here we demonstrate that RGS6 binds to a key nucleolar protein, Nucleolin, and controls its expression and activity in cardiomyocytes. In the human myocyte AC-16 cell line, induced pluripotent stem cell derived cardiomyocytes, primary murine cardiomyocytes, and the intact murine myocardium tuning RGS6 levels via overexpression or knockdown resulted in diametrically opposed impacts on Nucleolin mRNA, protein, and phosphorylation.RGS6 depletion provided marked protection against nucleolar stress-mediated cell death in vitro, and, conversely, RGS6 overexpression suppressed ribosomal RNA production, a key output of the nucleolus, and triggered death of myocytes. Importantly, overexpression of either Nucleolin or Nucleolin effector miRNA-21 counteracted the pro-apoptotic effects of RGS6. In both human and murine heart tissue, exposure to the genotoxic stressor doxorubicin was associated with an increase in the ratio of RGS6/Nucleolin. Preventing RGS6 induction via introduction of RGS6-directed shRNA via intracardiac injection proved cardioprotective in mice and was accompanied by restored Nucleolin/miRNA-21 expression, decreased nucleolar stress, and decreased expression of pro-apoptotic, hypertrophy, and oxidative stress markers in heart. Conclusion Together, these data implicate RGS6 as a driver of nucleolar stress-dependent cell death in cardiomyocytes via its ability to modulate Nucleolin. This work represents the first demonstration of a functional role for an RGS protein in the nucleolus and identifies the RGS6/Nucleolin interaction as a possible new therapeutic target in the prevention of cardiotoxicity.

Aims: The pathophysiological mechanism(s) underlying non-alcoholic fatty liver disease (NAFLD) have yet to be fully delineated and only a single drug, peroxisome proliferator-activated receptor (PPAR) α/γ agonist saroglitazar, has been approved. Here, we sought to investigate the role of Regulator of G Protein Signaling 7 (RGS7) in hyperlipidemia-dependent hepatic dysfunction. Results: RGS7 is elevated in the livers of NAFLD patients, particularly those with severe hepatic damage, pronounced insulin resistance, and high inflammation. In the liver, RGS7 forms a unique complex with transcription factor ATF3 and histone acetyltransferase Tip60, which is implicated in NAFLD. The removal of domains is necessary for ATF3/Tip60 binding compromises RGS7-dependent reactive oxygen species generation and cell death. Hepatic RGS7 knockdown (KD) prevented ATF3/Tip60 induction, and it provided protection against fibrotic remodeling and inflammation in high-fat diet-fed mice translating to improvements in liver function. Hyperlipidemia-dependent oxidative stress and metabolic dysfunction were largely reversed in RGS7 KD mice. Interestingly, saroglitazar failed to prevent RGS7/ATF3 upregulation but it did partially restore Tip60 levels. RGS7 drives the release of particularly tumor necrosis factor α (TNFα) from isolated hepatocytes, stellate cells and its depletion reverses steatosis, oxidative stress by direct TNFα exposure. Conversely, RGS7 overexpression in the liver is sufficient to trigger oxidative stress in hepatocytes that can be mitigated via TNFα inhibition. Innovation: We discovered a novel non-canonical function for an R7RGS protein, which usually functions to regulate G protein coupled receptor (GPCR) signaling. This is the first demonstration for a functional role of RGS7 outside the retina and central nervous system. Conclusion: RGS7 represents a potential novel target for the amelioration of NAFLD. Antioxid. Redox Signal. 38, 137-159.

Aberrant dopamine (DA) signaling has been implicated in various neuropsychiatric disorders, including attention-deficit/hyperactivity disorder (ADHD), autism spectrum disorder (ASD), schizophrenia, bipolar disorder (BPD) and addiction. The availability of extracellular DA is sculpted by exocytotic release of vesicular DA and subsequent transporter-mediated clearance, rendering the presynaptic DA transporter (DAT) a crucial regulator of DA neurotransmission. D2-type DA autoreceptors (D2ARs) regulate multiple aspects of DA homeostasis, including i) DA synthesis, ii) vesicular release, iii) DA neuron firing, and iv), the surface expression of DAT and DAT- mediated DA clearance. The DAT Val559 variant, identified in boys with ADHD or ASD, as well as in a girl with BPD, supports anomalous DA efflux (ADE), that we have shown drives tonic activation of D2ARs. Through ex vivo and in vivo studies of the DAT Val559 variant using transgenic knock-in mice, we have uncovered a circuit and sex-specific capacity of D2ARs to regulate DAT, which consequently disrupts DA signaling and behavior differently in males and females. Our studies reveal the ability of the construct valid DAT Val559 model to elucidate endogenous mechanisms that support DA signaling, findings that may be of translational and/or therapeutic importance.