Biology subjects

Mach, R. H.

Publications and source records attributed to Mach, R. H..

10 recordsLinked to original sources

bioRxiv2025

In vivo imaging of reactive oxygen species after myocardial ischemia-reperfusion injury: a large animal multimodal imaging and transcriptomic study

BackgroundReactive oxygen species (ROS) contribute to myocardial ischemia-reperfusion injury (IRI), but in-vivo data on the spatial myocardial distribution and systemic effects of ROS after IRI remain limited. This multimodal CMR and PET/CT study aimed to non-invasively image ROS activity in a clinically-relevant swine model of IRI using [18F]ROStrace, a fluorine-18-labeled analogue of dihydroethidium (DHE), and to investigate regional changes in ROS activity in the infarcted myocardium during the subacute post-IRI phase. MethodsIRI was induced by percutaneous occlusion of the left anterior descending artery for 90 minutes in swine (N=9). CMR and whole-body PET/CT imaging with [18F]ROStrace were performed before myocardial infarction (MI) and 3-5 days post-MI to assess ROS in non-infarct myocardium, lungs, bone marrow, spleen and skeletal muscle. Late gadolinium enhanced CMR was performed to structurally characterize infarct regions. Post-MI, in vivo [18F]ROStrace signal in infarcted myocardium was compared with remote, non-infarcted myocardium and validated via ex vivo DHE fluorescent imaging. Bulk RNA-sequencing (RNA-seq) and Gene Ontology pathway analysis were conducted on biopsies from infarct and remote myocardial tissue to identify differentially expressed genes and pathways connected to oxidative stress. ResultsDuring the subacute phase following MI, [18F]ROStrace fractional uptake rate (FUR; min-1) was significantly increased in skeletal muscle, compared to baseline (0.011{+/-}0.003 vs 0.016{+/-}0.005, p=0.04), with a trend toward increased FUR in bone marrow (0.046{+/-}0.009 vs 0.056{+/-}0.011, p=0.12) and the left ventricular free wall (0.067{+/-}0.007 vs 0.073{+/-}0.010, p=0.15). Within the myocardium, [18F]ROStrace FUR ((min-1)/(mL/min/g)) was significantly higher in infarcted compared to non-infarcted myocardium regions (0.110{+/-}0.034, vs 0.148{+/-}0.035, p=0.0005). DHE staining confirmed elevated ROS levels in the infarcted myocardium. RNA-seq identified 8,707 differentially expressed genes between infarct and remote myocardium, with downregulated pathways in the infarct associated with mitochondrial function, cellular respiration, and metabolic adaptation. ConclusionThis study demonstrated MI ROS imaging using [18F]ROStrace using a whole-body PET/CT scanner and structural assessment with CMR. Systemic and myocardial increases in ROS activity were observed post-MI, accompanied by substantial molecular alterations in infarcted tissue. These findings show potential imaging strategies to evaluate therapeutic targets that can mitigate oxidative stress after MI.

bioengineering↗

bioRxiv2025

Complementary Structural and Chemical Biology Methods Reveal the Basis for Selective Radioligand Binding to α-Synuclein in MSA Tissue

Fibrillar aggregation of -synuclein (Syn) is a hallmark of Parkinsons disease (PD) and related disorders, including multiple system atrophy (MSA) and dementia with Lewy bodies (DLB). Despite advances in Syn fibril structural characterization, the relevance of in vitro and ex vivo structures to patient aggregates remains unclear, particularly for developing therapeutic or diagnostic molecules. Cryo-electron microscopy (cryo-EM) studies of Syn fibrils with ligands often reveal binding at multiple sites, likely due to high ligand concentrations. Here, various structural and chemical biology techniques were used to characterize Syn fibrils in the presence of EX-6, a candidate ligand for positron emission tomography (PET) imaging of synucleinopathies. Transmission electron microscopy (TEM) and cryo-EM revealed no significant fibril core changes upon binding. Forster resonance energy transfer (FRET) further demonstrated that the disordered C-terminus was unaltered. Cryo-EM and crosslinking mass spectrometry (XL-MS) identified consistent binding sites, with one (Site 2*) providing a well-defined pocket for high-resolution analysis. Site 2* showed similar residue positioning in MSA patient-derived structures, suggesting MSA selectivity. [3H]-EX-6 binding assays demonstrated a 10-fold preference for MSA over PD tissue, with autoradiography further confirming MSA selectivity. Taken together, the combined use of structural and chemical biology techniques provides a comprehensive understanding of EX-6 binding that would not be possible with any single method. Optimization of ligand-protein and ligand-ligand interactions observed in the cryo-EM structure will enable the development of EX-6 as a PET imaging probe for MSA.

neuroscience↗

bioRxiv2024

A Small-molecule Antagonist Radiotracer for Positron Emission Tomography Imaging of the Mu Opioid Receptor.

The opioid crisis is a catastrophic health emergency catalyzed by the misuse of opioids that target and activate the mu opioid receptor. Traditional radioligands used to study the mu opioid receptor are often tightly regulated owing to their abuse and respiratory depression potential. In the present study, we sought to design and characterize a library of 24 non-agonist ligands for the mu opioid receptor. Ligands were evaluated for the binding affinity, intrinsic activity, and predicted blood-brain barrier permeability. Several ligands demonstrated single-digit nM binding affinity for the mu opioid receptor while also demonstrating selectivity over the delta and kappa opioid receptors. The antagonist behavior of 1A and 3A at the mu opioid receptor indicate that these ligands would likely not induce opioid-dependent respiratory depression. Therefore, these ligands can enable a safer means to interrogate the endogenous opioid system. Based on binding affinity, selectivity, and potential off-target binding, [11C]1A was prepared via metallophotoredox of the aryl-bromide functional group to [11C]methyl iodide. The nascent radiotracer demonstrated brain uptake in a rhesus macaque model and accumulation in the caudate and putamen. Naloxone was able to reduce [11C]1A binding, though the interactions were not as pronounced as naloxones ability to displace [11C]carfentanil. These results suggest that GSK1521498 and related congeners are amenable to radioligand design and can offer a safer way to query opioid neurobiology.

neuroscience↗

bioRxiv2022

Identification and Characterization of ML321: a Novel and Highly Selective D2 Dopamine Receptor Antagonist with Efficacy in Animal Models that Predict Atypical Antipsychotic Activity

We have developed and characterized a novel D2R antagonist with exceptional GPCR selectivity - ML321. In functional profiling screens of 168 different GPCRs, ML321 showed little activity beyond potent inhibition of the D2R, and to a lesser extent the D3R, demonstrating excellent receptor selectivity. The D2R selectivity of ML321 may be related to the fact that, unlike other monoaminergic ligands, ML321 lacks a positively charged amine group and adopts a unique binding pose within the orthosteric binding site of the D2R. PET imaging studies in non-human primates demonstrated that ML321 penetrates the CNS and occupies the D2R in a dose-dependent manner. Behavioral paradigms in rats demonstrate that ML321 can selectively antagonize a D2R-mediated response (hypothermia) while not affecting a D3R-mediated response (yawning) using the same dose of drug, thus indicating exceptional in vivo selectivity. We also investigated the effects of ML321 in animal models that are predictive of antipsychotic efficacy in humans. We found that ML321 attenuates both amphetamine- and phencyclidine-induced locomotor activity and restored pre-pulse inhibition (PPI) of acoustic startle in a dose-dependent manner. Surprisingly, using doses that were maximally effective in both the locomotor and PPI studies, ML321 was relatively ineffective in promoting catalepsy. Kinetic studies revealed that ML321 exhibits slow-on and fast-off receptor binding rates, similar to those observed with atypical antipsychotics with reduced extrapyramidal side effects. Taken together, these observations suggest that ML321, or a derivative thereof, may exhibit "atypical" antipsychotic activity in humans with significantly fewer side effects than observed with currently FDA-approved D2R antagonists.

pharmacology and toxicology↗

bioRxiv2022

ROStrace detects oxidative stress in vivo and predicts progression of Alzheimer's disease pathology in APP/PS1 mice.

PurposeOxidative stress is implicated in the pathogenesis of the most common neurodegenerative diseases, such as Alzheimers disease (AD). However, tracking oxidative stress in the brain has proven difficult and impeded its use as a biomarker. Herein, we investigate the utility of a novel positron emission tomography (PET) tracer, [18F]ROStrace, as a biomarker of oxidative stress throughout the course of AD in the well-established APP/PS1 double mutant mouse model. MethodsPET imaging studies were conducted in wild-type (WT) and APP/PS1 mice at 3 different time points, representing early (5 mo.), middle (10 mo.), and advanced (16 mo.) life (n = 6-12, per sex). Semi-quantitation SUVRs of the plateau phase (40-60min post-injection; SUVR40-60) of ten brain subregions were designated by the Mirrione atlas and analyzed by Pmod. Statistical parametric mapping (SPM) was used to distinguish brain regions with elevated ROS in APP/PS1 relative to WT in both sexes. The PET studies were validated by ex vivo autoradiography and immunofluorescence with the parent compound, dihydroethidium. Results[18F]ROStrace retention was increased in the APP/PS1 brain compared to age-matched controls by 10 mo. of age (p < 0.0001), and preceded the accumulation of oxidative damage in APP/PS1 neurons at 16mo. (p < 0.005). [18F]ROStrace retention and oxidative damages were higher and occurred earlier in female APP/PS1 mice as measured by PET (p < 0.001), autoradiography and immunohistochemistry (p < 0.05). [18F]ROStrace differences emerged mid-life, temporally and spatially correlating with increased A{beta} burden (r2 = 0.36; p = 0.0003), which was also greatest in the female brain (p < 0.001). Conclusions[18F]ROStrace identifies increased oxidative stress and neuroinflammation in APP/PS1 female mice, concurrent with increased amyloid burden mid-life. Differences in oxidative stress during this crucial time may partially explain the sexual dimorphism in AD. [18F]ROStrace may provide a long-awaited tool to stratify at-risk patients who may benefit from antioxidant therapy prior to irreparable neurodegeneration.

neuroscience↗

bioRxiv2021

Harnessing the Intrinsic Photochemistry of Isoxazoles for the Development of Chemoproteomic Crosslinking Methods

Photo-crosslinking is a powerful technique for identifying both coarse- and fine-grained information on protein binding by small molecules. However, the scope of useful functional groups remains limited, with most studies focusing on diazirine, aryl azide, or benzophenone-containing molecules. Here, we report a unique method for photo-crosslinking, employing the intrinsic photochemistry of the isoxazole, a common heterocycle in medicinal chemistry, to offer an alternative to existing strategies using more perturbing, extrinsic crosslinkers. In this initial report, this technique is applied both in vitro and ex vivo, used in a variety of common chemoproteomic workflows, and validated across multiple proteins, demonstrating the utility of isoxazole photo-crosslinking in a wide range of biologically relevant experiments.

biochemistry↗

bioRxiv2021

Differential gene expression by RNA-Seq in Sigma-2 Receptor/TMEM97 knockout cells reveals its role in complement activation and SARS-CoV-2 viral uptake

Our lab has recently shown that the Sigma-2 Receptor/Transmembrane Protein 97 (sigma-2R/TMEM97) interacts with the low-density lipoprotein receptor (LDLR) and facilitates the enhanced uptake of various ligands including lipoproteins and intrinsically disordered proteins. TMEM97 has been recently been shown to interact with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins, highlighting its potential involvement with viral entry into the cell. We hypothesized that sigma-2R/TMEM97 may play a role in facilitating viral uptake, and with the regulation of inflammatory and thrombotic pathways that are involved with viral infection. In this study, we identified the top differentially expressed genes upon the knockout of sigma-2R/TMEM97, and analyzed the genes involved with the inflammatory and thrombotic cascades, effects that are observed in patients infected with SARS-CoV-2. We found that the ablation of sigma-2R/TMEM97 resulted in an increase in Complement Component 4 Binding Protein (C4BP) proteins, at both the translational and transcriptional levels. We also showed that sigma-2R/TMEM97 interacts with the cellular receptor for SARS-CoV-2, the human angiotensin-converting enzyme 2 (ACE2) receptor, forming a protein complex, and that disruption of this complex results in the inhibition of viral uptake. The results of this study suggest that sigma-2R/TMEM97 may be a novel therapeutic target to inhibit SARS-CoV-2 viral uptake, as well as to decrease inflammatory and thrombotic effects through the modulation of the complement cascade.

cell biology↗

bioRxiv2020

ALC1 links chromatin accessibility to PARP inhibitor response in homologous recombination deficient cells

The response to Poly (ADP-ribose) polymerase inhibitors (PARPi) is dictated by homologous recombination (HR) DNA repair mechanisms and the abundance of lesions that trap PARP enzymes on chromatin. It remains unclear, however, if the established role of PARP in promoting chromatin accessibility impacts viability in these settings. Using a CRISPR based screen, we identify the PAR-binding Snf2-like ATPase, ALC1/CHD1L, as a key determinant of PARPi toxicity in HR-deficient cells. ALC1 loss reduced viability of BRCA mutant cells and enhanced their sensitivity to PARPi by up to 250-fold, while overcoming several known resistance mechanisms. ALC1 loss was not epistatic to other repair pathways that execute the PARPi response. Instead, ALC1 deficiency reduced chromatin accessibility concomitant with a decrease in the association of repair factors. This resulted in an accumulation of replication associated DNA damage and a reliance on HR. These findings establish PAR-dependent chromatin remodeling as a mechanistically distinct aspect of PARPi responses, implicating ALC1 inhibition as a new approach to overcome therapeutic resistance in HR-deficient cancers.

cancer biology↗

bioRxiv2020

Poly(ADP-ribose) induces α-synuclein aggregation in neuronal-like cells andinteracts with phosphorylated α-synuclein in post mortem PD samples

BackgroundPoly (ADP-ribose) (PAR) is a negatively charged polymer that is biosynthesized by Poly (ADP-ribose) Polymerase-1 (PARP-1) and regulates various cellular processes. Alpha-synuclein (Syn) is an intrinsically disordered protein (IDP) that has been directly implicated with driving the onset and progression of Parkinsons disease (PD). The mechanisms by which Syn elicits its neurotoxic effects remain unclear. Recent findings indicate that one of the key processes driving PD pathology are oligomeric species of Syn. Furthermore, it is well established that the main components of Lewy bodies (LBs) and Lewy neurites (LNs) in PD patients are aggregated hyperphosphorylated (S129) forms of Syn (pSyn). MethodsWe used biochemical and immunofluorescence-based assays to explore if PARP-1 enzymatic product (PAR) drives the conversion of monomeric Syn into aggregated assemblies. We performed quantitative measurements using in situ proximity ligation assays (PLA) on a transgenic murine model of -synucleinopathy (M83-SNCA*A53T) and post-mortem PD/PDD patient samples to characterize PAR-pSyn interactions. Additionally, we used bioinformatic approaches and site-directed mutagenesis to identify PAR-binding regions on fibrillar Syn. ResultsOur studies show that elevated intracellular levels of PAR promote the transition of Syn into higher molecular weight forms. We report that PAR-pSyn interactions are predominant in pathological states. Moreover, we confirm that the interactions between PAR and Syn involve electrostatic forces between negatively charged PAR and lysine residues on the N-terminal region of Syn. ConclusionsPAR plays a critical role in the early stages of monomeric Syn aggregation, thereby attributing to PD pathogenesis. Based on our results, we report that PAR seeds monomeric Syn aggregation and directly interacts with phosphorylated Syn in conditions that are pathologically relevant to PD.

neuroscience↗

bioRxiv2020

The Sigma-2 Receptor/TMEM97, PGRMC1, and LDL Receptor complex are responsible for the cellular uptake of Aβ42 and its protein aggregates

BackgroundOur lab has recently shown that the Sigma-2 Receptor/Transmembrane Protein 97 (TMEM97) and Progesterone Receptor Membrane Component 1 (PGRMC1) form a complex with the Low Density Lipoprotein Receptor (LDLR), and this intact complex is required for efficient uptake of lipoproteins such as LDL and apolipoprotein E (apoE). These receptors are expressed in the nervous system where they have implications in neurodegenerative diseases such as Alzheimers Disease (AD), where apoE is involved in neuronal uptake and accumulation of A{beta}42, eventually cascading into neurodegeneration, synaptic dysfunction, and ultimately, dementia. HypothesisWe hypothesize that the intact Sigma-2 receptor complex -TMEM97, PGRMC1, and LDLR-- is necessary for internalization of apoE and A{beta}42 monomers (mA{beta}42) and oligomers (oA{beta}42), and the disruption of the receptor complex inhibits uptake. ResultsThe results of this study suggest that the intact Sigma-2 receptor complex is a binding site for mA{beta}42 and oA{beta}42, in the presence or absence of apoE2, apoE3, and apoE4. The loss or pharmacological inhibition of one or both of these proteins results in the disruption of the complex leading to decreased uptake of mA{beta}42 and oA{beta}42 and apoE in primary neurons. ConclusionThe TMEM97, PGRMC1, and LDLR complex is a pathway for the cellular uptake of A{beta}42 via apoE dependent and independent mechanisms. This study suggests that the complex may potentially be a novel pharmacological target to decrease neuronal A{beta}42 internalization and accumulation, which may represent a new strategy for inhibiting the rate of neurotoxicity, neurodegeneration, and progression of AD.

neuroscience↗