Faculty Bibliography

Although many internalized G protein-coupled receptors (GPCRs) continue to signal, the mechanisms and outcomes of intracellular GPCR signaling are uncertain due to the challenges of measuring organelle-specific signals and of selectively antagonizing receptors in intracellular compartments. Herein, genetically encoded biosensors targeted to the plasma membrane and early endosomes were used to analyze compartmentalized signaling of protease-activated receptor 2 (PAR₂); the propensity of nanoparticles (NPs) to accumulate in endosomes was leveraged to preferentially antagonize intracellular PAR₂ signaling of pain. PAR₂ agonists evoked sustained activation of PAR₂, Gαq, and β-arrestin-1 in early endosomes and activated extracellular signal regulated kinase (ERK) in the cytosol and nucleus, measured with targeted biosensors. Fluorescent dendrimer and core-shell polymeric NPs accumulated in endosomes of HEK293T cells, colonic epithelial cells, and nociceptors, detected by confocal microscopy. NPs efficiently encapsulated and slowly released AZ3451, a negative allosteric PAR₂ modulator. NP-encapsulated AZ3451, but not unencapsulated AZ3451, rapidly and completely reversed PAR₂, Gαq, and β-arrestin-1 activation in early endosomes and ERK activation in the cytosol and nucleus. When administered into the mouse colon lumen, fluorescent dendrimer NPs accumulated in endosomes of colonocytes and polymeric NPs accumulated in neurons, sites of PAR₂ expression. Both NP formulations of AZ3451, but not unencapsulated AZ3451, caused long-lasting analgesia and normalized aberrant behavior in preclinical models of inflammatory bowel disease. These results provide evidence that PAR₂ endosomal signaling mediates pain and that nanomedicines that antagonize PAR₂ in endosomes effectively relieve pain. NP-mediated delivery may improve the efficacy of other GPCR antagonists for treatment of diverse diseases.

BACKGROUND:Glaucoma Research Foundation's third Catalyst for a Cure team (CFC3) was established in 2019 to uncover new therapies for glaucoma, a leading cause of blindness. In the 2021 meeting "Solving Neurodegeneration," (detailed in Mol Neurodegeneration 17(1), 2022) the team examined the failures of investigational monotherapies, issues with translatability, and other significant challenges faced when working with neurodegenerative disease models. They emphasized the need for novel, humanized models and proposed identifying commonalities across neurodegenerative diseases to support the creation of pan-neurodegenerative disease therapies. Since then, the fourth Catalyst for a Cure team (CFC4) was formed to explore commonalities between glaucoma and other neurodegenerative diseases. This review summarizes outcomes from the 2023 "Solving Neurodegeneration 2" meeting, a forum for CFC3 and CFC4 to share updates, problem solve, plan future research collaborations, and identify areas of unmet need or opportunity in glaucoma and the broader field of neurodegenerative disease research. MAIN BODY/METHODS:We summarize the recent progress in the field of neurodegenerative disease research and present the newest challenges and opportunities moving forward. While translatability and disease complexity continue to pose major challenges, important progress has been made in identifying neuroprotective targets and understanding neuron-glia-vascular cell interactions. New challenges involve improving our understanding of the disease microenvironment and timeline, identifying the optimal approach(es) to neuronal replacement, and finding the best drug combinations and synergies for neuroprotection. We propose solutions to common research questions, provide prescriptive recommendations for future studies, and detail methodologies, strategies, and approaches for addressing major challenges at the forefront of neurodegenerative disease research. CONCLUSIONS:This review is intended to serve as a research framework, offering recommendations and approaches to validating neuroprotective targets, investigating rare cell types, performing cell-specific functional characterizations, leveraging novel adaptations of scRNAseq, and performing single-cell sorting and sequencing across neurodegenerative diseases and disease models. We focus on modeling neurodegeneration using glaucoma and other neurodegenerative pathologies to investigate the temporal and spatial dynamics of neurodegenerative disease pathogenesis, suggesting researchers aim to identify pan-neurodegenerative drug targets and drug combinations leverageable across neurodegenerative diseases.

Theories propose that speech production can be approximated as a temporal reversal of speech perception. For example, phonological code is assumed to precede phonetic encoding in the motor system during speech production. However, empirical neural evidence directly testing the temporal order hypothesis remains scarce, mostly because of motor artifacts in non-invasive electrophysiology recordings and the requirements of both temporal and spatial precision. In this study, we investigated the neural dynamics of speech production using stereotactic electroencephalography (sEEG). In both onset latency analysis and representational similarity analysis (RSA), activation in the auditory region of the posterior superior temporal gyrus (pSTG) was observed before articulation, suggesting the availability of auditory phonological code before production. Surprisingly, the activation in the motor region of the inferior frontal gyrus (IFG) preceded that of pSTG, suggesting that the phonological code in the auditory domain may not necessarily be activated before the encoding in the motor domain during speech production.

Mothers exhibit an increased appetite to cope with the energetic demands of lactation. A new study has identified a neural circuit that interfaces between food seeking and pup caring.

By improving the delivery and tumor retention of chemotherapeutics, nanomedicines hold potential for cancer treatment. The usefulness of nanoparticle (NP)-encapsulated analgesics for the cancer pain treatment is comparatively unexplored. We investigated whether NPs encapsulating olcegepant (OCP), an antagonist of the calcitonin receptor-like receptor (CLR) for the calcitonin gene-related peptide (CGRP), effectively relieved oral cancer pain in mice. Because persistent endosomal CLR signaling in Schwann cells mediates craniofacial pain, we reasoned that the predisposition of NPs to accumulate in endosomes could be leveraged to effectively relieve oral cancer pain. By expressing biosensors for activated CLR, Gα proteins and β-arrestins in HEK293T and Schwann cells, we found that CGRP activates CLR signaling first at the plasma membrane and then in early, late and recycling endosomes and the cis- and trans-Golgi apparatus. We synthesized biocompatible NPs encapsulating OCP and fluorophores by integrating hydrophobic ion pairing nanoformulation with Flash NanoPrecipitation. NPs slowly released OCP and accumulated in early endosomes, leading to sustained inhibition of endosomal CLR signaling in HEK293T and Schwann cells. Oral cancers were established in mice, which led to heightened pain-like responses. After intra-tumoral injection, NPs were retained in tumors for at least one week. OCP-loaded NPs almost completely reversed allodynia and hyperalgesia for a prolonged period, whereas unencapsulated OCP had small and transient effects. The NP accumulation in endosomal sites of pain signaling, the sustained release of antagonist, and the retention of NPs in tumors explain their beneficial actions. Thus, NP-encapsulation holds promise for the relief of painful cancers that are inadequately treated by opioids.

OBJECTIVES/OBJECTIVE:Magnetization-prepared rapid gradient-echo (MP-RAGE) sequences are routinely acquired for brain exams, providing high conspicuity for enhancing lesions. Vessels, however, also appear bright, which can complicate the detection of small lesions. T1RESS (T1 relaxation-enhanced steady-state) sequences have been proposed as an alternative to MP-RAGE, offering improved lesion conspicuity and suppression of blood vessels. This work aims to evaluate the performance of radial T1RESS variants for motion-robust contrast-enhanced brain MRI. MATERIALS AND METHODS/METHODS:Radial stack-of-stars sampling was implemented for steady-state free-precession-based rapid T1RESS acquisition with saturation recovery preparation. Three variants were developed using a balanced steady-state free-precession readout (bT1RESS), an unbalanced fast imaging steady precession (FISP) readout (uT1RESS-FISP), and an unbalanced reversed FISP readout (uT1RESS-PSIF). Image contrast was evaluated in numerical simulations and phantom experiments. The motion robustness of radial T1RESS was demonstrated with a motion phantom. Four patients and six healthy volunteers were scanned at 3 T and 0.55 T. Extensions were developed combining T1RESS with GRASP for dynamic imaging, with GRAPPA for accelerated scans, and with Dixon for fat/water separation. RESULTS:In simulations and phantom scans, uT1RESS-FISP provided higher signal intensity for regions with lower T1 values (<500 ms) compared with MP-RAGE. In motion experiments, radial uT1RESS-FISP showed fewer artifacts than MP-RAGE and Cartesian uT1RESS-FISP. In patients, both unbalanced uT1RESS variants provided higher lesion conspicuity than MP-RAGE. Blood vessels appeared bright with MP-RAGE, gray with uT1RESS-FISP, and dark with uT1RESS-PSIF. At 0.55 T, bT1RESS provided high signal-to-noise ratio T1-weighted images without banding artifacts. Lastly, dynamic T1RESS images with a temporal resolution of 10.14 seconds/frame were generated using the GRASP algorithm. CONCLUSIONS:Radial T1RESS sequences offer improved lesion conspicuity and motion robustness and enable dynamic imaging for contrast-enhanced brain MRI. Both uT1RESS variants showed higher tumor-to-brain contrast than MP-RAGE and may find application as alternative techniques for imaging uncooperative patients with small brain lesions.

INTRODUCTION/BACKGROUND:The New York University Caregiver Intervention plus Enhanced Support Project is a randomized controlled trial of a family-based psychosocial intervention to enhance social support and reduce cardiometabolic risk for Chinese and Korean American dementia caregivers, using culturally tailored recruitment strategies. METHODS:We reviewed reflections from research staff, weekly meeting minutes, debriefing sessions, and progress reports, to identify key challenges and approaches to engaging participants. RESULTS:Key challenges included reluctance to involve family members, dementia stigma, and resistance to involving family. In response, we engaged online communities, partnered with local organizations, participated in events, and adapted recruitment messages to cultural norms. For the Chinese community, we focused on practical skills while for the Korean community, we emphasized caregiving strategies and the personal/social benefits of participation, reducing rejection rates. DISCUSSION/CONCLUSIONS:Our findings underscore the importance of culturally tailored recruitment strategies in dementia research. Respectful, sensitive, and culturally informed approaches can significantly enhance engagement and participation. HIGHLIGHTS/CONCLUSIONS:Culturally adapted recruitment strategies improve study engagement with Chinese and Korean American dementia caregivers. Community partnerships with local social services agencies are essential for recruitment success. Culturally relevant social media applications were integrated to increase accessibility for study participants. This study uniquely targets and recruits Chinese and Korean American dementia caregivers with metabolic syndrome-related symptoms, incorporating a psychological intervention alongside biomarker data collection. The iterative adaptation of recruitment methods and tailored messaging to specific ethnic groups ensure the intervention is culturally aligned, enhancing both participation and relevance to the caregivers' unique health and caregiving contexts.

Recent discoveries reveal that post-translational modifications (PTMs) do more than regulate protein activity - they encode conformational states that transform chaperones into epichaperomes: multimeric scaffolds that rewire protein-protein interaction networks. This emerging paradigm expands the framework of chaperone biology in disease and provides a structural basis for systems-level dysfunction in disorders such as cancer and Alzheimer's disease. This review explores how PTMs within intrinsically disordered regions drive epichaperome formation, how these scaffolds selectively regulate disease-enabling functions, and why their disruption normalizes pathological networks. By highlighting PTMs as molecular encoders of supramolecular assemblies, we propose a shift from targeting proteins to targeting network architectures that sustain and perpetuate disease - a concept with broad implications for cell biology, disease propagation, and therapeutic design.

Pooled single-cell CRISPR screens have profiled either gene expression or chromatin accessibility but not both modalities. Here we develop MultiPerturb-seq, a high-throughput CRISPR screening platform with joint single-nucleus chromatin accessibility, transcriptome and guide RNA capture using combinatorial indexing combined with droplet microfluidics to scale throughput and integrate all three modalities. We identify key differentiation genes in a rare pediatric cancer and establish ZNHIT1 as a potential target for cancer reprogramming therapy.

Tau protein truncated at Asp 421 is a characteristic feature of Alzheimer's disease and other tauopathies. Here, we show that a monoclonal antibody against Asp421, 5G2, cleared insoluble tau in the brains of JNPL3 mice, decreased tau levels in brain interstitial fluid in awake JNPL3 mice, improved in vivo neuronal function, and reduced microglial Iba-1 expression in PS19 mice, in which neuronal tau aggregation and dysfunction occurred earlier than microglial activation. For mechanistic insight using culture models, 5G2 prevented tau-mediated toxicity, cleared extra- and intracellular tau, and prevented microgliosis. TRIM21 knockdown reduced neuronal retention of tau antibodies and their acute but not longer-term efficacy. Inhibition of the endosomal/lysosomal pathway but not the proteasomal pathway blocked 5G2-mediated neuroprotection and tau clearance. These findings support targeting the Asp421 truncated tau protein to treat tauopathies, indicate that tau-associated neuronal dysfunction precedes microglial activation, and that intraneuronal antibody-mediated tau clearance is mostly via the lysosomes.