Publications

Hypoxic-ischemic (HI) brain injury is a common neurological problem in neonates. The postsynaptic density protein-95 (PSD-95) is an excitatory synaptic scaffolding protein that regulates synaptic function, and represents a potential therapeutic target to attenuate HI brain injury. Syn3 and d-Syn3 are novel high affinity cyclic peptides that bind the PDZ3 domain of PSD-95. We investigated the neuroprotective efficacy of Syn3 and d-Syn3 after exposure to HI in neonatal rodents. Postnatal (P) day-7 rats were treated with Syn3 and d-Syn3 at zero, 24, and 48-h after carotid artery ligation and 90-min of 8 % oxygen. Hemispheric volume atrophy and Iba-1 positive microglia were quantified by cresyl violet and immunohistochemical staining. Treatment with Syn3 and d-Syn3 reduced tissue volume loss by 47.0 % and 41.0 % in the male plus female, and by 42.1 % and 65.0 % in the male groups, respectively. Syn3 reduced tissue loss by 52.3 % in females. D-Syn3 reduced Iba-1 positive microglia/DAPI ratios in the pooled group, males, and females. Syn3 effects were observed in the pooled group and females. Changes in Iba-1 positive microglia/DAPI cellular ratios correlated directly with reduced hemispheric volume loss, suggesting that Syn3 and d-Syn3 provide neuroprotection in part by their effects on Iba-1 positive microglia. The pathogenic cis phosphorylated Thr231 in Tau (cis P-tau) is a marker of neuronal injury. Cis P-tau was induced in cortical cells of the placebo-treated pooled group, males and females after HI, and reduced by treatment with d-Syn3. Therefore, treatment with these peptidomimetic agents exert neuroprotective effects after exposure of neonatal subjects to HI related brain injury.

Induced oncoproteins degradation provides an attractive anti-cancer modality. Activation of anaphase-promoting complex (APC/CCDH1) prevents cell-cycle entry by targeting crucial mitotic proteins for degradation. Phosphorylation of its co-activator CDH1 modulates the E3 ligase activity, but little is known about its regulation after phosphorylation and how to effectively harness APC/CCDH1 activity to treat cancer. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1)-catalyzed phosphorylation-dependent cis-trans prolyl isomerization drives tumor malignancy. However, the mechanisms controlling its protein turnover remain elusive. Through proteomic screens and structural characterizations, we identify a reciprocal antagonism of PIN1-APC/CCDH1 mediated by domain-oriented phosphorylation-dependent dual interactions as a fundamental mechanism governing mitotic protein stability and cell-cycle entry. Remarkably, combined PIN1 and cyclin-dependent protein kinases (CDKs) inhibition creates a positive feedback loop of PIN1 inhibition and APC/CCDH1 activation to irreversibly degrade PIN1 and other crucial mitotic proteins, which force permanent cell-cycle exit and trigger anti-tumor immunity, translating into synergistic efficacy against triple-negative breast cancer.

Tauopathies are neurodegenerative diseases characterized by deposits of abnormal Tau protein in the brain. Conventional tauopathies are often defined by a limited number of Tau epitopes, notably neurofibrillary tangles, but emerging evidence suggests structural heterogeneity among tauopathies. The prolyl isomerase Pin1 isomerizes cis P-tau to inhibit the development of oligomers, tangles and neurodegeneration in multiple neurodegenerative diseases such as Alzheimer's disease, traumatic brain injury, vascular contribution to cognitive impairment and dementia (VCID) and preeclampsia (PE). Thus, cis P-tau has emerged as an early etiological driver, blood marker and therapeutic target for multiple neurodegenerative diseases, with clinical trials ongoing. The discovery of cis P-tau and other tau pathologies in VCID and PE calls attention for simplistic classification of tauopathy in neurodegenerative diseases. These recent advances have revealed the exciting novel role of the Pin1-cis P-tau axis in the development and treatment of vascular contribution to cognitive impairment and dementia and preeclampsia.

Catalysis and translocation of multisubunit DNA-directed RNA polymerases underlie all cellular mRNA synthesis. RNA polymerase II (Pol II) synthesizes eukaryotic pre-mRNAs from a DNA template strand buried in its active site. Structural details of catalysis at near-atomic resolution and precise arrangement of key active site components have been elusive. Here, we present the free-electron laser (FEL) structures of a matched ATP-bound Pol II and the hyperactive Rpb1 T834P bridge helix (BH) mutant at the highest resolution to date. The radiation-damage-free FEL structures reveal the full active site interaction network, including the trigger loop (TL) in the closed conformation, bonafide occupancy of both site A and B Mg2+, and, more importantly, a putative third (site C) Mg2+ analogous to that described for some DNA polymerases but not observed previously for cellular RNA polymerases. Molecular dynamics (MD) simulations of the structures indicate that the third Mg2+ is coordinated and stabilized at its observed position. TL residues provide half of the substrate binding pocket while multiple TL/BH interactions induce conformational changes that could allow translocation upon substrate hydrolysis. Consistent with TL/BH communication, a FEL structure and MD simulations of the T834P mutant reveal rearrangement of some active site interactions supporting potential plasticity in active site function and long-distance effects on both the width of the central channel and TL conformation, likely underlying its increased elongation rate at the expense of fidelity.

RNA polymerase II (Pol II) has a highly conserved domain, the trigger loop (TL), that controls transcription fidelity and speed. We previously probed pairwise genetic interactions between residues within and surrounding the TL for the purpose of understand functional interactions between residues and to understand how individual mutants might alter TL function. We identified widespread incompatibility between TLs of different species when placed in the Saccharomyces cerevisiae Pol II context, indicating species-specific interactions between otherwise highly conserved TLs and its surroundings. These interactions represent epistasis between TL residues and the rest of Pol II. We sought to understand why certain TL sequences are incompatible with S. cerevisiae Pol II and to dissect the nature of genetic interactions within multiply substituted TLs as a window on higher order epistasis in this system. We identified both positive and negative higher-order residue interactions within example TL haplotypes. Intricate higher-order epistasis formed by TL residues was sometimes only apparent from analysis of intermediate genotypes, emphasizing complexity of epistatic interactions. Furthermore, we distinguished TL substitutions with distinct classes of epistatic patterns, suggesting specific TL residues that potentially influence TL evolution. Our examples of complex residue interactions suggest possible pathways for epistasis to facilitate Pol II evolution.

The "dorsal pons", or "dorsal pontine tegmentum" (dPnTg), is part of the brainstem. It is a complex, densely packed region whose nuclei are involved in regulating many vital functions. Notable among them are the parabrachial nucleus, the Kölliker Fuse, the Barrington nucleus, the locus coeruleus, and the dorsal, laterodorsal, and ventral tegmental nuclei. In this study, we applied single-nucleus RNA-seq (snRNA-seq) to resolve neuronal subtypes based on their unique transcriptional profiles and then used multiplexed error robust fluorescence in situ hybridization (MERFISH) to map them spatially. We sampled  1 million cells across the dPnTg and defined the spatial distribution of over 120 neuronal subtypes. Our analysis identified an unpredicted high transcriptional diversity in this region and pinpointed the unique marker genes of many neuronal subtypes. We also demonstrated that many neuronal subtypes are transcriptionally similar between humans and mice, enhancing this study's translational value. Finally, we developed a freely accessible, GPU and CPU-powered dashboard ( http://harvard.heavy.ai:6273/ ) that combines interactive visual analytics and hardware-accelerated SQL into a data science framework to allow the scientific community to query and gain insights into the data.

Diabetic bladder dysfunction (DBD) is the most prevalent complication of diabetes mellitus (DM), affecting >50% of all patients. Currently, no specific treatment is available for this condition. In the early stages of DBD, patients typically complain of frequent urination and often have difficulty sensing when their bladders are full. Over time, bladder function deteriorates to a decompensated state in which incontinence develops. Based on studies of diabetic changes in the eye, kidney, heart, and nerves, it is now recognized that DM causes tissue damage by altering redox signaling in target organs. NADPH oxidase (NOX), whose sole function is the production of reactive oxygen species (ROS), plays a pivotal role in other well-known and bothersome diabetic complications. However, there is a substantial gap in understanding how NOX controls bladder function in health and the impact of NOX on DBD. The current review provides a thorough overview of the various NOX isoforms and their roles in bladder function and discusses the importance of further investigating the role of NOXs as a key contributor to DBD pathogenesis, either as a trigger and/or an effector and potentially as a target.

The targeted activation or inhibition of specific cell populations using chemogenetics allows the precise dissection of cellular signaling and function. Designer receptors exclusively activated by designer drugs (DREADDs) is a chemogenetic platform initially developed by mutating human muscarinic receptors to be unresponsive to endogenous acetylcholine but exclusively activated by an "inert" designer drug. Compound 21 (C21) is a new and potent DREADD agonist; however, radioligand assays from a recent report indicated its ability to bind to endogenous G protein-coupled receptors (GPCRs), including muscarinic M1-3 receptors. Whether this binding causes off-target effects is unclear. Renal innervation is important for the regulation of renal function, and the advent of chemogenetic tools provides significant opportunities for the mechanistic understanding of renal innervation and function. GPCRs such as adrenergic and muscarinic receptors play a role in renal function; thus, a careful pharmacological characterization of C21 in renal function is a prerequisite for this approach. Unexpectedly, an infusion of 1.0 mg/kg C21 in anesthetized mice caused an ∼4-fold increase in urine output and correspondingly increased the glomerular filtration rate (GFR), suggesting a C21-mediated acute diuretic effect. This acute diuresis effect was further confirmed in awake mice using voiding spot assays. The exact molecular mechanism for C21-mediated diuresis is unclear; however, we demonstrated by in vitro myography that C21 can effectively inhibit bladder smooth muscle contraction by antagonizing M3 receptors at the micromolar level, causing increased voiding size in vivo. In summary, C21 functions as a GPCR antagonist and has significant dose-dependent off-target effects in the renal system.

Caloric restriction extends healthy lifespan in multiple species1. Intermittent fasting, an alternative form of dietary restriction, is potentially more sustainable in humans, but its effectiveness remains largely unexplored2-8. Identifying the most efficacious forms of dietary restriction is key for developing interventions to improve human health and longevity9. Here we performed an extensive assessment of graded levels of caloric restriction (20% and 40%) and intermittent fasting (1 and 2 days fasting per week) on the health and survival of 960 genetically diverse female mice. We show that caloric restriction and intermittent fasting both resulted in lifespan extension in proportion to the degree of restriction. Lifespan was heritable and genetics had a larger influence on lifespan than dietary restriction. The strongest trait associations with lifespan included retention of body weight through periods of handling-an indicator of stress resilience, high lymphocyte proportion, low red blood cell distribution width and high adiposity in late life. Health effects differed between interventions and exhibited inconsistent relationships with lifespan extension. 40% caloric restriction had the strongest lifespan extension effect but led to a loss of lean mass and changes in the immune repertoire that could confer susceptibility to infections. Intermittent fasting did not extend the lifespan of mice with high pre-intervention body weight, and two-day intermittent fasting was associated with disruption of erythroid cell populations. Metabolic responses to dietary restriction, including reduced adiposity and lower fasting glucose, were not associated with increased lifespan, suggesting that dietary restriction does more than just counteract the negative effects of obesity. Our findings indicate that improving health and extending lifespan are not synonymous and raise questions about which end points are the most relevant for evaluating aging interventions in preclinical models and clinical trials.