Publications

The decision to urinate relies on assessing bladder fullness and context to determine an appropriate time and place to go. Any disruption in this interoceptive process results in frequent and sometimes debilitating consequences in daily life. Recent work has uncovered key pathways and brain regions that contribute to the sense of bladder stretch and the control of urinary reflexes, but many open questions remain. Here, we review the known mechanisms that convey sensory information from the bladder to the brain and back down again, and we highlight the knowledge gaps and opportunities for better understanding this system, which will be critical to develop effective therapies for urinary dysfunction.

Multi-subunit RNA Polymerases are responsible for transcription in all kingdoms of life. These enzymes rely on dynamic, highly conserved active site domains such as the so-called "trigger loop" to accomplish steps in the transcription cycle. Mutations in the RNA polymerase II trigger loop confer a spectrum of biochemical and genetic phenotypes that suggest two main classes, which decrease or increase catalysis or other nucleotide addition cycle events. The RNA polymerase II active site relies on networks of residue interactions to function, and mutations likely perturb these networks in ways that may alter mechanisms. Here, we take a structural genetics approach to reveal residue interactions within and surrounding the RNA polymerase II trigger loop - determining its "interaction landscape" - by deep mutational scanning in Saccharomyces cerevisiae RNA polymerase II. This analysis reveals connections between trigger loop residues and surrounding domains, demonstrating that trigger loop function is tightly coupled to its specific enzyme context.

Aquaporin 1 is a membrane water channel protein, which was studied here in spiny dogfish (Squalus acanthias) osmoregulatory tissues using a variety of techniques. The cloning of aquaporin 1 (AQP1) in the spiny dogfish identified a splice variant version of the mRNA/protein (AQP1SV1/AQP1SV1). Polymerase chain reaction (PCR) in a range of tissues showed AQP1 to be expressed at very high levels in the rectal gland with ubiquitous mRNA expression at lower levels in other tissues. Northern blotting showed that AQP1 had a mRNA size of 5.3 kb in kidney total RNA. The level of AQP1 mRNA was significantly lower in the rectal glands of fish acclimated to 120% seawater (SW; vs. 75% SW (p = 0.0007) and 100% SW (p = 0.0025)) but was significantly higher in those fish in the kidney (vs. 100% SW (p = 0.0178)) and intestine (vs. 75% SW (p= 0.0355) and 100% SW (p = 0.0285)). Quantitative PCR determined that AQP1SV1 mRNA levels were also significantly lower in the rectal glands of both 120% (p = 0.0134) and 100% SW (p = 0.0343) fish in comparison to 75% SW-acclimated dogfish. Functional expression in Xenopus oocytes showed that AQP1 exhibited significant apparent membrane water permeability (p = 0.000008-0.0158) across a range of pH values, whereas AQP1SV1 showed no similar permeability. Polyclonal antibodies produced against AQP1 (AQP1 and AQP1/2 antibodies) and AQP1SV1 had bands at the expected sizes of 28 kDa and 24 kDa, respectively, as well as some other banding. The weak AQP1 antibody and the stronger AQP1/2 antibody exhibited staining in the apical membranes of rectal gland secretory tubules, particularly towards the periphery of the gland. In the gill, the AQP1/2 antibody in particular showed staining in secondary-lamellar pavement-cell basal membranes, and in blood vessels and connective tissue in the gill arch. In the spiral valve intestine side wall and valve flap, the AQP1/2 antibody stained muscle tissue and blood vessel walls and, after tyramide signal amplification, showed some staining in the apical membranes of epithelial cells at the ends of the luminal surface of epithelial folds. In the rectum/colon, there was also some muscle and blood vessel staining, but the AQP1 and AQP1/2 antibodies both stained a layer of cells at the base of the surface epithelium. In the kidney convoluted bundle zone, all three antibodies stained bundle sheath membranes to variable extents, and the AQP1/2 antibody also showed staining in the straight bundle zone bundle sheath. In the kidney sinus zone, the AQP1/2 antibody stained the apical membranes of late distal tubule (LDT) nephron loop cells most strongly, with the strongest staining in the middle of the LDT loop and in patches towards the start of the LDT loop. There was also a somewhat less strong staining of segments of the first sinus zone nephron loop, particularly in the intermediate I (IS-I) tubule segment. Some tubules appeared to show no or only low levels of staining. The results suggest that AQP1 plays a role in rectal gland fluid secretion, kidney fluid reabsorption and gill pavement-cell volume regulation and probably a minor role in intestinal/rectal/colon fluid absorption.

The earliest molecular changes in Alzheimer's disease (AD) are poorly understood1-5. Here we show that endogenous lithium (Li) is dynamically regulated in the brain and contributes to cognitive preservation during ageing. Of the metals we analysed, Li was the only one that was significantly reduced in the brain in individuals with mild cognitive impairment (MCI), a precursor to AD. Li bioavailability was further reduced in AD by amyloid sequestration. We explored the role of endogenous Li in the brain by depleting it from the diet of wild-type and AD mouse models. Reducing endogenous cortical Li by approximately 50% markedly increased the deposition of amyloid-β and the accumulation of phospho-tau, and led to pro-inflammatory microglial activation, the loss of synapses, axons and myelin, and accelerated cognitive decline. These effects were mediated, at least in part, through activation of the kinase GSK3β. Single-nucleus RNA-seq showed that Li deficiency gives rise to transcriptome changes in multiple brain cell types that overlap with transcriptome changes in AD. Replacement therapy with lithium orotate, which is a Li salt with reduced amyloid binding, prevents pathological changes and memory loss in AD mouse models and ageing wild-type mice. These findings reveal physiological effects of endogenous Li in the brain and indicate that disruption of Li homeostasis may be an early event in the pathogenesis of AD. Li replacement with amyloid-evading salts is a potential approach to the prevention and treatment of AD.

OBJECTIVES: To examine the impact of overactive bladder (OAB) on all-cause and cardiovascular mortality in women in a real-world setting, and to examine the association of TyG-related indices with OAB.

METHODS: Data on 6580 women aged ≥20 years were collected from the National Health and Nutrition Examination Survey (NHANES) database. Kaplan-Meier curves and Cox survival analysis were used to evaluate the association between OAB and all-cause and cardiovascular mortality. Biomarkers for metabolic syndrome were assessed for their association with OAB, including triglyceride-glucose (TyG) and TyG-related indices. The association between TyG-related indices and OAB was evaluated using restricted cubic splines (RCS), receiver operating characteristic (ROC) curves and multivariate logistic regression, with propensity score matching (PSM) employed to balance confounders between OAB and non-OAB groups.

RESULTS: Kaplan-Meier curves showed that OAB was associated with a poorer prognosis, and multivariate Cox regression analyses indicated that OAB was an independent risk factor for both all-cause and cardiovascular mortality. RCS revealed a positive association between TyG-related indices and OAB. Both ROC curves and multivariate logistic regression analysis indicated that TyG-WHtR (TyG combined with waist-to-height ratio) was strongly associated with OAB, with a higher TyG-WHtR associated with an increased risk of OAB. The retrospective design and selection bias may be the potential limitations.

CONCLUSIONS: OAB is positively associated with all-cause and cardiovascular mortality in women. TyG-related indices are positively associated with OAB, with TyG-WHtR as the most effective index.

The activin-follistatin-inhibin (AFI) axis plays a crucial role in sexual development and reproduction. Recently it was demonstrated that these proteins are also synthesized by many local tissues and regulate different biological activities, including tissue regeneration and cancer metastasis. However, little is known about the expression profile of the AFI axis in the bladder and its role in bladder function and dysfunction. We have examined the expression profile of 11 AFI family members in the mouse bladder. INHA, INHBA, and follistatin are the major ligand subunits detected among the six examined in the bladder. ACVR1, ACVR1B, and ACVR2B are the major receptor subunits detected among the five examined in the bladder. Immunolocalization studies reveal unique cellular distributions of these ligands and receptors within the bladder. The urothelial-localized ACVR2B/ACVR1B receptor complex suggests a role of activin signaling in urothelial function. The stimulatory activin A is present only in a subset of interstitial cells, separated from the urothelial activin receptor ACVR2B/ACVR1B by a basement membrane containing accumulated inhibitory ligand FST and by a layer of activin-negative myofibroblasts. This spatial information on AFI signal molecules suggests that activin A-positive interstitial cells might regulate urothelial cell function via paracrine signaling through activin A-ACVR2B/ACVR1B interaction. Further analysis of the human bladder confirmed the expression profile of the AFI axis, and revealed significantly upregulated expression of INHBA-ACVR2B in bladder cancer. These data suggest roles for these molecules in the growth and metastasis of bladder cancer, and highlight their potential as diagnostic and prognostic biomarkers.

The void spot assay has gained popularity as a way of assessing functional bladder voiding parameters in mice, but analyzing the size and distribution of urine spot patterns on filter paper with software remains problematic due to inter-laboratory differences in image contrast and resolution quality and non-void artifacts. We have developed a machine learning algorithm based on Region-based Convolutional Neural Networks (Mask-RCNN) that was trained in object recognition to detect and quantitate urine spots across a broad range of sizes-ML-UrineQuant. The model proved extremely accurate at identifying urine spots in a wide variety of illumination and contrast settings. The overwhelming advantage it offers over current algorithms will be to allow individual labs to fine-tune the model on their specific images regardless of the image characteristics. This should be a valuable tool for anyone performing lower urinary tract research using mouse models.

Lower urinary tract symptoms (LUTS) affect approximately 50% of the population over 40 years of age and are strongly associated with obesity and metabolic syndrome. Adipose tissue plays a key role in obesity/metabolic syndrome by releasing adipokines that regulate systemic energy/lipid metabolism, insulin resistance, and inflammation. Adiponectin (ADPN), the most abundant adipokine, modulates energy/metabolism homeostasis through its insulin-sensitizing and antiinflammatory effects. Human plasma ADPN levels are inversely associated with obesity and diabetes. To the best of our knowledge, the role of adipokines such as ADPN in the LUTS associated with obesity/metabolic syndrome remains unknown. We have tested such a possible role in a global ADPN-knockout (Adpn-/-) mouse model. Adpn-/- mice exhibited increased voiding frequency, small voids, and reduced bladder smooth muscle (BSM) contractility, with absence of purinergic contraction. Molecular examination indicated significantly altered metabolic and purinergic pathways. The ADPN receptor agonist AdipoRon was found to abolish acute BSM contraction. Intriguingly, both AMPK activators and inhibitors also abolished BSM purinergic contraction. These data indicate the important contribution of what we believe is a novel ADPN signaling pathway to the regulation of BSM contractility. Dysregulation of this ADPN signaling pathway might be an important mechanism leading to LUTS associated with obesity/metabolic syndrome.

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.