Postnatal Dynamic Ciliary ARL13B and ADCY3 Localization in the Mouse Brain.

Primary cilia are hair-like structures found on nearly all mammalian cell types, including cells in the developing and adult brain. A diverse set of receptors and signaling proteins localize within cilia to regulate many physiological and developmental pathways, including the Hedgehog (Hh) pathway. Defects in cilia structure, protein localization, and function lead to genetic disorders called ciliopathies, which present with various clinical features that include several neurodevelopmental phenotypes and hyperphagia-associated obesity. Despite their dysfunction being implicated in several disease states, understanding their roles in central nervous system (CNS) development and signaling has proven challenging. We hypothesize that dynamic changes to ciliary protein composition contribute to this challenge and may reflect unrecognized diversity of CNS cilia. The proteins ARL13B and ADCY3 are established markers of cilia in the brain. ARL13B is a regulatory GTPase important for regulating cilia structure, protein trafficking, and Hh signaling, and ADCY3 is a ciliary adenylyl cyclase. Here, we examine the ciliary localization of ARL13B and ADCY3 in the perinatal and adult mouse brain. We define changes in the proportion of cilia enriched for ARL13B and ADCY3 depending on brain region and age. Furthermore, we identify distinct lengths of cilia within specific brain regions of male and female mice. ARL13B+ cilia become relatively rare with age in many brain regions, including the hypothalamic feeding centers, while ADCY3 becomes a prominent cilia marker in the mature adult brain. It is important to understand the endogenous localization patterns of these proteins throughout development and under different physiological conditions as these common cilia markers may be more dynamic than initially expected. Understanding regional- and developmental-associated cilia protein composition signatures and physiological condition cilia dynamic changes in the CNS may reveal the molecular mechanisms associated with the features commonly observed in ciliopathy models and ciliopathies, like obesity and diabetes.

Ciliary ARL13B prevents obesity in mice.

Cilia are near ubiquitous small, cellular appendages critical for cell-to-cell communication. As such, they are involved in diverse developmental and homeostatic processes, including energy homeostasis. ARL13B is a regulatory GTPase highly enriched in cilia. Mice expressing an engineered ARL13B variant, ARL13BV358A which retains normal biochemical activity, display no detectable ciliary ARL13B. Surprisingly, these mice become obese. Here, we measured body weight, food intake, and blood glucose levels to reveal these mice display hyperphagia and metabolic defects. We showed that ARL13B normally localizes to cilia of neurons in specific brain regions and pancreatic cells but is excluded from these cilia in the Arl13bV358A/V358A model. In addition to its GTPase function, ARL13B acts as a guanine nucleotide exchange factor (GEF) for ARL3. To test whether ARL13B's GEF activity is required to regulate body weight, we analyzed the body weight of mice expressing ARL13BR79Q, a variant that lacks ARL13B GEF activity for ARL3. We found no difference in body weight. Taken together, our results show that ARL13B functions within cilia to control body weight and that this function does not depend on its role as a GEF for ARL3. Controlling the subcellular localization of ARL13B in the engineered mouse model, ARL13BV358A, enables us to define the cilia-specific role of ARL13B in regulating energy homeostasis.

Physiological Condition-Dependent Changes in Ciliary GPCR Localization in the Brain.

Primary cilia are cellular appendages critical for diverse types of Signaling. They are found on most cell types, including cells throughout the CNS. Cilia preferentially localize certain G-protein-coupled receptors (GPCRs) and are critical for mediating the signaling of these receptors. Several of these neuronal GPCRs have recognized roles in feeding behavior and energy homeostasis. Cell and model systems, such as Caenorhabditis elegans and Chlamydomonas, have implicated both dynamic GPCR cilia localization and cilia length and shape changes as key for signaling. It is unclear whether mammalian ciliary GPCRs use similar mechanisms in vivo and under what conditions these processes may occur. Here, we assess two neuronal cilia GPCRs, melanin-concentrating hormone receptor 1 (MCHR1) and neuropeptide-Y receptor 2 (NPY2R), as mammalian model ciliary receptors in the mouse brain. We test the hypothesis that dynamic localization to cilia occurs under physiological conditions associated with these GPCR functions. Both receptors are involved in feeding behaviors, and MCHR1 is also associated with sleep and reward. Cilia were analyzed with a computer-assisted approach allowing for unbiased and high-throughput analysis. We measured cilia frequency, length, and receptor occupancy. We observed changes in ciliary length, receptor occupancy, and cilia frequency under different conditions for one receptor but not another and in specific brain regions. These data suggest that dynamic cilia localization of GPCRs depends on properties of individual receptors and cells where they are expressed. A better understanding of subcellular localization dynamics of ciliary GPCRs could reveal unknown molecular mechanisms regulating behaviors like feeding.

Neuronal cilia in energy homeostasis.

A subset of genetic disorders termed ciliopathies are associated with obesity. The mechanisms behind cilia dysfunction and altered energy homeostasis in these syndromes are complex and likely involve deficits in both development and adult homeostasis. Interestingly, several cilia-associated gene mutations also lead to morbid obesity. While cilia have critical and diverse functions in energy homeostasis, including their roles in centrally mediated food intake and peripheral tissues, many questions remain. Here, we briefly discuss syndromic ciliopathies and monogenic cilia signaling mutations associated with obesity. We then focus on potential ways neuronal cilia regulate energy homeostasis. We discuss the literature around cilia and leptin-melanocortin signaling and changes in ciliary G protein-coupled receptor (GPCR) signaling. We also discuss the different brain regions where cilia are implicated in energy homeostasis and the potential for cilia dysfunction in neural development to contribute to obesity. We close with a short discussion on the challenges and opportunities associated with studies looking at neuronal cilia and energy homeostasis. This review highlights how neuronal cilia-mediated signaling is critical for proper energy homeostasis.

Adult Onset Bilateral Cochlear Nerve Atrophy and Cochlear Implantation: A Case Report and Review of the Literature.

OBJECTIVE: To describe a case of idiopathic bilateral cochlear nerve atrophy acquired in adulthood. PATIENT: A 75-year-old male with acquired bilateral cochlear nerve atrophy. INTERVENTION(S): Unilateral cochlear implantation. MAIN OUTCOME AND RESULTS: Description of a patient with acquired bilateral cochlear nerve atrophy diagnosed at the age of 75. The patient had normal hearing and no communication deficits until the age of 66. At this point, the patient demonstrated a slight asymmetric hearing loss, which progressed to severe sensorineural hearing loss. Due to the resulting communication deficit, cochlear device implantation candidacy was pursued. Pre-operative magnetic resonance imaging (MRI) showed severe atrophy versus absence of the cochlear nerves bilaterally. After careful counseling regarding the expected communication outcomes given the MRI findings, the patient underwent left-sided cochlear implantation. The patient gained sound awareness, but no additional communication benefit compared to pre-operative baseline abilities. CONCLUSION: Cochlear nerve deficiency is a known finding in certain cases of congenital and acquired hearing loss, but no cases of idiopathic adult-onset bilateral nerve atrophy have been reported. Without MR imaging, the clinically significant finding would not have been identified. Thus, MRI is advantageous when compared with other imaging modalities in patients with progressive sensorineural hearing loss and enables improved patient counseling regarding expected auditory and communication outcomes.

A transgenic Alx4-CreER mouse to analyze anterior limb and nephric duct development.

BACKGROUND: Genetic tools to study gene function and the fate of cells in the anterior limb bud are very limited. RESULTS: We describe a transgenic mouse line expressing CreERT2 from the Aristaless-like 4 (Alx4) promoter that induces recombination in the anterior limb. Cre induction at embryonic day 8.5 revealed that Alx4-CreERT2 labeled cells using the mTmG Cre reporter contributed to anterior digits I to III as well as the radius of the forelimb. Cre activity is expanded further along the AP axis in the hindlimb than in the forelimb resulting in some Cre reporter cells contributing to digit IV. Induction at later time points labeled cells that become progressively restricted to more anterior digits and proximal structures. Comparison of Cre expression from the Alx4 promoter transgene with endogenous Alx4 expression reveals Cre expression is slightly expanded posteriorly relative to the endogenous Alx4 expression. Using Alx4-CreERT2 to induce loss of intraflagellar transport 88 (Ift88), a gene required for ciliogenesis, hedgehog signaling, and limb patterning, did not cause overt skeletal malformations. However, the efficiency of deletion, time needed for Ift88 protein turnover, and for cilia to regress may hinder using this approach to analyze cilia in the limb. Alx4-CreERT2 is also active in the mesonephros and nephric duct that contribute to the collecting tubules and ducts of the adult nephron. Embryonic activation of the Alx4-CreERT2 in the Ift88 conditional line results in cyst formation in the collecting tubules/ducts. CONCLUSION: Overall, the Alx4-CreERT2 line will be a new tool to assess cell fates and analyze gene function in the anterior limb, mesonephros, and nephric duct.

The Hedgehog Signaling Pathway is Expressed in the Adult Mouse Hypothalamus and Modulated by Fasting.

The hedgehog signaling pathway is best known for its role in developmental patterning of the neural tube and limb bud. More recently, hedgehog signaling has been recognized for its roles in growth of adult tissues and maintenance of progenitor cell niches. However, the role of hedgehog signaling in fully differentiated cells like neurons in the adult brain is less clear. In mammals, coordination of hedgehog pathway activity relies on primary cilia and patients with ciliopathies such as Bardet-Biedl and Alström syndrome exhibit clinical features clearly attributable to errant hedgehog such as polydactyly. However, these ciliopathies also present with features not clearly associated with hedgehog signaling such as hyperphagia-associated obesity. How hedgehog signaling may contribute to feeding behavior is complex and unclear, but cilia are critical for proper energy homeostasis. Here, we provide a detailed analysis of the expression of core components of the hedgehog signaling pathway in the adult mouse hypothalamus with an emphasis on feeding centers. We show that hedgehog pathway genes continue to be expressed in differentiated neurons important for the regulation of feeding behavior. Furthermore, we demonstrate for the first time that pathway activity is regulated at the transcriptional level by fasting. These data suggest that hedgehog signaling is involved in the proper functioning of brain regions that regulate feeding behavior and that hedgehog pathway dysfunction may play a role in the obesity observed in certain ciliopathies.

Artificial Intelligence Approaches to Assessing Primary Cilia.

Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. Cilia length is highly conserved, tightly regulated, and varies between different cell types and tissues and has been implicated in directly impacting their signaling capacity. For example, cilia have been shown to alter their lengths in response to activation of ciliary G protein-coupled receptors. However, accurately and reproducibly measuring the lengths of numerous cilia is a time-consuming and labor-intensive procedure. Current approaches are also error and bias prone. Artificial intelligence (Ai) programs can be utilized to overcome many of these challenges due to capabilities that permit assimilation, manipulation, and optimization of extensive data sets. Here, we demonstrate that an Ai module can be trained to recognize cilia in images from both in vivo and in vitro samples. After using the trained Ai to identify cilia, we are able to design and rapidly utilize applications that analyze hundreds of cilia in a single sample for length, fluorescence intensity and co-localization. This unbiased approach increased our confidence and rigor when comparing samples from different primary neuronal preps in vitro as well as across different brain regions within an animal and between animals. Moreover, this technique can be used to reliably analyze cilia dynamics from any cell type and tissue in a high-throughput manner across multiple samples and treatment groups. Ultimately, Ai-based approaches will likely become standard as most fields move toward less biased and more reproducible approaches for image acquisition and analysis.

Allosteric mechanism for KCNE1 modulation of KCNQ1 potassium channel activation.

The function of the voltage-gated KCNQ1 potassium channel is regulated by co-assembly with KCNE auxiliary subunits. KCNQ1-KCNE1 channels generate the slow delayed rectifier current, IKs, which contributes to the repolarization phase of the cardiac action potential. A three amino acid motif (F57-T58-L59, FTL) in KCNE1 is essential for slow activation of KCNQ1-KCNE1 channels. However, how this motif interacts with KCNQ1 to control its function is unknown. Combining computational modeling with electrophysiological studies, we developed structural models of the KCNQ1-KCNE1 complex that suggest how KCNE1 controls KCNQ1 activation. The FTL motif binds at a cleft between the voltage-sensing and pore domains and appears to affect the channel gate by an allosteric mechanism. Comparison with the KCNQ1-KCNE3 channel structure suggests a common transmembrane-binding mode for different KCNEs and illuminates how specific differences in the interaction of their triplet motifs determine the profound differences in KCNQ1 functional modulation by KCNE1 versus KCNE3.

Structures Illuminate Cardiac Ion Channel Functions in Health and in Long QT Syndrome.

The cardiac action potential is critical to the production of a synchronized heartbeat. This electrical impulse is governed by the intricate activity of cardiac ion channels, among them the cardiac voltage-gated potassium (Kv) channels KCNQ1 and hERG as well as the voltage-gated sodium (Nav) channel encoded by SCN5A. Each channel performs a highly distinct function, despite sharing a common topology and structural components. These three channels are also the primary proteins mutated in congenital long QT syndrome (LQTS), a genetic condition that predisposes to cardiac arrhythmia and sudden cardiac death due to impaired repolarization of the action potential and has a particular proclivity for reentrant ventricular arrhythmias. Recent cryo-electron microscopy structures of human KCNQ1 and hERG, along with the rat homolog of SCN5A and other mammalian sodium channels, provide atomic-level insight into the structure and function of these proteins that advance our understanding of their distinct functions in the cardiac action potential, as well as the molecular basis of LQTS. In this review, the gating, regulation, LQTS mechanisms, and pharmacological properties of KCNQ1, hERG, and SCN5A are discussed in light of these recent structural findings.

Upgraded molecular models of the human KCNQ1 potassium channel.

The voltage-gated potassium channel KCNQ1 (KV7.1) assembles with the KCNE1 accessory protein to generate the slow delayed rectifier current, IKS, which is critical for membrane repolarization as part of the cardiac action potential. Loss-of-function (LOF) mutations in KCNQ1 are the most common cause of congenital long QT syndrome (LQTS), type 1 LQTS, an inherited genetic predisposition to cardiac arrhythmia and sudden cardiac death. A detailed structural understanding of KCNQ1 is needed to elucidate the molecular basis for KCNQ1 LOF in disease and to enable structure-guided design of new anti-arrhythmic drugs. In this work, advanced structural models of human KCNQ1 in the resting/closed and activated/open states were developed by Rosetta homology modeling guided by newly available experimentally-based templates: X. leavis KCNQ1 and various resting voltage sensor structures. Using molecular dynamics (MD) simulations, the capacity of the models to describe experimentally established channel properties including state-dependent voltage sensor gating charge interactions and pore conformations, PIP2 binding sites, and voltage sensor-pore domain interactions were validated. Rosetta energy calculations were applied to assess the utility of each model in interpreting mutation-evoked KCNQ1 dysfunction by predicting the change in protein thermodynamic stability for 50 experimentally characterized KCNQ1 variants with mutations located in the voltage-sensing domain. Energetic destabilization was successfully predicted for folding-defective KCNQ1 LOF mutants whereas wild type-like mutants exhibited no significant energetic frustrations, which supports growing evidence that mutation-induced protein destabilization is an especially common cause of KCNQ1 dysfunction. The new KCNQ1 Rosetta models provide helpful tools in the study of the structural basis for KCNQ1 function and can be used to generate hypotheses to explain KCNQ1 dysfunction.

Interprofessional orientation for health professionals utilising simulated learning: Findings from a pilot study.

We describe a novel, interprofessional educational intervention pilot used to orient new health profession employees through the simulation laboratory. Health profession employees were recruited to engage in a simulation training session that focused on communication, collaboration, and healthcare roles and responsibilities. Learners (N = 11) were divided into two groups with representation from various health disciplines. Each group participated in a simulated patient scenario while the other group actively observed in another classroom. At the end of both sessions, the group reconvened for a debriefing session. Participants were given a survey before and after the training session, to evaluate the content, experience, and value to their practice. The pre- and post-evaluation survey analysis showed improvement in all objectives with a mean (SD) pre-evaluation score of 4.10 (0.40-1.01) and mean (SD) post-evaluation score of 4.73 (0.30-0.81). Results were favourable, and plans to expand this project are under way.

Variables explaining health-related quality of life in community-dwelling older adults.

BACKGROUND AND PURPOSE: Although health-related quality of life (HRQL) has been linked to numerous factors in older adults, limited or conflicting studies have investigated variables explaining HRQL in healthy, community-dwelling older adults. The purpose of this study was to determine whether physical activity, gait speed, balance, strength, endurance, and flexibility were associated with HRQL in healthy, community-dwelling older adults. METHODS: Participants of this cross-sectional, correlational research design study included residents of a senior living community, aged 60 years and older who were independent in at least unlimited household ambulation. These residents participated in tests of physical activity, gait speed, balance, strength, endurance, flexibility, and HRQL (Medical Outcomes Study Short-Form Health Survey, SF-36). The physical (PCS) and mental (MCS) component summary scores of the SF-36 were calculated. RESULTS: Data were collected on 84 participants (mean [SD] age = 78.6 (5.9) years, 54.8% women). Significant correlations were found between the PCS and fast gait speed (FGS) (r = 0.43; p < .001), the Fullerton Advanced Balance Scale (r = 0.44; p < .001), 8-ft up-and-go (r = -0.34; p = .002), and chair stand (r = 0.37; P = .001). Only body mass index (BMI) (r = 0.30; p = .007) was significantly correlated with MCS. Forward stepwise linear regression analyses were conducted, controlling for age, sex, and BMI, to identify factors associated with the PCS and MCS. In the model using PCS as the dependent variable, FGS accounted for 26% of the variance (R2 change) in PCS over and above age, sex, and BMI (R2 change = 0.03); for the full model, F = 5.37, p = .001. In the regression analysis using MCS as the dependent variable, only the 8-ft up-and-go was retained (R2 change = 0.06) over and above age, sex, and BMI (R2 change = 0.16); for the full model, F = 3.71, p = .01. DISCUSSION: Fast gait speed, balance, and lower body strength were associated with the PCS of the SF-36; however, FGS was the only variable that uniquely contributed to the variance in the PCS. Body mass index was associated with the MCS; however, only balance uniquely contributed to the variance in the MCS. Physical activity was not associated with the PCS or MCS. CONCLUSIONS: The results of this study support the assessment of FGS in community-dwelling older adults to gain insight into physical health status. Interventions directed toward FGS, balance, and BMI may contribute to optimum HRQL in this population.

Improved knee flexion following high-flexion total knee arthroplasty.

BACKGROUND: The application of new techniques and materials in total knee arthroplasty (TKA) continue to be a primary focus in orthopedic surgery. The primary aim of the present study is to evaluate post TKA total range of motion (ROM) among a group of patients who received a gender specific high-flexion design modification implant compared to a control group of patients who received non-gender specific implants. METHODS AND RESULTS: The control group was comprised of 39 TKAs that were recruited pre-operatively and received the non-gender specific implant while the study group consisted of 39 TKAs who received gender specific implants. The study group yielded an improvement in mean post-operative ROM of 21° at 12 months, whereas the mean improvement in ROM among the control group was 11°. Thus, the study group had a 10° increased ROM improvement (91%) over the control group (p = 0.00060). In addition, 100% of the subjects with gender specific high-flexion implants achieved greater or equal ROM post-operatively compared to 82% for the control cohort. Lastly, women who exhibited greater pre-operative ROM and lower body mass index (BMI) were found to benefit the most with the gender specific prosthesis. CONCLUSION: Our study demonstrates that among subjects with a normal BMI, the gender specific high-flexion knee implant is associated with increased ROM as compared to the non-gender specific non-high-flexion implant designs.

TDDFT investigation of surface-enhanced Raman scattering of HCN and CN(-) on Ag(20).

Density functional calculations at the BP86/TZP level of theory are employed to examine the structures, binding energies, and vibrational frequencies of HCN-Ag(20) and CN(-)-Ag(20) complexes. Excited states are calculated using time-dependent density functional theory. A short time approximation for the perturbed density matrix and an empirical damping factor are employed to calculate surface-enhanced Raman scattering (SERS) intensities that account for both chemical and electromagnetic enhancement effects. Chemical effects provide 1-2 orders of magnitude enhancement, whereas electromagnetic effects yield up to 6 orders of magnitude enhancement for CN(-) or 5 for HCN. The structures and SERS enhancement factors suggest that multiple bonding configurations may be observed in experiment.

Impact of conventional radiotherapy on health-related quality of life and critical functions of the head and neck.

PURPOSE: Head-and-neck radiotherapy is associated with significant morbidities. Our purpose was to document impact of morbidities by use of multiple objective measures and health-related quality of life (HR-QOL). METHODS AND MATERIALS: Ten head-and-neck cancer patients were evaluated before receiving conventional head-and-neck radiotherapy and at 1 month and 6 months after treatment. We evaluated weight, saliva production, diet, swallow function, auditory function, and HR-QOL. RESULTS: After radiotherapy, weight was reduced in 89% of subjects. Salivary function was significantly reduced and did not resolve by 6 months. Diet impairment and abnormalities in swallowing function persisted at 6 months. Perception of physical functioning was reduced after treatment, and swallowing, coughing, and dry-mouth symptoms increased. Very few changes were observed in auditory function. CONCLUSIONS: Conventional head-and-neck radiotherapy is associated with substantial functional deficits and diminished HR-QOL. Deficits reported here can serve as a baseline for comparison with results derived from new radiotherapy-treatment techniques.