SOX2 regulation by hedgehog signaling controls adult lingual epithelium homeostasis.

Adult tongue epithelium is continuously renewed from epithelial progenitor cells, a process that requires hedgehog (HH) signaling. In mice, pharmacological inhibition of the HH pathway causes taste bud loss within a few weeks. Previously, we demonstrated that sonic hedgehog (SHH) overexpression in lingual progenitors induces ectopic taste buds with locally increased SOX2 expression, suggesting that taste bud differentiation depends on SOX2 downstream of HH. To test this, we inhibited HH signaling in mice and observed a rapid decline in Sox2 and SOX2-GFP expression in taste epithelium. Upon conditional deletion of Sox2, differentiation of both taste and non-taste epithelial cells was blocked, and progenitor cell number increased. In contrast to basally restricted proliferation in controls, dividing cells were overabundant and spread to suprabasal epithelial layers in mutants. SOX2 loss in progenitors also led non-cell-autonomously to taste cell apoptosis, dramatically shortening taste cell lifespans. Finally, in tongues with conditional Sox2 deletion and SHH overexpression, ectopic and endogenous taste buds were not detectable; instead, progenitor hyperproliferation expanded throughout the lingual epithelium. In summary, we show that SOX2 functions downstream of HH signaling to regulate lingual epithelium homeostasis.

Sonic hedgehog from both nerves and epithelium is a key trophic factor for taste bud maintenance.

The integrity of taste buds is intimately dependent on an intact gustatory innervation, yet the molecular nature of this dependency is unknown. Here, we show that differentiation of new taste bud cells, but not progenitor proliferation, is interrupted in mice treated with a hedgehog (Hh) pathway inhibitor (HPI), and that gustatory nerves are a source of sonic hedgehog (Shh) for taste bud renewal. Additionally, epithelial taste precursor cells express Shh transiently, and provide a local supply of Hh ligand that supports taste cell renewal. Taste buds are minimally affected when Shh is lost from either tissue source. However, when both the epithelial and neural supply of Shh are removed, taste buds largely disappear. We conclude Shh supplied by taste nerves and local taste epithelium act in concert to support continued taste bud differentiation. However, although neurally derived Shh is in part responsible for the dependence of taste cell renewal on gustatory innervation, neurotrophic support of taste buds likely involves a complex set of factors.

The RNA-binding protein LIN28B regulates developmental timing in the mammalian cochlea.

Proper tissue development requires strict coordination of proliferation, growth, and differentiation. Strict coordination is particularly important for the auditory sensory epithelium, where deviations from the normal spatial and temporal pattern of auditory progenitor cell (prosensory cell) proliferation and differentiation result in abnormal cellular organization and, thus, auditory dysfunction. The molecular mechanisms involved in the timing and coordination of auditory prosensory proliferation and differentiation are poorly understood. Here we identify the RNA-binding protein LIN28B as a critical regulator of developmental timing in the murine cochlea. We show that Lin28b and its opposing let-7 miRNAs are differentially expressed in the auditory sensory lineage, with Lin28b being highly expressed in undifferentiated prosensory cells and let-7 miRNAs being highly expressed in their progeny-hair cells (HCs) and supporting cells (SCs). Using recently developed transgenic mouse models for LIN28B and let-7g, we demonstrate that prolonged LIN28B expression delays prosensory cell cycle withdrawal and differentiation, resulting in HC and SC patterning and maturation defects. Surprisingly, let-7g overexpression, although capable of inducing premature prosensory cell cycle exit, failed to induce premature HC differentiation, suggesting that LIN28B's functional role in the timing of differentiation uses let-7 independent mechanisms. Finally, we demonstrate that overexpression of LIN28B or let-7g can significantly alter the postnatal production of HCs in response to Notch inhibition; LIN28B has a positive effect on HC production, whereas let-7 antagonizes this process. Together, these results implicate a key role for the LIN28B/let-7 axis in regulating postnatal SC plasticity.

Minds on replay: musical hallucinations and their relationship to neurological disease.

The phenomenon of musical hallucinations, in which individuals perceive music in the absence of an external auditory stimulus, has been described sparingly in the literature through small case reports and series. Musical hallucinations have been linked to multiple associated conditions, including psychiatric and neurologic disease, brain lesions, drug effect, and hearing impairment. This study aimed to review the demographics of subjects with musical hallucinations and to determine the prevalence of neurological disorders, particularly neurodegenerative disease. Through the Mayo medical record, 393 subjects with musical hallucinations were identified and divided into five categories based on comorbid conditions that have been associated with musical hallucinations: neurological, psychiatric, structural, drug effect and not otherwise classifiable. Variables, including hearing impairment and the presence of visual and other auditory hallucinations, were evaluated independently in all five groups. The mean age at onset of the hallucinations was 56 years, ranging from 18 to 98 years, and 65.4% of the subjects were female. Neurological disease and focal brain lesions were found in 25% and 9% of the total subjects, respectively. Sixty-five subjects were identified with a neurodegenerative disorder, with the Lewy body disorders being the most common. Visual hallucinations were more common in the group with neurological disease compared to the psychiatric, structural, and not otherwise classifiable groups (P < 0.001), whereas auditory hallucinations were more common in the psychiatric group compared to all other groups (P < 0.001). Structural lesions associated with musical hallucinations involved both hemispheres with a preference towards the left, and all but two included the temporal lobe. Hearing impairment was common, particularly in the not otherwise classifiable category where 67.2% had documented hearing impairment, more than in any other group (P < 0.001). Those with an underlying neurodegenerative disorder or isolated hearing impairment tended to hear more persistent music, which was often religious and patriotic compared to those with a structural lesion, where more modern music was heard, and those with psychiatric disorders where music was mood-congruent. This case series shows that musical hallucinations can occur in association with a wide variety of conditions, of which neurological disease and brain lesions represent a substantial proportion, and that Lewy body disorders are the most commonly associated neurodegenerative diseases. A future prospective study would be helpful to further delineate an association between musical hallucinations and neurodegenerative disease.

A case of accidental self-enucleation caused by obstructive sleep apnea.

A novel form of injury associated with obstructive sleep apnea (OSA) that was comorbid with obesity hypoventilation syndrome, and severe daytime somnolence is reported in a 55 year-old woman, manifesting as severe ocular and extra-ocular muscle injuries sustained from suddenly falling asleep and colliding with a sharp object, resulting in surgical enucleation of the right eye and orbital implant. The literature on injuries (falls, motor vehicle accidents) related to OSA and excessive day time sleepiness (EDS) is reviewed, along with the literature on injuries from OSA-related parasomnias. The diverse health hazards, including physical injury, associated with OSA-EDS, are emphasized, further encouraging the need to educate primary care providers on early detection of OSA with prompt treatment intervention.

Euglycemic agent-mediated hypothalamic transcriptomic manipulation in the N171-82Q model of Huntington disease is related to their physiological efficacy.

Our aim was to employ novel analytical methods to investigate the therapeutic treatment of the energy regulation dysfunction occurring in a Huntington disease (HD) mouse model. HD is a neurodegenerative disorder that is characterized by progressive motor impairment and cognitive alterations. Changes in neuroendocrine function, body weight, energy metabolism, euglycemia, appetite function, and gut function can also occur. It is likely that the locus of these alterations is the hypothalamus. We determined the effects of three different euglycemic agents on HD progression using standard physiological and transcriptomic signature analyses. N171-82Q HD mice were treated with insulin, Exendin-4, and the newly developed GLP-1-Tf to determine whether these agents could improve energy regulation and delay disease progression. Blood glucose, insulin, metabolic hormone levels, and pancreatic morphology were assessed. Hypothalamic gene transcription, motor coordination, and life span were also determined. The N171-82Q mice exhibited significant alterations in hypothalamic gene transcription signatures and energy metabolism that were ameliorated, to varying degrees, by the different euglycemic agents. Exendin-4 or GLP-1-Tf (but not insulin) treatment also improved pancreatic morphology, motor coordination, and increased life span. Using hypothalamic transcription signature analyses, we found that the physiological efficacy variation of the drugs was evident in the degree of reversal of the hypothalamic HD pathological signature. Euglycemic agents targeting hypothalamic and energy regulation dysfunction in HD could potentially alter disease progression and improve quality of life in HD.

Treatment of severe morning sleep inertia with bedtime long-acting bupropion and/or long-acting methylphenidate in a series of 4 patients.

STUDY OBJECTIVES: To assess the benefit of bedtime long-acting bupropion and/or long-acting methylphenidate in the therapy of severe morning sleep inertia (SI), a chronic condition that has major adverse consequences on level of functioning and quality of life, and for which there is no recognized therapy. METHODS: Patients underwent clinical interviews and examinations and completed comprehensive questionnaires. They underwent overnight video-polysomnography and next-day multiple sleep latency testing (apart from 1 case with obstructive sleep apnea). Treatments are described in the case reports. RESULTS: Case 1, a 16-year-old girl who was very late to school every day from severe morning SI despite obstructive sleep apnea being fully controlled with continuous positive airway pressure therapy, responded to bedtime bupropion-extended release (xl) 150 mg, together with methylphenidate-sr (sustained release), 36 mg (along with 20 mg methylphenidate taken 1 hour before the alarm would go off). She woke up in a timely fashion and has started her classes on time, with benefit maintained at 6-month follow-up. Case 2, a 29-year-old female with idiopathic hypersomnia and major depression and associated severe morning SI while maintained on 20 mg twice-daily generic Adderall, responded immediately (first night) to bedtime bupropion-xl, 150 mg, with benefit maintained at the 4-month follow-up. Case 3, a 74-year-old man with idiopathic hypersomnia and major depression maintained on daily methylphenidate-sr and direct-release methylphenidate, along with 300 mg bupropion-xl, developed progressively severe morning SI that immediately responded to changing his bupropion-xl regimen to 150 mg nightly and 150 mg every morning, with benefit maintained at the 3-year follow-up. Case 4, a 60-year-old female with idiopathic hypersomnia and severe morning SI, was immediately intolerant to bedtime bupropion-xl, which was discontinued. CONCLUSIONS: Bedtime use of long-acting bupropion and/or long-acting methylphenidate can be effective in the therapy for severe morning SI and warrants further clinical use along with systematic research.

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells.

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is powered by local progenitor cells and renders taste function prone to disruption by a multitude of medical treatments, which in turn severely impacts the quality of life. Thus, studying this process in the context of drug treatment is vital to understanding if and how taste progenitor function and TRC production are affected. Given the ethical concerns and limited availability of human taste tissue, mouse models, which have a taste system similar to humans, are commonly used. Compared to in vivo methods, which are time-consuming, expensive, and not amenable to high throughput studies, murine lingual organoids can enable experiments to be run rapidly with many replicates and fewer mice. Here, previously published protocols have been adapted and a standardized method for generating taste organoids from taste progenitor cells isolated from the circumvallate papilla (CVP) of adult mice is presented. Taste progenitor cells in the CVP express LGR5 and can be isolated via EGFP fluorescence-activated cell sorting (FACS) from mice carrying an Lgr5EGFP-IRES-CreERT2 allele. Sorted cells are plated onto a matrix gel-based 3D culture system and cultured for 12 days. Organoids expand for the first 6 days of the culture period via proliferation and then enter a differentiation phase, during which they generate all three taste cell types along with non-taste epithelial cells. Organoids can be harvested upon maturation at day 12 or at any time during the growth process for RNA expression and immunohistochemical analysis. Standardizing culture methods for production of lingual organoids from adult stem cells will improve reproducibility and advance lingual organoids as a powerful drug screening tool in the fight to help patients experiencing taste dysfunction.

Onset of taste bud cell renewal starts at birth and coincides with a shift in SHH function.

Embryonic taste bud primordia are specified as taste placodes on the tongue surface and differentiate into the first taste receptor cells (TRCs) at birth. Throughout adult life, TRCs are continually regenerated from epithelial progenitors. Sonic hedgehog (SHH) signaling regulates TRC development and renewal, repressing taste fate embryonically, but promoting TRC differentiation in adults. Here, using mouse models, we show TRC renewal initiates at birth and coincides with onset of SHHs pro-taste function. Using transcriptional profiling to explore molecular regulators of renewal, we identified Foxa1 and Foxa2 as potential SHH target genes in lingual progenitors at birth and show that SHH overexpression in vivo alters FoxA1 and FoxA2 expression relevant to taste buds. We further bioinformatically identify genes relevant to cell adhesion and cell locomotion likely regulated by FOXA1;FOXA2 and show that expression of these candidates is also altered by forced SHH expression. We present a new model where SHH promotes TRC differentiation by regulating changes in epithelial cell adhesion and migration.

Voluntary exercise and caloric restriction enhance hippocampal dendritic spine density and BDNF levels in diabetic mice.

Diabetes may adversely affect cognitive function, but the underlying mechanisms are unknown. To investigate whether manipulations that enhance neurotrophin levels will also restore neuronal structure and function in diabetes, we examined the effects of wheel running and dietary energy restriction on hippocampal neuron morphology and brain-derived neurotrophic factor (BDNF) levels in db/db mice, a model of insulin resistant diabetes. Running wheel activity, caloric restriction, or the combination of the two treatments increased levels of BDNF in the hippocampus of db/db mice. Enhancement of hippocampal BDNF was accompanied by increases in dendritic spine density on the secondary and tertiary dendrites of dentate granule neurons. These studies suggest that diabetes exerts detrimental effects on hippocampal structure, and that this state can be attenuated by increasing energy expenditure and decreasing energy intake.

A counter gradient of Activin A and follistatin instructs the timing of hair cell differentiation in the murine cochlea.

The mammalian auditory sensory epithelium has one of the most stereotyped cellular patterns known in vertebrates. Mechano-sensory hair cells are arranged in precise rows, with one row of inner and three rows of outer hair cells spanning the length of the spiral-shaped sensory epithelium. Aiding such precise cellular patterning, differentiation of the auditory sensory epithelium is precisely timed and follows a steep longitudinal gradient. The molecular signals that promote auditory sensory differentiation and instruct its graded pattern are largely unknown. Here, we identify Activin A and its antagonist follistatin as key regulators of hair cell differentiation and show, using mouse genetic approaches, that a local gradient of Activin A signaling within the auditory sensory epithelium times the longitudinal gradient of hair cell differentiation. Furthermore, we provide evidence that Activin-type signaling regulates a radial gradient of terminal mitosis within the auditory sensory epithelium, which constitutes a novel mechanism for limiting the number of inner hair cells being produced.

Proteolysis of multiple myelin basic protein isoforms after neurotrauma: characterization by mass spectrometry.

Neurotrauma, as in the case of traumatic brain injury, promotes protease over-activation characterized by the select fragmentation of brain proteins. The resulting polypeptides are indicators of biochemical processes, which can be used to study post-injury dynamics and may also be developed into biomarkers. To this end, we devised a novel mass spectrometry approach to characterize post-injury calpain proteolytic processing of myelin basic protein (MBP), a biomarker of brain injury that denotes white matter damage and recovery. Our approach exceeds conventional immunological assays in its deconvolution of multiple protein isoforms, its absolute quantification of proteolytic fragments and its polypeptide selectivity. We quantified and characterized post-injury proteolytic processing of all MBP isoforms identified in adult rat cortex. Further, the translation of calpain-cleaved MBP into CSF was verified following brain injury. We ascertained that the exon-6 sequence of MBP resulted in a characteristic shift in gel migration for intact and fragmented protein alike. We also found evidence for a second post-TBI cleavage event within exon-2 and for the dimerization of the post-TBI 4.3 kDa fragment. Ultimately, the novel methodology described here can be used to study MBP dynamics and other similar proteolytic events of relevance to brain injury and other CNS processes.

Modulation of taste sensitivity by GLP-1 signaling.

In many sensory systems, stimulus sensitivity is dynamically modulated through mechanisms of peripheral adaptation, efferent input, or hormonal action. In this way, responses to sensory stimuli can be optimized in the context of both the environment and the physiological state of the animal. Although the gustatory system critically influences food preference, food intake and metabolic homeostasis, the mechanisms for modulating taste sensitivity are poorly understood. In this study, we report that glucagon-like peptide-1 (GLP-1) signaling in taste buds modulates taste sensitivity in behaving mice. We find that GLP-1 is produced in two distinct subsets of mammalian taste cells, while the GLP-1 receptor is expressed on adjacent intragemmal afferent nerve fibers. GLP-1 receptor knockout mice show dramatically reduced taste responses to sweeteners in behavioral assays, indicating that GLP-1 signaling normally acts to maintain or enhance sweet taste sensitivity. A modest increase in citric acid taste sensitivity in these knockout mice suggests GLP-1 signaling may modulate sour taste, as well. Together, these findings suggest a novel paracrine mechanism for the regulation of taste function.

Narcolepsy: Diagnosis and management.

Narcolepsy is a chronic disorder of hypersomnia that can have a significant impact on quality of life and livelihood. However, with appropriate treatment, its symptoms are manageable, and a satisfying personal, social, and professional life can still be enjoyed. Greater awareness of the disorder promotes accurate and efficient diagnosis. With ongoing research into its underlying biology, better treatments for narcolepsy will inevitably become available.

Sex-dependent metabolic, neuroendocrine, and cognitive responses to dietary energy restriction and excess.

Females and males typically play different roles in survival of the species and would be expected to respond differently to food scarcity or excess. To elucidate the physiological basis of sex differences in responses to energy intake, we maintained groups of male and female rats for 6 months on diets with usual, reduced [20% and 40% caloric restriction (CR), and intermittent fasting (IF)], or elevated (high-fat/high-glucose) energy levels and measured multiple physiological variables related to reproduction, energy metabolism, and behavior. In response to 40% CR, females became emaciated, ceased cycling, underwent endocrine masculinization, exhibited a heightened stress response, increased their spontaneous activity, improved their learning and memory, and maintained elevated levels of circulating brain-derived neurotrophic factor. In contrast, males on 40% CR maintained a higher body weight than the 40% CR females and did not change their activity levels as significantly as the 40% CR females. Additionally, there was no significant change in the cognitive ability of the males on the 40% CR diet. Males and females exhibited similar responses of circulating lipids (cholesterols/triglycerides) and energy-regulating hormones (insulin, leptin, adiponectin, ghrelin) to energy restriction, with the changes being quantitatively greater in males. The high-fat/high-glucose diet had no significant effects on most variables measured but adversely affected the reproductive cycle in females. Heightened cognition and motor activity, combined with reproductive shutdown, in females may maximize the probability of their survival during periods of energy scarcity and may be an evolutionary basis for the vulnerability of women to anorexia nervosa.

Impact of reduced meal frequency without caloric restriction on glucose regulation in healthy, normal-weight middle-aged men and women.

An unresolved issue in the field of diet and health is if and how changes in meal frequency affect energy metabolism in humans. We therefore evaluated the influence of reduced meal frequency without a reduction in energy intake on glucose metabolism in normal-weight, healthy male and female subjects. The study was a randomized crossover design, with two 8-week treatment periods (with an intervening 11-week off-diet period) in which subjects consumed all of their calories for weight maintenance distributed in either 3 meals or 1 meal per day (consumed between 4:00 pm and 8:00 pm). Energy metabolism was evaluated at designated time points throughout the study by performing morning oral glucose tolerance tests and measuring levels of glucose, insulin, glucagon, leptin, ghrelin, adiponectin, resistin, and brain-derived neurotrophic factor (BDNF). Subjects consuming 1 meal per day exhibited higher morning fasting plasma glucose levels, greater and more sustained elevations of plasma glucose concentrations, and a delayed insulin response in the oral glucose tolerance test compared with subjects consuming 3 meals per day. Levels of ghrelin were elevated in response to the 1-meal-per-day regimen. Fasting levels of insulin, leptin, ghrelin, adiponectin, resistin, and BDNF were not significantly affected by meal frequency. Subjects consuming a single large daily meal exhibit elevated fasting glucose levels and impaired morning glucose tolerance associated with a delayed insulin response during a 2-month diet period compared with those consuming 3 meals per day. The impaired glucose tolerance was reversible and was not associated with alterations in the levels of adipokines or BDNF.

Limb immobilization and corticobasal syndrome.

BACKGROUND: Recently, we evaluated two patients with corticobasal syndrome (CBS) who reported symptom onset after limb immobilization. Our objective was to investigate the association between trauma, immobilization and CBS. METHODS: The charts of forty-four consecutive CBS patients seen in the Mayo Clinic Alzheimer Disease Research Center were reviewed with attention to trauma and limb immobilization. RESULTS: 10 CBS patients (23%) had immobilization or trauma on the most affected limb preceding the onset or acceleration of symptoms. The median age at onset was 61. Six patients manifested their first symptoms after immobilization from surgery or fracture with one after leg trauma. Four patients had pre-existing symptoms of limb dysfunction but significantly worsened after immobilization or surgery. CONCLUSIONS: 23 percent of patients had immobilization or trauma of the affected limb. This might have implications for management of CBS, for avoiding injury, limiting immobilization and increasing movement in the affected limb.