Many neuromuscular conditions are characterized by an exaggerated exercise-induced fatigue response that is disproportionate to activity level. This fatigue does not necessarily correlate with elevated central or peripheral fatigue in patients1, and some patients experience severe fatigue without any demonstrable somatic disease2. Except in myopathies that are due to specific metabolic defects, the mechanism underlying this type of fatigue remains unknown2. With no treatment available, this form of inactivity is a major determinant of disability3. Here we show, using mouse models, that this exaggerated fatigue response is distinct from a loss in specific force production by muscle, and that sarcolemma-localized nNOS signaling in skeletal muscle is required to maintain activity after mild exercise. Of significance, we show that nNOS-null mice do not have muscle pathology and have no loss of muscle specific-force after exercise, but do display this exaggerated fatigue response to mild exercise. In mouse models of nNOS mislocalization from the sarcolemma, prolonged inactivity was only relieved by pharmacologically enhancing the cGMP signal that results from muscle nNOS activation during the nitric oxide signaling response to mild exercise. Our findings suggest that the mechanism underlying the exaggerated fatigue response to mild exercise is a lack of contraction-induced signaling from sarcolemma-localized nNOS, which reduces cGMP-mediated vasomodulation in the vessels that supply active muscle after mild exercise. Notably, sarcolemmal nNOS was reduced in patient biopsies from a large number of distinct myopathies, suggesting a common mechanism of fatigue. Our results suggest that patients with an exaggerated fatigue response to mild exercise would show clinical improvement in response to treatment strategies aimed at improving exercise-induced signaling.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder that results in retinal degeneration, obesity, cognitive impairment, polydactyly, renal abnormalities, and hypogenitalism. Of the 12 known BBS genes, BBS1 is the most commonly mutated, and a single missense mutation (M390R) accounts for Ϸ80% of BBS1 cases. To gain insight into the function of BBS1, we generated a Bbs1 M390R/M390R knockin mouse model. Mice homozygous for the M390R mutation recapitulated aspects of the human phenotype, including retinal degeneration, male infertility, and obesity. The obese mutant mice were hyperphagic and hyperleptinemic and exhibited reduced locomotor activity but no elevation in mean arterial blood pressure. Morphological evaluation of Bbs1 mutant brain neuroanatomy revealed ventriculomegaly of the lateral and third ventricles, thinning of the cerebral cortex, and reduced volume of the corpus striatum and hippocampus. Similar abnormalities were also observed in the brains of Bbs2 ؊/؊ , Bbs4 ؊/؊ , and Bbs6 ؊/؊ mice, establishing these neuroanatomical defects as a previously undescribed BBS mouse model phenotype. Ultrastructural examination of the ependymal cell cilia that line the enlarged third ventricle of the Bbs1 mutant brains showed that, whereas the 9 ؉ 2 arrangement of axonemal microtubules was intact, elongated cilia and cilia with abnormally swollen distal ends were present. Together with data from transmission electron microscopy analysis of photoreceptor cell connecting cilia, the Bbs1 M390R mutation does not affect axonemal structure, but it may play a role in the regulation of cilia assembly and/or function.B ardet-Biedl syndrome [BBS, Online Mendelian Inheritance in Man (OMIM) 209900] is a genetically heterogeneous autosomal recessive disorder characterized by obesity, retinal degeneration, polydactyly, cognitive impairment, hypogenitalism, and renal abnormalities, as well as susceptibility to hypertension, diabetes mellitus, olfaction deficits, and congenital cardiac defects. Twelve BBS genes (BBS1-12) have been identified to date (1-14). They encode a set of proteins thought to play a role in the structure or function of cilia, basal bodies, and intracellular transport (15, 16). Mutations in BBS1 are the most commonly observed in BBS. A single missense mutation that converts a methionine codon to an arginine codon (M390R) accounts for Ϸ80% of BBS1 mutations and is involved in 25% of all BBS cases (5). The M390R mutation occurs near predicted regions of coiled-coil protein domains and lies within a conserved predicted WD40-like protein motif. These protein motifs are involved in such basic biological processes as signal transduction, RNA synthesis/processing, chromatin assembly, vesicular trafficking, cytoskeletal assembly, cell cycle control, and apoptosis (17).Recently, BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, and BBS9 were shown to form a stable Ϸ450-kDa protein complex called the BBSome in cultured retinal pigment epithelial (RPE) cells and mouse testes. Depletion of some components of the BBSome, ...

Bardet-Biedl syndrome (BBS) is a heterogeneous genetic disorder characterized by many features, including obesity and cardiovascular disease. We previously developed knockout mouse models of 3 BBS genes: BBS2, BBS4, and BBS6. To dissect the mechanisms involved in the metabolic disorders associated with BBS, we assessed the development of obesity in these mouse models and found that BBS-null mice were hyperphagic, had low locomotor activity, and had elevated circulating levels of the hormone leptin. The effect of exogenous leptin on body weight and food intake was attenuated in BBS mice, which suggests that leptin resistance may contribute to hyperleptinemia. In other mouse models of obesity, leptin resistance may be selective rather than systemic; although mice became resistant to leptin's anorectic effects, the ability to increase renal sympathetic nerve activity (SNA) was preserved. Although all 3 of the BBS mouse models were similarly resistant to leptin, the sensitivity of renal SNA to leptin was maintained in Bbs4 -/-and Bbs6 -/-mice, but not in Bbs2 -/-mice. Consequently, Bbs4 -/-and Bbs6 -/-mice had higher baseline renal SNA and arterial pressure and a greater reduction in arterial pressure in response to ganglionic blockade. Furthermore, we found that BBS mice had a decreased hypothalamic expression of proopiomelanocortin, which suggests that BBS genes play an important role in maintaining leptin sensitivity in proopiomelanocortin neurons.

Summary The renin-angiotensin system (RAS), in addition to its endocrine functions, plays a role within individual tissues such as the brain. The brain RAS is thought to control blood pressure through effects on fluid intake, vasopressin release and sympathetic nerve activity (SNA), and may regulate metabolism through mechanisms which remain undefined. We used a double-transgenic mouse model that exhibits brain-specific RAS activity to examine mechanisms contributing to fluid and energy homeostasis. The mice exhibit high fluid turnover through increased adrenal steroids, which is corrected by adrenalectomy and attenuated by mineralocorticoid receptor blockade. They are also hyperphagic but lean because of a marked increase in body temperature and metabolic rate, mediated by increased SNA and suppression of the circulating RAS. β-adrenergic blockade or restoration of circulating angiotensin-II, but not adrenalectomy, normalized metabolic rate. Our data point to contrasting mechanisms by which the brain RAS regulates fluid intake and energy expenditure.

Localized pH changes have been suggested to occur in the brain during normal function. However, the existence of such pH changes has also been questioned. Lack of methods for noninvasively measuring pH with high spatial and temporal resolution has limited insight into this issue. Here we report that a magnetic resonance imaging (MRI) strategy, T 1 relaxation in the rotating frame (T 1 ρ), is sufficiently sensitive to detect widespread pH changes in the mouse and human brain evoked by systemically manipulating carbon dioxide or bicarbonate. Moreover, T 1 ρ detected a localized acidosis in the human visual cortex induced by a flashing checkerboard. Lactate measurements and pH-sensitive 31 P spectroscopy at the same site also identified a localized acidosis. Consistent with the established role for pH in blood flow recruitment, T 1 ρ correlated with blood oxygenation level-dependent contrast commonly used in functional MRI. However, T 1 ρ was not directly sensitive to blood oxygen content. These observations indicate that localized pH fluctuations occur in the human brain during normal function. Furthermore, they suggest a unique functional imaging strategy based on pH that is independent of traditional functional MRI contrast mechanisms. T o what degree pH changes during normal brain function is unclear (1). However, neuronal activity could cause transient, localized pH changes via several mechanisms. Increased neuronal activity enhances carbohydrate metabolism producing the pHlowering by-products lactic acid and CO 2 (2). Activity-evoked HCO 3 − transport can alter pH (3). Local field potentials produced by ion fluxes could change pH (4). In addition, acidic synaptic vesicles release protons during neurotransmission (5). Such dynamic pH fluctuations have the potential to dramatically alter physiology and behavior through a number of pH-sensitive receptors and channels (6). Acid-sensing ion channels, for example, play critical roles in synaptic plasticity, learning, memory, pain, and neurodegeneration (7-10). Superimposed on activity-dependent brain pH changes and the potential physiological effects are several buffering systems. Principal among these is the CO 2 /HCO 3 − system. In a reversible reaction, CO 2 combines with water to form carbonic acid, which readily dissociates into HCO 3 − and H + . Raising HCO 3− shifts the equilibrium away from H + and increases pH. Conversely, raising CO 2 shifts the equilibrium toward H + , thereby lowering pH. The ability to measure these pH changes in the functioning brain is key for gaining insight into this poorly understood dimension of CNS physiology and pathophysiology.Routinely measuring pH in the brain would require novel noninvasive methods. Traditionally, 31 P spectroscopy has been used to estimate brain pH (11); however, 31 P is limited by poor spatial resolution (typically 10-to 30-cm 3 volumes), long acquisition times (often 5-10 min for a single measurement), and the need for special hardware not typically available on clinical scanners. Recently, 1 H MRI pulse...

Technologies that enable the rapid detection and localization of bacterial infections in living animals could address an unmet need for infectious disease diagnostics. We describe a molecular imaging approach for the specific, non-invasive detection of S. aureus based on the activity of its secreted nuclease, micrococcal nuclease (MN). Several short, synthetic oligonucleotides, rendered resistant to mammalian serum nucleases by various chemical modifications, flanked with a fluorophore and quencher, were activated upon degradation by recombinant MN and in S. aureus culture supernatants. A probe consisting of a pair of deoxythymidines flanked by several 2′-O-methyl-modified nucleotides was activated in culture supernatants of S. aureus but not in culture supernatants of several other pathogenic bacteria. Systemic administration of this probe to mice bearing bioluminescent S. aureus muscle infections resulted in probe activation at the infection sites in an MN-dependent manner. This novel bacterial imaging approach has potential clinical applicability for S. aureus and several other medically significant pathogens.

Bardet-Biedl syndrome (BBS) is a highly pleiotropic autosomal recessive disorder associated with a wide range of phenotypes including obesity. However, the underlying mechanism remains unclear. Here, we show that neuronal BBSome is a critical determinant of energy balance through its role in the regulation of the trafficking of the long signaling form of the leptin receptor (LRb). Targeted disruption of the BBSome by deleting the Bbs1 gene from the nervous system causes obesity in mice, and this phenotype is reproduced by ablation of the Bbs1 gene selectively in the LRb-expressing cells, but not from adipocytes. Obesity developed as a consequence of both increased food intake and decreased energy expenditure in mice lacking the Bbs1 gene in LRb-expressing cells. Strikingly, the well-known role of BBS proteins in the regulation of ciliary formation and function is unlikely to account for the obesogenic effect of BBS1 loss as disruption of the intraflagellar transport (IFT) machinery required for ciliogenesis by deleting the Ift88 gene in LRb-expressing cells caused a marginal increase in body weight and adiposity. Instead, we demonstrate that silencing BBS proteins, but not IFT88, impair the trafficking of the LRb to the plasma membrane leading to central leptin resistance in a manner independent of obesity. Our data also demonstrate that postnatal deletion of the Bbs1 gene in the mediobasal hypothalamus can cause obesity in mice, arguing against an early neurodevelopmental origin of obesity in BBS. Our results depict a novel mechanism underlying energy imbalance and obesity in BBS with potential implications in common forms of human obesity.

Hydrocephalus is a common neurological disorder leading to expansion of the cerebral ventricles and is associated with significant morbidity and mortality. Most neonatal cases are of unknown etiology and are likely to display complex inheritance involving multiple genes and environmental factors. Identifying molecular mechanisms for neonatal hydrocephalus and developing non-invasive treatment modalities are high priorities. Here we employ a hydrocephalic mouse model of the human ciliopathy Bardet-Biedl Syndrome (BBS) and identify a role for neural progenitors in the pathogenesis of neonatal hydrocephalus. We found that hydrocephalus in this mouse model is caused by aberrant PDGFRα signaling, resulting in increased apoptosis and impaired proliferation of NG2+PDGFRα+ neural progenitors. Targeting this pathway with lithium treatment rescued NG2+PDGFRα+ progenitor cell proliferation in BBS mutant mice, reducing ventricular volume. Our findings demonstrate that neural progenitors are critical in the pathogenesis of neonatal hydrocephalus and we identify novel therapeutic targets for this common neurological disorder.