Role of α2δ3 in Cellular Synchronization of the Suprachiasmatic Nucleus Under Constant Light Conditions.

By the effort to identify candidate signaling molecules important for the formation of robust circadian rhythms in the suprachiasmatic nucleus (SCN), the mammalian circadian center, here we characterize the role of α2δ proteins, synaptic molecules initially identified as an auxiliary subunit of the voltage dependent calcium channel, in circadian rhythm formation. In situ hybridization study demonstrated that type 3 α2δ gene (α2δ3) was strongly expressed in the SCN. Mice without this isoform (Cacna2d3-/-) did not maintain proper circadian locomotor activity rhythms under a constant light (LL) condition, whereas under a constant dark (DD) condition, these mice showed a similar period length and similar light-responsiveness as compared to wild type mice. Reflecting this behavioral phenotype, Cacna2d3-/- mice showed a severely impaired Per1 expression rhythm in the SCN under LL, but not under DD. Cultured SCN slices from Per1-luc transgenic Cacna2d3-/- mice revealed reduced synchrony of Per1-luc gene expression rhythms among SCN neurons. These findings suggest that α2δ3 is essential for synchronized cellular oscillations in the SCN and thereby contributes to enhancing the sustainability of circadian rhythms in behavior.

Development of sheet-enhanced technique (Set) method for matrix-assisted laser desorption/ionization imaging mass spectrometry.

RATIONALE: The key to successful experiments in matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is to apply the matrix uniformly to the sample. With the development of automated equipment, uniform matrix application has made great progress while the sample preparation required to acquire a better image becomes complicated. METHODS: The approach is to apply the matrix uniformly to tape and adhere it to the tissue section. We call this the sheet-enhanced technique (Set) method. RESULTS: The Set method promotes ionization of biomolecules as well as the spray method. This procedure does not require the preparation and application of a matrix solution for each experiment, dramatically reducing the time and effort of matrix deposition. CONCLUSIONS: In the present study, we have developed the Set method as a new matrix application method. The method promotes ionization of biomolecules as well as the spray method for MALDI-IMS.

Analysis of potential anti-aging beverage Pru, a traditional Cuban refreshment, by desorption electrospray ionization-mass spectrometry and FTICR tandem mass spectrometry.

Aging has been established as a major risk factor for prevalent diseases and hence, the development of anti-aging medicines is of great importance. Recently, herbal fermented beverages have emerged as a promising source of potential anti-aging drug. Pru, a traditional Cuban refreshment produced by decoction and fermentation of multispecies plants with sugar, has been consumed for many years and is claimed to have multiple medicinal properties. Besides the traditional method, Pru is also manufactured industrially. The present study analyzed the major components of both traditional Pru (TP) and industrial Pru (IP) to reveal their potential application in promoting the health span. We performed desorption electrospray ionization-mass spectrometry (DESI-MS) and acquired mass spectra by scanning over the 50-1200 m/z range in both positive and negative ion modes. Fourier transform ion cyclotron resonance (FTICR) tandem mass spectrometry (MS/MS) was performed for validating the compound assignments. Three important compounds were identified by comparing the MS and MS/MS spectra with reported literature and the online database. One of the identified compounds, gluconic acid, was found to be the most abundant shared metabolite between TP and IP whereas the other two compounds, magnoflorine and levan were exclusively detected in TP. The present study is the first report of component profiling in Cuban traditional and industrial Pru using DESI-MS and FTICR MS/MS, and reveals the potential application of Pru as a health-promoting agent.

Unsupervised machine learning using an imaging mass spectrometry dataset automatically reassembles grey and white matter.

Current histological and anatomical analysis techniques, including fluorescence in situ hybridisation, immunohistochemistry, immunofluorescence, immunoelectron microscopy and fluorescent fusion protein, have revealed great distribution diversity of mRNA and proteins in the brain. However, the distributional pattern of small biomolecules, such as lipids, remains unclear. To this end, we have developed and optimised imaging mass spectrometry (IMS), a combined technique incorporating mass spectrometry and microscopy, which is capable of comprehensively visualising biomolecule distribution. We demonstrated the differential distribution of phospholipids throughout the cell body and axon of neuronal cells using IMS analysis. In this study, we used solarix XR, a high mass resolution and highly sensitive MALDI-FT-ICR-MS capable of detecting higher number of molecules than conventional MALDI-TOF-MS instruments, to create a molecular distribution dataset. We examined the diversity of biomolecule distribution in rat brains using IMS and hypothesised that unsupervised machine learning reconstructs brain structures such as the grey and white matters. We have demonstrated that principal component analysis (PCA) can reassemble the grey and white matters without assigning brain anatomical regions. Hierarchical clustering allowed us to classify the 10 groups of observed molecules according to their distributions. Furthermore, the group of molecules specifically localised in the cerebellar cortex was estimated to be composed of phospholipids.

Preferential Incorporation of Administered Eicosapentaenoic Acid Into Thin-Cap Atherosclerotic Plaques.

OBJECTIVE: n-3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have beneficial effects on atherosclerosis. Although specific salutary actions have been reported, the detailed distribution of n-3 polyunsaturated fatty acids in plaque and their relevance in disease progression are unclear. Our aim was to assess the pharmacodynamics of EPA and DHA and their metabolites in atherosclerotic plaques. Approach and Results: Apolipoprotein E-deficient (Apoe-/-) mice were fed a Western diet supplemented with EPA (1%, w/w) or DHA (1%, w/w) for 3 weeks. Imaging mass spectrometry analyses were performed in the aortic root and arch of the Apoe-/- mice to evaluate the distribution of EPA, DHA, their metabolites and the lipids containing EPA or DHA in the plaques. Liquid chromatography-mass spectrometry and histological analysis were also performed. The intima-media thickness of atherosclerotic plaque decreased in plaques containing free EPA and EPAs attached with several lipids. EPA was distributed more densely in the thin-cap plaques than in the thick-cap plaques, while DHA was more evenly distributed. In the aortic root, the distribution of total EPA level and cholesteryl esters containing EPA followed a concentration gradient from the vascular endothelium to the media. In the aortic arch, free EPA and 12-hydroxy-EPA colocalized with M2 macrophage. CONCLUSIONS: Administered EPA tends to be incorporated from the vascular lumen side and preferentially taken into the thin-cap plaque.

Change in Brain Plasmalogen Composition by Exposure to Prenatal Undernutrition Leads to Behavioral Impairment of Rats.

Epidemiological studies suggest that poor nutrition during pregnancy influences offspring predisposition to experience developmental and psychiatric disorders. Animal studies have shown that maternal undernutrition leads to behavioral impairment, which is linked to alterations in monoaminergic systems and inflammation in the brain. In this study, we focused on the ethanolamine plasmalogen of the brain as a possible contributor to behavioral disturbances observed in offspring exposed to maternal undernutrition. Maternal food or protein restriction between gestational day (GD) 5.5 and GD 10.5 resulted in hyperactivity of rat male adult offspring. Genes related to the phospholipid biosynthesis were found to be activated in the PFC, but not in the NAcc or striatum, in the offspring exposed to prenatal undernutrition. Corresponding to these gene activations, increased ethanolamine plasmalogen (18:0p-22:6) was observed in the PFC using mass spectrometry imaging. A high number of crossings and the long time spent in the center area were observed in the offspring exposed to prenatal undernutrition and were mimicked in adult rats via the intravenous injection of ethanolamine plasmalogen (18:0p-22:6) incorporated into the liposome. Additionally, plasmalogen (18:0p-22:6) increased only in the PFC, and not in the NAcc or striatum. These results suggest that brain plasmalogen is one of the key molecules to control behavior, and its injection using liposome is a potential therapeutic approach for cognitive impairment.SIGNIFICANCE STATEMENT Maternal undernutrition correlates to developmental and psychiatric disorders. Here, we found that maternal undernutrition in early pregnancy led to hyperactivity in rat male offspring and induced gene activation of phospholipid-synthesizing enzyme and elevation of ethanolamine plasmalogen (18:0p-22:6) level in the PFC. Intravenous injection of ethanolamine plasmalogen (18:0p-22:6) incorporated into the liposome maintained crossing activity and the activity was circumscribed to the center area for a long time period, as in prenatally undernourished offspring with aberrant behavior. Furthermore, the amount of ethanolamine plasmalogen (18:0p-22:6) increased in the PFC of the rat after injection. Our result suggests that brain plasmalogen is one of the key molecules to control behavior and that its injection using liposome is a potential therapeutic approach for cognitive impairment.

N-3 fatty acids modulate repeated stress-evoked pain chronicity.

N-3 fatty acids, including docosahexaenoic acid (DHA), have a beneficial effect in both pain and psychiatric disorders. In fact, we previously reported that stress-induced pain prolongation might be mediated through the suppression of the G-protein coupled-receptor 40/free fatty acid receptor 1 (GPR40/FFAR1), which is activated by DHA and long-chain fatty acids. However, the involvement of GPR40/FFAR1 ligands in the development of stress-induced chronic pain has not yet been described. In this study, we investigated the role of DHA in stress-evoked pain chronicity using diet-induced n-3 fatty acid deficient mice. The n-3 fatty acid deficient mice showed exacerbation of anxiety-like behavior after repeated exposure to social defeat stress. The intact n-3 fatty acid deficient mice showed a decrease in paw threshold values. On the other hand, paw withdrawal thresholds of defeated but not non-stressed, n-3 fatty acid deficient mice continued until day 49 after paw surgery. We evaluated changes in phosphatidylcholine composition in the brains of repeat stress-evoked chronic pain model mice which were not on n-3 fatty acid deficiency diets. On day 7 after paw surgery, phosphatidylcholines with DHA and other long-chain fatty acids were found to have decreased in the brains of stressed mice. Moreover, stress-induced persistent mechanical allodynia was improved by oral DHA supplementation. These results indicated that chronic stress may directly affect brain lipid composition; the related changes could be involved in chronic pain development. Our findings suggested that n-3 fatty acids, particularly DHA, are useful as a potential therapeutic target for stress-evoked chronic pain.

MALDI imaging mass spectrometry revealed atropine distribution in the ocular tissues and its transit from anterior to posterior regions in the whole-eye of rabbit after topical administration.

It is essential to elucidate drug distribution in the ocular tissues and drug transit in the eye for ophthalmic pharmaceutical manufacturers. Atropine is a reversible muscarinic receptor used to treat various diseases. However, its distribution in ocular tissues is still incompletely understood. Matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) evaluates drug distribution in biological samples. However, there have been few investigations of drug distribution in ocular tissues, including whole-eye segments. In the present study, we explored the spatial distribution of atropine in the whole-eye segment by MALDI-IMS, and then evaluated the changes in atropine level along the anterior-posterior and superior-inferior axes. A 1% atropine solution was administered to a rabbit and after 30 min, its eye was enucleated, sectioned, and analyzed by MALDI-IMS. Atropine accumulated primarily in the tear menisci but was found at substantially lower concentrations in the tissue surrounding the conjunctival sacs. Relative differences in atropine levels between the anterior and posterior regions provided insights into the post-instillation behavior of atropine. Atropine signal intensities differed among corneal layers and between the superior and inferior eyeball regions. Differences in signal intensity among tissues indicated that the drug migrated to the posterior regions via a periocular-scleral route. Line scan analysis elucidated atropine transit from the anterior to the posterior region. This information is useful for atropine delivery in the ocular tissues and indicates that MALDI-IMS is effective for revealing drug distribution in whole-eye sections.

Lysophosphatidic acid precursor levels decrease and an arachidonic acid-containing phosphatidylcholine level increases in the dorsal root ganglion of mice after peripheral nerve injury.

In the current study, we aimed to analyze the lipid changes in the dorsal root ganglion (DRG) after sciatic nerve transection (SNT) using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). We found that the arachidonic acid-containing phosphatidylcholine (AA-PC), PC(16:0/20:4) largely increased, while PC(16:0/18:1), PC(18:0/18:1) and phosphatidic acid (PA)(36:2) levels largely decreased in the DRG following nerve injury. Previous studies show that the increase in PC(16:0/20:4) was associated with neuropathic pain and that decrease in PC(16:0/18:1), PC(18:0/18:1), and PA(36:2) were due to producing lysophosphatidic acid (LPA), an initiator for neuropathic pain. These results suggest that the lipid changes in DRG after SNT could be the result of changes for the cause of neuropathic pain. Thus, blocking of LPA could be potential for treatment of neuropathic pain.

Profiling and Imaging of Phospholipids in Brains of Abcd1-Deficient Mice.

ABCD1 is a gene responsible for X-linked adrenoleukodystrophy (X-ALD), and is critical for the transport of very long-chain fatty acids (VLCFA) into peroxisomes and subsequent ß-oxidation. VLCFA-containing lipids accumulate in X-ALD patients, although the effect of ABCD1-deficiency on each lipid species in the central nervous system has not been fully characterized. In this study, each phospholipid and lysophospholipid species in Abcd1-deficient mice brains were profiled by liquid chromatography-mass spectrometry. Among the phospholipid and lysophospholipid species that are significantly more enriched in Abcd1-deficient mice brains, VLCFA were present in 75, 15, 5, 4, and 1 species of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, and lysophosphatidylethanolamine, respectively. Most VLCFA were incorporated at the sn-1 position of phosphatidylcholine and phosphatidylethanolamine. Among the phospholipid species that are significantly less enriched in Abcd1-deficient mice brains, odd-numbered saturated or mono-unsaturated fatty acyl moieties are contained in all phosphatidylcholine species. In addition, a number of phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine species contained highly unsaturated fatty acyl moieties. Intriguingly, 44:1 phosphatidylcholine with VLCFA was mainly distributed in the gray matter, such as the cortex, but not in the white matter in the cerebrum and cerebellum. These results show that ABCD1-deficiency causes metabolic alternation of long-chain fatty acids and VLCFA. Moreover, our results imply a molecular mechanism for the incorporation of saturated or monounsaturated VLCFA into the sn-1 position of phospholipids, and also indicate that the distribution of phospholipids with VLCFA may correlate with the development of X-ALD.

Imaging mass spectroscopy delineates the thinned and thickened walls of intracranial aneurysms.

OBJECT: The wall thickness of intracranial aneurysms (IAs) is heterogeneous. Although thinning of the IA wall is thought to contribute to IA rupture, the underlying mechanism remains poorly understood. Recently, imaging mass spectroscopy (IMS) has been used to reveal the distribution of phospholipids in vascular diseases. To investigate the feature of phospholipid composition of IA walls, we conducted IMS in a rat model of experimentally induced IA. MATERIAL AND METHODS: IAs were surgically induced in 7-week-old male rats and analyzed by IMS in negative-ion mode. RESULTS: A molecule at m/z 885.5 was more abundant in the thickened wall than in the thinned wall (P = 0.03). Multiple-stage mass spectroscopy revealed the molecule to be phosphatidylinositol containing stearic acid and arachidonic acid (PI 18:0/20:4). Immunohistochemistry indicated that vascular smooth muscle cells (SMCs) in the thickened wall had dedifferentiated phenotypes. To investigate the relationship between accumulation of PI (18:0/20:4) and phenotypic changes in SMCs, we subjected primary mouse aortic SMCs to liquid chromatography-tandem mass spectrometry. Notably, dedifferentiated SMCs had 1.3-fold more PI (18:0/20:4) than partly differentiated SMCs. CONCLUSIONS: Our study demonstrated the heterogeneity in phospholipid composition of the aneurysmal walls using experimentally induced IAs. PI (18:0/20:4) accumulated at high levels in the thickened aneurysmal wall where synthetic dedifferentiated SMCs exist, suggesting that this phospholipid may be involved in the phenotypic switching of medial SMCs in the IA wall.

[Imaging Mass Spectrometry in Histopathologic Analysis].

Matrix-assisted laser desorption/ionization (MALDI)-imaging mass spectrometry (IMS) enables visualization of the distribution of a range of biomolecules by integrating biochemical information from mass spectrometry with positional information from microscopy. IMS identifies a target molecule. In addition, IMS enables global analysis of biomolecules containing unknown molecules by detecting the ratio of the molecular weight to electric charge without any target, which makes it possible to identify novel molecules. IMS generates data on the distribution of lipids and small molecules in tissues, which is difficult to visualize with either conventional counter-staining or immunohistochemistry. In this review, we firstly introduce the principle of imaging mass spectrometry and recent advances in the sample preparation method. Secondly, we present findings regarding biological samples, especially pathological ones. Finally, we discuss the limitations and problems of the IMS technique and clinical application, such as in drug development.

The Lhx9 homeobox gene controls pineal gland development and prevents postnatal hydrocephalus.

Lhx9 is a member of the LIM homeobox gene family. It is expressed during mammalian embryogenesis in the brain including the pineal gland. Deletion of Lhx9 results in sterility due to failure of gonadal development. The current study was initiated to investigate Lhx9 biology in the pineal gland. Lhx9 is highly expressed in the developing pineal gland of the rat with transcript abundance peaking early in development; transcript levels decrease postnatally to nearly undetectable levels in the adult, a temporal pattern that is generally similar to that reported for Lhx9 expression in other brain regions. Studies with C57BL/6J Lhx9(-/-) mutant mice revealed marked alterations in brain and pineal development. Specifically, the superficial pineal gland is hypoplastic, being reduced to a small cluster of pinealocytes surrounded by meningeal and vascular tissue. The deep pineal gland and the pineal stalk are also reduced in size. Although the brains of neonatal Lhx9(-/-) mutant mice appear normal, severe hydrocephalus develops in about 70% of the Lhx9(-/-) mice at 5-8 weeks of age; these observations are the first to document that deletion of Lhx9 results in hydrocephalus and as such indicate that Lhx9 contributes to the maintenance of normal brain structure. Whereas hydrocephalus is absent in neonatal Lhx9(-/-)mutant mice, the neonatal pineal gland in these animals is hypoplastic. Accordingly, it appears that Lhx9 is essential for early development of the mammalian pineal gland and that this effect is not secondary to hydrocephalus.

Single-cell time-of-flight secondary ion mass spectrometry reveals that human breast cancer stem cells have significantly lower content of palmitoleic acid compared to their counterpart non-stem cancer cells.

Lipids comprise the primary component of cell membranes. Imaging mass spectrometry is increasingly being used to visualize membranous lipids in clinical specimens, and it has revealed that abnormal lipid metabolism is related to the development of diseases. To characterize cell populations which are rare and sparsely localized in tissues, we conducted time-of-flight secondary ion mass spectrometry (TOF-SIMS) analyses of individual cells sorted by fluorescence activated cell sorting (FACS) and applied the method to analyze breast cancer stem cells (CSCs). TOF-SIMS analyses visualized phosphoric acids and four fatty acid (FA) species in the sorted CD45(-)/CD44(+)/CD24(-) CSCs, and these ions are suspected to have originated from membranous phospholipids as they were uniformly detected from the locus where the cells attached. Integrated ion intensity of palmitoleic acids [FA(16:1)] normalized by phosphoric acid signals were decreased significantly in CSCs as compared to that of CD45(-)/CD44(-)/CD24(+) non-stem cancer cells (NSCCs). This finding was supported by liquid chromatography coupled electrospray ionization-tandem mass spectrometry analysis, which revealed phosphatidylcholine (PC)(16:0/16:1) to be less abundant and PC(16:0/16:0) to be more abundant in CSCs as compared to NSCCs. Therefore, our novel method successfully provided lipid composition analysis of individual cells classified by the expression of a complex combination of cell-surface markers. The lipid compositions of CSCs originating from the heterogeneous cellular populations of clinical specimens were successfully characterized by this method.

Angiotensin II triggers expression of the adrenal gland zona glomerulosa-specific 3ß-hydroxysteroid dehydrogenase isoenzyme through de novo protein synthesis of the orphan nuclear receptors NGFIB and NURR1.

The 3ß-hydroxysteroid dehydrogenase (3ß-HSD) is an enzyme crucial for steroid synthesis. Two different 3ß-HSD isoforms exist in humans. Classically, HSD3B2 was considered the principal isoform present in the adrenal. However, we recently showed that the alternative isoform, HSD3B1, is expressed specifically within the adrenal zona glomerulosa (ZG), where aldosterone is produced, raising the question of why this isozyme needs to be expressed in this cell type. Here we show that in both human and mouse, expression of the ZG isoform 3ß-HSD is rapidly induced upon angiotensin II (AngII) stimulation. AngII is the key peptide hormone regulating the capacity of aldosterone synthesis. Using the human adrenocortical H295R cells as a model system, we show that the ZG isoform HSD3B1 differs from HSD3B2 in the ability to respond to AngII. Mechanistically, the induction of HSD3B1 involves de novo protein synthesis of the nuclear orphan receptors NGFIB and NURR1. The HSD3B1 promoter contains a functional NGFIB/NURR1-responsive element to which these proteins bind in response to AngII. Knockdown of these proteins and overexpression of a dominant negative NGFIB both reduce the AngII responsiveness of HSD3B1. Thus, the AngII-NGFIB/NURR1 pathway controls HSD3B1. Our work reveals HSD3B1 as a new regulatory target of AngII.

pY RNA1-s2: a highly retina-enriched small RNA that selectively binds to Matrin 3 (Matr3).

The purpose of this study was to expand our knowledge of small RNAs, which are known to function within protein complexes to modulate the transcriptional output of the cell. Here we describe two previously unrecognized, small RNAs, termed pY RNA1-s1 and pY RNA1-s2 (processed Y RNA1-stem -1 and -2), thereby expanding the list of known small RNAs. pY RNA1-s1 and pY RNA1-s2 were discovered by RNA sequencing and found to be 20-fold more abundant in the retina than in 14 other rat tissues. Retinal expression of pY RNAs is highly conserved, including expression in the human retina, and occurs in all retinal cell layers. Mass spectrometric analysis of pY RNA1-S2 binding proteins in retina indicates that pY RNA1-s2 selectively binds the nuclear matrix protein Matrin 3 (Matr3) and to a lesser degree to hnrpul1 (heterogeneous nuclear ribonucleoprotein U-like protein). In contrast, pY RNA1-s1 does not bind these proteins. Accordingly, the molecular mechanism of action of pY RNA1-s2 is likely be through an action involving Matr3; this 95 kDa protein has two RNA recognition motifs (RRMs) and is implicated in transcription and RNA-editing. The high affinity binding of pY RNA1-s2 to Matr3 is strongly dependent on the sequence of the RNA and both RRMs of Matr3. Related studies also indicate that elements outside of the RRM region contribute to binding specificity and that phosphorylation enhances pY RNA-s2/Matr3 binding. These observations are of significance because they reveal that a previously unrecognized small RNA, pY RNA1-s2, binds selectively to Matr3. Hypothetically, pY RNA1-S2 might act to modulate cellular function through this molecular mechanism. The retinal enrichment of pY RNA1-s2 provides reason to suspect that the pY RNA1-s2/Matr3 interaction could play a role in vision.

Mice genetically deficient in vasopressin V1a and V1b receptors are resistant to jet lag.

Jet-lag symptoms arise from temporal misalignment between the internal circadian clock and external solar time. We found that circadian rhythms of behavior (locomotor activity), clock gene expression, and body temperature immediately reentrained to phase-shifted light-dark cycles in mice lacking vasopressin receptors V1a and V1b (V1a(-/-)V1b(-/-)). Nevertheless, the behavior of V1a(-/-)V1b(-/-) mice was still coupled to the internal clock, which oscillated normally under standard conditions. Experiments with suprachiasmatic nucleus (SCN) slices in culture suggested that interneuronal communication mediated by V1a and V1b confers on the SCN an intrinsic resistance to external perturbation. Pharmacological blockade of V1a and V1b in the SCN of wild-type mice resulted in accelerated recovery from jet lag, which highlights the potential of vasopressin signaling as a therapeutic target for management of circadian rhythm misalignment, such as jet lag and shift work.

Circadian regulation of intracellular G-protein signalling mediates intercellular synchrony and rhythmicity in the suprachiasmatic nucleus.

Synchronous oscillations of thousands of cellular clocks in the suprachiasmatic nucleus (SCN), the circadian centre, are coordinated by precisely timed cell-cell communication, the principle of which is largely unknown. Here we show that the amount of RGS16 (regulator of G protein signalling 16), a protein known to inactivate Gαi, increases at a selective circadian time to allow time-dependent activation of intracellular cyclic AMP signalling in the SCN. Gene ablation of Rgs16 leads to the loss of circadian production of cAMP and as a result lengthens circadian period of behavioural rhythm. The temporally precise regulation of the cAMP signal by clock-controlled RGS16 is needed for the dorsomedial SCN to maintain a normal phase-relationship to the ventrolateral SCN. Thus, RGS16-dependent temporal regulation of intracellular G protein signalling coordinates the intercellular synchrony of SCN pacemaker neurons and thereby defines the 24 h rhythm in behaviour.

Mammalian clock gene Cryptochrome regulates arthritis via proinflammatory cytokine TNF-alpha.

The mammalian clock genes, Period and Cryptochrome (Cry), regulate circadian rhythm. We show that circadian rhythmicity and rhythmic expression of Period in the nuclei of inflammatory synovial cells and spleen cells are disturbed in mouse models of experimental arthritis. Expressions of other clock genes, Bmal1 and Dbp, are also disturbed in spleen cells by arthritis induction. Deletion of Cry1 and Cry2 results in an increase in the number of activated CD3(+) CD69(+) T cells and a higher production of TNF-alpha from spleen cells. When arthritis is induced, Cry1(-/-)Cry2(-/-) mice develop maximal exacerbation of joint swelling, and upregulation of essential mediators of arthritis, including TNF-alpha, IL-1beta and IL-6, and matrix metalloproteinase-3. Wee-1 kinase is solely upregulated in Cry1(-/-)Cry2(-/-) mice, in line with upregulation of c-Fos and Wee-1 kinase in human rheumatoid arthritis. The treatment with anti-TNF-alpha Ab significantly reduced the severity and halted the progression of the arthritis of Cry1(-/-)Cry2(-/-) mice and vice versa, ectopic expression of Cry1 in the mouse embryonic fibroblast from Cry1(-/-)Cry2(-/-) mice significantly reduced the trans activation of TNF-alpha gene. Thus, the biological clock and arthritis influence each other, and this interplay can influence human health and disease.

Glucosamine induces autophagy via an mTOR-independent pathway.

Autophagy is a cellular process that nonspecifically degrades cytosolic components and is involved in many cellular responses. We found that amino sugars with a free amino group such as glucosamine, galactosamine and mannosamine induced autophagy via an mTOR-independent pathway. Glucosamine-induced autophagy at concentrations of at least 500 microM to over 40 mM. In the presence of 40 mM glucosamine, autophagy induction was initiated at 6h and reached a plateau at 36 h. Glucosamine-induced autophagy could remove accumulated ubiquitin-conjugated proteins as well as 79-glutamine repeats. Therefore, orally administered glucosamine could contribute to the prevention of neurodegenerative diseases and promotion of antiaging effects.