Macrocephaly and developmental delay caused by missense variants in RAB5C.

Rab GTPases are important regulators of intracellular vesicular trafficking. RAB5C is a member of the Rab GTPase family that plays an important role in the endocytic pathway, membrane protein recycling and signaling. Here we report on 12 individuals with nine different heterozygous de novo variants in RAB5C. All but one patient with missense variants (n = 9) exhibited macrocephaly, combined with mild-to-moderate developmental delay. Patients with loss of function variants (n = 2) had an apparently more severe clinical phenotype with refractory epilepsy and intellectual disability but a normal head circumference. Four missense variants were investigated experimentally. In vitro biochemical studies revealed that all four variants were damaging, resulting in increased nucleotide exchange rate, attenuated responsivity to guanine exchange factors and heterogeneous effects on interactions with effector proteins. Studies in C. elegans confirmed that all four variants were damaging in vivo and showed defects in endocytic pathway function. The variant heterozygotes displayed phenotypes that were not observed in null heterozygotes, with two shown to be through a dominant negative mechanism. Expression of the human RAB5C variants in zebrafish embryos resulted in defective development, further underscoring the damaging effects of the RAB5C variants. Our combined bioinformatic, in vitro and in vivo experimental studies and clinical data support the association of RAB5C missense variants with a neurodevelopmental disorder characterized by macrocephaly and mild-to-moderate developmental delay through disruption of the endocytic pathway.

A dominant negative variant of RAB5B disrupts maturation of surfactant protein B and surfactant protein C.

Pathogenic variants in surfactant proteins SP-B and SP-C cause surfactant deficiency and interstitial lung disease. Surfactant proteins are synthesized as precursors (proSP-B, proSP-C), trafficked, and processed via a vesicular-regulated secretion pathway; however, control of vesicular trafficking events is not fully understood. Through the Undiagnosed Diseases Network, we evaluated a child with interstitial lung disease suggestive of surfactant deficiency. Variants in known surfactant dysfunction disorder genes were not found in trio exome sequencing. Instead, a de novo heterozygous variant in RAB5B was identified in the Ras/Rab GTPases family nucleotide binding domain, p.Asp136His. Functional studies were performed in Caenorhabditis elegans by knocking the proband variant into the conserved position (Asp135) of the ortholog, rab-5 Genetic analysis demonstrated that rab-5[Asp135His] is damaging, producing a strong dominant negative gene product. rab-5[Asp135His] heterozygotes were also defective in endocytosis and early endosome (EE) fusion. Immunostaining studies of the proband's lung biopsy revealed that RAB5B and EE marker EEA1 were significantly reduced in alveolar type II cells and that mature SP-B and SP-C were significantly reduced, while proSP-B and proSP-C were normal. Furthermore, staining normal lung showed colocalization of RAB5B and EEA1 with proSP-B and proSP-C. These findings indicate that dominant negative-acting RAB5B Asp136His and EE dysfunction cause a defect in processing/trafficking to produce mature SP-B and SP-C, resulting in interstitial lung disease, and that RAB5B and EEs normally function in the surfactant secretion pathway. Together, the data suggest a noncanonical function for RAB5B and identify RAB5B p.Asp136His as a genetic mechanism for a surfactant dysfunction disorder.

Inverse association of plasma hydrogen sulfide levels with visceral fat area among Chinese young men: a cross-sectional study

ABSTRACT Objective: To investigate the association between plasma Hydrogen Sulfide (H2S) levels and visceral fat area (VFA) among Chinese young men. Subjects and methods: This cross-sectional study involved 156 Chinese male subjects, aged 18-45 years, who visited the First Hospital of Qinhuangdao (Hebei, China) in 2014 for annual health check-up. Participants were categorized into: low (VFA < 75.57 cm2), medium (75.57 cm2 ≤ VFA<100.37 cm2), and high (VFA ≥ 100.37 cm2) (n = 52/group). We estimated VFA and plasma H2S levels by using bioelectrical impedance analysis and a fluorescence probe-based approach, respectively. The associations of H2S with VFA and obesity anthropometric measures were assessed. Results: In the high VFA group, the body mass index (BMI, 30.4 ± 2.45 kg/m2), total body fat (TBF, 27.9 ± 3.23 kg), plasma H2S (3.5 μmol/L), free fatty acid (FFA, 0.6 ± 0.24 mmol/L), triglyceride (TG, 2.0 mmol/L), and total cholesterol (TC, 5.5 ± 1.02 mmol/L) levels were significantly higher than that of those of the low and medium VFA groups, respectively (P < 0.05). Plasma H2S levels were found to be inversely correlated with VFA, TBF, waist circumference, BMI, FFA, LnFINS, LnHOMA-IR, LnTG, TC, and LDL-C (P < 0.05). Multiple backward stepwise regression analysis revealed an inverse correlation of plasma H2S levels with FFA (β = −0.214, P = 0.005) and VFA (β = −0.429, P < 0.001), independent of adiposity measures and other confounding factors. Conclusion: VFA was independently and inversely associated with plasma H2S levels among Chinese young men. Therefore, determining plasma H2S levels could aid in the assessment of abnormal VAT distribution.

Reduction of phonetic mismatch negativity may depict illness course and predict functional outcomes in schizophrenia.

Schizophrenia (SZ) is characterized by a series of cognitive impairments, including automatic processing impairment of basic auditory information, indexed by mismatch negativity (MMN). Existing studies mainly focus on MMN induced by deviant of single acoustic features, and relatively few studies have focused on complex acoustic stimuli, especially speech-induced MMN. Many cognitive impairments in SZ are related to speech function. Thus, the present study aimed to examine the reduction of phonetic MMN in SZ as a potential biomarker and its relationship with illness course and functional outcomes. Electroencephalogram (EEG) signals were recorded from 32 SZ and 32 healthy controls (HC) in a double oddball paradigm, with /da/ as the standard stimulus and /ba/ and /du/ as the deviant stimuli. MMN was computed for vowel and consonant deviants separately. Clinical symptoms were assessed using the Positive and Negative Symptom Rating Scale (PANSS). Illness duration and illness relapse were acquired by combining clinical interviews and electronic medical records. Functional outcomes were assessed using the Global Assessment of Functioning scale (GAF). Compared with HC, SZ showed lower amplitudes of phonetic MMN, especially for vowel deviants. In addition, the MMN amplitude of the vowel deviant was significantly correlated with illness duration, illness relapse, and functional outcomes among patients with SZ. These findings indicate that the pre-attentive automatic phonetic processing of SZ was impaired for both consonants and vowels, while the vowel processing deficit may be the key speech processing deficit in SZ, which could depict the illness course and predict the functional outcomes.

Inverse association of plasma hydrogen sulfide levels with visceral fat area among Chinese young men: a cross-sectional study.

OBJECTIVE: To investigate the association between plasma Hydrogen Sulfide (H2S) levels and visceral fat area (VFA) among Chinese young men. METHODS: This cross-sectional study involved 156 Chinese male subjects, aged 18-45 years, who visited the First Hospital of Qinhuangdao (Hebei, China) in 2014 for annual health check-up. Participants were categorized into: low (VFA < 75.57 cm2), medium (75.57 cm2 ≤ VFA<100.37 cm2), and high (VFA ≥ 100.37 cm2) (n = 52/group). We estimated VFA and plasma H2S levels by using bioelectrical impedance analysis and a fluorescence probe-based approach, respectively. The associations of H2S with VFA and obesity anthropometric measures were assessed. RESULTS: In the high VFA group, the body mass index (BMI, 30.4 ± 2.45 kg/m2), total body fat (TBF, 27.9 ± 3.23 kg), plasma H2S (3.5 µmol/L), free fatty acid (FFA, 0.6 ± 0.24 mmol/L), triglyceride (TG, 2.0 mmol/L), and total cholesterol (TC, 5.5 ± 1.02 mmol/L) levels were significantly higher than that of those of the low and medium VFA groups, respectively (P < 0.05). Plasma H2S levels were found to be inversely correlated with VFA, TBF, waist circumference, BMI, FFA, LnFINS, LnHOMA-IR, LnTG, TC, and LDL-C (P < 0.05). Multiple backward stepwise regression analysis revealed an inverse correlation of plasma H2S levels with FFA (ß = -0.214, P = 0.005) and VFA (ß = -0.429, P < 0.001), independent of adiposity measures and other confounding factors. CONCLUSION: VFA was independently and inversely associated with plasma H2S levels among Chinese young men. Therefore, determining plasma H2S levels could aid in the assessment of abnormal VAT distribution.

GLP-1 Notch-LAG-1 CSL control of the germline stem cell fate is mediated by transcriptional targets lst-1 and sygl-1.

Stem cell systems are essential for the development and maintenance of polarized tissues. Intercellular signaling pathways control stem cell systems, where niche cells signal stem cells to maintain the stem cell fate/self-renewal and inhibit differentiation. In the C. elegans germline, GLP-1 Notch signaling specifies the stem cell fate, employing the sequence-specific DNA binding protein LAG-1 to implement the transcriptional response. We undertook a comprehensive genome-wide approach to identify transcriptional targets of GLP-1 signaling. We expected primary response target genes to be evident at the intersection of genes identified as directly bound by LAG-1, from ChIP-seq experiments, with genes identified as requiring GLP-1 signaling for RNA accumulation, from RNA-seq analysis. Furthermore, we performed a time-course transcriptomics analysis following auxin inducible degradation of LAG-1 to distinguish between genes whose RNA level was a primary or secondary response of GLP-1 signaling. Surprisingly, only lst-1 and sygl-1, the two known target genes of GLP-1 in the germline, fulfilled these criteria, indicating that these two genes are the primary response targets of GLP-1 Notch and may be the sole germline GLP-1 signaling protein-coding transcriptional targets for mediating the stem cell fate. In addition, three secondary response genes were identified based on their timing following loss of LAG-1, their lack of a LAG-1 ChIP-seq peak and that their glp-1 dependent mRNA accumulation could be explained by a requirement for lst-1 and sygl-1 activity. Moreover, our analysis also suggests that the function of the primary response genes lst-1 and sygl-1 can account for the glp-1 dependent peak protein accumulation of FBF-2, which promotes the stem cell fate and, in part, for the spatial restriction of elevated LAG-1 accumulation to the stem cell region.

CemOrange2 fusions facilitate multifluorophore subcellular imaging in C. elegans.

Due to its ease of genetic manipulation and transparency, Caenorhabditis elegans (C. elegans) has become a preferred model system to study gene function by microscopy. The use of Aequorea victoria green fluorescent protein (GFP) fused to proteins or targeting sequences of interest, further expanded upon the utility of C. elegans by labeling subcellular structures, which enables following their disposition during development or in the presence of genetic mutations. Fluorescent proteins with excitation and emission spectra different from that of GFP accelerated the use of multifluorophore imaging in real time. We have expanded the repertoire of fluorescent proteins for use in C. elegans by developing a codon-optimized version of Orange2 (CemOrange2). Proteins or targeting motifs fused to CemOrange2 were distinguishable from the more common fluorophores used in the nematode; such as GFP, YFP, and mKate2. We generated a panel of CemOrange2 fusion constructs, and confirmed they were targeted to their correct subcellular addresses by colocalization with independent markers. To demonstrate the potential usefulness of this new panel of fluorescent protein markers, we showed that CemOrange2 fusion proteins could be used to: 1) monitor biological pathways, 2) multiplex with other fluorescent proteins to determine colocalization and 3) gain phenotypic knowledge of a human ABCA3 orthologue, ABT-4, trafficking variant in the C. elegans model organism.

Combined fluvoxamine and extended-release methylphenidate improved treatment response compared to fluvoxamine alone in patients with treatment-refractory obsessive-compulsive disorder: A randomized double-blind, placebo-controlled study.

More effective, tolerable interventions for treatment-refractory obsessive-compulsive disorder (OCD) are needed. Preliminary findings encourage optimism that methylphenidate augmentation may be of benefit in the treatment of OCD. To test modulator methylphenidate (MPH) of extended-release formulations (MPH-ER) a safe and effective add-on therapy for refractory OCD, a pilot randomized, placebo-controlled, double-blind trial was conducted at an outpatient, single-center academic setting. Participants included 44 adults with serotonin reuptake inhibitor (SRI) treatment-refractory OCD and receiving a stable fluvoxamine pharmacotherapy with Yale-Brown Obsessive Compulsive Scale (Y-BOCS) scores higher than 20. Data were analyzed in the intention-to-treat sample. All subjects were randomized into two parallel groups to receive fluvoxamine (250 mg daily) plus MPH-ER (36 mg daily) or fluvoxamine (250 mg daily) plus identical placebo tablets under double-blind conditions and followed for 8 weeks. Forty-four patients (29 [66%] men), with a mean (SD) age of 24.7 (6) years participated; with a mean (SD) duration of episode 5.7 (3) were randomized and forty-one finished the trial. In the intention-to-treat analysis, the improvement in the Y-BOCS total score and Y-BOCS obsession subscale score was more prominent in the fluvoxamine and MPH-ER group compared with those receiving placebo (P < .001). Additionally, cumulative response rates were higher in the MPH-ER vs placebo groups (59% vs 5%; P  < .001). MPH-ER was well tolerated; No subjects dropped out due to side effects. In summary, combined treatment with MPH-ER demonstrated an enhanced clinical rate of response compared to placebo. Further trials should examine MPH-ER efficacy in a larger sample.

Gap Junctions and NCA Cation Channels Are Critical for Developmentally Timed Sleep and Arousal in Caenorhabditis elegans.

An essential characteristic of sleep is heightened arousal threshold, with decreased behavioral response to external stimuli. The molecular and cellular mechanisms underlying arousal threshold changes during sleep are not fully understood. We report that loss of UNC-7 or UNC-9 innexin function dramatically reduced sleep and decreased arousal threshold during developmentally timed sleep in Caenorhabditiselegans UNC-7 function was required in premotor interneurons and UNC-9 function was required in motor neurons in this paradigm. Simultaneous transient overexpression of UNC-7 and UNC-9 was sufficient to induce anachronistic sleep in adult animals. Moreover, loss of UNC-7 or UNC-9 suppressed the increased sleep of EGL-4 gain-of-function animals, which have increased cyclic-GMP-dependent protein kinase activity. These results suggest C. elegans gap junctions may act downstream of previously identified sleep regulators. In other paradigms, the NCA cation channels act upstream of gap junctions. Consistent with this, diminished NCA channel activity in C. elegans robustly increased arousal thresholds during sleep bouts in L4-to-adult developmentally timed sleep. Total time in sleep bouts was only modestly increased in animals lacking NCA channel auxiliary subunit UNC-79, whereas increased channel activity dramatically decreased sleep. Loss of EGL-4 or innexin proteins suppressed UNC-79 loss-of-function sleep and arousal defects. In Drosophila, the ion channel narrow abdomen, an ortholog of the C. elegans NCA channels, drive the pigment dispersing factor (PDF) neuropeptide release, regulating circadian behavior. However, in C. elegans, we found that loss of the PDF receptor PDFR-1 did not suppress gain-of-function sleep defects, suggesting an alternative downstream pathway. This study emphasizes the conservation and importance of neuronal activity modulation during sleep, and unequivocally demonstrates that gap junction function is critical for normal sleep.

Normal sleep bouts are not essential for C. elegans survival and FoxO is important for compensatory changes in sleep.

BACKGROUND: Sleep deprivation impairs learning, causes stress, and can lead to death. Notch and JNK-1 pathways impact C. elegans sleep in complex ways; these have been hypothesized to involve compensatory sleep. C. elegans DAF-16, a FoxO transcription factor, is required for homeostatic response to decreased sleep and DAF-16 loss decreases survival after sleep bout deprivation. Here, we investigate connections between these pathways and the requirement for sleep after mechanical stress. RESULTS: Reduced function of Notch ligand LAG-2 or JNK-1 kinase resulted in increased time in sleep bouts during development. These animals were inappropriately easy to arouse using sensory stimulation, but only during sleep bouts. This constellation of defects suggested that poor quality sleep bouts in these animals might activate homeostatic mechanisms, driving compensatory increased sleep bouts. Testing this hypothesis, we found that DAF-16 FoxO function was required for increased sleep bouts in animals with defective lag-2 and jnk-1, as loss of daf-16 reduced sleep bouts back to normal levels. However, loss of daf-16 did not suppress arousal thresholds defects. Where DAF-16 function was required differed; in lag-2 and jnk-1 animals, daf-16 function was required in neurons or muscles, respectively, suggesting that disparate tissues can drive a coordinated response to sleep need. Sleep deprivation due to mechanical stimulation can cause death in many species, including C. elegans, suggesting that sleep is essential. We found that loss of sleep bouts in C. elegans due to genetic manipulation did not impact their survival, even in animals lacking DAF-16 function. However, we found that sleep bout deprivation was often fatal when combined with the concurrent stress of mechanical stimulation. CONCLUSIONS: Together, these results in C. elegans confirm that Notch and JNK-1 signaling are required to achieve normal sleep depth, suggest that DAF-16 is required for increased sleep bouts when signaling decreases, and that failure to enter sleep bouts is not sufficient to cause death in C. elegans, unless paired with concurrent mechanical stress. These results suggest that mechanical stress may directly contribute to death observed in previous studies of sleep deprivation and/or that sleep bouts have a uniquely restorative role in C. elegans sleep.

[Efficacy of fluvoxamine combined with extended-release methylphenidate on treatment-refractory obsessive-compulsive disorder].

OBJECTIVE: To observe the clinical efficacy of dopamine modulator methylphenidate (MPH) of extended-release formulations (MPH-ER) augmentation of ongoing fluvoxamine treatment in refractory obsessive-compulsive disorder (OCD) and its effects on patient's anxiety and sleep quality.
 Methods: A pilot randomized, placebo-controlled, and double-blind trial was conducted at an outpatient, single-center academic setting. Participants included 44 adults with serotonin reuptake inhibitor treatment-refractory OCD and they received a stable fluvoxamine pharmacotherapy with Yale-Brown Obsessive Compulsive Scale (Y-BOCS) scores higher than 20. The 44 patients were randomly assigned into a study group and a control group, with 22 patiencs in each group. Fluvoxamine and MPH-ER were given to the study group, while fluvoxamine and placebo were given to the control group, with 8 weeks of the treatment course. Y-BOCS, Hamilton Anxiety Scale (HAMA) were used to assess the efficacy, Pittsburgh Sleep Quality Index (PSQI) was used to evaluate the sleep quality, and Treatment Emergent Symptom Scale (TESS) was used to evaluate the side effects. Data were analyzed in the intention-to-treat sample.
 Results: The improvement in the Y-BOCS total score, Y-BOCS obsession subscale score and HAMA score were more prominent in the study group than those in the control group (P<0.001). There was no significant difference in PSQI score and TESS score between the two groups. MPH-ER was well tolerated.
 Conclusion: Fluvoxamine combined with MPH-ER is effective in the treatment of refractory obsessive-compulsive disorder. It can improve anxiety and has no adverse effect on sleep quality.

Measuring Caenorhabditis elegans Sleep During the Transition to Adulthood Using a Microfluidics-based System.

C. elegans sleep during development is regulated by genes and cellular mechanisms that are conserved across the animal kingdom (Singh et al., 2014; Trojanowski & Raizen, 2016). C. elegans developmental sleep is usually assessed during the transition to adulthood, a 2.6 h time interval called lethargus (Raizen et al., 2008; Singh et al., 2011). During lethargus, animals cycle between periods of immobility (sleep bouts) and periods of active locomotion (motion bouts). Sleep bouts resemble sleep in other species based on behavioral criteria, including cessation of feeding and locomotion, increased arousal threshold for response to sensory stimulation, rapid reversibility, and homeostatic response to sleep loss. Several assays have been developed to study sleep in C. elegans (Belfer et al., 2013; Bringmann, 2011; Nelson et al., 2013; Raizen et al., 2008). Here, we contribute a detailed protocol for assessment of C. elegans sleep during lethargus, which has been used successfully by many research groups, incorporating simple microfluidic chambers, a low cost camera with lighting system, and computational analysis based on image subtraction. We note that this system could be easily adapted to assess sleep in any small animal.

Genome-Wide Screen for Genes Involved in Caenorhabditis elegans Developmentally Timed Sleep.

In Caenorhabditis elegans, Notch signaling regulates developmentally timed sleep during the transition from L4 larval stage to adulthood (L4/A) . To identify core sleep pathways and to find genes acting downstream of Notch signaling, we undertook the first genome-wide, classical genetic screen focused on C. elegans developmentally timed sleep. To increase screen efficiency, we first looked for mutations that suppressed inappropriate anachronistic sleep in adult hsp::osm-11 animals overexpressing the Notch coligand OSM-11 after heat shock. We retained suppressor lines that also had defects in L4/A developmentally timed sleep, without heat shock overexpression of the Notch coligand. Sixteen suppressor lines with defects in developmentally timed sleep were identified. One line carried a new allele of goa-1; loss of GOA-1 Gαo decreased C. elegans sleep. Another line carried a new allele of gpb-2, encoding a Gß5 protein; Gß5 proteins have not been previously implicated in sleep. In other scenarios, Gß5 GPB-2 acts with regulators of G protein signaling (RGS proteins) EAT-16 and EGL-10 to terminate either EGL-30 Gαq signaling or GOA-1 Gαo signaling, respectively. We found that loss of Gß5 GPB-2 or RGS EAT-16 decreased L4/A sleep. By contrast, EGL-10 loss had no impact. Instead, loss of RGS-1 and RGS-2 increased sleep. Combined, our results suggest that, in the context of L4/A sleep, GPB-2 predominantly acts with EAT-16 RGS to inhibit EGL-30 Gαq signaling. These results confirm the importance of G protein signaling in sleep and demonstrate that these core sleep pathways function genetically downstream of the Notch signaling events promoting sleep.

Combining Human Epigenetics and Sleep Studies in Caenorhabditis elegans: A Cross-Species Approach for Finding Conserved Genes Regulating Sleep.

Study Objectives: We aimed to test a combined approach to identify conserved genes regulating sleep and to explore the association between DNA methylation and sleep length. Methods: We identified candidate genes associated with shorter versus longer sleep duration in college students based on DNA methylation using Illumina Infinium HumanMethylation450 BeadChip arrays. Orthologous genes in Caenorhabditis elegans were identified, and we examined whether their loss of function affected C. elegans sleep. For genes whose perturbation affected C. elegans sleep, we subsequently undertook a small pilot study to re-examine DNA methylation in an independent set of human participants with shorter versus longer sleep durations. Results: Eighty-seven out of 485,577 CpG sites had significant differential methylation in young adults with shorter versus longer sleep duration, corresponding to 52 candidate genes. We identified 34 C. elegans orthologs, including NPY/flp-18 and flp-21, which are known to affect sleep. Loss of five additional genes alters developmentally timed C. elegans sleep (B4GALT6/bre-4, DOCK180/ced-5, GNB2L1/rack-1, PTPRN2/ida-1, ZFYVE28/lst-2). For one of these genes, ZFYVE28 (also known as hLst2), the pilot replication study again found decreased DNA methylation associated with shorter sleep duration at the same two CpG sites in the first intron of ZFYVE28. Conclusions: Using an approach that combines human epigenetics and C. elegans sleep studies, we identified five genes that play previously unidentified roles in C. elegans sleep. We suggest sleep duration in humans may be associated with differential DNA methylation at specific sites and that the conserved genes identified here likely play roles in C. elegans sleep and in other species.

C. elegans lifespan extension by osmotic stress requires FUdR, base excision repair, FOXO, and sirtuins.

Moderate stress can increase lifespan by hormesis, a beneficial low-level induction of stress response pathways. 5'-fluorodeoxyuridine (FUdR) is commonly used to sterilize Caenorhabditis elegans in aging experiments. However, FUdR alters lifespan in some genotypes and induces resistance to thermal and proteotoxic stress. We report that hypertonic stress in combination with FUdR treatment or inhibition of the FUdR target thymidylate synthase, TYMS-1, extends C. elegans lifespan by up to 30%. By contrast, in the absence of FUdR, hypertonic stress decreases lifespan. Adaptation to hypertonic stress requires diminished Notch signaling and loss of Notch co-ligands leads to lifespan extension only in combination with FUdR. Either FUdR treatment or TYMS-1 loss induced resistance to acute hypertonic stress, anoxia, and thermal stress. FUdR treatment increased expression of DAF-16 FOXO and the osmolyte biosynthesis enzyme GPDH-1. FUdR-induced hypertonic stress resistance was partially dependent on sirtuins and base excision repair (BER) pathways, while FUdR-induced lifespan extension under hypertonic stress conditions requires DAF-16, BER, and sirtuin function. Combined, these results demonstrate that FUdR, through inhibition of TYMS-1, activates stress response pathways in somatic tissues to confer hormetic resistance to acute and chronic stress. C. elegans lifespan studies using FUdR may need re-interpretation in light of this work.

A Conserved GEF for Rho-Family GTPases Acts in an EGF Signaling Pathway to Promote Sleep-like Quiescence in Caenorhabditis elegans.

Sleep is evolutionarily conserved and required for organism homeostasis and survival. Despite this importance, the molecular and cellular mechanisms underlying sleep are not well understood. Caenorhabditis elegans exhibits sleep-like behavioral quiescence and thus provides a valuable, simple model system for the study of cellular and molecular regulators of this process. In C. elegans, epidermal growth factor receptor (EGFR) signaling is required in the neurosecretory neuron ALA to promote sleep-like behavioral quiescence after cellular stress. We describe a novel role for VAV-1, a conserved guanine nucleotide exchange factor (GEF) for Rho-family GTPases, in regulation of sleep-like behavioral quiescence. VAV-1, in a GEF-dependent manner, acts in ALA to suppress locomotion and feeding during sleep-like behavioral quiescence in response to cellular stress. Additionally, VAV-1 activity is required for EGF-induced sleep-like quiescence and normal levels of EGFR and secretory dense core vesicles in ALA. Importantly, the role of VAV-1 in promoting cellular stress-induced behavioral quiescence is vital for organism health because VAV-1 is required for normal survival after cellular stress.

Abnormal Anterior Cingulate N-Acetylaspartate and Executive Functioning in Treatment-Resistant Depression After rTMS Therapy.

BACKGROUND: Cognitive impairment is a key feature of treatment-resistant depression (TRD) and can be related to the anterior cingulate cortex (ACC) function. Repetitive transcranial magnetic stimulation (rTMS) as an antidepressant intervention has increasingly been investigated in the last two decades. However, no studies to date have investigated the association between neurobiochemical changes within the anterior cingulate and executive dysfunction measured in TRD being treated with rTMS. METHODS: Thirty-two young depressed patients with treatment-resistant unipolar depression were enrolled in a double-blind, randomized study [active (n=18) vs. sham (n=14)]. ACC metabolism was investigated before and after high-frequency (15 Hz) rTMS using 3-tesla proton magnetic resonance spectroscopy (1H-MRS). The results were compared with 28 age- and gender-matched healthy controls. Executive functioning was measured with the Wisconsin Card Sorting Test (WCST) among 34 subjects with TRD and 28 healthy subjects. RESULTS: Significant reductions in N-acetylaspartate (NAA) and choline-containing Compound levels in the left ACC were found in subjects with TRD pre-rTMS when compared with healthy controls. After successful treatment, NAA levels increased significantly in the left ACC of subjects and were not different from those of age-matched controls. In the WCST, more perseverative errors and fewer correct numbers were observed in TRD subjects at baseline. Improvements in both perseverative errors and correct numbers occurred after active rTMS. In addition, improvement of perseverative errors was positively correlated with enhancement of NAA levels in the left ACC in the active rTMS group. CONCLUSIONS: Our results suggest that the NAA concentration in the left ACC is associated with an improvement in cognitive functioning among subjects with TRD response to active rTMS.

Visualizing mitochondrial lipids and fusion events in Mammalian cells.

Mitochondria are dynamic organelles that frequently undergo fusion and fission, the balance of which is critical for proper cellular functioning and viability. Most studies on mitochondrial fusion and fission mechanisms have focused on proteins thought to physically mediate the events. However, dynamic changes in membrane phospholipids also play roles in facilitating the fusion and fission events. This chapter will review the importance of lipids in mitochondrial dynamics and some of the methods that can be used to study the function of lipids in mitochondrial fusion and fission.

piRNA-associated germline nuage formation and spermatogenesis require MitoPLD profusogenic mitochondrial-surface lipid signaling.

The mammalian Phospholipase D MitoPLD facilitates mitochondrial fusion by generating the signaling lipid phosphatidic acid (PA). The Drosophila MitoPLD homolog Zucchini (Zuc), a proposed cytoplasmic nuclease, is required for piRNA generation, a critical event in germline development. We show that Zuc localizes to mitochondria and has MitoPLD-like activity. Conversely, MitoPLD(-/-) mice exhibit the meiotic arrest, DNA damage, and male sterility characteristic of mice lacking piRNAs. The primary function of MitoPLD seems to be the generation of mitochondrial-surface PA. This PA in turn recruits the phosphatase Lipin 1, which converts PA to diacylglycerol and promotes mitochondrial fission, suggesting a mechanism for mitochondrial morphology homeostasis. MitoPLD and Lipin 1 have opposing effects on mitochondria length and on intermitochondrial cement (nuage), a structure found between aggregated mitochondria that is implicated in piRNA generation. We propose that mitochondrial-surface PA generated by MitoPLD/Zuc recruits or activates nuage components critical for piRNA production.