Spatial selectivity of ATase inhibition in mouse models of Charcot-Marie-Tooth disease.

The endoplasmic reticulum acetylation machinery has emerged as a new branch of the larger endoplasmic reticulum quality control system. It regulates the selection of correctly folded polypeptides as well as reticulophagy-mediated removal of toxic protein aggregates with the former being a particularly important aspect of the proteostatic functions of endoplasmic reticulum acetylation. Essential to this function is the Nε-lysine acetyltransferase activity of acetyltransferase 1 and acetyltransferase 2, which regulates the induction of endoplasmic reticulum-specific autophagy through the acetylation of the autophagy-related protein 9A. Here, we used three mouse models of Charcot-Marie-Tooth disease, peripheral myelin protein 22/Tr-J, C3-peripheral myelin protein 22 and myelin protein zero/ttrr, to study spatial and translational selectivity of endoplasmic reticulum acetyltransferase inhibitors. The results show that inhibition of the endoplasmic reticulum acetyltransferases selectively targets misfolding/pro-aggregating events occurring in the lumen of the organelle. Therefore, they establish acetyltransferase 1 and acetyltransferase 2 as the first proven targets for disease-causing proteotoxic states that initiate within the lumen of the endoplasmic reticulum/secretory pathway.

Fluorescence quenching of deprotonated phenylurea through twisting motion induced by an electron-donating substituent group.

The excited-state proton transfer (ESPT) reaction between anthracen-2-yl-3-phenylurea (PUA) derivatives and tetrabutylammonium acetate (TBAAc) in dimethyl sulfoxide (DMSO) solvent was theoretically investigated using time-dependent density functional theory. The electron-donating methoxy group (OMe) and electron-withdrawing trifluoromethyl group (CF3) were bonded to 2PUA to form OMe-2PUA and CF3-2PUA, respectively. Two hydrogen bonds formed in the 1 : 1 hydrogen-bonded complexes between the 2PUA derivative and acetate ion (AcO-), namely N1-H1⋯O1 and N2-H2⋯O2. Strong charge transfer (CT) due to the electron-donating OMe group led to H1 transfer in the S1 state for the OMe-2PUA:AcO- hydrogen-bonded complex. On the contrary, weak CT due to the electron-withdrawing CF3 group led to H2 transfer in the S1 state for CF3-2PUA. After the ESPT reaction, the binding energies of the hydrogen-bonded complexes strongly decreased in both cases, and this promoted the separation of contact-ion pairs (CIPs*) and formed different types of anionic species. CF3-2PUA- could keep its nearly planar structure in the S1 state and emit "abnormal" fluorescence. On the other hand, the anionic OMe-2PUA- underwent a twisting motion to form a twisted structure in the S1 state with very low energy, and this led to a rapid internal conversion (IC) to quench long-wave fluorescence in the emission spectra.

Comprehension of the Synergistic Effect between m&t-BiVO4/TiO2-NTAs Nano-Heterostructures and Oxygen Vacancy for Elevated Charge Transfer and Enhanced Photoelectrochemical Performances.

Through the utilization of a facile procedure combined with anodization and hydrothermal synthesis, highly ordered alignment TiO2 nanotube arrays (TiO2-NTAs) were decorated with BiVO4 with distinctive crystallization phases of monoclinic scheelite (m-BiVO4) and tetragonal zircon (t-BiVO4), favorably constructing different molar ratios and concentrations of oxygen vacancies (Vo) for m&t-BiVO4/TiO2-NTAs heterostructured nanohybrids. Simultaneously, the m&t-BiVO4/TiO2-NTAs nanocomposites significantly promoted photoelectrochemical (PEC) activity, tested under UV-visible light irradiation, through photocurrent density testing and electrochemical impedance spectra, which were derived from the positive synergistic effect between nanohetero-interfaces and Vo defects induced energetic charge transfer (CT). In addition, a proposed self-consistent interfacial CT mechanism and a convincing quantitative dynamic process (i.e., rate constant of CT) for m&t-BiVO4/TiO2-NTAs nanoheterojunctions are supported by time-resolved photoluminescence and nanosecond time-resolved transient photoluminescence spectra, respectively. Based on the scheme, the m&t-BiVO4/TiO2-NTAs-10 nanohybrids exhibited a photodegradation rate of 97% toward degradation of methyl orange irradiated by UV-visible light, 1.14- and 1.04-fold that of m&t-BiVO4/TiO2-NTAs-5 and m&t-BiVO4/TiO2-NTAs-20, respectively. Furthermore, the m&t-BiVO4/TiO2-NTAs-10 nanohybrids showed excellent PEC biosensing performance with a detection limit of 2.6 µM and a sensitivity of 960 mA cm-2 M-1 for the detection of glutathione. Additionally, the gas-sensing performance of m&t-BiVO4/TiO2-NTAs-10 is distinctly superior to that of m&t-BiVO4/TiO2-NTAs-5 and m&t-BiVO4/TiO2-NTAs-20 in terms of sensitivity and response speed.

ATase inhibition rescues age-associated proteotoxicity of the secretory pathway.

Malfunction of autophagy contributes to the progression of many chronic age-associated diseases. As such, improving normal proteostatic mechanisms is an active target for biomedical research and a key focal area for aging research. Endoplasmic reticulum (ER)-based acetylation has emerged as a mechanism that ensures proteostasis within the ER by regulating the induction of ER specific autophagy. ER acetylation is ensured by two ER-membrane bound acetyltransferases, ATase1 and ATase2. Here, we show that ATase inhibitors can rescue ongoing disease manifestations associated with the segmental progeria-like phenotype of AT-1 sTg mice. We also describe a pipeline to reliably identify ATase inhibitors with promising druggability properties. Finally, we show that successful ATase inhibitors can rescue the proteopathy of a mouse model of Alzheimer's disease. In conclusion, our study proposes that ATase-targeting approaches might offer a translational pathway for many age-associated proteopathies affecting the ER/secretory pathway.

Menopause-specific quality of life during ovarian aging among Chinese women: A prospective cohort study.

OBJECTIVE: To describe the menopause-specific quality of life of Chinese urban women at midlife. STUDY DESIGN: Prospective cohort study. MAIN OUTCOME MEASURES: This study included 920 natural menopausal midlife Chinese women who were followed up for 10 years. The Menopause-Specific Quality of Life (MENQOL) questionnaire, which has four domains (vasomotor, psychosocial, physical, and sexual functioning symptoms), and other measures of physical and behavioral factors, were administered. Generalized estimating equations were used to assess the associations. RESULTS: The mean MENQOL scores in the four domains were 1.75 ± 1.32, 2.13 ± 1.16, 2.33 ± 1.11, and 2.20 ± 1.83, respectively. The occurrence of vasomotor symptoms (VMS) persisted in >50% of women in the perimenopausal and early postmenopausal stages. However, the prevalence of moderate/severe bothersome VMS was relatively low. More than 75% of the women presented with mild physical or psychological symptoms, whereas less than 5% of them had moderate/severe symptoms. Sexual problems were highly frequent and bothersome, and their occurrence increased with advancing menopausal stage and age. The prevalence of bothersome sexual symptoms and moderate/severe sexual symptoms ranged from 31.89 and 1.58% in premenopausal women to 78.09 and 39.35% in late postmenopausal women, respectively. Menopausal status, depressive symptoms, and poor health status were significantly associated with the four menopausal domains. CONCLUSION: VMS are among the menopausal symptoms most frequently rated as severe. Sexual problems become more prevalent with advancing age. Clinicians should have a broad understanding of changes that occur during the transition to maximize women's health.

ATG9A regulates proteostasis through reticulophagy receptors FAM134B and SEC62 and folding chaperones CALR and HSPB1.

The acetylation of ATG9A within the endoplasmic reticulum (ER) lumen regulates the induction of reticulophagy. ER acetylation is ensured by AT-1/SLC33A1, a membrane transporter that maintains the cytosol-to-ER flux of acetyl-CoA. Defective AT-1 activity, as caused by heterozygous/homozygous mutations and gene duplication events, results in severe disease phenotypes. Here, we show that although the acetylation of ATG9A occurs in the ER lumen, the induction of reticulophagy requires ATG9A to engage FAM134B and SEC62 on the cytosolic side of the ER. To address this conundrum, we resolved the ATG9A interactome in two mouse models of AT-1 dysregulation: AT-1 sTg, a model of systemic AT-1 overexpression with hyperacetylation of ATG9A, and AT-1S113R/+, a model of AT-1 haploinsufficiency with hypoacetylation of ATG9A. We identified CALR and HSPB1 as two ATG9A partners that regulate the induction of reticulophagy as a function of ATG9A acetylation and discovered that ATG9A associates with several proteins that maintain ER proteostasis.

Excited-state intramolecular proton transfer with and without the assistance of vibronic-transition-induced skeletal deformation in phenol-quinoline.

The excited-state intramolecular proton transfer (ESIPT) reaction of two phenol-quinoline molecules (namely PQ-1 and PQ-2) were investigated using time-dependent density functional theory. The five-(six-) membered-ring carbocycle between the phenol and quinolone moieties in PQ-1 (PQ-2) actually causes a relatively loose (tight) hydrogen bond, which results in a small-barrier (barrier-less) on an excited-state potential energy surface with a slow (fast) ESIPT process with (without) involving the skeletal deformation motion up to the electronic excitation. The skeletal deformation motion that is induced from the largest vibronic excitation with low frequency can assist in decreasing the donor-acceptor distance and lowering the reaction barrier in the excited-state potential energy surface, and thus effectively enhance the ESIPT reaction for PQ-1. The Franck-Condon simulation indicated that the low-frequency mode with vibronic excitation 0 → 1' is an original source of the skeletal deformation vibration. The present simulation presents physical insights for phenol-quinoline molecules in which relatively tight or loose hydrogen bonds can influence the ESIPT reaction process with and without the assistance of the skeletal deformation motion.

Deficient Endoplasmic Reticulum Acetyl-CoA Import in Pancreatic Acinar Cells Leads to Chronic Pancreatitis.

BACKGROUND & AIMS: Maintaining endoplasmic reticulum (ER) proteostasis is essential for pancreatic acinar cell function. Under conditions of severe ER stress, activation of pathogenic unfolded protein response pathways plays a central role in the development and progression of pancreatitis. Less is known, however, of the consequence of perturbing ER-associated post-translational protein modifications on pancreatic outcomes. Here, we examined the role of the ER acetyl-CoA transporter AT-1 on pancreatic homeostasis. METHODS: We used an AT-1S113R/+ hypomorphic mouse model, and generated an inducible, acinar-specific, AT-1 knockout mouse model, and performed histologic and biochemical analyses to probe the effect of AT-1 loss on acinar cell physiology. RESULTS: We found that AT-1 expression is down-regulated significantly during both acute and chronic pancreatitis. Furthermore, acinar-specific deletion of AT-1 in acinar cells induces chronic ER stress marked by activation of both the spliced x-box binding protein 1 and protein kinase R-like ER kinase pathways, leading to spontaneous mild/moderate chronic pancreatitis evidenced by accumulation of intracellular trypsin, immune cell infiltration, and fibrosis. Induction of acute-on-chronic pancreatitis in the AT-1 model led to acinar cell loss and glad atrophy. CONCLUSIONS: These results indicate a key role for AT-1 in pancreatic acinar cell homeostasis, the unfolded protein response, and that perturbations in AT-1 function leads to pancreatic disease.

Insomnia symptoms in relation to menopause among middle-aged Chinese women: Findings from a longitudinal cohort study.

OBJECTIVE: Our study aims to investigate the differences in insomnia between Chinese and Western women during menopause to fill the gaps in the research on menopause in Chinese women, and to examine the premenopausal factors predictive of moderate to severe insomnia during menopause. STUDY DESIGN: This is a longitudinal cohort study conducted in an urban Chinese community with a total of 458 participants. MAIN OUTCOME MEASURES: Presence of insomnia symptoms (trouble falling asleep, waking up early); vasomotor symptoms; anxiety and depression assessed by the Hospital Anxiety and Depression Scale; and menopausal stages. RESULTS: Multivariable analysis showed that compared with that in premenopause, the prevalence of trouble falling asleep was significantly higher in menopausal transition (P = 0.029) and postmenopause (P < 0.001), and the prevalence of early-morning awakenings also significantly increased in menopausal transition (P = 0.003) and postmenopause (P = 0.011). In multivariable analysis anxiety (P = 0.022) and depression (P = 0.005) were independently and significantly positively associated with trouble falling asleep. Anxiety (P < 0.001), depression (P = 0.018), and levels of follicle stimulating hormone (P-0.031) were independently and significantly positively associated with trouble falling asleep. Women who experienced insomnia in premenopause had a significantly higher risk of moderate to severe insomnia in menopausal transition (P = 0.003) and postmenopause (P = 0.047) than those who did not. CONCLUSION: This study showed that the prevalence of sleep disturbance significantly increased during and after menopause. Women with anxiety and depression had a higher risk of insomnia. Difficulty in sleep initiation in the premenopausal period was a strong predictor of moderate to severe insomnia at menopausal transition and postmenopause.

Relationships Between Vasomotor Symptoms and Mood in Midlife Urban Chinese Women: Observations in a Prospective Study.

CONTEXT: During the menopausal transition, there is a greater likelihood of the prevalence of various bothersome symptoms, including vasomotor symptoms (VMS) and mood symptoms. OBJECTIVE: To investigate the association among bothersome VMS and symptoms of anxiety and depression in Chinese women during perimenopause and early in menopause. DESIGN, PATIENTS, SETTING, AND INTERVENTIONS: This study included 430 midlife Chinese women who had experienced natural menopause and were followed up for 10 years. A structured questionnaire was provided annually, comprising the VMS Bother Score (range 1-8) from the Menopause-Specific Quality of Life questionnaire, the Hospital Anxiety and Depression Scale, and other physical and behavioral factors. RESULTS: Among the 430 women evaluated, 78.8% had experienced VMS during long-term follow-up. The overall level of VMS bother score was relatively low (1.92 ± 1.32). Both anxiety and depressive symptoms were significantly associated with VMS bother. After adjusting for potential covariates, the association between anxiety or depression symptoms and VMS bother remained highly significant. Menopausal stage, body mass index, general health, follicle-stimulating hormone, and estradiol were independent contributors to VMS. In time-lagged (1-year) models, VMS bother scores significantly predicted the risk of symptoms of both anxiety and depression the following year. In contrast, anxiety symptoms, rather than depressive symptoms, could predict VMS bother the following year. CONCLUSION: The prevalence of VMS in our cohort was higher than has been previously reported; however, the overall level of bother was relatively low. This study demonstrated a strong relationship between VMS bother and mood symptoms in Chinese women progressing from perimenopause through natural menopause.

The absorption and fluorescence spectra of 4-(3-methoxybenzylidene)-2-methyl-oxazalone interpreted by Franck-Condon simulation in various pH solvent environments.

The absorption and fluorescence spectra of 4-(3-methoxybenzylidene)-2-methyl-oxazalone (m-MeOBDI) dissolved in neutral, acidic, and basic solvent environments have been investigated and assigned by using Franck-Condon (FC) simulations at the quantum TDDFT level. Four different structures of m-MeOBDI in the ground and excited states are optimized and are found to be responsible for the observed absorption and fluorescence spectra. The (absorption) fluorescence of m-MeOBDI in pure methanol and neutral/basic methanol/water (1/9 vol) mixed solvent is found to arise from the (ground neutral N-I) excited neutral N-I* and cationic C-III* species, respectively. In acidic solvent, the absorption is found to arise from ground acidic C-II species, and the excited divalent cation DC-IV* is found to be formed in its excited state due to the excess H+ in the solution, and then it emits ∼560 nm fluorescence. FC simulations have also been employed to confirm our assignments as well as interpret the vibronic band profiles. As expected, the simulated emission spectrum of the divalent cationic species is in good agreement with the experimental observation. Therefore, within the present FC simulation, the observed absorption and fluorescence spectra have been reasonably interpreted and novel fluorescence mechanisms of m-MeOBDI in various pH solvent environments have been proposed.

Nectin-like molecule 2, a necessary sexual maturation regulator, participates in congenital hypogonadotropic hypogonadism.

BACKGROUND: Congenital hypogonadotropic hypogonadism (CHH) is a rare genetically heterogeneous disorder. We aimed to determine the prevalence and pathogenesis of NECL2 (Nectin-like molecule 2) variants in a cohort of female patients with CHH. METHODS: We sequenced and determined the prevalence of NECL2 variants in 68 female patients with CHH and 243 healthy controls collected from an academic medical center. Further cellular and animal studies were performed to verify the pathogenicity of the mutations. Necl2 knockout female mice were generated, and their puberty development was observed. RESULTS: A novel NECL2 variant (c.1052_1060del, p.Thr351_Thr353del) was detected in 4 of 68 (5.9%) patients with CHH. Its prevalence was significantly higher in CHH patients than in healthy controls (0%). At the cellular level, the necl2 variant leads to a decrease in gonadotropin-releasing hormone. In animal models, we found that the Necl2 protein was expressed in the hypothalamus, especially in the ventromedial hypothalamic nucleus of mice. Necl2 knockout female mice showed delayed puberty and an irregular estrous cycle, consistent with CHH patient phenotypes. CONCLUSIONS: Our findings predict that NECL2 may be a new candidate gene for CHH and that the NECL2 protein plays a critical role in the progression of puberty development.

Changes in ultrasound uterine morphology and endometrial thickness during ovarian aging and possible associated factors: findings from a prospective study.

OBJECTIVE: The aim of the study was to provide reference values for sonographic measurements of uterine morphology, quantify changes in uterine morphology across the menopausal transition, and identify possible factors associated with sonographic findings in uterine morphology. METHODS: This is a longitudinal cohort study conducted in middle-aged Chinese women. Using transvaginal ultrasound, we measured morphologic parameters of the uterus (volume and endometrial thickness) under standardized conditions every year for over a decade. RESULTS: Uterine volume begins to decrease before the final menstrual period and declines rapidly thereafter. Compared with a baseline measurement taken in the year of the final menstrual period, uterine volume decreased by 20% and 35% at the first year and second year of postmenopause, respectively. The rate of decrease was slower in the third year. Compared with endometrial thickness in the year of the final menstrual period, the figures for 2 and 3 years before the final menstrual period were 5% and 10% higher, while they decreased by 9% and 18% at the first and second year after the final menstrual period. Similarly, the endometrial thickness became relatively stable 3 years after the final menstrual cycle. These observations were fairly consistent across all women without uterine fibroids. Endometrial thickness was significantly positively associated with body mass index (P = 0.049) after adjusting for time and menopausal stage. CONCLUSIONS: The figures for uterine volume and endometrial thickness decrease around menopause using ultrasound measurments with large reductions in the first and second year after the final menstrual period. A higher body mass index is associated with increased endometrial thickness.

Symptoms of anxiety and depression among Chinese women transitioning through menopause: findings from a prospective community-based cohort study.

OBJECTIVE: To determine the prevalence of symptoms of anxiety and depression in Chinese women during and after menopause, and the associated risk factors. DESIGN: Prospective community-based cohort study. SETTING: An urban community in Beijing, People's Republic of China. PATIENT(S): Four hundred and thirty women who had transitioned through natural menopause. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Symptoms of anxiety and depression. RESULT(S): Symptoms of depression were more common than symptoms of anxiety. The prevalence of symptoms of depression rose from 14.5% during premenopause, to 18.2% during the menopausal transition, and 19.6% in the postmenopause period. The prevalence of symptoms of anxiety rose from 3.1% premenopause, to 7.0% during the menopausal transition, and 7.4% in the postmenopause period. Compared with women in the premenopausal stage, perimenopausal and postmenopausal women were more likely to have symptoms of anxiety and depression, but these differences were not statistically significant. Multivariable analysis showed that poor health status, trouble falling asleep, and early awakening were independently associated with symptoms of anxiety, and that a higher body mass index, poor health, low education status, and night sweats were independently associated with symptoms of depression. CONCLUSION(S): Symptoms of depression were more prevalent than symptoms of anxiety. Our findings suggest that symptoms of anxiety and depression are more common during and after menopause than in premenopausal women. These findings highlight the importance of screening and evaluation of women undergoing the menopausal transition for symptoms of anxiety and depression, especially those with risk factors.

Mass Spectrometry Imaging of N-Glycans from Formalin-Fixed Paraffin-Embedded Tissue Sections Using a Novel Subatmospheric Pressure Ionization Source.

N-linked glycosylation, featuring various glycoforms, is one of the most common and complex protein post-translational modifications (PTMs) controlling protein structures and biological functions. It has been revealed that abnormal changes of protein N-glycosylation patterns are associated with many diseases. Hence, unraveling the disease-related alteration of glycosylation, especially the glycoforms, is crucial and beneficial to improving our understanding about the pathogenic mechanisms of various diseases. In past decades, given the capability of in situ mapping of biomolecules and their region-specific localizations, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been widely applied to the discovery of potential biomarkers for many diseases. In this study, we coupled a novel subatmospheric pressure (SubAP)/MALDI source with a Q Exactive HF hybrid quadrupole-orbitrap mass spectrometer for in situ imaging of N-linked glycans from formalin-fixed paraffin-embedded (FFPE) tissue sections. The utility of this new platform for N-glycan imaging analysis was demonstrated with a variety of FFPE tissue sections. A total of 55 N-glycans were successfully characterized and visualized from a FFPE mouse brain section. Furthermore, 29 N-glycans with different spatial distribution patterns could be identified from a FFPE mouse ovarian cancer tissue section. High-mannose N-glycans exhibited elevated expression levels in the tumor region, indicating the potential association of this type of N-glycans with tumor progression.

Acetyl-CoA flux regulates the proteome and acetyl-proteome to maintain intracellular metabolic crosstalk.

AT-1/SLC33A1 is a key member of the endoplasmic reticulum (ER) acetylation machinery, transporting acetyl-CoA from the cytosol into the ER lumen where acetyl-CoA serves as the acetyl-group donor for Nε-lysine acetylation. Dysfunctional ER acetylation, as caused by heterozygous or homozygous mutations as well as gene duplication events of AT-1/SLC33A1, has been linked to both developmental and degenerative diseases. Here, we investigate two models of AT-1 dysregulation and altered acetyl-CoA flux: AT-1S113R/+ mice, a model of AT-1 haploinsufficiency, and AT-1 sTg mice, a model of AT-1 overexpression. The animals display distinct metabolic adaptation across intracellular compartments, including reprogramming of lipid metabolism and mitochondria bioenergetics. Mechanistically, the perturbations to AT-1-dependent acetyl-CoA flux result in global and specific changes in both the proteome and the acetyl-proteome (protein acetylation). Collectively, our results suggest that AT-1 acts as an important metabolic regulator that maintains acetyl-CoA homeostasis by promoting functional crosstalk between different intracellular organelles.

Hypothalamic mTORC2 is essential for metabolic health and longevity.

The mechanistic target of rapamycin (mTOR) is an evolutionarily conserved protein kinase that regulates growth and metabolism. mTOR is found in two protein complexes, mTORC1 and mTORC2, that have distinct components and substrates and are both inhibited by rapamycin, a macrolide drug that robustly extends lifespan in multiple species including worms and mice. Although the beneficial effect of rapamycin on longevity is generally attributed to reduced mTORC1 signaling, disruption of mTORC2 signaling can also influence the longevity of worms, either positively or negatively depending on the temperature and food source. Here, we show that loss of hypothalamic mTORC2 signaling in mice decreases activity level, increases the set point for adiposity, and renders the animals susceptible to diet-induced obesity. Hypothalamic mTORC2 signaling normally increases with age, and mice lacking this pathway display higher fat mass and impaired glucose homeostasis throughout life, become more frail with age, and have decreased overall survival. We conclude that hypothalamic mTORC2 is essential for the normal metabolic health, fitness, and lifespan of mice. Our results have implications for the use of mTORC2-inhibiting pharmaceuticals in the treatment of brain cancer and diseases of aging.

Golgi-resident TRIO regulates membrane trafficking during neurite outgrowth.

Neurite outgrowth requires coordinated cytoskeletal rearrangements in the growth cone and directional membrane delivery from the neuronal soma. As an essential Rho guanine nucleotide exchange factor (GEF), TRIO is necessary for cytoskeletal dynamics during neurite outgrowth, but its participation in the membrane delivery is unclear. Using co-localization studies, live-cell imaging, and fluorescence recovery after photobleaching analysis, along with neurite outgrowth assay and various biochemical approaches, we here report that in mouse cerebellar granule neurons, TRIO protein pools at the Golgi and regulates membrane trafficking by controlling the directional maintenance of both RAB8 (member RAS oncogene family 8)- and RAB10-positive membrane vesicles. We found that the spectrin repeats in Golgi-resident TRIO confer RAB8 and RAB10 activation by interacting with and activating the RAB GEF RABIN8. Constitutively active RAB8 or RAB10 could partially restore the neurite outgrowth of TRIO-deficient cerebellar granule neurons, suggesting that TRIO-regulated membrane trafficking has an important functional role in neurite outgrowth. Our results also suggest cross-talk between Rho GEF and Rab GEF in controlling both cytoskeletal dynamics and membrane trafficking during neuronal development. They further highlight how protein pools localized to specific organelles regulate crucial cellular activities and functions. In conclusion, our findings indicate that TRIO regulates membrane trafficking during neurite outgrowth in coordination with its GEF-dependent function in controlling cytoskeletal dynamics via Rho GTPases.

Distinct functions of Trio GEF domains in axon outgrowth of cerebellar granule neurons.

As a critical guanine nucleotide exchange factor (GEF) regulating neurite outgrowth, Trio coordinates multiple processes of cytoskeletal dynamics through activating Rac1, Cdc42 and RhoA small GTPases by two GEF domains, but the in vivo roles of these GEF domains and corresponding downstream effectors have not been determined yet. We established multiple lines of knockout mice and assessed the respective roles of Trio GEF domains and Rac1 in axon outgrowth. Knockout of total Trio in cerebellar granule neurons (CGNs) led to an impaired F-actin rearrangement of growth cone and hence a retarded neurite outgrowth. Such a retardation was reproduced by inhibition of GEF1 domain or knockdown of Cdc42 and restored apparently by introduction of active Cdc42. As Rac1 deficiency did not affect the neurite outgrowth of CGNs, we suggested that Trio GEF1-mediated Cdc42 activation was required for neurite outgrowth. We established a GEF2-knockout line with deletion of all Trio isoforms except a cerebella-specific Trio8, a short isoform of Trio without GEF2 domain, and used this line as a GEF2-deficient animal model. The GEF2-deficient CGNs had a normal neurite outgrowth but abolished Netrin-1-promoted growth, without affecting Netrin-1 induced Rac1 activation. We thus suggested that Trio GEF1-mediated Cdc42 activation rather than Rac1 activation drives the F-actin dynamics necessary for neurite outgrowth, while GEF2 functions in Netrin-1-promoted neurite elongation. Our results delineated the distinct roles of Trio GEF domains in neurite outgrowth, which is instructive to understand the pathogenesis of clinical Trio-related neurodevelopmental disorders.

Predicting the Initial Thermal Decomposition Path of Nitrobenzene Caused by Mode Vibration at Moderate-Low Temperatures: Temperature-Dependent Anti-Stokes Raman Spectra Experiments and First-Principals Calculations.

The lack of understanding of the initial decomposition micromechanism of energetic materials subjected to external stimulation has hindered its safe storage, usage, and development. The initial thermal decomposition path of nitrobenzene triggered by molecular thermal motion is investigated using temperature-dependent anti-Stokes Raman spectra experiments and first-principles calculations to clarify the initial thermal decomposition micromechanism. The experiment shows that the symmetric nitro stretching, antisymmetric nitro stretching, and phenyl ring stretching vibration modes are active as increasing temperature below 500 K. The DFT method is used to examine the effects of the three mode vibrations on the initial decomposition of nitrobenzene by relaxed scan for each relevant change in bond lengths and bond angles to obtain the optimal reaction channel leading to initial thermal decomposition of nitrobenzene. The results demonstrate that the initial thermal decomposition is the isomerization of nitrobenzene to phenyl nitrite. The optimal reaction channel leading to the initial isomerization is the increase or decrease of angle O-N-C from the antisymmetric nitro stretching vibration, which causes the torsion of nitro group and the subsequent oxygen atom attacking carbon atom. The scanning energy barrier related to angle O-N-C is about 62.1 kcal/mol, which is very consistent with the calculated activation barrier of isomerization of nitrobenzene. This proves the reliability of our conclusions.