Mice lacking DIO3 exhibit sex-specific alterations in circadian patterns of corticosterone and gene expression in metabolic tissues.

Disruption of circadian rhythms is associated with neurological, endocrine and metabolic pathologies. We have recently shown that mice lacking functional type 3 deiodinase (DIO3), the enzyme that clears thyroid hormones, exhibit a phase shift in locomotor activity, suggesting altered circadian rhythm. To better understand the physiological and molecular basis of this phenotype, we used Dio3+/+ and Dio3-/- mice of both sexes at different zeitgeber times (ZTs) and analyzed corticosterone and thyroxine (T4) levels, hypothalamic, hepatic, and adipose tissue expression of clock genes, as well as genes involved in the thyroid hormone action or physiology of liver and adipose tissues. Wild type mice exhibited sexually dimorphic circadian patterns of genes controlling thyroid hormone action, including Dio3. Dio3-/- mice exhibited altered hypothalamic expression of several clock genes at ZT12, but did not disrupt the overall circadian profile. Expression of clock genes in peripheral tissues was not disrupted by Dio3 deficiency. However, Dio3 loss in liver and adipose tissues disrupted circadian profiles of genes that determine tissue thyroid hormone action and physiology. We also observed circadian-specific changes in serum T4 and corticosterone as a result of DIO3 deficiency. The circadian alterations manifested sexual dimorphism. Most notable, the time curve of serum corticosterone was flattened in Dio3-/- females. We conclude that Dio3 exhibits circadian variations, influencing the circadian rhythmicity of thyroid hormone action and physiology in liver and adipose tissues in a sex-specific manner. Circadian disruptions in tissue physiology may then contribute to the metabolic phenotypes of DIO3-deficient mice.

CBX4 promotes antitumor immunity by suppressing Pdcd1 expression in T cells.

E3 SUMO-protein ligase CBX4 (CBX4), a key component of polycomb-repressive complexes 1 (PRC1), has been reported to regulate a variety of genes implicated in tumor growth, metastasis, and angiogenesis. However, its role in T-cell-mediated antitumor immunity remains elusive. To shed light on this issue, we generated mice with T-cell-specific deletion of Cbx4. Tumor growth was increased in the knockout mice. Additionally, their tumor-infiltrating lymphocytes exhibited impaired tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) production, with an elevated programmed cell death protein 1 (PD-1) level. In fact, dysregulated Pdcd1 expression was observed in all major subsets of peripheral T cells from the knockout mice, which was accompanied by a functional defect in response to T-cell receptor (TCR) stimulation. In support of a direct link between CBX4 and PD-1, Cbx4 overexpression resulted in the downregulation of Pdcd1 expression. Epigenetic analyses indicated that Cbx4 deficiency leads to diminished accumulation of inhibitory histone modifications at conserved region (CR)-C and CR-B sites of the Pdcd1 promoter, namely mono-ubiquitinated histone H2A at lysine 119 (H2AK119ub1) and trimethylated histone H3 at lysine 27 (H3K27me3). Moreover, inhibition of either the E3 ligase activity of polycomb-repressive complexes 1 (PRC1) or the methyltransferase activity of polycomb-repressive complexes 2 (PRC2) restores Pdcd1 expression in Cbx4-transfected cells. Cumulatively, this study reveals a novel function of CBX4 in the regulation of T-cell function and expands our understanding of the epigenetic control of Pdcd1 expression.

Paternal developmental thyrotoxicosis disrupts neonatal leptin leading to increased adiposity and altered physiology of the melanocortin system.

Background: The genetic code does not fully explain individual variability and inheritance of susceptibility to endocrine conditions, suggesting the contribution of epigenetic factors acting across generations. Methods: We used a mouse model of developmental thyrotoxicosis (Dio3-/- mouse) to analyze endocrine outcomes in the adult offspring of Dio3-/- males using standard methods for body composition, and baseline and fasting hormonal and gene expression determinations in serum and tissues of relevance to the control of energy balance. Results: Compared to controls, adult females with an exposed father (EF females) exhibited higher body weight and fat mass, but not lean mass, a phenotype that was much milder in EF males. After fasting, both EF females and males exhibited a more pronounced decrease in body weight than controls. EF females also showed markedly elevated serum leptin, increased white adipose tissue mRNA expression of leptin and mesoderm-specific transcript but decreased expression of type 2 deiodinase. EF females exhibited decreased serum ghrelin, which showed more pronounced post-fasting changes in EF females than in control females. EF female hypothalami also revealed significant decreases in the expression of pro-opiomelanocortin, agouti-related peptide, neuropeptide Y and melanocortin receptor 4. These markers also showed larger changes in response to fasting in EF females than in control females. Adult EF females showed no abnormalities in serum thyroid hormones, but pituitary expression of thyrotropin-releasing hormone receptor 1 and thyroid gland expression of thyroid-stimulating hormone receptor, thyroid peroxidase and iodotyrosine deiodinase were increased at baseline and showed differential regulation after fasting, with no increase in Trhr1 expression and more pronounced reductions in Tshr, Tpo and Iyd. In EF males, these abnormalities were generally milder. In addition, postnatal day 14 (P14) serum leptin was markedly reduced in EF pups. Discussion: A paternal excess of thyroid hormone during development modifies the endocrine programming and energy balance in the offspring in a sexually dimorphic manner, with baseline and dynamic range alterations in the leptin-melanocortin system and thyroid gland, and consequences for adiposity phenotypes. We conclude that thyroid hormone overexposure may have important implications for the non-genetic, inherited etiology of endocrine and metabolic pathologies.

Poleward shifts in the maximum of spring phenological responsiveness of Ginkgo biloba to temperature in China.

Global warming is advancing the timing of spring leaf-out in temperate and boreal plants, affecting biological interactions and global biogeochemical cycles. However, spatial variation in spring phenological responsiveness to climate change within species remains poorly understood. Here, we investigated variation in the responsiveness of spring phenology to temperature (RSP; days to leaf-out at a given temperature) in 2754 Ginkgo biloba twigs of trees distributed across subtropical and temperate regions in China from 24°N to 44°N. We found a nonlinear effect of mean annual temperature on spatial variation in RSP, with the highest response rate at c. 12°C and lower response rates at warmer or colder temperatures due to declines in winter chilling accumulation. We then predicted the spatial maxima in RSP under current and future climate scenarios, and found that trees are currently most responsive in central China, which corresponds to the species' main distribution area. Under a high-emission scenario, we predict a 4-degree latitude shift in the responsiveness maximum toward higher latitudes over the rest of the century. The identification of the nonlinear responsiveness of spring phenology to climate gradients and the spatial shifts in phenological responsiveness expected under climate change represent new mechanistic insights that can inform models of spring phenology and ecosystem functioning.

Different responses of alpine plants to natural climate change reduced coexistence through phenological niche overlap.

Plant phenology is the bridge between climate change and ecosystem functions. Time coordination of interspecific and intraspecific phenology changes overlap or separate can be regarded as an important characteristic of species coexistence. To confirm the hypothesis that plant phenological niche promotes species coexistence, three key alpine plants, Kobresia humilis (sedge), Stipa purpurea (grass), and Astragalus laxmannii (forb) were investigated in this study in the Qinghai-Tibet Plateau. Phenological niches represented as the duration of green up-flowering, flowering-fruiting, and fruiting-withering by 2-day intervals for phenological dynamics of three key alpine plants from 1997 to 2016. We found the role of precipitation on regulating the phenological niches of alpine plants was highlighted in the context of climate warming. The response of the intraspecific phenological niche of the three species to temperature and precipitation is different, and the phenological niche of Kobresia humilis and Stipa purpurea was separate, especially in the green up-flowering. But the overlapping degree of interspecific phenological niche of the three species has continued to increase in the past 20 years, reducing possibility of species coexistence. Our findings have profound implications for understanding the adaptation strategies of key alpine plants to climate change in the dimension of phenological niche.

The PRAK-NRF2 axis promotes the differentiation of Th17 cells by mediating the redox homeostasis and glycolysis.

Oxidative stress is a key feature in both chronic inflammation and cancer. P38 regulated/activated protein kinase (PRAK) deficiency can cause functional disorders in neutrophils and macrophages under high oxidative stress, but the precise mechanisms by which PRAK regulates reactive oxygen species (ROS) elimination and its potential impact on CD4+ T helper subset function are unclear. The present study reveals that the PRAK-NF-E2-related factor 2(NRF2) axis is essential for maintaining the intracellular redox homeostasis of T helper 17(Th17) cells, thereby promoting Th17 cell differentiation and antitumor effects. Through mechanistic analysis, we identify NRF2 as a novel protein substrate of PRAK and find that PRAK enhances the stability of the NRF2 protein through phosphorylation NRF2 Serine(S) 558 independent of protein ubiquitination. High accumulation of cellular ROS caused by loss of PRAK disrupts both glycolysis and PKM2-dependent phosphorylation of STAT3, which subsequently impairs the differentiation of Th17 cells. As a result, Prak knockout (KO) mice display significant resistance to experimental autoimmune encephalomyelitis (EAE) but impaired antitumor immunity in a MC38 tumor model. This work reveals that the PRAK-NRF2-mediated antioxidant pathway is a metabolic checkpoint that controls Th17-cell glycolysis and differentiation. Targeting PRAK is a promising strategy for maintaining an active ROS scavenging system and may lead to potent Th17 cell antitumor immunity.

Developmental thyroid hormone action on pro-opiomelanocortin-expressing cells programs hypothalamic BMPR1A depletion and brown fat activation.

Thyroid hormone excess secondary to global type 3 deiodinase (DIO3) deficiency leads to increased locomotor activity and reduced adiposity, but also to concurrent alterations in parameters of the leptin-melanocortin system that would predict obesity. To distinguish the underlying contributions to the energy balance phenotype of DIO3 deficiency, we generated mice with thyroid hormone excess targeted to pro-opiomelanocortin (POMC)-expressing cells via cell-specific DIO3 inactivation. These mice exhibit a male-specific phenotype of reduced hypothalamic Pomc expression, hyperphagia, and increased activity in brown adipose tissue, with adiposity and serum levels of leptin and thyroid hormones remained normal. These male mice also manifest a marked and widespread hypothalamic reduction in the expression of bone morphogenetic receptor 1a (BMPR1A), which has been shown to cause similar phenotypes when inactivated in POMC-expressing cells. Our results indicate that developmental overexposure to thyroid hormone in POMC-expressing cells programs energy balance mechanisms in a sexually dimorphic manner by suppressing adult hypothalamic BMPR1A expression.

Global warming is increasing the discrepancy between green (actual) and thermal (potential) seasons of temperate trees.

Over the past decades, global warming has led to a lengthening of the time window during which temperatures remain favorable for carbon assimilation and tree growth, resulting in a lengthening of the green season. The extent to which forest green seasons have tracked the lengthening of this favorable period under climate warming, however, has not been quantified to date. Here, we used remote sensing data and long-term ground observations of leaf-out and coloration for six dominant species of European trees at 1773 sites, for a total of 6060 species-site combinations, during 1980-2016 and found that actual green season extensions (GS: 3.1 ± 0.1 day decade-1 ) lag four times behind extensions of the potential thermal season (TS: 12.6 ± 0.1 day decade-1 ). Similar but less pronounced differences were obtained using satellite-derived vegetation phenology observations, that is, a lengthening of 4.4 ± 0.13 and 7.5 ± 0.13 day decade-1 for GS and TS, respectively. This difference was mainly driven by the larger advance in the onset of the thermal season compared to the actual advance of leaf-out dates (spring mismatch: 7.2 ± 0.1 day decade-1 ), but to a less extent caused by a phenological mismatch between GS and TS in autumn (2.4 ± 0.1 day decade-1 ). Our results showed that forest trees do not linearly track the new thermal window extension, indicating more complex interactions between winter and spring temperatures and photoperiod and a justification of demonstrating that using more sophisticated models that include the influence of chilling and photoperiod is needed to accurately predict spring phenological changes under warmer climate. They urge caution if such mechanisms are omitted to predict, for example, how vegetative health and growth, species distribution and crop yields will change in the future.

Soil moisture regulates warming responses of autumn photosynthetic transition dates in subtropical forests.

Autumn phenology plays a key role in regulating the terrestrial carbon and water balance and their feedbacks to the climate. However, the mechanisms underlying autumn phenology are still poorly understood, especially in subtropical forests. In this study, we extracted the autumn photosynthetic transition dates (APTD) in subtropical China over the period 2003-2017 based on a global, fine-resolution solar-induced chlorophyll fluorescence (SIF) dataset (GOSIF) using four fitting methods, and then explored the temporal-spatial variations of APTD and its underlying mechanisms using partial correlation analysis and machine learning methods. We further predicted the APTD shifts under future climate warming conditions by applying process-based and machine learning-based models. We found that the APTD was significantly delayed, with an average rate of 7.7 days per decade, in subtropical China during 2003-2017. Both partial correlation analysis and machine learning methods revealed that soil moisture was the primary driver responsible for the APTD changes in southern subtropical monsoon evergreen forest (SEF) and middle subtropical evergreen forest (MEF), whereas solar radiation controlled the APTD variations in the northern evergreen-broadleaf deciduous mixed forest (NMF). Combining the effects of temperature, soil moisture and radiation, we found a significantly delayed trend in APTD during the 2030-2100 period, but the trend amplitude (0.8 days per decade) was much weaker than that over 2003-2017. In addition, we found that machine learning methods outperformed process-based models in projecting APTD. Our findings generate from different methods highlight that soil moisture is one of the key players in determining autumn photosynthetic phenological processes in subtropical forests. To comprehensively understand autumn phenological processes, in-situ manipulative experiments are urgently needed to quantify the contributions of different environmental and physiological factors in regulating plants' response to ongoing climate change.

Spatial Difference of Interactive Effect Between Temperature and Daylength on Ginkgo Budburst.

Climate warming-induced shifts in spring phenology have substantially affected the structure and function of terrestrial ecosystems and global biogeochemical cycles. Spring phenology is primarily triggered by spring temperature and is also affected by daylength and winter chilling, yet the relative importance of these cues across spatial gradients remains poorly understood. Here, we conducted a manipulative experiment with two daylength and three temperature treatments to investigate spatial differences in the response of ginkgo budburst to temperature and daylength, using twigs collected at three sites across a spatial gradient: a control site at a low latitude and low elevation on Tianmu Mountain (TMlow), a low latitude and high elevation site on Tianmu Mountain (TMhigh), and a high latitude site on Jiufeng mountain (JF). The mechanisms were also tested using in situ phenological observations of ginkgo along latitudes in China. We found that, compared to TMlow individuals, budburst dates occurred 12.6 (JF) and 7.7 (TMhigh) days earlier in high-latitude and high-elevation individuals when exposed to the same temperature and daylength treatments. Importantly, daylength only affected budburst at low latitudes, with long days (16 h) advancing budburst in low-latitude individuals by, on average, 8.1 days relative to short-day (8 h) conditions. This advance was most pronounced in low-elevation/latitude individuals (TMlow = 9.6 days; TMhigh = 6.7 days; JF = 1.6 days). In addition, we found that the temperature sensitivity of budburst decreased from 3.4 to 2.4 days °C-1 along latitude and from 3.4 to 2.5 days °C-1 along elevation, respectively. The field phenological observations verified the experimental results. Our findings provide empirical evidence of spatial differences in the relative effects of spring temperature and daylength on ginkgo budburst, which improved our understanding of spatial difference in phenological changes and the responses of terrestrial ecosystem to climate change.

Climate warming shifts the time interval between flowering and leaf unfolding depending on the warming period.

The timing of flowering (FL) and leaf unfolding (LU) determine plants' reproduction and vegetative growth. Global warming has substantially advanced FL and LU of temperate and boreal plants, but their responses to warming differ, which may influence the time interval between FL and LU (∆LU-FL), thereby impacting plant fitness and intraspecific physiological processes. Based on twigs collected from two flowering-first tree species, Populus tomentosa and Amygdalus triloba, we conducted a manipulative experiment to investigate the effects of winter chilling, spring warming and photoperiod on the ∆LU-FL. We found that photoperiod did not affect the ∆LU-FL of Amygdalus triloba, but shortened ∆LU-FL by 5.1 d of Populus tomentosa. Interestingly, spring warming and winter chilling oppositely affected the ∆LU-FL of both species. Specifically, low chilling accumulation extended the ∆LU-FL by 3.8 and 9.4 d for Populus tomentosa and Amygdalus triloba, but spring warming shortened the ∆LU-FL by 4.1 and 0.2 d °C-1. Our results indicate that climate warming will decrease or increase the ∆LU-FL depending on the warming periods, i.e., spring or winter. The shifted time interval between flowering and leaf unfolding may have ecological effects including affecting pollen transfer efficiency and alter the structure and functioning of terrestrial ecosystem.

Modified Red Blue Vegetation Index for Chlorophyll Estimation and Yield Prediction of Maize from Visible Images Captured by UAV.

The vegetation index (VI) has been successfully used to monitor the growth and to predict the yield of agricultural crops. In this paper, a long-term observation was conducted for the yield prediction of maize using an unmanned aerial vehicle (UAV) and estimations of chlorophyll contents using SPAD-502. A new vegetation index termed as modified red blue VI (MRBVI) was developed to monitor the growth and to predict the yields of maize by establishing relationships between MRBVI- and SPAD-502-based chlorophyll contents. The coefficients of determination (R2s) were 0.462 and 0.570 in chlorophyll contents' estimations and yield predictions using MRBVI, and the results were relatively better than the results from the seven other commonly used VI approaches. All VIs during the different growth stages of maize were calculated and compared with the measured values of chlorophyll contents directly, and the relative error (RE) of MRBVI is the lowest at 0.355. Further, machine learning (ML) methods such as the backpropagation neural network model (BP), support vector machine (SVM), random forest (RF), and extreme learning machine (ELM) were adopted for predicting the yields of maize. All VIs calculated for each image captured during important phenological stages of maize were set as independent variables and the corresponding yields of each plot were defined as dependent variables. The ML models used the leave one out method (LOO), where the root mean square errors (RMSEs) were 2.157, 1.099, 1.146, and 1.698 (g/hundred grain weight) for BP, SVM, RF, and ELM. The mean absolute errors (MAEs) were 1.739, 0.886, 0.925, and 1.356 (g/hundred grain weight) for BP, SVM, RF, and ELM, respectively. Thus, the SVM method performed better in predicting the yields of maize than the other ML methods. Therefore, it is strongly suggested that the MRBVI calculated from images acquired at different growth stages integrated with advanced ML methods should be used for agricultural- and ecological-related chlorophyll estimation and yield predictions.

Thyroid hormone influences brain gene expression programs and behaviors in later generations by altering germ line epigenetic information.

Genetic factors do not fully account for the relatively high heritability of neurodevelopmental conditions, suggesting that non-genetic heritable factors contribute to their etiology. To evaluate the potential contribution of aberrant thyroid hormone status to the epigenetic inheritance of neurological phenotypes, we examined genetically normal F2 generation descendants of mice that were developmentally overexposed to thyroid hormone due to a Dio3 mutation. Hypothalamic gene expression profiling in postnatal day 15 F2 descendants on the paternal lineage of ancestral male and female T3-overexposed mice revealed, respectively, 1089 and 1549 differentially expressed genes. A large number of them, 675 genes, were common to both sets, suggesting comparable epigenetic effects of thyroid hormone on both the male and female ancestral germ lines. Oligodendrocyte- and neuron-specific genes were strongly overrepresented among genes showing, respectively, increased and decreased expression. Altered gene expression extended to other brain regions and was associated in adulthood with decreased anxiety-like behavior, increased marble burying and reduced physical activity. The sperm of T3-overexposed male ancestors revealed significant hypomethylation of CpG islands associated with the promoters of genes involved in the early development of the central nervous system. Some of them were candidates for neurodevelopmental disorders in humans including Nrg3, Nrxn1, Gabrb3, Gabra5, Apba2, Grik3, Reln, Nsd1, Pcdh8, En1, and Elavl2. Thus, developmental levels of thyroid hormone influence the epigenetic information of the germ line, disproportionately affecting genes with critical roles in early brain development, and leading in future generations to disease-relevant alterations in postnatal brain gene expression and adult behavior.

Type 3 Deiodinase Role on Central Thyroid Hormone Action Affects the Leptin-Melanocortin System and Circadian Activity.

The role of thyroid hormones (THs) in the central regulation of energy balance is increasingly appreciated. Mice lacking the type 3 deiodinase (DIO3), which inactivates TH, have decreased circulating TH levels relative to control mice as a result of defects in the hypothalamic-pituitary-thyroid axis. However, we have shown that the TH status of the adult Dio3-/- brain is opposite that of the serum, exhibiting enhanced levels of TH action. Because the brain, particularly the hypothalamus, harbors important circuitries that regulate metabolism, we aimed to examine the energy balance phenotype of Dio3-/- mice and determine whether it is associated with hypothalamic abnormalities. Here we show that Dio3-/- mice of both sexes exhibit decreased adiposity, reduced brown and white adipocyte size, and enhanced fat loss in response to triiodothyronine (T3) treatment. They also exhibit increased TH action in the hypothalamus, with abnormal expression and T3 sensitivity of genes integral to the leptin-melanocortin system, including Agrp, Npy, Pomc, and Mc4r. The normal to elevated serum levels of leptin, and elevated and repressed expression of Agrp and Pomc, respectively, suggest a profile of leptin resistance. Interestingly, Dio3-/- mice also display elevated locomotor activity and increased energy expenditure. This occurs in association with expanded nighttime activity periods, suggesting a disrupted circadian rhythm. We conclude that DIO3-mediated regulation of TH action in the central nervous system influences multiple critical determinants of energy balance. Those influences may partially compensate each other, with the result likely contributing to the decreased adiposity observed in Dio3-/- mice.

Hypothalamic Non-AgRP, Non-POMC GABAergic Neurons Are Required for Postweaning Feeding and NPY Hyperphagia.

The hypothalamus is critical for feeding and body weight regulation. Prevailing studies focus on hypothalamic neurons that are defined by selectively expressing transcription factors or neuropeptides including those expressing proopiomelanocortin (POMC) and agouti-related peptides (AgRP). The Cre expression driven by the pancreas-duodenum homeobox 1 promoter is abundant in several hypothalamic nuclei but not in AgRP or POMC neurons. Using this line, we generated mice with disruption of GABA release from a major subset of non-POMC, non-AgRP GABAergic neurons in the hypothalamus. These mice exhibited a reduction in postweaning feeding and growth, and disrupted hyperphagic responses to NPY. Disruption of GABA release severely diminished GABAergic input to the paraventricular hypothalamic nucleus (PVH). Furthermore, disruption of GABA-A receptor function in the PVH also reduced postweaning feeding and blunted NPY-induced hyperphagia. Given the limited knowledge on postweaning feeding, our results are significant in identifying GABA release from a major subset of less appreciated hypothalamic neurons as a key mediator for postweaning feeding and NPY hyperphagia, and the PVH as one major downstream site that contributes significantly to the GABA action. Significance statement: Prevalent studies on feeding in the hypothalamus focus on well characterized, selective groups neurons [e.g., proopiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons], and as a result, the role of the majority of other hypothalamic neurons is largely neglected. Here, we demonstrated an important role for GABAergic projections from non-POMC non-AgRP neurons to the paraventricular hypothalamic nucleus in promoting postweaning (mainly nocturnal) feeding and mediating NPY-induced hyperphagia. Thus, these results signify an importance to study those yet to be defined hypothalamic neurons in the regulation of energy balance and reveal a neural basis for postweaning (nocturnal) feeding and NPY-mediated hyperphagia.

GABAergic projections from lateral hypothalamus to paraventricular hypothalamic nucleus promote feeding.

Lesions of the lateral hypothalamus (LH) cause hypophagia. However, activation of glutamatergic neurons in LH inhibits feeding. These results suggest a potential importance for other LH neurons in stimulating feeding. Our current study in mice showed that disruption of GABA release from adult LH GABAergic neurons reduced feeding. LH GABAergic neurons project extensively to the paraventricular hypothalamic nucleus (PVH), and optogenetic stimulation of GABAergic LH → PVH fibers induced monosynaptic IPSCs in PVH neurons, and potently increased feeding, which depended on GABA release. In addition, disruption of GABA-A receptors in the PVH reduced feeding. Thus, we have identified a new feeding pathway in which GABAergic projections from the LH to the PVH promote feeding.

Mitochondrial toxic effects of Aß through mitofusins in the early pathogenesis of Alzheimer's disease.

Mitochondrial dysfunction has been implicated in the pathogenesis of Alzheimer's disease (AD). However, it is obscure how amyloid-beta (Aß) can impair mitochondria in the early stage of AD pathology. Using PrP-hAPP/hPS1 double-transgenic AD mouse model, we find that abnormal mitochondrial morphology and damaged mitochondrial structure in hippocampal neurons appear in the early stage of AD-like disease development. We also find consistent mitochondrial abnormalities in the SH-SY5Y cells, which express amyloid precursor protein (APP) Swedish mutation (APPsw) and have been used as a cell model of the early-onset AD. Significant changes of mitofusin GTPases (Mfn1 and Mfn2) were detected both in the PrP-hAPP/hPS1 brains and SH-SY5Y cells. Moreover, our results show that Aß accumulation in neurons of PrP-hAPP/hPS1 mice can affect the neurogenesis prior to plaque formation. These findings suggest that mitochondrial impairment is a very early event in AD pathogenesis and abnormal expression of Mfn1 and Mfn2 caused by excessive intracellular Aß is the possible molecular mechanism. Interestingly, L-theanine has significant effects on regulating mitochondrial fusion proteins in SH-SY5Y (APPsw) cells. Overall, our results not only suggest a new early mechanism of AD pathogenesis but also propose a preventive candidate, L-theanine, for the treatment of AD.

Glutamate mediates the function of melanocortin receptor 4 on Sim1 neurons in body weight regulation.

The melanocortin receptor 4 (MC4R) is a well-established mediator of body weight homeostasis. However, the neurotransmitter(s) that mediate MC4R function remain largely unknown; as a result, little is known about the second-order neurons of the MC4R neural pathway. Single-minded 1 (Sim1)-expressing brain regions, which include the paraventricular nucleus of hypothalamus (PVH), represent key brain sites that mediate melanocortin action. We conditionally restored MC4R expression in Sim1 neurons in the background of Mc4r-null mice. The restoration dramatically reduced obesity in Mc4r-null mice. The anti-obesity effect was completely reversed by selective disruption of glutamate release from those same Sim1 neurons. The reversal was caused by lower energy expenditure and hyperphagia. Corroboratively, selective disruption of glutamate release from adult PVH neurons led to rapid obesity development via reduced energy expenditure and hyperphagia. Thus, this study establishes glutamate as the primary neurotransmitter that mediates MC4Rs on Sim1 neurons in body weight regulation.

Ubiquitylation of phosphatidylinositol 4-phosphate 5-kinase type I γ by HECTD1 regulates focal adhesion dynamics and cell migration.

Phosphatidylinositol 4-phosphate 5-kinase type I γ (PIPKIγ90) binds talin and localizes at focal adhesions (FAs). Phosphatidylinositol (4,5)-bisphosphate (PIP2) generated by PIPKIγ90 is essential for FA formation and cell migration. On the other hand, PIPKIγ90 and the ß-integrin tail compete for overlapping binding sites on talin. Enhanced PIPKIγ90-talin interaction suppresses talin binding to the ß-integrin. It is unknown how PIPKIγ90 is removed from the PIPKIγ90-talin complex after on-site PIP2 production during cell migration. Here we show that PIPKIγ90 is a substrate for HECTD1, an E3 ubiquitin ligase regulating cell migration. HECTD1 ubiquitinated PIPKIγ90 at lysine 97 and resulted in PIPKIγ90 degradation. Expression of the mutant PIPKIγ90(K97R) enhanced PIP2 and PIP3 production, inhibited FA assembly and disassembly and inhibited cancer cell migration, invasion and metastasis. Interestingly, mutation at tryptophan 647 abolished the inhibition of PIPKIγ90(K97R) on FA dynamics and partially rescued cancer cell migration and invasion. Thus, cycling PIPKIγ90 ubiquitylation by HECTD1 and consequent degradation remove PIPKIγ90 from talin after on-site PIP2 production, providing an essential regulatory mechanism for FA dynamics and cell migration.