Calorie restriction (CR) improves health and extends life span in a variety of species. Despite many downstream molecules and physiological systems having been identified as being regulated by CR, the mechanism by which CR extends life span remains unclear. The Drosophila gene Indy (for I'm not dead yet), involved in the transport and storage of Krebs cycle intermediates in tissues important in fly metabolism, was proposed to regulate life span via an effect on metabolism that could overlap with CR. In this study, we report that CR down regulates Indy mRNA expression, and that CR and the level of Indy expression interact to affect longevity. Optimal life span extension is seen when Indy expression is decreased between 25 and 75% of normal. Indy long-lived flies show several phenotypes that are shared by long-lived CR flies, including decreased insulin-like signaling, lipid storage, weight gain, and resistance to starvation as well as an increase in spontaneous physical activity. We conclude that Indy and CR interact to affect longevity and that a decrease in Indy may induce a CR-like status that confers life span extension.Drosophila ͉ insulin ͉ physical activity ͉ triglyceride A ging is a complex biological process that causes deteriorative changes over time. It has been suggested that the interplay between environmental factors and genetic alterations may affect this near universal process. Calorie restriction (CR) is the most widely recognized life span-extending intervention, and it has been shown to extend lifespan in a variety of different organisms (1, 2). Progress has been made in identifying genes that regulate longevity, and many of them appear to belong to pathways related to nutrient sensing, metabolism or nutrient/ metabolic signaling (3-7). The life span extending effects of a subset of these longevity genes has been shown to be associated with, and in some cases, causally related to CR life span extension (chico, Sir2, p53). Studies have suggested that alterations in the activity of these genes may mediate elements of the normal CR life span extending effect. Despite these advances little is understood about the molecular and genetic mechanisms underlying the healthy life span extension of CR.In Drosophila melanogaster, mutations in the Indy gene dramatically extend life span (8). The INDY protein is a transmembrane transporter of Krebs cycle intermediates (citrate, succinate, fumarate, and alpha-ketoglutarate) predominantly found at the plasma membrane of cells in the midgut, fat body, and oenocytes, tissues important for the uptake, utilization, and storage of nutrients and the principal sites of intermediary metabolism in the fly (8-10). Several independently derived lines, each with a P-element in the non-coding region of the Indy locus leading to decreased expression of Indy mRNA, have been shown to extend life span. It has been reported that life span extension is seen even when the Indy mutation is crossed into different genetic backgrounds (e.g., Hyperkinetic, Shaker, drop dead, and the lo...

The survival of motor neurons is controlled by multiple factors that regulate different aspects of their physiology. The identification of these factors is important because of their relationship to motor neuron disease. We investigate here whether Mullerian Inhibiting Substance (MIS) is a motor neuron survival factor. We find that motor neurons from adult mice synthesize MIS and express its receptors, suggesting that mature motor neurons use MIS in an autocrine fashion or as a way to communicate with each other. MIS was observed to support the survival and differentiation of embryonic motor neurons in vitro. During development, male-specific MIS may have a hormone effect because the blood-brain barrier has yet to form, raising the possibility that MIS participates in generating sex-specific differences in motor neurons.Mullerian Inhibiting Substance type II receptor M otor neurons are particularly prone to age-related deterioration (1-3), which, in the extreme, leads to motor neuron disease and to death by paralysis. The survival of motor neurons is controlled by multiple factors, each of which appears to have a different physiological role. Motor neurons are, for instance, regulated by skeletal muscle fibers and Schwann cells via cardiotrophin-1 (4), TGF-␤2 (5, 6), and glial-cell-line-derived neurotrophic factor (GDNF) (7,8). Motor neurons also receive protection against viral-and hypoxic-induced damage through IL-6 (9) and VEGF (10, 11), respectively. Variations in the VEGF gene cause adult-onset motor-neuron degeneration in some mice and have been linked to ALS in some human populations (10, 11). These findings have renewed interest in identifying nonclassical neuronal survival factors.Mullerian Inhibiting Substance (MIS) is examined herein as a motor-neuron survival factor given that we found high expression of ligand and receptors in motor neurons. MIS is a member of the TGF-␤ superfamily, which includes motor-neuron survival factors, such as GDNF and TGF-␤2. The known physiological actions of MIS are thought to be limited to sexual differentiation of males and to the function of mature reproductive tissues of both sexes (12). These studies introduce a possible function for this interesting molecule and its known signaling pathway.TGF-␤ superfamily members signal through a complex of type I and type II receptors (13). MIS has a unique type II receptor (MISRII) but shares type I receptors with other members of the superfamily (12, 13). Genetic, organ culture, and cellular evidence implicates activin receptor-like kinase 3 (ALK3) (14) and ALK2 (Y. Zhan, D.T.M., and P.K.D., unpublished data) (15) as type I receptors for MIS in murine sexual differentiation, although ALK6 is likely to be involved in other cellular contexts (12, 16).We find that adult motor neurons from male and female mice synthesize MIS and its receptors, with the MIS receptor mRNA in motor neurons being much more abundant than the mRNAs for the GDNF and TGF-␤ receptors. Our experiments show that MIS supports the survival of embryonic motor n...

Many behavioral traits and most brain disorders are common to males and females but are more evident in one sex than the other. The control of these subtle sex-linked biases is largely unstudied and has been presumed to mirror that of the highly dimorphic reproductive nuclei. Sexual dimorphism in the reproductive tract is a product of Mü llerian inhibiting substance (MIS), as well as the sex steroids. Males with a genetic deficiency in MIS signaling are sexually males, leading to the presumption that MIS is not a neural regulator. We challenge this presumption by reporting that most immature neurons in mice express the MIS-specific receptor (MISRII) and that male Mis ؊/؊ and Misrii ؊/؊ mice exhibit subtle feminization of their spinal motor neurons and of their exploratory behavior. Consequently, MIS may be a broad regulator of the subtle sex-linked biases in the nervous system.anti-Mü llerian hormone ͉ exploratory behavior ͉ motor neuron ͉ sexual dimorphism

Decreased Indy activity extends lifespan in D. melanogaster without significant reduction in fecundity, metabolic rate, or locomotion. To understand the underlying mechanisms leading to lifespan extension in this mutant strain, we compared the genome-wide gene expression changes in the head and thorax of adult Indy mutant with control flies over the course of their lifespan. A signature enrichment analysis of metabolic and signaling pathways revealed that expression levels of genes in the oxidative phosphorylation pathway are significantly lower in Indy starting at day 20. We confirmed experimentally that complexes I and III of the electron transport chain have lower enzyme activity in Indy longlived flies by Day 20 and predicted that reactive oxygen species (ROS) production in mitochondria could be reduced. Consistently, we found that both ROS production and protein damage are reduced in Indy with respect to control. However, we did not detect significant differences in total ATP, a phenotype that could be explained by our finding of a higher mitochondrial density in Indy mutants. Thus, one potential mechanism by which Indy mutants extend life span could be through an alteration in mitochondrial physiology leading to an increased efficiency in the ATP/ROS ratio.electron transport chain ͉ mitochondria ͉ oxidative phosphorylation ͉ Drosophila ͉ aging U nderstanding the biological and physiological underpinnings of aging and the development of interventions to ameliorate its deleterious effects has been the subject of much interest. Identification and examination of specific genetic alterations that extend life span is one common approach for uncovering mechanisms underlying normal aging. A number of single gene alterations that extend healthy life span in model organisms have been isolated, but delineating the specific physiological changes responsible for their life span extending effects has been challenging (1).High throughput genomic analyses have emerged as an unbiased method for providing information of the physiological changes involved in mediating complex biological phenomena such as longevity. In particular, whole genome transcriptional studies (microarrays) to identify specific genes or physiological systems important in longevity determination have been successfully used in both nematodes and flies. Microarray studies of daf-2 long-lived nematodes identified specific genes that were then functionally verified as causally related to the daf-2 life span extension (2).Restricting the examination of microarrays to only those genes identically shared between different interventions may limit the ability to detect some of the physiologically relevant changes important in complex biological phenomena such as life span extension. As more data becomes available from high throughput gene expression studies, analyses have shifted from a gene centric model to a pathway centric approach. It has been realized that reproducibility of experiments, and comparison across interventions that should have resulted in simil...

Breast carcinoma amplified sequence 2 (BCAS2) is a core component of the hPrP19 complex that controls RNA splicing. Here, we performed an exon array assay and showed that β-catenin is a target of BCAS2 splicing regulation. The regulation of dendrite growth and morphology by β-catenin is well documented. Therefore, we generated conditional knockout (cKO) mice to eliminate the BCAS2 expression in the forebrain to investigate the role of BCAS2 in dendrite growth. BCAS2 cKO mice showed a microcephaly-like phenotype with a reduced volume in the dentate gyrus (DG) and low levels of learning and memory, as evaluated using Morris water maze analysis and passive avoidance, respectively. Golgi staining revealed shorter dendrites, less dendritic complexity and decreased spine density in the DG of BCAS2 cKO mice. Moreover, the cKO mice displayed a short dendrite length in newborn neurons labeled by DCX, a marker of immature neurons, and BrdU incorporation. To further examine the mechanism underlying BCAS2-mediated dendritic malformation, we overexpressed β-catenin in BCAS2-depleted primary neurons and found that the dendritic growth was restored. In summary, BCAS2 is an upstream regulator of β-catenin gene expression and plays a role in dendrite growth at least partly through β-catenin.

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BackgroundNuclear receptor interaction protein (NRIP) is a calcium/calmodulin (CaM) binding protein. Nuclear receptor interaction protein interacts with CaM to activate calcineurin and CaMKII signalling. The conventional NRIP knockout mice (global knockout) showed muscular abnormality with reduction of muscle oxidative functions and motor function defects.MethodsTo investigate the role of NRIP on neuromuscular system, we generated muscle‐restricted NRIP knockout mice [conditional knockout (cKO)]. The muscle functions (including oxidative muscle markers and muscle strength) and lumbar motor neuron functions [motor neuron number, axon denervation, neuromuscular junction (NMJ)] were tested. The laser‐captured microdissection at NMJ of skeletal muscles and adenovirus gene therapy for rescued effects were performed.ResultsThe cKO mice showed muscular abnormality with reduction of muscle oxidative functions and impaired motor performances as global knockout mice. To our surprise, cKO mice also displayed motor neuron degeneration with abnormal architecture of NMJ. Specifically, the cKO mice revealed reduced motor neuron number with small neuronal size in lumbar spinal cord as well as denervating change, small motor endplates, and decreased myonuclei number at NMJ in skeletal muscles. To explore the mechanisms, we screened various muscle‐derived factors and found that myogenin is a potential candidate that myogenin expression was lower in skeletal muscles of cKO mice than wild‐type mice. Because NRIP and myogenin were colocalized around acetylcholine receptors at NMJ, we extracted RNA from synaptic and extrasynaptic regions of muscles using laser capture microdissection and showed that myogenin expression was especially lower at synaptic region in cKO than wild‐type mice. Notably, overexpression of myogenin using intramuscular adenovirus encoding myogenin treatment rescued abnormal NMJ architecture and preserved motor neuron death in cKO mice.ConclusionsIn summary, we demonstrated that deprivation of NRIP decreases myogenin expression at NMJ, possibly leading to abnormal NMJ formation, denervation of acetylcholine receptor, and subsequent loss of spinal motor neuron. Overexpression of myogenin in cKO mice can partially rescue abnormal NMJ architecture and motor neuron death. Therefore, muscular NRIP is a novel trophic factor supporting spinal motor neuron via stabilization of NMJ by myogenin expression.

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