Nutrient Deprivation Induces Neuronal Autophagy and Implicates Reduced Insulin Signaling in Neuroprotective Autophagy Activation

Although autophagy maintains normal neural function by degrading misfolded proteins, little is known about how neurons activate this integral response. Furthermore, classical methods of autophagy induction used with nonneural cells, such as starvation, simply result in neuron death. To study neuronal autophagy, we cultured primary cortical neurons from transgenic mice that ubiquitously express green fluorescent protein-tagged LC3 and monitored LC3-I to LC3-II conversion by immunohistochemistry and immunoblotting. Evaluation of different culture media led us to discover that culturing primary neurons in Dulbecco''s modified Eagle''s medium without B27 supplementation robustly activates autophagy. We validated this nutrient-limited media approach for inducing autophagy by showing that 3-methyl-adenine treatment and Atg5 RNA interference knockdown each inhibits LC3-I to LC3-II conversion. Evaluation of B27 supplement components yielded insulin as the factor whose absence induced autophagy in primary neurons, and this activation was mammalian target of rapamycin-dependent. When we tested if nutrient-limited media could protect neurons expressing polyglutamine-expanded proteins against cell death, we observed a strong protective effect, probably due to autophagy activation. Our results indicate that nutrient deprivation can be used to understand the regulatory basis of neuronal autophagy and implicate diminished insulin signaling in the activation of neuronal autophagy.Most neurodegenerative disorders are characterized by the accumulation of misfolded proteins that coalesce into “inclusions” and become visible at the light microscope level in the brains and spinal cords of affected patients (1, 2). These inclusions manifest themselves pathologically in Alzheimer disease as extracellular plaques and neurofibrillary tangles, in Parkinson disease as Lewy bodies, and in poly(Q) repeat diseases as cytosolic and nuclear aggregates. A fundamental advance in our understanding of neurodegeneration has been the realization that protein misfolding is a common theme in many important neurological disorders, including Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, prion diseases, and poly(Q) diseases. The mechanistic underpinning of this “proteinopathy” hypothesis stems from the exquisite susceptibility of postmitotic cells in the central nervous system to misfolded protein stress, since neural cells are not continually replenished by cell division, unlike most of their nonneural counterparts.The ubiquitin-proteasome system is the main intracellular degradation pathway to remove short lived proteins and to eliminate peptides that exit from the protein-folding machinery of the endoplasmic reticulum with an aberrant conformation. However, many aggregate-prone proteins, such as poly(Q) proteins, are inefficiently degraded by the proteasome (3–5). Failure of adequate degradation of aggregate-prone proteins activates alternative protein turnover pathways in the cell, including macroautophagy (hereafter referred to as autophagy). Autophagy is a degradative process that begins with engulfment of cytosolic materials and/or organelles and progresses through a series of steps involving production of a double membrane bound structure, culminating in the delivery of the engulfed material to lysosomes (6). In the central nervous system, basal levels of autophagy are required for the continued health and normal function of neurons, since conditional inactivation of the autophagy pathway in neural cells in mice yields neuronal dysfunction and neurodegeneration characterized by the accumulation of proteinaceous material (7, 8). Furthermore, the presence of aggregate-prone proteins, not degraded by the proteasome, induces autophagy above basal levels, and activation of autophagy appears capable of clearing misfolded proteins, decreasing cytotoxicity, and preventing neurodegeneration in Drosophila and mouse models of misfolded protein stress (9–11).Although numerous reports have documented the protective effects of inducing autophagy in different areas of the diseased brain in model organisms (reviewed in Ref. 12), little is known about how neurons activate this integral response. Indeed, classical methods of autophagy induction used with cultured nonneural cells, such as starvation, simply result in the death of cultured primary neurons. Furthermore, starvation elicits quite different effects in neurons and nonneural cells, both in vitro and in vivo (13, 14). To directly study neuronal autophagy, we devised a primary neuron culture system where we can induce autophagy activation by withdrawal of a key supplement from the culture media. After independently validating the activation of autophagy in our system through pharmacological and genetic inhibition, we identified insulin as the factor responsible for autophagy induction in primary cortical neurons grown in nutrient-limited media. Further characterization of autophagy induction in primary neurons subjected to nutrient deprivation indicated that such autophagy activation is mammalian target of rapamycin (mTOR)²-dependent. We then tested if the autophagy response induced by nutrient deprivation could counter misfolded protein stress by expressing a poly(Q)-expanded protein in primary neurons and found that nutrient limitation prevented neuron cell death caused by misfolded protein stress.     

Arginine 104 is a key catalytic residue in leukotriene C4 synthase

Human leukotriene C₄ synthase (hLTC₄S) is an integral membrane enzyme that conjugates leukotriene (LT) A₄ with glutathione to form LTC₄, a precursor to the cysteinyl leukotrienes (LTC₄, LTD₄, and LTE₄) that are involved in the pathogenesis of human bronchial asthma. From the crystal structure of hLTC₄S, Arg-104 and Arg-31 have been implicated in the conjugation reaction. Here, we used site-directed mutagenesis, UV spectroscopy, and x-ray crystallography to examine the catalytic role of Arg-104 and Arg-31. Exchange of Arg-104 with Ala, Ser, Thr, or Lys abolished 94.3–99.9% of the specific activity against LTA₄. Steady-state kinetics of R104A and R104S revealed that the K_m for GSH was not significantly affected. UV difference spectra of the binary enzyme-GSH complex indicated that GSH ionization depends on the presence of Arg-104 because no thiolate signal, with λ_max at 239 nm, could be detected using R104A or R104S hLTC₄S. Apparently, the interaction of Arg-104 with the thiol group of GSH reduces its pK_a to allow formation of a thiolate anion and subsequent nucleophilic attack at C6 of LTA₄. On the other hand, exchange of Arg-31 with Ala or Glu reduced the catalytic activity of hLTC₄S by 88 and 70%, respectively, without significantly affecting the k_cat/K_m values for GSH, and a crystal structure of R31Q hLTC₄S (2.1 Å) revealed a Gln-31 side chain pointing away from the active site. We conclude that Arg-104 plays a critical role in the catalytic mechanism of hLTC₄S, whereas a functional role of Arg-31 seems more elusive. Because Arg-104 is a conserved residue, our results pertain to other homologous membrane proteins and represent a structure-function paradigm probably common to all microsomal GSH transferases.     

Carboxy terminal extended phytocystatins are bifunctional inhibitors of papain and legumain cysteine proteinases

Plant legumains are cysteine proteinases putatively involved in processing endogenous proteins. Phytocystatins (PhyCys) have been described as plant inhibitors of papain-like cysteine proteinases. Some PhyCys contain a carboxy terminal extension with an amino acid motif (SNSL) similar to that involved in the inhibition of legumain-like proteins by human cystatins. The role of these carboxy terminal extended PhyCys as inhibitors of legumain-like cysteine proteinases is here shown by in vitro inhibition of human legumain and legumain-like activities from barley extracts. Moreover, site-directed mutagenesis has demonstrated that the asparagine of the SNSL motif is essential in this inhibition. We prove for first time the existence of legumain inhibitors in plants.     

The high-resolution NMR structure of a single-chain chimeric protein mimicking a SH3-peptide complex

Here we present the high-resolution NMR structure of a chimera (SPCp41) between alpha-spectrin SH3 domain and the decapeptide p41. The tertiary structure mimics perfectly the interactions typically found in SH3-peptide complexes and is remarkably similar to that of the complex between the separate Spc-SH3 domain and ligand p41. Relaxation data confirm the tight binding between the ligand and SH3 part of the chimera. This chimera will serve as a tool for a deeper understanding of the relationship between structure and thermodynamics of binding using a combination of NMR, stability and site-directed mutagenesis studies, which can lead to an effective strategy for ligand design.     

<Emphasis Type="Italic">Turbinaria ornata</Emphasis> invasion in the Tuamotu Archipelago,French Polynesia: ocean drift connectivity

This paper focuses on the invasion by Turbinaria ornata (a brown algae) in the Tuamotu archipelago, French Polynesia [(5–35°S)/(200–230°E)]. Prior to 1980, this alga existed only in the Society and Austral archipelagoes. Between 1985 and 1990, it began to appear in the southern and northern parts of the Tuamotu archipelago. Genetic analyses have been shown not to be appropriate in determining the origin of this algae population. This study investigated the possible ocean drift of floating aggregates of algae. Ocean currents were calculated from satellite data from 1993 to 2001. Their spatial variations as well as their seasonal and interannual variations are described along with calculated drift trajectories. While it was found that mean currents cannot directly transport algae from the Society and Austral archipelagoes to the Tuamotu, the large interannual changes during the El Niño-Southern Oscillation phenomenon produce current reversals that are strong enough to create a transport pathway in a short enough time to allow their survival.     

A C-terminal aldehyde analog of the insect kinins inhibits diuresis in the housefly

The insect kinins are present in a wide variety of insects and function as potent diuretic peptides in flies. A C-terminal aldehyde insect kinin analog, Fmoc-RFFPWG-H (R-LK-CHO), demonstrates stimulation of Malpighian tubule fluid secretion in crickets, but shows inhibition of both in vitro and in vivo diuresis in the housefly. R-LK-CHO reduced the total amount of urine voided over 3 h from flies injected with 1 microL of distilled water by almost 50%. The analog not only inhibits stimulation of housefly fluid secretion by the native kinin Musdo-K, but also by thapsigargin, a SERCA inhibitor, and by ionomycin, a calcium ionophore. The activity of R-LK-CHO is selective, however, as related C-terminal aldehyde analogs do not demonstrate an inhibitory response on housefly fluid secretion. The selective inhibitory activity of R-LK-CHO on housefly tubules represents an important lead in the development of environmentally friendly insect management agents based on the insect kinins.