Journal of Neurochemistry

Rodnight, Richard Burnard; Gottfried, Carmem

doi: 10.1111/jnc.12087pmid: 23278277

In the past two decades, there has been an explosion of research on the role of neuroglial interactions in the control of brain homeostasis in both physiological and pathological conditions. Astrocytes, a subtype of glia in the central nervous system, are dynamic signaling elements that regulate neurogenesis and development of brain circuits, displaying intimate dynamic relationships with neurons, especially at synaptic sites where they functionally integrate the tripartite synapse. When astrocytes are isolated from the brain and maintained in culture, they exhibit a polygonal shape unlike their precursors in vivo. However, cultured astrocytes can be induced to undergo morphological plasticity leading to process formation, either by interaction with neurons or by the influence of pharmacological agents. This review highlights studies on the molecular mechanisms underlying morphological plasticity in astrocyte cultures and intact brain tissue, both in situ and in vivo.

McKelvey, Laura; Shorten, George D.; O'Keeffe, Gerard W.

doi: 10.1111/jnc.12093pmid: 23157347

Nerve growth factor (NGF) is the founding member of the neurotrophins family of proteins. It was discovered more than half a century ago through its ability to promote sensory and sympathetic neuronal survival and axonal growth during the development of the peripheral nervous system, and is the paradigmatic target‐derived neurotrophic factor on which the neurotrophic hypothesis is based. Since that time, NGF has also been shown to play a key role in the generation of acute and chronic pain and in hyperalgesia in diverse pain states. NGF is expressed at high levels in damaged or inflamed tissues and facilitates pain transmission by nociceptive neurons through a variety of mechanisms. Genetic mutations in NGF or its tyrosine kinase receptor TrkA, lead to a congenital insensitivity or a decreased ability of humans to perceive pain. The hereditary sensory autonomic neuropathies (HSANs) encompass a spectrum of neuropathies that affect one's ability to perceive sensation. HSAN type IV and HSAN type V are caused by mutations in TrkA and NGF respectively. This review will focus firstly on the biology of NGF and its role in pain modulation. We will review neuropathies and clinical presentations that result from the disruption of NGF signalling in HSAN type IV and HSAN type V and review current advances in developing anti‐NGF therapy for the clinical management of pain.

Zanin, Mark P.; Mackenzie, Kimberly D.; Peiris, Heshan; Pritchard, Melanie A.; Keating, Damien J.

doi: 10.1111/jnc.12086pmid: 23134420

We have previously shown that Regulator of Calcineurin 1 (RCAN1) regulates multiple stages of vesicle exocytosis. However, the mechanisms by which RCAN1 affects secretory vesicle exocytosis and quantal release kinetics remain unknown. Here, we use carbon fibre amperometry to detect exocytosis from chromaffin cells and identify these underlying mechanisms. We observe reduced exocytosis with repeated stimulations in chromaffin cells over‐expressing RCAN1 (RCAN1ox), but not in wild‐type (WT) cells, indicating a negative effect of RCAN1 on vesicle recycling and endocytosis. Acute exposure to calcineurin inhibitors, cyclosporine A and FK‐506, replicates this effect in WT cells but has no additional effect in RCAN1ox cells. When we chronically expose WT cells to cyclosporine A and FK‐506 we find that catecholamine release per vesicle and pre‐spike foot (PSF) signal parameters are decreased, similar to that in RCAN1ox cells. Inhibiting calcineurin activity in RCAN1ox cells has no additional effect on the amount of catecholamine release per vesicle but further reduces PSF signal parameters. Although electron microscopy studies indicate these changes are not because of altered vesicle number or distribution in RCAN1ox cells, the smaller vesicle and dense core size we observe in RCAN1ox cells may underlie the reduced quantal release in these cells. Thus, our results indicate that RCAN1 most likely affects vesicle recycling and quantal release kinetics via the inhibition of calcineurin activity.

Koperniak, Thomas M.; Garg, Brijesh K.; Boltax, Jay; Loring, Ralph H.

doi: 10.1111/jnc.12095pmid: 23157401

We tested whether surface α7 nicotinic acetylcholine receptor expression is dependent on an endogenous chaperone named Resistance to Inhibitors of Cholinesterase 3 (RIC3) by comparing RIC3 protein in rat GH4C1 and human SH‐EP1 cells, which express strikingly different surface receptor levels following α7 transfection. Cloned rat RIC3 exists in at least two isoforms because of an ambiguous splice site between exons 4 and 5. Both rat isoforms permit surface α7 expression in SH‐EP1 and human embryonic kidney (HEK) cells measured by α‐bungarotoxin binding. Contrary to expectations, endogenous RIC3 protein expression determined by immunoblots did not differ between untransfected GH4C1 or SH‐EP1 cells. siRNA against rat RIC3 exon 4 and shRNA against exons 2, 5 and 6 knocked down transfected rat RIC3 expression in SH‐EP1 cells and simultaneously blocked toxin binding. However, no RNAi construct blocked binding when co‐transfected with α7 into GH4C1 cells. shRNA against rat exons 2 and 5 knocked down rat RIC3 protein transfected into GH4C1 cells with a time course suggesting a protein half‐life of a few days. These results suggest GH4C1 cells may possess unknown chaperone(s) allowing high surface α7 expression in the absence of known RIC3 splice variants.

Lee, Young Jin; Baskakov, Ilia V.

doi: 10.1111/j.1471-4159.2012.07913.xpmid: 22860629

Prion protein (PrPC), is a glycoprotein that is expressed on the cell surface. The current study examines the role of PrPC in early human embryogenesis using human embryonic stem cells (hESCs) and tetracycline‐regulated lentiviral vectors that up‐regulate or suppresses PrPC expression. Here, we show that expression of PrPC in pluripotent hESCs cultured under self‐renewal conditions induced cell differentiation toward lineages of three germ layers. Silencing of PrPC in hESCs undergoing spontaneous differentiation altered the dynamics of the cell cycle and changed the balance between the lineages of the three germ layers, where differentiation toward ectodermal lineages was suppressed. Moreover, over‐expression of PrPC in hESCs undergoing spontaneous differentiation inhibited differentiation toward lineages of all three germ layers and helped to preserve high proliferation activity. These results illustrate that PrPC is involved in key activities that dictate the status of hESCs including regulation of cell cycle dynamics, controlling the switch between self‐renewal and differentiation, and determining the fate of hESCs differentiation. This study suggests that PrPC is at the crossroads of several signaling pathways that regulate the switch between preservation of or departure from the self‐renewal state, control cell proliferation activity, and define stem cell fate.

Kouzoukas, Dimitrios E.; Li, Guiying; Takapoo, Maysaam; Moninger, Thomas; Bhalla, Ramesh C.; Pantazis, Nicholas J.

doi: 10.1111/jnc.12076pmid: 23121601

Alcohol is a potent neuroteratogen that can trigger neuronal death in the developing brain. However, the mechanism underlying this alcohol‐induced neuronal death is not fully understood. Utilizing primary cultures of cerebellar granule neurons (CGN), we tested the hypothesis that the alcohol‐induced increase in intracellular calcium [Ca2+]i causes the death of CGN. Alcohol induced a dose‐dependent (200–800 mg/dL) neuronal death within 24 h. Ratiometric Ca2+ imaging with Fura‐2 revealed that alcohol causes a rapid (1–2 min), dose‐dependent increase in [Ca2+]i, which persisted for the duration of the experiment (5 or 7 min). The alcohol‐induced increase in [Ca2+]i was observed in Ca2+‐free media, suggesting intracellular Ca2+ release. Pre‐treatment of CGN cultures with an inhibitor (2‐APB) of the inositol‐triphosphate receptor (IP3R), which regulates Ca2+ release from the endoplasmic reticulum (ER), blocked both the alcohol‐induced rise in [Ca2+]i and the neuronal death caused by alcohol. Similarly, pre‐treatment with BAPTA/AM, a Ca2+‐chelator, also inhibited the alcohol‐induced surge in [Ca2+]i and prevented neuronal death. In conclusion, alcohol disrupts [Ca2+]i homeostasis in CGN by releasing Ca2+ from intracellular stores, resulting in a sustained increase in [Ca2+]i. This sustained increase in [Ca2+]i may be a key determinant in the mechanism underlying alcohol‐induced neuronal death.

Hall, Hélène; Cuellar‐Baena, Sandra; Denisov, Vladimir; Kirik, Deniz

doi: 10.1111/jnc.12025pmid: 23004566

Choline acetyltransferase (ChAT) is the key enzyme for acetylcholine (ACh) synthesis and constitutes a reliable marker for the integrity of cholinergic neurons. Cortical ChAT activity is decreased in the brain of patients suffering from Alzheimer's and Parkinson's diseases. The standard method used to measure the activity of ChAT enzyme relies on a very sensitive radiometric assay, but can only be performed on post‐mortem tissue samples. Here, we demonstrate the possibility to monitor ACh synthesis in rat brain homogenates in real time using NMR spectroscopy. First, the experimental conditions of the radiometric assay were carefully adjusted to produce maximum ACh levels. This was important for translating the assay to NMR, which has a low intrinsic sensitivity. We then used 15N‐choline and a pulse sequence designed to filter proton polarization by nitrogen coupling before 1H‐NMR detection. ACh signal was resolved from choline signal and therefore it was possible to monitor ChAT‐mediated ACh synthesis selectively over time. We propose that the present approach using a labeled precursor to monitor the enzymatic synthesis of ACh in rat brain homogenates through real‐time NMR represents a useful tool to detect neurotransmitter synthesis. This method may be adapted to assess the state of the cholinergic system in the brain in vivo in a non‐invasive manner using NMR spectroscopic techniques.

Llorente‐Folch, Irene; Sahún, Ignasi; Contreras, Laura; Casarejos, María José; Grau, Josep María; Saheki, Takeyori; Mena, María Angeles; Satrústegui, Jorgina; Dierssen, Mara; Pardo, Beatriz

doi: 10.1111/jnc.12096pmid:

Ward, Margaret S.; Lamb, Jonathan; May, James M.; Harrison, Fiona E.

doi: 10.1111/jnc.12069pmid: 23106783

Severe vitamin C deficiency (ascorbic acid; AA) was induced in gulo−/− mice incapable of synthesizing their own AA. A number of behavioral measures were studied before and during the deprivation period, including a scorbutic period, during which weight loss was observed in the mice. Mice were then resuscitated with AA supplements. During the scorbutic period, gulo−/− mice showed decreased voluntary locomotor activity, diminished physical strength, and increased preference for a highly palatable sucrose reward. These behaviors all returned to control levels following resuscitation. Altered trial times in subordinate mice in the tube test for social dominance in the AA‐deprived mice persisted following resuscitation and may signify a depressive‐like behavior in these mice. Biochemical analyses were undertaken following a second deprivation period. AA deficiency was accompanied by decreased blood glucose levels, oxidative damage to lipids and proteins in the cortex, and decreases in dopamine and serotonin metabolites in both the cortex and striatum. Given the reasonably high proportions of the population that do not consume sufficient AA in the diet, these data have important implications for physical and psychological function in the general population.

Modi, Hiren R.; Taha, Ameer Y.; Kim, Hyung‐Wook; Chang, Lisa; Rapoport, Stanley I.; Cheon, Yewon

doi: 10.1111/jnc.12078pmid: 23121637

Chronic administration of mood stabilizers to rats down‐regulates the brain arachidonic acid (AA) cascade. This down‐regulation may explain their efficacy against bipolar disorder (BD), in which brain AA cascade markers are elevated. The atypical antipsychotics, olanzapine (OLZ) and clozapine (CLZ), also act against BD. When given to rats, both reduce brain cyclooxygenase activity and prostaglandin E2 concentration; OLZ also reduces rat plasma unesterified and esterified AA concentrations, and AA incorporation and turnover in brain phospholipid. To test whether CLZ produces similar changes, we used our in vivo fatty acid method in rats given 10 mg/kg/day i.p. CLZ, or vehicle, for 30 days; or 1 day after CLZ washout. [1‐14C]AA was infused intravenously for 5 min, arterial plasma was collected and high‐energy microwaved brain was analyzed. CLZ increased incorporation coefficients ki * and rates Jin,i of plasma unesterified AA into brain phospholipids i, while decreasing plasma unesterified but not esterified AA. These effects disappeared after washout. Thus, CLZ and OLZ similarly down‐regulated kinetics and cyclooxygenase expression of the brain AA cascade, likely by reducing plasma unesterified AA availability. Atypical antipsychotics and mood stabilizers may be therapeutic in BD by down‐regulating, indirectly or directly respectively, the elevated brain AA cascade of that disease.