TauP301L disengages from the proteosome core complex and neurogranin coincident with enhanced neuronal network excitability.

Tauopathies are characterised by the pathological accumulation of misfolded tau. The emerging view is that toxic tau species drive synaptic dysfunction and potentially tau propagation before measurable neurodegeneration is evident, but the underlying molecular events are not well defined. Human non-mutated 0N4R tau (tauWT) and P301L mutant 0N4R tau (tauP301L) were expressed in mouse primary cortical neurons using adeno-associated viruses to monitor early molecular changes and synaptic function before the onset of neuronal loss. In this model tauP301L was differentially phosphorylated relative to tauwt with a notable increase in phosphorylation at ser262. Affinity purification - mass spectrometry combined with tandem mass tagging was used to quantitatively compare the tauWT and tauP301L interactomes. This revealed an enrichment of tauP301L with ribosomal proteins but a decreased interaction with the proteasome core complex and reduced tauP301L degradation. Differences in the interaction of tauP301L with members of a key synaptic calcium-calmodulin signalling pathway were also identified, most notably, increased association with CaMKII but reduced association with calcineurin and the candidate AD biomarker neurogranin. Decreased association of neurogranin to tauP301L corresponded with the appearance of enhanced levels of extracellular neurogranin suggestive of potential release or leakage from synapses. Finally, analysis of neuronal network activity using micro-electrode arrays showed that overexpression of tauP301L promoted basal hyperexcitability coincident with these changes in the tau interactome and implicating tau in specific early alterations in synaptic function.

Neurogranin modulates the rate of association between calmodulin and target peptides.

The best-known mode of action of calmodulin (CaM) is binding of Ca2+ to its N- and C-domains, followed by binding to target proteins. An underappreciated facet of this process is that CaM is typically bound to proteins at basal levels of free Ca2+, including the small, intrinsically disordered, neuronal IQ-motif proteins called PEP-19 and neurogranin (Ng). PEP-19 and Ng would not be effective competitive inhibitors of high-affinity Ca2+-dependent CaM targets at equilibrium because they bind to CaM with relatively low affinity, but they could influence the time course of CaM signaling by affecting the rate of association of CaM with high-affinity Ca2+-dependent targets. This mode of regulation may be domain specific because PEP-19 binds to the C-domain of CaM, whereas Ng binds to both N- and C-domains. In this report, we used a model CaM binding peptide (CKIIp) to characterize the preferred pathway of complex formation with Ca2+-CaM at low levels of free Ca2+ (0.25-1.5 µM), and how PEP-19 and Ng affect this process. We show that the dominant encounter complex involves association of CKIIp with the N-domain of CaM, even though the C-domain has a greater affinity for Ca2+. We also show that Ng greatly decreases the rate of association of Ca2+-CaM with CKIIp due to the relatively slow dissociation of Ng from CaM, and to interactions between the Gly-rich C-terminal region of Ng with the N-domain of CaM, which inhibits formation of the preferred encounter complex with CKIIp. These results provide the general mechanistic paradigms that binding CaM to targets can be driven by its N-domain, and that low-affinity regulators of CaM signaling have the potential to influence the rate of activation of high-affinity CaM targets and potentially affect the distribution of limited CaM among multiple targets during Ca2+ oscillations.

Cellular Iron Deficiency Disrupts Thyroid Hormone Regulated Gene Expression in Developing Hippocampal Neurons.

BACKGROUND: Developing neurons have high thyroid hormone and iron requirements to support their metabolically demanding growth. Early-life iron and thyroid-hormone deficiencies are prevalent and often coexist, and each independently increases risk of permanently impaired neurobehavioral function in children. Early-life dietary iron deficiency reduces thyroid-hormone concentrations and impairs thyroid hormone-responsive gene expression in the neonatal rat brain, but it is unclear whether the effect is cell-intrinsic. OBJECTIVES: This study determined whether neuronal-specific iron deficiency alters thyroid hormone-regulated gene expression in developing neurons. METHODS: Iron deficiency was induced in primary mouse embryonic hippocampal neuron cultures with the iron chelator deferoxamine (DFO) beginning at 3 d in vitro (DIV). At 11DIV and 18DIV, thyroid hormone-regulated gene messenger ribonucleic acid (mRNA)concentrations indexing thyroid hormone homeostasis (Hairless, mu-crystallin, Type II deiodinase, solute carrier family member 1c1, and solute carrier family member 16a2) and neurodevelopment (neurogranin, Parvalbumin, and Krüppel-like factor 9) were quantified. To assess the effect of iron repletion, DFO was removed at 14DIV from a subset of DFO-treated cultures, and gene expression and adenosine 5'-triphosphate (ATP) concentrations were quantified at 21DIV. RESULTS: At 11DIV and 18DIV, neuronal iron deficiency decreased neurogranin, Parvalbumin, and mu-crystallin, and by 18DIV, solute carrier family member 16a2, solute carrier family member 1c1, Type II deiodinase, and Hairless were increased, suggesting cellular sensing of a functionally abnormal thyroid hormone state. Dimensionality reduction with Principal component analysis reveals that thyroid hormone homeostatic genes strongly correlate with and predict iron status. Iron repletion from 14-21DIV did not restore ATP concentration, and Principal component analysis suggests that, after iron repletion, cultures maintain a gene expression signature indicative of previous iron deficiency. CONCLUSIONS: These novel findings suggest there is an intracellular mechanism coordinating cellular iron/thyroid hormone activities. We speculate this is a part of the homeostatic response to acutely match neuronal energy production and growth signaling. However, the adaptation to iron deficiency may cause permanent deficits in thyroid hormone-dependent neurodevelopmental processes even after recovery from iron deficiency.

Neurogranin inhibits calcineurin in murine soleus muscle: Effects of heterozygous knockdown on muscle adaptations to tenotomy and fatigue resistance.

Neurogranin (Ng) is a calmodulin (CaM) binding protein that negatively regulates calcineurin - a Ca2+/CaM-dependent phosphatase that can mitigate the slow-to-fast fibre type shift observed with muscle unloading. Here, we questioned whether heterozygous deletion of Ng (Ng+/-) would enhance calcineurin activity, thereby minimizing the slow-to-fast fibre type shift caused by muscle unloading. As expected, soleus muscles from young adult (3-4 months old) Ng± mice had lowered Ng content and enhanced calcineurin activity when compared to soleus muscles obtained from male age-matched wild-type (WT) mice. Two weeks after tenotomy surgery, where the soleus and gastrocnemius tendons were severed, soleus total fibre count were found to be similarly reduced across both genotypes. However, significant reductions in myofibre cross-sectional area were only found in WT mice and not Ng± mice. Furthermore, while soleus muscles from both WT and Ng± mice exhibited a slow-to-fast fibre type shift with tenotomy, soleus muscles from Ng± mice, in both sham and tenotomized conditions, had a greater proportion of oxidative fibres (type I and IIA) compared with that of WT mice. Corresponding well with this, we found that soleus muscles from Ng± mice were more fatigue resistant compared with those obtained from their WT counterparts. Collectively, these findings show that heterozygous Ng deletion increases calcineurin activation, preserves myofibre size in response to unloading, and promotes the oxidative fibre type to ultimately enhance fatigue resistance. This study demonstrates the role of Ng in regulating calcineurin in vivo and its influence on skeletal muscle form and function.

Low androgen level impairs erectile function of rat by regulating the Ng/CaN/AKT/eNOS pathway in penile corpus cavernosum.

BACKGROUND: The mechanism by which low androgen status inhibit erectile function has not yet been clearly elucidated. Neurogranin (Ng) is a Ca2+ -sensitive calmodulin binding protein that is expressed in endothelial cells and regulates eNOS function. OBJECTIVES: To investigate whether low androgen status inhibit erectile function by regulating the Ng/CaN/AKT/eNOS pathway in the penile cavernous tissue of rats. MATERIALS AND METHODS: Thirty-six 8-week-old male Sprague-Dawley rats were randomly divided into six groups as follows (n = 6): 4-week control group (4w-control), 4-week castration group (4w-cast), 4-week castration+testosterone replacement group (4w-cast+T), 8-week control group (8w-control), 8-week castration group (8w-cast), and 8-week castration+testosterone replacement group (8w-cast+T). Four weeks and eight weeks after surgery, the ratio of the maximum intracavernous pressure/mean arterial pressure (ICPmax/MAP) was examined. The level of NO and the expression of Ng, calcineurin (CaN), AKT, p-AKT(S473), eNOS, and p-eNOS(Ser1177) in the penile cavernous tissue of each group were determined. RESULTS: Ng and CaN were mainly expressed in the membrane and cytoplasm of endothelial cells and smooth muscle cells in the penile cavernous tissue of rats. The ICPmax/MAP and the concentration of NO in the cast group were significantly lower than those in the control group and cast+T replacement group (p < 0.01). The expression of Ng and the ratios of p-AKT/AKT and p-eNOS/eNOS in the penile cavernous tissue of rats in the cast group were significantly lower than those in the control group and cast+T replacement group (p < 0.01). The expression of CaN in the penile cavernous tissue of rats in the cast group was significantly increased compared with that in the control group and the cast+T replacement group (p < 0.01). CONCLUSION: Inhibiting the expression of Ng and subsequently upregulating the expression of CaN in the rat penile cavernous tissue was one of the upstream mechanisms of low androgen status inhibiting erectile function by inhibiting the AKT/eNOS signaling pathway.

Distribution of neurogranin-like immunoreactivity in the brain and sensory organs of the adult zebrafish.

We studied the expression of neurogranin in the brain and some sensory organs (barbel taste buds, olfactory organs, and retina) of adult zebrafish. Database analysis shows zebrafish has two paralog neurogranin genes (nrgna and nrgnb) that translate into three peptides with a conserved IQ domain, as in mammals. Western blots of zebrafish brain extracts using an anti-neurogranin antiserum revealed three separate bands, confirming the presence of three neurogranin peptides. Immunohistochemistry shows neurogranin-like expression in the brain and sensory organs (taste buds, neuromasts and olfactory epithelium), not being able to discern its three different peptides. In the retina, the most conspicuous positive cells were bipolar neurons. In the brain, immunopositive neurons were observed in all major regions (pallium, subpallium, preoptic area, hypothalamus, diencephalon, mesencephalon and rhombencephalon, including the cerebellum), a more extended distribution than in mammals. Interestingly, dendrites, cell bodies and axon terminals of some neurons were immunopositive, thus zebrafish neurogranins may play presynaptic and postsynaptic roles. Most positive neurons were found in primary sensory centers (viscerosensory column and medial octavolateral nucleus) and integrative centers (pallium, subpallium, optic tectum and cerebellum), which have complex synaptic circuitry. However, we also observed expression in areas not related to sensory or integrative functions, such as in cerebrospinal fluid-contacting cells associated with the hypothalamic recesses, which exhibited high neurogranin-like immunoreactivity. Together, these results reveal important differences with the patterns reported in mammals, suggesting divergent evolution from the common ancestor.

Neurogranin Regulates Alcohol Sensitivity through AKT Pathway in the Nucleus Accumbens.

Dysfunction of glutamate neurotransmission in the nucleus accumbens (NAc) has been implicated in the pathophysiology of alcohol use disorders (AUD). Neurogranin (Ng) is exclusively expressed in the brain and mediates N-methyl-d-aspartate receptor (NMDAR) hypo-function by regulating the intracellular calcium-calmodulin (Ca2+ -CaM) pathway. Ng null mice (Ng-/- mice) demonstrate increased alcohol drinking compared to wild-type mice, while also showing less tolerance to the effect of alcohol. To identify the molecular mechanism related to alcohol seeking, both in vivo microdialysis and label-free quantification proteomics comparing Ng genotype and effects of alcohol treatment on the NAc are utilized. There is significant difference in glutamate and gamma-aminobutyric acid (GABA) neurotransmission between genotypes; however, alcohol administration normalizes both glutamate and GABA levels in the NAc. Using label-free proteomics, 427 protein expression changes are identified against alcohol treatment in the NAc among 4347 total proteins detected. Bioinformatics analyses reveal significant molecular differences in Ng null mice in response to acute alcohol treatment. Ingenuity pathway analysis found that the AKT network is altered significantly between genotypes, which may increase the sensitivity of alcohol in Ng null mice. The pharmacoproteomics results presented here illustrate a possible molecular basis of the alcohol sensitivity through Ng signaling in the NAc.

Proteomic differences between focal and diffuse traumatic brain injury in human brain tissue.

The early molecular response to severe traumatic brain injury (TBI) was evaluated using biopsies of structurally normal-appearing cortex, obtained at location for intracranial pressure (ICP) monitoring, from 16 severe TBI patients. Mass spectrometry (MS; label free and stable isotope dimethyl labeling) quantitation proteomics showed a strikingly different molecular pattern in TBI in comparison to cortical biopsies from 11 idiopathic normal pressure hydrocephalus patients. Diffuse TBI showed increased expression of peptides related to neurodegeneration (Tau and Fascin, p < 0.05), reduced expression related to antioxidant defense (Glutathione S-transferase Mu 3, Peroxiredoxin-6, Thioredoxin-dependent peroxide reductase; p < 0.05) and increased expression of potential biomarkers (e.g. Neurogranin, Fatty acid-binding protein, heart p < 0.05) compared to focal TBI. Proteomics of human brain biopsies displayed considerable molecular heterogeneity among the different TBI subtypes with consequences for the pathophysiology and development of targeted treatments for TBI.

Neurogranin in the nucleus accumbens regulates NMDA receptor tolerance and motivation for ethanol seeking.

Dysfunction of N-methyl-d-aspartate receptor (NMDAR) signaling in the nucleus accumbens (NAc) has been implicated in the pathophysiology of alcohol use disorders (AUD). Neurogranin (Ng), a calmodulin-binding protein, is exclusively expressed in the post-synapse, and mediates NMDAR driven synaptic plasticity by regulating the calcium-calmodulin (Ca2+-CaM) pathway. To study the functional role of Ng in AUD, we administrated behavior tests including Pavlovian instrument transfer (PIT), operant conditioning, and rotarod test using Ng null mice (Ng-/- mice). We used adeno-associated virus (AAV)-mediated Ng expression and pharmacological manipulation to validate behavioral responses in Ng-/- mice. The results from our multidisciplinary approaches demonstrated that deficit of Ng increases tolerance to NMDAR inhibition and elicit faster cue reactivity during PIT without changes in ethanol reward. Operant conditioning results demonstrated that Ng-/- mice self-administered significantly more ethanol and displayed reduced sensitivity to aversive motivation. We identified that ethanol exposure decreases mGluR5 (metabotropic glutamate receptor 5) expression in the NAc of Ng-/- mice and pharmacological inhibition of mGluR5 reverses NMDAR desensitization in Ng-/- mice. Together these findings specifically suggest that accumbal Ng plays an essential role in the counterbalance between NMDAR and mGluR5 signaling; which alters NMDAR resistance, and thereby altering aversive motivation for ethanol and may ultimately contribute to susceptibility for alcohol addiction.

Daily rhythms of cognition-related factors are modified in an experimental model of Alzheimer's disease.

The accumulation of amyloid-ß (Aß) peptides in the brain of Alzheimer disease patients is associated to cognitive deficit, increased oxidative stress, and alterations in the circadian rhythms. Brain-derived neurotrophic factor (BDNF) and Neurogranin (RC3), play an important role in the synaptic plasticity underlying memory and learning. Previously, we observed BDNF and RC3 expression follow a daily rhythmic pattern in the hippocampus of young rats. The objective of this study was to investigate the effects of an intracerebroventricular (i.c.v) injection of aggregated Aß peptide (1-42) on temporal patterns of ApoE protein, Bdnf and Rc3 mRNA, lipid peroxidation (LPO) and reduced glutathione (GSH) levels, in the rat hippocampus. We observed an i.c.v. injection of Aß aggregates phase shifts daily BDNF and RC3 expression as well as LPO and decreased the mesor of GSH rhythms. ApoE protein levels vary rhythmically throughout the day. ApoE levels increase at ZT 03:39±00:22 in the hippocampus of control rats and at ZT 06:30±00:28 in the treated animals. Thus, elevated levels of Aß aggregates, characteristic of AD, altered temporal patterns of cognition related-factors, probably, as a consequence of changes in the daily variation of ApoE-mediated Aß aggregates clearance as well as in the 24h rhythms of the cellular redox state.

Nerve growth factor-mediated inhibition of apoptosis post-caspase activation is due to removal of active caspase-3 in a lysosome-dependent manner.

Nerve growth factor (NGF) is well characterised as an important pro-survival factor in neuronal cells that can inhibit apoptotic cell death upstream of mitochondrial outer membrane permeabilisation. Here we addressed the question of whether NGF can also protect against apoptosis downstream of caspase activation. NGF treatment promoted a rapid reduction in the level of the p17 subunit of active caspase-3 in PC12 cells that had been induced to undergo apoptosis by various cytotoxins. The mechanism involved TrkA-dependent activation of extracellular signal-regulated kinase (ERK1/2) but not phosphatidylinositol 3-kinase (PI3K)/Akt, and de novo protein synthesis. Involvement of inhibitor of apoptosis proteins (IAPs) and proteasomal degradation were ruled out. In contrast, inhibition of lysosome function using chloroquine and concanamycin A reversed NGF-induced removal of p17. Moreover, in NGF-treated cells, active caspases were found to be localised to lysosomes. The involvement of macroautophagy and chaperone-mediated autophagy were ruled out. Taken together, these findings suggest an anti-apoptotic mechanism by which NGF induces removal of active caspase-3 in a lysosome-dependent manner.

Zac1 plays a key role in the development of specific neuronal subsets in the mouse cerebellum.

BACKGROUND: The cerebellum is composed of a diverse array of neuronal subtypes. Here we have used a candidate approach to identify Zac1, a tumor suppressor gene encoding a zinc finger transcription factor, as a new player in the transcriptional network required for the development of a specific subset of cerebellar nuclei and a population of Golgi cells in the cerebellar cortex. RESULTS: We found that Zac1 has a complex expression profile in the developing cerebellum, including in two proliferating progenitor populations; the cerebellar ventricular zone and the external granular layer overlying posterior cerebellar lobules IX and X. Zac1 is also expressed in some postmitotic cerebellar neurons, including a subset of GABAergic interneurons in the medial cerebellar nuclei. Notably, GABAergic interneurons in the cerebellar nuclei are derived from the cerebellar ventricular zone, where Zac1 is also expressed, consistent with a lineage relationship between these two Zac1+ populations. Zac1 is also expressed in a small subset of cells in the posterior vermis, including some neurogranin-immunoreactive (NG+) Golgi cells, which, based on short-term birthdating, are derived from the EGL, where Zac1 is also expressed. However, Zac1+ cells and NG+ Golgi cells in the cerebellar cortex also display unique properties, as they are generated within different, albeit overlapping, time windows. Finally, consistent with the expression profile of Zac1, two conspicuous abnormalities were found in the cerebellum of Zac1 null mice: the medial cerebellar nuclei, and not the others, were significantly reduced in size; and the number of Golgi cells in cerebellar lobule IX was reduced by approximately 60% compared to wild-type littermates. CONCLUSIONS: The data presented here indicate that the tumor suppressor gene Zac1 is expressed in a complex fashion in the developing cerebellum, including in two dividing progenitor populations and in specific subsets of postmitotic neurons, including Golgi cells and GABAergic neurons in the medial nuclei, which require Zac1 for their differentiation. We thus conclude that Zac1 is a critical regulator of normal cerebellar development, adding a new transcriptional regulator to the growing list of factors involved in generating neuronal diversity in the developing cerebellum.

Depletion of Neuroguidin/CANu1 sensitizes human osteosarcoma U2OS cells to doxorubicin.

Osteosarcoma is a primary bone cancer which occurs mainly in children. Neuroguidin/CANu1 is a nucleolar protein involved in the maintenance of ribosomal structure. In this study, we investigated the effect of Neuroguidin/CANu1 depletion on the response of osteosarcoma cells to doxorubicin. In normal circumstances, Neuroguidin/CANu1 is localized at nucleoli, which translocates to nuclear foci in the presence of doxorubicin. shRNA knockdown of Neuroguidin/CANu1 did not affect cell viability in the absence of doxorubicin, but led to enhanced cytotoxicity in doxorubicin-treated cells. Doxorubicin increased the population of apoptotic cells by 3-fold in Neuroguidin/CANu1-depleted cells compared to that in control cells. Depletion of Neuroguidin/CANu1 mRNA induced the expression of p21 and the cleavage of PARP, leading to increased caspase-3/7 activity. Together, these results suggest that Neuroguidin/CANu1 is required for maintaining cellular homeostasis and may contribute to the improved efficiency of chemotherapy.

T3 administration in adult hypothyroid mice modulates expression of proteins involved in striatal synaptic plasticity and improves motor behavior.

Adult-onset hypothyroidism is associated with neurological changes such as cognitive dysfunction and impaired learning, which may be related to alterations of synaptic plasticity. We investigate the consequence of adult-onset hypothyroidism on thyroid-mediated transcription events in striatal synaptic plasticity, and the effect of triiodothyronine (T3) replacement. We used hypothyroid mice, treated with propylthiouracil (PTU) and methimazole (MMI), with or without subsequent administration of T3. We evaluated the amount of T3 nuclear receptors (TRalpha1, TRbeta) and striatal plasticity indicators: neurogranin (RC3), Ras homolog enriched in striatum (Rhes), Ca2+/calmodulin-dependent protein kinase (CaMKII), and dopamine- and cAMP-regulated phosphoprotein (DARPP-32). In addition, we assessed hypothyroid mice motor behavior as related to striatum synaptic functions. Hypothyroid mice exhibited significantly reduced TRbeta, RC3 and Rhes expression. T3 administration reversed the expression of TRbeta, RC3, and up-regulated CaMKII levels as well as motor behavior, and decreased DARPP-32 protein phosphorylation. We suggest that thyroid hormone modulation had a major impact on striatal synaptic plasticity of adult mice which produced in turn motor behavior modifications.

Polychlorinated biphenyls (Aroclor 1254) do not uniformly produce agonist actions on thyroid hormone responses in the developing rat brain.

Thyroid hormone (TH) is essential for normal brain development, and polychlorinated biphenyls (PCBs) are known to interfere with TH action in the developing brain. Thus, it is possible that the observed neurotoxic effects of PCB exposure in experimental animals and humans are mediated in part by their ability to interfere with TH signaling. PCBs may interfere with TH signaling by reducing circulating levels of TH, acting as TH receptor analogs, or both. If PCBs act primarily by reducing serum TH levels, then their effects should mimic those of low TH. In contrast, if PCBs act primarily as TH agonists in the developing brain, then they should mimic the effect of T(4) in hypothyroid animals. We used a two-factor design to test these predictions. Both hypothyroidism (Htx) and/or PCB treatment reduced serum free and total T(4) on postnatal d 15. However, only Htx increased pituitary TSHbeta expression. RC3/neurogranin expression was decreased by Htx and increased by PCB treatment. In contrast, Purkinje cell protein-2 expression was reduced in hypothyroid animals and restored by PCB treatment. Finally, PCB treatment partially ameliorated the effect of Htx on the thickness of the external granule layer of the cerebellum. These studies demonstrate clearly that PCB exposure does not mimic the effect of low TH on several important TH-sensitive measures in the developing brain. However, neither did PCBs mimic T(4) in hypothyroid animals on all end points measured. Thus, PCBs exert a complex action on TH signaling in the developing brain.

Expression of PKC substrate proteins, GAP-43 and neurogranin, is downregulated by cAMP signaling and alterations in synaptic activity.

Growth-associated protein 43 (GAP-43) and neurogranin are protein kinase C substrate proteins that are thought to play an important role in synaptic plasticity, but little is currently known about the mechanisms that may regulate their function at the synapse. In this study, we show that long-term elevation of intracellular cAMP levels in rat primary cortical cultures results in a persistent downregulation of GAP-43 and neurogranin, most likely at the transcriptional level. This effect may be at least partially mediated by protein kinase A, but is independent of protein kinase C activation. Moreover, it is mimicked and occluded by manipulations that alter the levels of spontaneous synaptic activity in primary cultures, such as bicuculline and tetrodotoxin. These data suggest that levels of GAP-43 and neurogranin are regulated by factors known to modulate synaptic strength, thus providing a potential mechanism by which protein kinase C signaling pathways and their substrates might contribute to synaptic function and/or plasticity.

Proteomics analysis of the expression of neurogranin in murine neuroblastoma (Neuro-2a) cells reveals its involvement for cell differentiation.

Neurogranin (Ng) is a neural-specific, calmodulin (CaM)-binding protein that is phosphorylated by protein kinase C (PKC). Although its biochemical property has been well characterized, the physiological function of Ng needs to be elucidated. In the present study, we performed proteomics analysis of the induced compositional changes due to the expression of Ng in murine neuroblastoma (Neuro-2a) cells using isotope coded affinity tags (ICAT) combined with 2-dimensional liquid chromatography/tandem mass spectrometry (2D-LC/MS/MS). We found that 40% of identified proteins were down-regulated and most of these proteins are microtubule components and associated proteins that mediated neurite outgrowth. Western blot experiments confirmed the expression of alpha-tubulin and microtubule- associated protein 1B (MAP 1B) was dramatically reduced in Neuro-2a-Ng cells compared to control. Cell morphology of Neuro-2a-Ng showed far less neurites than the control. Serum deprivation induced the extension of only one or two long neurites per cell in Neuro-2a-Ng, contrasting to the extension of multiple neurites per control cell. Ng may be linked to neurite formation by affecting expression of several microtubule related proteins. Furthermore, the PKC activator (PMA) induced an enhanced ERK1/2 activity in the cells that expressed Ng. The mutation of Ng at S36A caused sustained increase of ERK1/2 activity, whereas the ERK1/2 activity in mutation at I33Q showed no difference compared to wild type Ng, suggesting the phosphorylation of Ng but not the CaM /Ng interaction plays an important role in ERK activation. Ng may be involved in neuronal growth and differentiation via PKC and ERK1/2 signaling pathways.

Effects of perinatal exposure to low-dose cadmium on thyroid hormone-related and sex hormone receptor gene expressions in brain of offspring.

Perinatal cadmium (Cd) exposure has been shown to alter behaviors and reduce learning ability of offspring. A few studies have shown that Cd reduced serum thyroid hormones (THs), which are important for brain development during the perinatal period. Brain specific genes, neurogranin (RC3) and myelin basic protein (BMP), are known to be regulated by TH through TH receptors (TR). It has been suggested that RC3 may play roles in memory and learning. In addition, Cd has been suggested to have estrogen-like activity. To evaluate the effects of perinatal low-dose exposure to Cd on thyroid hormone-related gene (RC3, TR-beta1, MBP, RAR-beta) and sex hormone receptor gene (ER-alpha, ER-beta and PgR) expressions in the brain and on behaviors of offspring, mice were administered with 10ppm Cd (from gestational day 1 to postnatal day 10) and/or 0.025% methimazole (MMI; anti-thyroid drug) (from gestational day 12 to postnatal day 10) in drinking water. Also, 0.1% MMI was administered as a positive control (high MMI group). RC3 mRNA expression was reduced in the female brain of combined exposure and high MMI groups and was negatively correlated with the activity in the open-field. ER-alpha, ER-beta and PgR mRNA expressions were decreased in male and female Cd, and female Cd+MMI groups, respectively; among these changes the reduced expression of PgR was opposite to estrogenic action. These results suggested that perinatal exposure to Cd disrupted the gene expressions of sex hormone receptors, which could not be considered to be a result of estrogenic action. Our study indicates that alteration in the gene expressions of RC3 and sex hormone receptors in the brain induced by perinatal Cd and MMI exposure might be one mechanism of developmental toxicity of Cd.

Molecular studies on the protective effect of nicotine in adult-onset hypothyroidism-induced impairment of long-term potentiation.

We have recently shown that chronic nicotine treatment reverses hypothyroidism-induced learning and memory impairment. Chronic nicotine treatment also reverses the hypothyroidism-induced impairment of long-term potentiation (LTP). Analysis of LTP associated key signaling molecules revealed that chronic nicotine treatment prevented the hypothyroidism-induced reduction of the basal phosphotransferase activity of CaMKII and protein levels of P-CaMKII. In addition, the failure of high frequency stimulation to increase the levels of P-CaMKII in hypothyroid rats was reversed by nicotine treatment, suggesting that the neuroprotective effect of nicotine during hypothyroidism involved activation of CaMKII. Furthermore, chronic nicotine treatment reverses the hypothyroidism-induced elevated phosphatase activity and protein levels of calcineurin, a phosphatase that regulates CaMKII activation. We conclude that the neuroprotective effects of nicotine in adult-onset hypothyroidism may result from restoration of CaMKII and calcineurin activity.