Identification of a novel aromatic-turmerone analog that activates chaperone-mediated autophagy through the persistent activation of p38.

Introduction: Aromatic (Ar)-turmerone is a bioactive component of turmeric oil obtained from Curcuma longa. We recently identified a novel analog (A2) of ar-turmerone that protects dopaminergic neurons from toxic stimuli by activating nuclear factor erythroid 2-related factor 2 (Nrf2). D-cysteine increases Nrf2, leading to the activation of chaperone-mediated autophagy (CMA), a pathway in the autophagy-lysosome protein degradation system, in primary cultured cerebellar Purkinje cells. In this study, we attempted to identify novel analogs of ar-turmerone that activate Nrf2 more potently and investigated whether these analogs activate CMA. Methods: Four novel analogs (A4-A7) from A2 were synthesized. We investigated the effects of A2 and novel 4 analogs on Nrf2 expression via immunoblotting and CMA activity via fluorescence observation. Results: Although all analogs, including A2, increased Nrf2 expression, only A4 activated CMA in SH-SY5Y cells. Additionally, A4-mediated CMA activation was not reversed by Nrf2 inhibition, indicating that A4 activated CMA via mechanisms other than Nrf2 activation. We focused on p38, which participates in CMA activation. Inhibition of p38 significantly prevented A4-mediated activation of CMA. Although all novel analogs significantly increased the phosphorylation of p38 6 h after drug treatment, only A4 significantly increased phosphorylation 24 h after treatment. Finally, we revealed that A4 protected SH-SY5Y cells from the cytotoxicity of rotenone, and that this protection was reversed by inhibiting p38. Conclusion: These findings suggest that the novel ar-turmerone analog, A4, activates CMA and protects SH-SY5Y cells through the persistent activation of p38.

Wild-type and pathogenic forms of ubiquilin 2 differentially modulate components of the autophagy-lysosome pathways.

Missense mutations of ubiquilin 2 (UBQLN2) have been identified to cause X-linked amyotrophic lateral sclerosis (ALS). Proteasome-mediated protein degradation is reported to be impaired by ALS-associated mutations of UBQLN2. However, it remains unknown how these mutations affect autophagy-lysosome protein degradation, which consists of macroautophagy (MA), microautophagy (mA), and chaperone-mediated autophagy (CMA). Using a CMA/mA fluorescence reporter we found that overexpression of wild-type UBQLN2 impairs CMA. Conversely, knockdown of endogenous UBQLN2 increases CMA activity, suggesting that normally UBQLN2 negatively regulates CMA. ALS-associated mutant forms of UBQLN2 exacerbate this impairment of CMA. Using cells stably transfected with wild-type or ALS-associated mutant UBQLN2, we further determined that wild-type UBQLN2 increased the ratio of LAMP2A (a CMA-related protein) to LAMP1 (a lysosomal protein). This could represent a compensatory reaction to the impairment of CMA by wild-type UBQLN2. However, ALS-associated mutant UBQLN2 failed to show this compensation, exacerbating the impairment of CMA by mutant UBQLN2. We further demonstrated that ALS-associated mutant forms of UBQLN2 also impair MA, but wild-type UBQLN2 does not. These results support the view that ALS-associated mutant forms of UBQLN2 impair both CMA and MA which may contribute to the neurodegeneration observed in patients with UBQLN2-mediated ALS.

Proteomic analysis of the heart in normal aging mice.

Aging induces pathological cardiovascular changes such as cardiac dysfunction and arteriosclerosis. With aging, heart cells, especially, become more susceptible to lethal damage. In this report, we tried to understand the precise mechanism of myocardial change resulting from aging by examining the heart proteome in aging mice using two-dimensional gel electrophoresis (2DE). The proteins were stained with fluorescence dyes (SYPRO Ruby and Pro-Q Diamond) and identified by subsequent MALDI-TOF-MS / MS. As a result, markedly altered levels of 14 proteins and 7 phosphoproteins were detected in the hearts of 3-, 7-, 11-, and 20-month-old mice. The functions of these identified proteins and phosphoproteins were energy metabolism, muscle contraction, glycolysis, and cytoskeletal support. Additionally, the results of Western blotting confirmed changes in the expression of FTH, CPNE5, and SUCLA2. These findings showed that aging modified the expression of proteins and phosphoproteins in the heart. We suggest that changes in the expression of these proteins are critical to the development of cardiac dysfunction resulting from aging. J. Med. Invest. 69 : 217-223, August, 2022.

In Vitro and In Vivo Antiglycation Effects of Connarus ruber Extract.

Accumulation of advanced glycation end products (AGEs) of the Maillard reaction has been implicated in the pathogenesis of diabetes and its complications. Connarus ruber has been used as a folk remedy for several diseases, including diabetes; however, its underlying mechanism has not yet been investigated. This study investigated the effects of C. ruber extract against glycation on collagen-linked AGEs in vitro and streptozotocin-induced diabetic rats (STZ-DM rats) in vivo. The antiglycation activities of C. ruber extract and aminoguanidine (AG) were examined using a collagen glycation assay kit. Nonfluorescent AGE, Nε-carboxymethyl lysine (CML), Nω-carboxymethyl arginine, and Nε-carboxyethyl lysine levels were measured via electrospray ionization-liquid chromatography-tandem mass spectrometry. The effect of the extract on the cytotoxicity of methylglyoxal (MG), a precursor of AGEs, was examined in HL60 cells. STZ-DM rats were treated with the extract for 4 wk, and the effect was assessed using biochemical markers in the serum and CML-positive cells in renal tissues. C. ruber extract dose-dependently inhibited the glycation of collagen and formation of nonfluorescent AGEs, which was comparable to AG, and it significantly attenuated MG-induced cytotoxicity in HL60 cells. Furthermore, the glycated albumin levels in STZ-DM rats decreased, the increase in serum lipid levels was reversed, and immunohistochemistry demonstrated that CML deposition in the glomerulus of STZ-DM rats significantly decreased. Although further studies are needed, C. ruber could be a potential therapeutic for preventing and progressing many pathological conditions, including diabetes.

Proteomic analysis involved with synaptic plasticity improvement by GABAA receptor blockade in hippocampus of a mouse model of Alzheimer's disease.

GABAergic system plays a part in synaptic plasticity in the hippocampus. We had reported a long-term potentiation (LTP)-like facilitation in vivo, known as synaptic plasticity, through GABAA receptor blockade by bicuculline and the expression of proteins involved with this synaptic plasticity in mouse hippocampus. In the present study, we aimed to show improvement of impaired synaptic plasticity through GABAA receptor blockade and to clarify the molecular mechanisms involved with this improvement in the hippocampus of mice overexpressing human amyloid precursor protein with the E693Δ mutation (APPOSK-Tg) as an Alzheimer's disease model showing impaired synaptic plasticity. Electrophysiological study showed that the LTP-like facilitation expressed with application of bicuculline in vivo was significantly greater than impaired tetanic LTP in APPOSK-Tg mice, which was improved by bicuculline. Proteomic analysis showed that the expression of 11 proteins in the hippocampus was significantly changed 8 h after bicuculline application to APPOSK-Tg mice. The identified proteins could be functionally classified as chaperone, cytoskeletal protein, energy metabolism, metabolism, neuronal development, and synaptic component. Additionally, western blotting validated the changes in four proteins. We therefore propose that the improvement of impaired synaptic plasticity through GABAA receptor blockade could be mediated by the changed expression of these proteins.

Changes in the expression of prefoldin subunit 5 depending on synaptic plasticity in the mouse hippocampus.

Prefoldin is a molecular chaperone that assists the folding of newly synthesized polypeptide chains and prevents aggregation of misfolded proteins. Dysfunction of prefoldin is one of the causes of neurodegenerative diseases such as Alzheimer's disease. The aim of this study was to clarify the involvement of prefoldin subunit 5 (PFDN5) in synaptic plasticity. PFDN5 protein expressed in the hippocampus was predominantly localized in the pyramidal cell layer of CA1-CA3 regions. Nicotine application caused a long-term potentiation (LTP)-like facilitation in vivo, that is synaptic plasticity, in the mouse hippocampus. The levels of PFDN5 mRNA and protein were increased 2-24 h and 4-24 h, respectively, after intraperitoneal application of nicotine (3 mg/kg, i.p.), finally returning to the basal level. This increase of PFDN5 protein was significantly inhibited by mecamylamine (0.5 mg/kg, i.p.), a non-selective nicotinic acetylcholine receptors (nAChRs) antagonist, and required combined application of ABT-418 (10 mg/kg, i.p.), a selective α4ß2 nAChR agonist, and choline (30 mg/kg, i.p.), a selective α7 nAChR agonist. In transgenic mice overexpressing human tau with N279 K mutation as a model of Alzheimer's disease that showed impaired synaptic plasticity, the levels of PFDN5 mRNA and protein in the hippocampus were significantly decreased in an age-dependent manner as compared with age-matched control. The findings demonstrated that the level of PFDN5 protein in the hippocampus was changed depending on the situation of synaptic plasticity. We propose that PFDN5 could be one of the important components of synaptic plasticity.

Proteomic Analysis of Hippocampus and Cortex in Streptozotocin-Induced Diabetic Model Mice Showing Dementia.

AIM: Diabetes with its associated hyperglycemia induces various type of peripheral damage and also impairs the central nervous system (CNS). This study is aimed at clarifying the precise mechanism of diabetes-induced dementia as an impairment of CNS. METHODS: The proteomic analysis of the hippocampus and cortex in streptozotocin- (STZ-) treated mouse diabetic model showing dementia was performed using two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (n = 3/group). RESULTS: Significant changes in the expression of 32 proteins and 7 phosphoproteins were observed in the hippocampus and cortex. These identified proteins and phosphoproteins could be functionally classified as cytoskeletal protein, oxidoreductase, protein deubiquitination, energy metabolism, GTPase activation, heme binding, hydrolase, iron storage, neurotransmitter release, protease inhibitor, transcription, glycolysis, antiapoptosis, calcium ion binding, heme metabolic process, protein degradation, vesicular transport, and unknown in the hippocampus or cortex. Additionally, Western blotting validated the changes in translationally controlled tumor protein, ATP-specific succinyl-CoA synthetase beta subunit, and gamma-enolase isoform 1. CONCLUSIONS: These findings showed that STZ-induced diabetes changed the expression of proteins and phosphoproteins in the hippocampus and cortex. We propose that alterations in expression levels of these proteins play an important role in diabetes-induced dementia.

The influence of chronic nicotine treatment on proteins expressed in the mouse hippocampus and cortex.

Chronic treatment with nicotine, the primary psychoactive substance in tobacco smoke, affects central nervous system functions, such as synaptic plasticity. Here, to clarify the effects of chronic nicotine treatment on the higher brain functions, proteomic analysis of the hippocampus and cortex of mice treated for 6 months with nicotine was performed using two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry. There was significant change in the expression of 16 proteins and one phosphoprotein in the hippocampus (increased tubulin ß-5, atp5b, MDH1, cytochrome b-c1 complex subunit 1, Hsc70, dynamin, profilin-2, 4-aminobutyrate aminotransferase, mitochondrial isoform 1 precursor, calpain small subunit 1, and vacuolar adenosine triphosphatase subunit B and decreased γ-actin, α-tubulin isotype M-α-2, putative ß-actin, tubulin ß-2A, NDUFA10, and G6PD) and 24 proteins and two phosphoproteins in the cortex (increased spectrin α chain, non-erythrocytic 1 isoform 1, tubulin ß-5, γ-actin, creatine kinase B-type, LDH-B, secernin-1, UCH-L1, 14-3-3 γ, type II peroxiredoxin 1, PEBP-1, and unnamed protein product and decreased tubulin α-1C, α-internexin, γ-enolase, PDHE1-B, DPYL2, vacuolar adenosine triphosphatase subunit A, vacuolar adenosine triphosphatase subunit B, TCTP, NADH dehydrogenase Fe-S protein 1, protein disulfide-isomerase A3, hnRNP H2, γ-actin, atp5b, and unnamed protein product). Additionally, Western blotting validated the changes in dynamin, Hsc70, MDH1, NDUFA10, α-internexin, tubulin ß-5 chain, and secernin-1. Thus, these findings indicate that chronic nicotine treatment changes the expression of proteins and phosphoproteins in the hippocampus and cortex. We propose that effect of smoking on higher brain functions could be mediated by alterations in expression levels of these proteins.

Changes in the expression of collapsin response mediator protein-2 during synaptic plasticity in the mouse hippocampus.

We have previously reported that nicotine application to the adult mouse causing long-term potentiation-like facilitation in vivo in the hippocampus can serve as a model of synaptic plasticity. The present study clarifies the involvement of collapsin response mediator protein-2 (CRMP2) in synaptic plasticity. CRMP2 was detected in hippocampal neurons of adult mice. The levels of CRMP2 mRNA and protein were increased 2-24 hr and 4-24 hr, respectively, after application of nicotine (3 mg/kg, i.p.), finally returning to the basal level by 48 hr. Furthermore, the ratio of phosphorylated CRMP2 (pCRMP2) at Thr514 residue, an inactive form, to total CRMP2 levels was not changed during synaptic plasticity expressed by nicotine, indicating an enhanced level of non-pCRMP2. This increase of CRMP2 was inhibited by blockade of nicotinic acetylcholine receptors (nAChRs) and required activation of both α4ß2 and α7 nAChRs. Although the level of ubiquitinated CRMP2 was increased 8 hr after nicotine treatment, the ratio of ubiquitinated CRMP2 to total CRMP2 protein was similar for nicotine-treated and nontreated mice. This study demonstrates that the expression of CRMP2 increases in hippocampal neurons during synaptic plasticity and that the increment is due mainly to mRNA expression. We propose that CRMP2, particularly non-pCRMP2, could contribute to long-lasting synaptic plasticity.

Lipid A-activated inducible nitric oxide synthase expression via nuclear factor-κB in mouse choroid plexus cells.

Choroid plexus (CP) which is responsible for the inflammatory mediators including nitric oxide (NO) are thought to play a crucial role in the process of bacterial meningitis. The present study investigated the mechanisms regulating inducible nitric oxide synthase (iNOS) expression in the choroid plexus epithelium (CPe) in mice. Initially, the expression of iNOS in mouse CPe was strengthened by intracerebroventriclar (i.c.v.) administration of lipid A, which is part of a Gram-negative bacterial endotoxin located at one end of the lipopolysaccharide (LPS) molecule. Next, the expression of iNOS in the CP epithelial cell line ECPC-4 cells was increased from 24 to 48h after lipid A treatment, although mRNA and proteins of toll-like receptor (TLR)-2 and -4 expressed in ECPC-4 cells were not changed by lipid A. The expression of total nuclear factor κB (NFκB), an inflammatory transcriptional factor, in ECPC-4 cells was not changed for 72 h after lipid A treatment, while cytoplasmic NFκB was decreased and nuclear NFκB was increased from 1 to 2 h. In addition, the phosphorylation of inhibitor κB (IκB) was peaked at 10 min, and the level of IκB was attenuated from 10 to 45 min after lipid A treatment. Moreover, the RNA interference (RNAi) of NFκB suppressed the expression of iNOS induced by lipid A. We demonstrated that lipid A-induced iNOS expression in ECPC-4 cells was mainly regulated by the activation of NFκB-IκB intracellular signaling pathway. Thus, we propose that the CPe plays a pivotal role in innate immunity responses of the brain, that is, the signal pathway TLRs on the CPe following inflammatory stimulation such as meningitis is activated, leading to iNOS expression through NFκB.

The influence of chronic ibuprofen treatment on proteins expressed in the mouse hippocampus.

Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID), treatment with which has been shown to delay the onset, slows the cognitive decline, and decreases the incidence of Alzheimer׳s disease (AD) in epidemiological and clinical studies. However, a comprehensive understanding of its mechanism of action remains unclear. To elucidate the prophylactic effect of ibuprofen on the onset of the learning and memory disturbances of AD, we performed proteomic analysis of the hippocampus of chronic ibuprofen-treated mice using two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry. Twenty-eight proteins and seven phosphoproteins were identified to be significantly changed in the hippocampus of chronic ibuprofen-treated mice: translationally controlled tumor protein, thioredoxin-dependent peroxide reductase, and peroxiredoxin 6 were increased, and glial fibrillary acidic protein, dihydropyrimidinase-related protein 2, EF-hand domain-containing protein D2, and 14-3-3ζ were decreased. These identified proteins and phosphoproteins could be classified as cytoskeletal, neuronal development, chaperone, metabolic, apoptosis, neurotransmitter release, ATP synthase, deubiquitination, proteasome, NOS inhibitor, adapter, vesicle transport, signal transduction, antioxidant enzyme, proton transport, synaptogenesis, and serine/threonine phosphatase types. Western blot analysis showed the changes in dihydropyrimidinase-related protein 2, heat shock protein 8, ubiquitin carboxyl-terminal hydrolase PGP9.5, and γ-enolase levels in the hippocampus of chronic ibuprofen-treated mice. These findings showed that the chronic treatment with ibuprofen changed the levels of some proteins and phosphoproteins in the hippocampus. We propose that these identified proteins and phosphoproteins play an important role in decreasing the incidence of AD, especially impaired learning and memory functions.

Proteomic analysis of time-dependent changes in proteins expressed in mouse hippocampus during synaptic plasticity induced by GABAA receptor blockade.

Protein synthesis is required for long-lasting synaptic plasticity. We examined the time-dependent changes in protein expression that occurred in the hippocampus during synaptic plasticity using two-dimensional gel electrophoresis followed by mass spectrometry. The levels of 15 proteins were significantly changed in mouse hippocampus 8h after bicuculline application (1.0mg/kg, i.p.). Expression of 14 proteins (i.e., dihydropyrimidinase-related protein 2, α-tubulin isotype M-α-2, tubulin ß-1 chain, tubulin ß-2A chain, protein disulfide-isomerase ERp61 precursor, chaperonin-containing T complex polypeptide 1 ß subunit, T complex polypeptide 1 [partial], creatine kinase B-type, cytosolic malate dehydrogenase [partial], vacuolar adenosine triphosphatase subunit A, and uncharacterized protein LOC433182) was increased and expression of one protein (i.e., actin γ, cytoplasmic 1) was decreased. Western blotting also validated the changes in dihydropyrimidinase-related protein 2, creatine kinase B-type, and vacuolar adenosine triphosphatase subunit A levels in mouse hippocampus 8h after bicuculline application. The identified proteins were effectors of cellular functions including neuronal differentiation, cytoskeletal dynamics, folding of proteins, stress response, energy metabolism, synapse formation, and unknown function. Taken together, these findings indicate that the identified proteins play an important role in synaptic plasticity in the hippocampus.

The expression changes of EphA3 receptor during synaptic plasticity in mouse hippocampus through activation of nicotinic acetylcholine receptor.

We have reported that systemic application of nicotinic agonists results in expression of a long-term potentiation-like facilitation, a model of synaptic plasticity, in the mouse hippocampus in vivo. Eph receptors and their ephrin ligands, are thought to participate in synaptic plasticity. The present study was conducted to clarify the involvement of EphA3 receptor in synaptic plasticity by investigating the time-dependent change of the expression levels of EphA3 receptor during long-term potentiation-like facilitation in the mouse hippocampus. EphA3 receptor mRNA and protein expression was found in adult mouse hippocampus. EphA3 receptor was localized in neuronal cells but not astrocytes or microglia of hippocampus. After intraperitoneal application of nicotine (3 mg/kg), the protein expression of EphA3 receptor in hippocampus increased during 2-24-h period, significantly increasing during 2-12-h period, and finally returned to the basal level in 72 h, although the mRNA expression of EphA3 receptor was not changed for 24 h. This enhanced expression of EphA3 receptor protein at 4 h was inhibited by pretreatment of mecamylamine (0.5 mg/kg, intraperitoneally), a nonselective nicotinic acetylcholine receptor antagonist. Our findings demonstrated that EphA3 receptor localized only in neuronal cells of the hippocampus was enhanced without transcriptional regulation during synaptic plasticity through activation of the nicotinic acetylcholine receptor. These results suggest that the enhancement of EphA3 receptor after synaptic plasticity may contribute to long-lasting synaptic plasticity through positive, feedforward mechanisms.

Hepatocyte growth factor overexpression in the nervous system enhances learning and memory performance in mice.

Hepatocyte growth factor (HGF) and its receptor, c-Met, play pivotal roles in the nervous system during development and in disease states. However, the physiological roles of HGF in the adult brain are not well understood. In the present study, to assess its role in learning and memory function, we used transgenic mice that overexpress HGF in a neuron-specific manner (HGF-Tg) to deliver HGF into the brain without injury. HGF-Tg mice displayed increased alternation rates in the Y-maze test compared with age-matched wild-type (WT) controls. In the Morris water maze (MWM) test, HGF-Tg mice took less time to find the platform on the first day, whereas the latency to escape to the hidden platform was decreased over training days compared with WT mice. A transfer test revealed that the incidence of arrival at the exact location of the platform was higher for HGF-Tg mice compared with WT mice. These results demonstrate that overexpression of HGF leads to an enhancement of both short- and long-term memory. Western blot analyses revealed that the levels of N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B, but not NR1, were increased in the hippocampus of HGF-Tg mice compared with WT controls, suggesting that an upregulation of NR2A and NR2B could represent one mechanism by which HGF enhances learning and memory performance. These results demonstrate that modulation of learning and memory performance is an important physiological function of HGF that contributes to normal CNS plasticity, and we propose HGF as a novel regulator of higher brain functions.

Loss of dopaminoreceptive neuron causes L-dopa resistant parkinsonism in tauopathy.

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is a family of inherited dementias caused by tauopathy. A mutation in exon 10 of the tau gene, N279K, causes a particular kindred of FTDP-17, which is predominant for parkinsonism. The disease initially presents as L-dopa resistant parkinsonism which then rapidly progresses. The final pathological features reveal disappearing dopamine (DA) neurons, but the causes remain poorly understood. We previously established a transgenic mouse with human N279K mutant tau as a model for FTDP-17, which showed cognitive dysfunctions caused by the mutant. Here we analyze L-dopa resistant parkinsonism by several behavioral tests, and focus on the distributions and accumulations of the mutant tau in the DA system by immunohistochemistry and Western blot. Interestingly, dopaminoreceptive (DAr) neurons in the striatum showed neurofibrils degeneration and apoptosis through caspase-3 activation by mutant tau accumulation. The DAr neuron loss in the caudoputamen, the target of the nigrostriatal system occurred before DA neuron loss in young symptomatic mice. Residual DA neurons in the mouse functioned in DA transportation, whereas dysregulation of intracellular DA compartmentalization implied an excess level of DA caused by DAr neuron loss. In the final stages, both DAr and DA neurons decreased equally, unlike Parkinson's disease. Therefore, DAr neurons were fundamentally vulnerable to the mutation indicating a critical role for the L-dopa resistant parkinsonism in tauopathy.

Phosphoproteome profiling using a fluorescent phosphosensor dye in two-dimensional polyacrylamide gel electrophoresis.

We validated the novel PhosphoQUANTI SolidBlue Complex (PQSC) dye for the sensitive fluorescent detection of phosphorylated proteins in polyacrylamide- and two-dimensional gel electrophoresis (PAGE and 2DE, respectively). PQSC can detect as little as 15.6 ng of ß-casein, a pentaphosphorylated protein, and 61.3 ng of ovalbumin, a diphosphorylated protein. Fluorescence intensity correlates with the number of phosphorylated residues on the protein. To demonstrate the specificity of PQSC for phosphoproteins, enzymatically dephosphorylated lysates of Swiss 3T3 cells were separated in 2DE gels and stained by PQSC. The fluorescence signals in these gels were markedly reduced following dephosphorylation. When the phosphorylated proteins in Swiss 3T3 cell lysates were concentrated using a phosphoprotein enrichment column, the majority of phosphoproteins showed fluorescence signals in the pI 4-5 range. Finally, we performed phosphoproteome analysis to study differences in the protein phosphorylation profiles of proliferating and quiescent Swiss 3T3 cells. Over 135 discernible protein spots were detected, from which a selection of 15 spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). The PQSC staining procedure for phosphoprotein detection is simple, reversible, and fully compatible with MALDI TOF-MS.

Synaptotagmin1 synthesis induced by synaptic plasticity in mouse hippocampus through activation of nicotinic acetylcholine receptors.

We have reported that systemic application of nicotinic agonists expresses a long-term potentiation (LTP)-like facilitation, a model of synaptic plasticity, in vivo in the mouse hippocampus. The present study conducted to clarify the involvement of synaptotagmin1 in synaptic plasticity by investigating the time-dependent change of the mRNA and protein levels of synaptotagmin1 during LTP-like facilitation in the mouse hippocampus. The mRNA expression of synaptotagmin1 increased during 2- to 8-h period by intraperitoneal application of nicotine (3mg/kg), returning to the basal level in 12-h. Also, the protein level of synaptotagmin1, but not synaptophysin, in a total fraction from hippocampus increased during 4- to 12-h period by the same treatment, returning to the basal level in 24-h. The protein level of synaptotagmin1 in a membrane fraction from hippocampus also increased during 4- to 8-h period by nicotine, returning to the basal level in 12-h. This nicotine-enhanced synaptotagmin1 protein in a membrane fraction was inhibited by pretreatment of mecamylamine (0.3mg/kg, i.p.), a nonselective nicotinic acetylcholine receptors (nAChRs) antagonist. Furthermore, choline (30mg/kg, i.p.), a selective α7 nAChR agonist, or ABT-418 (10mg/kg, i.p.), a selective α4ß2 nAChR agonist, enhanced the level of synaptotagmin1 in a membrane fraction. Our findings demonstrate that synaptotagmin1 protein following mRNA which is enhanced without increasing the number of synapse gathers around pre-synaptic membrane during hippocampal LTP-like facilitation through activation of α7 and/or α4ß2 nAChRs in the brain. These results suggest that new-synthesized synaptotagmin1 following synaptic plasticity may contribute to long-lasting synaptic plasticity via positive, feedfoward mechanisms.

SJLB mice develop tauopathy-induced parkinsonism.

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is an inherited dementia caused by tauopathy. Recently, we established the N279K mutant human tau transgenic mice SJLB. Although SJLB mice show cognitive dysfunction with insoluble tau in the brain, it has remained unclear whether they show signs of parkinsonism. To clarify this issue, we studied whether SJLB mice in fact develop parkinsonism. Behavioral analysis showed shorter stride length than that of non-transgenic control mice in the footprint test and movement disorder in the pole test, thus mimicking some features of human parkinsonism. We also found that these symptoms were not affected by dopamine treatment. These results indicate that SJLB mice show signs of parkinsonism and they could be of usefulness not only for studies of dementing disease but also of parkinsonism induced by tauopathy.

Use of a phosphosensor dye in proteomic analysis of human mutant tau transgenic mice.

Recently, we have generated transgenic mice (designated as SJLB) carrying human N279K mutant tau, one of the tau mutations causing parkinsonism linked to chromosome 17 (FTDP-17). SJLB mice mimic some features of behavioral alterations and neuronal pathology of patients with Alzheimer's disease. To investigate how tau dysfunctions cause these features, we examined the expression and phosphorylation levels in SJLB mouse hippocampal proteins using a phosphosensor dye in two-dimensional poly acrylamide gel electrophoresis analysis and mass spectrometry. Calreticulin and tubulin beta4 are significantly more phosphorylated, and heat shock cognate 71 kDa protein, tubulin beta2, vacuolar ATP synthase catalytic subunit A, alpha-internexin, alpha-enolase, ubiquitin carboxyl-terminal hydrolase isozyme L1, and complexin-2 are significantly less phosphorylated in SJLB mice than control mice. These proteins could be new targets for elucidating underlying mechanisms and therapeutic intervention in neurodegenerative diseases.

Disease-dependent reciprocal phosphorylation of serine and tyrosine residues of c-Met/HGF receptor contributes disease retardation of a transgenic mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by progressive degeneration of motoneurons. We have demonstrated that hepatocyte growth factor (HGF) attenuates loss of both spinal and brainstem motoneurons of ALS model mice expressing mutated human SOD1(G93A) (G93A). This study was designed to assess disease-dependent regulatory mechanisms of c-Met/HGF receptor (c-Met) activation in the facial motoneurons of G93A mice. Using double transgenic mice expressing HGF and mutated SOD1(G93A) (G93A/HGF), we showed that phosphorylation of c-Met tyrosine residues at positions 1230, 1234 and 1235 (phospho-Tyr), and thereby its activation, was slightly evident in G93A and highly obvious in G93A/HGF mice (but absent in WT and HGF-Tg mice). Phosphorylation of the c-Met serine residue at position 985 (phospho-Ser), a residue involved in the negative regulation of its activation, was evident in WT and HGF-Tg mice. Protein phosphatase 2A (PP2A), which is capable of dephosphorylating c-Met phospho-serine, is upregulated in the facial motoneurons of G93A and G93A/HGF mice compared with WT and HGF-Tg mice. Thus, c-Met activation is reciprocally regulated by phosphorylation between c-Met serine and tyrosine residues through PP2A induction in the presence or absence of mutant SOD1 expression, and HGF functions more efficiently in ALS and ALS-related diseases.