Al pha1- chi maeri n,  a Rac1 GTPase- act i vat i ngprot ei n,  i s expressed at  reduced mRNA l evel si n t he brai n of  Al zhei mer' s di sease pat i ent s.
著者KATO Tomoko
学位授与機関滋賀医科大学
学位授与年度平成27年度
学位授与番号14202乙第420号
j ournal  orpubl i cat i on t i t l e
Neurosci ence l et t ers
vol ume591
page range19- 24
year2015- 03- 30
URLht t p: //hdl . handl e. net /10422/11671
doi: 10.1016/j.neulet.2015.02.013(https://doi.org/10.1016/j.neulet.2015.02.013)
Neuroscience Letters 591 (2015) 19–24
Contents lists available at ScienceDirect
Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet
Research article
Alpha1-chimaerin, a Rac1 GTPase-activating protein, is expressed atreduced mRNA levels in the brain of Alzheimer’s disease patients
Tomoko Kato a, Yoshihiro Konishi b, Shun Shimohama c, Thomas G. Beach d,Hiroyasu Akatsu e, Ikuo Tooyama a,∗
a Molecular Neuroscience Research Center, Shiga University of Medical Science, Otsu 520-2192, Japanb Department of Clinical Research, Nishi-tottori National Hospital, Tottori 689-0202, Japanc Department of Neurology, Sapporo Medical University, Sapporo 060-8556, Japand Banner Sun Health Research Institute, Sun City, AZ 85372, USAe Department of Community-Based Medical Education, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan
h i g h l i g h t s
• Alpha1-chimaerin mRNA was localized to neurons in postmortem human brain.• Reduced mRNA levels of ˛1-chiamerin in the temporal cortex and hippocampus of AD.• Expression of ˛2-chimaerin mRNA was not reduced in the temporal cortex of AD.
a r t i c l ei n f o
Article history:Received 23 October 2014Received in revised form 3 February 2015Accepted 6 February 2015Available online 9 February 2015
Keywords:Alzheimer’s disease˛1-chimaerinRac1In situ hybridizationReal-time PCR
a b s t r a c t
Alpha1-chimaerin is a GTPase-activating protein (GAP) for Rac1, a member of the Rho small GTPase fam-ily, whose action leads to the inactivation of Rac1. Rac1 activity is upregulated in Alzheimer’s disease,but little is known about the role of ˛1-chimaerin. In this study, we investigated the expression andlocalization of ˛1-chimaerin mRNA in postmortem human brains from patients with Alzheimer’s diseaseand control subjects. In situ hybridization studies demonstrated that ˛1-chimaerin was expressed byneurons in the neo-cortex of the temporal lobe and the hippocampus of both controls and Alzheimer’sdisease cases, with the signal intensity dramatically decreased in patients with Alzheimer’s disease. Real-time PCR analysis conﬁrmed a signiﬁcant reduction of ˛1-chimaerin mRNA expression in the temporalcortex of Alzheimer’s disease cases. In contrast, ˛2-chimaerin mRNA levels showed no signiﬁcant dif-ference between the groups. The present study showed reduced ˛1-chimaerin expression in the brainof Alzheimer’s disease cases, suggesting a role in the upregulation of Rac1 activity during the diseaseprocess.© 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Alpha1-chimaerin is a GTPase-activating protein (GAP) for Ras-related C3 botulinum toxin substrate 1 (Rac1), a member of theRho small GTPase family [1,2]. Alpha1-chimaerin is selectivelyexpressed in the brain, where its Rac1 GAP activity mediates Rac1inactivation. Rac1 regulates actin polymerization, actin reorgani-zation, cell migration, and cell cycle progression [3]. In neurons,Rac1 mediates dendritic spine formation and other morphological
∗ Correspondence to. Molecular Neuroscience Research Center, Shiga Universityof Medical Science, Seta Tsukinowa-cho, Otsu 520-2192, Japan.Tel.: +81 77 548 2330; fax: +81 77 548 2331.E-mail address: kinchan@belle.shiga-med.ac.jp (I. Tooyama).
changes [4,5]. By inactivating Rac1, ˛1-chimaerin plays a signif-icant role in the regulation of dendritic growth during neuronaldevelopment in the brain [6].Alpha-chimaerin consists of two splice variants (˛1 and ˛2).The ˛1 variant lacks the N-terminal SH2 domains and is moreabundant in the adult brain than during development, whereasthe ˛2 variant is mainly expressed in the developing brain andtestes [7,8]. Increased ˛1-chimaerin promotes the pruning ofdendritic branches and arbors, and ˛1-chimaerin overexpressioncauses loss of spines in the mouse brain [6]. In cultured hip-pocampus, ˛1-chimaerin inhibits the formation of new spines andremoves existing spines [9]. Conversely, the down-regulation of ˛1-chimaerin increases protrusive activity from the dendrite, resultingin an increased abundance of neurons with morphologically atypi-cal spines [6]. Therefore, ˛1-chimaerin is thought to be a regulator
http://dx.doi.org/10.1016/j.neulet.2015.02.0130304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
20T. Kato et al. / Neuroscience Letters 591 (2015) 19–24
of dendritic spine growth, branching, and morphology that exertsits function by increasing synaptic activity via muscarinic acetyl-choline receptors [6] and N-methyl-d-aspartate (NMDA) receptors[9], leading to pruning of dendritic arbors for precisely selectedsynaptic formation [6].Alzheimer’s disease (AD) is the most common cause of demen-tia associated with the accumulation of ß-amyloid (Aß) plaques[10–13], formation of neuroﬁbrillary tangles [14], and neuronaldeath. In addition, synaptic loss is implicated as a major struc-tural cause of cognitive dysfunction in AD [15], probably involvingloss or alterations of dendritic spine formation [16]. Aß oligomersdisrupt synaptic plasticity in vivo [17] and cause synaptic dysfunc-tion in an animal model of AD [18]. A previous study has reportedthat Rac1 activity is upregulated in the hippocampus of AD patients[19]. However, little information is available about the expressionof ˛1-chimaerin in the brain of AD patients. In this study, we there-fore investigated the expression and localization of ˛1-chimaerinin postmortem brains of patients with AD and of age-matched,neuropathologically normal controls at the mRNA level.
2. Materials and methods
2.1. Subjects
All subjects were from the Banner Sun Health Research InstituteBrain and Body Donation Program [20]. The tissue was processedfollowing previously described methods [21,22]. The clinicopatho-logical data are summarized in Table 1.For quantitative real-time PCR, total RNA was extracted fromthe temporal cortex of seven sporadic AD cases (mean age ± SD,86.3 ± 4.8 years) and eight control subjects without neurologicaldisease (mean age ± SD, 81.3 ± 4.8 years). The mean postmortemdelays for the AD cases and control subjects were 2.67 h and 2.51 h,respectively.
For in situ hybridization histochemistry, we examined the hip-pocampus and the temporal cortex of three sporadic AD cases(mean age ± SD, 71.7 ± 10.1 years) and three control subjects (meanage ± SD, 83.0 ± 4.4 years).
2.2. In situ hybridization
The hybridization probe was designed to detect the N-terminalregion speciﬁc to ˛1-chimaerin (bases 1–175 from the initiationcodon; accession number S75654). The N-terminal regions of ˛1-chimaerin were subcloned into the pGEM-T easy vector (Promega,Madison, WI, USA), and the digoxigenin-UTP-labeled sense andantisense riboprobes were synthesized, according to the manufac-turer’s protocol.Frozen, ﬁxed 40 �m sections of the postmortem brain weremounted on RNase-free silane-coated glass slides (Dako JapanCo., Ltd., Tokyo, Japan) and air-dried before immersion in diethylpyrocarbonate-treated phosphate-buffered saline (0.1 M, pH 7.4)for 10 min. The sections were then treated for 10 min at room tem-perature with proteinase K (5 �g/ml) in 10 mM tris-HCl buffer (pH8.0) containing 150 mM NaCl at 37 ◦C, and then post-ﬁxed with4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at roomtemperature for 10 min. The sections were pre-hybridized for 2 hat 37 ◦C in a hybridization buffer [50% formamide, 5 × denhardt’ssolution, 3 × saline/sodium citrate (SSC), 0.5 mg/ml yeast tRNA,and 0.5 mg/ml heat-denatured salmon sperm DNA]. The probeswere diluted in the hybridization buffer to a ﬁnal concentra-tion of 2 �g/ml, and hybridization was performed for 16 h at60 ◦C. After hybridization, the sections were washed for 2 hin 0.2 × SSC buffer at 60 ◦C, followed by rinsing in 0.1 M tris-HCl (pH 7.5) containing 150 mM NaCl (NT buffer) for 5 min atroom temperature. The sections were blocked in 1% skim milkin NT buffer for 60 min, and incubated overnight at 4 ◦C with
Table 1Clinicopathological data of study subjects.
Case #PathologicaldiagnosisAge (years)GenderPostmortemdelay (h)Clinical diagnosisand complicationsCERAD neuriticplaque scoreBraakneuroﬁbrillarytangle stage
Analysis
1Control81F3.0Myocardial infarction, congestiveheart failure, renal failureAIIqPCR
2Control78M2.7Coronary artery disease, chronic,obstructive pulmonary disease,congestive heart failure, diabetesmellitus, hip bone fracture, cardiacpacemaker
0IIqPCR
3Control82F2.0Lung cancer, myocardiac infarction AIIqPCR4Control73F1.5Ovarian cancer0IqPCR5Control85F2.5Cardiac and respiratory failure,atrial ﬁbrillation, cerebrovascular,accident (right hemiplegia)
0IIIqPCR
6Control78M1.7Lung cancer, heart failure0IqPCR, ISH7Control85M3.2Congestive heart failure0IIqPCR, ISH8Control88F3.0Chronic renal failure0IIqPCR9Control86M2.5Renal failure, attrial fabrillation,coronary artery disease, congestiveheart failure
0IIISH
10AD89F3.0AD, osteoarthritis, anxiety,depression, aspiration pneumoniaCVqPCR
11AD79M2.0ADCVqPCR12AD89F3.0AD bronchopneumoniaCVqPCR13AD80F2.2AD, parkinsonismCVIqPCR14AD91F3.0ADCVqPCR15AD87M3.0AD parkinsonismCVqPCR16AD89F2.5ADCVIqPCR17AD61F2.5AD, uterus cancer, bronchitisCVIISH18AD73F2.0AD, atrial ﬁbrillation,cerebrovascular accidentCVISH
19AD81M3.0AD, cardiac and respiratory failure, CVISH
AD: Alzheimer’s disease, qPCR: quantitative polymerase chain reaction, ISH: in situ hybridization histochemistry.
T. Kato et al. / Neuroscience Letters 591 (2015) 19–2421
Fig. 1. In situ hybridization of ˛1-chimaerin mRNA in the temporal cortex of control subjects (A, C and D) and AD cases (B and E) using an antisense probe (A, B, D and E) ora sense probe (C). (A) and (B): positive signals were detected in the cortex of both control subjects and AD cases. (C): no signals were detected using the sense probe withthe exception of nuclear staining in areas near the cortical surface of layer 1. (D) and (E): high magniﬁcation of the boxed area in layer 3 of a control (A) and an AD case (B).Positive cells appear to be neurons. Scale bar = 200 �m in A–C, and 50 �m in D and E.
alkaline phosphatase-labeled anti-digoxigenin antibody (1:200;Roche Diagnostics, Basel, Switzerland). The signal was detectedusing the substrates nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt.
2.3. Quantitative real-time PCR
Five micrograms of each total RNA sample was reverse-transcribed for ﬁrst-strand cDNA synthesis using 80 units ofsuperscript II (Gibco BRL, Gaithersburg, MD, USA) and 500 pmol ofoligo dT12–18 (Amersham Biosciences Corp., Piscataway, NJ, USA).The reaction mixture consisted of 1 × LC-FastStart mixture,4 mM MgCl2, 0.5 �M of primer, and 200 ng of cDNA. We designeda set of primers to amplify ˛1-chimaerin mRNA, but not ˛2-chimaerin, and conﬁrmed on agarose gels that the PCR productwas a single band. Real-time PCR primers for the reactionswere as follows: ˛1-chimaerin, 5′-AAAATGCCATCCAAAGAGTCT-3′(sense,2170–2190inGenBankaccessionnumberS75654)and5′-GAAATTGTGAATCTTTTCATATTT-3′
(antisense,2398–2421inS75654);˛2-chimaerin,5′-GGCTCTACTACGATGGCAAGC-3′(sense,297–317inZ22641)and 5′-CTGTAGAATCTCTCTCATCATGT-3′ (antisense, 511–533 inZ22641); ß-actin, 5′-TGGTGGGCATGGGTCAGAAGGATTC-3′ (sense,172–196 in X00351) and 5′-CATGGCTGGGGTGTTGAAGGTCTCA-3′
(antisense,413–437inX00351);andMAP2,5′-CTGTAGCAGTCCTGAAAGGTGA-3′ (sense, 454–475 in BC027583)and 5′-TGCTAGGGCAGGCTGAGCTGTATC-3′ (antisense, 718–741in BC027583). Cycling conditions comprised an initial 10 min ofincubation at 95 ◦C followed by 1–40 cycles of denaturation for15 s at 95 ◦C, annealing for 8 s at 54 ◦C for ˛1-chimaerin, for 5 s at56 ◦C for ˛2-chimaerin, or for 5 s at 58 ◦C for ß-actin and MAP2,and extension for 15 s at 72 ◦C. Standard curves were obtainedfrom plasmids containing ˛1-chimaerin or ˛2-chimaerin cDNA.According to the ﬁt points method of lightcycler, we calculatedmRNA content using the standard curves. The expression levelsof ˛1- and ˛2-chimaerin mRNAs in neurons were normalized toß-actin and MAP2, respectively. Statistical analysis was performedusing student’s unpaired t-test.
3. Results
3.1. ˛1-chimaerin mRNA was downregulated in the temporalcortex of AD patients, as demonstrated by in situ hybridization
In situ hybridization detected ˛1-chimaerin mRNA in the tem-poral cortex of both control subjects (Fig. 1A) and AD cases (Fig. 1B).
No signal was detected using the sense probe with the exception ofnuclear staining in areas near the cortical surface of layer 1 (Fig. 1C).At high magniﬁcation, positive signals were localized to neurons(Fig. 1D and E, supplementary Fig. S1). Although there was no dif-ference in the localization of ˛1-chimaerin mRNA between controlsubjects and AD cases, the signal intensity dramatically decreasedin the cerebral cortex of AD patients (Fig. 1B and E), compared withthose of control subjects (Fig. 1A and D).In the hippocampus of control cases (Fig. 2), ˛1-chimaerinmRNA was strongly expressed in the pyramidal neurons of thecornu ammonis (CA) (Fig. 2A and B) and in the granule cells ofthe dentate gyrus (DG) (Fig. 2C); however, this signal intensity wasdramatically decreased in pyramidal neurons (Fig. 2D and E) andgranule cells (Fig. 2F) of AD cases.
3.2. Reduced ˛1-chimaerin mRNA levels in the temporal cortex ofAD patients were conﬁrmed by real-time PCR analysis
Real-time PCR was used to compare the expression levelsof ˛1-chimaerin mRNA between AD cases and control subjects(Fig. 3). Statistical data are presented in Table 2. We used 200 ngof cDNA from the temporal cortex for the real-time PCR, and calcu-lated the contents according to a standard curve generated usingthe ˛1-chimaerin plasmid. The expression levels of ˛1-chimaerinmRNA in control subjects and AD cases were 1185.49 ± 163.97 fg(mean ± SEM, n = 8) and 263.75 ± 134.12 fg (mean ± SEM, n = 7),respectively. When normalized to ß-actin and MAP2, ˛1-chimaerinmRNA levels in the AD cases were reduced to 37.3% and 31.3%,respectively, compared to control levels (P < 0.01, Table 2 andFig. 3A).We also examined the expression of ˛2-chimaerin mRNA usingreal-time PCR. The relative expression levels of ˛2-chimaerinmRNA in the control subjects and AD cases were 18.53 ± 1.22(mean ± SEM, n = 8) and 8.95 ± 2.22 (mean ± SEM, n = 7), respec-tively, with no signiﬁcant difference detected between the
Table 2Alpha-chimaerin mRNA expression levels in the temporal cortex of Alzheimer’sdisease cases and control subjects.
Control (n = 8)Alzeimer’s diseaseP-value
˛1-chimaerin/ß-actin0.50 ± 0.080.18 ± 0.040.0053**
˛1-chimaerin /MAP2553.09 ± 70.00173.1 ± 94.70.0060**
˛2-chimaerin/ß-actin0.0051 ± 0.00040.0058 ± 0.00080.407˛2-chimaerin/MAP29.40 ± 1.566.13 ± 1.610.169
Values are presented as mean ± SEM.
** P < 0.01.
22T. Kato et al. / Neuroscience Letters 591 (2015) 19–24
Fig. 2. In situ hybridization of ˛1-chimaerin mRNA in the hippocampus of a control subject (A–C) and AD case (D–F). (A) and (D): at low magniﬁcation, positive signals weremainly visible in the pyramidal layers of the cornu ammonis (CA) and granular cell layer of the dentate gyrus (DG) in both control subjects and AD cases. (B) and (E): highmagniﬁcation of the pyramidal layer of the CA2 region. (C) and (F): high magniﬁcation of the granular cell layer of the dentate gyrus. Signal intensity is reduced in the ADcase (D–F) relative to that in controls (A–C). Scale bar = 200 �m in (A) and (D); 100 �m in (B, C, E and F).
Fig. 3. mRNA expression levels of ˛1-chimaerin (A) and ˛2-chimaerin (B) in the temporal cortex of patients with AD (n = 7) and control cases (n = 8) using real-time PCR.The mRNA expression levels of ˛1-chimaerin and ˛2-chimaerin are normalized to the ß-actin and MAP2 mRNA levels. The mRNA expression level of ˛1-chimaerin, but not˛2-chimaerin, is signiﬁcantly reduced in the temporal cortex of AD cases compared to controls. Results are presented as mean ± SEM. Statistical analysis was performedusing student’s t-test: **P < 0.01 vs. control cases.
groups after normalization to ß-actin and MAP2 (Table 2 andFig. 3B).
4. Discussion
This study is the ﬁrst to demonstrate the localization of ˛1-chimaerin mRNA in the brains of human AD patients and controlsubjects, and to compare the expression levels of ˛1-chimaerinmRNA between temporal cortex samples from both groups. In situ
hybridization histochemistry demonstrated ˛1-chimaerin mRNAin the temporal cortex neurons of both control subjects and ADcases, with the AD brains showing a reduced signal intensity of˛1-chimaerin mRNA compared to controls. The neuronal local-ization of ˛1-chimaerin is consistent with previous reports usingrats [2,7,8]. In the rat brain, ˛1-chimaerin mRNA is expressedspeciﬁcally in neurons and expression rapidly increases post-natally [8], although interestingly, expression was high in thecortex, including the entorhinal cortex and hippocampus, and
T. Kato et al. / Neuroscience Letters 591 (2015) 19–2423
the amygdala [8]. These regions are known to be vulnerable inAD.In agreement with the in situ hybridization results, the real-time PCR analysis conﬁrmed a signiﬁcantly reduced expressionlevel of ˛1-chimaerin mRNA in the temporal cortex of AD brainscompared to controls. When the expression level was normalizedto that of MAP2 mRNA, ˛1-chimaerin mRNA expression was sig-niﬁcantly reduced in the AD brains. These results suggest that thereduction of ˛1-chimaerin is not due simply to neuronal loss, butthat it could reﬂect a pathological mechanism.When normalized to ß-actin and MAP2, the expression of ˛2-chimaerin mRNA showed no signiﬁcant difference between the ADcases and control subjects. These results are in good agreementwith a previous study using the rat brain [8], and suggest that ˛1-chimaerin is the main form of this GAP protein in the adult brain.The precise roles of ˛1-chimaerin in the pathology of AD arenot revealed in this study. However, there are several possibilities.Synapse development and plasticity are controlled by Rho GTPaseregulatory proteins [23], and chimaerins are one class of Rho GAPswith a GAP domain speciﬁc for Rac. GAPs are generally thought todownregulate the activity of small GTPases such as Rac1, as activeGTP-bound forms become inactive GDP-bound forms. Therefore,a reduction in ˛1-chimaerin would be expected to increase Rac1activity at that location. This is in agreement with a previous papershowing that Rac1 activity was increased in the brain of AD patients[19]. In addition, increased expression of Rac1b, a constitutivelyactive splice variant of Rac1, increased only within neurons in AD[24]. Taking together, these ﬁndings suggest that the alterations in˛1-chimaerin and Rac1 in AD brains could be one of the mecha-nisms underlying synaptic dysfunction.The other possibility is that ˛1-chimaerin is associated in someway with cyclin-dependent kinase 5 (Cdk5). Cdk5 is a neuron-speciﬁc Rac effector [25], and ˛1-chimaerin exists in a functionalcomplex with Cdk5 in the brain [26]. Cdk5 is thought to be involvedin the phosphorylation and aggregation of tau protein, tangle for-mation, and Aß neurotoxicity in the brain of AD patients [27,28].Thus, it will be of great interest to clarify the interaction between˛1-chimaerin and Cdk5.
5. Conclusion
In this study, we investigated the expression and localizationof ˛1-chimaerin mRNA in postmortem brains from patients withAD and control subjects. In situ hybridization studies demonstratedthat ˛1-chimaerin was expressed by neurons in the temporal lobeand the hippocampus, and staining intensity was reduced in ADcases. Real-time PCR analysis conﬁrmed a signiﬁcant reductionof ˛1-chimaerin mRNA expression in the brain of AD cases com-pared to controls, while there was no signiﬁcant difference in˛2-chimaerin mRNA levels between the groups.
Disclosure statement for authors
Dr. Thomas G. Beach is a paid consultant with GE Healthcareand Avid Radiopharmaceuticals. The ethics committee at ShigaUniversity of Medical Science veriﬁed that appropriate approvaland procedures were used in this study including human subjects(′3#55).
Acknowledgements
This study was supported in part by a Grant-in-Aid for ScientiﬁcResearch on Innovative Areas (“Brain Environment”) (26111709)and (B) (2629002) from the Ministry of Education, Science, Sportsand Culture of Japan. We are grateful to the Banner Sun Health
Research Institute Brain and Body Donation Program of Sun City,Arizona for the provision of human brain tissue. The Brain andBody Donation Program is supported by the National Instituteof Neurological Disorders and Stroke (U24 NS072026 NationalBrain and Tissue Resource for Parkinson’s Disease and RelatedDisorders), the National Institute on Aging (P30 AG19610 Ari-zona Alzheimer’s Disease Core Center), the Arizona Departmentof Health Services (contract 211002, Arizona Alzheimer’s ResearchCenter), the Arizona Biomedical Research Commission (contracts4001, 0011, 05-901 and 1001 to the Arizona Parkinson’s DiseaseConsortium) and the Michael J. Fox Foundation for Parkinson’sResearch.
Appendix A. Supplementary data
Supplementarydataassociatedwiththisarticlecanbefound,intheonlineversion,athttp://dx.doi.org/10.1016/j.neulet.2015.02.013.
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