713
Journal of Alzheimer’s Disease 40 (2014) 713–726 DOI 10.3233/JAD-132033 IOS Press
CO
PY
Hyperhomocysteinemia and Bleomycin Hydrolase Modulate the Expression of Mouse Brain Proteins Involved in Neurodegeneration Joanna Suszy´nska-Zajczyka , Magdalena Łuczaka , Łukasz Marczaka and Hieronim Jakubowskia,b,c,∗ a Institute
of Bioorganic Chemistry, Pozna´n, Poland of Biochemistry and Biotechnology, University of Life Sciences, Pozna´n, Poland c Department of Microbiology & Molecular Genetics, Rutgers-New Jersey Medical School, International Center for Public Health, Newark, NJ, USA
Accepted 18 December 2013
OR
b Department
AU
TH
Abstract. Homocysteine (Hcy) is a risk factor for Alzheimer’s disease (AD). Bleomycin hydrolase (BLMH) participates in Hcy metabolism and is also linked to AD. The inactivation of the Blmh gene in mice causes accumulation of Hcy-thiolactone in the brain and increases susceptibility to Hcy-thiolactone-induced seizures. To gain insight into brain-related Blmh function, we used two-dimensional IEF/SDS-PAGE gel electrophoresis and MALDI-TOF/TOF mass spectrometry to examine brain proteomes of Blmh–/– mice and their Blmh+/+ littermates fed with a hyperhomocysteinemic high-Met or a control diet. We found that: 1) proteins involved in brain-specific function (Ncald, Nrgn, Stmn1, Stmn2), antioxidant defenses (Aop1), cell cycle (RhoGDI1, Ran), and cytoskeleton assembly (Tbcb, CapZa2) were differentially expressed in brains of Blmh-null mice; 2) hyperhomocysteinemia amplified effects of the Blmh–/– genotype on brain protein expression; 3) proteins involved in brainspecific function (Pebp1), antioxidant defenses (Sod1, Prdx2, DJ-1), energy metabolism (Atp5d, Ak1, Pgam-B), and iron metabolism (Fth) showed differential expression in Blmh-null brains only in hyperhomocysteinemic animals; 4) most proteins regulated by the Blmh–/– genotype were also regulated by high-Met diet, albeit in the opposite direction; and 5) the differentially expressed proteins play important roles in neural development, learning, plasticity, and aging and are linked to neurodegenerative diseases, including AD. Taken together, our findings suggest that Blmh interacts with diverse cellular processes from energy metabolism and anti-oxidative defenses to cell cycle, cytoskeleton dynamics, and synaptic plasticity essential for normal brain homeostasis and that modulation of these interactions by hyperhomocysteinemia underlies the involvement of Hcy in AD. Keywords: Alzheimer’s disease, bleomycin hydrolase, Blmh-null mouse, brain proteome, dietary hyperhomocysteinemia, homocysteine, neurodegenerative diseases
INTRODUCTION Bleomycin hydrolase (BLMH), named for its ability to deaminate and inactivate the anticancer glycopep∗ Correspondence
to: Hieronim Jakubowski, Department of Microbiology & Molecular Genetics, Rutgers-New Jersey Medical School, International Center for Public Health, 225 Warren Street Newark, New Jersey 07101-1709, USA. Tel.: +973 972 8733; Fax: +973 972 8982; E-mail:
[email protected].
tide drug bleomycin, is a thiol-dependent cytoplasmic aminopeptidase ubiquitously expressed in human and rodent organs, including the brain [1, 2]. It belongs to the papain protease family which is characterized by the conserved active site cysteine, histidine, and asparagine residues. In addition to the aminopeptidase activity, BLMH has a hydrolase activity toward a homocysteine (Hcy) metabolite, Hcy-thiolactone [3, 4].
ISSN 1387-2877/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
J. Suszy´nska-Zajczyk et al. / Blmh-null Mouse, Hyperhomocysteinemia, and Neurodegeneration
MATERIALS AND METHODS Mice and diets Colonies of Blmh–/– mice on the C57BL/6J genetic background [6] and wild type Blmh+/+ littermates were bred and housed at the New Jersey Medical School Animal Facility. The mice were fed a normal rodent chow (LabDiet 5010, Purina Mills International, St. Louis, MO) [4]. Hyperhomocysteinemia was induced by providing 4-week-old mice with 1% methionine in drinking water for 8 weeks (high-Met diet) [4, 25]. Four experimental groups of animals were studied (4 animals/group): 1) Blmh–/– mice, control diet; 2) Blmh+/+ mice, control diet; 3) Blmh–/– mice, high-Met diet; and 4) Blmh+/+ mice, high-Met diet. Animal procedures were approved by the Institutional Animal Care and Use Committee at the New Jersey Medical School.
AU
TH
OR
CO
BLMH has been implicated in Alzheimer’s disease (AD), Huntington disease (HD), and cognitive function. In mice, deletion of the Blmh gene results in several phenotypes, including a global reactive astrogliosis in the aged animals, indicative of undefined brain pathology [5]. Increased neonatal mortality, tail dermatitis [6], and impaired presentation of some antigens [7] are also observed in Blmh–/– mice. In human brain, the BLMH protein is localized in neocortical neurons and in dystrophic neurites of senile plaques [8]. In some but not all [9, 10] studies, a polymorphism in the human BLMH gene, resulting in Ile443->Val substitution in the BLMH protein, is associated with an increased risk of AD [11, 12]. Furthermore, the BLMH protein has the ability to process amyloid- protein precursor and amyloid- in vitro [13]. In cell culture models, BLMH has also the ability to generate N-terminal fragments of huntingtin, thought to be important mediators of HD pathogenesis [14]. We found that human BLMH is a major Hcythiolactonase that protects cells against Hcy toxicity [3]. Our more recent studies show that the Hcythiolactonase activity is decreased in brains of AD patients, suggesting that the diminished functional activity of BLMH could contribute to the pathology of AD [15]. Furthermore, we found that the Blmh-null mice show impaired metabolic conversion of Hcythiolactone to Hcy, elevated brain Hcy-thiolactone levels, and increased susceptibility to the neurotoxic effects of intraperitoneally-injected Hcy-thiolactone [4]. Taken together, these findings suggest that BLMH plays important roles in the central nervous system. Human clinical studies show that elevated Hcy is a risk factor for stroke [16], cognitive impairment, and AD [17, 18]. Elevated Hcy has been linked to cognitive impairment and amyloidosis also in mouse models [19, 20]. Hcy lowering by B-vitamin treatment has a significant protective effect on stroke [16, 21], improves cognitive performance [22], and slows the rate of brain atrophy [23], particularly in the gray matter regions related to cognitive decline and AD, including the medial temporal lobe [24]. Hcy lowering improves brain function also in a mouse model of AD by ameliorating brain amyloidosis and improving cognitive deficits [20]. How hyperhomocysteinemia or the alterations of Hcy metabolism in Blmh-null mice affect brain homeostasis is not known. To gain insight into the role of BLMH in the central nervous system and to identify metabolic pathways regulated by BLMH, we examined the brain proteome in Blmh-null mice in the absence and presence of diet-induced hyperhomocysteinemia.
PY
714
Genotyping To establish the status of the Blmh locus, genomic DNA was isolated and genotyped by PCR using the Blmh intron 2 forward primer p1 (CACTGTAGCTGTACTCACAC), Blmh exon 3 reverse primer p2 (GCGACAGAGTACCATGTAGG) and neomycin cassette reverse primer p3 (ATTTGTCACGTCCTGCACGACG) as described by Schwartz et al. [6]. Briefly, the 10 L PCR mixture contained 100 ng purified mouse DNA, 5 l PCR MasterMix (Fermentas), 0.5 L primer p1, p2, p3, 0.5 units of Taq polymerase (Fermentas) and water to 10 L. The thermal cycling reaction was run for 34 cycles of 92◦ C for 30 s, 65◦ C for 40 s, and 72◦ C for 90 s. The 0.7 kb amplicon from the Blmh+/+ wild-type allele (obtained with p1, p2 primers) and the 0.95 kb amplicon from the Blmh–/– knockout allele (obtained with p1, p3 primers) were distinguished on a 1.5% agarose gel stained with SYBRSafe (Invitrogen). Hcy assays Total Hcy and N-Hcy-protein were assayed by HPLC-based methods with post-column derivatization and fluorescence detection as described previously [26, 27]. Protein extraction Brain proteins were extracted using the phenol method [28]. Briefly, brain tissue was disintegrated by
J. Suszy´nska-Zajczyk et al. / Blmh-null Mouse, Hyperhomocysteinemia, and Neurodegeneration
TH
Two-dimensional IEF/SDS-PAGE
PY
manually to ensure the correctness of spot matching. For each identified protein, the relative abundance (% volume) was calculated from its area and intensity divided by the total volume of all protein spots on a gel [31]. This procedure corrects for small variations between individual gels due to protein loading and staining. Mass spectrometry
CO
Protein spots were manually excised from gels using Pasteur pipets, transferred to Eppendorf tubes, destained by series of washes with 50 mM ammonium bicarbonate, 25 mM ammonium bicarbonate/50% acetonitrile, and dehydrated with neat acetonitrile according to a procedure previously described [32]. The dried gel pieces were digested with 10 l 20 ng/l trypsin (Promega), 25 mM ammonium bicarbonate ◦ (37 C, 16 h). Tryptic peptides were recovered from gel pieces by adding acetonitrile (to 10%), sonication in an ultrasound bath for 5 min, followed by 0.5 h incubation at 4◦ C. The proteins were identified using UltrafleXtreme MALDI-TOF/TOF (Bruker Daltonics, Germany) mass spectrometer operating in reflector mode. Positively charged ions in the m/z range 850–3500 were analyzed. 0.5 L of the sample was co-crystallized with a CHCA matrix and spotted directly on MALDI AnchorChip 800 nm target (Bruker Daltonics). For data validation, external calibration was performed with a standard mixture of peptides with masses from 700 to 3500 Da (Peptide Calibration Standards 1 – Bruker). Standards were spotted on calibration spots and calibration was performed after each four samples (samples surrounding calibration spot). Flex control v 3.3 was used for the acquisition of spectra and all further data processing was carried out using Flex analysis v 3.3. Monoisotopic peptide masses were assigned and used for databases search. Additionally five most intensive peaks for each sample were chosen to be fragmented in LIFT mode. MS and MS/MS spectra acquired for each sample were combined and used for Mascot MS/MS Ion Search. For data processing and Mascot (Matrix Science, London, UK) analysis Bruker BioTools 3.2 package was employed. The proteins were identified against UniProtKB/SwissProt protein database. The protein search was done using the following search parameters: MS mass tolerance ± 0.2 Da, MS/MS mass tolerance 0.5 Da, one allowed missed cleavage, cysteine treated with iodoacetamide to form carbamidomethyl-cysteine and methionine in the oxidized form.
OR
grinding with dry ice using a mortar and a pestle. A 100 mg portion of the pulverized brain material was extracted with 0.9 mL of extraction buffer (0.5 M TrisHCl pH 7.5, 50 mM EDTA, 0.1 M KCl, 0.7 M sucrose, 2% w/v DTT) containing protease inhibitors (Protease Inhibitor Mix, GE Healthcare) and 1 mL phenol containing 0.1 % hydroxyquinoline with vigorous shaking (10 min, 4◦ C). The mixture was centrifuged (12 000 g, 10 min, 4◦ C), the phenol layer collected, and extracted again with an equal volume of the extraction buffer. The phenol layer was separated by centrifugation, collected, and the proteins precipitated with 5 volumes of 0.1 M ammonium acetate in methanol (–80◦ C, 2 days). The protein pellet was collected by centrifugation (12 000 × g, 10 min, 4◦ C), washed 3 times with 0.1 M ammonium acetate in methanol, followed by 5min washes with 80% and 100% acetone, and allowed to dry in the air. Brain protein samples were dissolved in IEF rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS). Insoluble material was removed by centrifugation (16 000 g, 20 min). Protein concentration was determined using a commercial 2-D Quant kit (GE Healthcare).
AU
Protein separations and image analysis were carried out as previously described [29]. IPG strips (11 cm, pH 4–7, GE Healthcare) were rehydrated overnight in IEF buffer containing brain protein samples (0.3 mg/strip), 55 mM DTT, 0.5% (v/v) ampholite pH 4–7 (GE Healthcare). The strips were subjected to IEF on IPGphor III apparatus (GE Healthcare) using a ramping voltage (50–6000 V) to final 25 000 Vh. After IEF, IPG strips were incubated for 15 min in an equilibration buffer (6 M urea, 2% w/v SDS, 30% v/v glycerol, 50 mM Tris/HCl, pH 8.8) containing 1% w/v DTT during the first equilibration step and 2.5% iodoacetamide w/v during the second equilibration step. The second dimension was carried out using 11% polyacrylamide gels (24 × 24 cm) on an Ettan DALT six system (GE Healthcare) according to the manufacturer’s instructions. For each sample, a 2D analysis was repeated three times. After electrophoresis, gels were stained with Blue Silver overnight [30] and scanned using an Umax scanner and LabScan software (GE Healthcare). The images were analyzed using the Image Master Platinum software version 7.0 (GE Healthcare). Spots were detected automatically without filtering. Gel patterns were automatically matched between groups. In addition, all individual matched spots were validated
715
J. Suszy´nska-Zajczyk et al. / Blmh-null Mouse, Hyperhomocysteinemia, and Neurodegeneration
Western blotting
Identification of proteins differentially expressed in Blmh-null mouse brain
Statistical analysis
AU
TH
OR
CO
Mouse brains were homogenized with 400 L 0.1 M K-HEPES buffer, pH 7.4, containing 2 mM CaCl2 , 1 mM DTT, and protease inhibitor cocktail using MP Biomedicals FastPrep24 apparatus and Lysing Matrics M tubes (shaker speed 6 m/s, 40 s, 4◦ C). The extract was clarified by centrifugation (14 000 g, 4◦ C, 15 min), the supernatant collected, and protein concentration quantified using the Bradford assay (Bio-Rad). Extracts containing identical amount of protein (10 g) were added to the sample buffer (125 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol), denatured (100◦ C, 5 min), and subjected to SDS-PAGE on 12%, 10 × 10 cm gels. The separated proteins were transferred to a PVDF membrane using ECL Semi-dry Blotter TE 77 (GE Healthcare) (60 V, 1 h). The PVDF membrane was blocked with 1% BSA in PBS, 0.1% Tween-20 and the blots were incubated overnight with mouse anti-peroxiredoxin-2 or anti-actin primary antibodies (Santa Cruz Biotechnology). Anti-mouse IgG conjugated to horseradish peroxidase (Sigma) was used as a secondary antibody. After washing away the excess of the secondary antibody, the membrane was incubated with ChemiFast Chemiluminescence Substrate (2 min in the dark; Syngene) and the resulting chemiluminescent bands recorded using Gbox-Chemi apparatus (Syngen).
type and hyperhomocysteinemia. Plasma tHcy levels in Blmh–/– and Blmh+/+ mice fed the standard chow diet were 6.8 ± 2.2 M and 7.4 ± 2.2 M, and increased to 39 ± 19 M and 77 ± 45 M, respectively, in animals fed with a high-Met diet [4]. N-Hcy-protein increased to 8.4 ± 2.8 M and 5.4 ± 2.9 M in hyperhomocysteinemic Blmh–/– and Blmh+/+ mice, respectively, from the corresponding basal levels of 2.8 ± 0.8 M and 1.2 ± 0.4 M in non-homocysteinemic animals [4].
PY
716
The relative abundance of each protein spot (% vol) was calculated as its volume divided by the total volume of all spots. Data are expressed as mean ± SD. Data for each protein spot had a normal distribution. The differences between the groups were analyzed by ANOVA. Unpaired Student’s t-test was used to test differences between two groups. Statistical analyses were carried out using Statistica 8.0 software. RESULTS Dietary hyperhomocysteinemia in Blmh–/– and Blmh+/+ mice We studied brain proteomes of Blmh–/– mice and their Blmh+/+ littermates fed a standard chow diet. We also studied how brain proteome is affected by hyperhomocysteinemia induced by a high-Met diet and examined the interaction between Blmh geno-
Mouse brain protein separation by IEF/SDS-PAGE yielded several hundred distinct protein spots (Supplementary Fig. 1), 67 of which have been identified by MALDI-TOF/TOF mass spectrometry (Supplementary Table 1). Ten of these proteins were found to have significantly changed expression in Blmh–/– mice relative to Blmh+/+ littermates, while twelve had significantly changed expression in response to high-Met diet (Table 1). The expression levels of the other 53 identified proteins were not affected by the Blmh genotype or the high-Met diet. Close-up views of representative IEF/SDS-PAGE separations of differentially expressed proteins are shown in Fig. 1. Quantification of the levels (% volume) for each of the differentially expressed proteins is shown in Fig. 2. Validation of the IEF/SDS-PAGE by western blotting for one of the differentially expressed proteins is shown in Fig. 3. Brain proteins regulated by Blmh genotype
In mice fed with a standard chow the differential expression (Blmh–/– versus Blmh+/+ mice) of nine brain proteins was higher (1.23–1.68, p < 0.01; Table 1). The proteins upregulated in Blmh–/– mice include those involved in brain specific function (neurocalcin delta – Ncald, neurogranin – Nrgn, and stathmins – Stmn1, Stmn2), cell cycle (rho GDP dissociation inhibitor alpha – Rho GDI1, and GTPbinding nuclear protein – Ran), and cytoskeleton assembly (tubulin-folding cofactor B – Tbcb, and Factin-capping protein subunit ␣-2 – CapZa2). Haloacid dehalogenase-like hydrolase domain-containing protein – Hdhd2, was also upregulated by the Blmh–/– genotype. The differential expression of one brain protein (peroxiredoxin 3 – Prdx3) was lower (−1.18-fold, p < 0.05; Table 1).
717
TH
OR
CO
PY
J. Suszy´nska-Zajczyk et al. / Blmh-null Mouse, Hyperhomocysteinemia, and Neurodegeneration
Brain proteins regulated by the hyperhomocysteinemic diet
AU
Fig. 1. Close-up views of representative IEF/SDS-PAGE gels showing mouse brain proteins whose expression was affected by Blmh genotype and/or high-Met diet. Analyses were carried out for the following four groups of mice: Upper left: Blmh+/+ , control diet; Upper right: Blmh+/+ , high-Met diet; Lower left: Blmh– / – , control diet; Lower right Blmh– / – , high-Met diet. Arrows indicate the direction of the change, up or down. Arrows indicate the changes dependent of Blmh genotype (lower left panel) and high-Met diet (upper and lower right panels).
In wild type Blmh+/+ mice, the high-Met diet caused lower expression of ten brain proteins and higher expression of two brain proteins (Table 1). The down-regulated proteins include those involved in brain specific function (Ncald, Nrgn, phosphatidylethanolamine-binding protein1 – Pebp1, and Stmn isoform 1; −1.25–−1.35-fold, p < 0.05–0.01), antioxidant defenses (peroxiredoxin 2 – Prdx2, peroxiredoxin 3 – Prdx3, and protein DJ-1; −1.39–−1.55-fold, p < 0.01–0.001), energy metabolism (adenylate kinase – Ak1, −1.27-fold, p < 0.05), and cell cycle (RhoGDI1 and Ran; −1.20–−1.27-fold, p < 0.05–0.01). The two proteins that were upregulated by the high-Met diet (−1.25–−1.30-fold, p < 0.001) are the cytoskeleton assembly proteins Tbcb and CapZa2. While most proteins were regulated in the opposite direction, only three, Tbcb, CapZa2, and Prdx3, were regulated in the same direction by high-Met-diet and Blmh–/– genotype (Table 1).
Brain proteins regulated by Blmh genotype and hyperhomocysteinemic diet The magnitude of protein upregulation by the Blmh–/– genotype was increased in mice fed with high-Met diet (Table 1). This increase was observed for brain-specific proteins (Ncald, Nrgn, Stmn1, Stmn2; 1.51–2.33-fold, p < 0.001), antioxidant defense proteins (Sod1, Prdx2, Prdx3, DJ-1; 1.38–2.13-fold, p < 0.001), energy metabolism proteins (Atp5d, Ak1, Pgam-B; 1.37–1.63-fold, p < 0.001), cell cycle proteins (1.69–2.12-fold, p < 0.001), and Hdhd2 (1.48-fold, p < 0.001). For three proteins (Prdx3, CapZa2, Tbcb), the direction of the regulation by the Blmh–/– genotype was dependent on the diet. For example, the upregulation of the cytoskeleton assembly proteins by the Blmh–/– genotype, observed in mice fed with the standard chow diet, was absent (for Tbcb) or changed to down-regulation (for CapZa2) in animals fed with high-Met diet (Table 1). In contrast, the down-regulation of the antioxidant defense protein Prdx3 by the Blmh–/– genotype, observed in
J. Suszy´nska-Zajczyk et al. / Blmh-null Mouse, Hyperhomocysteinemia, and Neurodegeneration
AU
TH
OR
CO
PY
718
Fig. 2. Mouse brain protein expression levels (% volume) as a function of Blmh genotype and/or high-Met diet. The following groups of mice were studies: 1) Blmh+/+ , control diet; 2) Blmh– / – , control diet; 3) Blmh+/+ , high-Met diet; 4) Blmh– / – , high-Met diet. Symbols # and * indicate significant effects of the Blmh– / – genotype and high-Met diet (p < 0.05), respectively.
mice fed with the standard chow diet, was changed to upregulation in animals fed with high-Met diet (Table 1).
The expression of four proteins, three involved in antioxidant defense (Sod1, Prdx2, DJ-1), and one involved in iron metabolism (ferritin heavy chain, Fth),
719
OR
CO
PY
J. Suszy´nska-Zajczyk et al. / Blmh-null Mouse, Hyperhomocysteinemia, and Neurodegeneration
TH
Fig. 2. (Continued).
AU
became dependent on the Blmh genotype only in mice fed with high-Met diet and was increased 1.70–2.49fold (p < 0.001) in Blmh–/– animals (Table 1). Brain proteins regulated by Blmh genotype and hyperhomocysteinemia in mice are differentially expressed in AD and other neurodegenerative diseases
Of the eighteen brain proteins that we found to be differentially expressed in response to the Blmh–/– genotype and/or hyperhomocysteinemia, sixteen (89%) are also known to be differentially expressed in AD and other neurodegenerative diseases (Table 2). For example, proteins involved in brainspecific function (Ncald, Nrgn, Pebp1, Stmn1, Stmn2) and antioxidant defense (Prdx3), were down-regulated both in hyperhomocysteinemic mice and human AD (or animal model). Prdx3 was also down-regulated in Blmh–/– mice (Table 2). Stmn1 that we found to be upregulated in Blmh–/– mice is also known to be upregulated in multiple sclerosis, temporal lobe epilepsy, spinal muscular atrophy, and schizophrenia. The cytoskeleton assembly protein Tbcb that was upregulated by hyperhomocysteinemia and Blmh–/– genotype in mice was also upregulated in giant axon
neuropathy in humans and in giant axon neuropathy mouse model. The iron metabolism protein Fth that was upregulated in hyperhomocysteinemic Blmh–/– mice was also upregulated in human patients with AD, Parkinson’s disease, or HD. Antioxidant defense proteins Prdx2 and DJ-1 and the cell cycle protein Ran were upregulated both by Blmh–/– genotype in mice and in AD patients. The antioxidant defense protein Sod1 that we found to be upregulated in hyperhomocysteinemic Blmh–/– mice is known to be upregulated in human patients with amyotrophic lateral sclerosis (Table 2). DISCUSSION In this work, we used Blmh-null mice in a proteomic study to discover metabolic pathways regulated by the Blmh genotype in the brain. We also examined how brain proteome is affected by dietary hyperhomocysteinemia and studied the interaction between Blmh genotype and hyperhomocysteinemia. We found that: 1) proteins involved in brain-specific function (Ncald, Nrgn, Stmn1, Stmn2), antioxidant defenses (Prdx3), cell cycle (RhoGDI1, Ran), and cytoskeleton assembly (CapZa2, Tbcb) were differentially expressed in brains of Blmh-null mice; 2) hyperho-
720
J. Suszy´nska-Zajczyk et al. / Blmh-null Mouse, Hyperhomocysteinemia, and Neurodegeneration Table 1 Differentially expressed brain proteins regulated by Blmh– / – genotype and/or high-Met diet
Protein description (function) (spot#)
Gene name
Fold change Blmh– / – versus Blmh+/+
1%-Met versus std. diet Blmh– / –
1%-Met diet
Blmh+/+
Ncald
1.43b
2.33a
–1.35b
1.21b
Nrgn
1.68a
1.98a
–1.35b
–1.14 c
Pebp1
1.12
1.83a
–1.27c
1.3a
1.27 a 1.23b
1.94a 1.51a
–1.25c –1.08
1.22b 1.13
1.08 1.20 –1.18c 1.01
1.70a 2.13a 1.38c 1.96a
–1.01 –1.64b –1.16c –1.35b
1.55b 1.09 1.39b 1.44a
1.37a 1.63a 1.60a
–1.01 –1.27c –1.28
1.18c 1.16c 1.29c 1.24a
OR
Sod1 Prdx 2 Prdx3 Park7
CO
Stmn1 Stmn2
Atp5d Ak1 Pgam-B
PY
Std. Diet
1.15 1.11 –1.03
1.41a
2.12a
–1.20b
Ran
1.38b
1.69a
–1.27c
–1.04
1.55a 1.37a
–1.04 –1.39a
1.25a 1.30a
–1.28a –1.45a
Fth
1.04
2.49a
–1.06
2.25b
Hdhd2
1.28c
1.48a
–1.14
1.01
TH
RhoGDI1
Tbcb CapZa2
AU
Brain-specific Neurocalcin delta (rhodopsin phosphorylation) (#59) Neurogranin (synaptic plasticity, spatial learning) (#67) Phosphatidylethanolaminebinding protein 1 (hippocampal cholinergic neurostimulation) (#57) Stathmin 1 (neurite growth) (#62) Stathmin2 (#66) Antioxidant defense Superoxide dismutase 1 (#61) Peroxiredoxin 2 (#56) Peroxiredoxin 3 (#47) Protein DJ-1 (#46) Energy metabolism ATP synthase subunit d (#55) Adenylate kinase 1 (#54) Phosphoglycerate mutase 1 (#44) Cell cycle proteins Rho GDP dissociation inhibitor (GDI) alpha (#51) GTP-binding nuclear protein Ran (#48) Cytoskeleton assembly Tubulin-folding cofactor B (#41) F-actin-capping protein subunit ␣-2 (#26) Iron metabolism Ferritin heavy chain (#58) Other proteins Haloacid dehalogenase-like hydrolase domain-containing protein (#43)
Fold change
Significantly different: a p < 0.001, b p < 0.01, c p < 0.05.
mocysteinemia amplified effects of Blmh–/– genotype on brain protein expression; 3) a group of proteins involved in brain-specific function (Pebp1), antioxidant defenses (Sod1, Prdx2, DJ-1), energy metabolism (Atp5d, Ak1, Pgam-B), and iron metabolism (Fth) showed differential expression in brains of Blmh–/– mice only in animals fed with high-Met diet; 4) proteins regulated by Blmh–/– genotype were also regulated by high-Met diet, albeit in the opposite direction for most proteins; and 5) the only proteins regulated in the same direction by the Blmh–/– genotype and high-Met diet are Prdx3 (down-regulated), CapZa2 and Tbcb (upregulated). These findings suggest that Blmh interacts with diverse cellular pathways that are essential for normal brain homeosta-
sis and that hyperhomocysteinemia modulates these interactions. Previous studies have found no gross abnormalities in the central nervous system in Blmh–/– mice but revealed an increased immunostaining for glial fibrillary acidic protein in the CA1 and CA3 regions of the hippocampus in old null animals, indicating a global reactive astrogliosis and undefined brain pathology [6]. Blmh–/– mice also exhibited poorer performance in water maze trials without detectable effect of the Blmh-null genotype on sensorimotor function [6]. Furthermore, Blmh–/– animals are more sensitive to seizures induced by Hcy-thiolactone [4]. Taken together, these previous findings suggested an important function of Blmh in the brain.
J. Suszy´nska-Zajczyk et al. / Blmh-null Mouse, Hyperhomocysteinemia, and Neurodegeneration
721
Table 2 Brain proteins differentially expressed in response to the Blmh– / – genotype and/or hyperhomocysteinemia are also differentially expressed in AD and other neurodegenerative diseases (see text for discussion and references)
↑ ↑ –
↑ ↑ ↑
Stmn1 Stmn2
↑ ↑
↑ ↑
Other proteins Hdhd2
– – ↓ –
↑ ↑ ↑ ↑
– – –
↑ ↑ ↑
↑ ↑
↑ ↑
↑ ↑
– ↓
–
↑
↑
↓ ↓ ↓
↓, (↓ in Gls– /– mouse) ↓ ↓, (↓ mouse model of AD), (↓ rat chronic stress model) ↓, (↑ in MS, TLE, SMA, schizophrenia),
↓ –
TH
Antioxidant defense Sod1 Prdx2 Prdx3 DJ-1 Energy metabolism Atp5d Ak1 Pgam-B Cell cycle RhoGDI1 Ran Cytoskeleton assembly Tbcb CapZa2 Iron metabolism Fth
Change in AD brain (other neuropathy or animal model)
CO
Brain-specific Ncald Nrgn Pebp1
Change in 1%-Met versus std. diet brain* Blmh+/+
PY
Change Blmh– / – versus Blmh+/+ brain* Std. diet 1%-Met diet
OR
Protein name
↑
(↑ in HD4 mouse model)
– ↓ ↓ ↓
(↑ in ALS) ↑ ↓ ↑
– ↓ –
(↓ in rat model of AD) ↑ ↓, (↓ in rat model of AD)
↓ ↓
(↑ in rat ischemic brain) ↑
↑ ↑
(↑ in GAN) ↑ CapZb2#
–
↑, (↑ in PD), (↑ in HD), (↑ in mouse model of HD)
–
AU
*The up ‘↑’ and down ‘↓’ arrows indicate upregulated and down-regulated proteins, respectively. The dash ‘–‘ indicates no significant change; # The b2 subunit of the CapZ heterodimer; AD, Alzheimer’s disease; GAN, giant axon neuropathy; HD, Huntington disease; MS, multiple sclerosis; PD, Parkinson’s disease; SMA, spinal muscular atrophy; TLE, temporal lobe epilepsy.
As shown in the present work, in Blmh-null mice fed with a control diet nine brain proteins were identified with increased expression (Ncald, Nrgn, Stmn1, Stmn2, RhoGDI1, Ran, Tbcb, CapZa2, Hdhd2) and one brain protein was identified with decreased expression (Prdx3). Thus, in the absence of hyperhomocysteinemia, Blmh interacts with proteins involved in brain-specific function (Ncald, Nrgn, Stmn1, Stmn2), antioxidant defense (Prdx3), cell cycle (RhoGDI1, Ran), and cytoskeleton assembly (CapZa2, Tbcb). The deregulated expression of proteins important for normal brain function provides a possible explanation for astrogliosis and poorer cognitive performance observed in Blmh–/– mice [6] and increased susceptibility of the Blmh-null animals to Hcy-thiolactone-induced seizures [4]. In brains of Blmh-null mice fed with high-Met diet, sixteen proteins were identified with increased expression (Ncald, Nrgn, Pebp1, Stmn1, Stmn2, Sod1, Prdx2, Prdx3, DJ-1, Atp5d, Ak1, Pgam-B, RhoGDI1,
Ran, Fth, Hdad2), one was identified with decreased expression (CapZa2), and the expression of one was unaffected (Tbcb). Effects of the Blmh–/– genotype on expression of the seventeen affected proteins were more pronounced and had a greater magnitude (39–233%) in mice fed with high-Met diet, compared with a control diet (23–68%). High-Met diet alone changed the expression of ten proteins (Ncald, Nrgn, Stmn1, Prdx3, RhoGDI1, Ran, CapZa2, Tbcb) that were also affected by the Blmh–/– genotype, as well as of five proteins (Pebp1, Prdx2, DJ-1, Ak1) that were not affected by the Blmh–/– genotype. Taken together, these findings indicate that Blmh genotype and hyperhomocysteinemia induced by high-Met-diet have distinct effects on protein expression, and that there is an interaction between high-Met-diet and Blmh–/– genotype that modulates protein expression. Previous studies have linked both Blmh [15] and hyperhomocysteinemia [18] to neurodegenera-
J. Suszy´nska-Zajczyk et al. / Blmh-null Mouse, Hyperhomocysteinemia, and Neurodegeneration
of proteins important for normal brain function could account for detrimental effects of hyperhomocysteinemia on cognitive performance in humans [22] and experimental animals [19, 33]. We found that two cytoskeletal proteins, CapZa2 and Tbcb, were upregulated by hyperhomocysteinemia and the Blmh–/– genotype. CapZa2 together with CapZb2 forms a heterodimer actin capping protein (CapZ) that regulates F-actin assembly. The role of the CapZ subunits in mammalian neurons was unknown until 2009, when it was shown that the beta2 subunit, CapZb2, binds tubulin and, in the presence of tau, affects microtubule polymerization necessary for neurite outgrowth and normal growth cone morphology [34]. Tbcb is one of the tubulin-binding cofactors that are believed to be involved in tubulin dimer biogenesis and degradation and therefore to contribute to microtubule functional diversity and regulation. Tbcb interacts with the dynactin p150(Glued) subunit, that is part of the dynein-dynactin motor protein complex responsible for retrograde axonal transport in motor neurons [35]. Cytoskeletal networks made up of F-actin microfilaments and tubulin microtubules are indispensable for normal neurite development and regenerative responses to injury and neurodegenerative stimuli, while cytoskeletal abnormalities characterize neurodegenerative diseases including AD [36]. Elevated Hcy is a risk factor for AD [17] and upregulation of CapZb2 protein is observed in hippocampal neurons of AD patients (Table 2) where it possibly reflects cytoskeletal reorganization and regenerative response [36]. Our findings that hyperhomocysteinemic diet and the Blmh–/– genotype upregulate CapZa2 and Tbcb suggest that disturbed homeostasis of cytoskeletal proteins in response to disturbed Hcy metabolism may contribute to the development of AD. Because increased CapZb2 expression is accompanied by increased expression of brain derived neurotrophic factor receptor tyrosine kinase B [36], mediator of synaptic plasticity in hippocampal neurons, important both for neuronal regeneration and memory formation [37], upregulation of cytoskeletal proteins by highMet diet may also contribute to Hcy-induced cognitive impairment associated with AD. Our findings also suggest that Blmh interacts with CapZa2 and Tbcb to maintain cytoskeletal homeostasis. Excess Tbcb causes abnormalities in growth cone microtubules, such as microtubule depolymerization, growth cone retraction, and axonal damage that lead to neuronal degeneration [38]. These features characterize neuronal changes in giant axonal neuropathy, a
AU
TH
OR
CO
PY
722
Fig. 3. Western blot validation of IEF/SDS-PAGE results. Western blots are shown for Prdx2 (B) and actin (A) as a control that shows equal loading in the four lanes. C) ‘% Volume’ for Prdx2 (spot #56) from IEF/SDS-PAGE analyses of the same samples. The following groups of mice were studied: 1) Blmh+/+ , control diet; 2) Blmh+/+ , high-Met diet; 3) Blmh– / – , control diet; 4) Blmh– / – , high-Met diet.
tive diseases, including AD, although the underlying mechanisms are not understood. Majority of the brain proteins that we found to be differentially expressed in response to Blmh–/– genotype and/or high-Met diet, are known to play important roles in neural development, learning, plasticity, and aging, and have been linked to neurodegenerative diseases, including AD (Table 2). Thus, our findings point to a novel role for BLMH as a modulator of the brain proteome and suggest mechanistic explanation for the association between hyperhomocysteinemia and brain diseases: hyperhomocysteinemia alters the expression of proteins whose homeostasis is crucial for cytoskeletal dynamics, neural development, learning, aging, degeneration, and plasticity. Alterations in the expression
J. Suszy´nska-Zajczyk et al. / Blmh-null Mouse, Hyperhomocysteinemia, and Neurodegeneration
AU
TH
PY
OR
mice are more sensitive than their wild type littermates to Hcy-thiolactone neurotoxicity [4]. Altered expression of proteins important for brainspecific function, such as Ncald, Nrgn, Pebp1, Stmn1, and Stmn2 that we found to be differentially expressed in response to the Blmh–/– genotype or hyperhomocysteinemia, are common findings in neurodegenerative diseases (Table 2). Specifically, reduced expression of these proteins that we observed in hyperhomocysteinemia is also observed in patients with AD [40–44] or Down syndrome [43], while enhanced expression that we observed in Blmh–/– mice is also observed in multiple sclerosis [45], temporal lobe epilepsy [46], or schizophrenia patients [47] (Table 2). Expression of Ncald is reduced in Gls–/– mice (Table 2) that are deficient in glutaminase, a multifunctional enzyme involved in energy metabolism, ammonia trafficking, and regeneration of neurotransmitter glutamate [48]. Nrgn is upregulated in cerebrospinal fluid of AD patients compared with controls [49], while Nrgn mRNA is down-regulated in the caudate and BA4 cortex regions in brains of HD patients [50]. Decreased hippocampal PEBP1 expression and memory dysfunction are observed in chronically stressed rats [51]. Stmn is upregulated in spinal muscular atrophy, a motor neuron degeneration disorder caused by mutations in the survival motor neuron 1 (Smn1) gene [52]. Increased Stmn1 expression in the dentate gyrus causes abnormal axonal sprouting and arborization in the mouse brain [47]. Stmn1 is upregulated also in ischemic brain injury in rats [53]. Thus, our findings show that hyperhomocysteinemia, a risk factor for AD, is detrimental to brain homeostasis because it alters expression of Ncald, Nrgn, Pebp1, Stmn1, and Stmn2, whose deregulation is a hallmark of neurodegenerative diseases, including AD. Our findings also suggest that the altered expression of Ncald, Nrgn, Pebp1, Stmn1, and Stmn2 is a contributing mechanism by which hyperhomocysteinemia causes neurodegeneration and that Blmh interacts with these proteins to maintain their
homeostasis and thus has a protective role in the brain. We identified four antioxidant defense proteins Sod1, Prdx2, Prdx3, and DJ-1 that were upregulated by the Blmh–/– genotype only in the presence of hyperhomocysteinemia and three of them, Prdx2, Prdx3, and DJ-1, were down-regulated by hyperhomocysteinemia alone. The upregulation of Sod1, Prdx2, and DJ-1 is known to be associated with AD [54, 55]. Sod1 is also responsible for ALS characterized by progressive loss of upper and lower motor neurons, which results in paralysis and finally death from respiratory failure [56]. Mutated versions of protein DJ-1 are involved in Parkinson’s disease, a neurodegenerative motor system disorder affecting brainstem structures and cortical areas [57]. Down-regulation of Prdx2, Prdx3, and DJ-1 in brains of mice fed with high-Met diet (Table 1) suggests that hyperhomocysteinemia alone compromises antioxidant defenses in the brain. Thus, our results suggest that BLMH deficiency and/or hyperhomocysteinemia contribute to neurodegeneration by affecting antioxidant defense proteins. Energy metabolism proteins, Atp5d, Ak1, and Pgam-B, that play important roles in cellular energy homeostasis and adenine nucleotide metabolism, were found to be upregulated by the Blmh–/– genotype only in the presence of hyperhomocysteinemia, while two of them, Ak1 and Pgam-B, were down-regulated by hyperhomocysteinemia alone (Table 1). The changes in the expression of Atp5d, Ak1, and Pgam-b in the brain in response to the Blmh–/– genotype and hyperhomocysteinemia observed in the present work are similar to the changes in expression that have been reported for Ak1 and Pgam-B in AD brains [58] and for Pgam-B and Atp5d in a rat model of AD [59], suggesting that Blmh deficiency and/or hyperhomocysteinemia contribute to neurodegeneration by affecting brain energy metabolism. Our findings that Ak1 and Pgam-B are downregulated by the high-Met diet suggest that glycolysis is less efficient in the brains of hyperhomocysteinemic mice compared with animals without hyperhomocystenemia. On the other hand, our findings that Atp5d, Ak1, and Pgam-b are upregulated by the Blmh–/– genotype only in the presence of hyperhomocystienemia suggest that BLMH interacts with these proteins to upregulate energy generation under hyperhomocysteinemic conditions. Because BLMH is not known to be present in mitochondria, the interaction with Atp5d is most likely indirect. Consistent with the role of Hcy in energy metabolism, the growth of wild type mice on a high-Met diet is
CO
devastating autosomal recessive disorder characterized by a severe early-onset peripheral motor and sensory neuropathy [39]. Our findings that CapZa2 and Tbcb are upregulated by the Blmh–/– genotype, in conjunction with findings of other investigators showing that CapZb2 and Tbcb are upregulated in neurodegenerative diseases, giant axon neuropathy, and AD, respectively (Table 3), suggest that BLMH plays a neuroprotective role. This suggestion is consistent with our previous findings showing that BLMH activity is reduced in brains of AD patients [15] and that Blmh–/–
723
J. Suszy´nska-Zajczyk et al. / Blmh-null Mouse, Hyperhomocysteinemia, and Neurodegeneration
In conclusion, our findings showing that Blmh deletion changes the expression of brain proteins involved in brain-specific function, antioxidant defenses, energy metabolism, cell cycle, and cytoskeleton dynamics suggest that Blmh is an important neuroprotective protein. Our results also suggest that hyperhomocysteinemia contributes to neurodegeneration by affecting expression of proteins involved in processes important for brain homeostasis, such as cytoskeleton assembly, cell, cycle, energy metabolism, antioxidant defenses, neural development, learning, aging, degeneration, and plasticity. The differentially expressed proteins identified in our study are implicated in neurodegenerative diseases, including AD.
AU
TH
OR
CO
retarded [25], suggesting that they are at a metabolic disadvantage. We found that the cell cycle proteins Rho-GDI1 and Ran are upregulated by the Blmh–/– genotype and down-regulated by hyperhomocysteinemia. Rho-GDI factors regulate the GDP/GTP exchange reaction of the small GTPases by inhibiting the dissociation of GDP. Two RhoGDIs, RhoGDI1 and 3, are expressed in the brain cerebral cortex, striatum, and hippocampus [60]. The Rho family of small GTPases controls cytoskeletal remodeling as well as cell cycle and are important in dendritic and axonal growth as well as in synaptic plasticity [61]. Dendritic abnormalities are the most consistent neuroanatomical finding in mental retardation [61]. RhoGDI1 is upregulated in ischemic brain injury in rats [53]. Ran is a GTP-binding protein that is involved in nucleocytoplasmic transport. Reduced Ran expression that we found in hyperhomocysteinemic mice is also observed in AD brains, where it is accompanied by reduced transport of transcription regulators (DNMT1 and RNA pol II) to the nucleus, that may lead to cellular manifestations of AD [62]; these findings suggest that hyperhomocysteinemia contributes to AD by downregulating Ran. Upregulation of Ran and Rho-GDI1 that we found in Blmh–/– mice suggests that Blmh protects against brain injury by maintaining homeostasis of these cell cycle proteins. We found that the iron metabolism protein Fth is upregulated in brains of Blmh–/– mice in mice fed with a high-Met diet, while hyperhomocysteinemia alone or Blmh deficiency alone did not affect the Fth expression. These findings show that only the simultaneous presence of Blmh deficiency and hyperhomocysteinemia, which lead to increased protein N-homocysteinylation [4], causes perturbations in iron metabolism. Previous work has established that perturbations in iron homeostasis are linked to neurodegenerative diseases. For example, Fth is known to be upregulated in the hippocampus of patients with AD [58] and substantia nigra of Parkinson’s disease patients [57]. Furthermore, ferritin accumulation in dystrophic microglia is an early event in the development of HD, which is characterized by neuropathological changes in the striatum, including loss of medium-spiny neurons, nuclear inclusions of the huntingtin protein, gliosis, and abnormally high iron levels. The accumulation occurs both in HD patients and in a mouse model of HD [63]. Ferritin is also known to contain the highest levels of N-linked Hcy [27]. Whether N-homocysteinylation contributes to upregulation of Fth expression in the brain remains to be investigated.
PY
724
ACKNOWLEDGMENTS We thank John Lazo for providing a pair of Blmhnull mice. This work was supported in part by grants from the American Heart Association, the National Science Center (2011/01/B/NZ1/03417, 2011/02/ A/NZ1/00010, and 2012/07/B/NZ7/01178), and MNiSW, Poland (N401 065321504 and N N302 434439). Authors’ disclosures available online (http://www.jalz.com/disclosures/view.php?id=2067). SUPPLEMENTARY MATERIAL The supplementary figure and table are available in the electronic version of this article: http://dx.doi.org/ 10.3233/JAD-132033. REFERENCES [1]
[2]
[3]
[4]
[5]
[6]
Bromme D, Rossi AB, Smeekens SP, Anderson DC, Payan DG (1996) Human bleomycin hydrolase: Molecular cloning, sequencing, functional expression, and enzymatic characterization. Biochemistry 35, 6706-6714. Kamata Y, Itoh Y, Kajiya A, Karasawa S, Sakatani C, Takekoshi S, Osamura RY, Takeda A (2007) Quantification of neutral cysteine protease bleomycin hydrolase and its localization in rat tissues. J Biochem 141, 69-76. Zimny J, Sikora M, Guranowski A, Jakubowski H (2006) Protective mechanisms against homocysteine toxicity: The role of bleomycin hydrolase. J Biol Chem 281, 22485-22492. Borowczyk K, Tisonczyk J, Jakubowski H (2012) Metabolism and neurotoxicity of homocysteine thiolactone in mice: Protective role of bleomycin hydrolase. Amino Acids 43, 1339-1348. Montoya SE, Thiels E, Card JP, Lazo JS (2007) Astrogliosis and behavioral changes in mice lacking the neutral cysteine protease bleomycin hydrolase. Neuroscience 146, 890900. Schwartz DR, Homanics GE, Hoyt DG, Klein E, Abernethy J, Lazo JS (1999) The neutral cysteine protease
J. Suszy´nska-Zajczyk et al. / Blmh-null Mouse, Hyperhomocysteinemia, and Neurodegeneration
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[25]
[26]
PY
[24]
trial: A randomised, double blind, controlled trial. Lancet 369, 208-216. Smith AD, Smith SM, de Jager CA, Whitbread P, Johnston C, Agacinski G, Oulhaj A, Bradley KM, Jacoby R, Refsum H (2010) Homocysteine-lowering by B vitamins slows the rate of accelerated brain atrophy in mild cognitive impairment: A randomized controlled trial. PloS one 5, e12244. Douaud G, Refsum H, de Jager CA, Jacoby R, Nichols TE, Smith SM, Smith AD (2013) Preventing Alzheimer’s diseaserelated gray matter atrophy by B-vitamin treatment. Proc Natl Acad Sci U S A 110, 9523-9528. Velez-Carrasco W, Merkel M, Twiss CO, Smith JD (2008) Dietary methionine effects on plasma homocysteine and HDL metabolism in mice. J Nutr Biochem 19, 362-370. Chwatko G, Jakubowski H (2005) The determination of homocysteine-thiolactone in human plasma. Anal Biochem 337, 271-277. Jakubowski H (2008) New method for the determination of protein N-linked homocysteine. Anal Biochem 380, 257-261. Faurobert M, Pelpoir E, Chaib J (2007) Phenol extraction of proteins for proteomic studies of recalcitrant plant tissues. Methods Mol Biol 355, 9-14. Luczak M, Formanowicz D, Pawliczak E, Wanic-Kossowska M, Wykretowicz A, Figlerowicz M (2011) Chronic kidney disease-related atherosclerosis - proteomic studies of blood plasma. Proteome Sci 9, 25. Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri GM, Carnemolla B, Orecchia P, Zardi L, Righetti PG (2004) Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25, 1327-1333. Luczak M, Kazmierczak M, Handschuh L, Lewandowski K, Komarnicki M, Figlerowicz M (2012) Comparative proteome analysis of acute myeloid leukemia with and without maturation. J Proteomics 75, 5734-5748. Shevchenko A, Shevchenko A (2001) Evaluation of the efficiency of in-gel digestion of proteins by peptide isotopic labeling and MALDI mass spectrometry. Anal Biochem 296, 279-283. Bernardo A, McCord M, Troen AM, Allison JD, McDonald MP (2007) Impaired spatial memory in APP-overexpressing mice on a homocysteinemia-inducing diet. Neurobiol Aging 28, 1195-1205. Davis DA, Wilson MH, Giraud J, Xie Z, Tseng HC, England C, Herscovitz H, Tsai LH, Delalle I (2009) Capzb2 interacts with beta-tubulin to regulate growth cone morphology and neurite outgrowth. PLoS Biol 7, e1000208. Kuh GF, Stockmann M, Meyer-Ohlendorf M, Linta L, Proepper C, Ludolph AC, Bockmann J, Boeckers TM, Liebau S (2012) Tubulin-binding cofactor B is a direct interaction partner of the dynactin subunit p150(Glued). Cell Tissue Res 350, 13-26. Kao PF, Davis DA, Banigan MG, Vanderburg CR, Seshadri S, Delalle I (2010) Modulators of cytoskeletal reorganization in CA1 hippocampal neurons show increased expression in patients at mid-stage Alzheimer’s disease. PLoS One 5, e13337. Rex CS, Lin CY, Kramar EA, Chen LY, Gall CM, Lynch G (2007) Brain-derived neurotrophic factor promotes long-term potentiation-related cytoskeletal changes in adult hippocampus. J Neurosci 27, 3017-3029. Lopez-Fanarraga M, Carranza G, Bellido J, Kortazar D, Villegas JC, Zabala JC (2007) Tubulin cofactor B plays a role in the neuronal growth cone. J Neurochem 100, 16801687.
CO
[10]
[23]
[27]
[28]
[29]
OR
[9]
TH
[8]
AU
[7]
bleomycin hydrolase is essential for epidermal integrity and bleomycin resistance. Proc Natl Acad Sci U S A 96, 46804685. Towne CF, York IA, Watkin LB, Lazo JS, Rock KL (2007) Analysis of the role of bleomycin hydrolase in antigen presentation and the generation of CD8 T cell responses. J Immunol 178, 6923-6930. Namba Y, Ouchi Y, Takeda A, Ueki A, Ikeda K (1999) Bleomycin hydrolase immunoreactivity in senile plaque in the brains of patients with Alzheimer’s disease. Brain Res 830, 200-202. Farrer LA, Abraham CR, Haines JL, Rogaeva EA, Song Y, McGraw WT, Brindle N, Premkumar S, Scott WK, Yamaoka LH, Saunders AM, Roses AD, Auerbach SA, Sorbi S, Duara R, Pericak-Vance MA, St George-Hyslop PH (1998) Association between bleomycin hydrolase and Alzheimer’s disease in caucasians. Ann Neurol 44, 808-811. Thome J, Gewirtz JC, Sakai N, Zachariou V, Retz-Junginger P, Retz W, Duman RS, Rosler M (1999) Polymorphisms of the human apolipoprotein E promoter and bleomycin hydrolase gene: Risk factors for Alzheimer’s dementia? Neurosci Lett 274, 37-40. Papassotiropoulos A, Bagli M, Jessen F, Frahnert C, Rao ML, Maier W, Heun R (2000) Confirmation of the association between bleomycin hydrolase genotype and Alzheimer’s disease. Mol Psychiatry 5, 213-215. Montoya SE, Aston CE, DeKosky ST, Kamboh MI, Lazo JS, Ferrell RE (1998) Bleomycin hydrolase is associated with risk of sporadic Alzheimer’s disease. Nat Genet 18, 211-212. Kajiya A, Kaji H, Isobe T, Takeda A (2006) Processing of amyloid beta-peptides by neutral cysteine protease bleomycin hydrolase. Protein Peptide Lett 13, 119-123. Ratovitski T, Chighladze E, Waldron E, Hirschhorn RR, Ross CA (2011) Cysteine proteases bleomycin hydrolase and cathepsin Z mediate N-terminal proteolysis and toxicity of mutant huntingtin. J Biol Chem 286, 12578-12589. Suszynska J, Tisonczyk J, Lee HG, Smith MA, Jakubowski H (2010) Reduced homocysteine-thiolactonase activity in Alzheimer’s disease. J Alzheimers Dis 19, 1177-1183. Huang T, Chen Y, Yang B, Yang J, Wahlqvist ML, Li D (2012) Meta-analysis of B vitamin supplementation on plasma homocysteine, cardiovascular and all-cause mortality. Clin Nutr 31, 448-454. Seshadri S, Beiser A, Selhub J, Jacques PF, Rosenberg IH, D’Agostino RB, Wilson PW, Wolf PA (2002) Plasma homocysteine as a risk factor for dementia and Alzheimer’s disease. N Engl J Med 346, 476-483. Seshadri S (2006) Elevated plasma homocysteine levels: Risk factor or risk marker for the development of dementia and Alzheimer’s disease? J Alzheimers Dis 9, 393-398. Troen AM, Shea-Budgell M, Shukitt-Hale B, Smith DE, Selhub J, Rosenberg IH (2008) B-vitamin deficiency causes hyperhomocysteinemia and vascular cognitive impairment in mice. Proc Natl Acad Sci U S A 105, 12474-12479. Zhuo JM, Pratico D (2010) Normalization of hyperhomocysteinemia improves cognitive deficits and ameliorates brain amyloidosis of a transgenic mouse model of Alzheimer’s disease. FASEB J 24, 3895-3902. Ji Y, Tan S, Xu Y, Chandra A, Shi C, Song B, Qin J, Gao Y (2013) Vitamin B supplementation, homocysteine levels, and the risk of cerebrovascular disease: A meta-analysis. Neurology 81, 1298-1307. Durga J, van Boxtel MP, Schouten EG, Kok FJ, Jolles J, Katan MB, Verhoef P (2007) Effect of 3-year folic acid supplementation on cognitive function in older adults in the FACIT
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
725
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[52]
Feldmann RE Jr, Maurer MH, Hunzinger C, Lewicka S, Buergers HF, Kalenka A, Hinkelbein J, Broemme JO, Seidler GH, Martin E, Plaschke K (2008) Reduction in rat phosphatidylethanolamine binding protein-1 (PEBP1) after chronic corticosterone treatment may be paralleled by cognitive impairment: A first study. Stress 11, 134-147. Wen HL, Ting CH, Liu HC, Li H, Lin-Chao S (2013) Decreased stathmin expression ameliorates neuromuscular defects but fails to prolong survival in a mouse model of spinal muscular atrophy. Neurobiol Dis 52, 94-103. Koh PO (2010) Proteomic analysis of focal cerebral ischemic injury in male rats. J Vet Med Sci 72, 181-185. Schonberger SJ, Edgar PF, Kydd R, Faull RL, Cooper GJ (2001) Proteomic analysis of the brain in Alzheimer’s disease: Molecular phenotype of a complex disease process. Proteomics 1, 1519-1528. Chen XH, Wang LN, Wang HL, Liu BY (2009) The expression of DJ-1 protein in proteomic analysis of late-onset Alzheimer disease. Zhonghua Nei Ke Za Zhi 48, 277-279. Graffmo KS, Forsberg K, Bergh J, Birve A, Zetterstrom P, Andersen PM, Marklund SL, Brannstrom T (2013) Expression of wild-type human superoxide dismutase-1 in mice causes amyotrophic lateral sclerosis. Hum Mol Genet 22, 51-60. Werner CJ, Heyny-von Haussen R, Mall G, Wolf S (2008) Proteome analysis of human substantia nigra in Parkinson’s disease. Proteome Sci 6, 8. Sultana R, Boyd-Kimball D, Cai J, Pierce WM, Klein JB, Merchant M, Butterfield DA (2007) Proteomics analysis of the Alzheimer’s disease hippocampal proteome. J Alzheimers Dis 11, 153-164. Wilson KE, Marouga R, Prime JE, Pashby DP, Orange PR, Crosier S, Keith AB, Lathe R, Mullins J, Estibeiro P, Bergling H, Hawkins E, Morris CM (2005) Comparative proteomic analysis using samples obtained with laser microdissection and saturation dye labelling. Proteomics 5, 3851-3858. Ferland RJ, Li X, Buhlmann JE, Bu X, Walsh CA, Lim B (2005) Characterization of Rho-GDIgamma and RhoGDIalpha mRNA in the developing and mature brain with an analysis of mice with targeted deletions of Rho-GDIgamma. Brain Res 1054, 9-21. Kaufmann WE, Moser HW (2000) Dendritic anomalies in disorders associated with mental retardation. Cereb Cortex 10, 981-991. Mastroeni D, Chouliaras L, Grover A, Liang WS, Hauns K, Rogers J, Coleman PD (2013) Reduced RAN expression and disrupted transport between cytoplasm and nucleus; a key event in Alzheimer’s disease pathophysiology. PLoS One 8, e53349. Simmons DA, Casale M, Alcon B, Pham N, Narayan N, Lynch G (2007) Ferritin accumulation in dystrophic microglia is an early event in the development of Huntington’s disease. Glia 55, 1074-1084.
PY
[42]
[51]
[53] [54]
CO
[41]
OR
[40]
Bomont P, Cavalier L, Blondeau F, Ben Hamida C, Belal S, Tazir M, Demir E, Topaloglu H, Korinthenberg R, Tuysuz B, Landrieu P, Hentati F, Koenig M (2000) The gene encoding gigaxonin, a new member of the cytoskeletal BTB/kelch repeat family, is mutated in giant axonal neuropathy. Nat Genet 26, 370-374. Shimohama S, Chachin M, Taniguchi T, Hidaka H, Kimura J (1996) Changes of neurocalcin, a calcium-binding protein, in the brain of patients with Alzheimer’s disease. Brain Res 716, 233-236. Reddy PH, Mani G, Park BS, Jacques J, Murdoch G, Whetsell W, Jr., Kaye J, Manczak M. (2005) Differential loss of synaptic proteins in Alzheimer’s disease: Implications for synaptic dysfunction. J Alzheimers Dis 7, 103-117; discussion 173-180. George AJ, Gordon L, Beissbarth T, Koukoulas I, Holsinger RM, Perreau V, Cappai R, Tan SS, Masters CL, Scott HS, Li QX (2010) A serial analysis of gene expression profile of the Alzheimer’s disease Tg2576 mouse model. Neurotox Res 17, 360-379. Cheon MS, Fountoulakis M, Cairns NJ, Dierssen M, Herkner K, Lubec G (2001) Decreased protein levels of stathmin in adult brains with Down syndrome and Alzheimer’s disease. J Neural Transm Suppl 281-288. Saetre P, Jazin E, Emilsson L (2011) Age-related changes in gene expression are accelerated in Alzheimer’s disease. Synapse 65, 971-974. Liu A, Stadelmann C, Moscarello M, Bruck W, Sobel A, Mastronardi FG, Casaccia-Bonnefil P (2005) Expression of stathmin, a developmentally controlled cytoskeletonregulating molecule, in demyelinating disorders. J Neurosci 25, 737-747. Zhao F, Hu Y, Zhang Y, Zhu Q, Zhang X, Luo J, Xu Y, Wang X (2012) Abnormal expression of stathmin 1 in brain tissue of patients with intractable temporal lobe epilepsy and a rat model. Synapse 66, 781-791. Yamada K, Matsuzaki S, Hattori T, Kuwahara R, Taniguchi M, Hashimoto H, Shintani N, Baba A, Kumamoto N, Yamada K, Yoshikawa T, Katayama T, Tohyama M (2010) Increased stathmin1 expression in the dentate gyrus of mice causes abnormal axonal arborizations. PLoS One 5, e8596. Bae N, Wang Y, Li L, Rayport S, Lubec G (2013) Network of brain protein level changes in glutaminase deficient fetal mice. J Proteomics 80, 236-249. Thorsell A, Bjerke M, Gobom J, Brunhage E, Vanmechelen E, Andreasen N, Hansson O, Minthon L, Zetterberg H, Blennow K (2010) Neurogranin in cerebrospinal fluid as a marker of synaptic degeneration in Alzheimer’s disease. Brain Res 1362, 13-22. Hodges A, Strand AD, Aragaki AK, Kuhn A, Sengstag T, Hughes G, Elliston LA, Hartog C, Goldstein DR, Thu D, Hollingsworth ZR, Collin F, Synek B, Holmans PA, Young AB, Wexler NS, Delorenzi M, Kooperberg C, Augood SJ, Faull RL, Olson JM, Jones L, Luthi-Carter R (2006) Regional and cellular gene expression changes in human Huntington’s disease brain. Hum Mol Genet 15, 965-977.
TH
[39]
J. Suszy´nska-Zajczyk et al. / Blmh-null Mouse, Hyperhomocysteinemia, and Neurodegeneration
AU
726
[55]
[56]
[57]
[58]
[59]
[60]
[61]
[62]
[63]