Abstract
A rapidly progressing aging society has raised attention to white matter lesions in Alzheimer’s disease. In the present study, we applied an AD plus cerebral hypoperfusion (HP) mouse model and investigated the alternation of key protein molecules in the nodal, paranodal, and intermodal sites in the white matter as well as the efficacy of galantamine. Cerebral HP was induced in APP23 mice by bilateral common carotid arteries stenosis with ameroid constrictors. Compared with the wild type and simple APP23 mice, APP23 + HP mice showed a progressive loss of MAG and NF186 from 6 to 12 months, broken misdistribution of MBP, and extended relocation of Nav1.6 and AnkG beyond the primary nodal region in the corpus callosum. Such abnormal neuropathological processes were retrieved with galantamine treatment. The present study demonstrated that cerebral HP strongly disrupted white matter integrity (WMI) at intermodal, paranodal, and Ranvier’s nodal sites which may be associated with cognitive decline. Galantamine treatment significantly protected such WMI probably by allosterically potentiating ligand action.
INTRODUCTION
White matter integrity (WMI), which is comprised mostly of myelinated axons, is critical in neural conduction between brain regions and cognitive performance [1]. On myelinated fibers, the nodes of Ranvier are flanked by paranodes (axon-glial junctions) and internodes (juxtaparanodal region), which are characterized by specific protein complexes [2]. The molecular components of the node are voltage gated sodium channels (Nav), ankyrin G (AnkG), and Nfasc186 (NF186), those of the paranode are Caspr and myelin-associated glycoprotein (MAG), which are critical for axon-glia junctions, and those of the internode are MAG and myelin basic protein (MBP), which are localized on glial cells (Fig. 1i). Disruption of such architecture of myelinated axons could impede neuronal conduction and finally lead to a decline in cognitive function.
A rapidly progressing aging society has raised attention to white matter lesions (WMLs) in Alzheimer’s disease (AD) [3 –6]. Cerebral hypoperfusion (HP) has been suggested to contribute to the development of WML and cognitive impairment in aged brains [7] (Zhai et al., unpublished data). Galantamine, which is currently used for treatment of AD, inhibits acetylcholinesterase [8], modulates nicotinic acetylcholine receptors (nAChRs) [9], and exerts anti-oxidative and anti-inflammatory effects [10 –12]. Our previous report showed the suppression of both microglial activation and WML by galantamine using AD plus HP model mice. However, the specific molecular mechanism related to WMI involved in neuronal conduction has not yet been identified.
In the present study, therefore, we applied the AD mouse model with long-standing cerebral HP for studying WML in aged AD brains (Zhai et al., unpublished data), focusing on the key protein molecules in nodal, paranodal, and intermodal sites as well as the efficacy of galantamine.
MATERIALS AND METHODS
Experimental model and drug treatment
All animal experiments were performed in compliance with a protocol approved by the Animal Committee of the Graduate School of Medicine and Dentistry, Okayama University (approval #OKU-2014095). The present study was a part of whole project that is focusing on white matter change in AD model mice with chronic hypoperfusion. Male mice were divided into 4 groups in this study: wild type mice (WT, C57BL/6J, only sham surgery, n = 10), APP23 group (APP23 + sham surgery, n = 17), chronic hypoperfusion group (APP23 + HP, n = 12), and chronic hypoperfusion + galantamine-treated group (APP23 + HP + Gal, n = 10). The generation of the B6, D2-TgN (Thy1-APPSwe) transgenic mouse line (APP23) was previously described [13, 14]. Chronic cerebral HP was induced by applying ameroid constrictors (0.75 mm internal diameter; Research Instruments NW, Lebanon, OR, USA). To conduct surgery of cerebral HP, a cervical incision was made and ameroid constrictors were applied to bilateral common carotid (BCC) arteries for chronic progressive stenosis (BCCS) at 4 months (M) of age in APP23 mice (body weight 20–25 g). 15 days (d) after surgery, when the cervical incision had completely healed, APP23 + HP + Gal group mice began to receive galantamine (5 mg/kg; 1 mg/ml in ultrapure water, 0.15 ml; Takeda Pharmaceutical Co. Ltd., Osaka, Japan) once daily by oral gavage untilsacrifice.
Tissue preparation and immunohistochemistry
At 6 and 12 M of age, WT, APP23, APP23 + HP, and APP23 + HP + Gal mice were deeply anesthetized by intraperitoneal injection of pentobarbital (40 mg/kg) and transcardially perfused with 20 ml of ice-cold phosphate-buffered saline (PBS) and then 20 ml of ice-cold 4% paraformaldehyde (PFA) in 0.1 mol/L phosphate buffer. The brains were removed and post-fixed in 4% PFA overnight. Floating coronal sections (50 μm thickness) were produced with a vibrating blade microtome (LEICA VT1000S; Leica, Nussloch, Germany). For immunohistochemistry, brain sections were immersed in 0.6% periodic acid to block intrinsic peroxidase, and were treated with 5% bovine serum in 50 mM PBS, pH 7.4, containing 0.1% triton to block any non-specific antibody responses, and were incubated at 4°C overnight with primary antibody. We used the following primary antibodies: MAG (1 : 100, ab89780; Abcam, Cambridge, UK), MBP (1 : 100, MAB386; Millipore, Billerica, MA, USA), Nav1.6 (1 : 200, AB5580; Millipore, Billerica, MA, USA), Caspr (1 : 100, clone K65/35; UC Davies NIH NeuroMab, Davis, CA, USA), Nfasc186 (NF186, 1 : 100, ab31719; Abcam, Cambridge, UK), ankyrin G (AnkG, 1 : 100, H-215, sc-28561; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunoreactions were visualized using fluorescent secondary antibody with the diaminobenzidine reaction.
Quantitative analysis
All images for immunostaining analysis were taken with a laser scanning confocal microscope (LSM510, CarlZeiss, Oberkochen, Germany), and were analyzed using image processing software (ImageJ; National Institutes of Health, Bethesda, USA). For each measurement, we analyzed separated sections from three levels of the caudate putamen (1.0, 0.5, and 0 mm rostral to the bregma) per brain and 3 or 4 randomly selected regions per section (i.e., n = 9–12 measurements per mouse) [15]. Fluorescent intensity of MAG and MBP immunohistochemistry in the corpus callosum region was measured. To analyze MBP distribution, we ranked MBP-positive fiber sizes in ascending order, i.e., 0~20 μm2, 21~40 μm2, 41~60 μm2, 61~80 μm2, and 81~100 μm2, and then measured the number of MBP-positive fibers in each size. The length of Nav1.6, NF186, and AnkG aggregates were analyzed in the corpus callosum by double immunolabeling (Na1.6-Caspr, NF186-Caspr, and AnkG-Caspr), respectively. Similarly, the length of the gap between Caspr signals was measured by contrasting with the nodal signal (Nav1.6, NF186, and AnkG).
Statistical analysis
All data were expressed as mean ± SD. Statistical analyses were performed using one way analysis of variance followed by a Tukey–Kramer postcomparison. Differences with a probability value of p < 0.05 were considered statistically significant.
RESULTS
Breakdown of axon-glial connections MAG and MBP
No significant reduction was observed in the fluorescent intensity of MAG both in WT and APP23 groups at 6 and 12 M (Fig. 2A, a, b and 2B). However, APP23 + HP group showed a progressive reduction in MAG immunofluorescence from 6 to 12 M (p < 0.05 versus WT) with a small recovery by galantamine treatment (Fig. 2A, c, d and 2B, p < 0.05 versus WT).
On the other hand, there was no difference in MBP fluorescent intensity among four groups of mice at 6 and 12 M (Fig. 2A, e-h and 2C). However, size-dependent analysis of MBP immunofluorescence showed a significant increase in the number of large MBP-positive fibers (21~100 μm2) in APP23 + HP group compared the smallest MBP-positive fibers (0~20 μm2) at 12 M (Fig. 2D and E, * p < 0.05). The APP23 + HP + Gal group showed ameliorations of such increases in APP + HP group at 12 M (Fig. 2E).
Spreading of Nav1.6 channel and Caspr disruption
The Nav1.6 channel cluster was located in the node of Ranvier (0.71~0.84 μm long), and was flanked by paranodal protein Caspr in both WT and APP23 at 6 and 12 M of corpus callosum (Fig. 3A, a, b, e, f and 3B). APP23 + HP progressively extended the length of the Nav1.6 cluster into both sides of the primary paranodal area from 6 (0.93 ± 0.32 μm, §§ p < 0.01 versus WT) to 12 M (1.26 ± 0.44 μm, §§ p < 0.01 versus WT), which was significantly recovered by galantamine treatment (6 M, 0.84 ± 0.33 μm; 12 M, 0.91 ± 0.44 μm, both # # p < 0.01 versus APP23 + HP) (Fig. 3A, c, d, g, h, arrowheads and 3B), with a corresponding extension in Caspr gap length (Fig. 3A, c, g and 3C). As a result, overlapping areas of Nav1.6 + Caspr increased at 6 and 12 M in APP23 + HP group (yellow in Fig. 3Ac and 3Ag). The number of nodes in the corpus callosum was not different among four experimental groups both at 6 and 12 M (Fig. 3A, D).
Alternation of nodal anchoring proteins NF186 and ankyrin G
APP23 group showed a progressive reduction in the staining and length of NF186 from 6 to 12 M in the corpus callosum (Fig. 4A, b, f, arrowheads and 4B, WT 0.78 ± 0.31 μm versus APP23 0.68 ± 0.21 μm at 12 M, §§ p < 0.01). Compared with WT and APP23 groups, the length of NF186 decreased significantly in APP23 + HP group both at 6 (* p < 0.05) and 12 M (** p < 0.01), but recovered significantly after galantamine treatment (Fig. 4A, c, d, g, h, arrowheads and 4B, # # p < 0.01 versus APP23 + HP).
AnkG staining was not different between WT and APP23 groups. However, APP23 + HP group showed a significant extension in the length of AnkG beyond the primary nodal area of Ranvier at 6 M (0.80 ± 0.21 versus 0.67 ± 0.12 μm, §§ p < 0.01 versus WT). Such an extension was significantly retrieved after galantamine treatment (0.73 ± 0.20 μm, # # p < 0.01 versus APP23 + HP, Fig. 5A, c, d, arrowheads and 5B).
DISCUSSION
In the present study, we applied an AD plus HP mouse model, which mimicked a mild but sustained reduction of cerebral blood flow in aging brains with AD [16, 17] (Zhai et al, unpublished data) to examine key proteins at intermodal, paranodal, and nodal regions that are responsible for WMI. Our results showed the disruption of WMI in this AD mouse model with a significant decrease of MAG intensity, MBP breakdown (Fig. 2), extended relocation of Nav1.6 and AnkG (Figs. 3 and 5), and the loss of NF186 (Fig. 4). On the other hand, galantamine retrieved such abnormal key proteins expressions and distributions (Figs. 2 –5). Figure 1 presents a schematic summary of the changes of internodal, paranodal, and nodal constituents in each experimental group.
MAG normally localizes in the periaxonal membranes of glial internodes and paranodes, and plays an important role in maintenance of the axon-glial junction [18, 19]. As previously reported, the protein level of MAG decreased in WML after stroke, multiple sclerosis, and other inflammatory brain diseases [20]. In agreement with previous reports, a progressive loss of MAG was also shown in the present study in APP23 + HP group from 6 to 12 M (Fig. 2A, c and 2B), suggesting the disruption of the axon-glia junction. On the other hand, MBP is normally located on the compact myelin sheath (Fig. 1i). APP23 + HP group showed that the distribution of MBP protein changed but that the protein level of MBP was not altered at 12 M (Fig. 2A, g, 2C-2E). This difference may be derived from characteristics of each animal model. In a focal cerebral ischemia model, cerebral blood flow was reduced to 30% of normal levels, showing a marked loss of MBP in the ipsilateral ischemic hemisphere [21]. These findings strongly suggest that chronic cerebral HP did not result in a simple reduction of MBP, but accelerated axon-glia damage and myelin sheath alteration (Fig. 1).
At the node of Ranvier, Nav channels are normally associated with both anchor proteins NF186 and AnkG, to form the nodal Nav1.6 complex [22]. Previous reports showed that Nav channels and AnkG were dispersed along the axon membrane under demyelination and did not cluster at the node of Ranvier in mice lacking NF186 [23 –26]. In the present study, the loss of NF186 distribution was accompanied by extended relocations of Nav1.6 and AnkG in APP23 + HP group (Figs. 3 –5), suggesting that HP exacerbated the breakdown of the Nav1.6 complex together with NF186 and AnkG, leading to disruption of the node of Ranvier (Fig. 1iii). Caspr is an integral component of paranodal septate-like junctions [27]. In the present study, Caspr gap length was extended in APP23 + HP mice (Fig. 3Ac, g, 3C), suggesting the breakdown of paranodal septate-like junctions (Fig. 1iii).
Galantamine is an acetylcholinesterase inhibitor, acts as an allosterically potentiating ligand (APL) for nAChRs at the synaptic terminals of most neurotransmitters, and also exerts anti-oxidative/anti-inflammatory effects [10, 11]. It is well recognized that microglial cells play an important role in the pathogenesis of inflammatory demyelination [28]. As previously demonstrated, activated microglia within demyelinating lesions can produce a number of cytotoxic molecules to induce myelin and/or axonal injury [29, 30], probably leading to disruption of key proteins of myelinated axons such as Nav1.6, Caspr, and NF186 [31, 32]. Other reports demonstrated that galantamine improved spatial working memory and significantly rescued WML by suppressing microglial activation through APL action [12 , 33–35], further suggesting the potential anti-inflammatory effect of galantamine. Therefore, in the present study, the breakdown of MAG/MBP/NF186 and the extension of Nav1.6/AnkG were significantly rescued by galantamine treatment (Figs. 2 –5), probably by an anti-inflammatory effect (Fig. 1iv).
In summary, the present study demonstrated that chronic cerebral HP exacerbated the expression and redistribution of key proteins at intermodal, paranodal and Ranvier’s nodal sites in white matter, and that such neuropathological abnormalities were significantly ameliorated by galantamine treatment, most likely in association with nAChR-mediated neuroprotection by APL action.
Footnotes
ACKNOWLEDGMENTS
This work was partly supported by Grant-in-Aid for Scientific Research (B) 25293202, (C) 15K09316 and Challenging Research 15K15527 and Young Research 15K21181, and by Grants-in-Aid from the Research Committees (Mizusawa H, Nakashima K, Nishizawa M, Sasaki H, and Aoki M) from the Ministry of Health, Labour and Welfare of Japan.
