Cortical Cholinergic Denervation Elicits Vascular A> Deposition ALEX E. ROHER,a,d YU-MIN KUO,a PAMELA E. POTTER,a MARK R. EMMERLING,b ROBERT A. DURHAM,b DOUGLAS G. WALKER,a LUCIA I. SUE,a WILLIAM G. HONER,c AND THOMAS G. BEACHa aSun
Health Research Institute, Sun City, Arizona 85351, USA
bParke-Davis
Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105, USA
cDepartment
of Psychiatry, University of British Columbia, Vancouver, British Columbia, Canada
ABSTRACT: Selective destruction of the cholinergic nucleus basalis magnocellularis (nbm) in the rabbit by the p75 neurotrophin receptor (NTR) immunoglobulin G (IgG) complexed to the toxin saporin leads to the deposition of amyloid-beta (A>) in and around cerebral blood vessels. In some instances, the perivascular A> resemble the diffuse deposits observed in Alzheimer’s disease (AD). We propose that cortical cholinergic deprivation results, among other perturbations, in the loss of vasodilation mediated by acetylcholine. In addition to a dysfunctional cerebral blood flow, alterations in vascular chemistry affecting endothelial and smooth muscle cells may result in cerebral hypoperfusion and a breached blood-brain barrier (BBB). The selective removal of the rabbit nbm and A> accumulation may serve as an important nontransgenic, and more physiological, model for the testing of pharmacological and immunological agents designed to control the deposition and the deleterious effects of A> in AD.
INTRODUCTION The neuropathology of Alzheimer’s disease (AD) is characterized by the abundant accumulation of amyloid in cortical neuritic plaques and vascular walls, and by the intraneuronal deposition of neurofibrillary tangles.1,2 The neuritic plaques represent a collection of dystrophic neurites and reactive glial cells that surround a core of fibrillar amyloid-beta (Aβ) peptide.1 The neurofibrillary tangles are mainly composed of the microtubule-associated protein tau and membrane-derived glycolipids.3,4 In addition to these lesions, there is an extensive vascular pathology affecting the microcirculation of the brain and leptomeninges.5,6 The capillary and arteriolar tree in AD is extensively damaged, far beyond the anatomical changes observed during normal aging.7 There are important changes in the blood-brain barrier (BBB) endothelium associated with AD such as basement membrane fenestration and duplication, loss of perivascular plexus, loss of endothelial cells, decrease in gludAddress for correspondence: Dr. Alex E Roher, Sun Health Research Institute, 10515 West Santa Fe Dr., Sun City, Arizona, 85351. Tel.: (602) 876-5465; fax: (602) 876-5698. e-mail:
[email protected]
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cose transporter activity, decrease in α-adrenergic receptors, decrease in protein kinase C (PKC) activity, increase in cyclic adenosine-5′,3′-monophosphate (cAMP) and increased nitric oxide levels.7–14 In AD, the presence of Aβ deposits around cortical vessels appears to obliterate capillaries and arterioles causing vascular stenosis resulting in focal hypoxia and ischemia.6,15,16 These perivascular deposits are dramatically exacerbated in those AD individuals carrying the apolipoprotein E (ApoE) ε4 allele.17–19 Aβ deposition in larger cerebral and leptomeningeal vessels results in the destruction of the myocytes of the tunica media leading to failure in the control of cerebral blood flow (CBF).7,16 The cerebral blood vessels are innervated by the cholinergic neurons of the nucleus basalis of Meynert (nbM) which effect vasodilation and by the noradrenergic locus ceruleous and serotonergic dorsal raphe nucleus which mediate vasoconstriction.20,21 The neurons in these nuclei are extensively damaged in AD.22,23 There are decreased neuronal numbers, and the remaining cells contain heavy deposits of paired helical filaments (PHF). Injury to these nuclei results in vascular denervation causing alterations in cerebral perfusion and disturbances in BBB integrity. We hypothesized that during normal senescence, and more critically during the early, preclinical stages of AD, cholinergic denervation among other cellular perturbations may lead to vascular deposition of Aβ. To test this hypothesis we induced selective cholinergic denervation in the rabbit’s brain using the ribosomal toxin saporin conjugated to an antibody raised against the p75 neurotrophin receptor (NTR). We chose the rabbit as a model because its nucleus basalis magnocellularis (nbm), which is equivalent to nbM in the human, is well developed and because the rabbit’s Aβ amino acid sequence is 100% homologous to that of the human species.
METHODS A specific hybridoma line expressing the monoclonal antibody ME20.2 recognizing the p75 NTR was obtained from the American Type Culture Collection (Rockville, MD). The immunoglobulin G (IgG) was produced, purified and tagged with saporin by Advanced Targeting Systems (Carlsbad, CA). In the following experiments eleven 12-week-old New Zealand white rabbits were utilized. Six rabbits, weighing from 2.09 to 2.27 kg, received a unilateral intracerebroventricular injection of 12 µl immunotoxin in sterile saline at a concentration of 2.7 µg/µl. Five rabbits serving as control animals received 12 µl sterile saline alone. The stereotactic coordinates used in both groups of animals were relative to bregma: AP = 0, L = 2.2 mm, and D = 7.5 mm. Six months after surgery, the rabbits were killed and brain slices (4 mm) ipsilateral and immediate to the site of injection were fixed in 4% paraformaldehyde and used for immunohistological studies. The remaining of the brain slices were immediately frozen at −85°C for biochemical studies. Aβ was detected using the antibodies 10D5, raised against Aβ residues 1–16 (Athena Neurosciences, South San Francisco, CA) and 4G8 raised against Aβ residues 17–24 (Senetek PLC, Maryland Heights, MO). The antibody ME20.4 (Advanced Targeting Systems, San Diego, CA) was used for the detection of p75 NTR. Forty-µm sections were cut on a freezing microtome and reacted with the antibodies, and the color reaction was developed using 3,3′-diaminobenzidine as the
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substrate.24 Acetylcholinesterase enzyme histochemistry was performed following the method of Tago et al.25 Cortical choline acetyltransferase (ChAT) was determined on frontal pole cerebral cortex ipsilateral to the injection site, using the method of Fonnum.26 For the detection of microglia, the lectin Griffonia simplicifolia (Sigma, St. Louis, MO) was utilized as previously described.27 For Aβ quantification, cerebral cortex (100 mg) from rabbit brain was homogenized in 10 volumes of 2% diethylamine prepared in 0.9% NaCl and centrifuged at 12,000 × g for 20 min. The supernatant was neutralized with an equal volume of 0.5 M Tris-HCl, pH 7.5. The levels of Aβ peptides were measured by enzyme-linked immunosorbent assay (ELISA) using the C-terminal specific polyclonal antibodies R163 and R165, raised against Aβ40 and Aβ42, respectively, as capture antibodies. Antibody 4G8, recognizing Aβ17–24, was used as reporter antibody.28 Amyloid-β precursor protein (AβPP) was quantified by Western blotting using the monoclonal antiboby 22C11 (Roche Molecular Biochemical, Indianapolis, IN). In the 6-monthold control and immunotoxin-treated rabbits the levels of two synaptic proteins were determined by ELISA.29 In this study we employed the polyclonal antibodies EP10 and SP15 (raised against synaptophysin) and SP12 (raised against SNAP-25).
RESULTS Intraventricular injection of the IgG-saporin complex resulted in the selective ablation of the cholinergic neurons of the nucleus basalis magnocellularis (FIGS. 1A and 1B), with the concomitant loss of their cortical afferents (FIGS. 1C and 1D). The demise of the cholinergic neurons was determined by p75 NTR immunohistochemistry and acetylcholinesterase histochemistry. In the immunotoxin-treated rabbits, there were abundant small and large cortical vessels with immunopositive Aβ deposits (FIGS. 2A, 2B, 2C, and 2D), which were not present in the control animals. In addition, there were diffuse deposits of immunopositive Aβ around some blood vessels (FIGS. 2E and 2F) and along the subpial region of the cortex (data not shown). Congo red and thioflavine-S staining were negative suggesting that the deposited Aβ peptide is not in the β-sheet conformation. ELISA revealed that the immunotoxin-treated animals contained 2.5 and 8 times more Aβ40 and Aβ42, respectively, than the control rabbits (TABLE 1). These differences were statistically significant: Aβ40 , p = 0.020; Aβ42 , p = 0.033 (Student t-test, 2 tailed). In reference to the levels of soluble and full-length AβPP, no significant statistical differences were found between the immunotoxin-treated and the control rabbits. TABLE 1. Choline acetyltransferase (ChAT) enzyme activity, synaptophysin and Aβ (N-40, N-42) in cerebral cortex of immunotoxin- and saline-injected animals Treatment Immunotoxin Saline
ChAT (pmol/mg protein/min) 0.21 ± 0.05* 0.68 ± 0.08*
Synaptophysina 0.485 ± 0.060 0.421 ± 0.029
N-40 N-42 (pmol/g) (pmol/g) 13.14 ± 6.32 12.65 ± 9.72 5.09 ± 0.64 1.58 ± 1.15
aValues represent the amount of protein in µg required to generate an optical density value of 0.50. * Value represents mean ± standard deviations.
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FIGURE 1. Selective immunotoxic elimination of cholinergic neurons. (A) The rabbit nucleus basalis magnocellularis (nbM) stained by p75 NTR immunohistochemistry. (B) The cholinergic neurons of the nbm annihilated by the p75 NTR IgG-saporin complex. Only a few scattered neurons remain in the nbm area. (C) Acetylcholinesterase staining of the cholinergic terminals in the cortical area of the rabbit brain. (D) Acetylcholinesterase histochemistry shows the complete demise of the cholinergic cortical afferents after treatment with the IgG-saporin complex.
In the immunotoxin-treated rabbits, there was a significant reduction in cortical cholinergic innervation, since the average levels of ChAT represented only 31% of the values observed in the control rabbits (TABLE 1). As expected, there was a modest decrease in the amount of synaptic proteins. In the immunotoxin-treated rabbits, synaptophysin was reduced by an average of 10% (TABLE 1) and SNAP-25 decreased by 13% when compared to the control values (not shown).
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FIGURE 2. Vascular deposition of Aβ peptide immunostained by the 10D5 antibody. (A) A large number of cortical microvessels immunopositive for Aβ peptide. (B, C and D) Other cortical vessels immunopositive for the Aβ peptide. (E and F) Condensed and diffused Aβ deposition around cortical blood vessels in the immunotoxin treated rabbits.
DISCUSSION The present study demonstrates for the first time that ablation of the cholinergic neurons of the nbm in the rabbit causes Aβ deposition around cerebral blood vessels and in the pia limitans. Aβ deposition was not observed in the sham-operated rabbits, nor in studies on aged rabbits.27 The loss of the cholinergic innervation from the nbm may be a seminal event in AD pathogenesis. In a recent study our group demonstrated that in the nondemented elderly population the presence of neurofibrillary degeneration is common in the
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nbM.30 In this study, all individuals (n = 31; average age 79.0 years) exhibited neurofibrillary tangles (NFT) as detected by thioflavine-S, PHF immunochemistry, and double staining by PHF-1 and acetylcholinesterase. This observation suggests that degeneration of the nbM is the most probable cause of the cortical cholinergic denervation seen in the elderly and may be an important preclinical event in the pathogenesis of AD. In line with this hypothesis, it has been shown that cholinergic deficits antedate the emergence of senile plaques in normal aging,31and that there is an elevation in Aβ levels starting at age 50, as detected by Western blots.32 The elimination of cholinergic afferents may be the traumatic event that stimulates the production of Aβ in cortical neurons. In this scenario, the chronic reduction of m1 and m3 muscarinic receptor activation may reduce the nonamyloidogenic processing of AβPP and thereby increase the production of Aβ.33 In the absence of AβPP overproduction, which in the present study appears not to differ from the control animals at least at 6 months postlesion, an alteration of AβPP processing needs to be contemplated. However, more AβPP and Aβ estimations are necessary in the immunotoxin-treated rabbits at earlier dates to distinguish between these possibilities. The loss of cholinergic innervation of the nbm may also promote the sequestration of Aβ around blood vessels due to compromised vasomotor activity. Damage to the vasoactive neurons of the nbM would have serious repercussions. Endothelial cells and vascular myocytes have muscarinic receptors that on acetylcholine stimulation result in vasodilation possibly mediated by nitric oxide release. Perturbations in this chain of chemical events are likely to produce alterations in the control of CBF, and cerebral perfusion as well as changes in the integrity of the BBB. In a similar experiment, rats receiving intraventricular injections of 192 IgG-saporin, to elicit cortical cholinergic denervation, exhibited a marked decrease in CBF.34 In the rat the most affected areas were the temporal and parietal cortices, which are also the most affected in terms of CBF failure in AD, especially in those patients who are ApoE ε4. Those who are ApoE ε4 may be more at risk for the vascular pathology seen in AD, since they appear to be hypocholinergic naturally.35 One would also expect that the vasomotor dysfunction resulting from cholinergic deprivation of the brain vasculature would be exacerbated by factors such as: hypertension, hypercholesterolemia, hyperhomocysteinemia, and estrogen loss, all of which decrease vasomotor function and increase the risk for AD.36–39 Vascular Aβ may also lead to decreased responsiveness of cerebral vessels by cholinergic stimulation as recently shown by Mullan’s group.40 It has been recently demonstrated that antibodies against the human Aβ42 peptide produced in the human AβPP transgenic mice apparently prevent the amyloid deposition and toxicity, and perhaps facilitate the clearance of Aβ peptide from the brain.41 The rabbit cholinergic model of Aβ deposition presented here thus becomes a very useful system to test the effectiveness of these antibodies in animals possessing an Aβ amino acid sequence identical to the human species. ACKNOWLEDGMENTS This work was partially supported by the State of Arizona Center for Alzheimer’s Disease Research and by the Alzheimer’s Association. This paper is dedicated to the memory of our colleague W. Harold Civin, M.D.
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