Yoshie Takaki

Identification of the major Aβ1-42 degrading catabolic pathway in brain parenchyma : suppression leads to biochemical and pathological deposition

Alzheimer amyloid β-peptide (Aβ) is a physiological peptide constantly anabolized and catabolized under normal conditions. We investigated the mechanism of catabolism by tracing multiple-radiolabeled synthetic peptide injected into rat hippocampus. The Aβ1–42 peptide underwent full degradation through limited proteolysis conducted by neutral endopeptidase (NEP) similar or identical to neprilysin as biochemically analyzed. Consistently, NEP inhibitor infusion resulted in both biochemical and pathological deposition of endogenous Aβ42 in brain. This NEP-catalyzed proteolysis therefore limits the rate of Aβ42 catabolism, up-regulation of which could reduce the risk of developing Alzheimers disease by preventing Aβ accumulation.

Reply to: 'Clearance of amyloid β-peptide from brain: transport or metabolism?'

To the editor—We read with interest the article by Iwata et al. in the February 2000 issue of Nature Medicine suggesting a major degrading catabolic pathway for Alzheimer amyloid β-peptide (Aβ) in brain. Our own work, however, has indicated that, in addition to production of the peptide, transport of Aβ across the microvascular endothelium (that is, the site of the blood–brain barrier) is essential in controlling Aβ levels in the brain. The physiological relevance of degradation mechanisms of Aβ described by Iwata et al. remains unclear, as the peptide was studied at very high concentrations of about 240 μm/l, which may impair blood–brain barrier integrity. It is likely that Aβ at a concentration of 240 μm/l will cause plasma leakage into brain tissue. The size of the spot of Evans blue dye in the hippocampus and along the needle track shown by Iwata et al. (ref. 1, Fig. 1d) may translate into a volume of about 20–30 μl, much larger than the reported infusion volume of 0.5 μl. The invasive nature of the acute placement of 26-gauge needle used by Iwata et al. very likely damages the blood–brain barrier. There is injury to blood vessels and microbleeding. Also, staining along the needle track indicates substantial reflux of injected material back to subarchnoid space, where contamination with cerebrospinal fluid is obvious. It is essential to place a small 33-gauge guiding cannula at least 3–5 days before infusing peptides to allow the tissue to recover from the trauma. Finally, the affinity of neprilysin for its physiological substrates (such as enkephalins, tachykinins and atrial natriuretic peptide) and/or different synthetic peptides is in the low millimolar to micromolar range. Normal levels of Aβ in the brain are in the low nanomolar ranges. Even in transgenic models of brain amyloidosis, brain levels of Aβ range from 40 nm/kg to 250 nm/kg from 3 to 12 months. Several cell surface receptors in brain endothelial cells, microglia and/or neurons, including the receptor specific for advanced glycosylation end-products (RAGE), the scavenger type A receptor (SR-A; refs. 2,8), LRP-1 (low density lipoprotein receptor related protein) (ref. 9) and LRP-2 (ref. 3) bind Aβ with high nanomolar affinity directly (RAGE and SR-A) or indirectly (LRP-1 and LRP-2) through ligands such as α2M and apolipoproteins E and J, which act as high-affinity transport binding proteins for Aβ. Thus, in the intact brain, Aβ probably binds to several high-affinity cell surface receptors and/or binding proteins before being recognized by potential degrading enzymes.

Somatostatin regulates brain amyloid beta peptide Abeta42 through modulation of proteolytic degradation.

Expression of somatostatin in the brain declines during aging in various mammals including apes and humans. A prominent decrease in this neuropeptide also represents a pathological characteristic of Alzheimer disease. Using in vitro and in vivo paradigms, we show that somatostatin regulates the metabolism of amyloid β peptide (Aβ), the primary pathogenic agent of Alzheimer disease, in the brain through modulating proteolytic degradation catalyzed by neprilysin. Among various effector candidates, only somatostatin upregulated neprilysin activity in primary cortical neurons. A genetic deficiency of somatostatin altered hippocampal neprilysin activity and localization, and increased the quantity of a hydrophobic 42-mer form of Aβ, Aβ42, in a manner similar to presenilin gene mutations that cause familial Alzheimer disease. These results indicate that the aging-induced downregulation of somatostatin expression may be a trigger for Aβ accumulation leading to late-onset sporadic Alzheimer disease, and suggest that somatostatin receptors may be pharmacological-target candidates for prevention and treatment of Alzheimer disease.

Presynaptic Localization of Neprilysin Contributes to Efficient Clearance of Amyloid-β Peptide in Mouse Brain

A local increase in amyloid-β peptide (Aβ) is closely associated with synaptic dysfunction in the brain in Alzheimers disease. Here, we report on the catabolic mechanism of Aβ at the presynaptic sites. Neprilysin, an Aβ-degrading enzyme, expressed by recombinant adeno-associated viral vector-mediated gene transfer, was axonally transported to presynaptic sites through afferent projections of neuronal circuits. This gene transfer abolished the increase in Aβ levels in the hippocampal formations of neprilysin-deficient mice and also reduced the increase in young mutant amyloid precursor protein transgenic mice. In the latter case, Aβ levels in the hippocampal formation contralateral to the vector-injected side were also significantly reduced as a result of transport of neprilysin from the ipsilateral side, and in both sides soluble Aβ was degraded more efficiently than insoluble Aβ. Furthermore, amyloid deposition in aged mutant amyloid precursor protein transgenic mice was remarkably decelerated. Thus, presynaptic neprilysin has been demonstrated to degrade Aβ efficiently and to retard development of amyloid pathology.

Region-specific reduction of A beta-degrading endopeptidase, neprilysin, in mouse hippocampus upon aging

Metabolism of amyloid‐β peptide (Aβ) is closely associated with the pathology and etiology of Alzheimers disease (AD). Neprilysin is the only rate‐limiting catabolic peptidase proven by means of reverse genetics to participate in Aβ metabolism in vivo. The aim of the present study is to assess whether possible spatial changes in neprilysin level in the brain with aging correlate to AD‐vulnerable regions. When neprilysin levels in various brain regions of 10‐, 80‐ and 132‐week‐old mice were evaluated by neprilysin‐dependent endopeptidase activity assay and Western blot‐based quantitative analysis, a clear change in neprilysin level with aging was observed in the hippocampal formation, in which the level was reduced by 20% at 132 weeks, compared to the 10‐week group. Quantitative immunohistochemical analysis confirmed a marked local reduction of neprilysin levels with aging at the outer molecular layer and polymorphic layer of the dentate gyrus, and the stratum lucidum of the hippocampus, where the densities were reduced by 56%, 82% and 83%, respectively, at 132 weeks compared to the 10‐week group. Thus, neprilysin was decreased selectively at the terminal zones and on axons of the lateral perforant path and the mossy fibers. These are the sites that show Aβ pathology in mutant amyloid precursor protein (APP) transgenic mice, and that show synaptic loss in AD. The immunoreactivities to synaptic vesicle protein‐2 and synaptophysin in the stratum lucidum and the dentate gyrus were unchanged, suggesting that a loss or decrease of synapses was not responsible for the decrease in the neprilysin levels. These observations suggest that downregulation of neprilysin is likely to be related to AD pathology and to the Aβ deposition associated with normal aging in humans.

Cerebrospinal fluid neprilysin is reduced in prodromal Alzheimer's disease

Amyloid β peptide (Aβ) has been implicated in Alzheimers disease (AD) as an initiator of the pathological cascades. Several lines of compelling evidence have supported major roles of Aβ‐degrading enzyme neprilysin in the pathogenesis of sporadic AD. Here, we have shown a substantial reduction of cerebrospinal fluid (CSF) neprilysin activity (CSF‐NEP) in patients with AD‐converted mild cognitive impairment and early AD as compared with age‐matched control subjects. The altered CSF‐NEP likely reflects changes in neuronal neprilysin, since transfer of neprilysin from brain tissue into CSF was demonstrated by injecting neprilysin‐carrying viral vector into the brains of neprilysin‐deficient mice. Interestingly, CSF‐NEP showed an elevation with the progression of AD. Along with a close association of CSF‐NEP with CSF tau proteins, this finding suggests that presynaptically located neprilysin can be released into CSF as a consequence of synaptic disruption. The impact of neuronal damages on CSF‐NEP was further demonstrated by a prominent increase of CSF‐NEP in rats exhibiting kainate‐induced neurodegeneration. Our results unequivocally indicate significance of CSF‐NEP as a biochemical indicator to pursue a pathological process that involves decreased neprilysin activity and Aβ‐induced synaptic toxicity, and the support the potential benefits of neprilysin up‐regulation in ameliorating neuropathology in prodromal and early AD. Ann Neurol 2005;57:832–842

Molecular cloning and expression of aminopeptidase A isoforms from rat hippocampus.

The full-length cDNA encoding aminopeptidase A (APAL) was cloned from a rat hippocampus cDNA library. A short variant aminopeptidase A (APAS), produced by deletion, was also cloned. In the case of APAL, the longest open reading frame encodes 945 amino acid residues with a calculated molecular mass of 108 kDa, and the deduced amino acid sequence shows 76, 86 and 78% identity with its human, murine and porcine counterparts, respectively. Rat aminopeptidase A mRNAs were detected in the kidney, liver, heart and brain by Northern blot analysis. When overexpressed in COS-1 cells, APAL shows apparent aminopeptidase A activity, whereas APAS does not.

Disulphide linkage in mouse ST6Gal-I: determination of linkage positions and mutant analysis

All cloned sialyltransferases from vertebrates are classified into four subfamilies and are characterized as having type II transmembrane topology. The catalytic domain has highly conserved motifs known as sialylmotifs. Besides sialylmotifs, each family has several unique conserved cysteine (Cys) residues mainly in the catalytic domain. The number and loci of conserved amino acids, however, differ with each subfamily, suggesting that the conserved Cys-residues and/or disulphide linkages they make may contribute to linkage specificity. Using Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF)-mass spectrometry, the present study performed disulphide linkage analysis on soluble mouse ST6Gal-I, which has six Cys-residues. Results confirmed that there were no free Cys-residues, and all six residues contributed to disulphide linkage formation, C(139)-C(403), C(181)-C(332) and C(350)-C(361). Study of single amino acid-substituted mutants revealed that the disulphide linkage C(181)-C(332) was necessary for molecular expression of the enzyme, and that the disulphide linkage C(350)-C(361) was necessary for enzyme activity. The remaining disulphide linkage C(139)-C(403) was not necessary for enzyme expression or for activity, including substrate specificity. Crystallographic study of pig ST3Gal I has recently been reported. Interestingly, the loci of disulphide linkages in ST6Gal-I differ from those in ST3Gal I, suggesting that the linkage specificity of sialyltransferase may results from significant structural differences, including the loci of disulphide linkages.