Keiro Shirotani

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.

Transgenic mice with Alzheimer presenilin 1 mutations show accelerated neurodegeneration without amyloid plaque formation.

Familial Alzheimer disease mutations of presenilin 1 (PS-1) enhance the generation of Aβ1–42, indicating that PS-1 is involved in amyloidogenesis. However, PS-1 transgenic mice have failed to show amyloid plaques in their brains. Because PS-1 mutations facilitate apoptotic neuronal death in vitro, we did careful quantitative studies in PS-1 transgenic mice and found that neurodegeneration was significantly accelerated in mice older than 13 months (aged mice) with familial Alzheimer disease mutant PS-1, without amyloid plaque formation. However, there were significantly more neurons containing intracellularly deposited Aβ42 in aged mutant transgenic mice. Our data indicate that the pathogenic role of the PS-1 mutation is upstream of the amyloid cascade.

MUC1 mucin expression as a marker of progression and metastasis of human colorectal carcinoma.

BACKGROUND/AIMS The MUC1 mucin distributes among a variety of epithelial tissues (except the intestinal epithelia) and is often detectable in colorectal carcinoma tissues and cell lines. This study aimed to elucidate whether MUC1 mucin expression correlated to the progression of colorectal carcinomas. METHODS We collected 113 tissue specimens, including primary colorectal carcinoma, normal mucosa, liver metastases, lymph node metastases, and normal livers from 58 patients with colorectal carcinoma. Immunohistochemical staining and Western blotting analysis with mature MUC1 mucin-specific monoclonal antibodies were performed. RESULTS The levels of mature MUC1 mucins were significantly higher in carcinoma tissues than those in normal colonic mucosa (P < 0.001). Furthermore, the levels of mature MUC1 mucins were significantly higher in primary tumors from patients having metastasis or metastatic tumors than in primary tumors from patients without metastasis (P < 0.001). In the primary sites, mature MUC1 mucin levels apparently increased according to progression of the stages (P < 0.001). CONCLUSIONS These results strongly suggest that mature MUC1 mucins become ectopically expressed in colorectal carcinoma progressed to the metastatic stages and that mature MUC1 mucins may be a useful marker for advanced colorectal carcinoma.

Alzheimer's β-secretase, β-site amyloid precursor protein-cleaving enzyme, is responsible for cleavage secretion of a Golgi-resident sialyltransferase

The deposition of amyloid β-peptide (Aβ) in the brain is closely associated with the development of Alzheimers disease. Aβ is generated from the amyloid precursor protein (APP) by sequential action of β-secretase (BACE1) and γ-secretase. Although BACE1 is distributed among various other tissues, its physiological substrates other than APP have yet to be identified. ST6Gal I is a sialyltransferase that produces a sialylα2,6galactose residue, and the enzyme is secreted out of the cell after proteolytic cleavage. We report here that BACE1 is involved in the proteolytic cleavage of ST6Gal I, on the basis of the following observations. ST6Gal I was colocalized with BACE1 in the Golgi apparatus by immunofluorescence microscopy, suggesting that BACE1 acts on ST6Gal I within the same intracellular compartment. When BACE1 was overexpressed with ST6Gal I in COS cells, the secretion of ST6Gal I markedly increased. When APPSW (Swedish familial Alzheimers disease mutation), a preferable substrate for BACE1, was coexpressed with ST6Gal I in COS cells, the secretion of ST6Gal I significantly decreased, suggesting that that the β-cleavage of overexpressed APPSW competes with ST6Gal I processing. In addition, BACE1-Fc (Fc, the hinge and constant region of IgG) chimera cleaved protein A-ST6Gal I fusion protein in vitro. Thus, we conclude that BACE1 is responsible for the cleavage and secretion of ST6Gal I.

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.

Distinct Mechanistic Roles of Calpain and Caspase Activation in Neurodegeneration as Revealed in Mice Overexpressing Their Specific Inhibitors

Enzymatic proteolysis has been implicated in diverse neuropathological conditions, including acute/subacute ischemic brain injuries and chronic neurodegeneration such as Alzheimer disease and Parkinson disease. Calcium-dependent proteases, calpains, have been intensively analyzed in relation to these pathological conditions, but in vivo experiments have been hampered by the lack of appropriate experimental systems for a selective regulation of the calpain activity in animals. Here we have generated transgenic (Tg) mice that overexpress human calpastatin, a specific and the only natural inhibitor of calpains. In order to clarify the distinct roles of these cell death-associated cysteine proteases, we dissected neurodegenerative changes in these mice together with Tg mice overexpressing a viral inhibitor of caspases after intrahippocampal injection of kainic acid (KA), an inducer of neuronal excitotoxicity. Immunohistochemical analyses using endo-specific antibodies against calpain- and caspase-cleaved cytoskeletal components revealed that preclusion of KA-induced calpain activation can rescue the hippocampal neurons from disruption of the neuritic cytoskeletons, whereas caspase suppression has no overt effect on the neuritic pathologies. In addition, progressive neuronal loss between the acute and subacute phases of KA-induced injury was largely halted only in human calpastatin Tg mice. The animal models and experimental paradigm employed here unequivocally demonstrate their usefulness for clarifying the distinct contribution of calpain and caspase systems to molecular mechanisms governing neurodegeneration in adult brains, and our results indicate the potentials of specific calpain inhibitors in ameliorating excitotoxic neuronal damages.

Novel γ-Secretase Enzyme Modulators Directly Target Presenilin Protein

Background: γ-Secretase modulators (GSMs) hold great potential as anti-Alzheimer disease drugs, but their molecular target(s) are not established. Results: The catalytic subunit of γ-secretase, presenilin, was identified as a direct target of novel GSMs. Conclusion: Enzyme-targeting GSMs establish allosteric modulation as a mechanism of GSM action. Significance: The identification of presenilin as GSM target may contribute to the development of therapeutically active GSMs. γ-Secretase is essential for the generation of the neurotoxic 42-amino acid amyloid β-peptide (Aβ42). The aggregation-prone hydrophobic peptide, which is deposited in Alzheimer disease (AD) patient brain, is generated from a C-terminal fragment of the β-amyloid precursor protein by an intramembrane cleavage of γ-secretase. Because Aβ42 is widely believed to trigger AD pathogenesis, γ-secretase is a key AD drug target. Unlike inhibitors of the enzyme, γ-secretase modulators (GSMs) selectively lower Aβ42 without interfering with the physiological function of γ-secretase. The molecular target(s) of GSMs and hence the mechanism of GSM action are not established. Here we demonstrate by using a biotinylated photocross-linkable derivative of highly potent novel second generation GSMs that γ-secretase is a direct target of GSMs. The GSM photoprobe specifically bound to the N-terminal fragment of presenilin, the catalytic subunit of γ-secretase, but not to other γ-secretase subunits. Binding was differentially competed by GSMs of diverse structural classes, indicating the existence of overlapping/multiple GSM binding sites or allosteric alteration of the photoprobe binding site. The β-amyloid precursor protein C-terminal fragment previously implicated as the GSM binding site was not targeted by the compound. The identification of presenilin as the molecular target of GSMs directly establishes allosteric modulation of enzyme activity as a mechanism of GSM action and may contribute to the development of therapeutically active GSMs for the treatment of AD.

Pro-apoptotic effect of presenilin 2 (PS2) overexpression is associated with down-regulation of Bcl-2 in cultured neurons

Presenilin 2 (PS2) is a polytopic membrane protein that is mutated in some cases of familial Alzheimers disease (AD). The normal functions of PS2 and its pathogenic role in AD remain unclear. We investigated the biological role of this protein in neurons, using adenovirus‐mediated transduction of the PS2 gene into rat primary cortical neurons. Immunocytochemical analyses demonstrated increased PS2 immunoreactivity in most neurons infected with recombinant adenoviruses expressing PS2. Neurons infected with wild‐type or mutant (N141I) PS2‐expressing adenoviruses showed a significant increase in basal cell death, compared with those infected with control β‐galactosidase‐expressing adenovirus. Moreover, PS2 overexpression markedly increased neuronal susceptibility to staurosporine‐induced apoptosis. Mutant PS2 was more effective in enhancing apoptosis than its wild‐type counterpart. Staurosporine‐induced death was significantly inhibited by a specific caspase 3 inhibitor. Western analyses revealed that Bcl‐2 protein expression was specifically down‐regulated in neurons overexpressing PS2, which temporally corresponded to the accumulation of C‐ and N‐terminal fragments of PS2. Additionally, expression of mutant, but not wild‐type PS2, increased the production of β‐amyloid protein (Aβ) 42. These data collectively suggest that the pro‐apoptotic effect of PS2 is mediated by down‐regulation of Bcl‐2. PS2 mutations may increase the susceptibility of neurons to apoptotic stimuli by perturbing the regulation of cell death.

Overexpression of Presenilin-2 Enhances Apoptotic Death of Cultured Cortical Neurons

Abstract: Presenilin‐2 (PS2) is a gene of unknown function linked with some forms of familial Alzheimers disease. To investigate the biological role of PS2 in neurons, we overexpressed PS2 in primary cortical neurons using recombinant adenoviral vectors. Western blot and immunohistochemical analyses showed enhanced expression of PS2 proteins in infected neurons after infection of recombinant adenoviruses containing the human wild‐type or mutant PS2 gene. Neuronal survival was decreased by approximately 30% in cultures infected with adenovirus expressing either wild‐type or mutant PS2, as compared with those infected with adenovirus expressing the LacZ gene. Fragmented nuclei were frequently observed in dying neurons. These data suggest that apoptotic death of cultured cortical neurons is enhanced by PS2 over‐expression.

In vivo role of caspases in excitotoxic neuronal death: generation and analysis of transgenic mice expressing baculoviral caspase inhibitor, p35, in postnatal neurons.

Caspases, a family of cysteine proteases, are thought to be critical mediators of apoptosis. To examine the role of neuronal caspases in excitotoxic neurodegeneration in vivo, we have generated transgenic mice expressing the baculovirus protein p35, a potent viral caspase inhibitor, using the neuron-specific calmodulin dependent kinase-II alpha (CaMKII-alpha) promoter. The expression of p35 was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. We analyzed caspase activation and cell death by employing an experimental paradigm, in which the excitotoxin kainate (KA) was injected into CA1 of hippocampus and the distribution of the caspase-generated actin fragment was detected immunohistochemically. While kainate treatment led to selective neuronal death in the CA1, CA3 and CA4 of non-transgenic control mice, we observed restricted caspase activation only in the CA3 sector. The transgenic expression of p35 consistently inhibited the kainate-induced caspase activation, but failed to influence the death of neurons to any extent. In addition, we observed concomitant early calpain activation in the specific areas where neurons underwent degeneration in both the transgenic and non-transgenic mice. These results indicate that p35-inhibitable caspases play rather minor roles in the kainate-induced excitotoxicity and that the relative contribution of calpain is likely to be greater than that of caspases.