Robert Crescentini

Role of CD40 ligand in amyloidosis in transgenic Alzheimer's mice

We have shown that interaction of CD40 with CD40L enables microglial activation in response to amyloid-β peptide (Aβ), which is associated with Alzheimers disease (AD)-like neuronal tau hyperphosphorylation in vivo. Here we report that transgenic mice overproducing Aβ, but deficient in CD40L, showed decreased astrocytosis and microgliosis associated with diminished Aβ levels and β-amyloid plaque load. Furthermore, in the PSAPP transgenic mouse model of AD, a depleting antibody against CD40L caused marked attenuation of Aβ/β-amyloid pathology, which was associated with decreased amyloidogenic processing of amyloid precursor protein (APP) and increased circulating levels of Aβ. Conversely, in neuroblastoma cells overexpressing wild-type human APP, the CD40–CD40L interaction resulted in amyloidogenic APP processing. These findings suggest several possible mechanisms underlying mitigation of AD pathology in response to CD40L depletion, and validate the CD40–CD40L interaction as a target for therapeutic intervention in AD.

Vasoactive effects of Aβ in isolated human cerebrovessels and in a transgenic mouse model of Alzheimer's disease: Role of inflammation

Abstract Aβ peptides are the major protein constituents of Alzheimers disease (AD) senile plaques and also form some deposits in the cerebrovasculature leading to cerebral amyloid angiopathy and hemorrhagic stroke. Functional vascular abnormalities are one of the earlier clinical manifestations in both sporadic and familial forms of AD. Most of the cardiovascular risk factors (for instance, diabetes, hypertension, high cholesterol levels, atherosclerosis and smoking) constitute risk factors for AD as well, suggesting that functional vascular abnormalities may contribute to AD pathology. We studied the effect of Aβ on endothelin-1 induced vasoconstriction in isolated human cerebral arteries collected following rapid autopsies. We report that freshly solubilized Aβ enhances endothelin-1 induced vasoconstriction in isolated human middle cerebral and basilar arteries. The vasoactive effect of Aβ in these large human cerebral arteries is inhibited by NS-398, a selective cyclooxygenase-2 inhibitor and by SB202190, a specific p38 Mitogen Activated Protein Kinase inhibitor suggesting the involvement of a pro-inflammatory pathway. Using a scanner laser Doppler imager, we observed that cerebral blood flow is decreased in the double transgenic APPsw Alzheimer mouse (PS1/APPsw) compared to PS1 littermates and can be improved by chronic treatment with either NS-398 or SB202190. Altogether, our data suggest a link between inflammation and the compromised cerebral hemodynamics in AD.

p35/Cdk5 pathway mediates soluble amyloid-β peptide-induced tau phosphorylation in vitro

Alzheimers disease (AD) is pathologically characterized by deposition of amyloid‐β peptides (Aβ) as senile plaques and by the occurrence of neurofibrillary tangles (NFTs) composed primarily of hyperphosphorylated tau protein. Activation of cyclin‐dependent kinase 5 (Cdk5) via its potent activator p25 has recently been shown to promote phosphorylation of tau at AD‐specific phosphoepitopes, and increased cleavage of p35 to p25 has been demonstrated in AD patients, suggesting that Cdk5 may represent a pathogenic tau protein kinase. We were interested in the potential effect of soluble forms of Aβ on Cdk5‐mediated AD‐like tau phosphorylation, insofar as previous studies of human biopsies and aged canine and primate brains have shown that dystrophic neurites appear before the formation of neuritic plaques. We transfected N2a cells with a p35 vector (N2a/p35 cells) and, after differentiation, challenged these cells with Aβ1–42 peptide in soluble form (sAβ1–42). Results show that sAβ1–42 at relatively low levels (1–5 μM) dose‐dependently increases tau phosphorylation at AD‐specific phosphoepitopes in differentiated N2a/p35 cells compared with controls, an effect that is blocked by antisense oligonucleotides against p35. sAβ1–42‐induced tau phosphorylation is concomitant with an increase in both p25 to p35 ratio and Cdk5 activity (but not protein levels). Additionally, blockade of L‐type calcium channels or inhibition of calpain completely abolishes this effect. Taken together, these data indicate that sAβ is a potent activator of the p25/Cdk5 pathway, resulting in promotion of AD‐like tau phosphorylation in vitro.

Inhibition of angiogenesis by Abeta peptides.

Aβ peptides are naturally occurring peptides forming β-sheet aggregates that constitute an integral component of senile plaques and vascular deposits in Alzheimers disease. Since several peptides adopting a β-sheet conformation have been shown to be anti-angiogenic, we investigated the effect of Aβ on angiogenesis. We show that in vitro, Aβ dose-dependently inhibits the formation of capillaries by human brain endothelial cells plated on Matrigel and stimulates capillary degeneration at high doses. Preparations of Aβ peptides containing a higher content of β-sheet structures are more potently anti-angiogenic in vitro. Ex vivo, Aβ dose-dependently opposes angiogenesis in rat aortae and in human middle cerebral arteries. In vivo, Aβ dose dependently inhibits angiogenesis in the chick chorioallantoic membrane assay and suppresses bFGF-induced blood vessel formation in the corneal micropocket and Matrigel plug assays. Since angiogenesis is required for tumor growth, we explored the effect of Aβ on human glioblastoma (U87MG) and human lung adenocarcinoma (A549) tumors. We show that intra-tumoral injection of Aβ potently inhibits the growth and vascularization of human glioblastoma and human lung adenocarcinoma tumor xenografts in nude mice. Similarly to the intra-tumoral injection regimen, Aβ delivered intraperitoneally also suppressed the growth of human lung adenocarcinoma tumor xenografts. Altogether our data show that Aβ is an angiogenesis inhibitor.

Inhibition of Angiogenesis by Aβ Peptides

Aβ peptides are naturally occurring peptides forming β-sheet aggregates that constitute an integral component of senile plaques and vascular deposits in Alzheimers disease. Since several peptides adopting a β-sheet conformation have been shown to be anti-angiogenic, we investigated the effect of Aβ on angiogenesis. We show that in vitro, Aβ dose-dependently inhibits the formation of capillaries by human brain endothelial cells plated on Matrigel and stimulates capillary degeneration at high doses. Preparations of Aβ peptides containing a higher content of β-sheet structures are more potently anti-angiogenic in vitro. Ex vivo, Aβ dose-dependently opposes angiogenesis in rat aortae and in human middle cerebral arteries. In vivo, Aβ dose dependently inhibits angiogenesis in the chick chorioallantoic membrane assay and suppresses bFGF-induced blood vessel formation in the corneal micropocket and Matrigel plug assays. Since angiogenesis is required for tumor growth, we explored the effect of Aβ on human glioblastoma (U87MG) and human lung adenocarcinoma (A549) tumors. We show that intra-tumoral injection of Aβ potently inhibits the growth and vascularization of human glioblastoma and human lung adenocarcinoma tumor xenografts in nude mice. Similarly to the intra-tumoral injection regimen, Aβ delivered intraperitoneally also suppressed the growth of human lung adenocarcinoma tumor xenografts. Altogether our data show that Aβ is an angiogenesis inhibitor.

Nilvadipine antagonizes both Aβ vasoactivity in isolated arteries, and the reduced cerebral blood flow in APPsw transgenic mice

The development of Alzheimers disease (AD) is generally thought to correlate with cerebral accumulation of Abeta. It has previously been shown that Abeta peptides enhance vasoconstriction in isolated arteries and oppose certain vasorelaxants. Moreover, exogenous application of Abeta peptides causes cerebral vasoconstriction in rodents and in transgenic mouse models of AD that overexpress Abeta there is reduced cerebral blood flow. In the present study, we investigated the effect of nilvadipine, a dihydropyridine-type calcium channel blocker, on Abeta induced vasoconstriction in isolated arteries and in vivo on cerebral blood flow (CBF) of an AD transgenic mouse model overexpressing Abeta (Tg APPsw line 2576). Nilvadipine completely inhibited the vasoactivity elicited by Abeta in rat aortae and in human middle cerebral arteries. The effect of a short treatment duration (2 weeks) with nilvadipine on regional CBF was investigated in 13-month-old Tg APPsw mice and control littermates using a laser Doppler imager. Additionally, CBF was also measured in 20-month-old Tg APPsw mice and control littermates that were chronically treated with nilvadipine for 7 months. Untreated Tg APPsw mice showed a reduction of regional CBF compared to their untreated control littermates. Nilvadipine restored cortical perfusion levels in Tg APPsw to values similar to those observed in control littermates without notably affecting the CBF of control mice. All together, these data suggest that nilvadipine might be useful for the treatment of oligemia associated with AD.

Behavioral effects of CD40–CD40L pathway disruption in aged PSAPP mice

We have shown that, when an amyloid-beta peptide (Abeta) overproducing transgenic mouse model (PSAPP) of Alzheimers disease (AD) is treated with a depleting antibody against CD40L, it causes marked attenuation of Abeta pathology associated with decreased amyloidogenic processing of amyloid precursor protein (APP) and increased cerebral clearance of Abeta. Here, we report that, when PSAPP mice receive a regimen of anti-CD40L antibody commencing at an age associated with initial Abeta deposition, they demonstrate superior spatial memory on the standard water maze and radial arm water maze tasks, as well as exhibiting superior non-spatial memory in the object recognition test, as compared to control PSAPP mice. Furthermore, PSAPP mice treated with an anti-CD40L antibody regimen commencing at an age associated with extensive Abeta deposition demonstrate superior spatial memory on the standard water maze task, as compared to control PSAPP mice. Disruption of CD40L activity has beneficial effects on pathology and cognitive behavior in the PSAPP mouse model, providing support for the therapeutic potential of interrupting the CD40-CD40L interaction in AD.

Confirmation of association between D10S583 and Alzheimer's disease in a case--control sample.

Several independent studies have reported that loci on chromosome 10 are associated/linked with Alzheimers disease (AD), including a family-based study demonstrating an association between the marker D10S583 and AD. We have examined the D10S583 polymorphic marker and apolipoprotein E (APOE) gene in a case-control study. We observed the expected association of the APOE allele varepsilon4 with AD, and an inverse association between the D10S583 allele 209 and AD. These data support the original findings that suggest the presence of a candidate gene for AD in this region of chromosome 10. The nearby insulin degrading enzyme gene has been previously proposed as a candidate gene; however, a number of other putative candidate genes are also located in this region. The ongoing investigation of the genetic source of association and linkage in this region is clearly warranted.

Inhibition of Angiogenesis by A Peptides

Aβ peptides are naturally occurring peptides forming β-sheet aggregates that constitute an integral component of senile plaques and vascular deposits in Alzheimers disease. Since several peptides adopting a β-sheet conformation have been shown to be anti-angiogenic, we investigated the effect of Aβ on angiogenesis. We show that in vitro, Aβ dose-dependently inhibits the formation of capillaries by human brain endothelial cells plated on Matrigel and stimulates capillary degeneration at high doses. Preparations of Aβ peptides containing a higher content of β-sheet structures are more potently anti-angiogenic in vitro. Ex vivo, Aβ dose-dependently opposes angiogenesis in rat aortae and in human middle cerebral arteries. In vivo, Aβ dose dependently inhibits angiogenesis in the chick chorioallantoic membrane assay and suppresses bFGF-induced blood vessel formation in the corneal micropocket and Matrigel plug assays. Since angiogenesis is required for tumor growth, we explored the effect of Aβ on human glioblastoma (U87MG) and human lung adenocarcinoma (A549) tumors. We show that intra-tumoral injection of Aβ potently inhibits the growth and vascularization of human glioblastoma and human lung adenocarcinoma tumor xenografts in nude mice. Similarly to the intra-tumoral injection regimen, Aβ delivered intraperitoneally also suppressed the growth of human lung adenocarcinoma tumor xenografts. Altogether our data show that Aβ is an angiogenesis inhibitor.

Increased TNFα production and Cox-2 activity in organotypic brain slice cultures from APPsw transgenic mice

beta-Amyloid (Abeta) peptides are the principal component of senile plaques and vascular deposits in Alzheimers disease and are derived from the proteolytic cleavage of the beta-amyloid precursor protein (APP). We have previously shown that synthetic Abeta can stimulate cyclooxygenase-2 (Cox-2) activity in brain organotypic slice cultures. In the present study, we used brain slices from transgenic APP Swedish (TgAPPsw) mice and control littermates of different age groups to determine the effect of APP overexpression on the levels of prostaglandin and tumor necrosis factor alpha (TNFalpha) release. The production of eicosanoid and TNFalpha was increased as a function of age in organotypic brain slice culture from TgAPPsw mice compared to age matched control littermates. We also showed that the selective Cox-2 inhibitor NS-398 reduces the production of eicosanoid and TNFalpha in organotypic brain slice cultures of TgAPPsw mice. In conclusion, our data suggest that either activity or expression of Cox-2 is increased in TgAPPsw mice brains as a function of age, contributing to an increased production of pro-inflammatory eicosanoids and TNFalpha.