Trevor Atkinson

Involvement of Insulin-Like Growth Factor 1 Receptor Signaling in the Amyloid-β Peptide Oligomers-Induced p75 Neurotrophin Receptor Protein Expression in Mouse Hippocampus

The p75 neurotrophin receptor (p75NTR) has been thought to play a critical role in amyloid-β peptide (Aβ)-mediated neurodegeneration and Aβ metabolism in Alzheimers disease (AD) brains. Our previous report showed that membrane-associated p75NTR protein expression was significantly increased in the hippocampi of two different strains of transgenic AD mice and was associated with the age-dependent elevation of Aβ1-42 levels. Here, we provide evidence that the Aβ1-42 oligomers known as ADDLs (Aβ-derived diffusible ligands) induce p75NTR protein expression through insulin-like growth factor 1 receptor (IGF-1R) phosphorylation in SH-SY5Y human neuroblastoma cells. An in vivo microinjection study demonstrated that microinjected ADDLs increased the p75NTR protein expression by 1.4-fold in the ipsilateral hippocampus compared to the contralateral hippocampus. In addition, ADDLs microinjected into mouse hippocampi facilitated IGF-1R phosphorylation within 30 min and the co-administration of picropodophyllin, an IGF-1R kinase inhibitor, blocked ADDLs-induced p75NTR expression. We examined the possible involvement of IGF-1R in the increased p75NTR protein expression in the hippocampi of 6-month-old AβPPswe/PS1dE9 AD model mice that had accumulated significant amounts of Aβ1-42 and showed significantly higher p75NTR expression than age-matched wild-type mice. We found that IGF-1R phosphorylation in these transgenic mice was higher than that in the wild-type mice. These findings indicate that Aβ1-42 oligomers stimulate the p75NTR protein expression in the hippocampus through IGF-1R signaling. Thus, Aβ1-42 oligomers-mediated IGF-1R activation may trigger an increase in p75NTR protein expression in the hippocampus of AD brain during the early stages of disease development.

Reduction of the immunostainable length of the hippocampal dentate granule cells' primary cilia in 3xAD-transgenic mice producing human Aβ1-42 and tau

The hippocampal dentate gyrus is one of the two sites of continuous neurogenesis in adult rodents and humans. Virtually all dentate granule cells have a single immobile cilium with a microtubule spine or axoneme covered with a specialized cell membrane loaded with receptors such as the somatostatin receptor 3 (SSTR3), and the p75 neurotrophin receptor (p75(NTR)). The signals from these receptors have been reported to stimulate neuroprogenitor proliferation and the post-mitotic maturation of newborn granule cells into functioning granule cells. We have found that in 6-24-months-old triple transgenic Alzheimers disease model mice (3xTg-AD) producing both Aβ(1-42) and the mutant human tau protein tau(P301L,) the dentate granule cells still had immunostainable SSTR3- and p75(NTR)-bearing cilia but they were only half the length of the immunostained cilia in the corresponding wild-type mice. However, the immunostainable length of the granule cell cilia was not reduced either in 2xTg-AD mice accumulating large amounts of Aβ(1-42) or in mice accumulating only a mutant human tau protein. Thus it appears that a combination of Aβ(1-42) and tau protein accumulation affects the levels of functionally important receptors in 3xTg-AD mice. These observations raise the important possibility that structural and functional changes in granule cell cilia might have a role in AD.

Identification of protein kinase C inhibitory activity associated with a polypeptide isolated from a phage display system with homology to PCM-1, the pericentriolar material-1 protein

We had previously identified a protein kinase C (PKC) inhibitor in murine neuroblastoma cells (Chakravarthy et al. [1]). Similar PKC inhibitory activity was also found in adult rat brain. Using polyclonal antibodies raised against the partially purified PKC inhibitor from rat brain as bait, we isolated a putative brain PKC inhibitor using a T-7 phage display system expressing human brain cDNA library. After enriching the phage population expressing the putative PKC inhibitor with four rounds of biopanning using ELISA and in vitro PKC binding assays, we identified a phage clone that expressed a product with significant PKC inhibitory activity. We have cloned and expressed this cDNA in a bacterial system and purified the recombinant protein. This polypeptide (174 amino acids) is highly homologous to a region of the 228-kDa PCM-1, the human pericentriolar material 1 protein. We have mapped this polypeptides PKC-inhibitory domain and shown its PKC inhibitory activity in vitro. However, it will need to be determined whether the full-length PCM-1 protein possesses PKC inhibitory activity in vivo, and if so, how this might contribute to PCM-1s recently demonstrated roles in ciliogenesis and neurogenesis.

A synthetic peptide corresponding to a region of the human pericentriolar material 1 (PCM‐1) protein binds β‐amyloid (Aβ1‐42) oligomers

We have recently reported that a ~19‐kDa polypeptide, rPK‐4, is a protein kinase Cs inhibitor that is 89% homologous to the 1171–1323 amino acid region of the 228‐kDa human pericentriolar material‐1 (PCM‐1) protein (Chakravarthy et al. 2012). We have now discovered that rPK‐4 binds oligomeric amyloid‐β peptide (Aβ)1‐42 with high affinity. Most importantly, a PCM‐1‐selective antibody co‐precipitated Aβ and amyloid β precursor protein (AβPP) from cerebral cortices and hippocampi from AD (Alzheimers disease) transgenic mice that produce human AβPP and Aβ1‐42, suggesting that PCM‐1 may interact with amyloid precursor protein/Aβ in vivo. We have identified rPK‐4′s Aβ‐binding domain using a set of overlapping synthetic peptides. We have found with ELISA, dot‐blot, and polyacrylamide gel electrophoresis techniques that a ~ 5 kDa synthetic peptide, amyloid binding peptide (ABP)‐p4‐5 binds Aβ1‐42 at nM levels. Most importantly, ABP‐p4‐5, like rPK‐4, appears to preferentially bind Aβ1‐42 oligomers, believed to be the toxic AD‐drivers. As expected from these observations, ABP‐p4‐5 prevented Aβ1‐42 from killing human SH‐SY5Y neuroblastoma cells via apoptosis. These findings indicate that ABP‐p4‐5 is a possible candidate therapeutic for AD.

BRAIN DELIVERY OF AN AMYLOID-β OLIGOMER (AβO)-TARGETING PEPTIDE-THERAPEUTIC BY A NOVEL BLOOD-BRAIN BARRIER (BBB)-CROSSING DOMAIN ANTIBODY

Background:To decrease the levels of toxic Amyloid-beta (Ab) species in the brain, we have developed and designed D-enantiomeric peptides. Here, we investigated a cyclic D-enantiomeric peptide’s (cycP) aptitude as an Alzheimer’s disease (AD) drug by in vitro, ex vivo and in vivo experiments. Especially, cycP’s ability to cross the blood brain barrier (BBB) to a higher extent than its linear equivalent (linP) is of advantage. Furthermore, cycP’s pharmacokinetic profile, proteolytic stability and plasma protein binding properties were characterized. Methods:CycP’s impact on cell viability (MTT test), Ab fibril formation (Thioflavin T assay), proteolytic stability in murine plasma, brain-homogenate and human liver microsomes (thin layer chromatography (TLC)) and plasma protein binding to human serum albumin (HSA) and a1-acid-glycoprotein (AGP) (TRANSIL Binding Kits) were investigated. To determine cycP’s concentration-time-profile, a mixture of H-labelled and non-labelled peptide was administered to wildtype mice. Results: The negative impact of Ab on cell viability could be reduced and Ab fibril formation could be clearly decelerated by cycP. Proteolytic stability tests showed that cycP was not degraded within 24 h. Plasma protein binding of cycP was stronger to AGP than to HSA and the fraction unbound after binding to HSA and AGP was about 2-3 %. Two hours after administration, cycP concentrations in the brain were up to ten times higher than linP concentrations although concentrations in plasma were similar for both peptides. Additionally, cycP showed longer half-life than linP. Oral cycP administration resulted in a constant concentration-time-profile in plasma and brain with high bioavailability. Conclusions: In vitro and ex vivo experiments showed promising results for cycP as a nontoxic, Ab-targeting AD drug. As cycP concentrations in the brain were considerably higher than linP concentrations while plasma concentrations were in the same range, cycP seems to pass the BBB more efficiently than linP. This is fundamental for an Abtargeting AD-drug. In fact, an increased mobility to pass the BBB and high oral bioavailability probably allow administration of lower doses for cycP treatment, further reducing the risk of adverse side effects.

Evidence that a synthetic amyloid-ß oligomer-binding peptide (ABP) targets amyloid-ß deposits in transgenic mouse brain and human Alzheimer's disease brain.

The synthetic ~5 kDa ABP (amyloid-ß binding peptide) consists of a region of the 228 kDa human pericentrioloar material-1 (PCM-1) protein that selectively and avidly binds in vitro Aβ1-42 oligomers, believed to be key co-drivers of Alzheimers disease (AD), but not monomers (Chakravarthy et al., (2013) [3]). ABP also prevents Aß1-42 from triggering the apoptotic death of cultured human SHSY5Y neuroblasts, likely by sequestering Aß oligomers, suggesting that it might be a potential AD therapeutic. Here we support this possibility by showing that ABP also recognizes and binds Aβ1-42 aggregates in sections of cortices and hippocampi from brains of AD transgenic mice and human AD patients. More importantly, ABP targets Aβ1-42 aggregates when microinjected into the hippocampi of the brains of live AD transgenic mice.

Aβ binding properties of a synthetic peptide corresponding to an amino acid sequence in human pericentriolar material 1 (PCM-1) protein

role and underlying mechanisms has not been fully understood. Hyperphosphorylated tau is the major component of Neurofibrillary Tangles (NFTs), which is one of the chracteristic hallmarks of AD. In the present study, the direct effect of Helicobacter pylori on tau phosphorylation, learning and memory are investigated. Methods: C57BLmice (3 months) were received Morris Water Maze training for 7 days and then divided into experimental group for intraperitoneal injection with conditional components of Helicobacter pylori (bacteria were incubated with culture medium for 30 min, then all the bacteria were removed to get the conditional medium), and control group for intraperitoneal injection with culture medium for 7 days. Then, the memory and learning abilities were detected by recording the latency (memory) and swimming strategy for the hidden platform with altered location. The mice were sacrificed and the hippocampus was homogenated, the phosphorylation levels of tau were detected by Western blotting. The same experiment was further repeated in N2a cells, cells were cultured in conditional medium for 24, 36, and 48 hours, the phosphorylation levels of tau were detected. Results: Conditional components of Helicobacter pylori treatment-induced cognitive impairment and tau hyperphosphorylation at several AD-tau hyperphosphorylation sites such as Ser214,Ser404, Thr205 and Thr231 both in mice brains and N2a cells, no change of total-tau level was observed. Conclusions: We conclude that conditional components of Helicobacter pylori impair the learning and memory ability, and directly induce tau hyperphosphorylation both in vivo and in vitro, peripheral infection with Helicobacter pylori may induce AD like pathogenesis through the release of toxins which could be sensed by the neurons.

Changes in the expression of somatostatin 3 receptor (SSTR3) and p75NTR in the hippocampal primary cilia of Alzheimer's disease transgenic mice

Background: APOE e4 is the strongest genetic risk factor for late-onset, sporadic Alzheimer’s disease, strongly increasing risk and decreasing age of onset, whereas the APOE e2 allele decreases risk. Convincing evidence has revealed accelerated onset of amyloid deposition in APOE e4-carriers, leading to the hypothesis that APOE genotype differentially regulates AD risk and onset via effects on As accumulation. Using PDAPP/human apoE targeted replacement (PDAPP/TRE) mice that exhibit apoE isoform-dependent levels of As burden (E4 > E3 > E2), we have found that human apoE isoforms differentially modulate endogenous As clearance, but not synthesis, in a manner that corresponds to the apoE isoform-dependent pattern of As accumulation in older mice. Here, we sought to assess the contribution of both the concentration and isoform of apoE in the regulation of ISF As clearance by acute infusion of synthetic human apoE particles during in vivo microdialysis. Methods: Synthetic particles containing apoE2, apoE3, and apoE4 were prepared, verifying functionality in vitro using cholesterol efflux assays. We next bred PDAPP/E2, PDAPP/E3, and PDAPP/E4 mice and aged them to 3 months of age for in vivo microdialysis experiments. To acutely assess the effect of each apoE isoform, apoE particles were directly infused around the implanted 38kDa microdialysis probe during simultaneous sampling of the brain interstitial fluid of freely behaving mice. To assess the effect of each apoE infusion on As clearance, each mouse was then given an intraperitoneal injection of a potent gamma secretase inhibitor in order to halt As production, allowing for the determination of As half-life after measurement with a sandwich ELISA. Results:We report that synthetically prepared apoE particles composed of phospholipids, cholesterol, and human apoE are functional and can be utilized to assess acute effects of human apoE on As metabolism in vivo. Specifically, in PDAPP/TRE mice, we report that acute infusions of synthetic human apoE particles increase ISFAs levels and decrease As clearance in a manner that depends on the isoform of apoE infused. Conclusions: These results have important implications for understanding how both the concentration and isoform of apoE influence As clearance and may be informative in designing therapeutic strategies that target apoE.