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 Aß1-42 oligomers, believed to be key co-drivers of Alzheimer's 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 Aß1-42 aggregates in sections of cortices and hippocampi from brains of AD transgenic mice and human AD patients. More importantly, ABP targets Aß1-42 aggregates when microinjected into the hippocampi of the brains of live AD transgenic mice.

A synthetic peptide corresponding to a region of the human pericentriolar material 1 (PCM-1) protein binds ß-amyloid (Aß1-42 ) 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-ß peptide (Aß)1-42 with high affinity. Most importantly, a PCM-1-selective antibody co-precipitated Aß and amyloid ß precursor protein (AßPP) from cerebral cortices and hippocampi from AD (Alzheimer's disease) transgenic mice that produce human AßPP and Aß1-42 , suggesting that PCM-1 may interact with amyloid precursor protein/Aß in vivo. We have identified rPK-4's Aß-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 Aß1-42 at nM levels. Most importantly, ABP-p4-5, like rPK-4, appears to preferentially bind Aß1-42 oligomers, believed to be the toxic AD-drivers. As expected from these observations, ABP-p4-5 prevented Aß1-42 from killing human SH-SY5Y neuroblastoma cells via apoptosis. These findings indicate that ABP-p4-5 is a possible candidate therapeutic for 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 polypeptide's 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-1's recently demonstrated roles in ciliogenesis and neurogenesis.

Reduction of the immunostainable length of the hippocampal dentate granule cells' primary cilia in 3xAD-transgenic mice producing human Aß(1-42) 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 Alzheimer's disease model mice (3xTg-AD) producing both Aß(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 Aß(1-42) or in mice accumulating only a mutant human tau protein. Thus it appears that a combination of Aß(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.

Can Ratios Between Prognostic Factors Predict the Clinical Pregnancy Rate in an IVF/ICSI Program with a GnRH Agonist-FSH/hMG Protocol? An Assessment of 2421 Embryo Transfers, and a Review of the Literature.

None of the models developed in in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) is sufficiently good predictors of pregnancy. The aim of this study was to determine whether ratios between prognostic factors could predict the clinical pregnancy rate in IVF/ICSI. We analyzed IVF/ICSI cycles (based on long GnRH agonist-FSH protocols) at two ART centers (the second to validate externally the data). The ratios studied were (i) the total FSH dose divided by the serum estradiol level on the hCG trigger day, (ii) the total FSH dose divided by the number of mature oocytes, (iii) the serum estradiol level on the trigger day divided by the number of mature oocytes, (iv) the serum estradiol level on the trigger day divided by the endometrial thickness on the trigger day, (v) the serum estradiol level on the trigger day divided by the number of mature oocytes and then by the number of grade 1 or 2 embryos obtained, and (vi) the serum estradiol level on the trigger day divided by the endometrial thickness on the trigger day and then by the number of grade 1 or 2 embryos obtained. The analysis covered 2421 IVF/ICSI cycles with an embryo transfer, leading to 753 clinical pregnancies (31.1% per transfer). Four ratios were significantly predictive in both centers; their discriminant power remained moderate (area under the receiver operating characteristic curve between 0.574 and 0.610). In contrast, the models' calibration was excellent (coefficients: 0.943-0.978; p < 0.001). Our ratios were no better than existing models in IVF/ICSI programs. In fact, a strongly discriminant predictive model will be probably never be obtained, given the many factors that influence the occurrence of a pregnancy.

The p75 neurotrophin receptor is localized to primary cilia in adult murine hippocampal dentate gyrus granule cells.

The densely ciliated granule cell layer of the adult murine hippocampal dentate gyrus is one of two sites of adult neurogenesis. The granule cells have already been proven to localize their SSTR3 (somatostatin receptor 3) receptors to their so-called primary cilia. Here we show for the first time that 70-90% of these cells in 7-18 months-old wild-type and 3×Tg-AD (Alzheimer disease transgenic) mice also load p75(NTR) receptors into the structures containing SSTR3, i.e., their primary cilia. On the other hand, p75(NTR')s TrkA co-receptors were not localized to cilia but conventionally distributed throughout the cell surface. Significantly fewer cells (20-40%) in the hippocampal CA1 and CA3 regions and cerebral cortex have p75(NTR) containing cilia. While we don't know what the impact of the cilial localization of p75(NTR) on dentate gyral adult neurogenesis and memory encoding might be, the cilia's amyloid ß-activatable p75(NTR) receptors could be damaging or lethal to the hippocampal functioning of amyloid ß-accumulating Alzheimer brain.

Association of Gap-43 (neuromodulin) with microtubule-associated protein MAP-2 in neuronal cells.

Gap-43 (B-50, neuromodulin) is a presynaptic protein implicated in axonal growth, neuronal differentiation, plasticity, and regeneration. Its activities are regulated by its dynamic interactions with various neuronal proteins, including actin and brain spectrin. Recently we have shown that Gap-43 co-localizes with an axonal protein DPYSL-3 in primary cortical neurons. In the present study we provide evidence that Gap-43 co-localizes and potentially interacts with microtubule-associated protein MAP-2 in adult and fetal rat brain, as well as in primary neuronal cultures. Our studies suggest that this interaction may be developmentally regulated.

Estrogen withdrawal-induced NF-kappaB activity and bcl-3 expression in breast cancer cells: roles in growth and hormone independence.

About one-third of breast cancers express a functional estrogen (beta-estradiol [E2]) receptor (ER) and are initially dependent on E2 for growth and survival but eventually progress to hormone independence. We show here that ER(+), E2-independent MCF-7/LCC1 cells derived from E2-dependent MCF-7 cells contain elevated basal NF-kappaB activity and elevated expression of the transcriptional coactivator Bcl-3 compared with the parental MCF-7 line. LCC1 NF-kappaB activity consists primarily of p50 dimers, although low levels of a p65/p50 complex are also present. The ER(-) breast cancer cell lines harbor abundant levels of both NF-kappaB complexes. In contrast, nuclear extracts from MCF-7 cells contain a significantly lower level of p50 and p65 than do LCC1 cells. Estrogen withdrawal increases both NF-kappaB DNA binding activity and expression of Bcl-3 in MCF-7 and LCC1 cells in vitro and in vivo. Tumors derived from MCF-7 cells ectopically expressing Bcl-3 remain E2 dependent but display a markedly higher tumor establishment and growth rate compared to controls. Expression of a stable form of IkappaBalpha in LCC1 cells severely reduced nuclear expression of p65 and the p65/p50 DNA binding heterodimer. Whereas LCC1 tumors in nude mice were stable or grew, LCC1(IkappaBalpha) tumors regressed after E2 withdrawal. Thus, both p50/Bcl-3- and p65/p50-associated NF-kappaB activities are activated early in progression and serve differential roles in growth and hormone independence, respectively. We propose that E2 withdrawal may initiate selection for hormone independence in breast cancer cells by activation of NF-kappaB and Bcl-3, which could then supplant E2 by providing both survival and growth signals.

Relative expression of the p75 neurotrophin receptor, tyrosine receptor kinase A, and insulin receptor in SH-SY5Y neuroblastoma cells and hippocampi from Alzheimer's disease patients.

We have previously shown in SH-SY5Y human neuroblastoma cells that the expressions of basal (75 kDa) and high molecular weight (HMW; 85 kDa) isoforms of the p75 neurotrophic receptor (p75NTR) are stimulated by amyloid-ß peptide1-42 oligomers (AßOs) via the insulin-like growth factor-1 receptor (IGF-1R). On the other hand, it is known that AßOs inhibit insulin receptor (IR) signaling. The purpose of the present study was to determine the involvement of IR signaling in the regulation of p75 neurotrophin receptor (p75NTR) protein isoform expression in cultured SH-SY5Y cells and in hippocampi from late-stage human Alzheimer's disease (AD) brains. Interestingly, insulin induced the expression of basal and HMW p75NTR isoforms in SH-SY5Y cells, suggesting the presence of cross-talk between the IR and IGF-1R for the regulation of p75NTR expression. Reducing IR signaling with an IR kinase inhibitor (AG 1024) or IR-targeted siRNAs increased HMW p75NTR expression and reduced tyrosine receptor kinase-A (Trk-A) expression as well as postsynaptic density protein 95 (PSD95) expression in SH-SY5Y cells. Both basal and HMW p75NTR isoforms were increased in the hippocampi of post-mortem late-stage human AD brains (relative to non-AD brains), and the protein expression of HMW p75NTR was negatively associated with Trk-A expression, PSD95 expression, and IR expression. Thus, increased p75NTR expression, specifically an increased p75NTR-to-Trk-A ratio, is likely to play a role in synaptic loss and neuronal cell death in late-stage AD. Collectively, these findings suggest that increased expression of the p75NTR due to IR signaling inhibition by AßOs might be involved in the pathology of AD.

Challenges in ovarian stimulation.

In 2016, ovarian stimulation faces two main challenges: how to obtain good quality oocytes while not endangering the patients treated, but also limited by maternal age and poor ovarian responders (POR). The first IVF birth, Louise Brown, was obtained from a natural cycle. With the introduction, in the 1980s of gonadotropin releasing hormone agonists (GnRHa) and in the 2000s of GnRH antagonists (GnRHant), stimulation became plurifollicular (and source of consequences). Today, only about 50% of the transferred blastocysts after IVF lead to a pregnancy. The purpose of this review was to describe the current challenges and limits of ovarian stimulation.

Involvement of insulin-like growth factor 1 receptor signaling in the amyloid-ß 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-ß peptide (Aß)-mediated neurodegeneration and Aß metabolism in Alzheimer's 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 Aß1-42 levels. Here, we provide evidence that the Aß1-42 oligomers known as ADDLs (Aß-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 AßPPswe/PS1dE9 AD model mice that had accumulated significant amounts of Aß1-42 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 Aß1-42 oligomers stimulate the p75NTR protein expression in the hippocampus through IGF-1R signaling. Thus, Aß1-42 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.

Hippocampal membrane-associated p75NTR levels are increased in Alzheimer's disease.

The pan-specific p75 neurotrophin receptor (p75(NTR)) is believed to play an important role in the pathogenesis of Alzheimer's disease (AD). It is involved in mediating amyloid-ß (Aß) toxicity and stimulating amyloidogenesis. In addition, we have recently shown that stimulating cultured SH-SY5Y human neuroblastoma cells with Aß(42) increases the level of membrane-associated p75(NTR) and that Aß(42)-accumation in two strains of transgenic AD model mice is accompanied by an increased level of hippocampal membrane-associated p75(NTR) (Chakravarthy et al. J Alzheimers Dis 19, 915-925, 2010). This raised an important question whether accumulating Aß(42) in human AD is also accompanied by an increased hippocampal membrane-associated p75(NTR). In this study, using polyclonal and monoclonal antibodies against the p75(NTR) receptor's intra- and extracellular domains, we show that indeed the mean level of membrane-associated p75(NTR) in the hippocampal formation is significantly higher (~two-fold, p < 0.03) in human AD brains than in identical samples of hippocampal formation in age-matched non-AD human brains. The possible relation of this elevated hippocampal p75(NTR) to AD cognitive decline is discussed.

Amyloid-beta peptides stimulate the expression of the p75(NTR) neurotrophin receptor in SHSY5Y human neuroblastoma cells and AD transgenic mice.

The progression toward end-stage Alzheimer's disease (AD) in the aging brain is driven by accumulating amyloid-beta (Abeta)(1-42) oligomers that is accompanied by the downregulation of the Trk A neurotrophin receptor and by either upregulation or at least maintenance of the p75 neurotrophin receptor (p75(NTR)), which can be stimulated by the accumulating Abeta(1-42) peptides. Here we show that Abeta(1-42) and its active fragment Abeta(25-35), but not Abeta(42-1), can at least double the level of p75(NTR) receptors in the membranes of model SH-SY5Y human neuroblastoma cells. We also show that p75(NTR) is upregulated in the hippocampi of two strains of AD transgenic mice. Specifically, the level of the p75(NTR) receptor in the hippocampal membranes from 12-15 month old AD-triple transgenic mice (3xTg-AD) harboring PS1 (M146V), AbetaPP (Swe), and tau (P301L) was nearly twice that in hippocampal membranes from age-matched wild-type mice. Similarly, the level of p75 (NTR) receptor in 7 month-old B6.Cg-Tg AD mice harboring PSEN1dE9 and AbetaPP (Swe) was also increased above the level in the corresponding wild-type mice. This increase correlated with the age-dependent rise in Abeta(1-42) levels in the AD mice. Thus, it appears that it could be the accumulating Abeta(1-42) that increases or at least prevents the downregulation of p75 (NTR) receptors in key parts of AD brains. It is possible that when the Abeta (1-42) accumulation reaches a critical level in the brain on the way to late-onset AD, the Abeta (1-42) induced p75 (NTR) receptor signaling starts a vicious cycle that accelerates AD development because of the activated receptors' recently shown ability to stimulate Abeta(1-42) production.

Co-localization and interaction of DPYSL3 and GAP43 in primary cortical neurons.

Dihydropyrimidinase-like 3 (DPYSL3) and GAP43 are both involved in neurite outgrowth, a crucial process for the differentiation of neurons. The present study shows for the first time that DPYSL3 co-localizes with GAP43 in primary cortical neurons. Further co-immunoprecipitation and overlay assay showed the ability of both recombinant and endogenous DPYSL3 to bind GAP43, indicating a specific interaction between DPYSL3 and GAP43 in primary cortical neurons.

A comparison of image deconvolution algorithms applied to the detection of endocytic vesicles in fluorescence images of neural proteins.

In this work we present a comparative study of three image deconvolution methods applied to fluorescence images of neural proteins. The purpose of this work is to compare the efficiency of these methods, in order to establish which one performs better the restoration of this type o image. Moreover we show that image deconvolution improve not only image quality, but detection capabilities and thus the counting of endocytic vesicles. Image deconvolution was performed by Gold-Meinel (GM) and Lucy-Richardson Maximum likelihood (LRML) non-blind methods and by Lucy-Richardson Maximum likelihood blind method (LRMLB). These methods were tested in 120 images from two different experiments. Computed theoretical point spread function (psf) was used for non-blind deconcovolution methods. Twenty five iterations were performed to restore each image using GM and LRML algorithms. In the case of LRMLB, 10 cycles were performed with 15 psf iterations and 5 image iterations per cycle to deconvolve each image. Endocytic vessels' counting was manually made in deconvolved and non-deconvolved images by a trained observer. Results showed an increase of 22% and 24% in the detection of endocytic vessels using LRML and LRMLB methods respectively and a decrease of 6% using GM method, against detection with non deconvolved images.