Elucidation of conserved multi-epitope vaccine against Leishmania donovani using reverse vaccinology.

Visceral leishmaniasis (VL) is a tropical disease that causes severe public health problems in humans when untreated. As no licensed vaccine exists against VL, we aimed to formulate a potential MHC-restricted chimeric vaccine construct against this dreadful parasitic disease. Amastin-like protein derived from L. donovani is considered to be stable, immunogenic and non-allergic. A comprehensive established framework was used to explore the set of immunogenic epitopes with estimated population coverage of 96.08% worldwide. The rigorous assessment revealed 6 promiscuous T-epitopes which can plausibly be presented by more than 66 diverse HLA alleles. Further docking and simulation study of peptide receptor complexes identified a strong and stable binding interaction with better structural compactness. The predicted epitopes were combined with appropriate linkers and adjuvant molecules and their translation efficiency was evaluated in pET28+(a), an bacterial expression vector using in-silico cloning. Molecular docking followed by MD simulation study revealed a stable interaction between chimeric vaccine construct with TLRs. Immune simulation of the chimeric vaccine constructs showed an elevated Th1 immune response against both B and T epitopes. With this, the detailed computational analysis suggested that the chimeric vaccine construct can evoke a robust immune response against Leishmania donovani infection. Future studies are required to validate the role of amastin as a promising vaccine target.Communicated by Ramaswamy H. Sarma.

Naphthyl bearing 1,3,4-thiadiazoleacetamides targeting the parasitic folate pathway as anti-infectious agents: in silico, synthesis, and biological approach.

Malaria is still a complex and lethal parasitic infectious disease, despite the availability of effective antimalarial drugs. Resistance of malaria parasites to current treatments necessitates new antimalarials targeting P. falciparum proteins. The present study reported the design and synthesis of a series of a 2-(4-substituted piperazin-1-yl)-N-(5-((naphthalen-2-yloxy)methyl)-1,3,4-thiadiazol-2-yl)acetamide hybrids for the inhibition of Plasmodium falciparum dihydrofolate reductase (PfDHFR) using computational biology tools followed by chemical synthesis, structural characterization, and functional analysis. The synthesized compounds were evaluated for their in vitro antimalarial activity against CQ-sensitive PfNF54 and CQ-resistant PfW2 strain. Compounds T5 and T6 are the most active compounds having anti-plasmodial activity against PfNF54 with IC50 values of 0.94 and 3.46 µM respectively. Compound T8 is the most active against the PfW2 strain having an IC50 of 3.91 µM. Further, these active hybrids (T5, T6, and T8) were also evaluated for enzyme inhibition assay against PfDHFR. All the tested compounds were non-toxic against the Hek293 cell line with good selectivity indices. Hemolysis assay also showed non-toxicity of these compounds on normal uninfected human RBCs. In silico molecular docking studies were carried out in the binding pocket of both the wild-type and quadruple mutant Pf-DHFR-TS to gain further insights into probable modes of action of active compounds. ADME prediction and physiochemical properties support their drug-likeness. Additionally, they were screened for antileishmanial activity against L. donovani promastigotes to explore broader applications. Thus, this study provides molecular frameworks for developing potent antimalarials and antileishmanial agents.

Simultaneous Sustained Drug Delivery, Tracking, and On-Demand Photoactivation of DNA-Hydrogel Formulated from a Biomass-Derived DNA Nanoparticle.

Conversion of biomass into nanoparticles for meaningful biomedical applications is a formidable proposition with excellent prospects but fewer patrons. A lack of general methodology for upscaled production and limited versatility of those nanoparticles are the main drawbacks. Herein, we report the creation of a DNA nanoparticle (DNA Dots) from onion genomic DNA (gDNA), a plant biomass source, through controlled hydrothermal pyrolysis in water without any chemicals. The DNA Dots are further formulated into a stimuli-responsive hydrogel through hybridization-mediated self-assembly with untransformed precursor gDNA. The versatility of the DNA Dots is recognized through its crosslinking ability with gDNA through its dangling DNA strands on the surface resulting from incomplete carbonization during annealing without the need for any external organic, inorganic, or polymeric crosslinkers. The gDNA-DNA Dots hybrid hydrogel is shown to be an excellent drug delivery vehicle for sustained release trackable through the inherent fluorescence of the DNA Dots. Interestingly, the DNA Dots are photoexcited with normal visible light to generate on-demand reactive oxygen species, making them exciting candidates for combination therapeutics. Most importantly, the ease with which the hydrogel is internalized in fibroblast cells with minimal cytotoxicity should encourage the nanotization of biomass as a tool for interesting sustainable biomedical applications.

Transcriptome Analysis of Dermal Fibroblasts Derived From Visceral Leishmaniasis and Post-Kala-Azar Dermal Leishmaniasis Patients Reveal Disease-Specific Gene Expression and Pathological Regulation.

BACKGROUND: Post-kala-azar dermal leishmaniasis (PKDL), a dermal form of the disease, occurs in some visceral leishmaniasis (VL) patients following treatment. The PKDL disease mechanism is not yet clearly understood. Here we have studied the role of dermal fibroblasts in VL and PKDL disease mechanism. METHODS: Dermal fibroblasts were grown from skin biopsy explants collected from individual VL and PKDL patients and healthy controls. Fibroblasts from the third passage were subjected to RNA sequencing to analyze differentially expressed genes (DEGs). Significantly important genes were further validated by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: Transcriptome analysis of PKDL versus VL identified 516 DEGs (263 were overrepresented and 253 were underrepresented in PKDL). Among the top hub genes, MMP2, IL1B, CXCL8, IFIH1, NFKB1A, IL6, ISG15, and EGFR were underexpressed and ACTB, HSP90AA1, RAB7A, and RPS27A were overexpressed in PKDL compared to VL. CONCLUSIONS: Our data indicate that PKDL fibroblasts may present antigens through the MHC I pathway activating CD8+ T-cell mediated response, while VL fibroblasts express nuclear factor-κB (NFκB)-mediated chemokines, IL1B, IL6, and IL8, resulting in the recruitment of natural killer (NK)-cells and monocytes to the site of infection, leading to the clearance of parasite from the skin and visceralization of the disease.

Chemically induced crosslinked enhanced emission of carbon polymer dots discerning healthy and cancer cells through pH-dependent tunable photoluminescence.

Chemically induced crosslinked enhanced emission (CEE) of urea and citric acid-derived carbon polymer dot (CPD) nanoparticles is established here with a rare zero linker approach, i.e. without the use of any separate crosslinkers. Such chemical CEE like any chemical reaction was achieved through amide bond formation using carbodiimide chemistry, pointing towards the feasibility of developing a general methodology for their formation through engineering the nanoparticle surface functionality. Exhaustive characterization was done to pinpoint the structure, morphology, and photophysics of the CPDs and concurrently eliminate the possibility of the involvement and interference by molecular fluorophores for the unique optical tuning of the CPDs. The structure-photophysics relation was further restated through theoretical studies involving density functional theory (DFT) that correlated significantly well with the experimental findings. Most interestingly, the CPDs revealed pH responsiveness due to the formation or hydrolysis of amide bonds with acid or base, respectively, which was manifested through a spectacular change in fluorescence emission visible to the naked eye through UV illumination. This distinct pH-dependent photoluminescence properties of CPDs opens up an enormous opportunity for interesting applications, including discriminating normal and cancerous cells, which we demonstrate herein as a proof of concept through in vitro imaging.

Mevalonate kinase of Leishmania donovani protects parasite against oxidative stress by modulating ergosterol biosynthesis.

Leishmaniasis comprises of a wide variety of diseases, caused by protozoan parasite belonging to the genus Leishmania. Leishmania parasites undergo different types of stress during their lifetime and have developed strategies to overcome this damage. Identifying the mechanistic approach used by the parasite in dealing with the stress is of immense importance for unfolding the survival strategy adopted by the parasite. Mevalonate kinase (MVK) is an important regulatory factor in the mevalonate pathway in both bacteria and eukaryotes. In this study, we explored the role of Leishmania donovani mevalonate kinase (LdMVK) in parasite survival under stress condition. Hydrogen peroxide (H2O2) and menadione, the two known oxidants were used to carry out the experiments. The MVK expression was found to be up regulated ∼2.1 fold and ∼2.3 fold under oxidative stress condition and under the effect of anti-Leishmania drug, AmBisome respectively. The cell viability declined under the effect of MVK inhibitor viz: vanadyl sulfate (VS). The level of intracellular ROS was also found to be increased under the effect of MVK inhibitor. To confirm the findings, LdMVK over expression (LdMVK OE) and LdMVK knockdown (LdMVK KD) parasites were generated. The level of ergosterol, an important component of plasma membrane in L. donovani, was observed and found to be reduced by nearly 60 % in LdMVK KD parasite and increased by nearly 30 % in LdMVK OE parasites as compared to wild type. However, the ergosterol content was found to be elevated under oxidative stress. Furthermore, LdMVK was also found to be associated with maintaining the plasma membrane integrity and also in preventing the peroxidation of cellular lipids when exposed to oxidative stress. The above data clearly suggests that MVK has a vital role in protecting the parasite from oxidative stress. These findings may also explore the contribution of LdMVK in drug unresponsiveness which may help in future rational drug designing for leishmaniasis.

Isoform-Specific Effects of Apolipoprotein E on Markers of Inflammation and Toxicity in Brain Glia and Neuronal Cells In Vitro.

Mutations to the cholesterol transport protein apolipoprotein E (ApoE) have been identified as a major risk factor for the development of sporadic or late-onset Alzheimer's disease (AD), with the e4 allele representing an increased risk and the rare e2 allele having a reduced risk compared to the primary e3 form. The reasons behind the change in risk are not entirely understood, though ApoE4 has been connected to inflammation and toxicity in both the brain and the periphery. The goal of this study was to better understand how the ApoE isoforms (ApoE2/3/4) confer differential AD-related risk by assessing cell-specific ApoE-related neuroinflammatory and neurotoxic effects. We compared the effects of ApoE isoforms in vitro on human astrocytes, a human immortalized microglia cell line (HMC3), and the human neuroblastoma cell line SH-SY5Y. Cells were treated for 24 h with or without recombinant ApoE2, ApoE3, or ApoE4 (20 nM) and inflammation and toxicity markers assessed. Our results indicated the expression of inflammatory cytokines IL-1ß, TNFα, and IL-6 in human astrocytes was increased in response to all ApoE isoforms, with ApoE4 evoking the highest level of cytokine expression. In response to ApoE2 or ApoE3, microglial cells showed reduced levels of microglial activation markers TREM2 and Clec7a, while ApoE4 induced increased levels of both markers. ApoE2 promoted neuron survival through increased BDNF release from astrocytes. In addition, ApoE2 promoted, while ApoE4 reduced, neuronal viability. Overall, these results suggest that ApoE4 acts on cells in the brain to promote inflammation and neuronal injury and that the deleterious effects of ApoE4 on these cells may, in part, contribute to its role as a risk factor for AD.

Oxidant activated soluble adenylate cyclase of Leishmania donovani regulates the cAMP-PKA signaling axis for its intra-macrophage survival during infection.

Adenosine 3',5'-cyclic monophosphate (cAMP) is a stress sensor molecule that transduces the cellular signal when Leishmania donovani moves from insect vector to mammalian host. At this stage, the parasite membrane-bound receptor adenylate cyclase predominantly produces cAMP to cope with the oxidative assault imposed by host macrophages. However, the role of soluble adenylate cyclase of L. donovani (LdHemAC) has not been investigated fully. In the present investigation, we monitored an alternative pool of cAMP, maintained by LdHemAC. The elevated cAMP effectively transmits signals by binding to Protein Kinase A (PKA) present in the cytosol and regulates antioxidant gene expression and phosphorylates several unknown PKA substrate proteins. Menadione-catalyzed production of reactive oxygen species (ROS) mimics host oxidative condition in vitro in parasites where cAMP production and PKA activity were found increased by ~1.54 ± 0.35, and ~1.78 ± 0.47-fold, respectively while expression of LdHemAC gene elevated by ~2.18 ± 0.17-fold. The LdHemAC sense these oxidants and became activated to cyclize ATP to enhance the cAMP basal level that regulates antioxidant gene expression to rescue parasites from oxidative stress. In knockdown parasites (LdHemAC-KD), the downregulated antioxidant genes expression, namely, Sod (2.30 ± 0.46), Pxn (2.73 ± 0.15), Tdr (2.7 ± 0.12), and Gss (1.57 ± 0.15) results in decreased parasite viability while in overexpressed parasites (LdHemAC-OE), the expression was upregulated by ~5.7 ± 0.35, ~2.57 ± 0.56, ~4.7 ± 0.36, and ~2.4 ± 0.83, respectively, which possibly overcomes ROS accumulation and enhances viability. Furthermore, LdHemAC-OE higher PKA activity regulates phosphorylation of substrate proteins (~56 kDs in membrane fraction and ~25 kDs in the soluble fraction). It reduced significantly when treated with inhibitors like DDA, Rp-cAMP, and H-89 and increased by ~2.1 ± 0.28-fold, respectively under oxidative conditions. The LdHemAC-KD was found less infective to RAW 264.7 macrophages and more prone to oxidative damage as compared to LdHemAC-OE and control parasites. Together, this study demonstrates mechanistic links among LdHemAC, cAMP, and PKA in parasite survival and invasion under host oxidative condition.

Leishmania donovani Secretory Mevalonate Kinase Regulates Host Immune Response and Facilitates Phagocytosis.

Leishmania secretes over 151 proteins during in vitro cultivation. Cellular functions of one such novel protein: mevalonate kinase is discussed here; signifying its importance in Leishmania infection. Visceral Leishmaniasis is a persistent infection, caused by Leishmania donovani in Indian subcontinent. This persistence is partly due to phagocytosis and evasion of host immune response. The underlying mechanism involves secretory proteins of Leishmania parasite; however, related studies are meagre. We have identified a novel secretory Leishmania donovani glycoprotein, Mevalonate kinase (MVK), and shown its importance in parasite internalization and immuno-modulation. In our studies, MVK was found to be secreted maximum after 1 h temperature stress at 37°C. Its secretion was increased by 6.5-fold in phagolysosome-like condition (pH ~5.5, 37°C) than at pH ~7.4 and 25°C. Treatment with MVK modulated host immune system by inducing interleukin-10 and interleukin-4 secretion, suppressing host's ability to kill the parasite. Peripheral blood mononuclear cell (PBMC)-derived macrophages infected with mevalonate kinase-overexpressing parasites showed an increase in intracellular parasite burden in comparison to infection with vector control parasites. Mechanism behind the increase in phagocytosis and immunosuppression was found to be phosphorylation of mitogen-activated protein (MAP) kinase pathway protein, Extracellular signal-regulated kinases-1/2, and actin scaffold protein, cortactin. Thus, we conclude that Leishmania donovani Mevalonate kinase aids in parasite engulfment and subvert the immune system by interfering with signal transduction pathways in host cells, which causes suppression of the protective response and facilitates their persistence in the host. Our work elucidates the involvement of Leishmania in the process of phagocytosis which is thought to be dependent largely on macrophages and contributes towards better understanding of host pathogen interactions.

TLR4 and TLR9 polymorphism: Probable role in susceptibility among the population of Bihar for Indian visceral leishmaniasis.

Genetic variations in the host TLRs genes play an important role in susceptibility and/or resistance to visceral leishmaniasis by altering the host-pathogen interaction. In this study, we investigated the association between polymorphisms of TLR4 (Asp299Gly, Thr399Ile) and TLR-9 (T-1237C), with susceptibility to visceral leishmaniasis. A bi-directional PCR amplification of specific alleles technique was used to characterize the distribution of TLR4 (Asp299Gly and Thr399Ile) and TLR9 (T-1237C) polymorphisms. A total of 60 samples were randomly selected from confirmed visceral leishmaniasis patients and 24 endemic healthy volunteers. The samples were genotyped and allele frequencies were determined. We observed that TLR4 Asp299Gly and Thr399Ile genotypes were more frequent in visceral leishmaniasis patients (10% and 15% respectively) compared to controls (4.2% and 8.3% respectively). However, the differences were not significant in TLR4 Asp299Gly and Thr399Ile alleles and genotypes. In the case of TLR9, we observed the frequency of T1237C genotype was higher in visceral leishmaniasis patients (43.3%) than in healthy controls (33.3%). Statistically significant differences were observed in TLR9 T1237C alleles and genotypes. We concluded that TLR9 T1237C, but not TLR4, gene polymorphisms can be regarded as contributors to visceral leishmaniasis susceptibility among the Indian population of Bihar state.

Dabigatran reduces thrombin-induced neuroinflammation and AD markers in vitro: Therapeutic relevance for Alzheimer's disease.

Background: Vascular risk factors such as atherosclerosis, diabetes, and elevated homocysteine levels are strongly correlated with onset of Alzheimer's disease (AD). Emerging evidence indicates that blood coagulation protein thrombin is associated with vascular and non-vascular risk factors of AD. Here, we examined the effect of thrombin and its direct inhibitor dabigatran on key mediators of neuro-inflammation and AD pathology in the retinoic acid (RA)-differentiated human neuroblastoma cell line SH-SY5Y. Methods: SH-SY5Y cells exposed to thrombin concentrations (10-100 nM) +/- 250 nM dabigatran for 24 h were analyzed for protein and gene expression. Electrophoretic mobility shift assay (EMSA) was used to determine DNA binding of NFkB. Western blotting, qRT-PCR and ELISA were used to measure the protein, mRNA, and activity levels of known AD hallmarks and signaling molecules. Results: Dabigatran treatment attenuated thrombin-induced increase in DNA binding of NFκB by 175% at 50 nM and by 77% at 100 nM thrombin concentration. Thrombin also augmented accumulation of Aß protein expression and phosphorylation of p38 MAPK, a downstream molecule in the signaling cascade, expression of pro-apoptotic mediator caspase 3, APP, tTau and pTau. Additionally, thrombin increased BACE1 activity, GSK3ß expression, and APP, BACE1, Tau and GSK3ß mRNA levels. Co-incubation with dabigatran attenuated thrombin-induced increases in the protein, mRNA, and activities of the aforesaid molecules to various extents (between -31% and -283%). Conclusion: Our data demonstrates that thrombin promotes AD-related pathological changes in neuronal cultures and suggests that use of direct oral anticoagulants may provide a therapeutic benefit against thrombin-driven neuroinflammation and downstream pathology in AD.

Thrombin Signaling Contributes to High Glucose-Induced Injury of Human Brain Microvascular Endothelial Cells.

BACKGROUND: Diabetes is one of the strongest disease-related risk factors for Alzheimer's disease (AD). In diabetics, hyperglycemia-induced microvascular complications are the major cause of end-organ injury, contributing to morbidity and mortality. Microvascular pathology is also an important and early feature of AD. The cerebral microvasculature may be a point of convergence of both diseases. Several lines of evidence also implicate thrombin in AD as well as in diabetes. OBJECTIVE: Our objective was to investigate the role of thrombin in glucose-induced brain microvascular endothelial injury. METHODS: Cultured Human brain microvascular endothelial cells (HBMVECs) were treated with 30 mM glucose±100 nM thrombin and±250 nM Dabigatran or inhibitors of PAR1, p38MAPK, MMP2, or MMP9. Cytotoxicity and thrombin activity assays on supernatants and western blotting for protein expression in lysates were performed. RESULTS: reatment of HBMVECs with 30 mM glucose increased thrombin activity and expression of inflammatory proteins TNFα, IL-6, and MMPs 2 and 9; this elevation was reduced by the thrombin inhibitor dabigatran. Direct treatment of brain endothelial cells with thrombin upregulated p38MAPK and CREB, and induced TNFα, IL6, MMP2, and MMP9 as well as oxidative stress proteins NOX4 and iNOS. Inhibition of thrombin, thrombin receptor PAR1 or p38MAPK decrease expression of inflammatory and oxidative stress proteins, implying that thrombin may play a central role in glucose-induced endothelial injury. CONCLUSION: Since preventing brain endothelial injury would preserve blood-brain barrier integrity, prevent neuroinflammation, and retain intact functioning of the neurovascular unit, inhibiting thrombin, or its downstream signaling effectors, could be a therapeutic strategy for mitigating diabetes-induced dementia.

Short-term treatment with dabigatran alters protein expression patterns in a late-stage tau-based Alzheimer's disease mouse model.

Proteins that regulate the coagulation cascade, including thrombin, are elevated in the brains of Alzheimer's disease (AD) patients. While studies using amyloid-based AD transgenic mouse models have implicated thrombin as a protein of interest, the role of thrombin in tau-based animal models has not been explored. The current study aims to determine how inhibiting thrombin could alter oxidative stress, inflammation, and AD-related proteins in a tau-based mouse model, the Tg4510. Aged Tg4510 mice were treated with the direct thrombin inhibitor dabigatran or vehicle for 7 days, brains collected, and western blot and data-independent proteomics using mass spectrometry with SWATH-MS acquisition performed to evaluate proteins related to oxidative stress, intracellular signaling, inflammation, and AD pathology. Dabigatran reduced iNOS, NOX4, and phosphorylation of tau (S396, S416). Additionally, dabigatran treatment increased expression of several signaling proteins related to cell survival and synaptic function. Increasing evidence supports a chronic procoagulant state in AD, highlighting a possible pathogenic role for thrombin. Our data demonstrate that inhibiting thrombin produces alterations in the expression of proteins involved in oxidative stress, inflammation, and AD-related pathology, suggesting that thrombin-mediated signaling affects multiple AD-related pathways providing a potential future therapeutic target.

Differential Regulation of miRNA Profiles of Human Cells Experimentally Infected by Leishmania donovani Isolated From Indian Visceral Leishmaniasis and Post-Kala-Azar Dermal Leishmaniasis.

MicroRNAs are small ribonucleic acid that act as an important regulator of gene expression at the molecular level. However, there is no comparative data on the regulation of microRNAs (miRNAs) in visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL). In this current study, we compared the expression miRNA profile in host cells (GTHP), with VL strain (GVL) and PKDL strain-infected host cell (GPKDL). Normalized read count comparison between different conditions revealed that the miRNAs are indeed differentially expressed. In GPKDL with respect to GVL and GTHP, a total of 798 and 879 miRNAs were identified, out of which 349 and 518 are known miRNAs, respectively. Comparative analysis of changes in miRNA expression suggested that the involvement of differentially expressed miRNAs in various biological processes like PI3K pathway activation, cell cycle regulation, immunomodulation, apoptosis inhibition, different cytokine production, T-cell phenotypic transitions calcium regulation, and so on. A pathway enrichment study using in silico predicted gene targets of differentially expressed miRNAs showed evidence of potentially universal immune signaling pathway effects. Whereas cytokine-cytokine receptor interaction, phagocytosis, and transforming growth factor beta (TGF-ß) signaling pathways were more highly enriched using targets of miRNAs upregulated in GPKDL. These findings could contribute to a better understanding of PKDL pathogenesis. Furthermore, the identified miRNAs could also be used as biomarkers in diagnosis, prognosis, and therapeutics of PKDL infection control.

Leishmania donovani infection induce differential miRNA expression in CD4+ T cells.

Visceral leishmaniasis is characterized by mixed production of Th1/2 cytokines and the disease is established by an enhanced level of Th2 cytokine. CD4+ T cells are main cell type which produces Th1/2 cytokine in the host upon Leishmania infection. However, the regulatory mechanism for Th1/2 production is not well understood. In this study, we co-cultured mice CD4+ T cells with Leishmania donovani infected and uninfected macrophage for the identification of dysregulated miRNAs in CD4+ T cells by next-generation sequencing. Here, we identified 604 and 613 known miRNAs in CD4+ T cells in control and infected samples respectively and a total of only 503 miRNAs were common in both groups. The expression analysis revealed that 112 miRNAs were up and 96 were down-regulated in infected groups, compared to uninfected control. Nineteen up-regulated and 17 down-regulated miRNAs were statistically significant (p < 0.05), which were validated by qPCR. Further, using insilco approach, we identified the gene targets of significant miRNAs on the basis of CD4+ T cell biology. Eleven up-regulated miRNAs and 9 down-regulated miRNAs were associated with the cellular immune responses and Th1/2 dichotomy upon Leishmania donovani infection. The up-regulated miRNAs targeted transcription factors that promote differentiation of CD4+ T cells towards Th1 phenotype. While down-regulated miRNAs targeted the transcription factors that facilitate differentiation of CD4+ T cells towards Th2 populations. The GO and pathway enrichment analysis also showed that the identified miRNAs target the pathway and genes related to CD4+ T cell biology which plays important role in Leishmania donovani infection.

Apolipoprotein E particle size is increased in Alzheimer's disease.

INTRODUCTION: Apolipoprotein E4 (apoE4) is the predominant risk factor for late-onset Alzheimer's disease (AD), but the question of which structural differences might explain its effect remains unclear. METHODS: We compared high-density lipoprotein-like apoE particles from 12 AD and 10 control patients using size-exclusion chromatography. RESULTS: ApoE particles from patients genotyped as ε4/ε4 were 2.2 ± 0.3 times as massive as particles from ε3/ε3 control subjects and 1.4 ± 0.1 times as massive as particles from ε3/ε3 AD patients. The increased particle size was not because of incorporation of amyloid ß or apoE proteolysis products. Particles from AD patients genotyped as ε3/ε3 were 1.59 ± 0.27 times as massive as ε3/ε3 control subjects. DISCUSSION: Increased particle size in AD is affected by A PO E genotype and by disease-related differences in assembly or stability. These differences suggest that lipoprotein assembly or stability in AD brain plays an important role in determining apoE4 pathogenicity.

Loss in PKC Epsilon Causes Downregulation of MnSOD and BDNF Expression in Neurons of Alzheimer's Disease Hippocampus.

Oxidative stress and amyloid-ß (Aß) oligomers have been implicated in Alzheimer's disease (AD). The growth and maintenance of neuronal networks are influenced by brain derived neurotrophic factor (BDNF) expression, which is promoted by protein kinase C epsilon (PKCɛ). We investigated the reciprocal interaction among oxidative stress, Aß, and PKCɛ levels and subsequent PKCɛ-dependent MnSOD and BDNF expression in hippocampal pyramidal neurons. Reduced levels of PKCɛ, MnSOD, and BDNF and an increased level of Aß were also found in hippocampal neurons from autopsy-confirmed AD patients. In cultured human primary hippocampal neurons, spherical aggregation of Aß (amylospheroids) decreased PKCɛ and MnSOD. Treatment with t-butyl hydroperoxide (TBHP) increased superoxide, the oxidative DNA/RNA damage marker, 8-OHG, and Aß levels, but reduced PKCɛ, MnSOD, BDNF, and cultured neuron density. These changes were reversed with the PKCɛ activators, bryostatin and DCPLA-ME. PKCɛ knockdown suppressed PKCɛ, MnSOD, and BDNF but increased Aß. In cultured neurons, the increase in reactive oxygen species (ROS) associated with reduced PKCɛ during neurodegeneration was inhibited by the SOD mimetic MnTMPyP and the ROS scavenger NAc, indicating that strong oxidative stress suppresses PKCɛ level. Reduction of PKCɛ and MnSOD was prevented with the PKCɛ activator bryostatin in 5-6-month-old Tg2576 AD transgenic mice. In conclusion, oxidative stress and Aß decrease PKCɛ expression. Reciprocally, a depression of PKCɛ reduces BDNF and MnSOD, resulting in oxidative stress. These changes can be prevented with the PKCɛ-specific activators.

Hippocampal microvasculature changes in association with oxidative stress in Alzheimer's disease.

Vascular endothelial dysfunction is a primary phenotype of aging, and microvascular (MV) lesion is mainly associated with Alzheimer's disease (AD). Here we have studied the correlation of MV wall thickness and CA1 pyramidal neuronal pathology in autopsy-confirmed AD brains. Both hyaline (h-MV) and increased cell number (c-MV) associated MV wall thickening was found in age-matched control (AC) hippocampus without significant change in Aß level (Braak stages 0-III). AC neurons neighboring the h-MV showed lower levels of oxidative DNA/RNA damage and Aß precursor protein (APP), while the neurons around c-MV showed higher oxidative DNA/RNA damage with increased APP expression. Neurons in AC hippocampus without MV wall thickening (thin wall) showed increased DNA/RNA damage and APP levels compared to AC cases with h-MV and c-MV walls. In the AD hippocampus neurons neighboring h-MV walls showed increased levels of Aß and decreased number of dendritic spines (at Braak stages IV-VI). C-MV neighboring neurons in the AD cases showed higher levels of DNA/RNA damage with increased APP at stages II - III, followed by lower levels of oxidative DNA/RNA damage, decreased APP and increased Aß levels with loss of dendritic spines at stages IV-VI. Prolonged treatment of primary human fetal hippocampal neurons with tert-butyl hydroperoxide (TBHP) induced oxidative DNA damage with a sustained increase in APP. Aß increased rapidly and then decreased overtime. Short-term TBHP treated neurons showed lower levels of superoxide (O2• -) without significant DNA damage. Short-term TBHP treatment induced a gradual decrease in APP but an increase in Aß levels over time. In conclusion this study indicates that AD hippocampus at Braak stages II-III are characterized by strong oxidative DNA/RNA damage with increased APP in neurons associated with c-MV, while stages IV-VI are characterized by a slow increase in Aß in neurons neighboring both h-MV and c-MV.

ApoE isoforms differentially regulates cleavage and secretion of BDNF.

Apolipoprotein E4 (ApoE4) is a major genetic risk factor for sporadic or late onset Alzheimer's disease (AD). Brain-derived neurotrophic factor (BDNF) is decreased by 3 to 4-fold in the brains of AD patients at autopsy. ApoE4 mice also have reduced BDNF levels. However, there have been no reports relating the different ApoE isoforms or AD to differential regulation of BDNF. Here we report that in the hippocampal regions of AD patients both prepro-BDNF and pro-BDNF expression showed a 40 and 60% decrease respectively compared to that expression in the hippocampi of age-matched control patients. We further report that ApoE isoforms differentially regulate maturation and secretion of BDNF from primary human astrocytes. After 24 h, ApoE3 treated astrocytes secreted 1.75- fold higher pro-BDNF than ApoE2-treated astrocytes, and ApoE2-treated astrocytes secreted 3-fold more mature-BDNF (m-BDNF) than ApoE3-treated astrocytes. In contrast, ApoE4-treated cells secreted negligible amounts of m-BDNF or pro-BDNF. ApoE2 increased the level of intracellular pre-pro BDNF by 19.04 ± 6.68%, while ApoE4 reduced the pre-pro BDNF by 21.61 ± 5.9% compared to untreated cells. Similar results were also seen in ApoE2, ApoE3 or ApoE4 treated cells at 4 h. Together, these results indicate that an ApoE2 or ApoE3 mediated positive regulation of BDNF may be protective while ApoE4 related defects in BDNF processing could lead to AD pathophysiology. These interactions of the ApoE isoforms with BDNF may help explain the increased risk of AD associated with the ApoE4 isoform.

Bryostatin Effects on Cognitive Function and PKCɛ in Alzheimer's Disease Phase IIa and Expanded Access Trials.

Bryostatin 1, a potent activator of protein kinase C epsilon (PKCɛ), has been shown to reverse synaptic loss and facilitate synaptic maturation in animal models of Alzheimer's disease (AD), Fragile X, stroke, and other neurological disorders. In a single-dose (25 µg/m2) randomized double-blind Phase IIa clinical trial, bryostatin levels reached a maximum at 1-2 h after the start of infusion. In close parallel with peak blood levels of bryostatin, an increase of PBMC PKCɛ was measured (p = 0.0185) within 1 h from the onset of infusion. Of 9 patients with a clinical diagnosis of AD, of which 6 received drug and 3 received vehicle within a double-blind protocol, bryostatin increased the Mini-Mental State Examination (MMSE) score by +1.83±0.70 unit at 3 h versus -1.00±1.53 unit for placebo. Bryostatin was well tolerated in these AD patients and no drug-related adverse events were reported. The 25 µg/m2 administered dose was based on prior clinical experience with three Expanded Access advanced AD patients treated with bryostatin, in which return of major functions such as swallowing, vocalization, and word recognition were noted. In one Expanded Access patient trial, elevated PKCɛ levels closely tracked cognitive benefits in the first 24 weeks as measured by MMSE and ADCS-ADL psychometrics. Pre-clinical mouse studies showed effective activation of PKCɛ and increased levels of BDNF and PSD-95. Together, these Phase IIa, Expanded Access, and pre-clinical results provide initial encouragement for bryostatin 1 as a potential treatment for AD.