Iron-export ferroxidase activity of ß-amyloid precursor protein is inhibited by zinc in Alzheimer's disease.

Alzheimer's Disease (AD) is complicated by pro-oxidant intraneuronal Fe(2+) elevation as well as extracellular Zn(2+) accumulation within amyloid plaque. We found that the AD ß-amyloid protein precursor (APP) possesses ferroxidase activity mediated by a conserved H-ferritin-like active site, which is inhibited specifically by Zn(2+). Like ceruloplasmin, APP catalytically oxidizes Fe(2+), loads Fe(3+) into transferrin, and has a major interaction with ferroportin in HEK293T cells (that lack ceruloplasmin) and in human cortical tissue. Ablation of APP in HEK293T cells and primary neurons induces marked iron retention, whereas increasing APP695 promotes iron export. Unlike normal mice, APP(-/-) mice are vulnerable to dietary iron exposure, which causes Fe(2+) accumulation and oxidative stress in cortical neurons. Paralleling iron accumulation, APP ferroxidase activity in AD postmortem neocortex is inhibited by endogenous Zn(2+), which we demonstrate can originate from Zn(2+)-laden amyloid aggregates and correlates with Aß burden. Abnormal exchange of cortical zinc may link amyloid pathology with neuronal iron accumulation in AD.

N-Terminomic Changes in Neurons During Excitotoxicity Reveal Proteolytic Events Associated With Synaptic Dysfunctions and Potential Targets for Neuroprotection.

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIß (CaMKIIß). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIß, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.

Bacterial Membrane Vesicles: The Missing Link Between Bacterial Infection and Alzheimer Disease.

Periodontitis is a common chronic inflammatory disease, affecting approximately 19% of the global adult population. A relationship between periodontal disease and Alzheimer disease has long been recognized, and recent evidence has been uncovered to link these 2 diseases mechanistically. Periodontitis is caused by dysbiosis in the subgingival plaque microbiome, with a pronounced shift in the oral microbiota from one consisting primarily of Gram-positive aerobic bacteria to one predominated by Gram-negative anaerobes, such as Porphyromonas gingivalis. A common phenomenon shared by all bacteria is the release of membrane vesicles to facilitate biomolecule delivery across long distances. In particular, the vesicles released by P gingivalis and other oral pathogens have been found to transport bacterial components across the blood-brain barrier, initiating the physiologic changes involved in Alzheimer disease. In this review, we summarize recent data that support the relationship between vesicles secreted by periodontal pathogens to Alzheimer disease pathology.

Chronic Oral Inoculation of Porphyromonas gingivalis and Treponema denticola Induce Different Brain Pathologies in a Mouse Model of Alzheimer Disease.

Periodontitis is a chronic inflammatory disease driven by dysbiosis in subgingival microbial communities leading to increased abundance of a limited number of pathobionts, including Porphyromonas gingivalis and Treponema denticola. Oral health, particularly periodontitis, is a modifiable risk factor for Alzheimer disease (AD) pathogenesis, with components of both these bacteria identified in postmortem brains of persons with AD. Repeated oral inoculation of mice with P. gingivalis results in brain infiltration of bacterial products, increased inflammation, and induction of AD-like biomarkers. P. gingivalis displays synergistic virulence with T. denticola during periodontitis. The aim of the current study was to determine the ability of P. gingivalis and T. denticola, grown in physiologically relevant conditions, individually and in combination, to induce AD-like pathology following chronic oral inoculation of female mice over 12 weeks. P. gingivalis alone significantly increased all 7 brain pathologies examined: neuronal damage, activation of astrocytes and microglia, expression of inflammatory cytokines interleukin 1ß (IL-1ß) and interleukin 6 and production of amyloid-ß plaques and hyperphosphorylated tau, in the hippocampus, cortex and midbrain, compared to control mice. T. denticola alone significantly increased neuronal damage, activation of astrocytes and microglia, and expression of IL-1ß, in the hippocampus, cortex and midbrain, compared to control mice. Coinoculation of P. gingivalis with T. denticola significantly increased activation of astrocytes and microglia in the hippocampus, cortex and midbrain, and increased production of hyperphosphorylated tau and IL-1ß in the hippocampus only. The host brain response elicited by oral coinoculation was less than that elicited by each bacterium, suggesting coinoculation was less pathogenic.

Sex-dependent effects of amyloid precursor-like protein 2 in the SOD1-G37R transgenic mouse model of MND.

Motor neurone disease (MND) is a neurodegenerative disorder characterised by progressive destruction of motor neurons, muscle paralysis and death. The amyloid precursor protein (APP) is highly expressed in the central nervous system and has been shown to modulate disease outcomes in MND. APP is part of a gene family that includes the amyloid precursor-like protein 1 (APLP1) and 2 (APLP2) genes. In the present study, we investigated the role of APLP2 in MND through the examination of human spinal cord tissue and by crossing APLP2 knockout mice with the superoxide dismutase 1 (SOD1-G37R) transgenic mouse model of MND. We found the expression of APLP2 is elevated in the spinal cord from human cases of MND and that this feature of the human disease is reproduced in SOD1-G37R mice at the End-stage of their MND-like phenotype progression. APLP2 deletion in SOD1-G37R mice significantly delayed disease progression and increased the survival of female SOD1-G37R mice. Molecular and biochemical analysis showed female SOD1-G37R:APLP2-/- mice displayed improved innervation of the neuromuscular junction, ameliorated atrophy of muscle fibres with increased APP protein expression levels in the gastrocnemius muscle. These results indicate a sex-dependent role for APLP2 in mutant SOD1-mediated MND and further support the APP family as a potential target for further investigation into the cause and regulation of MND.

Amyloid precursor protein and amyloid precursor-like protein 2 have distinct roles in modulating myelination, demyelination, and remyelination of axons.

The identification of factors that regulate myelination provides important insight into the molecular mechanisms that coordinate nervous system development and myelin regeneration after injury. In this study, we investigated the role of amyloid precursor protein (APP) and its paralogue amyloid precursor-like protein 2 (APLP2) in myelination using APP and APLP2 knockout (KO) mice. Given that BACE1 regulates myelination and myelin sheath thickness in both the peripheral and central nervous systems, we sought to determine if APP and APLP2, as alternate BACE1 substrates, also modulate myelination, and therefore provide a better understanding of the events regulating axonal myelination. In the peripheral nervous system, we identified that adult, but not juvenile KO mice, have lower densities of myelinated axons in their sciatic nerves while in the central nervous system, axons within both the optic nerves and corpus callosum of both KO mice were significantly hypomyelinated compared to wild-type (WT) controls. Biochemical analysis demonstrated significant increases in BACE1 and myelin oligodendrocyte glycoprotein and decreased NRG1 and proteolipid protein levels in both KO brain tissue. The acute cuprizone model of demyelination/remyelination revealed that whereas axons in the corpus callosum of WT and APLP2-KO mice underwent similar degrees of demyelination and subsequent remyelination, the myelinated callosal axons in APP-KO mice were less susceptible to cuprizone-induced demyelination and showed a failure in remyelination after cuprizone withdrawal. These data identified APP and APLP2 as modulators of normal myelination and demyelination/remyelination conditions. Deletion of APP and APLP2 identifies novel interplays between the BACE1 substrates in the regulation of myelination.

Analysis of Motor Function in Amyloid Precursor-Like Protein 2 Knockout Mice: The Effects of Ageing and Sex.

The amyloid precursor protein (APP) is a member of a conserved gene family that includes the amyloid precursor-like proteins 1 (APLP1) and 2 (APLP2). APP and APLP2 share a high degree of similarity, and have overlapping patterns of spatial and temporal expression in the central and peripheral tissues, in particular at the neuromuscular junction. APP-family knockout (KO) studies have helped elucidate aspects of function and functional redundancy amongst the APP-family members. In the present study, we investigated motor performance of APLP2-KO mice and the effect sex differences and age-related changes have on motor performance. APLP2-KO and WT (on C57Bl6 background) littermates control mice from 8 (young adulthood) to 48 weeks (middle age) were investigated. Analysis of motor neuron and muscle morphology showed APLP2-KO females but not males, had less age-related motor function impairments. We observed age and sex differences in both motor neuron number and muscle fiber size distribution for APLP2-KO mice compared to WT (C57Bl6). These alterations in the motor neuron number and muscle fiber distribution pattern may explain why female APLP2-KO mice have far better motor function behaviour during ageing.

Minocycline attenuates colistin-induced neurotoxicity via suppression of apoptosis, mitochondrial dysfunction and oxidative stress.

Background: Neurotoxicity is an adverse effect patients experience during colistin therapy. The development of effective neuroprotective agents that can be co-administered during polymyxin therapy remains a priority area in antimicrobial chemotherapy. The present study investigates the neuroprotective effect of the synergistic tetracycline antibiotic minocycline against colistin-induced neurotoxicity. Methods: The impact of minocycline pretreatment on colistin-induced apoptosis, caspase activation, oxidative stress and mitochondrial dysfunction were investigated using cultured mouse neuroblastoma-2a (N2a) and primary cortical neuronal cells. Results: Colistin-induced neurotoxicity in mouse N2a and primary cortical cells gives rise to the generation of reactive oxygen species (ROS) and subsequent cell death via apoptosis. Pretreatment of the neuronal cells with minocycline at 5, 10 and 20 µM for 2 h prior to colistin (200 µM) exposure (24 h), had an neuroprotective effect by significantly decreasing intracellular ROS production and by upregulating the activities of the anti-ROS enzymes superoxide dismutase and catalase. Minocycline pretreatment also protected the cells from colistin-induced mitochondrial dysfunction, caspase activation and subsequent apoptosis. Immunohistochemical imaging studies revealed colistin accumulates within the dendrite projections and cell body of primary cortical neuronal cells. Conclusions: To our knowledge, this is first study demonstrating the protective effect of minocycline on colistin-induced neurotoxicity by scavenging of ROS and suppression of apoptosis. Our study highlights that co-administration of minocycline kills two birds with one stone: in addition to its synergistic antimicrobial activity, minocycline could potentially ameliorate unwanted neurotoxicity in patients undergoing polymyxin therapy.

Site of fluorescent label modifies interaction of melittin with live cells and model membranes.

The mechanism of membrane disruption by melittin (MLT) of giant unilamellar vesicles (GUVs) and live cells was studied using fluorescence microscopy and two fluorescent synthetic analogues of MLT. The N-terminus of one of these was acylated with thiopropionic acid to enable labeling with maleimido-AlexaFluor 430 to study the interaction of MLT with live cells. It was compared with a second analogue labeled at P14C. The results indicated that the fluorescent peptides adhered to the membrane bilayer of phosphatidylcholine GUVs and inserted into the plasma membrane of HeLa cells. Fluorescence and light microscopy revealed changes in cell morphology after exposure to MLT peptides and showed bleb formation in the plasma membrane of HeLa cells. However, the membrane disruptive effect was dependent upon the location of the fluorescent label on the peptide and was greater when MLT was labeled at the N-terminus. Proline at position 14 appeared to be important for antimicrobial activity, hemolysis and cytotoxicity, but not essential for cell membrane disruption.

Membrane-bound tetramer and trimer Aß oligomeric species correlate with toxicity towards cultured neurons.

Amyloid beta (Aß) peptide is the major constituent of the extracellular amyloid plaques deposited in the brains of Alzheimer's disease patients and is central to the pathogenic pathway causing this disease. The identity of the neurotoxic Aß species remains elusive. We previously reported that Aß toxicity correlates strongly with its neuronal cell binding leading us to hypothesize that neuronal cell death is caused by the binding of a specific oligomeric Aß species. To identify the specific oligomeric Aß species that is associated with cell death, we treated mouse cortical neuronal cultures with synthetic Aß40 and Aß42 peptides and identified that the cellular Aß binding and neurotoxicity were time and concentration dependent. We found a significant correlation between the amount of trimer and tetramer species bound to neurons with increasing neurotoxicity. We prepared Aß40 oligomers (up to tetramers) using photo-induced cross-linking of unmodified peptides to confirm this oligomer-specific neurotoxic activity. Our results identify the Aß tetramer, followed by the trimer, as the most toxic low-order oligomers Aß species. Our findings suggested that binding of amyloid-ß (Aß) tetramer and trimer, not monomer or dimer, to neurons is critical to induce neuronal cell death associated with Alzheimer's Disease. We proposed that Aß trimer and tetramer are the potential neurotoxic Aß species. This would provide more specific therapeutic target for Alzheimer's Disease.

Aggregation distributions on cells determined by photobleaching image correlation spectroscopy.

The organization of molecules into macromolecular (nanometer scale), supramolecular complexes (submicron-to-micron scale), and within subcellular domains, is an important architectural principle of cellular biology and biochemistry. Determining the precise nature and distribution of complexes within the cellular milieu is a challenging biophysical problem. Time-series analysis of laser scanning confocal microscopy images by image correlation spectroscopy (ICS) or fluctuation moments methods provides information on aggregation, flow, and dynamics of fluorescently tagged macromolecules. All the methods to date require a brightness standard to relate the experimental data to absolute aggregation. In this article, we show that ICS as a function of gradual photobleaching is a sensitive indicator of aggregation distribution on the submicron scale. Specifically, in photobleaching ICS, the extent of nonlinearity of the apparent cluster density as a function of bleaching is related to the size of clusters. The analysis is tested using computer simulations on model aggregate systems and then applied to an experimental determination of Aß peptide aggregation on nerve cells. The analysis reveals time-dependent increases in Aß1-42 peptide aggregation. Globally, the datasets could be described by a monomer-dimer-tetramer-hexamer or a monomer-dimer-trimer-pentamer model. The results demonstrate the utility of photobleaching with ICS for determining aggregation states on the supramolecular scale in intact cells without the requirement for a brightness standard.

Suppression of amyloid beta A11 antibody immunoreactivity by vitamin C: possible role of heparan sulfate oligosaccharides derived from glypican-1 by ascorbate-induced, nitric oxide (NO)-catalyzed degradation.

Amyloid ß (Aß) is generated from the copper- and heparan sulfate (HS)-binding amyloid precursor protein (APP) by proteolytic processing. APP supports S-nitrosylation of the HS proteoglycan glypican-1 (Gpc-1). In the presence of ascorbate, there is NO-catalyzed release of anhydromannose (anMan)-containing oligosaccharides from Gpc-1-nitrosothiol. We investigated whether these oligosaccharides interact with Aß during APP processing and plaque formation. anMan immunoreactivity was detected in amyloid plaques of Alzheimer (AD) and APP transgenic (Tg2576) mouse brains by immunofluorescence microscopy. APP/APP degradation products detected by antibodies to the C terminus of APP, but not Aß oligomers detected by the anti-Aß A11 antibody, colocalized with anMan immunoreactivity in Tg2576 fibroblasts. A 50-55-kDa anionic, sodium dodecyl sulfate-stable, anMan- and Aß-immunoreactive species was obtained from Tg2576 fibroblasts using immunoprecipitation with anti-APP (C terminus). anMan-containing HS oligo- and disaccharide preparations modulated or suppressed A11 immunoreactivity and oligomerization of Aß42 peptide in an in vitro assay. A11 immunoreactivity increased in Tg2576 fibroblasts when Gpc-1 autoprocessing was inhibited by 3-ß[2(diethylamino)ethoxy]androst-5-en-17-one (U18666A) and decreased when Gpc-1 autoprocessing was stimulated by ascorbate. Neither overexpression of Gpc-1 in Tg2576 fibroblasts nor addition of copper ion and NO donor to hippocampal slices from 3xTg-AD mice affected A11 immunoreactivity levels. However, A11 immunoreactivity was greatly suppressed by the subsequent addition of ascorbate. We speculate that temporary interaction between the Aß domain and small, anMan-containing oligosaccharides may preclude formation of toxic Aß oligomers. A portion of the oligosaccharides are co-secreted with the Aß peptides and deposited in plaques. These results support the notion that an inadequate supply of vitamin C could contribute to late onset AD in humans.

Alleviating transcriptional inhibition of the norepinephrine slc6a2 transporter gene in depolarized neurons.

Recent studies have brought to light additional experimental information, namely, that the MeCP2 protein complex is not only capable of associating with members of the ATPase-dependent bromodomain family, but also found on nonmethylated genomic sequences. These unexpected results are indicative of a multifunctional role for MeCP2, more importantly; our view of the molecular mechanisms that regulate gene activity may not be necessarily distinguishable. Depolarized mouse neuronal cortical cells were examined for increased Slc6a2 mRNA synthesis, changes in CpG methylation status using bisulfite sequencing, and binding of MeCP2 and Smarca2 on the Slc6a2 promoter sequence by chromatin immunopurification strategies. Increased Slc6a2 gene expression in response to membrane depolarization was strongly correlated with the dissociation of MeCP2 and Smarca2 complex on the unmethylated gene. We identified that gene expression in neuronal cortical cells involves increased histone hyperacetylation on the Slc6a2 promoter, which is commensurate with the recruitment of SP1 and RNA Polymerase II and is inversely correlated with H3K9 trimethylation. We hypothesize that the MeCP2 corepressor is capable of associating with multiple forms of SWI/SNF to remodel chromatin for important regulatory roles. The results of our experiments indicate that these proteins are asymmetrically bound to chromatin independent of DNA methylation and not inevitably diametrically opposed. These results now begin to offer a new perspective on the mechanism of Slc6a2 gene regulation.

Oligomerization and toxicity of Aß fusion proteins.

This study has found that the Maltose binding protein Aß42 fusion protein (MBP-Aß42) forms soluble oligomers while the shorter MBP-Aß16 fusion and control MBP did not. MBP-Aß42, but neither MBP-Aß16 nor control MBP, was toxic in a dose-dependent manner in both yeast and primary cortical neuronal cells. This study demonstrates the potential utility of MBP-Aß42 as a reagent for drug screening assays in yeast and neuronal cell cultures and as a candidate for further Aß42 characterization.

APP involvement in retinogenesis of mice.

Very few studies have examined expression and function of amyloid precursor protein (APP) in the retina. We showed that APP mRNA and protein are expressed according to the different waves of retinal differentiation. Depletion of App led to an absence of amacrine cells, a 50% increase in the number of horizontal cells and alteration of the synapses. The retinas of adult APP(-/-) mice showed only half as many glycinergic amacrine cells as wild-type retinas. We identified Ptf1a, which plays a role in controlling both amacrine and horizontal cell fates, as a downstream effector of APP. The observation of a similar phenotype in sorLA knockout mice, a major regulator of APP processing, suggests that regulation of APP functions via sorLA controls the determination of amacrine and horizontal cell fate. These findings provide novel insights that indicate that APP plays an important role in retinal differentiation.

Platinum-based inhibitors of amyloid-beta as therapeutic agents for Alzheimer's disease.

Amelyoid-beta peptide (Abeta) is a major causative agent responsible for Alzheimer's disease (AD). Abeta contains a high affinity metal binding site that modulates peptide aggregation and toxicity. Therefore, identifying molecules targeting this site represents a valid therapeutic strategy. To test this hypothesis, a range of L-PtCl(2) (L = 1,10-phenanthroline derivatives) complexes were examined and shown to bind to Abeta, inhibit neurotoxicity and rescue Abeta-induced synaptotoxicity in mouse hippocampal slices. Coordination of the complexes to Abeta altered the chemical properties of the peptide inhibiting amyloid formation and the generation of reactive oxygen species. In comparison, the classic anticancer drug cisplatin did not affect any of the biochemical and cellular effects of Abeta. This implies that the planar aromatic 1,10-phenanthroline ligands L confer some specificity for Abeta onto the platinum complexes. The potent effect of the L-PtCl(2) complexes identifies this class of compounds as therapeutic agents for AD.

The Alzheimer's Disease Amyloid Precursor Protein and its Neuritogenic Actions.

The Amyloid Precursor Protein (APP) is principally known and studied for its involvement in Alzheimer's disease as the source of the amyloid ß peptide; however, its physiological actions within the nervous system are also important as it is involved in a range of neuronal activities, including neurogenesis, synaptic plasticity, neurite outgrowth, and neuroprotection. Of the different neuronal functions that APP can affect, some may be relevant to APP's role in Alzheimer's disease, while others can be primarily related to its physiological roles. This review will focus on APP's neuritogenic actions and surmise the key molecular mechanisms, as well as the structural and signaling requirements, which form the basis for APP's neuritogenic effects. Deciphering the normal function(s) of APP is valuable to properly understanding its role in health as well as Alzheimer's disease.

A domain level interaction network of amyloid precursor protein and Abeta of Alzheimer's disease.

The primary constituent of the amyloid plaque, beta-amyloid (Abeta), is thought to be the causal "toxic moiety" of Alzheimer's disease. However, despite much work focused on both Abeta and its parent protein, amyloid precursor protein (APP), the functional roles of APP and its cleavage products remain to be fully elucidated. Protein-protein interaction networks can provide insight into protein function, however, high-throughput data often report false positives and are in frequent disagreement with low-throughput experiments. Moreover, the complexity of the CNS is likely to be under represented in such databases. Therefore, we curated the published work characterizing both APP and Abeta to create a protein interaction network of APP and its proteolytic cleavage products, with annotation, where possible, to the level of APP binding domain and isoform. This is the first time that an interactome has been refined to domain level, essential for the interpretation of APP due to the presence of multiple isoforms and processed fragments. Gene ontology and network analysis were used to identify potentially novel functional relationships among interacting proteins.

Genetic Modulators of Traumatic Brain Injury in Animal Models and the Impact of Sex-Dependent Effects.

Traumatic brain injury (TBI) is a major health problem causing disability and death worldwide. There is no effective treatment, due in part to the complexity of the injury pathology and factors affecting its outcome. The extent of brain injury depends on the type of insult, age, sex, lifestyle, genetic risk factors, socioeconomic status, other co-injuries, and underlying health problems. This review discusses the genes that have been directly tested in TBI models, and whether their effects are known to be sex-dependent. Sex differences can affect the incidence, symptom onset, pathology, and clinical outcomes following injury. Adult males are more susceptible at the acute phase and females show greater injury in the chronic phase. TBI is not restricted to a single sex; despite variations in the degree of symptom onset and severity, it is important to consider both female and male animals in TBI pre-clinical research studies.

Amyloid-beta peptide (Abeta) neurotoxicity is modulated by the rate of peptide aggregation: Abeta dimers and trimers correlate with neurotoxicity.

Alzheimer's disease is an age-related neurodegenerative disorder with its toxicity linked to the generation of amyloid-beta peptide (Abeta). Within the Abeta sequence, there is a systemic repeat of a GxxxG motif, which theoretical studies have suggested may be involved in both peptide aggregation and membrane perturbation, processes that have been implicated in Abeta toxicity. We synthesized modified Abeta peptides, substituting glycine for leucine residues within the GxxxG repeat motif (GSL peptides). These GSL peptides undergo beta-sheet and fibril formation at an increased rate compared with wild-type Abeta. The accelerated rate of amyloid fibril formation resulted in a decrease in the presence of small soluble oligomers such as dimeric and trimeric forms of Abeta in solution, as detected by mass spectrometry. This reduction in the presence of small soluble oligomers resulted in reduced binding to lipid membranes and attenuated toxicity for the GSL peptides. The potential role that dimer and trimer species binding to lipid plays in Abeta toxicity was further highlighted when it was observed that annexin V, a protein that inhibits Abeta toxicity, specifically inhibited Abeta dimers from binding to lipid membranes.