[Show abstract][Hide abstract] ABSTRACT: Activated microglia/macrophages significantly contribute to the secondary inflammatory damage in ischemic stroke. Cultured neonatal microglia express the K+ channels Kv1.3 and KCa3.1, both of which have been reported to be involved in microglia-mediated neuronal killing, oxidative burst and cytokine production. However, it is questionable whether neonatal cultures accurately reflect the K+ channel expression of activated microglia in the adult brain. We here subjected mice to middle cerebral artery occlusion with eight days of reperfusion and patch-clamped acutely isolated microglia/macrophages. Microglia from the infarcted area exhibited higher densities of K+ currents with the biophysical and pharmacological properties of Kv1.3, KCa3.1 and Kir2.1 than microglia from non-infarcted control brains. Similarly, immunohistochemistry on human infarcts showed strong Kv1.3 and KCa3.1 immunoreactivity on activated microglia/macrophages. We next investigated the effect of genetic deletion and pharmacological blockade of KCa3.1 in reversible middle cerebral artery occlusion. KCa3.1−/− mice and wild-type mice treated with the KCa3.1 blocker TRAM-34 exhibited significantly smaller infarct areas on day-8 after middle cerebral artery occlusion and improved neurological deficit. Both manipulations reduced microglia/macrophage activation and brain cytokine levels. Our findings suggest KCa3.1 as a pharmacological target for ischemic stroke. Of potential, clinical relevance is that KCa3.1 blockade is still effective when initiated 12 h after the insult.
Preview · Article · Nov 2015 · Journal of Cerebral Blood Flow & Metabolism
[Show abstract][Hide abstract] ABSTRACT: Amyloid-β (Aβ) protein causes neurotoxicity and its abnormal aggregation into amyloid is a pathological hallmark of Alzheimer's disease (AD). Cellular proteins able to interact with Aβ or its precursor, AβPP (amyloid-β protein precursor), may regulate Aβ production and neurotoxicity. We identified a brain-enriched type I transmembrane protein, tomoregulin (TR), that directly binds Aβ and Aβ oligomers (AβO). TR co-immunoprecipitated with Aβ and AβO in cultured cells and co-localized with amyloid plaques and intraneuronal Aβ in the 5xFAD AD mouse model. TR was also enriched in astrocytic processes reactive to amyloid plaques. Surface plasmon resonance spectroscopy studies showed that the extracellular domain of TR binds to AβO with a high affinity (KD = 76.8 nM). Electron paramagnetic resonance spectroscopy also demonstrated a physical interaction between spin-labeled Aβ and the TR extracellular domain in solution. Furthermore, TR also interacted with AβPP and enhanced its cleavage by α-secretase. Both cellular expression of TR and application of recombinant TR extracellular domain protected N2a neurons from AβO-induced neuronal death. These data provide first evidence that neuronal and astrocytic expression of TR is intimately related to Aβ metabolism and toxicity, and could be neuroprotective through its direct interaction with Aβ and AβPP.
[Show abstract][Hide abstract] ABSTRACT: Alzheimer's disease is characterized by the presence of extracellular plaques comprised of amyloid beta (Aβ) peptides. Soluble oligomers of the Aβ peptide underlie a cascade of neuronal loss and dysfunction associated with Alzheimer's disease. Single particle analyses of Aβ oligomers in solution by fluorescence correlation spectroscopy (FCS) were used to provide real-time descriptions of how spin-labeled fluorenes (SLFs; bi-functional small molecules that block the toxicity of Aβ) prevent and disrupt oligomeric assemblies of Aβ in solution. Furthermore, the circular dichroism (CD) spectrum of untreated Aβ shows a continuous, progressive change over a 24-hour period, while the spectrum of Aβ treated with SLF remains relatively constant following initial incubation. These findings suggest the conformation of Aβ within the oligomer provides a complementary determinant of Aβ toxicity in addition to oligomer growth and size. Although SLF does not produce a dominant state of secondary structure in Aβ, it does induce a net reduction in beta secondary content compared to untreated samples of Aβ. The FCS results, combined with electron paramagnetic resonance spectroscopy and CD spectroscopy, demonstrate SLFs can inhibit the growth of Aβ oligomers and disrupt existing oligomers, while retaining Aβ as a population of smaller, yet largely disordered oligomers.
No preview · Article · Sep 2015 · Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics
[Show abstract][Hide abstract] ABSTRACT: Development of therapeutic strategies to prevent Alzheimer's disease (AD) is of great importance. We show that mild inhibition of mitochondrial complex I with small molecule CP2 reduces levels of amyloid beta and phospho-Tau and averts cognitive decline in three animal models of familial AD. Low-mass molecular dynamics simulations and biochemical studies confirmed that CP2 competes with flavin mononucleotide for binding to the redox center of complex I leading to elevated AMP/ATP ratio and activation of AMP-activated protein kinase in neurons and mouse brain without inducing oxidative damage or inflammation. Furthermore, modulation of complex I activity augmented mitochondrial bioenergetics increasing coupling efficiency of respiratory chain and neuronal resistance to stress. Concomitant reduction of glycogen synthase kinase 3β activity and restoration of axonal trafficking resulted in elevated levels of neurotrophic factors and synaptic proteins in adult AD mice. Our results suggest that metabolic reprogramming induced by modulation of mitochondrial complex I activity represents promising therapeutic strategy for AD.
[Show abstract][Hide abstract] ABSTRACT: Various condensation and ring-closing reactions were used for the syntheses of 3-[(alkylamino)methylene]-6-methylpyri-dine-2,4(1H,3H)-diones, bicyclic pyridinones, and tricyclic morpholinopyrones. For instance, 3-[(dialkylamino)methylene]-6-methylpyridine-2,4(1H,3H)-diones were synthesized from the condensation of dialkylamines and 3-formyl-4-hydroxy-6-methylpyridin-2(1H)-one. 3-Formyl-4-hydroxy-6-methylpyridin-2(1H)-one, derived from 3-formyl-4-hydroxy-6-methylpyridin-2(1H)-one, was used to construct a number of bicyclic pyridinones via a one-pot Knoevenagal and intramolecular lactonization reaction. Tricyclic morpholinopyrones were assembled from a dialkylation reaction involving a dinucleophile, 3-amino-4-hydroxy-6-methyl-2H-pyran-2-one, and a dielectrophile, trans-3,6-dibromocyclohexene. Depending on the reaction conditions, isomers of the tricyclic molecules can be selectively produced, and their chemical structures were unequivocally determined using single-crystal X-ray analyses and 2D COSY spectroscopy. The fluorescently active bicyclic pyridinone compounds show longer absorption (368-430 nm; maximum) and emission wavelengths (450-467 nm) than those of 7-amino-4-methylcoumarin (AMC; λabs,max = 350 nm; λem = 430 nm) suggesting these molecules, such as 3-(2-aminoacetyl)-7-methyl-2H-pyrano[3,2-c]pyridine-2,5(6H)-dione, can be employed as fluorescence activity based probes for tracing biological pathways.
[Show abstract][Hide abstract] ABSTRACT: A new series of fifteen 5-, 6-, and 8-appended 4-methylquinolines were synthesized and evaluated for their neural protective activities. Selected compounds were further examined for their inhibition of glycogen synthase kinase-3β (GSK-3β) and protein kinase C (PKC). Two most potent analogs, compounds 3 and 10, show nanomolar protective activities in amyloid β-induced MC65 cells and enzymatic inhibitory activities against GSK-3β, but poor PKC inhibitory activities. Using normal mouse model, the distribution of the most potent analog 3 in various tissues and possible toxic effects in the locomotors and inhibition of liver transaminases activities were carried out. No apparent decline of locomotor activity and no inhibition of liver transaminases were found. The compound appears to be safe for long-term use in Alzheimer's disease mouse model.
[Show abstract][Hide abstract] ABSTRACT: A series of new Tacrine analogs modified with nitroxides or pre-nitroxides on 9-amino group via methylene or piperazine spacers were synthesized; the nitroxide or its precursors were incorporated into the Tacrine scaffold. The new compounds were tested for their hydroxyl radical and peroxyl radical scavenging ability, acetylcholinesterase inhibitor activity and protection against Aβ-induced cytotoxicity. Based on these assays, we conclude that Tacrine analogs connected to five and six-membered nitroxides via piperazine spacers (9b, 9b/HCl and 12) exhibited the best activity, providing direction for further development of additional candidates with dual functionality (anti Alzheimer's and antioxidant).
Full-text · Article · Apr 2014 · European Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: Background
Mutations in MECP2 encoding methyl-CpG-binding protein 2 (MeCP2) cause the X-linked neurodevelopmental disorder Rett syndrome. Rett syndrome patients exhibit neurological symptoms that include irregular breathing, impaired mobility, stereotypic hand movements, and loss of speech. MeCP2 protein epigenetically modulates gene expression through genome-wide binding to methylated CpG dinucleotides. While neurons have the highest level of MeCP2 expression, astrocytes and other cell types also express detectable levels of MeCP2. Recent studies suggest that astrocytes likely control the progression of Rett syndrome. Thus, the object of these studies was to identify gene targets that are affected by loss of MeCP2 binding in astrocytes.
To identify gene targets of MeCP2 in astrocytes, combined approaches of expression microarray and chromatin immunoprecipitation of MeCP2 followed by sequencing (ChIP-seq) were compared between wild-type and MeCP2-deficient astrocytes. MeCP2 gene targets were compared with genes in the top 10% of MeCP2 binding levels in gene windows either within 2 kb upstream of the transcription start site, or the ‘gene body’ that extended from transcription start to end site, or 2 kb downstream of the transcription end site.
A total of 118 gene transcripts surpassed the highly significant threshold (P < 0.005, fold change > 1.2) in expression microarray analysis from triplicate cultures. The top 10% of genes with the highest levels of MeCP2 binding were identified in two independent ChIP-seq experiments. Together this integrated, genome-wide screen for MeCP2 target genes provided an overlapping list of 19 high-confidence MeCP2-responsive gene transcripts in astrocytes. Validation of candidate target gene transcripts by RT-PCR revealed that expression of Apoc2, Cdon, Csrp and Nrep were consistently responsive to MeCP2 deficiency in astrocytes.
The first MeCP2 ChIP-seq and gene expression microarray analysis in astrocytes reveals a set of potential MeCP2 target genes that may contribute to normal astrocyte signaling, cell division and neuronal support functions, the loss of which may contribute to the Rett syndrome phenotype.
[Show abstract][Hide abstract] ABSTRACT: A major effort in Alzheimer's disease therapeutic development has targeted Aβ and downstream events. We have synthesized a small library of tricyclic pyrone compounds. Their protective action in MC65 cells and inhibition of ACAT along with the upregulation of cholesterol transporter gene were investigated. Five active compounds exhibited potencies in the nanomolar ranges. The multiple effects of the compounds on Aβ and cellular cholesterol pathways could be potential mechanisms underlying the protective effects in vivo.
No preview · Article · Oct 2012 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: There exists an urgent need for new target discovery to treat Alzheimer's disease (AD); however, recent clinical trials based on anti-Aβ and anti-inflammatory strategies have yielded disappointing results. To expedite new drug discovery, we propose reposition targets which have been previously pursued by both industry and academia for indications other than AD. One such target is the calcium-activated potassium channel KCa3.1 (KCNN4), which in the brain is primarily expressed in microglia and is significantly upregulated when microglia are activated. We here review the existing evidence supporting that KCa3.1 inhibition could block microglial neurotoxicity without affecting their neuroprotective phagocytosis activity and without being broadly immunosuppressive. The anti-inflammatory and neuroprotective effects of KCa3.1 blockade would be suitable for treating AD as well as cerebrovascular and traumatic brain injuries, two well-known risk factors contributing to the dementia in AD patients presenting with mixed pathologies. Importantly, the pharmacokinetics and pharmacodynamics of several KCa3.1 blockers are well known, and a KCa3.1 blocker has been proven safe in clinical trials. It is therefore promising to reposition old or new KCa3.1 blockers for AD preclinical and clinical trials.
Full-text · Article · May 2012 · International Journal of Alzheimer's Disease
[Show abstract][Hide abstract] ABSTRACT: The deposition and oligomerization of amyloid β (Aβ) peptide plays a key role in the pathogenesis of Alzheimer's disease (AD). Aβ peptide arises from cleavage of the membrane-associated domain of the amyloid precursor protein (APP) by β and γ secretases. Several lines of evidence point to the soluble Aβ oligomer (AβO) as the primary neurotoxic species in the etiology of AD. Recently, we have demonstrated that a class of fluorene molecules specifically disrupts the AβO species.
To achieve a better understanding of the mechanism of action of this disruptive ability, we extend the application of electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels in the Aβ peptide to investigate the binding and influence of fluorene compounds on AβO structure and dynamics. In addition, we have synthesized a spin-labeled fluorene (SLF) containing a pyrroline nitroxide group that provides both increased cell protection against AβO toxicity and a route to directly observe the binding of the fluorene to the AβO assembly. We also evaluate the ability of fluorenes to target multiple pathological processes involved in the neurodegenerative cascade, such as their ability to block AβO toxicity, scavenge free radicals and diminish the formation of intracellular AβO species.
Fluorene modified with pyrroline nitroxide may be especially useful in counteracting Aβ peptide toxicity, because they possess both antioxidant properties and the ability to disrupt AβO species.
[Show abstract][Hide abstract] ABSTRACT: Autism spectrum disorders (ASDs) including classic autism is a group of complex developmental disabilities with core deficits of impaired social interactions, communication difficulties and repetitive behaviors. Although the neurobiology of ASDs has attracted much attention in the last two decades, the role of microglia has been ignored. Existing data are focused on their recognized role in neuroinflammation, which only covers a small part of the pathological repertoire of microglia. This review highlights recent findings on the broader roles of microglia, including their active surveillance of brain microenvironments and regulation of synaptic connectivity, maturation of brain circuitry and neurogenesis. Emerging evidence suggests that microglia respond to pre- and postnatal environmental stimuli through epigenetic interface to change gene expression, thus acting as effectors of experience-dependent synaptic plasticity. Impairments of these microglial functions could substantially contribute to several major etiological factors of autism, such as environmental toxins and cortical underconnectivity. Our recent study on Rett syndrome, a syndromic autistic disorder, provides an example that intrinsic microglial dysfunction due to genetic and epigenetic aberrations could detrimentally affect the developmental trajectory without evoking neuroinflammation. We propose that ASDs provide excellent opportunities to study the influence of microglia on neurodevelopment, and this knowledge could lead to novel therapies.
[Show abstract][Hide abstract] ABSTRACT: Accumulating evidence indicates that white matter degeneration contributes to the neural disconnections that underlie Alzheimer's disease pathophysiology. Although this white matter degeneration is partly attributable to axonopathy associated with neuronal degeneration, amyloid β (Aβ) protein-mediated damage to oligodendrocytes could be another mechanism. To test this hypothesis, we studied effects of soluble Aβ in oligomeric form on survival and differentiation of cells of the oligodendroglial lineage using highly purified oligodendroglial cultures from rats at different developmental stages. Aβ oligomer at 10 μM or higher reduced survival of mature oligodendrocytes, whereas oligodendroglial progenitor cells (OPCs) were relatively resistant to the Aβ oligomer-mediated cytotoxicity. Further study revealed that Aβ oligomer even at 1 μM accelerated 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) formazan exocytosis in mature oligodendrocytes, and, more significantly, inhibited myelin sheet formation after induction of in vitro differentiation of OPCs. These results imply a novel pathogenetic mechanism underlying Aβ oligomer-mediated white matter degeneration, which could impair myelin maintenance and remyelination by adult OPCs, resulting in accumulating damage to myelinating axons thereby contributing to neural disconnections.
Preview · Article · Mar 2012 · Neurobiology of aging
[Show abstract][Hide abstract] ABSTRACT: Fragile X syndrome (FXS) is the most common inherited form of intellectual disability, and is the most common single-gene disorder known to be associated with autism. Despite recent advances in functional neuroimaging and our understanding of the molecular pathogenesis, only limited neuropathologic information on FXS is available.
Neuropathologic examinations were performed on post-mortem brain tissue from three older men (aged 57, 64 and 78 years) who had received a clinical or genetic diagnosis of FXS. In each case, physical and cognitive features were typical of FXS, and one man was also diagnosed with autism. Guided by reports of clinical and neuroimaging abnormalities of the limbic system and cerebellum of individuals with FXS, the current analysis focused on neuropathologic features present in the hippocampus and the cerebellar vermis.
Histologic and immunologic staining revealed abnormalities in both the hippocampus and cerebellar vermis. Focal thickening of hippocampal CA1 and irregularities in the appearance of the dentate gyrus were identified. All lobules of the cerebellar vermis and the lateral cortex of the posterior lobe of the cerebellum had decreased numbers of Purkinje cells, which were occasionally misplaced, and often lacked proper orientation. There were mild, albeit excessive, undulations of the internal granular cell layer, with patchy foliar white matter axonal and astrocytic abnormalities. Quantitative analysis documented panfoliar atrophy of both the anterior and posterior lobes of the vermis, with preferential atrophy of the posterior lobule (VI to VII) compared with age-matched normal controls.
Significant morphologic changes in the hippocampus and cerebellum in three adult men with FXS were identified. This pattern of pathologic features supports the idea that primary defects in neuronal migration, neurogenesis and aging may underlie the neuropathology reported in FXS.