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α-Synuclein (αSN) aggregation is central to the etiology of Parkinson's disease (PD). Large-scale screening of compounds to identify aggregation inhibitors is challenged by stochastic αSN aggregation and difficulties in detecting early-stage oligomers (αSOs). We developed a high-throughput screening assay combining SDS-stimulated αSN aggregation with FRET to reproducibly detect initial stages in αSN aggregation. We screened 746,000 compounds, leading to 58 hits that markedly inhibit αSN aggregation and reduce αSOs' membrane permeabilization activity. The most effective aggregation inhibitors were derivatives of (4-hydroxynaphthalen-1-yl)sulfonamide. They interacted strongly with the N-terminal part of monomeric αSN and reduced αSO-membrane interactions, possibly by affecting electrostatic interactions. Several compounds reduced αSO toxicity toward neuronal cell lines. The inhibitors introduced chemical modifications of αSN that were, however, not a prerequisite for inhibitory activity. We also identified several phenyl-benzoxazol compounds that promoted αSN aggregation (proaggregators). These compounds may be useful tools to modulate αSN aggregation in cellula.

Amyloid plaques associated with Alzheimer's disease (AD) induce inflammatory responses associated with activated microglia and reactive astrocytes, which exacerbate neurodegeneration through release of inflammatory cytokines, reactive oxygen species, and other factors. Inflammation contributes to neurodegeneration at later stages of AD, but it may also play a role in early disease pathogenesis. We found that before plaque deposition, amyloid precursor protein (APP)/presenilin 1 (PSEN1) transgenic mice (PSAPP mice), a well-characterized model of AD, exhibit evidence of cerebrovascular inflammation. Expression of the endothelial cell-specific antigen MECA-32 (mouse endothelial cell antigen-32) was upregulated in the cerebrovasculature of young PSAPP mice (3 months old) and was similar to that observed in mice with experimental autoimmune encephalomyelitis, a model of multiple sclerosis characterized by neuroinflammation. MECA-32 is normally expressed in central and peripheral vasculature throughout development, but expression in the cerebrovasculature is downregulated on establishment of the blood-brain barrier (BBB). However, CNS inflammation triggers re-expression of MECA-32 in compromised cerebrovasculature. Our study indicates that MECA-32 may be a robust marker of cerebrovascular inflammation and compromised BBB integrity, triggered by soluble amyloid-β early in disease pathogenesis.

Huntington’s disease (HD) is a late-onset, neurodegenerative disease for which there are currently no cures nor disease-modifying treatments. Here we report the identification of several potential anti-inflammatory targets for HD using an ex vivo model of HD that involves the acute transfection of human mutant huntingtin-based constructs into rat brain slices. This model recapitulates key components of the human disease, including the formation of intracellular huntingtin protein (HTT)-containing inclusions and the progressive neurodegeneration of striatal neurons—both occurring within the native tissue context of these neurons. Using this "high-throughput biology" screening platform, we conducted a hypothesis-neutral screen of a collection of drug-like compounds which identified several anti-inflammatory targets that provided neuroprotection against HTT fragment-induced neurodegeneration. The nature of these targets provide further support for non-cell autonomous mechanisms mediating significant aspects of neuropathogenesis induced by mutant HTT fragment proteins.

The identification of small molecule aminohydantoins as potent and selective human β-secretase inhibitors is reported. These analogs exhibit good brain permeability (40-70%), low nanomolar potency for BACE1, and demonstrate >100-fold selectivity for the structurally related aspartyl proteases cathepsin D, renin and pepsin. Alkyl and alkoxy groups at the meta-position of the P1 phenyl, which extend toward the S3 region of the enzyme, have contributed to the ligand's reduced affinity for the efflux transporter protein P-gp, and decreased topological polar surface area, thus resulting in enhanced brain permeability. A fluorine substitution at the para-position of the P1 phenyl has contributed to 100-fold decrease of CYP3A4 inhibition and enhancement of compound metabolic stability. The plasma and brain protein binding properties of these new analogs are affected by substitutions at the P1 phenyl moiety. Higher compound protein binding was observed in the brain than in the plasma. Two structurally diverse potent BACE1 inhibitors (84 and 89) reduced 30% plasma Aβ40 in the Tg2576 mice in vivo model at 30 mg/kg p.o..

8,8-Diphenyl-2,3,4,8-tetrahydroimidazo[1,5-a]pyrimidin-6-amine (1) was identified through HTS, as a weak (micromolar) inhibitor of BACE1. X-Ray crystallographic studies indicate the 2-aminoimidazole ring forms key H-bonding interactions with Asp32 and Asp228 in the catalytic site of BACE1. Lead optimization using structure-based focused libraries led to the identification of low nanomolar BACE1 inhibitors such as 20b with substituents which extend from the S1 to the S3 pocket.

Inheritance of the apoE4 allele (epsilon4) increases the risk of developing Alzheimer's disease; however, the mechanisms underlying this association remain elusive. Recent data suggest that inheritance of epsilon4 may lead to reduced apoE protein levels in the CNS. We therefore examined apoE protein levels in the brains, CSF and plasma of epsilon2/2, epsilon3/3, and epsilon4/4 targeted replacement mice. These apoE mice showed a genotype-dependent decrease in apoE levels; epsilon2/2 >epsilon3/3 >epsilon4/4. Next, we sought to examine the relative contributions of apoE4 and apoE3 in the epsilon3/4 mouse brains. ApoE4 represented 30-40% of the total apoE. Moreover, the absolute amount of apoE3 per allele was similar between epsilon3/3 and epsilon3/4 mice, implying that the reduced levels of total apoE in epsilon3/4 mice can be explained by the reduction in apoE4 levels. In culture medium from epsilon3/4 human astrocytoma or epsilon3/3, epsilon4/4 and epsilon3/4 primary astrocytes, apoE4 levels were consistently lower than apoE3. Secreted cholesterol levels were also lower from epsilon4/4 astrocytes. Pulse-chase experiments showed an enhanced degradation and reduced half-life of newly synthesized apoE4 compared with apoE3. Together, these data suggest that astrocytes preferentially degrade apoE4, leading to reduced apoE4 secretion and ultimately to reduced brain apoE levels. Moreover, the genotype-dependent decrease in CNS apoE levels, mirror the relative risk of developing AD, and suggest that low levels of total apoE exhibited by epsilon4 carriers may directly contribute to the disease progression, perhaps by reducing the capacity of apoE to promote synaptic repair and/or Abeta clearance.

Estrogens have long been implicated in influencing cognitive processes, yet the molecular mechanisms underlying these effects and the roles of the estrogen receptors alpha (ERalpha) and beta (ERbeta) remain unclear. Using pharmacological, biochemical and behavioral techniques, we demonstrate that the effects of estrogen on hippocampal synaptic plasticity and memory are mediated through ERbeta. Selective ERbeta agonists increased key synaptic proteins in vivo, including PSD-95, synaptophysin and the AMPA-receptor subunit GluR1. These effects were absent in ERbeta knockout mice. In hippocampal slices, ERbeta activation enhanced long-term potentiation, an effect that was absent in slices from ERbeta knockout mice. ERbeta activation induced morphological changes in hippocampal neurons in vivo, including increased dendritic branching and increased density of mushroom-type spines. An ERbeta agonist, but not an ERalpha agonist, also improved performance in hippocampus-dependent memory tasks. Our data suggest that activation of ERbeta can regulate hippocampal synaptic plasticity and improve hippocampus-dependent cognition.

Rapamycin is an immunosuppressive immunophilin ligand reported as having neurotrophic activity. We show that modification of rapamycin at the mammalian target of rapamycin (mTOR) binding region yields immunophilin ligands, WYE-592 and ILS-920, with potent neurotrophic activities in cortical neuronal cultures, efficacy in a rodent model for ischemic stroke, and significantly reduced immunosuppressive activity. Surprisingly, both compounds showed higher binding selectivity for FKBP52 versus FKBP12, in contrast to previously reported immunophilin ligands. Affinity purification revealed two key binding proteins, the immunophilin FKBP52 and the β1-subunit of L-type voltage-dependent Ca 2+ channels (CACNB1). Electrophysiological analysis indicated that both compounds can inhibit L-type Ca 2+ channels in rat hippocampal neurons and F-11 dorsal root ganglia (DRG)/neuroblastoma cells. We propose that these immunophilin ligands can protect neurons from Ca 2+ -induced cell death by modulating Ca 2+ channels and promote neurite outgrowth via FKBP52 binding.

Activation of glycogen synthase kinase 3beta (Gsk3beta) has been shown to be a key component in signaling pathways that underlie neurodegeneration and neurodegenerative disease. Conversely, inactivation of Gsk3beta by phosphoinositide 3-kinase (PI3K)/Akt is an important neuroprotective mechanism. Previous studies have shown that agonist activation of group I metabotropic glutamate receptors (mGluRs) can increase neuronal survival and prevent apoptosis. However, little is known about the signaling pathways that couple mGluR5 to neuroprotection. In this report, we investigated whether activation of the PI3K/Akt/Gsk3beta pathway, which has been shown to have an important neuroprotective mechanism, is required for mGluR5 activation mediated neuroprotection against beta-amyloid. We found that brief incubations of mouse hippocampal slices with (R,S)-3,5-dihydroxyphenylglycine (DHPG) resulted in increased phosphorylation of Akt and Gsk3beta. The PI3K inhibitors, LY294002 and wortmannin, blocked the DHPG-induced increased phosphorylation of Akt and Gsk3beta. Similar results were observed in rat primary hippocampal cultures. Finally, we found that the PI3K inhibitor LY294002 can block (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) mediated neuroprotection against beta-amyloid. Thus, these findings suggest that mGluR5 can modulate the PI3K/Akt/Gsk3beta pathway in the hippocampus, and that modulation of this signaling pathway can reverse beta-amyloid-induced neuronal toxicity.

Summary: Alzheimer’s disease is a progressive neurodegenerative disorder and the leading cause of dementia in the Western world. Postmortem, it is characterized neuropathologically by the presence of amyloid plaques, neurofibrillary tangles, and a profound gray matter loss. Neurofibrillary tangles are composed of an abnormally hyperphosphorylated intracellular protein called tau, tightly wound into paired helical filaments and thought to impact microtubule assembly and protein trafficking, resulting in the eventual demise of neuronal viability. The extracellular amyloid plaque deposits are composed of a proteinacious core of insoluble aggregated amyloid-β (Aβ) peptide and have led to the foundation of the amyloid hypothesis. This hypothesis postulates that Aβ is one of the principal causative factors of neuronal death in the brains of Alzheimer’s patients. With multiple drugs now moving through clinical development for the treatment of Alzheimer’s disease, we will review current and future treatment strategies aimed at improving both the cognitive deficits associated with the disease, as well as more novel approaches that may potentially slow or halt the deadly neurodegenerative progression of the disease.

AD (Alzheimer's disease) is characterized neuropathologically by the presence of amyloid plaques, neurofibrillary tangles and profound grey matter loss. The ‘amyloid’ hypothesis postulates that the toxic Aβ (amyloid β) peptide, enzymatically derived from the proteolytic processing of a larger protein called APP (amyloid precursor protein), is one of the principal causative factors of neuronal cell death in the brains of AD patients. As such, methods for lowering Aβ levels in the brain are of significant interest with regard to identifying novel disease modifying therapies for the treatment of AD. In this review, we will review a variety of approaches and mechanisms capable of modulating levels of Aβ.

Transient receptor potential (TRP) genes encode a family of related ion-channel subunits. This family consists of cation-selective, calcium-permeable channels that include a group of vanilloid receptor channels (TRPV) implicated in pain and inflammation. These channels are activated by diverse stimuli, including capsaicin, lipids, membrane deformation, heat, and protons. Six members of the TRPV family have been identified that differ predominantly in their activation properties. However, in neurons, TRPV channels do not account for the observed diversity of responses to activators. By probing human and rat brain cDNA libraries to identify TRPV subunits, we identified a novel human TRPV1 RNA splice variant, TRPV1b, which forms functional ion channels that are activated by temperature (threshold, approximately 47 degrees C), but not by capsaicin or protons. Channels with similar activation properties were found in trigeminal ganglion neurons, suggesting that TRPV1b receptors are expressed in these cells and contribute to thermal nociception.

Transcriptional dysregulation by mutant huntingtin (Htt) protein has been implicated in the pathogenesis of Huntington's disease (HD). We find that cultured cells expressing mutant Htt and striatal cells from HD transgenic mice have elevated nuclear factor-kappaB (NF-kappaB) activity. Furthermore, NF-kappaB is concentrated in the nucleus of neurons in the brains of HD transgenic mice. In inducible PC12 cells and in HD transgenic mice, mutant Htt activates the IkappaB kinase complex (IKK), a key regulator of NF-kappaB. Activation of IKK is likely mediated by direct interaction with mutant Htt, because the expanded polyglutamine stretch and adjacent proline-rich motifs in mutant Htt interact with IKKgamma, a regulatory subunit of IKK. Activation of IKK may also influence the toxicity of mutant Htt, because expression of IKKgamma promotes aggregation and nuclear localization of mutant Htt exon-1. Moreover, in acute striatal slice cultures, inhibition of IKK activity with an N-terminally truncated form of IKKgamma blocks mutant Htt-induced toxicity in medium-sized spiny neurons (MSNs). In addition, blocking degradation of NF-kappaB inhibitors with a dominant-negative ubiquitin ligase beta-transducin repeat-containing protein also reduces the toxicity of mutant Htt in MSNs. Therefore, aberrant NF-kappaB activation may contribute to the neurodegeneration induced by mutant Htt.

Intermediate-conductance (IK) Ca(2+)-activated K(+) channels are expressed in many different cell types where they perform a variety of functions including cell volume regulation, transepithelial secretion, lymphocyte activation and cell cycle progression. IK channels are thought to be regulated by phosphorylation; however, whether kinases act directly on the channel is unclear. Using IK channels heterologously expressed in Xenopus oocytes, we demonstrate that IK channels are potently inhibited (60%) by the catalytic subunit of protein kinase A (PKA). Inhibition of IK channel current by PKA is abolished by mutation of four phosphorylation residues (S312, T327, S332, and T348) in the putative calmodulin-binding region of the channel. Evidence for direct modulation of the IK channel by PKA was further demonstrated using GST fusion proteins. The major site of phosphorylation was found to be serine 332; however, other residues were also phosphorylated. We conclude that IK channels can be directly regulated by the cAMP second-messenger system. The mechanism appears to involve direct phosphorylation by PKA of a modulatory locus in the cytoplasmic region of the channel, the site at which calmodulin is thought to interact. Modulation of IK channels by protein kinases may be an important mechanism regulating cell function.

The cerebellum is important for many aspects of behaviour, from posture maintenance and goal-oriented reaching movements to timing tasks and certain forms of learning. In every case, information flowing through the cerebellum passes through Purkinje neurons, which receive input from the two primary cerebellar afferents and generate continuous streams of action potentials that constitute the sole output from the cerebellar cortex to the deep nuclei. The tonic firing behaviour observed in Purkinje neurons in vivo is maintained in brain slices even when synaptic inputs are blocked, suggesting that Purkinje neuron activity relies to a significant extent on intrinsic conductances. Previous research has suggested that the interplay between Ca2+ currents and Ca2+-activated K+ channels (KCa channels) is important for Purkinje cell activity, but how many different KCa channel types are present and what each channel type contributes to cell behaviour remains unclear. In order to better understand the ionic mechanisms that control the behaviour of these neurons, we investigated the effects of different Ca2+ channel and KCa channel antagonists on Purkinje neurons in acute slices of rat cerebellum. Our data show that Ca2+ entering through P-type voltage-gated Ca2+ channels activates both small-conductance (SK) and large-conductance (BK) KCa channels. SK channels play a role in setting the intrinsic firing frequency, while BK channels regulate action potential shape and may contribute to the unique climbing fibre response.

Voltage-dependent large-conductance Ca 2+ -activated K + channels (BK channels) are widely expressed in excitable and nonexcitable cells. BK channels exhibit diverse electrophysiological properties, which are attributable in part to alternative splicing of their α-subunits. BK currents have been implicated in the growth control of glial cells, and BK channels with novel biophysical properties have recently been characterized in human glioma cells. Here we report the isolation, cloning, and functional characterization of glioma BK (gBK), a novel splice isoform of hSlo , the gene that encodes the α-subunits of human BK channels. The primary sequence of gBK is 97% identical to its closest homolog hbr5, but it contains an additional 34-amino-acid exon at splice site 2 in the C-terminal tail of BK channels. hSlo transcripts containing this novel exon are expressed ubiquitously in various normal tissues as well as in neoplasmic samples, suggesting that the novel exon may modulate important physiological functions of BK channels. Expression of gBK in Xenopus oocytes gives rise to iberiotoxin-sensitive (IbTX) currents, with an IC 50 for IbTX of 5.7 nm and a Hill coefficient of 0.76. Single gBK channels have a unitary conductance of ∼250 pS, and the currents show significantly slower activation and higher Ca 2+ sensitivity than hbr5. Ca 2+ sensitivity was enhanced specifically at physiologically relevant [Ca 2+ ] i (100–500 nm). Examination of biopsies from patients with malignant gliomas has revealed specific overexpression of BK channels in gliomas compared with nonmalignant human cortical tissues. Importantly, tumor malignancy grades have correlated positively with BK channel expression, suggesting an important role for the gBK channel in glioma biology.

More than 50 genes are known to encode K + channel monomers and can coassemble to form hetero-tetrameric K + channels. However, only a subset of possible monomer combinations come together to form functional ion channels. The assembly and tetramerization of appropriate channel monomers is mediated by association domains (ADs). To identify such domains in human large-conductance Ca 2+ -activated K + channels (hSlo1), we screened hSlo1 domains for self-association using yeast two-hybrid assays. Putative ADs were subjected to functional assays in Xenopus oocytes and further characterized by coprecipitation, native gel electrophoresis, and sucrose density gradient centrifugation assays. This led to the identification of a single intracellular association domain localized near the channel pore and required for channel function. We conclude that this novel tetramerization domain, referred to as BK-T1, promotes the assembly of hSlo1 monomers into functional K Ca channels.

The modulation of ion channel proteins by cellular redox potential has emerged recently as a significant determinant of channel function. We have investigated the influence of sulfhydryl redox reagents on human brain Ca2+-activated K+channels (hslo) expressed in both human embryonic kidney 293 cells andXenopusoocytes using macropatch and single-channel analysis. Intracellular application of the reducing agent dithiothreitol (DTT): (1) shifts the voltage of half-maximal channel activation (V0.5) ≈18 mV to more negative potentials without affecting the maximal conductance or the slope of the voltage dependence; (2) slows by ≈10-fold a time-dependent right-shift inV0.5values (“run-down”); (3) speeds macroscopic current activation kinetics by ≈33%; and (4) increases the single-channel open probability without affecting the unitary conductance. In contrast to DTT treatment, oxidation with hydrogen peroxide shifts macropatchV0.5values to more positive potentials, increases the rate of channel run-down, and decreases the single-channel open probability. KCachannels cloned fromDrosophiladiffer fromhslochannels in that they show very little run-down and are not modulated by the addition of DTT. These data indicate thathsloCa2+-activated K+channels may be modulated by changes in the cellular redox potential as well as by the transmembrane voltage and the cytoplasmic Ca2+concentration.

Iberiotoxin (IbTX), a selective peptidyl ligand for high-conductance Ca2(+)-activated K+ (maxi-K) channels cannot be radioiodinated in biologically active form due to the importance of Y36 in interacting with the channel pore. Therefore, an IbTX double mutant (IbTX-D19Y/Y36F) was engineered, expressed in Escherichia coli, purified to homogeneity, and radiolabeled to high specific activity with 125I. IbTX-D19Y/Y36F and [127I]IbTX-D19Y/Y36F block maxi-K channels expressed in Xenopus laevis oocytes with equal potency as wild-type IbTX (Kd approximately 1 nM). Under low ionic strength conditions, [125I]IbTX-D19Y/Y36F binds with high affinity to smooth muscle sarcolemmal maxi-K channels (Kd of 5 pM as determined by either equilibrium binding or kinetic binding analysis), and with a binding site density of 0.45 pmol/mg of protein. Competition studies with wild-type IbTX, IbTX-D19Y/Y36F or charybdotoxin (ChTX) result in complete inhibition of binding whereas toxins selective for voltage-gated K+ channels (margatoxin (MgTX) or alpha-dendrotoxin (alpha-DaTX) do not have any effect on IbTX binding. Indole diterpene alkaloids, which are selective inhibitors of maxi-K channels, and potassium ions both modulate [125I]IbTX-D19Y/Y36F binding in a complex manner. This pattern is also reflected during covalent incorporation of the radiolabeled toxin into the 31 kDa beta-subunit of the maxi-K channel in the presence of a bifunctional cross-linking reagent. In rat brain membranes, IbTX-D19Y/Y36F does not displace binding of [125I]MgTX or [125I]-alpha-DaTX to sites associated with voltage-gated K+ channels, nor do these latter toxins inhibit [125I]IbTX-D19Y/Y36F binding. Taken together, these results demonstrate that [125I]IbTX-D19Y/Y36F is the first selective radioligand for maxi-K channels with high specific activity.

BACE1 is the rate-limiting enzyme that cleaves amyloid precursor protein (APP) to produce the amyloid β peptides that accumulate in Alzheimer's disease (AD). BACE1, which is elevated in AD patients and APP transgenic mice, also cleaves the β2-subunit of voltage-gated sodium channels (Navβ2). Although increased BACE1 levels are associated with Navβ2 cleavage in AD patients, whether Navβ2 cleavage occurs in APP mice had not yet been examined. Such a finding would be of interest because of its potential impact on neuronal activity: previous studies demonstrated that BACE1-overexpressing mice exhibit excessive cleavage of Navβ2 and reduced sodium current density, but the phenotype associated with loss of function mutations in either Navβ-subunits or pore-forming α-subunits is epilepsy. Because mounting evidence suggests that epileptiform activity may play an important role in the development of AD-related cognitive deficits, we examined whether enhanced cleavage of Navβ2 occurs in APP transgenic mice, and whether it is associated with aberrant neuronal activity and cognitive deficits. We found increased levels of BACE1 expression and Navβ2 cleavage fragments in cortical lysates from APP transgenic mice, as well as associated alterations in Nav1.1α expression and localization. Both pyramidal neurons and inhibitory interneurons exhibited evidence of increased Navβ2 cleavage. Moreover, the magnitude of alterations in sodium channel subunits was associated with aberrant EEG activity and impairments in the Morris water maze. Together, these results suggest that altered processing of voltage-gated sodium channels may contribute to aberrant neuronal activity and cognitive deficits in AD.

Xenopus laevis oocytes have become a pre-eminent tool for studying cloned ion channels, primarily because they intrinsically express low levels of most types of ion channels. However, when these cells are used for single channel studies, it is essential to determine whether or not oocytes contain even low levels of endogenous ion channels with properties similar to the channel being investigated. We show here that X. laevis oocytes express endogenous large-conductance Ca2(+)-activated K+ channels with properties similar to mammalian isoforms of this channel. The endogenous channels exhibit a voltage-dependence of 12-14 mV per e-fold change in open probability (po), can be activated by micromolar Ca2+ concentrations, and have a single channel conductance of approximately 200 pS in symmetrical 110 mM K+ solutions. Patch clamp experiments indicate that this endogenous channel is present at low densities (approximately 1 channel/3000 microns2). If endogenous channel subunits can form functional tetramers with other exogenous potassium channel subunits, then they will give rise to the expression of a heterogeneous channel population. Therefore, studies involving the heterologous expression of large-conductance Ca2(+)-activated K+ channels in Xenopus laevis oocytes require careful analysis and interpretation.

Type-2 calcium-dependent potassium (KCa) channels from mammalian brain, reconstituted into planar phospholipid bilayers, are modulated by ATP or ATP analogs via an endogenous protein kinase activity intimately associated with the channel (Chung et al., 1991). We show here that the endogenous protein kinase activity is protein kinase C (PKC)-like because (1) modulation by ATP can be mimicked by exogenous PKC, and (2) the effects of ATP can be blocked by PKC(19–36), a specific peptide inhibitor of PKC. Furthermore, adding the PKC inhibitor peptide after the addition of ATP reverses the modulation produced by ATP, suggesting that there is a phosphoprotein phosphatase activity closely associated with type-2 KCa channels. Consistent with this idea is the finding that microcystin, a non-specific phosphatase inhibitor, enhances the modulation of KCa channel activity by ATP. Inhibitor-1, a specific protein inhibitor of phosphoprotein phosphatase-1, also enhances the effect of ATP, suggesting that the endogenous phosphatase activity is phosphatase-1-like. The results imply that type-2 KCa channels exist as part of a regulatory complex that includes a PKC-like protein kinase and a phosphatase-1-like phosphoprotein phosphatase, both of which participate in the modulation of channel function.

Summary We have cloned and expressed nine Ca 2+ -activated K + channel isoforms from human brain. The open reading frames encode proteins ranging from 1154 to 1195 amino acids, and all possess significant identity with the slowpoke gene products in Drosophila and mouse. All isoforms are generated by alternative RNA splicing of a single gene on chromosome 10 at band q22.3 ( hslo ). RNA splicing occurs at four sites located in the carboxy-terminal portion of the protein and gives rise to at least nine ion channel constructs (hbr1–hbr9). hslo mRNA is expressed abundantly in human brain, and individual isoforms show unique expression patterns. Expression of hslo mRNA in Xenopus oocytes produces robust voltage and Ca 2+ -activated K + currents. Splice variants differ significantly in their Ca 2+ sensitivity, suggesting a broad functional role for these channels in the regulation of neuronal excitability.

Mutations in LRRK2 (leucine-rich repeat kinase 2) have been identified as major genetic determinants of Parkinson's disease (PD). The most prevalent mutation, G2019S, increases LRRK2's kinase activity, therefore understanding the sites and substrates that LRRK2 phosphorylates is critical to understanding its role in disease aetiology. Since the physiological substrates of this kinase are unknown, we set out to reveal potential targets of LRRK2 G2019S by identifying its favored phosphorylation motif. A non-biased screen of an oriented peptide library elucidated F/Y-x-T-x-R/K as the core dependent substrate sequence. Bioinformatic analysis of the consensus phosphorylation motif identified several novel candidate substrates that potentially function in neuronal pathophysiology. Peptides corresponding to the most PD relevant proteins were efficiently phosphorylated by LRRK2 in vitro. Interestingly, the phosphomotif was also identified within LRRK2 itself. Autophosphorylation was detected by mass spectrometry and biochemical means at the only F-x-T-x-R site (Thr 1410) within LRRK2. The relevance of this site was assessed by measuring effects of mutations on autophosphorylation, kinase activity, GTP binding, GTP hydrolysis, and LRRK2 multimerization. These studies indicate that modification of Thr1410 subtly regulates GTP hydrolysis by LRRK2, but with minimal effects on other parameters measured. Together the identification of LRRK2's phosphorylation consensus motif, and the functional consequences of its phosphorylation, provide insights into downstream LRRK2-signaling pathways.

Background: Parkinson’s disease (PD) is a progressive neurodegenerative condition characterized by an increasing loss of dopaminergic neurons resulting in motor dysfunction. However, cognitive impairments in PD patients are a common clinical feature that has gained increased attention. Objective: The purpose of the current study was to evaluate the effects of an MPTP-induced dopaminergic lesion in mice on social odor recognition (SOR) memory. Methods: Mice were acutely treated with MPTP and evaluated for memory impairments in the SOR assay and characterized using biochemical and immunohistochemical methods approximately 2 weeks later. Results: Here we demonstrate that SOR memory is sensitive to MPTP treatment and that it correlates with multiple measures of nigrostriatal integrity. MPTP treatment of C57BL/6N mice produced a profound decrease in dopamine levels, dopamine transporter binding and tyrosine hydroxylase immunoreactivity in the striatum. These impairments in stratial dopaminergic function were blocked by pretreatment with the MAO-B inhibitor deprenyl. Changes in the dopaminergic system parallel those observed in SOR with MPTP treatment impairing recognition memory in the absence of a deficit in odor discrimination during learning. Deprenyl pretreatment blocked the MPTP-induced impairment of SOR memory. Conclusion: The use of the SOR memory model may provide a preclinical method for evaluating cognitive therapies for PD.

The proteolytic enzyme beta-secretase (BACE1) plays a central role in the synthesis of the pathogenic beta-amyloid in Alzheimer's disease. Recently, we reported small molecule acylguanidines as potent BACE1 inhibitors. However, many of these acylguanidines have a high polar surface area (e.g. as measured by the topological polar surface area or TPSA), which is unfavorable for crossing the blood-brain barrier. Herein, we describe the identification of the 2-aminopyridine moiety as a bioisosteric replacement of the acylguanidine moiety, which resulted in inhibitors with lower TPSA values and superior brain penetration. X-ray crystallographic studies indicated that the 2-aminopyridine moiety interacts directly with the catalytic aspartic acids Asp32 and Asp228 via a hydrogen-bonding network.

α-synuclein is a 140-residue natively unfolded protein, whose aggregation is implicated in the development of Parkinson's Disease. It is thought that the cytotoxic species is not the mature fibril, but rather a prefibrillar aggregate which has membrane-permeabilizing properties. We have used Small Angle X-ray Scattering (SAXS) to determine the low-resolution structures of the different species formed during α-synuclein fibrillation in a non-invasive fashion. In addition to the starting monomer-dimer equilibrium and two bona fide fibril types accumulating towards the end of the aggregation process, we have identified a wreath-shaped oligomeric state which has a very distinct central hole. Both its structure and the kinetics of its formation are consistent with an on-pathway role, while its membrane-permeabilizing properties identify it as a putative cytotoxic species. We have also used SAXS to monitor the fibrillation of α-synuclein in the presence of the surfactant SDS and find that the fibrillar aggregates grow in a continuous fashion, forming beads on a string where the individual beads are stabilized by intermolecular α-synuclein contacts. The high reproducibility of this aggregative behaviour has formed the basis for a high-throughput screening assay involving 746.000 compounds that has allowed us to identify a significant number of compounds with the ability to inhibit early-stage aggregation of α-synuclein. This distinguishes the assay from previous assays that have focused mainly on the ability to prevent formation of α-synuclein fibrils. The hits from our assay may form the basis for a therapeutic intervention against Parkinson's Disease.

Mice transgenic for production of excessive or mutant forms of beta-amyloid differ from patients with Alzheimer's disease in the degree of inflammation, oxidative damage, and alteration of intermediary metabolism, as well as the paucity or absence of neuronal atrophy and cognitive impairment. Previous observers have suggested that differences in inflammatory response reflect a discrepancy in the state of the locus coeruleus (LC), loss of which is an early change in Alzheimer's disease but which is preserved in the transgenic mice. In this paper, we extend these observations by examining the effects of the LC on markers of oxidative stress and intermediary metabolism. We compare four groups: wild-type or Tg2576 A transgenic mice injected with DSP4 or vehicle. Of greatest interest were metabolites different between ablated and intact transgenics, but not between ablated and intact wild-type animals. The Tg2576_DSP4 mice were distinguished from the other three groups by oxidative stress and altered energy metabolism. These observations provide further support for the hypothesis that Tg2576 A transgenic mice with this ablation may be a more congruent model of Alzheimer's disease than are transgenics with an intact LC.

The identification of highly selective small molecule di-substituted pyridinyl aminohydantoins as β-secretase inhibitors is reported. The more potent and selective analogs demonstrate low nanomolar potency for the BACE1 enzyme as measured in a FRET assay, and exhibit comparable activity in a cell-based (ELISA) assay. In addition, these pyridine-aminohydantoins are highly selectivity (>500×) against the other structurally related aspartyl proteases BACE2, cathepsin D, pepsin and renin. Our design strategy followed a traditional SAR approach and was supported by molecular modeling studies based on the previously reported aminohydantoin 3a. We have taken advantage of the amino acid difference between the BACE1 and BACE2 at the S2′ pocket (BACE1 Pro70 changed to BACE2 Lys86) to build ligands with >500-fold selectivity against BACE2. The addition of large substituents on the targeted ligand at the vicinity of this aberration has generated a steric conflict between the ligand and these two proteins, thus impacting the ligand’s affinity and selectivity. These ligands have also shown an exceptional selectivity against cathepsin D (>5000-fold) as well as the other aspartyl proteases mentioned. One of the more potent compounds (S)-39 displayed an IC50 value for BACE1 of 10 nM, and exhibited cellular activity with an EC50 value of 130 nM in the ELISA assay.

The presenilin containing gamma-secretase complex is responsible for the regulated intramembraneous proteolysis of the amyloid precursor protein (APP), the Notch receptor, and a multitude of other substrates. gamma-Secretase catalyzes the final step in the generation of Abeta(40) and Abeta(42) peptides from APP. Amyloid beta-peptides (Abeta peptides) aggregate to form neurotoxic oligomers, senile plaques, and congophilic angiopathy, some of the cardinal pathologies associated with Alzheimer's disease. Although inhibition of this protease acting on APP may result in potentially therapeutic reductions of neurotoxic Abeta peptides, nonselective inhibition of the enzyme may cause severe adverse events as a result of impaired Notch receptor processing. Here, we report the preclinical pharmacological profile of GSI-953 (begacestat), a novel thiophene sulfonamide gamma-secretase inhibitor (GSI) that selectively inhibits cleavage of APP over Notch. This GSI inhibits Abeta production with low nanomolar potency in cellular and cell-free assays of gamma-secretase function, and displaces a tritiated analog of GSI-953 from enriched gamma-secretase enzyme complexes with similar potency. Cellular assays of Notch cleavage reveal that this compound is approximately 16-fold selective for the inhibition of APP cleavage. In the human APP-overexpressing Tg2576 transgenic mouse, treatment with this orally active compound results in a robust reduction in brain, plasma, and cerebral spinal fluid Abeta levels, and a reversal of contextual fear-conditioning deficits that are correlated with Abeta load. In healthy human volunteers, oral administration of a single dose of GSI-953 produces dose-dependent changes in plasma Abeta levels, confirming pharmacodynamic activity of GSI-953 in humans.