Artemisinin-treatment in pre-symptomatic APP-PS1 mice increases gephyrin phosphorylation at Ser270: a modification regulating postsynaptic GABAAR density.

Artemisinins, a group of plant-derived sesquiterpene lactones, are efficient antimalarial agents. They also share anti-inflammatory and anti-viral activities and were considered for treatment of neurodegenerative disorders like Alzheimer's disease (AD). Additionally, artemisinins bind to gephyrin, the multifunctional scaffold of GABAergic synapses, and modulate inhibitory neurotransmission in vitro. We previously reported an increased expression of gephyrin and GABAA receptors in early pre-symptomatic stages of an AD mouse model (APP-PS1) and in parallel enhanced CDK5-dependent phosphorylation of gephyrin at S270. Here, we studied the effects of artemisinin on gephyrin in the brain of young APP-PS1 mice. We detected an additional increase of gephyrin protein level, elevated gephyrin phosphorylation at Ser270, and an increased amount of GABAAR-γ2 subunits after artemisinin-treatment. Interestingly, the CDK5 activator p35 was also upregulated. Moreover, we demonstrate decreased density of postsynaptic gephyrin and GABAAR-γ2 immunoreactivities in cultured hippocampal neurons expressing gephyrin with alanine mutations at two CDK5 phosphorylation sites. In addition, the activity-dependent modulation of synaptic protein density was abolished in neurons expressing gephyrin lacking one or both of these phosphorylation sites. Thus, our results reveal that artemisinin modulates expression as well as phosphorylation of gephyrin at sites that might have important impact on GABAergic synapses in AD.

Artesunate restores the levels of inhibitory synapse proteins and reduces amyloid-ß and C-terminal fragments (CTFs) of the amyloid precursor protein in an AD-mouse model.

Alzheimer's disease (AD) is the most frequent form of dementia, characterized histopathologically by the formation of amyloid plaques and neurofibrillary tangles in the brain. Amyloid ß-peptide (Aß) is a major component of amyloid plaques and is released together with carboxy-terminal fragments (CTFs) from the amyloid precursor protein (APP) through proteolytic cleavage, thought to contribute to synapse dysfunction and loss along the progression of AD. Artemisinins, primarily antimalarial drugs, reduce neuroinflammation and improve cognitive capabilities in mouse models of AD. Furthermore, artemisinins were demonstrated to target gephyrin, the main scaffold protein of inhibitory synapses and modulate GABAergic neurotransmission in vitro. Previously, we reported a robust decrease of inhibitory synapse proteins in the hippocampus of 12-month-old double transgenic APP-PS1 mice which overexpress in addition to the Swedish mutated form of the human APP a mutated presenilin 1 (PS1) gene and are characterized by a high plaque load at this age. Here, we provide in vivo evidence that treating these mice with artemisinin or its semisynthetic derivative artesunate in two different doses (10 mg/kg and 100 mg/kg), these compounds affect differently inhibitory synapse components, amyloid plaque load and APP-processing. Immunofluorescence microscopy demonstrated the rescue of gephyrin and γ2-GABAA-receptor protein levels in the brain of treated mice with both, artemisinin and artesunate, most efficiently with a low dose of artesunate. Remarkably, artemisinin reduced only in low dose the amyloid plaque load correlating with lower levels of mutated human APP (hAPPswe) whereas artesunate treatment in both doses resulted in significantly lower plaque numbers. Correspondingly, the level of APP-cleavage products, specifically the amount of CTFs in hippocampus homogenates was reduced significantly only by artesunate, in line with the findings in hAPPswe expressing cultured hippocampal neurons evidencing a concentration-dependent inhibition of CTF-release by artesunate already in the nanomolar range. Thus, our data support artemisinins as neuroprotective multi-target drugs, exhibiting a potent anti-amyloidogenic activity and reinforcing key proteins of inhibitory synapses.

Amyloid-ß Fosters p35/CDK5 Signaling Contributing to Changes of Inhibitory Synapses in Early Stages of Cerebral Amyloidosis.

Early changes in inhibitory synapse connectivities are thought to contribute to the excitation/inhibition imbalance preceding neurodegeneration in Alzheimer's disease (AD). Recently, we reported a robust increase in the level of different key-proteins of inhibitory synapses in hippocampal subregions of pre-symptomatic APPswe-PS1 mice, a model of cerebral amyloidosis. Besides increased inhibitory synaptic clusters on parvalbumin-positive projections in CA1 and CA3, we observed impaired communication between these two hippocampal areas of young APP-PS1 mice. Interestingly, the phosphorylation of gephyrin, a major organizer of inhibitory synapses, was also increased. Here, we demonstrate that the protein levels of CDK5, a kinase involved in the phosphorylation of gephyrin, and its regulatory protein p35 are also significantly increased in hippocampal subregions of young APP-PS1 mice. Consistently, the expression of hAPP-swe in cultured hippocampal neurons resulted in higher p35-protein levels, indicating a possible molecular link between increased Aß-production and the elevated p35/CDK5 levels seen in vivo. Further, a shRNA mediated downregulation of p35-expression in hippocampal neurons correlated with a decrease in gephyrin phosphorylation and in a reduced density of synaptic γ2-GABAA-receptor clusters. These findings, together with the detection of gephyrin colocalization with CDK5 and p35 by immunostaining and proximity ligation experiments in vivo and in vitro, are supporting our hypothesis that Aß has a profound impact on inhibitory network properties, likely mediated at least in part by p35/CDK5 signaling. This further underscores the impact of altered inhibitory synaptic transmission in AD.

Biphasic Alteration of the Inhibitory Synapse Scaffold Protein Gephyrin in Early and Late Stages of an Alzheimer Disease Model.

The pathogenesis of Alzheimer disease (AD) is thought to begin many years before the diagnosis of dementia. Accumulating evidence indicates the involvement of GABAergic neurotransmission in the physiopathology of AD. However, in comparison to excitatory synapses, the structural and functional alterations of inhibitory synapses in AD are less well characterized. We studied the expression and distribution of proteins specific for inhibitory synapses in hippocampal areas of APPPS1 mice at different ages. Interestingly, by immunoblotting and confocal fluorescence microscopy, we disclosed a robust increase in the expression of gephyrin, an organizer of ligand-gated ion channels at inhibitory synapses in hippocampus CA1 and dentate gyrus of young presymptomatic APPPS1 mice (1 to 3 months) as compared to controls. The postsynaptic γ2-GABA(A)-receptor subunit and the presynaptic vesicular inhibitory amino acid transporter protein showed similar expression patterns. In contrast, adult transgenic animals (12 months) displayed decreased levels of these proteins in comparison to wild type in hippocampus areas devoid of amyloid plaques. Within most plaques, strong gephyrin immunoreactivity was detected, partially colocalizing with vesicular amino acid transporter and GABA(A)-receptor γ2 subunit immunoreactivities. Our results indicate a biphasic alteration in expression of hippocampal inhibitory synapse components in AD. Altered inhibition of neurotransmission might be an early prognostic marker and might even be involved in the pathogenesis of AD.

KCC2 knockdown impairs glycinergic synapse maturation in cultured spinal cord neurons.

Synaptic inhibition in the spinal cord is mediated mainly by strychnine-sensitive glycine (GlyRs) and by γ-aminobutyric acid type A receptors (GABAAR). During neuronal maturation, neonatal GlyRs containing α2 subunits are replaced by adult-type GlyRs harboring α1 and α3 subunits. At the same time period of postnatal development, the transmembrane chloride gradient is changed due to increased expression of the potassium-chloride cotransporter (KCC2), thereby shifting the GABA- and glycine-mediated synaptic currents from mostly excitatory depolarization to inhibitory hyperpolarization. Here, we used RNA interference to suppress KCC2 expression during in vitro maturation of spinal cord neurons. Morphological analysis revealed reduced numbers and size of dendritic GlyR clusters containing α1 subunits but not of clusters harboring neonatal α2 subunits. The morphological changes were accompanied by decreased frequencies and amplitudes of glycinergic miniature inhibitory currents, whereas GABAergic synapses appeared functionally unaltered. Our data indicate that KCC2 exerts specific functions for the maturation of glycinergic synapses in cultured spinal cord neurons.

Cyclin-dependent kinase 5 is involved in the phosphorylation of gephyrin and clustering of GABAA receptors at inhibitory synapses of hippocampal neurons.

CDK5 has been implicated in neural functions including growth, neuronal migration, synaptic transmission and plasticity of excitatory chemical synapses. Here we report robust effects of CDK5 on phosphorylation of the postsynaptic scaffold protein gephyrin and clustering of inhibitory GABAA receptors in hippocampal neurons. shRNA-mediated knockdown of CDK5 and pharmacological inhibition of cyclin-dependent kinases reduced phosphorylated gephyrin clusters and postsynaptic γ2-containing GABAA receptors. Phosphorylation of S270 is antagonized by PP1/PP2a phosphatase and site-directed mutagenesis and in vitro phosphorylation experiments indicate that S270 is a putative CDK5 phosphorylation site of gephyrin. Our data suggest that CDK5 plays an essential role for the stability of gephyrin-dependent GABAA receptor clusters in hippocampal neurons.

Phosphorylation of gephyrin in hippocampal neurons by cyclin-dependent kinase CDK5 at Ser-270 is dependent on collybistin.

Gephyrin is a scaffold protein essential for the postsynaptic clustering of inhibitory glycine and different subtypes of GABA(A) receptors. The cellular and molecular mechanisms involved in gephyrin-mediated receptor clustering are still not well understood. Here we provide evidence that the gephyrin-binding protein collybistin is involved in regulating the phosphorylation of gephyrin. We demonstrate that the widely used monoclonal antibody mAb7a is a phospho-specific antibody that allows the cellular and biochemical analysis of gephyrin phosphorylation at Ser-270. In addition, another neighbored epitope determinant was identified at position Thr-276. Analysis of the double mutant gephyrin(T276A,S277A) revealed significant reduction in gephyrin cluster formation and altered oligomerization behavior of gephyrin. Moreover, pharmacological inhibition of cyclin-dependent kinases in hippocampal neurons reduced postsynaptic gephyrin mAb7a immunoreactivities. In vitro phosphorylation assays and phosphopeptide competition experiments revealed a phosphorylation at Ser-270 depending on enzyme activities of cyclin-dependent kinases CDK1, -2, or -5. These data indicate that collybistin and cyclin-dependent kinases are involved in regulating the phosphorylation of gephyrin at postsynaptic membrane specializations.

Effects of distinct collybistin isoforms on the formation of GABAergic synapses in hippocampal neurons.

Collybistin (Cb) is a brain specific guanine nucleotide exchange factor that interacts with the inhibitory postsynaptic scaffold protein gephyrin. Cb is essential for the postsynaptic clustering of gephyrin and major GABA(A) receptor subtypes during the formation and maintenance of GABAergic synapses in the hippocampus and other areas of the forebrain. In the rat, four distinct splice variants (Cb1, Cb2(SH3-), Cb2(SH3+) and Cb3), have been described, which differ in their C-termini (Cb1-3) and in respect of the SH3-domain that is absent in Cb2(SH3-). In the human brain, only a single isoform (hPEM2) corresponding to Cb3, was found to be expressed. This has been implicated in neurological defects such as hyperekplexia, epilepsy, anxiety, aggression and mental retardation. In this study, we address the functional significance of the differentially spliced Cb isoforms by generating a shRNA-mediated knock-down of endogenous Cb in hippocampal cultured neurons that is subsequently rescued by the expression of distinct Cb isoforms. We found that the Cb knock-down induced impairment in GABAergic neurotransmission could be rescued by the expression of any of the Cb isoforms, independent of their C-termini or the presence of the SH3-domain in the N-terminal region. Thus, the different Cb isoforms all confer basic functionality.

Activity-dependent shedding of the NMDA receptor glycine binding site by matrix metalloproteinase 3: a PUTATIVE mechanism of postsynaptic plasticity.

Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.

Components of the translational machinery are associated with juvenile glycine receptors and are redistributed to the cytoskeleton upon aging and synaptic activity.

The translation eukaryotic elongation factor 1alpha (eEF1A) is a monomeric GTPase involved in protein synthesis. In addition, this protein is thought to participate in other cellular functions such as actin bundling, cell cycle regulation, and apoptosis. Here we show that eEF1A is associated with the alpha2 subunit of the inhibitory glycine receptor in pulldown experiments with rat brain extracts. Moreover, additional proteins involved in translation like ribosomal S6 protein and p70 ribosomal S6 protein kinase as well as ERK1/2 and calcineurin were identified in the same pulldown approaches. Glycine receptor activation in spinal cord neurons cultured for 1 week resulted in an increased phosphorylation of ribosomal S6 protein. Immunocytochemistry showed that eEF1A and ribosomal S6 protein are localized in the soma, dendrites, and at synapses of cultured hippocampal and spinal cord neurons. Consistent with our biochemical data, immunoreactivities of both proteins were partially overlapping with glycine receptor immunoreactivity in cultured spinal cord and hippocampal neurons. After 5 weeks in culture, eEF1A immunoreactivity was redistributed to the cytoskeleton in about 45% of neurons. Interestingly, the degree of redistribution could be increased at earlier stages of in vitro differentiation by inhibition of either the ERK1/2 pathway or glycine receptors and simultaneous N-methyl-D-aspartate receptor activation. Our findings suggest a functional coupling of eEF1A with both inhibitory and excitatory receptors, possibly involving the ERK-signaling pathway.

Human BACE forms dimers and colocalizes with APP.

Beta-site APP-cleaving enzyme (BACE) is a membrane-bound aspartyl protease with no strict primary preference for cleavage. The molecular mechanisms that link the gamma-secretase multicomponent amyloid precursor protein (APP) processing complex to biochemical properties of BACE generating the N terminus of the amyloid beta-peptide have not, as yet, been identified. We found that in human brain tissue, BACE occurred as a dimer. The overall stability of the BACE homodimer was based on intermolecular interactions that were not affected by high salt, nonionic detergents or reducing conditions. BACE homodimers could only partially be separated even under strong denaturing conditions and revealed dramatic differences in the surface charge distribution compared with the monomer. In contrast, the soluble ectodomain of truncated BACE revealed a seemingly lower avidity to the prototypic aspartate protease inhibitor pepstatin and exclusively occurred in the monomeric form. Immunocytochemical studies colocalized APP and BACE in the plasma membrane of cells expressing endogenous levels of BACE and overexpressing APP. In cells that were cotransfected with APP and a putative active site D289A mutant of BACE, colocalization persisted. Remaining enzyme activity was found to be attributable to the mutant protease. Accordingly, inactivation of the carboxyl-terminal active site motif of BACE without an impairment of overall enzyme activity suggests that the enzyme may act as a dimer. Thus, homodimerization of BACE may help the enzyme to acquire specific mechanisms to associate with its substrates to exert catalytic activity.

Kinetic analysis of the interaction of the copper chaperone Atox1 with the metal binding sites of the Menkes protein.

Excess copper is effluxed from mammalian cells by the Menkes or Wilson P-type ATPases (MNK and WND, respectively). MNK and WND have six metal binding sites (MBSs) containing a CXXC motif within their N-terminal cytoplasmic region. Evidence suggests that copper is delivered to the ATPases by Atox1, one of three cytoplasmic copper chaperones. Attempts to monitor a direct Atox1-MNK interaction and to determine kinetic parameters have not been successful. Here we investigated interactions of Atox1 with wild-type and mutated pairs of the MBSs of MNK using two different methods: yeast two-hybrid analysis and real-time surface plasmon resonance (SPR). A copper-dependent interaction of Atox1 with the MBSs of MNK was observed by both approaches. Cys to Ser mutations of conserved CXXC motifs affected the binding of Atox1 underlining the essentiality of Cys residues for the copper-induced interaction. Although the yeast two-hybrid assay failed to show an interaction of Atox1 with MBS5/6, SPR analysis clearly demonstrated a copper-dependent binding with all six MBSs highlighting the power and sensitivity of SPR as compared with other, more indirect methods like the yeast two-hybrid system. Binding constants for copper-dependent chaperone-MBS interactions were determined to be 10-5-10-6 m for all the MBSs representing relatively low affinity binding events. The interaction of Atox1 with pairs of the MBSs was non-cooperative. Therefore, a functional difference of the MBSs in the MNK N terminus cannot be attributed to cooperativity effects or varying affinities of the copper chaperone Atox1 with the MBSs.

Identification of a beta-secretase activity, which truncates amyloid beta-peptide after its presenilin-dependent generation.

The beta-amyloid precursor protein (beta APP) is proteolytically processed by two secretase activities to produce the pathogenic amyloid beta-peptide (A beta). N-terminal cleavage is mediated by beta-secretase (BACE) whereas C-terminal intramembraneous cleavage is exerted by the presenilin (PS) gamma-secretase complex. The A beta-generating gamma-secretase cleavage principally occurs after amino acid 40 or 42 and results in secretion of A beta-(1-40) or A beta-(1-42). Upon overexpression of BACE in cultured cells we unexpectedly noticed a reduction of secreted A beta-(1-40/42). However, mass spectrometry revealed a truncated A beta species, which terminates at amino acid 34 (A beta-(1-34)) suggesting an alternative gamma-secretase cut. Indeed, expression of a loss-of-function variant of PS1 inhibited not only the production of A beta-(1-40) and A beta-(1-42) but also that of A beta-(1-34). However, expression levels of BACE correlate with the amount of A beta-(1-34), and A beta-(1-34) is produced at the expense of A beta-(1-40) and A beta-(1-42). Since this suggested that BACE is involved in a C-terminal truncation of A beta, we incubated purified BACE with A beta-(1-40) in vitro. Under these conditions A beta-(1-34) was generated. Moreover, when conditioned media containing Abeta-(1-40) and A beta-(1-42) were incubated with cells expressing a loss-of-function PS1 variant together with BACE, A beta-(1-34) was efficiently produced in vivo. These data demonstrate that an apparently gamma-secretase-dependent A beta derivative is produced after the generation of the non-truncated A beta via an additional and unexpected activity of BACE.

Evidence for a copper-binding superfamily of the amyloid precursor protein.

The amyloid precursor protein (APP) copper-binding domain (CuBD) has been shown to reduce Cu(II) to Cu(I) and to mediate copper-induced oxidation in vitro. However, little is known about copper binding to the homologous domains of APP and APP family paralogs and orthologs (including amyloid precursor-like proteins from Drosophila melanogaster, Xenopus laevis, and Caenorhabditis elegans) and their effects on Cu-induced oxidation and Cu(I) formation. Here, we show that APP homologues with and without conserved histidine residues at positions 147, 149, and 151 all bind Cu(II). Oxidized peptides were the kinetically favored products of the redox reaction of CuBDs promoting the reduction of Cu(II) to Cu(I). These results reveal a molecular phylogeny-based divergence that has taken place between the ancestral Drosophila APPL and C. elegans APL-1 and the recently evolved APP lineage of CuBDs. Whereas higher species CuBDs have a decreased affinity for Cu(II) and high Cu(II) reducing activities, ancestral CuBDs form very tight binding sites for Cu(II) ions and have low Cu(II) reducing activities. Thus, the APP lineage displays a gain in activity toward promoting Cu(II) reduction and Cu(I) release. The findings suggests that the Cu(II)-binding equilibrium at the phylogenetic stage of Drosophila APPL and C. elegans APL-1 is shifted from the exchangeable Cu(II) pool to the tightly bound, nonexchangeable pool and that ancestral CuBDs may exert antioxidation activities in vivo. The more recently evolved homologues of human APP appear to take advantage of unique redox properties for yet unknown biological functions.

Possible mechanisms of APP-mediated oxidative stress in Alzheimer's disease.

Oxidative stress was presented to play an important role in the pathogenesis of Alzheimer's disease (AD), especially in the early evolution of AD amyloidogenesis and not only as a consequence thereof. The effect of oxidative stress catalysed by transition metals appears to have a critical relevance in AD. Metal-ion homeostasis is severely dysregulated in AD and it was found that experimentally induced disturbances in the homeostasis of Zn(II) and Cu(II) affect the amyloid precursor protein (APP) metabolism. APP itself binds Zn(II) and Cu(II) at nanomolar concentrations and an altered APP metabolism or expression level is believed to result in neurotoxic processes.