Protection of primary neurons and mouse brain from Alzheimer's pathology by molecular tweezers.

Alzheimer's disease is a devastating cureless neurodegenerative disorder affecting >35 million people worldwide. The disease is caused by toxic oligomers and aggregates of amyloid ß protein and the microtubule-associated protein tau. Recently, the Lys-specific molecular tweezer CLR01 has been shown to inhibit aggregation and toxicity of multiple amyloidogenic proteins, including amyloid ß protein and tau, by disrupting key interactions involved in the assembly process. Following up on these encouraging findings, here, we asked whether CLR01 could protect primary neurons from Alzheimer's disease-associated synaptotoxicity and reduce Alzheimer's disease-like pathology in vivo. Using cell culture and brain slices, we found that CLR01 effectively inhibited synaptotoxicity induced by the 42-residue isoform of amyloid ß protein, including ∼80% inhibition of changes in dendritic spines density and long-term potentiation and complete inhibition of changes in basal synaptic activity. Using a radiolabelled version of the compound, we found that CLR01 crossed the mouse blood-brain barrier at ∼2% of blood levels. Treatment of 15-month-old triple-transgenic mice for 1 month with CLR01 resulted in a decrease in brain amyloid ß protein aggregates, hyperphosphorylated tau and microglia load as observed by immunohistochemistry. Importantly, no signs of toxicity were observed in the treated mice, and CLR01 treatment did not affect the amyloidogenic processing of amyloid ß protein precursor. Examining induction or inhibition of the cytochrome P450 metabolism system by CLR01 revealed minimal interaction. Together, these data suggest that CLR01 is safe for use at concentrations well above those showing efficacy in mice. The efficacy and toxicity results support a process-specific mechanism of action of molecular tweezers and suggest that these are promising compounds for developing disease-modifying therapy for Alzheimer's disease and related disorders.

Evidence that behavioral phenotypes of morphine in ß-arr2-/- mice are due to the unmasking of JNK signaling.

The altered behavioral effects of morphine, but not most other mu agonists, in mice lacking ß-arrestin 2, suggest that this scaffolding protein regulates the signaling cascade of this commonly used analgesic. One of the cascades that could be regulated by ß-arrestin 2 is cJun-N-terminal kinase (JNK), which binds with ß-arrestin 2 and modulates the analgesic effects of morphine. Using neurons lacking ß-arrestin 2 (ß-arr2-/-) to examine this interaction, we found that ß-arr2-/- neurons show altered intracellular distribution of JNK and cJun, and that morphine, but not fentanyl, increased the nuclear localization of the phosphorylated, therefore activated, form of cJun, a JNK target in dorsal root ganglia neurons. This suggests that deleting ß-arrestin 2 affects the JNK cascade. We therefore examined whether some of the behavioral phenotypes of mice lacking ß-arrestin 2 could be a result of altered JNK signaling. Indeed, two different JNK inhibitors reversed the enhanced analgesic effect of morphine, a known phenotype of ß-arr2-/- mice, to +/+ levels. Both the reduced locomotor effect of morphine and the psychomotor sensitization to repeated morphine administration in ß-arr2-/- mice were also returned to +/+ levels by inhibiting JNK. In contrast, the behavioral effects of fentanyl were neither genotype-dependent nor affected by JNK inhibition. Furthermore, a PKC inhibitor had a similar effect as inhibiting JNK in reducing the enhanced analgesic effect of morphine in ß-arr2-/- mice to +/+ levels. In summary, removing ß-arrestin 2 reveals mu receptor activation of the JNK cascade in a ligand-specific manner explaining several behavioral phenotypes of ß-arr2-/- mice.

Epigenetic enhancement of BDNF signaling rescues synaptic plasticity in aging.

Aging-related cognitive declines are well documented in humans and animal models. Yet the synaptic and molecular mechanisms responsible for cognitive aging are not well understood. Here we demonstrated age-dependent deficits in long-term synaptic plasticity and loss of dendritic spines in the hippocampus of aged Fisher 344 rats, which were closely associated with reduced histone acetylation, upregulation of histone deacetylase (HDAC) 2, and decreased expression of a histone acetyltransferase. Further analysis showed that one of the key genes affected by such changes was the brain-derived neurotrophic factor (Bdnf) gene. Age-dependent reductions in H3 and H4 acetylation were detected within multiple promoter regions of the Bdnf gene, leading to a significant decrease in BDNF expression and impairment of downstream signaling in the aged hippocampus. These synaptic and signaling deficits could be rescued by enhancing BDNF and trkB expression via HDAC inhibition or by directly activating trkB receptors with 7,8-dihydroxyflavone, a newly identified, selective agonist for trkB. Together, our findings suggest that age-dependent declines in chromatin histone acetylation and the resulting changes in BDNF expression and signaling are key mechanisms underlying the deterioration of synaptic function and structure in the aging brain. Furthermore, epigenetic or pharmacological enhancement of BDNF-trkB signaling could be a promising strategy for reversing cognitive aging.

Enhanced neuronal expression of major histocompatibility complex class I leads to aberrations in neurodevelopment and neurorepair.

Mice deficient in classical major histocompatibility complex class I (MHCI) have aberrations in neurodevelopment. The consequences of upregulated neuronal MHCI expression have not been examined. We found that transgenic C57Bl/6 mice that are engineered to express higher levels of self-D(b) on their CNS neurons have alterations in their hippocampal morphology and retinogeniculate projections, as well as impaired neurorepair responses. Thus, enhanced neuronal classical MHCI expression can lead to aberrations in neural circuitry and neurorepair. These findings complement a growing body of knowledge concerning the neurobiological activities of MHCI and may have potential clinical relevance.

Neurotrophins enhance CaMKII activity and rescue amyloid-ß-induced deficits in hippocampal synaptic plasticity.

Amyloid-ß (Aß) peptide-induced impairment of hippocampal synaptic plasticity is considered an underlying mechanism for memory loss in the early stages of Alzheimer's disease and its animal models. We previously reported inhibition of long-term potentiation (LTP) and miniature excitatory postsynaptic currents by oligomeric Aß(1-42) at hippocampal synapses. While multiple cellular mechanisms could be involved in Aß-induced synaptic dysfunction, blockade of activity-dependent autophosphorylation of Ca2+ and calmodulin-dependent protein kinase II (CaMKII) appeared to be a major component of Aß action in our studies. The present study further tested this hypothesis and examined the therapeutic potential of trkB receptor-acting neurotrophins in rescuing Aß-induced synaptic and signaling impairments. As expected, treatment of rat hippocampal slices with Aß(1-42) significantly reduced LTP in the Schaffer collateral-CA1 pathway and dentate medial perforant path. LTP-associated CaMKII activation and AMPA receptor phosphorylation were blocked by Aß(1-42) at the same concentration that inhibited LTP. Aß-induced LTP impairment, however, was prevented when slices were co-treated with neurotrophin 4 (NT4). Western blotting and immunohistochemical analyses confirmed that treatment with NT4 or brain-derived neurotrophic factor, another trkB-acting neurotrophin, could oppose Aß action, enhancing autophosphorylation of CaMKII, and AMPA receptor phosphorylation at a CaMKII-dependent site. These findings support the view that CaMKII is a key synaptic target of Aß toxicity as well as a potential therapeutic site of neurotrophins for Alzheimer's disease.

Mechanistic investigation of the inhibition of Abeta42 assembly and neurotoxicity by Abeta42 C-terminal fragments.

Oligomeric forms of amyloid beta-protein (Abeta) are key neurotoxins in Alzheimer's disease (AD). Previously, we found that C-terminal fragments (CTFs) of Abeta42 interfered with assembly of full-length Abeta42 and inhibited Abeta42-induced toxicity. To decipher the mechanism(s) by which CTFs affect Abeta42 assembly and neurotoxicity, here, we investigated the interaction between Abeta42 and CTFs using photoinduced cross-linking and dynamic light scattering. The results demonstrate that distinct parameters control CTF inhibition of Abeta42 assembly and Abeta42-induced toxicity. Inhibition of Abeta42-induced toxicity was found to correlate with stabilization of oligomers with a hydrodynamic radius (R(H)) of 8-12 nm and attenuation of formation of oligomers with an R(H) of 20-60 nm. In contrast, inhibition of Abeta42 paranucleus formation correlated with CTF solubility and the degree to which CTFs formed amyloid fibrils themselves but did not correlate with inhibition of Abeta42-induced toxicity. Our findings provide important insight into the mechanisms by which different CTFs inhibit the toxic effect of Abeta42 and suggest that stabilization of nontoxic Abeta42 oligomers is a promising strategy for designing inhibitors of Abeta42 neurotoxicity.

p38 MAPK and beta-arrestin 2 mediate functional interactions between endogenous micro-opioid and alpha2A-adrenergic receptors in neurons.

Formation of receptor complexes between micro-opioid and alpha2A-adrenergic receptors has been demonstrated in transfected cells. The functional significance and underlying mechanisms of such receptor interactions remain to be determined in neuronal systems. We examined functional interactions between endogenous micro and alpha2A receptors in mouse dorsal root ganglion neurons. Acute application of the micro agonist [D-Ala2,N-MePhe4, Gly-ol5]enkephalin (DAMGO) or the alpha2 agonist clonidine inhibited voltage-gated Ca2+ currents in these neurons. Prolonged treatment with either DAMGO or clonidine induced a mutual cross-desensitization between micro and alpha2A receptor-mediated current inhibition. The cross-desensitization was closely associated with simultaneous internalization of micro and alpha2A receptors. Morphine, a mu agonist triggering little mu receptor endocytosis, induced neither cross-desensitization nor internalization of alpha2A receptors. Furthermore, inhibition of p38 MAPK prevented the cross-desensitization as well as cointernalization of micro and alpha2A receptors. Changes in receptor trafficking profiles suggested that p38 MAPK activity was required for initiating micro receptor internalization and maintaining possible micro-alpha2A association during their cointernalization. Finally, the micro-alpha2A cross-desensitization was absent in dorsal root ganglion neurons lacking beta-arrestin 2. These findings demonstrated p38 MAPK- and beta-arrestin 2-dependent cross-regulation between neuronal micro and alpha2A receptors. By promoting receptor cross-desensitization and cointernalization, such functional interactions may serve as negative feedback mechanisms triggered by prolonged agonist exposure to modulate the signaling of functionally related G protein-coupled receptors.

C-terminal peptides coassemble into Abeta42 oligomers and protect neurons against Abeta42-induced neurotoxicity.

Alzheimer's disease (AD) is an age-related disorder that threatens to become an epidemic as the world population ages. Neurotoxic oligomers of Abeta42 are believed to be the main cause of AD; therefore, disruption of Abeta oligomerization is a promising approach for developing therapeutics for AD. Formation of Abeta42 oligomers is mediated by intermolecular interactions in which the C terminus plays a central role. We hypothesized that peptides derived from the C terminus of Abeta42 may get incorporated into oligomers of Abeta42, disrupt their structure, and thereby inhibit their toxicity. We tested this hypothesis using Abeta fragments with the general formula Abeta(x-42) (x = 28-39). A cell viability screen identified Abeta(31-42) as the most potent inhibitor. In addition, the shortest peptide, Abeta(39-42), also had high activity. Both Abeta(31-42) and Abeta(39-42) inhibited Abeta-induced cell death and rescued disruption of synaptic activity by Abeta42 oligomers at micromolar concentrations. Biophysical characterization indicated that the action of these peptides likely involved stabilization of Abeta42 in nontoxic oligomers. Computer simulations suggested a mechanism by which the fragments coassembled with Abeta42 to form heterooligomers. Thus, Abeta(31-42) and Abeta(39-42) are leads for obtaining mechanism-based drugs for treatment of AD using a systematic structure-activity approach.

Dopamine transporter, gender, and number of sexual partners among young adults.

The dopamine transporter gene (DAT1) codes for a dopamine transporter protein, which limits the level and duration of dopamine receptor activation. The DAT1 gene is a strong candidate gene for reward-seeking behavior. This article reports compelling evidence for the association between the 40 bp variable number of tandem repeats in the DAT1 gene and the self-reported number of sexual partners among young adults in the United States using the sibling subsample of more than 2500 individuals who participated in the National Longitudinal Study of Adolescent Health. We performed tests of genotype-gender interaction as well as analyses stratified by gender. Among the males, possessing one or two alleles of the 10 repeat is associated with an 80-100% increase (P<0.0001, 2df) in the number of sexual partners as compared with the homozygotes for the 9 repeat. The association holds in race/ethnicity-stratified analyses, in Allison's procedure that tests population stratification, and in within-family fixed-effects models. Covariate adjustment for a standard set of socioeconomic factors including religiosity, family structure, parental education, marital and cohabitation history, and neighborhood poverty did not attenuate these associations. Discussion is provided why this finding is absent among females.

Deletion of the neuron-specific protein delta-catenin leads to severe cognitive and synaptic dysfunction.

Delta-catenin (delta-catenin) is a neuron-specific catenin, which has been implicated in adhesion and dendritic branching. Moreover, deletions of delta-catenin correlate with the severity of mental retardation in Cri-du-Chat syndrome (CDCS), which may account for 1% of all mentally retarded individuals. Interestingly, delta-catenin was first identified through its interaction with Presenilin-1 (PS1), the molecule most frequently mutated in familial Alzheimer's Disease (FAD). We investigated whether deletion of delta-catenin would be sufficient to cause cognitive dysfunction by generating mice with a targeted mutation of the delta-catenin gene (delta-cat(-/-)). We observed that delta-cat(-/-) animals are viable and have severe impairments in cognitive function. Furthermore, mutant mice display a range of abnormalities in hippocampal short-term and long-term synaptic plasticity. Also, N-cadherin and PSD-95, two proteins that interact with delta-catenin, are significantly reduced in mutant mice. These deficits are severe but specific because delta-cat(-/-) mice display a variety of normal behaviors, exhibit normal baseline synaptic transmission, and have normal levels of the synaptic adherens proteins E-cadherin and beta-catenin. These data reveal a critical role for delta-catenin in brain function and may have important implications for understanding mental retardation syndromes such as Cri-du-Chat and neurodegenerative disorders, such as Alzheimer's disease, that are characterized by cognitive decline.

Amyloid beta prevents activation of calcium/calmodulin-dependent protein kinase II and AMPA receptor phosphorylation during hippocampal long-term potentiation.

Accumulation of amyloid beta-peptides (Abeta) in the brain has been linked with memory loss in Alzheimer's disease and its animal models. However, the synaptic mechanism by which Abeta causes memory deficits remains unclear. We previously showed that acute application of Abeta inhibited long-term potentiation (LTP) in the hippocampal perforant path via activation of calcineurin, a Ca2+ -dependent protein phosphatase. This study examined whether Abeta could also inhibit Ca2+/calmodulin dependent protein kinase II (CaMKII), further disrupting the dynamic balance between protein kinase and phosphatase during synaptic plasticity. Immunoblot analysis was conducted to measure autophosphorylation of CaMKII at Thr286 and phosphorylation of the GluR1 subunit of AMPA receptors in single rat hippocampal slices. A high-frequency tetanus applied to the perforant path significantly increased CaMKII autophosphorylation and subsequent phosphorylation of GluR1 at Ser831, a CaMKII-dependent site, in the dentate area. Acute application of Abeta1-42 inhibited dentate LTP and associated phosphorylation processes, but was without effect on phosphorylation of GluR1 at Ser845, a protein kinase A-dependent site. These results suggest that activity-dependent CaMKII autophosphorylation and AMPA receptor phosphorylation are essential for dentate LTP. Disruption of such mechanisms could directly contribute to Abeta-induced deficits in hippocampal synaptic plasticity and memory.

Calcium-regulated signaling pathways: role in amyloid beta-induced synaptic dysfunction.

Amyloid beta (Abeta) peptides have been shown to impair synaptic function, especially long-term synaptic plasticity, in transgenic mouse models of Alzheimer's disease (AD) and in acute hippocampal preparations. In the transgenic mice overexpressing mutant forms of human amyloid precursor protein (APP), the deficits in hippocampal long-term potentiation (LTP) occur prior to synaptic loss and cell death, suggesting early functional changes at these synapses. Recent studies demonstrate that Abeta-induced synaptic dysfunction is linked with altered Ca2+ signaling in hippocampal neurons. While reducing Ca2+ influx through NMDA receptors, Abeta peptides elevate intracellular Ca2+ concentration by enhancing Ca2+ influx from voltage-gated Ca2+ channels or nonselective cation channels, or by stimulating Ca2+ release from intracellular stores. Interestingly, acute application of Abeta or APP overexpression inhibits activity-dependent regulation of several protein kinase pathways that require Ca2+ influx via NMDA receptors for activation, including Ca2+/calmodulin-dependent protein kinase II, protein kinase A, and extracellular regulated kinases (Erk). On the other hand, activation of Ca2+-dependent protein phosphatase 2B (calcineurin) is implicated in Abeta inhibition of LTP. Thus, multiple Ca2+-regulated signaling pathways are involved in the synaptic action of Abeta, and malfunction of these pathways may underlie the synaptic dysfunction in early AD.

Phosphoinositide 3-kinase cascade facilitates mu-opioid desensitization in sensory neurons by altering G-protein-effector interactions.

Signaling via G-protein-coupled receptors undergoes desensitization after prolonged agonist exposure. Here we investigated the role of phosphoinositide 3-kinase (PI3K) and its downstream pathways in desensitization of micro-opioid inhibition of neuronal Ca2+ channels. In cultured mouse dorsal root ganglion neurons, two mechanistically different forms of desensitization were observed after acute or chronic treatment with the micro agonist [D-Ala2, N-MePhe4, Gly-ol5]-enkephalin (DAMGO). Chronic DAMGO desensitization was heterologous in nature and significantly attenuated by blocking the activity of PI3K or mitogen-activated protein kinase (MAPK). A combined application of PI3K and MAPK inhibitors showed no additive effect, suggesting that these two kinases act in a common pathway to facilitate chronic desensitization. Acute DAMGO desensitization, however, was not affected by the inhibitors. Furthermore, upregulation of the PI3K-Akt pathway in mutant mice lacking phosphatase and tensin homolog, a lipid phosphatase counteracting PI3K, selectively enhanced chronic desensitization in a PI3K- and MAPK-dependent manner. Using the prepulse facilitation (PPF) test, we further examined changes in the voltage-dependent component of DAMGO action that requires direct interactions between betagamma subunits of G-proteins and Ca2+ channels. DAMGO-induced PPF was diminished after chronic treatment, suggesting disruption of G-protein-channel interactions. Such disruption could occur at the postreceptor level, because chronic DAMGO also reduced GTPgammaS-induced PPF that was independent of receptor activation. Again, inhibition of PI3K or MAPK reduced desensitization of PPF. Our data suggest that the PI3Kcascade involving MAPK and Akt enhances micro-opioid desensitization via postreceptor modifications that interfere with G-protein-effector interactions.

Alzheimer amyloid beta-peptide inhibits the late phase of long-term potentiation through calcineurin-dependent mechanisms in the hippocampal dentate gyrus.

The perforant path projecting from the entorhinal cortex to the hippocampal dentate gyrus is a particularly vulnerable target to the early deposition of amyloid beta (Abeta) peptides in Alzheimer's brain. The authors previously showed that brief applications of Abeta at subneurotoxic concentrations suppressed the early-phase long-term potentiation (E-LTP) in rat dentate gyrus. The current study further examines the effect of Abeta on the late-phase LTP (L-LTP) in this area. Using multiple high-frequency stimulus trains, a stable L-LTP lasting for at least 3 h was induced in the medial perforant path of rat hippocampal slices. Bath application of Abeta(1-42) (0.2-1.0 microM) during the induction trains attenuated both the initial and late stages of L-LTP. On the other hand, Abeta(1-42) perfusion within the first hour following the induction primarily impaired the late stage of L-LTP, which resembled the action of the protein synthesis inhibitor emetine. Blockade of calcineurin activity with FK506 or cyclosporin A completely prevented Abeta-induced L-LTP deficits. These results suggest that Abeta(1-42) impaired both the induction and maintenance phase of dentate L-LTP through calcineurin-dependent mechanisms. In the concentration range effective for inhibiting L-LTP, Abeta(1-42) also reduced the amplitude of NMDA receptor-mediated synaptic currents in dentate granule cells via a postsynaptic mechanism. In addition, concurrent applications of Abeta(1-42) with the protein synthesis inhibitor caused no additive reduction of L-LTP, indicating a common mechanism underlying the action of both. Thus, inhibition of NMDA receptor channels and disruption of protein synthesis were two possible mechanisms contributing to Abeta-induced L-LTP impairment.