A peptide inhibitor of Tau-SH3 interactions ameliorates amyloid-ß toxicity.

The microtubule-associated protein Tau is strongly implicated in Alzheimer's disease (AD) and aggregates into neurofibrillary tangles in AD. Genetic reduction of Tau is protective in several animal models of AD and cell culture models of amyloid-ß (Aß) toxicity, making it an exciting therapeutic target for treating AD. A variety of evidence indicates that Tau's interactions with Fyn kinase and other SH3 domain-containing proteins, which bind to PxxP motifs in Tau's proline-rich domain, may contribute to AD deficits and Aß toxicity. Thus, we sought to determine if inhibiting Tau-SH3 interactions ameliorates Aß toxicity. We developed a peptide inhibitor of Tau-SH3 interactions and a proximity ligation assay (PLA)-based target engagement assay. Then, we used membrane trafficking and neurite degeneration assays to determine if inhibiting Tau-SH3 interactions ameliorated Aß oligomer (Aßo)-induced toxicity in primary hippocampal neurons from rats. We verified that Tau reduction ameliorated Aßo toxicity in neurons. Using PLA, we identified a peptide inhibitor that reduced Tau-SH3 interactions in HEK-293 cells and primary neurons. This peptide reduced Tau phosphorylation by Fyn without affecting Fyn's kinase activity state. In primary neurons, endogenous Tau-Fyn interaction was present primarily in neurites and was reduced by the peptide inhibitor, from which we inferred target engagement. Reducing Tau-SH3 interactions in neurons ameliorated Aßo toxicity by multiple outcome measures, namely Aßo-induced membrane trafficking abnormalities and neurite degeneration. Our results indicate that Tau-SH3 interactions are critical for Aßo toxicity and that inhibiting them is a promising therapeutic target for AD.

Synaptotoxicity in Alzheimer's Disease Involved a Dysregulation of Actin Cytoskeleton Dynamics through Cofilin 1 Phosphorylation.

Amyloid-ß (Aß) drives the synaptic impairment and dendritic spine loss characteristic of Alzheimer's disease (AD), but how Aß affects the actin cytoskeleton remains unknown and contentious. The actin-binding protein, cofilin-1 (cof1), is a major regulator of actin dynamics in dendritic spines, and is subject to phospho-regulation by multiple pathways, including the Rho-associated protein kinase (ROCK) pathway. While cof1 is implicated as a driver of the synaptotoxicity characteristic of the early phases of AD pathophysiology, questions remain about the molecular mechanisms involved. Cofilin-actin rods are observed in neurons exposed to Aß oligomers (Aßo) and in tissue from AD patients, and others have described an increased cofilin phosphorylation (p-cof1) in AD patients. Here, we report elevated p-cof1 of the postsynaptic enriched fraction of synaptosomes from cortical samples of male APP/PS1 mice and human AD cases of either sex. In primary cortical neurons, Aßo induced rapid actin stabilization and increased p-cof1 in the postsynaptic compartment of excitatory synapses within 30 min. Fluorescence recovery after photobleaching of actin-GFP and calcium imaging in live neurons expressing active or inactive cof1 mutants suggest that cof1 phosphorylation is necessary and sufficient for Aßo-induced synaptic impairment via actin stabilization before the reported formation of cofilin-actin rods. Moreover, the clinically available and well-tolerated ROCK inhibitor, fasudil, prevented Aßo-induced actin stabilization, synaptic impairment, and synaptic loss by blocking cofilin phosphorylation. Aßo also blocked the LTP-induced insertion of the AMPAR subunit, GluA1, at the postsynaptic density, in a fasudil-sensitive manner. These data support an important role for ROCKs and cofilin in mediating Aß-induced synaptic impairment.SIGNIFICANCE STATEMENT We report that amyloid-ß oligomers rapidly induce aberrant stabilization of F-actin within dendritic spines, which impairs synaptic strength and plasticity. Activation of the Rho-associated protein kinase (ROCK) pathway results in phosphorylation of cof1 and is sufficient to mediate Aßo-induced actin stabilization synaptic impairment and synaptic loss. Further, the ROCK inhibitor, fasudil, prevents cofilin phosphorylation, acute synaptic disruption, and synaptotoxicity in primary cortical neurons. Together, the herein presented data provide strong support for further study of the ROCK pathway as a therapeutic target for the cognitive decline and synaptotoxicity in Alzheimer's disease.

The Alzheimer's disease risk factor CD2AP maintains blood-brain barrier integrity.

CD2-associated protein (CD2AP) is a leading genetic risk factor for Alzheimer's disease, but little is known about the function of CD2AP in the brain. We studied CD2AP(-/-) mice to address this question. Because CD2AP(-/-) mice normally die by 6 weeks from nephrotic syndrome, we used mice that also express a CD2AP transgene in the kidney, but not brain, to attenuate this phenotype. CD2AP-deficient mice had no behavioral abnormalities except for mild motor and anxiety deficits in a subset of CD2AP(-/-) mice exhibiting severe nephrotic syndrome, associated with systemic illness. Pentylenetetrazol (PTZ)-induced seizures occurred with shorter latency in CD2AP(-/-) mice, but characteristics of these seizures on electroencephalography were not altered. As CD2AP is expressed in brain-adjacent endothelial cells, we hypothesized that the shorter latency to seizures without detectably different seizure characteristics may be due to increased penetration of PTZ related to compromised blood-brain barrier integrity. Using sodium fluorescein extravasation, we found that CD2AP(-/-) mice had reduced blood-brain barrier integrity. Neither seizure severity nor blood-brain barrier integrity was correlated with nephrotic syndrome, indicating that these effects are dissociable from the systemic illness associated with CD2AP deficiency. Confirming this dissociation, wild-type mice with induced nephrotic syndrome maintained an intact blood-brain barrier. Taken together, our results support a role of CD2AP in mediating blood-brain barrier integrity and suggest that cerebrovascular roles of CD2AP could contribute to its effects on Alzheimer's disease risk.

Rho-associated protein kinase 1 (ROCK1) is increased in Alzheimer's disease and ROCK1 depletion reduces amyloid-ß levels in brain.

Alzheimer's disease (AD) is the leading cause of dementia and mitigating amyloid-ß (Aß) levels may serve as a rational therapeutic avenue to slow AD progression. Pharmacologic inhibition of the Rho-associated protein kinases (ROCK1 and ROCK2) is proposed to curb Aß levels, and mechanisms that underlie ROCK2's effects on Aß production are defined. How ROCK1 affects Aß generation remains a critical barrier. Here, we report that ROCK1 protein levels were elevated in mild cognitive impairment due to AD (MCI) and AD brains compared to controls. Aß42 oligomers marginally increased ROCK1 and ROCK2 protein levels in neurons but strongly induced phosphorylation of Lim kinase 1 (LIMK1), suggesting that Aß42 activates ROCKs. RNAi depletion of ROCK1 or ROCK2 suppressed endogenous Aß40 production in neurons, and Aß40 levels were reduced in brains of ROCK1 heterozygous knock-out mice compared to wild-type littermate controls. ROCK1 knockdown decreased amyloid precursor protein (APP), and treatment with bafilomycin accumulated APP levels in neurons depleted of ROCK1. These observations suggest that reduction of ROCK1 diminishes Aß levels by enhancing APP protein degradation. Collectively, these findings support the hypothesis that both ROCK1 and ROCK2 are therapeutic targets to combat Aß production in AD. Mitigating amyloid-ß (Aß) levels is a rational strategy for Alzheimer's disease (AD) treatment, however, therapeutic targets with clinically available drugs are lacking. We hypothesize that Aß accumulation in mild cognitive impairment because of AD (MCI) and AD activates the RhoA/ROCK pathway which in turn fuels production of Aß. Escalation of this cycle over the course of many years may contribute to the buildup of amyloid pathology in MCI and/or AD.

Activity-dependent tau protein translocation to excitatory synapse is disrupted by exposure to amyloid-beta oligomers.

Tau is a microtubule-associated protein well known for its stabilization of microtubules in axons. Recently, it has emerged that tau participates in synaptic function as part of the molecular pathway leading to amyloid-beta (Aß)-driven synaptotoxicity in the context of Alzheimer's disease. Here, we report the implication of tau in the profound functional synaptic modification associated with synaptic plasticity. By exposing murine cultured cortical neurons to a pharmacological synaptic activation, we induced translocation of endogenous tau from the dendritic to the postsynaptic compartment. We observed similar tau translocation to the postsynaptic fraction in acute hippocampal slices subjected to long-term potentiation. When we performed live confocal microscopy on cortical neurons transfected with human-tau-GFP, we visualized an activity-dependent accumulation of tau in the postsynaptic density. Coprecipitation using phalloidin revealed that tau interacts with the most predominant cytoskeletal component present, filamentous actin. Finally, when we exposed cortical cultures to 100 nm human synthetic Aß oligomers (Aßo's) for 15 min, we induced mislocalization of tau into the spines under resting conditions and abrogated subsequent activity-dependent synaptic tau translocation. These changes in synaptic tau dynamics may rely on a difference between physiological and pathological phosphorylation of tau. Together, these results suggest that intense synaptic activity drives tau to the postsynaptic density of excitatory synapses and that Aßo-driven tau translocation to the spine deserves further investigation as a key event toward synaptotoxicity in neurodegenerative diseases.

Reciprocal disruption of neuronal signaling and Aß production mediated by extrasynaptic NMDA receptors: a downward spiral.

It is becoming increasingly clear that aberrant neuronal activity can be the cause and the result of amyloid beta production. Synaptic activation facilitates non-amyloidogenic processing of amyloid precursor protein (APP) and cell survival, primarily through synaptic NMDA receptors (NMDARs) and perhaps specifically those containing GluN2A-subunits. In contrast, extrasynaptic and GluN2B-containing NMDARs promote beta-secretase cleavage of APP into amyloid-beta (Aß). The opposing nature of these NMDAR populations is reflected in their control over cell survival and death pathways. Subtle changes in glutamate homeostasis may shift the balance between these pathways and could play a role in Alzheimer's disease (AD). Indeed, Aß production, regional loss of brain connectivity and neurodegeneration correlate with neuronal activity in AD patients. From another perspective, Aß oligomers (Aßo) alter neuronal signaling through several mechanisms involving NMDARs and intracellular calcium mishandling. While Aßo affect multiple receptors, GluN2B-NMDARs have emerged as primary mediators of altered synaptic plasticity and neurotoxicity. Memantine and its successor, NitroMemantine, are efficient at blocking or reversing the deleterious actions of Aßo largely due to their selectivity for extrasynaptic NMDARs. Recently, Aßo were shown to trigger astrocytic release of glutamate to the extrasynaptic space where it activates NMDARs to promote further Aß production and synaptic depression. Combined with the reciprocal regulation between neuronal activity and Aß production, extrasynaptic glutamate release adds to a maladaptive model and ultimately results in synaptotoxicity and neurodegeneration of AD. Extrasynaptic NMDAR antagonists remain as a promising therapeutic avenue by interfering with this cascade.

Development of small-molecule Tau-SH3 interaction inhibitors that prevent amyloid-ß toxicity and network hyperexcitability.

Alzheimer's disease (AD) is the leading cause of dementia and lacks highly effective treatments. Tau-based therapies hold promise. Tau reduction prevents amyloid-ß-induced dysfunction in preclinical models of AD and also prevents amyloid-ß-independent dysfunction in diverse disease models, especially those with network hyperexcitability, suggesting that strategies exploiting the mechanisms underlying Tau reduction may extend beyond AD. Tau binds several SH3 domain-containing proteins implicated in AD via its central proline-rich domain. We previously used a peptide inhibitor to demonstrate that blocking Tau interactions with SH3 domain-containing proteins ameliorates amyloid-ß-induced dysfunction. Here, we identify a top hit from high-throughput screening for small molecules that inhibit Tau-FynSH3 interactions and describe its optimization with medicinal chemistry. The resulting lead compound is a potent cell-permeable Tau-SH3 interaction inhibitor that binds Tau and prevents amyloid-ß-induced dysfunction, including network hyperexcitability. These data support the potential of using small molecule Tau-SH3 interaction inhibitors as a novel therapeutic approach to AD.

Alzheimer's disease risk gene BIN1 induces Tau-dependent network hyperexcitability.

Genome-wide association studies identified the BIN1 locus as a leading modulator of genetic risk in Alzheimer's disease (AD). One limitation in understanding BIN1's contribution to AD is its unknown function in the brain. AD-associated BIN1 variants are generally noncoding and likely change expression. Here, we determined the effects of increasing expression of the major neuronal isoform of human BIN1 in cultured rat hippocampal neurons. Higher BIN1 induced network hyperexcitability on multielectrode arrays, increased frequency of synaptic transmission, and elevated calcium transients, indicating that increasing BIN1 drives greater neuronal activity. In exploring the mechanism of these effects on neuronal physiology, we found that BIN1 interacted with L-type voltage-gated calcium channels (LVGCCs) and that BIN1-LVGCC interactions were modulated by Tau in rat hippocampal neurons and mouse brain. Finally, Tau reduction prevented BIN1-induced network hyperexcitability. These data shed light on BIN1's neuronal function and suggest that it may contribute to Tau-dependent hyperexcitability in AD.

Effects of chelators on mercury, iron, and lead neurotoxicity in cortical culture.

Chelation therapy for the treatment of acute, high dose exposure to heavy metals is accepted medical practice. However, a much wider use of metal chelators is by alternative health practitioners for so called "chelation therapy". Given this widespread and largely unregulated use of metal chelators it is important to understand the actions of these compounds. We tested the effects of four commonly used metal chelators, calcium disodium ethylenediaminetetraacetate (CaNa2EDTA), D-penicillamine (DPA), 2,3 dimercaptopropane-1-sulfonate (DMPS), and dimercaptosuccinic acid (DMSA) for their effects on heavy metal neurotoxicity in primary cortical cultures. We studied the toxicity of three forms of mercury, inorganic mercury (HgCl2), methyl mercury (MeHg), and ethyl mercury (thimerosal), as well as lead (PbCl2) and iron (Fe-citrate). DPA had the worst profile of effects, providing no protection while potentiating HgCl2, thimerosal, and Fe-citrate toxicity. DMPS and DMSA both attenuated HgCl2 toxicity and potentiated thimerosal and Fe toxicity, while DMPS also potentiated PbCl2 toxicity. CaNa2EDTA attenuated HgCl2 toxicity, but caused a severe potentiation of Fe-citrate toxicity. The ability of these chelators to attenuate the toxicity of various metals is quite restricted, and potentiation of toxicity is a serious concern. Specifically, protection is provided only against inorganic mercury, while it is lacking against the common form of mercury found in food, MeHg, and the form found in vaccines, thimerosal. The potentiation of Fe-citrate toxicity is of concern because of iron's role in oxidative stress in the body. Potentiation of iron toxicity could have serious health consequences when using chelation therapy.

The amyloid-ß oligomer Aß*56 induces specific alterations in neuronal signaling that lead to tau phosphorylation and aggregation.

Oligomeric forms of amyloid-forming proteins are believed to be the principal initiating bioactive species in many neurodegenerative disorders, including Alzheimer's disease (AD). Amyloid-ß (Aß) oligomers are implicated in AD-associated phosphorylation and aggregation of the microtubule-associated protein tau. To investigate the specific molecular pathways activated by different assemblies, we isolated various forms of Aß from Tg2576 mice, which are a model for AD. We found that Aß*56, a 56-kDa oligomer that is detected before patients develop overt signs of AD, induced specific changes in neuronal signaling. In primary cortical neurons, Aß*56 interacted with N-methyl-d-aspartate receptors (NMDARs), increased NMDAR-dependent Ca2+ influx, and consequently increased intracellular calcium concentrations and the activation of Ca2+-dependent calmodulin kinase IIα (CaMKIIα). In cultured neurons and in the brains of Tg2576 mice, activated CaMKIIα was associated with increased site-specific phosphorylation and missorting of tau, both of which are associated with AD pathology. In contrast, exposure of cultured primary cortical neurons to other oligomeric Aß forms (dimers and trimers) did not trigger these effects. Our results indicate that distinct Aß assemblies activate neuronal signaling pathways in a selective manner and that dissecting the molecular events caused by each oligomer may inform more effective therapeutic strategies.

Iron overload accelerates neuronal amyloid-ß production and cognitive impairment in transgenic mice model of Alzheimer's disease.

Iron dyshomeostasis is proving increasingly likely to be involved in the pathology of Alzheimer's disease (AD); yet, its mechanism is not well understood. Here, we investigated the AD-related mechanism(s) of iron-sulfate exposure in vitro and in vivo, using cultured primary cortical neurons and APP/PS1 AD-model mice, respectively. In both systems, we observed iron-induced disruptions of amyloid precursor protein (APP) processing, neuronal signaling, and cognitive behavior. Iron overload increased production of amyloidogenic KPI-APP and amyloid beta. Further, this APP misprocessing was blocked by MK-801 in vitro, suggesting the effect was N-methyl-D-aspartate receptor (NMDAR) dependent. Calcium imaging confirmed that 24 hours iron exposure led to disrupted synaptic signaling by augmenting GluN2B-containing NMDAR expression-GluN2B messenger RNA and protein levels were increased and promoting excessing extrasynaptic NMDAR signaling. The disrupted GluN2B expression was concurrent with diminished expression of the splicing factors, sc35 and hnRNPA1. In APP/PS1 mice, chronic iron treatment led to hastened progression of cognitive impairment with the novel object recognition discrimination index, revealing a deficit at the age of 4 months, concomitant with augmented GluN2B expression. Together, these data suggest iron-induced APP misprocessing and hastened cognitive decline occur through inordinate extrasynaptic NMDAR activation.

Synergistic toxicity of the environmental neurotoxins methylmercury and ß-N-methylamino-L-alanine.

Determination of the environmental factors involved in neurodegenerative diseases has been elusive. Methylmercury and ß-N-methylamino-L-alanine (BMAA) have both been implicated in this role. Exposure of primary cortical cultures to these compounds independently induced concentration-dependent neurotoxicity. Importantly, concentrations of BMAA (10-100 µM) that caused no toxicity alone potentiated methylmercury (3 µM) toxicity. In addition, concentrations of BMAA and methylmercury that had no effect by themselves on the main cellular antioxidant glutathione together decreased glutathione levels. Furthermore, the combined toxicity of methylmercury and BMAA was attenuated by the cell permeant form of glutathione, glutathione monoethyl ester. The results indicate a synergistic toxic effect of the environmental neurotoxins BMAA and methylmercury, and that the interaction is at the level of glutathione depletion.

Functional upregulation of system xc- by fibroblast growth factor-2.

The cystine/glutamate antiporter (system xc-) is a Na(+)-independent amino acid transport system. Disruption of this system may lead to multiple effects in the CNS including decreased cellular glutathione. Since multiple neurological diseases involve glutathione depletion, and disruption of growth factor signaling has also been implicated in these diseases, it is possible that some growth factors effects are mediated by regulation of system xc-. We tested the growth factors fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1 (IGF-1), neuregulin-1 (NRG), neurotrophin-4 (NT-4), and brain derived neurotrophic factor (BDNF) on system xc- mediated 14C-cystine uptake in mixed neuronal and glial cortical cultures. Only FGF-2 significantly increased cystine uptake. The effect was observed in astrocyte-enriched cultures, but not in cultures of neurons or microglia. The increase was blocked by the system xc- inhibitor (s)-4-carboxyphenylglycine, required at least 12 h FGF-2 treatment, and was prevented by the protein synthesis inhibitor cycloheximide. Kinetic analysis indicated FGF-2 treatment increased the V(max) for cystine uptake while the K(m) remained the same. Quantitative PCR showed an increase in mRNA for xCT, the functional subunit of system xc-, beginning at 3 h of FGF-2 treatment, with a dramatic increase after 12 h. Blocking FGFR1 with PD 166866 blocked the FGF-2 effect. Treatment with a PI3-kinase inhibitor (LY-294002) or a MEK/ERK inhibitor (U0126) for 1 h prior to and during the FGF-2 treatment, each partially blocked the increased cystine uptake. The upregulation of system xc- by FGF-2 may be responsible for some of the known physiological actions of FGF-2. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

Glutathione-mediated neuroprotection against methylmercury neurotoxicity in cortical culture is dependent on MRP1.

Methylmercury (MeHg) exposure at high concentrations poses significant neurotoxic threat to humans worldwide. The present study investigated the mechanisms of glutathione-mediated attenuation of MeHg neurotoxicity in primary cortical culture. MeHg (5 µM) caused depletion of mono- and disulfide glutathione in neuronal, glial and mixed cultures. Supplementation with exogenous glutathione, specifically glutathione monoethyl ester (GSHME) protected against the MeHg induced neuronal death. MeHg caused increased reactive oxygen species (ROS) formation measured by dichlorodihydrofluorescein (DCF) fluorescence with an early increase at 30 min and a late increase at 6h. This oxidative stress was prevented by the presence of either GSHME or the free radical scavenger, trolox. While trolox was capable of quenching the ROS, it showed no neuroprotection. Exposure to MeHg at subtoxic concentrations (3 µM) caused an increase in system x(c)(-) mediated (14)C-cystine uptake that was blocked by the protein synthesis inhibitor, cycloheximide (CHX). Interestingly, blockade of the early ROS burst prevented the functional upregulation of system x(c)(-). Inhibition of multidrug resistance protein-1 (MRP1) potentiated MeHg neurotoxicity and increased cellular MeHg. Taken together, these data suggest glutathione offers neuroprotection against MeHg toxicity in a manner dependent on MRP1-mediated efflux.

Selective death of cholinergic neurons induced by beta-methylamino-L-alanine.

Beta-N-methylamino-L-alanine (BMAA) is a nonprotein amino acid that may be involved in neurodegenerative diseases. It is produced by a large variety of cyanobacteria and is found at high levels in the brains of Alzheimer's disease and amyotrophic lateral sclerosis patients. Although BMAA is clearly a neurotoxin, previous studies using cortical cultures indicated that millimolar concentrations were required to cause toxicity. We tested the toxicity of BMAA in septal cultures containing cholinergic neurons and mesencephalic cultures containing dopaminergic neurons. We found that cholinergic, but not dopaminergic, neurons were selectively vulnerable to BMAA toxicity, with toxicity occurring at 30 microM. The toxicity of BMAA to total septal neurons involved activation of N-methyl D-aspartate receptors, whereas the death of cholinergic neurons was mediated by AMPA/kainate receptors.

beta-N-methylamino-l-alanine induces oxidative stress and glutamate release through action on system Xc(-).

beta-N-methylamino-l-alanine (BMAA) is a non-protein amino acid implicated in the neurodegenerative disease amyotrophic lateral sclerosis/Parkinson-dementia complex (ALS/PDC) on Guam. BMAA has recently been discovered in the brains of Alzheimer's patients in Canada and is produced by various species of cyanobacteria around the world. These findings suggest the possibility that BMAA may be of concern not only for specific groups of Pacific Islanders, but for a much larger population. Previous studies have indicated that BMAA can act as an excitotoxin by acting on the NMDA receptor. We have shown that the mechanism of neurotoxicity is actually three-fold; it involves not only direct action on the NMDA receptor, but also activation of metabotropic glutamate receptor 5 (mGluR5) and induction of oxidative stress. We now explore the mechanism by which BMAA activates the mGluR5 receptor and induces oxidative stress. We found that BMAA inhibits the cystine/glutamate antiporter (system Xc(-)) mediated cystine uptake, which in turn leads to glutathione depletion and increased oxidative stress. BMAA also appears to drive glutamate release via system Xc(-) and this glutamate induces toxicity through activation of the mGluR5 receptor. Therefore, the oxidative stress and mGluR5 activation induced by BMAA are both mediated through action at system Xc(-). The multiple mechanisms of BMAA toxicity, particularly the depletion of glutathione and enhanced oxidative stress, may account for its ability to induce complex neurodegenerative diseases.