Tau is not necessary for amyloid-ß-induced synaptic and memory impairments.

The amyloid hypothesis posits that the amyloid-beta (Aß) protein precedes and requires microtubule-associated protein tau in a sort of trigger-bullet mechanism leading to Alzheimer's disease (AD) pathology. This sequence of events has become dogmatic in the AD field and is used to explain clinical trial failures due to a late start of the intervention when Aß already activated tau. Here, using a multidisciplinary approach combining molecular biological, biochemical, histopathological, electrophysiological, and behavioral methods, we demonstrated that tau suppression did not protect against Aß-induced damage of long-term synaptic plasticity and memory, or from amyloid deposition. Tau suppression could even unravel a defect in basal synaptic transmission in a mouse model of amyloid deposition. Similarly, tau suppression did not protect against exogenous oligomeric tau-induced impairment of long-term synaptic plasticity and memory. The protective effect of tau suppression was, in turn, confined to short-term plasticity and memory. Taken together, our data suggest that therapies downstream of Aß and tau together are more suitable to combat AD than therapies against one or the other alone.

Synaptic and memory dysfunction induced by tau oligomers is rescued by up-regulation of the nitric oxide cascade.

BACKGROUND: Soluble aggregates of oligomeric forms of tau protein (oTau) have been associated with impairment of synaptic plasticity and memory in Alzheimer's disease. However, the molecular mechanisms underlying the synaptic and memory dysfunction induced by elevation of oTau are still unknown. METHODS: This work used a combination of biochemical, electrophysiological and behavioral techniques. Biochemical methods included analysis of phosphorylation of the cAMP-responsive element binding (CREB) protein, a transcriptional factor involved in memory, histone acetylation, and expression immediate early genes c-Fos and Arc. Electrophysiological methods included assessment of long-term potentiation (LTP), a type of synaptic plasticity thought to underlie memory formation. Behavioral studies investigated both short-term spatial memory and associative memory. These phenomena were examined following oTau elevation. RESULTS: Levels of phospho-CREB, histone 3 acetylation at lysine 27, and immediate early genes c-Fos and Arc, were found to be reduced after oTau elevation during memory formation. These findings led us to explore whether up-regulation of various components of the nitric oxide (NO) signaling pathway impinging onto CREB is capable of rescuing oTau-induced impairment of plasticity, memory, and CREB phosphorylation. The increase of NO levels protected against oTau-induced impairment of LTP through activation of soluble guanylyl cyclase. Similarly, the elevation of cGMP levels and stimulation of the cGMP-dependent protein kinases (PKG) re-established normal LTP after exposure to oTau. Pharmacological inhibition of cGMP degradation through inhibition of phosphodiesterase 5 (PDE5), rescued oTau-induced LTP reduction. These findings could be extrapolated to memory because PKG activation and PDE5 inhibition rescued oTau-induced memory impairment. Finally, PDE5 inhibition re-established normal elevation of CREB phosphorylation and cGMP levels after memory induction in the presence of oTau. CONCLUSIONS: Up-regulation of CREB activation through agents acting on the NO cascade might be beneficial against tau-induced synaptic and memory dysfunctions.

Role of Amyloid-ß and Tau Proteins in Alzheimer's Disease: Confuting the Amyloid Cascade.

The "Amyloid Cascade Hypothesis" has dominated the Alzheimer's disease (AD) field in the last 25 years. It posits that the increase of amyloid-ß (Aß) is the key event in AD that triggers tau pathology followed by neuronal death and eventually, the disease. However, therapeutic approaches aimed at decreasing Aß levels have so far failed, and tau-based clinical trials have not yet produced positive findings. This begs the question of whether the hypothesis is correct. Here we have examined literature on the role of Aß and tau in synaptic dysfunction, memory loss, and seeding and spreading of AD, highlighting important parallelisms between the two proteins in all of these phenomena. We discuss novel findings showing binding of both Aß and tau oligomers to amyloid-ß protein precursor (AßPP), and the requirement for the presence of this protein for both Aß and tau to enter neurons and induce abnormal synaptic function and memory. Most importantly, we propose a novel view of AD pathogenesis in which extracellular oligomers of Aß and tau act in parallel and upstream of AßPP. Such a view will call for a reconsideration of therapeutic approaches directed against Aß and tau, paving the way to an increased interest toward AßPP, both for understanding the pathogenesis of the disease and elaborating new therapeutic strategies.

LTP and memory impairment caused by extracellular Aß and Tau oligomers is APP-dependent.

The concurrent application of subtoxic doses of soluble oligomeric forms of human amyloid-beta (oAß) and Tau (oTau) proteins impairs memory and its electrophysiological surrogate long-term potentiation (LTP), effects that may be mediated by intra-neuronal oligomers uptake. Intrigued by these findings, we investigated whether oAß and oTau share a common mechanism when they impair memory and LTP in mice. We found that as already shown for oAß, also oTau can bind to amyloid precursor protein (APP). Moreover, efficient intra-neuronal uptake of oAß and oTau requires expression of APP. Finally, the toxic effect of both extracellular oAß and oTau on memory and LTP is dependent upon APP since APP-KO mice were resistant to oAß- and oTau-induced defects in spatial/associative memory and LTP. Thus, APP might serve as a common therapeutic target against Alzheimer's Disease (AD) and a host of other neurodegenerative diseases characterized by abnormal levels of Aß and/or Tau.

Time-dependent reversal of synaptic plasticity induced by physiological concentrations of oligomeric Aß42: an early index of Alzheimer's disease.

The oligomeric amyloid-ß (Aß) peptide is thought to contribute to the subtle amnesic changes in Alzheimer's disease (AD) by causing synaptic dysfunction. Here, we examined the time course of synaptic changes in mouse hippocampal neurons following exposure to Aß42 at picomolar concentrations, mimicking its physiological levels in the brain. We found opposite effects of the peptide with short exposures in the range of minutes enhancing synaptic plasticity, and longer exposures lasting several hours reducing it. The plasticity reduction was concomitant with an increase in the basal frequency of spontaneous neurotransmitter release, a higher basal number of functional presynaptic release sites, and a redistribution of synaptic proteins including the vesicle-associated proteins synapsin I, synaptophysin, and the post-synaptic glutamate receptor I. These synaptic alterations were mediated by cytoskeletal changes involving actin polymerization and p38 mitogen-activated protein kinase. These in vitro findings were confirmed in vivo with short hippocampal infusions of picomolar Aß enhancing contextual memory and prolonged infusions impairing it. Our findings provide a model for initiation of synaptic dysfunction whereby exposure to physiologic levels of Aß for a prolonged period of time causes microstructural changes at the synapse which result in increased transmitter release, failure of synaptic plasticity, and memory loss.

Novel Selective Calpain 1 Inhibitors as Potential Therapeutics in Alzheimer's Disease.

Alzheimer's disease, one of the most important brain pathologies associated with neurodegenerative processes, is related to overactivation of calpain-mediated proteolysis. Previous data showed a compelling efficacy of calpain inhibition against abnormal synaptic plasticity and memory produced by the excess of amyloid-ß, a distinctive marker of the disease. Moreover, a beneficial effect of calpain inhibitors in Alzheimer's disease is predictable by the occurrence of calpain hyperactivation leading to impairment of memory-related pathways following abnormal calcium influxes that might ensue independently of amyloid-ß elevation. However, molecules currently available as effective calpain inhibitors lack adequate selectivity. This work is aimed at characterizing the efficacy of a novel class of epoxide-based inhibitors, synthesized to display improved selectivity and potency towards calpain 1 compared to the prototype epoxide-based generic calpain inhibitor E64. Both functional and preliminary toxicological investigations proved the efficacy, potency, and safety of the novel and selective calpain inhibitors NYC438 and NYC488 as possible therapeutics against the disease.

Re-engineering a neuroprotective, clinical drug as a procognitive agent with high in vivo potency and with GABAA potentiating activity for use in dementia.

BACKGROUND: Synaptic dysfunction is a key event in pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD) where synapse loss pathologically correlates with cognitive decline and dementia. Although evidence suggests that aberrant protein production and aggregation are the causative factors in familial subsets of such diseases, drugs singularly targeting these hallmark proteins, such as amyloid-ß, have failed in late stage clinical trials. Therefore, to provide a successful disease-modifying compound and address synaptic dysfunction and memory loss in AD and mixed pathology dementia, we repurposed a clinically proven drug, CMZ, with neuroprotective and anti-inflammatory properties via addition of nitric oxide (NO) and cGMP signaling property. RESULTS: The novel compound, NMZ, was shown to retain the GABAA potentiating actions of CMZ in vitro and sedative activity in vivo. Importantly, NMZ restored LTP in hippocampal slices from AD transgenic mice, whereas CMZ was without effect. NMZ reversed amnestic blockade of acetylcholine receptors by scopolamine as well as NMDA receptor blockade by a benzodiazepine and a NO synthase inhibitor in the step-through passive avoidance (STPA) test of learning and working memory. A PK/PD relationship was developed based on STPA analysis coupled with pharmacokinetic measures of drug levels in the brain: at 1 nM concentration in brain and plasma, NMZ was able to restore memory consolidation in mice. CONCLUSION: Our findings show that NMZ embodies a promising pharmacological approach targeting synaptic dysfunction and opens new avenues for neuroprotective intervention strategies in mixed pathology AD, neurodegeneration, and dementia.

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus.

Alzheimer's disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer's disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer's disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Synaptic therapy in Alzheimer's disease: a CREB-centric approach.

Therapeutic attempts to cure Alzheimer's disease (AD) have failed, and new strategies are desperately needed. Motivated by this reality, many laboratories (including our own) have focused on synaptic dysfunction in AD because synaptic changes are highly correlated with the severity of clinical dementia. In particular, memory formation is accompanied by altered synaptic strength, and this phenomenon (and its dysfunction in AD) has been a recent focus for many laboratories. The molecule cyclic adenosine monophosphate response element-binding protein (CREB) is at a central converging point of pathways and mechanisms activated during the processes of synaptic strengthening and memory formation, as CREB phosphorylation leads to transcription of memory-associated genes. Disruption of these mechanisms in AD results in a reduction of CREB activation with accompanying memory impairment. Thus, it is likely that strategies aimed at these mechanisms will lead to future therapies for AD. In this review, we will summarize literature that investigates 5 possible therapeutic pathways for rescuing synaptic dysfunction in AD: 4 enzymatic pathways that lead to CREB phosphorylation (the cyclic adenosine monophosphate cascade, the serine/threonine kinases extracellular regulated kinases 1 and 2, the nitric oxide cascade, and the calpains), as well as histone acetyltransferases and histone deacetylases (2 enzymes that regulate the histone acetylation necessary for gene transcription).

Regulation of synaptic plasticity and cognition by SUMO in normal physiology and Alzheimer's disease.

Learning and memory and the underlying cellular correlate, long-term synaptic plasticity, involve regulation by posttranslational modifications (PTMs). Here we demonstrate that conjugation with the small ubiquitin-like modifier (SUMO) is a novel PTM required for normal synaptic and cognitive functioning. Acute inhibition of SUMOylation impairs long-term potentiation (LTP) and hippocampal-dependent learning. Since Alzheimer's disease (AD) prominently features both synaptic and PTM dysregulation, we investigated SUMOylation under pathology induced by amyloid-ß (Aß), a primary neurotoxic molecule implicated in AD. We observed that SUMOylation is dysregulated in both human AD brain tissue and the Tg2576 transgenic AD mouse model. While neuronal activation normally induced upregulation of SUMOylation, this effect was impaired by Aß42 oligomers. However, supplementing SUMOylation via transduction of its conjugating enzyme, Ubc9, rescued Aß-induced deficits in LTP and hippocampal-dependent learning and memory. Our data establish SUMO as a novel regulator of LTP and hippocampal-dependent cognition and additionally implicate SUMOylation impairments in AD pathogenesis.

Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers I: Abeta 42 oligomer binding to specific neuronal receptors is displaced by drug candidates that improve cognitive deficits.

Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1-42 oligomers is proposed to underlie cognitive decline in Alzheimer's disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer's disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors--i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models and sustain improvement long-term, representing a novel mechanism of action for disease-modifying Alzheimer's therapeutics.

Dynamin 1 is required for memory formation.

Dynamin 1-3 isoforms are known to be involved in endocytotic processes occurring during synaptic transmission. No data has directly linked dynamins yet with normal animal behavior. Here we show that dynamin pharmacologic inhibition markedly impairs hippocampal-dependent associative memory. Memory loss was associated with changes in synaptic function occurring during repetitive stimulation that is thought to be linked with memory induction. Synaptic fatigue was accentuated by dynamin inhibition. Moreover, dynamin inhibition markedly reduced long-term potentiation, post-tetanic potentiation, and neurotransmitter released during repetitive stimulation. Most importantly, the effect of dynamin inhibition onto memory and synaptic plasticity was due to a specific involvement of the dynamin 1 isoform, as demonstrated through a genetic approach with siRNA against this isoform to temporally block it. Taken together, these findings identify dynamin 1 as a key protein for modulation of memory and release evoked by repetitive activity.

Design, synthesis, and optimization of novel epoxide incorporating peptidomimetics as selective calpain inhibitors.

Hyperactivation of the calcium-dependent cysteine protease calpain 1 (Cal1) is implicated as a primary or secondary pathological event in a wide range of illnesses and in neurodegenerative states, including Alzheimer's disease (AD). E-64 is an epoxide-containing natural product identified as a potent nonselective, calpain inhibitor, with demonstrated efficacy in animal models of AD. By use of E-64 as a lead, three successive generations of calpain inhibitors were developed using computationally assisted design to increase selectivity for Cal1. First generation analogues were potent inhibitors, effecting covalent modification of recombinant Cal1 catalytic domain (Cal1cat), demonstrated using LC-MS/MS. Refinement yielded second generation inhibitors with improved selectivity. Further library expansion and ligand refinement gave three Cal1 inhibitors, one of which was designed as an activity-based protein profiling probe. These were determined to be irreversible and selective inhibitors by kinetics studies comparing full length Cal1 with the general cysteine protease papain.

Design and synthesis of neuroprotective methylthiazoles and modification as NO-chimeras for neurodegenerative therapy.

Learning and memory deficits in Alzheimer's disease (AD) result from synaptic failure and neuronal loss, the latter caused in part by excitotoxicity and oxidative stress. A therapeutic approach is described that uses NO-chimeras directed at restoration of both synaptic function and neuroprotection. 4-Methylthiazole (MZ) derivatives were synthesized, based upon a lead neuroprotective pharmacophore acting in part by GABA(A) receptor potentiation. MZ derivatives were assayed for protection of primary neurons against oxygen-glucose deprivation and excitotoxicity. Selected neuroprotective derivatives were incorporated into NO-chimera prodrugs, coined nomethiazoles. To provide proof of concept for the nomethiazole drug class, selected examples were assayed for restoration of synaptic function in hippocampal slices from AD-transgenic mice, reversal of cognitive deficits, and brain bioavailability of the prodrug and its neuroprotective MZ metabolite. Taken together, the assay data suggest that these chimeric nomethiazoles may be of use in treatment of multiple components of neurodegenerative disorders, such as AD.

Furoxans (1,2,5-oxadiazole-N-oxides) as novel NO mimetic neuroprotective and procognitive agents.

Furoxans (1,2,5-oxadiazole-N-oxides) are thiol-bioactivated NO-mimetics that have not hitherto been studied in the CNS. Incorporation of varied substituents adjacent to the furoxan ring system led to modulation of reactivity toward bioactivation, studied by HPLC-MS/MS analysis of reaction products. Attenuated reactivity unmasked the cytoprotective actions of NO in contrast to the cytotoxic actions of higher NO fluxes reported previously for furoxans. Neuroprotection was observed in primary neuronal cell cultures following oxygen glucose deprivation (OGD). Neuroprotective activity was observed to correlate with thiol-dependent bioactivation to produce NO(2)(-), but not with depletion of free thiol itself. Neuroprotection was abrogated upon cotreatment with a sGC inhibitor, ODQ, thus supporting activation of the NO/sGC/CREB signaling cascade by furoxans. Long-term potentiation (LTP), essential for learning and memory, has been shown to be potentiated by NO signaling, therefore, a peptidomimetic furoxan was tested in hippocampal slices treated with oligomeric amyloid-ß peptide (Aß) and was shown to restore synaptic function. The novel observation of furoxan activity of potential therapeutic use in the CNS warrants further studies.

Endogenous amyloid-ß is necessary for hippocampal synaptic plasticity and memory.

OBJECTIVE: The goal of this study was to investigate the role of endogenous amyloid-ß peptide (Aß) in healthy brain. METHODS: Long-term potentiation (LTP), a type of synaptic plasticity that is thought to be associated with learning and memory, was examined through extracellular field recordings from the CA1 region of hippocampal slices, whereas behavioral techniques were used to assess contextual fear memory and reference memory. Amyloid precursor protein (APP) expression was reduced through small interfering RNA (siRNA) technique. RESULTS: We found that both antirodent Aß antibody and siRNA against murine APP reduced LTP as well as contextual fear memory and reference memory. These effects were rescued by the addition of human Aß42, suggesting that endogenously produced Aß is needed for normal LTP and memory. Furthermore, the effect of endogenous Aß on plasticity and memory was likely due to regulation of transmitter release, activation of α7-containing nicotinic acetylcholine receptors, and Aß42 production. INTERPRETATION: Endogenous Aß42 is a critical player in synaptic plasticity and memory within the normal central nervous system. This needs to be taken into consideration when designing therapies aiming at reducing Aß levels to treat Alzheimer disease.

Danish dementia mice suggest that loss of function and not the amyloid cascade causes synaptic plasticity and memory deficits.

According to the prevailing "amyloid cascade hypothesis," genetic dementias such as Alzheimer's disease and familial Danish dementia (FDD) are caused by amyloid deposits that trigger tauopathy, neurodegeneration, and behavioral/cognitive alterations. To efficiently reproduce amyloid lesions, murine models of human dementias invariably use transgenic expression systems. However, recent FDD transgenic models showed that Danish amyloidosis does not cause memory defects, suggesting that other mechanisms cause Danish dementia. We studied an animal knock-in model of FDD (FDD(KI/+)) genetically congruous with human cases. FDD(KI/+) mice present reduced Bri2 levels, impaired synaptic plasticity and severe hippocampal memory deficits. These animals show no cerebral lesions that are reputed characteristics of human dementia, such as tangles or amyloid plaques. Bri2(+/-) mice exhibit synaptic and memory deficits similar to FDD(KI/+) mice, and memory loss of FDD(KI/+) mice is prevented by expression of WT BRI2, indicating that Danish dementia is caused by loss of BRI2 function. Together, the data suggest that clinical dementia in Danish patients occurs via a loss of function mechanism and not as a result of amyloidosis and tauopathy.