Multi-Target Neuroprotective Compound Exhibits EAAT2-Modulatingand Alzheimer\u2019s Pathology\u2013Attenuating Effects in InVitro and In Vivo Models

Effects of vanillic acid on Aβ1-40-induced oxidative stress and learning and memory deficit in male rats

Synaptic loss is the earliest pathological change in Alzheimer disease (AD) and is the pathological change most directly correlated with the degree of dementia. ApoE4 is the major genetic risk factor for the age-dependent form of AD, which accounts for 95% of cases. Here we show that in synaptic networks formed from primary hippocampal neurons in culture, apoE3, but not apoE4, prevents the loss of synaptic networks produced by amyloid β oligomers (amylospheroids). Specific activators of PKCε, such as 8-(2-(2-pentyl-cyclopropylmethyl)-cyclopropyl)-octanoic acid methyl ester and bryostatin 1, protected against synaptic loss by amylospheroids, whereas PKCε inhibitors blocked this synaptic protection and also blocked the protection by apoE3. Blocking LRP1, an apoE receptor on the neuronal membrane, also blocked the protection by apoE. ApoE3, but not apoE4, induced the synthesis of PKCε mRNA and expression of the PKCε protein. Amyloid β specifically blocked the expression of PKCε but had no effect on other isoforms. These results suggest that protection against synaptic loss by apoE is mediated by a novel intracellular PKCε pathway. This apoE pathway may account for much of the protective effect of apoE and reduced risk for the age-dependent form of AD. This finding supports the potential efficacy of newly developed therapeutics for AD.

Alzheimer's disease (AD) is characterized by the progressive neuronal and synapse loss in brain regions associated with cognitive dysfunction as well as the deposition of amyloid plaques and neurofibrillary tangles. Synapse loss occurs early in the pathogenesis of AD and is regarded as the basis of dementia as observed in both patients and related transgenic mouse models. Therefore, restoration, prevention and/or reduction of synapse loss represents a major therapeutic target for AD. Excitatory (glutamatergic) synapses are formed between presynaptic axon terminals and highly specialized protrusions on the postsynaptic dendrites known as dendritic spines. In AD, a significant reduction of spine density and number has been observed. Filamentous actin (F-actin) is the major structural element of dendritic spines. Drebrin, a neuron-specific F-actin binding protein, is concentrated in dendritic spines and is known to play a role in dendritic spine morphology via modulation of actin dynamics. In AD, a significant downregulation of drebrin has been observed. Loss of drebrin has been suggested to destabilize actin cytoskeleton leading to dendritic pathology. The mechanism responsible for drebrin loss remains to be elucidated. Western blotting analysis has been used to measure drebrin protein level in Egr-1 KO mice as well primary hippocampal nuerons overexpressing Egr-1 via lentivirus-mediated infection. Drebrin mRNA level in primary hippocampal neurons was measured using qRT-PCR. In this study, we have found significant elevation of drebrin protein level in mice brain deficient in Early Growth Response 1 (Egr-1) transcription factor known to be upregulated in AD brain. When overexpression in primary hippocampal neurons in culture, Egr-1 causes reduction in drebrin level and decrease in the number of dendritic spines. In silico RVista analysis revealed three putative Egr-1 binding sites within the drebrin promoter. In vitro qRT-PCR showed a dose-dependent decrease of drebrin mRNA level in Egr-1 overexpressing neurons. Our data demonstrate that Egr-1 is an in vivo transcription suppressor of drebrin gene expression and suggest that Egr-1 plays a role in AD synaptic pathology by reducing drebrin level in the brain.

The neural cell adhesion molecule (NCAM) plays a key role in morphogenesis of the nervous system and in remodeling of neuronal connections accompanying regenerative and cognitive processes. Recently, a new synthetic ligand of NCAM, the C3-peptide, which binds to the NCAM IgI module, has been identified by means of combinatorial chemistry (Rønn, L. C. B, Olsen, M., Ostergaard, S., Kiselyov, V., Berezin, V., Mortensen, M. T., Lerche, M. H., Jensen, P. H., Soroka, V., Saffell, J. L., Doherty, P., Poulsen, F. M., Bock, E., Holm, A., and Saffells, J. L. (1999) Nat. Biotechnol. 17, 1000-1005). In vitro, the dendrimeric form of C3, termed C3d, disrupts NCAM-mediated cell adhesion, induces neurite outgrowth, and triggers intracellular signaling cascades similar to those activated by homophilic NCAM binding. The peptide may therefore be expected to regulate regeneration and synaptic plasticity. Here we demonstrate that in primary cultures of hippocampal neurons: 1) C3d induces a sustained neuritogenic response, the neuritogenic activity of the compound being dependent on the dose, starting time, and duration of peptide application; 2) the peptide triggers the neuritogenic response by forming an adhesive substratum necessary for NCAM-mediated neurite formation and elongation; 3) C3d promotes synapse formation; and 4) C3d modulates the presynaptic function, causing a transient increase of the function at low (2 and 5 microm) doses and a reduction when applied at a higher concentration (10 microm). The effect of the peptide is dependent on the activation of the fibroblast growth factor receptor. We suggest that C3d may constitute a useful lead for the development of compounds for treatment of various neurodegenerative disorders.

In the normal brain, tau protein is phosphorylated at a number of proline- and non-proline directed sites, which reduce tau microtubule binding and thus regulate microtubule dynamics. In Alzheimer disease (AD), tau is abnormally hyperphosphorylated, leading to neurofibrillary tangle formation and microtubule disruption, suggesting a loss of regulatory mechanisms controlling tau phosphorylation. Early growth response 1 (Egr-1) is a transcription factor that is significantly up-regulated in AD brain. The pathological significance of this up-regulation is not known. In this study, we found that lentivirus-mediated overexpression of Egr-1 in rat brain hippocampus and primary neurons in culture activates proline-directed kinase Cdk5, inactivates PP1, promotes tau phosphorylation at both proline-directed Ser(396/404) and non-proline-directed Ser(262) sites, and destabilizes microtubules. Furthermore, in Egr-1(-/-) mouse brain, Cdk5 activity was decreased, PP1 activity was increased, and tau phosphorylation was reduced at both proline-directed and non-proline-directed sites. By using nerve growth factor-exposed PC12 cells, we determined that Egr-1 activates Cdk5 to promote phosphorylation of tau and inactivates PP1 via phosphorylation. When Cdk5 was inhibited, tau phosphorylation at both proline- and non-proline directed sites and PP1 phosphorylation were blocked, indicating that Egr-1 acts through Cdk5. By using an in vitro kinase assay and HEK-293 cells transfected with tau, PP1, and Cdk5, we found that Cdk5 phosphorylates Ser(396/404) directly. In addition, by phosphorylating and inactivating PP1, Cdk5 promotes tau phosphorylation at Ser(262) indirectly. Our results indicate that Egr-1 is an in vivo regulator of tau phosphorylation and suggest that in AD brain increased levels of Egr-1 aberrantly activate an Egr-1/Cdk5/PP1 pathway, leading to accumulation of hyperphosphorylated tau, thus destabilizing the microtubule cytoskeleton.

The amyloid precursor protein (APP) and its proteolytic product amyloid beta (Abeta) are associated with both familial and sporadic forms of Alzheimer disease (AD). Aberrant expression and function of microRNAs has been observed in AD. Here, we show that in rat hippocampal neurons cultured in vitro, the down-regulation of Argonaute-2, a key component of the RNA-induced silencing complex, produced an increase in APP levels. Using site-directed mutagenesis, a microRNA responsive element (RE) for miR-101 was identified in the 3'-untranslated region (UTR) of APP. The inhibition of endogenous miR-101 increased APP levels, whereas lentiviral-mediated miR-101 overexpression significantly reduced APP and Abeta load in hippocampal neurons. In addition, miR-101 contributed to the regulation of APP in response to the proinflammatory cytokine interleukin-1beta (IL-lbeta). Thus, miR-101 is a negative regulator of APP expression and affects the accumulation of Abeta, suggesting a possible role for miR-101 in neuropathological conditions.

Endocytosis of Trk (tropomyosin-related kinase) receptors is critical for neurotrophin signal transduction and biological functions. However, the mechanism governing endocytosis of TrkB (tropomyosin-related kinase B) and the specific contributions of TrkB endocytosis to downstream signaling are unknown. In this study, we report that blocking clathrin, dynamin, or AP2 in cultured neurons of the central nervous system inhibited brain-derived neurotrophic factor (BDNF)-induced activation of Akt but not ERK. Treating neurons with the clathrin inhibitor monodansylcadaverine or a peptide that blocks dynamin function specifically abrogated Akt pathway activation in response to BDNF but did not affect the response of other downstream effectors or the up-regulation of immediate early genes neuropeptide Y and activity-regulated cytoskeleton-associated protein. Similar effects were found in neurons expressing small interfering RNA to silence AP2 or a dominant negative form of dynamin that inhibits clathrin-mediated endocytosis. In PC12 cells, ERK but not Akt activation required TrkA endocytosis following stimulation with nerve growth factor, whereas the opposite was true when TrkA-expressing neurons were stimulated with nerve growth factor in the central nervous system. Thus, the specific effects of internalized Trk receptors probably depend on the presence of cell type-specific modulators of neurotrophin signaling and not on differences inherent to Trk receptors themselves. Endocytosis-dependent activation of Akt in neurons was found to be critical for BDNF-supported survival and dendrite outgrowth. Together, these results demonstrate the functional requirement of clathrin- and dynamin-dependent endocytosis in generating the full intracellular response of neurons to BDNF in the central nervous system.

Chronic exposure to morphine decreases the expression of EAAT3 via opioid receptors in hippocampal neurons

The phenomenon of population aging, caused by increased life expectancy and declining birth rates, has resulted in a high, and continuously rising proportion of elderly people in the population. This means it is critical to improve our understanding of age-associated diseases and to develop new therapies for them. One of the conditions most commonly associated with aging is a decline in memory, which occurs at different rates in elderly individuals. At one end of the spectrum are cognitively healthy individuals who retain good memory capacities in old age. On the other are patients who suffer from Alzheimer’s disease and suffer a sharp decline in cognitive capabilities, with memory usually being one of the earliest-affected functions. There relationship between the various kinds of age-associated memory impairment and the degree to which they share molecular mechanisms is unclear. The aim of the experiments described in this thesis is to gain an understanding of age-associated memory impairment and Alzheimer's disease by studying RNA expression profiles. Cellular RNA undergoes constant turnover, being transcribed, translated and degraded at varying rates. It thus provides a dynamic index of cellular status, being required for housekeeping functions as well as response to stimuli, stress or changes in the cellular environment. A learning stimulus is associated with a change in expression levels of large number of genes, and learning is impaired in the absence of these gene expression changes. In addition to protein-coding genes, there are a number of regulatory RNAs that likely play a role in the process, but their functions are not yet clear. Thus studying the coding and non-coding RNA under situations of disease or impaired function gives an insight not only into cellular protein expression but also into the regulatory or compensatory mechanisms that occur in response to stimuli or to disease. Clearly, the RNA profile of a cell at any given time depends both on the underlying genome and on the environment of the cell, allowing the study of their interaction. For these reasons, next generation RNA sequencing, followed by analysis of coding and non-coding RNA expression profiles was the method chosen for the study. The first major aim of this project was to develop a mouse model that the inter-individual variation in age-associated memory impairment. We show that wild type mice show an age-dependent increase in intra-group variability that parallels the decline in memory. Thus, as in humans, a group of aging mice consists of some individuals who perform well on memory tests, at levels comparable to young mice and a some individuals who decline faster. These differences cannot be attributed to loss of motor or visual abilities and individual mice show remarkable consistency in day-to-day performance. This means that by testing over a number of days on the water maze task, ‘good learners’ can be distinguished from ‘poor learners’. This model was set up in a group of genetically identical wild-type mice which had been raised in the same environment, enabling us to study the hippocampal gene expression profiles and correlate them with memory performance in the absence of confounding factors. A large number of genes were found to have a significant correlation with performance, and a gene set enrichment analysis was performed on this dataset to look for functions or pathways that were over- or under-represented in the best- and worst-performing individuals. We found that high expression of the components of the translational machinery, including ribosomes, RNA-binding proteins, and translation initiation factors were associated with good memory performance in aging. Another finding was that high expression of components of the glutamatergic pathway was associated with age-associated memory impairment. These pathways merit further investigation as potential therapeutic targets in age-associated memory impairment. Alzheimer's disease, a condition that has a very strong connection with aging, is one of the age-associated diseases for which no effective therapies exist. One possible reason for this is that our knowledge about the underlying molecular mechanisms is still incomplete, relying mainly on the pathological evidence of amyloid-beta accumulation and to genetic linkage studies. However, the majority of patients with AD suffer from a sporadic form of the disease, with the strongest risk factor being age. There is still no clear explanation for this strong relationship between aging and AD. While aging is often associated with a mild degree of memory loss, the rapid decline characteristic of Alzheimer’s disease is probably associated with distinct pathomechanisms. Since the current methods to study the living brain in AD are limited to neuroimaging approaches, our knowledge of RNA expression in AD comes from post-mortem analyses and mouse models. In particular, we lack methods to study the progress of AD in live patients in the early stages of disease. Recently, it has been shown that cells secrete RNA into the extracellular space, enclosed in vesicles. If this RNA can be isolated and profiled from cerebrospinal fluid, it could serve as a “nanobiopsy” for brain tissue. Thus the second major aim of this project was to determine if extracellular RNA isolated from cerebrospinal fluid could be used to help understand the underlying molecular mechanisms in live patients with AD. To this end, we isolated the vesicle fractions from human cerebrospinal fluid and tested for RNA yield. We were able to obtain a robust and reproducible RNA expression profile from CSF extracellular vesicles (EVs) that was distinct from the cellular RNA profile. Primary hippocampal neurons in culture were used to compare the RNA derived from EVs with the RNA in the neurons of origin. Interestingly, when the cells were challenged by application of amyloid-beta, there was a detectable response in the secreted vesicular RNA. Before using this method to study CSF from patients with AD, the scalability of the RNA isolation and profiling protocol was tested for the small amounts of RNA typically available from 1‒2 ml of CSF. We show that it is possible to distinguish between subjects using as little as 0.25 ng of cerebrospinal EV-derived RNA. Finally we compared EV RNA between the cerebrospinal fluid of patients with Alzheimer's disease and control subjects. One potential application of CSF RNA profiling is the development of biomarkers that allow early disease diagnosis. We used a machine learning approach to predict AD based on CSF microRNA and other non-coding RNA and were able to achieve a prediction accuracy of over 80%. If these findings can be extended to a larger set of patients, RNA isolated from cerebrospinal fluid could provide a new method not only for further research into early Alzheimer's disease, but also be useful as a diagnostic marker.

Glycogen synthase kinase 3-beta (GSK3B) is overactive in Alzheimer's disease (AD), favoring the hyperphosphorylation of microtubule-associated protein Tau and the formation of neurofibrillary tangles. For this reason, GSK3B inhibition may be considered as a candidate disease-modifying approach in AD. In addition to the direct enzymatic inhibition (eg. by lithium), GSK3B can be also indirectly inactivated by phosphorylation at the Ser9 residue. This is a downstream effect of several intracellular cascades, including Akt and CaMKII signaling. Phospholipase A 2 (PLA 2) are key enzymes in membrane homeostasis and cell signaling. Reduced PLA 2 activity has been demonstrated in cerebral and peripheral tissues of patients with AD, and recent studies suggested that PLA 2 is further implicated in GSK3B homeostasis. The objective of this study is to evaluate the effect of the inhibition of PLA 2 on the GSK3B activity and to determine whether this effect involves CAMKII and Akt. Primary cultures of cortical and hippocampal neurons started from embryonic rats (E18) were treated after 4 four days in culture with different concentrations (5-250 μM) of methyl-aracdonyl-fluorophosphonate (MAFP), which is an irreversible, dual inhibitor of calcium-dependent and -independent cytosolic PLA 2. Treatment effects were evaluated by Western-blot, addressing the expression of total- and phosphorylated (Ser9) GSK3B, phosphorylated Akt (Ser473), and total CAMKII at protein level. The inhibition of GSK3B is a downstream effect of cytosolic PLA 2 inhibition, which apparently involves the activation of Akt and CAMKII. This effect seems to be specific to hippocampal neurons.

Author response: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

Transforming growth factors-β protect primary rat hippocampal neuronal cultures from degeneration induced by β-amyloid peptide

Anxiolytic and antidepressant-like activities of UFP-512, a novel selective delta opioid receptor agonist

Excitatory aminoacids (EAAs) are stored in glutamatergic neurons and related into synaptic cleft, where they can activate inotropic or metabotropic receptors. Their action ends due to transport mechanisms performed by EAAT transporters (EAAT1/GLAST, EAAT2/GLT1, EAAT3/EAAC1, and EAAT4 or EAAT5). Glutamate neurotoxicity has been described in several neurodegenerative diseases such as Alzheimer’s disease (AD), Huntington’s disease (HD), Parkinson’s disease (PD) and amyotropic lateral sclerosis ALS). Some drugs, such as paclitaxel, are able to increase translation of microRNA and could be possible used as regulatory against glutamate neurotoxicity. Abbreviations: AD: Alzheimer’s disease; ALS: amyotrophic lateral sclerosis; AMPA: α-amino-3-hydroxy-5-methyl-isoxazole-4propionate; Asp: aspartate; CSF: cerebral spinal fluid; EAA: excitatory amino acid; EAAC1 (EAAT3): excitatory amino acid carrier; EAAT: excitatory amino acid transporter; GABA: gamma-aminobutyric acid; GDH: glutamate dehydrogenase; GLAST (EAAT1): glutamate – aspartate transporter; GLT1 (EAAT2): glutamate transporter; iGluR: ionotropic glutamate receptor; KA: kaninic acid; L-Glu: L-glutamate; mGluR: metabotropic glutamate receptor; miR: micro RNA; NMDA: N-methyl-D-aspartate; PD: Parkinson’s disease Introduction Some amino acids act as neurotransmitters in the nervous system, being glutamate and aspartate the common excitatory amino acids and GABA, glycine and taurine the inhibitory ones. Of these amino acids, glutamate and GABA are intimately associated, as their metabolism is associated through glutamic acid decarboxylase (E.C. 4.1.1.15.) (Figure 1). Furthermore, GABA and glutamic acid effects are antagonic and they are related with CO2 fixation (relevant to central ventilation). Glutamic acid metabolism is also related with NH3 detoxification (due to a reduction in α-ketoglutarate and glutamate contents and an increase in glutamine). The efflux of glutamate from brain across the hemato-encephalic barrier is much higher than the influx [1-3], meaning that metabolism of glutamate must play an important role in regulating the brain glutamate levels. Studies on metabolic generation of glutamate/glutamine by using radioactive substrates in brain shows that two pathways are involved. Glucose, glycerol, lactate, pyruvate, α-ketoglutarate and β-hydroxybutyrate seem to be metabolized to glutamate in neurons [4,5], as a low specific radioactivity of glutamine is obtained. In glial cells [6], where higher glutamine synthase is present [7,8], low radiolabelled glutamate and higher glutamine marked are obtained. This is the case of acetate, propionate, butyrate, citrate, leucine, GABA, aspartate and ammonia [9]. In order to decrease glutamic acid in synaptic cleft, excitatory amino acids transporters have an important effect. Therefore, in this paper we present some aspects of these proteins. Glutamatergic neurotransmission The excitatory amino acids (EAAs) are stored in synaptic vesicles in glutamatergic neurons and, upon an action potential, are released via exocytosis into the synaptic cleft where they can activate two different families of receptors: ionotropic (ligand-gated ion channels) and metabotropic (GTP-binding protein coupled) receptors. The ligandgated ion channels are further divided into three families: α-amino3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), kainate (KA) and N–methyl-D-aspartate (NMDA). While AMPA and kainate receptors mediate rapid depolarizing responses at most synapses in the mammalian central nervous system [10], the NMDA receptor participates in synaptic plasticity and synapse formation [11]. The family of metabotropic receptors consists of at least eight subtypes and are involved in the modulation of synaptic signaling by EAAs and other neurotransmitters [12]. The termination of the EAA action takes place by an uptake mechanism that uses the Na+, K+ and pH gradients as a driving force to translocate the neurotransmitters against their concentration gradients, keeping their concentration below the level that activates their receptors (~1 μM) [13-15] (Figure 2). Excessive activation of EAA receptors contributes to brain injury through a process known as excitotoxicity. Therefore, this transport mechanism is not only important for ensuring accurate synaptic signaling but also for limiting the EAA-mediated excitotoxicity Excitatory amino acid (EAA) transporters Three broad subtypes of EAA transport activities have been Correspondence to: Josep J Centelles, Departament de Bioquimica i Biologia Molecular (Biologia), Facultat de Biologia, Universitat de Barcelona, Avda Diagonal, 643. Edifici Prevosti, planta-2, 08028, Barcelona, Spain, Tel: 934021870; E-mail: josepcentelles@ub.edu Received: January 16, 2016; Accepted: January 27, 2016; Published: January 30, 2016 Centelles JJ (2016) Glutamate transporters: the regulatory proteins for excitatory/excitotoxic glutamate in brain J Transl Sci, 2016 doi: 10.15761/JTS.1000123 Volume 2(1): 92-99 extracellular concentrations of EAAs by reducing the driving force required to transport an EAA into the cytoplasm. The second activity is a chloride-dependent transport which exchanges amino acids identified in brain preparations. One type, which is directly coupled to ATP hydrolysis, introduces glutamate into vesicles for release upon depolarization of the synaptic terminal [16]. It indirectly ensures low Glutamine ADP + Pi Glutamine synthetase (GS) (E.C. 6.3.1.2.) Glutaminase (Gase) (E.C. 3.5.1.2.) NH4 H2O Glutamate GABA Glutamate decarboxylase (GD) (E.C. 4.1.1.15.) Oxalacetate Glutamate dehydrogenase (GDH) (E.C. 1.4.1.2.) Glutamate oxalacetate transaminase (GOT) (E.C. 2.6.1.1.) NH4 + NADH Aspartate α-Ketoglutarate CO2 NH4 + ATP

Alzheimer's disease (AD), a chronic, progressive, neurodegenerative disorder, is the most common type of dementia. Beta amyloid (Aβ) peptide aggregation and phosphorylated tau protein accumulation are considered as one of the causes for AD. Our previous studies have demonstrated the neuroprotective effect of the Rho kinase inhibitor fasudil, but the mechanism remains elucidated. In the present study, we examined the effects of fasudil on Aβ1-42 aggregation and apoptosis and identified the intracellular signaling pathways involved in these actions in primary cultures of mouse hippocampal neurons. The results showed that fasudil increased neurite outgrowth (52.84%), decreased Aβ burden (46.65%), Tau phosphorylation (96.84%), and ROCK-II expression. In addition, fasudil reversed Aβ1-42-induced decreased expression of Bcl-2 and increases in caspase-3, cleaved-PARP, phospho-JNK(Thr183/Tyr185), and phospho-ASK1(Ser966). Further, fasudil decreased mitochondrial membrane potential and intracellular calcium overload in the neurons treated with Aβ1-42. These results suggest that inhibition of Rho kinase by fasudil reverses Aβ1-42-induced neuronal apoptosis via the ASK1/JNK signal pathway, calcium ions, and mitochondrial membrane potential. Fasudil could be a drug of choicefor treatment of Alzheimer's disease.