Neuromodulatory Action of Picomolar Extracellular Aß42 Oligomers on Presynaptic and Postsynaptic Mechanisms Underlying Synaptic Function and Memory.

Failure of anti-amyloid-ß peptide (Aß) therapies against Alzheimer's disease (AD), a neurodegenerative disorder characterized by high amounts of the peptide in the brain, raised the question of the physiological role of Aß released at low concentrations in the healthy brain. To address this question, we studied the presynaptic and postsynaptic mechanisms underlying the neuromodulatory action of picomolar amounts of oligomeric Aß42 (oAß42) on synaptic glutamatergic function in male and female mice. We found that 200 pm oAß42 induces an increase of frequency of miniature EPSCs and a decrease of paired pulse facilitation, associated with an increase in docked vesicle number, indicating that it augments neurotransmitter release at presynaptic level. oAß42 also produced postsynaptic changes as shown by an increased length of postsynaptic density, accompanied by an increased expression of plasticity-related proteins such as cAMP-responsive element binding protein phosphorylated at Ser133, calcium-calmodulin-dependent kinase II phosphorylated at Thr286, and brain-derived neurotrophic factor, suggesting a role for Aß in synaptic tagging. These changes resulted in the conversion of early into late long-term potentiation through the nitric oxide/cGMP/protein kinase G intracellular cascade consistent with a cGMP-dependent switch from short- to long-term memory observed in vivo after intrahippocampal administration of picomolar amounts of oAß42 These effects were present upon extracellular but not intracellular application of the peptide and involved α7 nicotinic acetylcholine receptors. These observations clarified the physiological role of oAß42 in synaptic function and memory formation providing solid fundamentals for investigating the pathological effects of high Aß levels in the AD brains.SIGNIFICANCE STATEMENT High levels of oligomeric amyloid-ß42 (oAß42) induce synaptic dysfunction leading to memory impairment in Alzheimer's disease (AD). However, at picomolar concentrations, the peptide is needed to ensure long-term potentiation (LTP) and memory. Here, we show that extracellular 200 pm oAß42 concentrations increase neurotransmitter release, number of docked vesicles, postsynaptic density length, and expression of plasticity-related proteins leading to the conversion of early LTP into late LTP and of short-term memory into long-term memory. These effects require α7 nicotinic acetylcholine receptors and are mediated through the nitric oxide/cGMP/protein kinase G pathway. The knowledge of Aß function in the healthy brain might be useful to understand the causes leading to its increase and detrimental effect in AD.

Amyloid-ß Peptide Is Needed for cGMP-Induced Long-Term Potentiation and Memory.

High levels of amyloid-ß peptide (Aß) have been related to Alzheimer's disease pathogenesis. However, in the healthy brain, low physiologically relevant concentrations of Aß are necessary for long-term potentiation (LTP) and memory. Because cGMP plays a key role in these processes, here we investigated whether the cyclic nucleotide cGMP influences Aß levels and function during LTP and memory. We demonstrate that the increase of cGMP levels by the phosphodiesterase-5 inhibitors sildenafil and vardenafil induces a parallel release of Aß due to a change in the approximation of amyloid precursor protein (APP) and the ß-site APP cleaving enzyme 1. Moreover, electrophysiological and behavioral studies performed on animals of both sexes showed that blocking Aß function, by using anti-murine Aß antibodies or APP knock-out mice, prevents the cGMP-dependent enhancement of LTP and memory. Our data suggest that cGMP positively regulates Aß levels in the healthy brain which, in turn, boosts synaptic plasticity and memory.SIGNIFICANCE STATEMENT Amyloid-ß (Aß) is a key pathogenetic factor in Alzheimer's disease. However, low concentrations of endogenous Aß, mimicking levels of the peptide in the healthy brain, enhance hippocampal long-term potentiation (LTP) and memory. Because the second messenger cGMP exerts a central role in LTP mechanisms, here we studied whether cGMP affects Aß levels and function during LTP. We show that cGMP enhances Aß production by increasing the APP/BACE-1 convergence in endolysosomal compartments. Moreover, the cGMP-induced enhancement of LTP and memory was disrupted by blockade of Aß, suggesting that the physiological effect of the cyclic nucleotide on LTP and memory is dependent upon Aß.

Nitric oxide/cGMP/CREB pathway and amyloid-beta crosstalk: From physiology to Alzheimer's disease.

The nitric oxide (NO)/cGMP pathway has been extensively studied for its pivotal role in synaptic plasticity and memory processes, resulting in an increase of cAMP response element-binding (CREB) phosphorylation, and consequent synthesis of plasticity-related proteins. The NO/cGMP/CREB signaling is downregulated during aging and neurodegenerative disorders and is affected by Amyloid-ß peptide (Aß) and tau protein, whose increase and deposition is considered the key pathogenic event of Alzheimer's disease (AD). On the other hand, in physiological conditions, the crosstalk between the NO/cGMP/PKG/CREB pathway and Aß ensures long-term potentiation and memory formation. This review summarizes the current knowledge on the interaction between the NO/cGMP/PKG/CREB pathway and Aß in the healthy and diseased brain, offering a new perspective to shed light on AD pathophysiology. We will focus on the synaptic mechanisms underlying Aß physiological interplay with cGMP pathway and how this balance is corrupted in AD, as high levels of Aß interfere with NO production and cGMP molecular signaling leading to cognitive impairment. Finally, we will discuss results from preclinical and clinical studies proposing the increase of cGMP signaling as a therapeutic strategy in the treatment of AD.

Innate Preferences Affect Results of Object Recognition Task in Wild Type and Alzheimer's Disease Mouse Models.

BACKGROUND: Object recognition task (ORT) is a widely used behavioral paradigm to assess memory in rodent models, due to its easy technical execution, the lack of aversive stressful stimuli, and the possibility to repeat the test on the same animals. However, mouse exploration might be strongly influenced by a variety of variables. OBJECTIVE: To study whether innate preferences influenced exploration in male and female wild type mice and the Alzheimer's disease (AD) model 3xTg. METHODS: We first evaluated how object characteristics (material, size, and shape) influence exploration levels, latency, and exploration modality. Based on these findings, we evaluated whether these innate preferences biased the results of ORT performed in wild type mice and AD models. RESULTS: Assessment of Exploration levels, i.e., the time spent in exploring a certain object in respect to the total exploration time, revealed an innate preference for objects made in shiny materials, such as metal and glass. A preference for bigger objects characterized by higher affordance was also evident, especially in male mice. When performing ORT, exploration was highly influenced by these innate preferences. Indeed, both wild type and AD mice spent more time in exploring the metal object, regardless of its novelty. Furthermore, the use of objects with higher affordance such as the cube was a confounding factor leading to "false" results that distorted ORT interpretation. CONCLUSION: When designing exploration-based behavioral experiments aimed at assessing memory in healthy and AD mice, object characteristics should be carefully evaluated to improve scientific outcomes and minimize possible biases.

Application of 3D Printing Technology to Produce Hippocampal Customized Guide Cannulas.

Implantation of guide cannulas is a widely used technique to access specific brain areas. Although commercially available, the need to personalize these implants and the high cost prompted us to design open-source customized devices taking advantage of 3D printing technology. Our cannulas consisted in a 3D-printed head mount designed according to the Paxinos coordinates to reach the CA1 area of the hippocampus. To cut guide cannulas to the proper length, we designed and realized an original 3D-printed linear motion apparatus. Polylactic acid thermoplastic polymer was used as printing material. Homemade or commercial cannulas were implanted in 4- to 6-month-old wild-type mice and intrahippocampal injections of amyloid-ß peptide at different concentrations were performed. In vivo behavioral studies of novel object recognition indicated that results obtained with homemade versus commercial devices were comparable. Methylene blue injections and Nissl staining confirmed the correct localization of cannulas in the CA1 area of mouse hippocampus. Our method allows a fast manufacturing of hippocampal cannulas preserving the required precision at very low cost. Furthermore, this system can be easily modified to produce cannulas to target other brain areas. In conclusion, 3D printing might be used as a useful and versatile technology to realize open-source customized devices in neuroscience laboratories.

Genetic deletion of α7 nicotinic acetylcholine receptors induces an age-dependent Alzheimer's disease-like pathology.

The accumulation of amyloid-beta peptide (Aß) and the failure of cholinergic transmission are key players in Alzheimer's disease (AD). However, in the healthy brain, Aß contributes to synaptic plasticity and memory acting through α7 subtype nicotinic acetylcholine receptors (α7nAChRs). Here, we hypothesized that the α7nAChR deletion blocks Aß physiological function and promotes a compensatory increase in Aß levels that, in turn, triggers an AD-like pathology. To validate this hypothesis, we studied the age-dependent phenotype of α7 knock out mice. We found that α7nAChR deletion caused an impairment of hippocampal synaptic plasticity and memory at 12 months of age, paralleled by an increase of Amyloid Precursor Protein expression and Aß levels. This was accompanied by other classical AD features such as a hyperphosphorylation of tau at residues Ser 199, Ser 396, Thr 205, a decrease of GSK-3ß at Ser 9, the presence of paired helical filaments and neurofibrillary tangles, neuronal loss and an increase of GFAP-positive astrocytes. Our findings suggest that α7nAChR malfunction might precede Aß and tau pathology, offering a different perspective to interpret the failure of anti-Aß therapies against AD and to find novel therapeutical approaches aimed at restoring α7nAChRs-mediated Aß function at the synapse.

Antioxidant Activity of Fluoxetine and Vortioxetine in a Non-Transgenic Animal Model of Alzheimer's Disease.

Depression is a risk factor for the development of Alzheimer's disease (AD). A neurobiological and clinical continuum exists between AD and depression, with neuroinflammation and oxidative stress being involved in both diseases. Second-generation antidepressants, in particular selective serotonin reuptake inhibitors (SSRIs), are currently investigated as neuroprotective drugs in AD. By employing a non-transgenic AD model, obtained by intracerebroventricular (i.c.v.) injection of amyloid-ß (Aß) oligomers in 2-month-old C57BL/6 mice, we recently demonstrated that the SSRI fluoxetine (FLX) and the multimodal antidepressant vortioxetine (VTX) reversed the depressive-like phenotype and memory deficits induced by Aß oligomers rescuing the levels of transforming growth factor-ß1 (TGF-ß1). Aim of our study was to test FLX and VTX for their ability to prevent oxidative stress in the hippocampus of Aß-injected mice, a brain area strongly affected in both depression and AD. The long-term intraperitoneal (i.p.) administration of FLX (10 mg/kg) or VTX (5 and 10 mg/kg) for 24 days, starting 7 days before Aß injection, was able to prevent the over-expression of inducible nitric oxide synthase (iNOS) and NADPH oxidase 2 (Nox2) induced by Aß oligomers. Antidepressant pre-treatment was also able to rescue the mRNA expression of glutathione peroxidase 1 (Gpx1) antioxidant enzyme. FLX and VTX also prevented Aß-induced neurodegeneration in mixed neuronal cultures treated with Aß oligomers. Our data represent the first evidence that the long-term treatment with the antidepressants FLX or VTX can prevent the oxidative stress phenomena related to the cognitive deficits and depressive-like phenotype observed in a non-transgenic animal model of AD.

Fluoxetine and Vortioxetine Reverse Depressive-Like Phenotype and Memory Deficits Induced by Aß1-42 Oligomers in Mice: A Key Role of Transforming Growth Factor-ß1.

Depression is a risk factor for the development of Alzheimer's disease (AD), and the presence of depressive symptoms significantly increases the conversion of mild cognitive impairment (MCI) into AD. A long-term treatment with antidepressants reduces the risk to develop AD, and different second-generation antidepressants such as selective serotonin reuptake inhibitors (SSRIs) are currently being studied for their neuroprotective properties in AD. In the present work, the SSRI fluoxetine and the new multimodal antidepressant vortioxetine were tested for their ability to prevent memory deficits and depressive-like phenotype induced by intracerebroventricular injection of amyloid-ß (1-42) (Aß1-42) oligomers in 2-month-old C57BL/6 mice. Starting from 7 days before Aß injection, fluoxetine (10 mg/kg) and vortioxetine (5 and 10 mg/kg) were intraperitoneally injected daily for 24 days. Chronic treatment with fluoxetine and vortioxetine (both at the dose of 10 mg/kg) was able to rescue the loss of memory assessed 14 days after Aß injection by the passive avoidance task and the object recognition test. Both antidepressants reversed the increase in immobility time detected 19 days after Aß injection by forced swim test. Vortioxetine exerted significant antidepressant effects also at the dose of 5 mg/kg. A significant deficit of transforming growth factor-ß1 (TGF-ß1), paralleling memory deficits and depressive-like phenotype, was found in the hippocampus of Aß-injected mice in combination with a significant reduction of the synaptic proteins synaptophysin and PSD-95. Fluoxetine and vortioxetine completely rescued hippocampal TGF-ß1 levels in Aß-injected mice as well as synaptophysin and PSD-95 levels. This is the first evidence that a chronic treatment with fluoxetine or vortioxetine can prevent both cognitive deficits and depressive-like phenotype in a non-transgenic animal model of AD with a key contribution of TGF-ß1.

Sub-efficacious doses of phosphodiesterase 4 and 5 inhibitors improve memory in a mouse model of Alzheimer's disease.

Cyclic nucleotides cAMP and cGMP cooperate to ensure memory acquisition and consolidation. Increasing their levels by phosphodiesterase inhibitors (PDE-Is) enhanced cognitive functions and rescued memory loss in different models of aging and Alzheimer's disease (AD). However, side effects due to the high doses used limited their application in humans. Based on previous studies suggesting that combinations of sub-efficacious doses of cAMP- and cGMP-specific PDE-Is improved synaptic plasticity and memory in physiological conditions, here we aimed to study whether this treatment was effective to counteract the AD phenotype in APPswe mice. We found that a 3-week chronic treatment with a combination of sub-efficacious doses of the cAMP-specific PDE4-I roflumilast (0.01 mg/kg) and the cGMP-specific PDE5-I vardenafil (0.1 mg/kg) improved recognition, spatial and contextual fear memory. Importantly, the cognitive enhancement persisted for 2 months beyond administration. This long-lasting action, and the possibility to minimize side effects due to the low doses used, might open feasible therapeutic strategies against AD.

Activation of Serotonin 5-HT7 Receptors Modulates Hippocampal Synaptic Plasticity by Stimulation of Adenylate Cyclases and Rescues Learning and Behavior in a Mouse Model of Fragile X Syndrome.

We have previously demonstrated that activation of serotonin 5-HT7 receptors (5-HT7R) reverses metabotropic glutamate receptor-mediated long term depression (mGluR-LTD) in the hippocampus of wild-type (WT) and Fmr1 Knockout (KO) mice, a model of Fragile X Syndrome (FXS) in which mGluR-LTD is abnormally enhanced. Here, we have investigated intracellular mechanisms underlying the effect of 5-HT7R activation using patch clamp on hippocampal slices. Furthermore, we have tested whether in vivo administration of LP-211, a selective 5-HT7R agonist, can rescue learning and behavior in Fmr1 KO mice. In the presence of an adenylate cyclase blocker, mGluR-LTD was slightly enhanced in WT and therefore the difference between mGluR-LTD in WT and Fmr1 KO slices was no longer present. Conversely, activation of adenylate cyclase by either forskolin or Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) completely reversed mGluR-LTD in WT and Fmr1 KO. 5-HT7R activation reversed mGluR-LTD in WT and corrected exaggerated mGluR-LTD in Fmr1 KO; this effect was abolished by blockade of either adenylate cyclase or protein kinase A (PKA). Exposure of hippocampal slices to LP-211 caused an increased phosphorylation of extracellular signal regulated kinase (ERK), an intracellular effector involved in mGluR-LTD, in WT mice. Conversely, this effect was barely detectable in Fmr1 KO mice, suggesting that 5-HT7R-mediated reversal of mGluR-LTD does not require ERK stimulation. Finally, an acute in vivo administration of LP-211 improved novel object recognition (NOR) performance in WT and Fmr1 KO mice and reduced stereotyped behavior in Fmr1 KO mice. Our results indicate that mGluR-LTD in WT and Fmr1 KO slices is bidirectionally modulated in conditions of either reduced or enhanced cAMP formation. Activation of 5-HT7 receptors reverses mGluR-LTD by activation of the cAMP/PKA intracellular pathway. Importantly, a systemic administration of a 5-HT7R agonist to Fmr1 KO mice corrected learning deficits and repetitive behavior. We suggest that selective 5-HT7R agonists might become novel pharmacological tools for FXS therapy.

The effect of amyloid-ß peptide on synaptic plasticity and memory is influenced by different isoforms, concentrations, and aggregation status.

The increase of oligomeric amyloid-beta (oAß) has been related to synaptic dysfunction, thought to be the earliest event in Alzheimer's disease pathophysiology. Conversely, the suppression of endogenous Aß impaired synaptic plasticity and memory, suggesting that the peptide is needed in the healthy brain. However, different species, aggregation forms and concentrations of Aß might differently influence synaptic function/dysfunction. Here, we have tested the contribution of monomeric and oligomeric Aß42 and Aß40 at 200 nM and 200 pM concentrations on hippocampal long-term potentiation and spatial memory. We found that, when at 200 nM, oAß40, oAß42, and monomeric Aß42 impaired long-term potentiation and memory, whereas only oAß42 200 pM enhanced synaptic plasticity and memory and rescued the detrimental effect due to depletion of endogenous Aß. Interestingly, quantification of monomer-like and oligomer-like species carried out by transmission electron microscopy revealed an increase of the monomer/oligomer ratio in the oAß42 200 pM preparation, suggesting that the content of monomers and oligomers depends on the final concentration of the solution.

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.

Salidroside, a Bioactive Compound of Rhodiola Rosea, Ameliorates Memory and Emotional Behavior in Adult Mice.

Rhodiola Rosea (R. Rosea) is a plant used in traditional popular medicine to enhance cognition and physical performance. R. Rosea medicinal properties have been related to its capability to act as an adaptogen, i.e., a substance able to increase the organism's resistance to a variety of chemical, biological, and physical stressors in a non-specific way. These adaptogen properties have been mainly attributed to the glycoside salidroside, one of the bioactive compounds present in the standardized extracts of R. Rosea. Here, we aimed to investigate whether a single dose of salidroside is able to affect memory and emotional behavior in wild type adult mice. We performed fear conditioning to assess cued and contextual memory, elevated plus maze and open field to evaluate anxiety, and tail suspension test to evaluate depression. Our results showed that a single i.p. administration of salidroside was able to enhance fear memory and exerted an anxiolytic and antidepressant effect. These data confirmed the adaptogenic effect of R. Rosea bioactive compounds in animal models and suggest that salidroside might represent an interesting pharmacological tool to ameliorate cognition and counteract mood disorders.