The aim of this thesis was to study the pathogenetic mechanisms underlying Alzheimer s disease (A... more The aim of this thesis was to study the pathogenetic mechanisms underlying Alzheimer s disease (AD), a neurodegenerative disorder affecting the elderly and characterized by memory loss, personality changes and cognitive dysfunction leading to dementia. I will discuss the main projects in which I participated aimed at understanding the role of the main molecular interactors involved in AD pathogenesis, i.e. Amyloid-beta peptide and tau protein, on hippocampal synaptic plasticity and memory in animal models. After reviewing the pathophysiological models that have been developed so far, our general purpose was to study novel aspects of Amyloid-beta peptide and tau involvement in physiological and pathological conditions to give a different interpretation of the disease
Growing evidence points to altered metabolic signals as a risk factor for Alzheimer's disease... more Growing evidence points to altered metabolic signals as a risk factor for Alzheimer's disease (AD). The high fatty acid levels and brain insulin resistance (BIR) found in AD brains may impinge on protein palmitoylation (P-S-palm), a post-translational modification critically involved in the regulation of neuronal protein localization and synaptic function. Our previous findings highlighted the critical role of aberrant palmitoylation in BIR-dependent memory impairment (Spinelli et al., 2017). We tested the effect of chronic intranasal injection of the palmitoylation inhibitor 2-bromopalmitate (2BP) on a large cohort of male and female 3xTg-AD mice, starting from 3 months of age, by performing cognitive (novel object recognition and object displacement tests), electrophysiological (LTP), immunohistochemical (Abeta deposition) and molecular analyses (Abeta measurement by ELISA). We also analyzed the palmitoyl-proteome (by acyl biotin exchange assay and mass-spectrometry) in the hippocampus of 9-month-old wild type, 3xTg-AD and 2BP-treated 3xTg-AD mice. 2BP delayed the onset of memory deficits in both male and female 3xTg-AD mice. More importantly, 2BP significantly enhanced cognitive performances in 6-, 9- and 12-month-old animals. Accordingly, electrophysiological analyses on hippocampal brain slices from 2BP-treated 3xTg-AD mice revealed greater long-term potentiation at CA3-CA1 synapses. In addition, 2BP mice showed lower Abeta deposition in hippocampus. Finally, palmitoyl-proteome analysis revealed a large number of proteins involved in APP and tau metabolism, synaptic plasticity and brain metabolism aberrantly palmitoylated in the AD mouse model and reverted by 2BP. Our data indicate that aberrant palmitoylation plays a critical role in the onset and progression of AD and reveal novel targets of protein palmitoylation potentially involved in the development of neurodegeneration and dementia. This is also the first preclinical study on the effect of 2BP on AD-related cognitive decline.
Scopo del presente documento è quello di fornire una descrizione dettagliata degli "use case... more Scopo del presente documento è quello di fornire una descrizione dettagliata degli "use case" già identificati in fase di preparazione della proposta progettuale, inclusiva di un'analisi dei loro requisiti in termini di infrastruttura necessaria per la loro implementazione. A questo proposito, con riferimento a Technopedia2 e alla tassonomia introdotta da IEEE Computer Society, è stata elaborata una tassonomia di tutti i possibili requisiti dei casi d'uso del progetto già identificati.<br> Lo schema del documento è il seguente. La Sezione 2 introduce la metodologia e l'analisi dei requisiti dei casi d'uso. I lemmi della tassonomia sono riportati nella in quella Sezione e sono poi riassunti per completezza nell'Appendice 1, nella quale ad ogni lemma è associato un codice numerico che è quello poi usato come riferimento nelle Sezioni successive del presente documento. La Sezione 3 presenta il modello di definizione dei casi d'uso in termini dei ...
cGMP elevation does not influence motor, visible or exploratory behavior in mice treated with oTa... more cGMP elevation does not influence motor, visible or exploratory behavior in mice treated with oTau. Evaluation of speed, latency, sensory threshold and exploratory behavior in mice treated with vehicle, oTau, 7a, 7a + oTau, 8pCPT-cGMP, 8pCPT-cGMP, +oTau. (PDF 171 kb)
Background: Object recognition task (ORT) is a widely used behavioral paradigm to assess memory i... more 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...
The accumulation of amyloid-beta peptide (Aβ) and the failure of cholinergic transmission are key... more The accumulation of amyloid-beta peptide (Aβ) and the failure of cholinergic transmission are key players in Alzheimer's disease (AD). However, in the healthy brain, Aβ contributes to synaptic plasticity and memory acting through α7 subtype nicotinic acetylcholine receptors (α7nAChRs). Here, we hypothesized that the α7nAChR deletion blocks Aβ physiological function and promotes a compensatory increase in Aβ 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 Aβ 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-3β 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 Aβ and tau pathology, offering a different perspective to interpret the failure of anti-Aβ therapies against AD and to find novel therapeutical approaches aimed at restoring α7nAChRs-mediated Aβ function at the synapse.
ABSTRACTThe accumulation of amyloid-beta peptide (Aβ) and the failure of cholinergic transmission... more ABSTRACTThe accumulation of amyloid-beta peptide (Aβ) and the failure of cholinergic transmission are key players in Alzheimer’s disease (AD). However, in the healthy brain, Aβ contributes to synaptic plasticity and memory acting through α7 subtype nicotinic acetylcholine receptors (α7nAChRs). Here, we hypothesized that the α7nAChR deletion blocks Aβ physiological function and promotes a compensatory increase in Aβ 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 Aβ 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-3β at Ser 9, the presence of paired helical filaments and neurofibrillary tangles, neuron...
Abstract The response of a biological system to an endogenous or exogenous molecule depends upon ... more Abstract The response of a biological system to an endogenous or exogenous molecule depends upon the dose. For this reason, performing dose‐response curves is crucial to understand physiological and pathophysiological phenomena, and to predict the effect of a drug. Most of the studies in pharmacological research have been performed according to the classical threshold model, focusing on higher doses able to ensure a biological effect. However, recent evidences pointed out the need to investigate the effect of low doses. Indeed, several molecules behave in a hormetic fashion, i.e. low‐doses stimulate whereas high‐doses inhibit a biological response. This is particularly interesting in CNS, where several physiological molecules involved in neuronal transmission during learning and memory have shown a biphasic effect that might represent the link between physiology and pathology. In this review we will focus on cholinergic, glutamatergic and nitrinergic transmission, because of their central role in learning and memory and their impairment in neurodegenerative disorders such as Alzheimer’s disease. Pre‐clinical studies performed on healthy adult animals and aged animals, as well as transgenic animal models of AD, have suggested a biphasic DR for acetylcholine, glutamate and nitric oxide. This stresses the relevance to perform DR curves when studying the mechanisms underlying synaptic plasticity and memory, the pharmacological profile of cognitive‐enhancing drugs acting on these systems, and the possibility to combine low/ineffective doses of drugs that might have additive/synergistic effects, reducing the unwanted side effects associated to the high doses.
The concurrent application of subtoxic doses of soluble oligomeric forms of human amyloid-beta (o... more The concurrent application of subtoxic doses of soluble oligomeric forms of human amyloid-beta (oAβ) 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 oAβ and oTau share a common mechanism when they impair memory and LTP in mice. We found that as already shown for oAβ, also oTau can bind to amyloid precursor protein (APP). Moreover, efficient intra-neuronal uptake of oAβ and oTau requires expression of APP. Finally, the toxic effect of both extracellular oAβ and oTau on memory and LTP is dependent upon APP since APP-KO mice were resistant to oAβ- 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 Aβ and/or Tau.
The aim of this thesis was to study the pathogenetic mechanisms underlying Alzheimer s disease (A... more The aim of this thesis was to study the pathogenetic mechanisms underlying Alzheimer s disease (AD), a neurodegenerative disorder affecting the elderly and characterized by memory loss, personality changes and cognitive dysfunction leading to dementia. I will discuss the main projects in which I participated aimed at understanding the role of the main molecular interactors involved in AD pathogenesis, i.e. Amyloid-beta peptide and tau protein, on hippocampal synaptic plasticity and memory in animal models. After reviewing the pathophysiological models that have been developed so far, our general purpose was to study novel aspects of Amyloid-beta peptide and tau involvement in physiological and pathological conditions to give a different interpretation of the disease
Growing evidence points to altered metabolic signals as a risk factor for Alzheimer's disease... more Growing evidence points to altered metabolic signals as a risk factor for Alzheimer's disease (AD). The high fatty acid levels and brain insulin resistance (BIR) found in AD brains may impinge on protein palmitoylation (P-S-palm), a post-translational modification critically involved in the regulation of neuronal protein localization and synaptic function. Our previous findings highlighted the critical role of aberrant palmitoylation in BIR-dependent memory impairment (Spinelli et al., 2017). We tested the effect of chronic intranasal injection of the palmitoylation inhibitor 2-bromopalmitate (2BP) on a large cohort of male and female 3xTg-AD mice, starting from 3 months of age, by performing cognitive (novel object recognition and object displacement tests), electrophysiological (LTP), immunohistochemical (Abeta deposition) and molecular analyses (Abeta measurement by ELISA). We also analyzed the palmitoyl-proteome (by acyl biotin exchange assay and mass-spectrometry) in the hippocampus of 9-month-old wild type, 3xTg-AD and 2BP-treated 3xTg-AD mice. 2BP delayed the onset of memory deficits in both male and female 3xTg-AD mice. More importantly, 2BP significantly enhanced cognitive performances in 6-, 9- and 12-month-old animals. Accordingly, electrophysiological analyses on hippocampal brain slices from 2BP-treated 3xTg-AD mice revealed greater long-term potentiation at CA3-CA1 synapses. In addition, 2BP mice showed lower Abeta deposition in hippocampus. Finally, palmitoyl-proteome analysis revealed a large number of proteins involved in APP and tau metabolism, synaptic plasticity and brain metabolism aberrantly palmitoylated in the AD mouse model and reverted by 2BP. Our data indicate that aberrant palmitoylation plays a critical role in the onset and progression of AD and reveal novel targets of protein palmitoylation potentially involved in the development of neurodegeneration and dementia. This is also the first preclinical study on the effect of 2BP on AD-related cognitive decline.
Scopo del presente documento è quello di fornire una descrizione dettagliata degli "use case... more Scopo del presente documento è quello di fornire una descrizione dettagliata degli "use case" già identificati in fase di preparazione della proposta progettuale, inclusiva di un'analisi dei loro requisiti in termini di infrastruttura necessaria per la loro implementazione. A questo proposito, con riferimento a Technopedia2 e alla tassonomia introdotta da IEEE Computer Society, è stata elaborata una tassonomia di tutti i possibili requisiti dei casi d'uso del progetto già identificati.<br> Lo schema del documento è il seguente. La Sezione 2 introduce la metodologia e l'analisi dei requisiti dei casi d'uso. I lemmi della tassonomia sono riportati nella in quella Sezione e sono poi riassunti per completezza nell'Appendice 1, nella quale ad ogni lemma è associato un codice numerico che è quello poi usato come riferimento nelle Sezioni successive del presente documento. La Sezione 3 presenta il modello di definizione dei casi d'uso in termini dei ...
cGMP elevation does not influence motor, visible or exploratory behavior in mice treated with oTa... more cGMP elevation does not influence motor, visible or exploratory behavior in mice treated with oTau. Evaluation of speed, latency, sensory threshold and exploratory behavior in mice treated with vehicle, oTau, 7a, 7a + oTau, 8pCPT-cGMP, 8pCPT-cGMP, +oTau. (PDF 171 kb)
Background: Object recognition task (ORT) is a widely used behavioral paradigm to assess memory i... more 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...
The accumulation of amyloid-beta peptide (Aβ) and the failure of cholinergic transmission are key... more The accumulation of amyloid-beta peptide (Aβ) and the failure of cholinergic transmission are key players in Alzheimer's disease (AD). However, in the healthy brain, Aβ contributes to synaptic plasticity and memory acting through α7 subtype nicotinic acetylcholine receptors (α7nAChRs). Here, we hypothesized that the α7nAChR deletion blocks Aβ physiological function and promotes a compensatory increase in Aβ 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 Aβ 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-3β 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 Aβ and tau pathology, offering a different perspective to interpret the failure of anti-Aβ therapies against AD and to find novel therapeutical approaches aimed at restoring α7nAChRs-mediated Aβ function at the synapse.
ABSTRACTThe accumulation of amyloid-beta peptide (Aβ) and the failure of cholinergic transmission... more ABSTRACTThe accumulation of amyloid-beta peptide (Aβ) and the failure of cholinergic transmission are key players in Alzheimer’s disease (AD). However, in the healthy brain, Aβ contributes to synaptic plasticity and memory acting through α7 subtype nicotinic acetylcholine receptors (α7nAChRs). Here, we hypothesized that the α7nAChR deletion blocks Aβ physiological function and promotes a compensatory increase in Aβ 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 Aβ 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-3β at Ser 9, the presence of paired helical filaments and neurofibrillary tangles, neuron...
Abstract The response of a biological system to an endogenous or exogenous molecule depends upon ... more Abstract The response of a biological system to an endogenous or exogenous molecule depends upon the dose. For this reason, performing dose‐response curves is crucial to understand physiological and pathophysiological phenomena, and to predict the effect of a drug. Most of the studies in pharmacological research have been performed according to the classical threshold model, focusing on higher doses able to ensure a biological effect. However, recent evidences pointed out the need to investigate the effect of low doses. Indeed, several molecules behave in a hormetic fashion, i.e. low‐doses stimulate whereas high‐doses inhibit a biological response. This is particularly interesting in CNS, where several physiological molecules involved in neuronal transmission during learning and memory have shown a biphasic effect that might represent the link between physiology and pathology. In this review we will focus on cholinergic, glutamatergic and nitrinergic transmission, because of their central role in learning and memory and their impairment in neurodegenerative disorders such as Alzheimer’s disease. Pre‐clinical studies performed on healthy adult animals and aged animals, as well as transgenic animal models of AD, have suggested a biphasic DR for acetylcholine, glutamate and nitric oxide. This stresses the relevance to perform DR curves when studying the mechanisms underlying synaptic plasticity and memory, the pharmacological profile of cognitive‐enhancing drugs acting on these systems, and the possibility to combine low/ineffective doses of drugs that might have additive/synergistic effects, reducing the unwanted side effects associated to the high doses.
The concurrent application of subtoxic doses of soluble oligomeric forms of human amyloid-beta (o... more The concurrent application of subtoxic doses of soluble oligomeric forms of human amyloid-beta (oAβ) 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 oAβ and oTau share a common mechanism when they impair memory and LTP in mice. We found that as already shown for oAβ, also oTau can bind to amyloid precursor protein (APP). Moreover, efficient intra-neuronal uptake of oAβ and oTau requires expression of APP. Finally, the toxic effect of both extracellular oAβ and oTau on memory and LTP is dependent upon APP since APP-KO mice were resistant to oAβ- 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 Aβ and/or Tau.
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