Double-stranded RNA-mediated interference (RNAi) is a new simple and fast research tool for shutt... more Double-stranded RNA-mediated interference (RNAi) is a new simple and fast research tool for shutting down genes and characterizes function of their respective proteins. Many strategies for design and delivery of siRNA to target cells are available. Here, we describe the use of lentiviral short hairpinRNA (shRNA) RNA silencing to identify the involvement of D-serineracemase (SR)- an enzyme that syntheses D-serine to modulate glutamate-N-methyl-D-aspartate receptor- in regulating ratcerebellar granule neurons (CGN) apoptosis. Apoptosis is induced by serum and KCl withdrawal and is detected with fluorometric caspase 3assay.
Alzheimer’s disease (AD) is an incurable neurodegenerative disorder with a few early detection st... more Alzheimer’s disease (AD) is an incurable neurodegenerative disorder with a few early detection strategies. We previously proposed the amyloid precursor protein (APP) tyrosine 682 (Tyr682) residue as a valuable target for the development of new innovative pharmacologic or diagnostic interventions in AD. Indeed, when APP is phosphorylated at Tyr682, it is forced into acidic neuronal compartments where it is processed to generate neurotoxic amyloid β peptides. Of interest, Fyn tyrosine kinase (TK) interaction with APP Tyr682 residue increases in AD neurons. Here we proved that when Fyn TK was overexpressed it elicited APP Tyr682 phosphorylation in neurons from healthy donors and promoted the amyloidogenic APP processing with Aβ peptides accumulation and neuronal death. Phosphorylation of APP at Tyr (pAPP-Tyr) increased in neurons of AD patients and AD neurons that exhibited high pAPP-Tyr also had higher Fyn TK activity. Fyn TK inhibition abolished the pAPP-Tyr and reduced Aβ42 secretio...
Basal forebrain cholinergic neurons (BFCN) are key modulators of learning and memory and are high... more Basal forebrain cholinergic neurons (BFCN) are key modulators of learning and memory and are high energy-demanding neurons. Impaired neuronal metabolism and reduced insulin signaling, known as insulin resistance, has been reported in the early phase of Alzheimer's disease (AD), which has been suggested to be "Type 3 Diabetes." We hypothesized that BFCN may develop insulin resistance and their consequent failure represents one of the earliest event in AD. We found that a condition reminiscent of insulin resistance occurs in the medial septum of 3 months old 3×Tg-AD mice, reported to develop typical AD histopathology and cognitive deficits in adulthood. Further, we obtained insulin resistant BFCN by culturing them with high insulin concentrations. By means of these paradigms, we observed that nerve growth factor (NGF) reduces insulin resistance in vitro and in vivo. NGF activates the insulin receptor substrate 1 (IRS) and rescues c-Fos expression and glucose metabolism. ...
Amyotrophic Lateral Sclerosis (ALS) is a chronic neurodegenerative disease affecting upper and lo... more Amyotrophic Lateral Sclerosis (ALS) is a chronic neurodegenerative disease affecting upper and lower motor neurons, with unknown aetiology. Lipid rafts, cholesterol enriched microdomains of the plasma membrane, have been linked to neurodegenerative disorders like ALS. The NMDA-receptor subcellular localization in lipid rafts is known to play many roles, from modulating memory strength to neurotoxicity. In this study, performed on the widely used G93A mouse model of ALS, we have shown an equal content of total membrane cholesterol in Control and G93A cortical cultures. Moreover, by electrophysiological studies, we have recorded NMDA- and AMPA-evoked currents which were not significantly different between the two neuronal populations. To study the role of membrane cholesterol on glutamate receptor functionality, we have analysed NMDA and AMPA receptors following cholesterol membrane depletion by methyl-β-cyclodextrin (MβCD). Interestingly, MβCD chronic treatment has provoked a signifi...
International journal of molecular sciences, Jan 20, 2017
Dysfunction of nerve growth factor (NGF) and its high-affinity Tropomyosin receptor kinase A (Trk... more Dysfunction of nerve growth factor (NGF) and its high-affinity Tropomyosin receptor kinase A (TrkA) receptor has been suggested to contribute to the selective degeneration of basal forebrain cholinergic neurons (BFCN) associated with the progressive cognitive decline in Alzheimer's disease (AD). The aim of this review is to describe our progress in elucidating the molecular mechanisms underlying the dynamic interplay between NGF/TrkA signaling and amyloid precursor protein (APP) metabolism within the context of AD neuropathology. This is mainly based on the finding that TrkA receptor binding to APP depends on a minimal stretch of ~20 amino acids located in the juxtamembrane/extracellular domain of APP that carries the α- and β-secretase cleavage sites. Here, we provide evidence that: (i) NGF could be one of the "routing" proteins responsible for modulating the metabolism of APP from amyloidogenic towards non-amyloidogenic processing via binding to the TrkA receptor; (i...
The amyloid precursor protein (APP) interacts with the tropomyosin receptor kinase A (TrkA) in no... more The amyloid precursor protein (APP) interacts with the tropomyosin receptor kinase A (TrkA) in normal rat, mouse, and human brain tissue but not in Alzheimer's disease (AD) brain tissue. However, it has not been reported whether the two proteins interact directly, and if so, which domains are involved. Clarifying these points will increase our understanding of the role and regulation of the TrkA/APP interaction in normal brain functioning as well as in AD. Here we addressed these questions using bimolecular fluorescence complementation (BiFC) and the proximity ligation assay (PLA). We demonstrated that exogenously expressed APP and TrkA associate through their juxtamembrane/transmembrane domains, to form a complex that localizes mainly to the plasma membrane, endoplasmic reticulum (ER) and Golgi. Formation of the complex was inhibited by p75NTR, ShcC and Mint-2. Importantly, we demonstrated that the association between endogenous APP and TrkA in primary septal neurons were modif...
Disconnection between membrane signalling and actin networks may have catastrophic effects depend... more Disconnection between membrane signalling and actin networks may have catastrophic effects depending on cell size and polarity. The Survival Motor Neuron (SMN) protein is ubiquitously involved in assembly of spliceosomal small nuclear ribonucleoprotein particles. Other SMN functions could, however, affect cellular activities driving asymmetrical cell surface expansions. Genes able to mitigate SMN deficiency operate within pathways as part of which SMN can act: mRNA translation, actin network, and endocytosis. Here, we found that SMN accumulates at membrane protrusions during dynamic rearrangement of the actin filament. In addition to localization data, we show that SMN interacts with caveolin-1, which mediates anchoring of translation machinery components. Importantly, SMN deficiency depletes the plasma membrane of ribosomes, and this correlates with the failure of fibroblasts to extend membrane protrusions. These findings strongly support a relationship between SMN and membrane dyn...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 2000
We investigated the potential role of the ubiquitin proteolytic system in the death of cerebellar... more We investigated the potential role of the ubiquitin proteolytic system in the death of cerebellar granule neurons induced by reduction of extracellular potassium. Inhibitors of proteasomal function block apoptosis if administered at onset of this process, but they do not exert such effect when added 2-3 hr later. The same inhibitors also prevent caspase-3 activity and calpain-caspase-3-mediated processing of tau protein, suggesting that proteasomes are involved upstream of the caspase activation. Although the proteasomes seem to play an early primary role in programmed cell death, we found that with progression of apoptosis, during the execution phase, a perturbation in normal ubiquitin-proteasome function occurs, and high levels of ubiquitinated proteins accumulate in the cytoplasm of dying cells. Such accumulation correlates with a progressive decline of proteasome chymotrypsin and trypsin-like activities and, to a lower extent, of postacidic-like activity. Both intracytoplasmic a...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 1998
Cerebellar granule cells undergo apoptosis in culture after deprivation of potassium and serum. D... more Cerebellar granule cells undergo apoptosis in culture after deprivation of potassium and serum. During this process we found that tau, a neuronal microtubule-associated protein that plays a key role in the maintenance of neuronal architecture, and the pathology of which correlates with intellectual decline in Alzheimer's disease, is cleaved. The final product of this cleavage is a soluble dephosphorylated tau fragment of 17 kDa that is unable to associate with microtubules and accumulates in the perikarya of dying cells. The appearance of this 17 kDa fragment is inhibited by both caspase and calpain inhibitors, suggesting that tau is an in vivo substrate for both of these proteases during apoptosis. Tau cleavage is correlated with disruption of the microtubule network, and experiments with colchicine and taxol show that this is likely to be a cause and not a consequence of tau cleavage. These data indicate that tau cleavage and change in phosphorylation are important early facto...
Diabetes often correlates with tau phosphorylation and the development of Alzheimer's disease... more Diabetes often correlates with tau phosphorylation and the development of Alzheimer's disease. Both are associated with brain cholinergic dysfunction that could benefit from nerve growth factor (NGF)-based therapies. Electroacupuncture (EA) improves brain NGF availability and action. Here we assessed the variations of NGF and tau phosphorylation in the cortex and hippocampus, as well as the expression of choline acetyltransferase in the basal forebrain following diabetes induction and EA in adult rats. We found that EA counteracts diabetes-associated tau hyperphosphorylation and decreases in NGF and choline acetyltransferase, suggesting a possible beneficial effect of EA on brain cholinergic system in diabetes.
The microtubule associated protein tau plays a crucial role in Alzheimer's disease and in man... more The microtubule associated protein tau plays a crucial role in Alzheimer's disease and in many neurodegenerative disorders collectively known as tauopathies. Recently, tau pathology has been also documented in prion diseases although the possible molecular events linking these two proteins are still unknown. We have investigated the fate of normal cellular prion protein (PrP(C)) in primary cortical neurons overexpressing tau protein. We found that overexpression of tau reduces PrP(C) expression at the cell surface and causes its accumulation and aggregation in the cell body but does not affect its maturation and glycosylation. Trapped PrP(C) forms detergent-insoluble aggregates, mainly composed of un-glycosylated and mono-glycosylated forms of prion protein. Interestingly, co-transfection of tau gene in cortical neurons with a proteasome activity reporter, consisting of a short peptide degron fused to the carboxyl-terminus of green fluorescent protein (GFP-CL1), results in down-...
Biochemical studies on the process of neuronal differentiation have largely profited from the est... more Biochemical studies on the process of neuronal differentiation have largely profited from the establishment of cell lines which acquire a neuronal phenotype in response to various agents. In the last decade PC12 cells, derived from a rat pheochromocytoma, have become the first choice for studies of neuronal differentiation in tissue culture (Greene and Tishler 1982, Guroff 1985, Levi and Alema 1991), especially since these cells differentiate in response to a well defined growth factor: nerve growth factor, NGF (Levi-Montalcini 1987). As a result, the molecular characterization of the structure of the two kinds of NGF receptor, and recent theories on the possible mechanism of action of NGF have relied heavily on the use of these cells and mutant subclones derived from them (Hempstead et al. 1989, Hempstead et al. 1991, Kaplan et al. 1991). These studies have been the basis for understanding, in general, how the specificity of action of the various member of the neurotrophin family is achieved (Bothwell M. 1991, Hallbook et al. 1991). NGF, like its close relatives, is supposed to act not only on subpopulations of cells derived from the neural crest (Levi Montacini 1987, Whittemore et al.1987, Thoenen et al. 1987, Vantini et al. 1989), but also on several cell types which display high affinity binding for this growth factor (Cattaneo 1986, Otten et al. 1989, Aloe and Levi Montalcini 1979, Levi-Montalcini et al. 1990).
Double-stranded RNA-mediated interference (RNAi) is a new simple and fast research tool for shutt... more Double-stranded RNA-mediated interference (RNAi) is a new simple and fast research tool for shutting down genes and characterizes function of their respective proteins. Many strategies for design and delivery of siRNA to target cells are available. Here, we describe the use of lentiviral short hairpinRNA (shRNA) RNA silencing to identify the involvement of D-serineracemase (SR)- an enzyme that syntheses D-serine to modulate glutamate-N-methyl-D-aspartate receptor- in regulating ratcerebellar granule neurons (CGN) apoptosis. Apoptosis is induced by serum and KCl withdrawal and is detected with fluorometric caspase 3assay.
Alzheimer’s disease (AD) is an incurable neurodegenerative disorder with a few early detection st... more Alzheimer’s disease (AD) is an incurable neurodegenerative disorder with a few early detection strategies. We previously proposed the amyloid precursor protein (APP) tyrosine 682 (Tyr682) residue as a valuable target for the development of new innovative pharmacologic or diagnostic interventions in AD. Indeed, when APP is phosphorylated at Tyr682, it is forced into acidic neuronal compartments where it is processed to generate neurotoxic amyloid β peptides. Of interest, Fyn tyrosine kinase (TK) interaction with APP Tyr682 residue increases in AD neurons. Here we proved that when Fyn TK was overexpressed it elicited APP Tyr682 phosphorylation in neurons from healthy donors and promoted the amyloidogenic APP processing with Aβ peptides accumulation and neuronal death. Phosphorylation of APP at Tyr (pAPP-Tyr) increased in neurons of AD patients and AD neurons that exhibited high pAPP-Tyr also had higher Fyn TK activity. Fyn TK inhibition abolished the pAPP-Tyr and reduced Aβ42 secretio...
Basal forebrain cholinergic neurons (BFCN) are key modulators of learning and memory and are high... more Basal forebrain cholinergic neurons (BFCN) are key modulators of learning and memory and are high energy-demanding neurons. Impaired neuronal metabolism and reduced insulin signaling, known as insulin resistance, has been reported in the early phase of Alzheimer's disease (AD), which has been suggested to be "Type 3 Diabetes." We hypothesized that BFCN may develop insulin resistance and their consequent failure represents one of the earliest event in AD. We found that a condition reminiscent of insulin resistance occurs in the medial septum of 3 months old 3×Tg-AD mice, reported to develop typical AD histopathology and cognitive deficits in adulthood. Further, we obtained insulin resistant BFCN by culturing them with high insulin concentrations. By means of these paradigms, we observed that nerve growth factor (NGF) reduces insulin resistance in vitro and in vivo. NGF activates the insulin receptor substrate 1 (IRS) and rescues c-Fos expression and glucose metabolism. ...
Amyotrophic Lateral Sclerosis (ALS) is a chronic neurodegenerative disease affecting upper and lo... more Amyotrophic Lateral Sclerosis (ALS) is a chronic neurodegenerative disease affecting upper and lower motor neurons, with unknown aetiology. Lipid rafts, cholesterol enriched microdomains of the plasma membrane, have been linked to neurodegenerative disorders like ALS. The NMDA-receptor subcellular localization in lipid rafts is known to play many roles, from modulating memory strength to neurotoxicity. In this study, performed on the widely used G93A mouse model of ALS, we have shown an equal content of total membrane cholesterol in Control and G93A cortical cultures. Moreover, by electrophysiological studies, we have recorded NMDA- and AMPA-evoked currents which were not significantly different between the two neuronal populations. To study the role of membrane cholesterol on glutamate receptor functionality, we have analysed NMDA and AMPA receptors following cholesterol membrane depletion by methyl-β-cyclodextrin (MβCD). Interestingly, MβCD chronic treatment has provoked a signifi...
International journal of molecular sciences, Jan 20, 2017
Dysfunction of nerve growth factor (NGF) and its high-affinity Tropomyosin receptor kinase A (Trk... more Dysfunction of nerve growth factor (NGF) and its high-affinity Tropomyosin receptor kinase A (TrkA) receptor has been suggested to contribute to the selective degeneration of basal forebrain cholinergic neurons (BFCN) associated with the progressive cognitive decline in Alzheimer's disease (AD). The aim of this review is to describe our progress in elucidating the molecular mechanisms underlying the dynamic interplay between NGF/TrkA signaling and amyloid precursor protein (APP) metabolism within the context of AD neuropathology. This is mainly based on the finding that TrkA receptor binding to APP depends on a minimal stretch of ~20 amino acids located in the juxtamembrane/extracellular domain of APP that carries the α- and β-secretase cleavage sites. Here, we provide evidence that: (i) NGF could be one of the "routing" proteins responsible for modulating the metabolism of APP from amyloidogenic towards non-amyloidogenic processing via binding to the TrkA receptor; (i...
The amyloid precursor protein (APP) interacts with the tropomyosin receptor kinase A (TrkA) in no... more The amyloid precursor protein (APP) interacts with the tropomyosin receptor kinase A (TrkA) in normal rat, mouse, and human brain tissue but not in Alzheimer's disease (AD) brain tissue. However, it has not been reported whether the two proteins interact directly, and if so, which domains are involved. Clarifying these points will increase our understanding of the role and regulation of the TrkA/APP interaction in normal brain functioning as well as in AD. Here we addressed these questions using bimolecular fluorescence complementation (BiFC) and the proximity ligation assay (PLA). We demonstrated that exogenously expressed APP and TrkA associate through their juxtamembrane/transmembrane domains, to form a complex that localizes mainly to the plasma membrane, endoplasmic reticulum (ER) and Golgi. Formation of the complex was inhibited by p75NTR, ShcC and Mint-2. Importantly, we demonstrated that the association between endogenous APP and TrkA in primary septal neurons were modif...
Disconnection between membrane signalling and actin networks may have catastrophic effects depend... more Disconnection between membrane signalling and actin networks may have catastrophic effects depending on cell size and polarity. The Survival Motor Neuron (SMN) protein is ubiquitously involved in assembly of spliceosomal small nuclear ribonucleoprotein particles. Other SMN functions could, however, affect cellular activities driving asymmetrical cell surface expansions. Genes able to mitigate SMN deficiency operate within pathways as part of which SMN can act: mRNA translation, actin network, and endocytosis. Here, we found that SMN accumulates at membrane protrusions during dynamic rearrangement of the actin filament. In addition to localization data, we show that SMN interacts with caveolin-1, which mediates anchoring of translation machinery components. Importantly, SMN deficiency depletes the plasma membrane of ribosomes, and this correlates with the failure of fibroblasts to extend membrane protrusions. These findings strongly support a relationship between SMN and membrane dyn...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 2000
We investigated the potential role of the ubiquitin proteolytic system in the death of cerebellar... more We investigated the potential role of the ubiquitin proteolytic system in the death of cerebellar granule neurons induced by reduction of extracellular potassium. Inhibitors of proteasomal function block apoptosis if administered at onset of this process, but they do not exert such effect when added 2-3 hr later. The same inhibitors also prevent caspase-3 activity and calpain-caspase-3-mediated processing of tau protein, suggesting that proteasomes are involved upstream of the caspase activation. Although the proteasomes seem to play an early primary role in programmed cell death, we found that with progression of apoptosis, during the execution phase, a perturbation in normal ubiquitin-proteasome function occurs, and high levels of ubiquitinated proteins accumulate in the cytoplasm of dying cells. Such accumulation correlates with a progressive decline of proteasome chymotrypsin and trypsin-like activities and, to a lower extent, of postacidic-like activity. Both intracytoplasmic a...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 1998
Cerebellar granule cells undergo apoptosis in culture after deprivation of potassium and serum. D... more Cerebellar granule cells undergo apoptosis in culture after deprivation of potassium and serum. During this process we found that tau, a neuronal microtubule-associated protein that plays a key role in the maintenance of neuronal architecture, and the pathology of which correlates with intellectual decline in Alzheimer's disease, is cleaved. The final product of this cleavage is a soluble dephosphorylated tau fragment of 17 kDa that is unable to associate with microtubules and accumulates in the perikarya of dying cells. The appearance of this 17 kDa fragment is inhibited by both caspase and calpain inhibitors, suggesting that tau is an in vivo substrate for both of these proteases during apoptosis. Tau cleavage is correlated with disruption of the microtubule network, and experiments with colchicine and taxol show that this is likely to be a cause and not a consequence of tau cleavage. These data indicate that tau cleavage and change in phosphorylation are important early facto...
Diabetes often correlates with tau phosphorylation and the development of Alzheimer's disease... more Diabetes often correlates with tau phosphorylation and the development of Alzheimer's disease. Both are associated with brain cholinergic dysfunction that could benefit from nerve growth factor (NGF)-based therapies. Electroacupuncture (EA) improves brain NGF availability and action. Here we assessed the variations of NGF and tau phosphorylation in the cortex and hippocampus, as well as the expression of choline acetyltransferase in the basal forebrain following diabetes induction and EA in adult rats. We found that EA counteracts diabetes-associated tau hyperphosphorylation and decreases in NGF and choline acetyltransferase, suggesting a possible beneficial effect of EA on brain cholinergic system in diabetes.
The microtubule associated protein tau plays a crucial role in Alzheimer's disease and in man... more The microtubule associated protein tau plays a crucial role in Alzheimer's disease and in many neurodegenerative disorders collectively known as tauopathies. Recently, tau pathology has been also documented in prion diseases although the possible molecular events linking these two proteins are still unknown. We have investigated the fate of normal cellular prion protein (PrP(C)) in primary cortical neurons overexpressing tau protein. We found that overexpression of tau reduces PrP(C) expression at the cell surface and causes its accumulation and aggregation in the cell body but does not affect its maturation and glycosylation. Trapped PrP(C) forms detergent-insoluble aggregates, mainly composed of un-glycosylated and mono-glycosylated forms of prion protein. Interestingly, co-transfection of tau gene in cortical neurons with a proteasome activity reporter, consisting of a short peptide degron fused to the carboxyl-terminus of green fluorescent protein (GFP-CL1), results in down-...
Biochemical studies on the process of neuronal differentiation have largely profited from the est... more Biochemical studies on the process of neuronal differentiation have largely profited from the establishment of cell lines which acquire a neuronal phenotype in response to various agents. In the last decade PC12 cells, derived from a rat pheochromocytoma, have become the first choice for studies of neuronal differentiation in tissue culture (Greene and Tishler 1982, Guroff 1985, Levi and Alema 1991), especially since these cells differentiate in response to a well defined growth factor: nerve growth factor, NGF (Levi-Montalcini 1987). As a result, the molecular characterization of the structure of the two kinds of NGF receptor, and recent theories on the possible mechanism of action of NGF have relied heavily on the use of these cells and mutant subclones derived from them (Hempstead et al. 1989, Hempstead et al. 1991, Kaplan et al. 1991). These studies have been the basis for understanding, in general, how the specificity of action of the various member of the neurotrophin family is achieved (Bothwell M. 1991, Hallbook et al. 1991). NGF, like its close relatives, is supposed to act not only on subpopulations of cells derived from the neural crest (Levi Montacini 1987, Whittemore et al.1987, Thoenen et al. 1987, Vantini et al. 1989), but also on several cell types which display high affinity binding for this growth factor (Cattaneo 1986, Otten et al. 1989, Aloe and Levi Montalcini 1979, Levi-Montalcini et al. 1990).
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