Roberto Cappai - Academia.edu (original) (raw)
Papers by Roberto Cappai
Mol. BioSyst., 2014
The 140 residue intrinsically disordered protein a-synuclein (a-syn) self-associates to form fibr... more The 140 residue intrinsically disordered protein a-synuclein (a-syn) self-associates to form fibrils that are the major constituent of the Lewy body intracellular protein inclusions, and neurotoxic oligomers. Both of these macromolecular structures are associated with a number of neurodegenerative diseases, including Parkinson's disease and dementia with Lewy bodies. Using ensemble optimisation modelling (EOM) and small angle X-ray scattering (SAXS) on a size-exclusion column equipped beamline, we studied how the distribution of structural conformers in a-syn may be influenced by the presence of the familial early-onset mutations A30P, E45K and A53T, by substituting the four methionine residues with alanines and by reaction with copper (Cu 2+ ) or an anti-fibril organic platinum (Pt) complex. We found that the WT had two major conformer groups, representing ensembles of compact and extended structures. The population of the extended group was increased in the more rapidly fibril-forming E45K and A53T mutants, while the compact group was enlarged in the oligomer-forming A30P mutant.
Neurobiology of Aging, 2004
15-fold increase in xCT RNA levels in the cell. Furthermore, we show that the anti-inflammatory i... more 15-fold increase in xCT RNA levels in the cell. Furthermore, we show that the anti-inflammatory interleukins (IL)-4, IL-10 and IL-13 are capable of attenuating the AIMnduced microglial glutamate secretion by more than half. Conclusions: Given these results on glutamate release and xCT gene regulation together with our earlier reports that IL-4, IL-10, and IL-13 are found in the brain and suppress cytokine and chemokine release from A~activated microglia, we propose that glutamatergic disruption in AD may in part be mediated by chronic inflammation and microglial-derived glutamate release inadequately countered by endogenous anti-inflammatory cytokines.
Journal of Biological Chemistry, 2003
The amyloid  peptide is toxic to neurons, and it is believed that this toxicity plays a central ... more The amyloid  peptide is toxic to neurons, and it is believed that this toxicity plays a central role in the progression of Alzheimer's disease. The mechanism of this toxicity is contentious. Here we report that an A peptide with the sulfur atom of Met-35 oxidized to a sulfoxide (Met(O)A) is toxic to neuronal cells, and this toxicity is attenuated by the metal chelator clioquinol and completely rescued by catalase implicating the same toxicity mechanism as reduced A. However, unlike the unoxidized peptide, Met(O)A is unable to penetrate lipid membranes to form ion channel-like structures, and -sheet formation is inhibited, phenomena that are central to some theories for A toxicity. Our results show that, like the unoxidized peptide, Met(O)A will coordinate Cu 2؉ and reduce the oxidation state of the metal and still produce H 2 O 2 . We hypothesize that Met(O)A production contributes to the elevation of soluble A seen in the brain in Alzheimer's disease.
Brain Research, 2011
The amyloid precursor protein (APP) is known to increase following traumatic brain injury (TBI). ... more The amyloid precursor protein (APP) is known to increase following traumatic brain injury (TBI). This increase in levels of APP may be deleterious to outcome due to the production of neurotoxic Aβ. Conversely, this upregulation may be beneficial as cleavage of APP via the alternative non-amyloidogenic pathway produces the soluble α form of APP (sAPPα), which is known to have many neuroprotective and neurotrophic functions. Indeed it has previously been shown that treatment with sAPPα following a diffuse injury in rats improves outcome. However, the exact location within the sAPPα molecule which contains this neuroprotective activity has yet to be determined. The sAPPα peptide can consist of up to 6 domains, with the main isoform in the brain missing the 4th and 5th. Of the remaining domains, the D1 and D6a domains seem the most likely as they have been shown to have beneficial actions in vitro. This present study examined the effects of in vivo posttraumatic administration via an intracerebroventricular injection of the D1, D2 and D6a domains of sAPPα on outcome following moderate-impact acceleration TBI in rats. While treatment with either the D1 or D6a domains was found to significantly improve motor and cognitive outcome, as assessed on the rotarod and Y maze, treatment with the D2 domain had no effect. Furthermore axonal injury was reduced in D1 and D6a domain treated animals, but not those that received the D2 domain. As the D1 and D6a domains contain a heparin binding region while the D2 domain does not, this suggests that sAPPα mediates its neuroprotective response through its ability to bind to heparin sulfate proteoglycans.
Brain Research, 2012
The amyloid precursor protein (APP) increases following traumatic brain injury (TBI), although th... more The amyloid precursor protein (APP) increases following traumatic brain injury (TBI), although the functional significance of this remains unclear largely because the functions of the subsequent APP metabolites are so different: Aβ is neurotoxic whilst sAPPα is neuroprotective.
Neuroreport, 2002
We have analyzed the expression of Alzheimer&... more We have analyzed the expression of Alzheimer's disease-associated presenilin 1 (PS1) in various neurodegenerative disorders. Western blotting identified PS1 N- and C-terminal fragments similarly in the cortex of controls, Parkinson, Huntington and schizophrenia subjects. Additional PS1 immunoreactive species of 42 and 46 kDa were present in six out of seven cases of sporadic frontotemporal dementia (FTD) and these were particularly prominent in two cases. RT-PCR analysis using nested primers showed the presence of PS1 gene products with deletions within the exon 4-8 region. Our results suggest that alternative transcription of PS1 may be associated with FTD.
Journal of Neurochemistry, 2002
To study amyloid -protein (A) production and aggregation in vivo, we created two transgenic (Tg... more To study amyloid -protein (A) production and aggregation in vivo, we created two transgenic (Tg) mouse lines expressing the C-terminal 100 amino acids of human amyloid precursor protein (APP): Tg C100.V717F and Tg C100.WT. Western blot analysis showed that human APP-C100 and A were produced in brain and some peripheral tissues and A was produced in serum. Using antibodies specific for the A C terminus we found that Tg C100.V717F produced a 1.6-fold increase in A42/A40 compared with Tg C100.WT. Approximately 30% of total brain A (ϳ122 ng/g of wet tissue) was water-soluble. The remaining 70% of A partitioned into the particulate fraction and was completely sodium dodecyl sulfate-soluble. In contrast, human Alzheimer's disease brain has predominantly sodium dodecyl sulfate-insoluble A. Immunohistochemistry with an A(5-8) antibody showed that A or A-containing fragments accumulated intracellularly in the hippocampus of aged Tg C100.V717F mice. The soluble A levels in Tg brain are similar to those in normal human brain, and this may explain the lack of microscopic amyloid deposits in the Tg mice. However, this mouse model provides a system to study the intracellular processing and accumulation of A or A-containing fragments and to screen for compounds directed at the ␥-secretase activity. Key Words: Alzheimer's disease -Amyloid -protein-Amyloid precursor protein-C100 -Transgenic mouse -Processing.
Neurobiology of Disease, 2000
The ␣-synuclein (␣SN) protein is thought to play a central role in the pathogenesis of neurodegen... more The ␣-synuclein (␣SN) protein is thought to play a central role in the pathogenesis of neurodegenerative diseases where it aggregates to form intracellular inclusions. We have used Western blotting to examine the expression levels and solubility of ␣SN in brain homogenates from dementia with Lewy bodies (DLB), Parkinson's disease (PD), Alzheimer's disease (AD), and normal controls using samples from the parahippocampus/transentorhinal cortex. Compared to controls, DLB brains accumulate significantly greater amounts of sodium dodecyl sulfate (SDS)-soluble and SDS-insoluble ␣SN but levels of TBS-soluble ␣SN did not change. Levels of synaptophysin, a marker of synaptic integrity, were significantly lower in DLB cases than in normal aged controls regardless of whether concurrent changes of AD were present. This limbic synaptic dysfunction may contribute to cognitive impairment in DLB. Whether aggregated ␣SN is a cause or effect of the disease process in DLB and PD remains to be determined, but the presence of aggregated ␣SN is consistent with a pathogenesis similar to that associated with aggregates of A amyloid in AD.
Experimental Neurology, 2007
α-Synuclein (αSN) is implicated in Parkinson's disease (PD) and is the major component of Lewy bo... more α-Synuclein (αSN) is implicated in Parkinson's disease (PD) and is the major component of Lewy bodies (LBs). Although αSN is mainly expressed in neuronal cells and exists as a cytoplasmic protein, it has been found in body fluids including cerebrospinal fluid and blood. This study explored plasma αSN as a diagnostic marker for PD. Western blot analysis was used to characterize plasma αSN compared to brain αSN. Plasma αSN of 16 kDa migrates with the same mobility as its brain counterpart and recombinant αSN on denatured polyacrylamide gels and reacted with three different antibodies against the C-terminal and NAC regions of the αSN protein. The αSN levels in plasma from PD subjects are significantly lower than that in age-matched controls (p = 0.001), and the αSN levels in patients with early-onset PD are lower than that in both late-onset PD and controls. This initial study indicates that measurement of αSN in plasma can provide support for a clinical diagnosis of Parkinson's disease and warrants further study in a larger population.
The FASEB Journal, 2005
Dopamine (DA) and α-synuclein (α-SN) are two key molecules associated with Parkinson's disease (P... more Dopamine (DA) and α-synuclein (α-SN) are two key molecules associated with Parkinson's disease (PD). We have identified a novel action of DA in the initial phase of α-SN aggregation and demonstrate that DA induces α-SN to form soluble, SDS-resistant oligomers. The DA:α-SN oligomeric species are not amyloidogenic as they do not react with thioflavin T and lack the typical amyloid fibril structures as visualized with electron microscopy. Circular dichroism studies indicate that in the presence of lipid membranes DA interacts with α-SN, causing an alteration to the structure of the protein. Furthermore, DA inhibited the formation of ironinduced α-SN amyloidogenic aggregates, suggesting that DA acts as a dominant modulator of α-SN aggregation. These observations support the paradigm emerging for other neurodegenerative diseases that the toxic species is represented by a soluble oligomer and not the insoluble fibril.
Neuroreport, 2000
Presenilin (PS) mutations are associated with early-onset Alzheimer's disease and PS prot... more Presenilin (PS) mutations are associated with early-onset Alzheimer's disease and PS proteins are involved with gamma-secretase cleavage of the amyloid precursor protein, APP. We have shown previously that alpha-, beta- and gamma-secretase cleavages of APP are conserved in Pichia pastoris. Here, we report co-expression of APP and PSI in P. pastoris and show by immunoelectron microscopy colocalization of these two proteins in expanded endoplasmic reticulum. Western blot analysis indicates a drastic reduction of both alpha- and beta-secretase products. A relative increase in beta-secretase product derived from immature APP is also observed, pointing to a beta-secretase activity of P. pastoris associated with the early secretory pathway.
Mol. BioSyst., 2014
The 140 residue intrinsically disordered protein a-synuclein (a-syn) self-associates to form fibr... more The 140 residue intrinsically disordered protein a-synuclein (a-syn) self-associates to form fibrils that are the major constituent of the Lewy body intracellular protein inclusions, and neurotoxic oligomers. Both of these macromolecular structures are associated with a number of neurodegenerative diseases, including Parkinson's disease and dementia with Lewy bodies. Using ensemble optimisation modelling (EOM) and small angle X-ray scattering (SAXS) on a size-exclusion column equipped beamline, we studied how the distribution of structural conformers in a-syn may be influenced by the presence of the familial early-onset mutations A30P, E45K and A53T, by substituting the four methionine residues with alanines and by reaction with copper (Cu 2+ ) or an anti-fibril organic platinum (Pt) complex. We found that the WT had two major conformer groups, representing ensembles of compact and extended structures. The population of the extended group was increased in the more rapidly fibril-forming E45K and A53T mutants, while the compact group was enlarged in the oligomer-forming A30P mutant.
Neurobiology of Aging, 2004
15-fold increase in xCT RNA levels in the cell. Furthermore, we show that the anti-inflammatory i... more 15-fold increase in xCT RNA levels in the cell. Furthermore, we show that the anti-inflammatory interleukins (IL)-4, IL-10 and IL-13 are capable of attenuating the AIMnduced microglial glutamate secretion by more than half. Conclusions: Given these results on glutamate release and xCT gene regulation together with our earlier reports that IL-4, IL-10, and IL-13 are found in the brain and suppress cytokine and chemokine release from A~activated microglia, we propose that glutamatergic disruption in AD may in part be mediated by chronic inflammation and microglial-derived glutamate release inadequately countered by endogenous anti-inflammatory cytokines.
Journal of Biological Chemistry, 2003
The amyloid  peptide is toxic to neurons, and it is believed that this toxicity plays a central ... more The amyloid  peptide is toxic to neurons, and it is believed that this toxicity plays a central role in the progression of Alzheimer's disease. The mechanism of this toxicity is contentious. Here we report that an A peptide with the sulfur atom of Met-35 oxidized to a sulfoxide (Met(O)A) is toxic to neuronal cells, and this toxicity is attenuated by the metal chelator clioquinol and completely rescued by catalase implicating the same toxicity mechanism as reduced A. However, unlike the unoxidized peptide, Met(O)A is unable to penetrate lipid membranes to form ion channel-like structures, and -sheet formation is inhibited, phenomena that are central to some theories for A toxicity. Our results show that, like the unoxidized peptide, Met(O)A will coordinate Cu 2؉ and reduce the oxidation state of the metal and still produce H 2 O 2 . We hypothesize that Met(O)A production contributes to the elevation of soluble A seen in the brain in Alzheimer's disease.
Brain Research, 2011
The amyloid precursor protein (APP) is known to increase following traumatic brain injury (TBI). ... more The amyloid precursor protein (APP) is known to increase following traumatic brain injury (TBI). This increase in levels of APP may be deleterious to outcome due to the production of neurotoxic Aβ. Conversely, this upregulation may be beneficial as cleavage of APP via the alternative non-amyloidogenic pathway produces the soluble α form of APP (sAPPα), which is known to have many neuroprotective and neurotrophic functions. Indeed it has previously been shown that treatment with sAPPα following a diffuse injury in rats improves outcome. However, the exact location within the sAPPα molecule which contains this neuroprotective activity has yet to be determined. The sAPPα peptide can consist of up to 6 domains, with the main isoform in the brain missing the 4th and 5th. Of the remaining domains, the D1 and D6a domains seem the most likely as they have been shown to have beneficial actions in vitro. This present study examined the effects of in vivo posttraumatic administration via an intracerebroventricular injection of the D1, D2 and D6a domains of sAPPα on outcome following moderate-impact acceleration TBI in rats. While treatment with either the D1 or D6a domains was found to significantly improve motor and cognitive outcome, as assessed on the rotarod and Y maze, treatment with the D2 domain had no effect. Furthermore axonal injury was reduced in D1 and D6a domain treated animals, but not those that received the D2 domain. As the D1 and D6a domains contain a heparin binding region while the D2 domain does not, this suggests that sAPPα mediates its neuroprotective response through its ability to bind to heparin sulfate proteoglycans.
Brain Research, 2012
The amyloid precursor protein (APP) increases following traumatic brain injury (TBI), although th... more The amyloid precursor protein (APP) increases following traumatic brain injury (TBI), although the functional significance of this remains unclear largely because the functions of the subsequent APP metabolites are so different: Aβ is neurotoxic whilst sAPPα is neuroprotective.
Neuroreport, 2002
We have analyzed the expression of Alzheimer&... more We have analyzed the expression of Alzheimer's disease-associated presenilin 1 (PS1) in various neurodegenerative disorders. Western blotting identified PS1 N- and C-terminal fragments similarly in the cortex of controls, Parkinson, Huntington and schizophrenia subjects. Additional PS1 immunoreactive species of 42 and 46 kDa were present in six out of seven cases of sporadic frontotemporal dementia (FTD) and these were particularly prominent in two cases. RT-PCR analysis using nested primers showed the presence of PS1 gene products with deletions within the exon 4-8 region. Our results suggest that alternative transcription of PS1 may be associated with FTD.
Journal of Neurochemistry, 2002
To study amyloid -protein (A) production and aggregation in vivo, we created two transgenic (Tg... more To study amyloid -protein (A) production and aggregation in vivo, we created two transgenic (Tg) mouse lines expressing the C-terminal 100 amino acids of human amyloid precursor protein (APP): Tg C100.V717F and Tg C100.WT. Western blot analysis showed that human APP-C100 and A were produced in brain and some peripheral tissues and A was produced in serum. Using antibodies specific for the A C terminus we found that Tg C100.V717F produced a 1.6-fold increase in A42/A40 compared with Tg C100.WT. Approximately 30% of total brain A (ϳ122 ng/g of wet tissue) was water-soluble. The remaining 70% of A partitioned into the particulate fraction and was completely sodium dodecyl sulfate-soluble. In contrast, human Alzheimer's disease brain has predominantly sodium dodecyl sulfate-insoluble A. Immunohistochemistry with an A(5-8) antibody showed that A or A-containing fragments accumulated intracellularly in the hippocampus of aged Tg C100.V717F mice. The soluble A levels in Tg brain are similar to those in normal human brain, and this may explain the lack of microscopic amyloid deposits in the Tg mice. However, this mouse model provides a system to study the intracellular processing and accumulation of A or A-containing fragments and to screen for compounds directed at the ␥-secretase activity. Key Words: Alzheimer's disease -Amyloid -protein-Amyloid precursor protein-C100 -Transgenic mouse -Processing.
Neurobiology of Disease, 2000
The ␣-synuclein (␣SN) protein is thought to play a central role in the pathogenesis of neurodegen... more The ␣-synuclein (␣SN) protein is thought to play a central role in the pathogenesis of neurodegenerative diseases where it aggregates to form intracellular inclusions. We have used Western blotting to examine the expression levels and solubility of ␣SN in brain homogenates from dementia with Lewy bodies (DLB), Parkinson's disease (PD), Alzheimer's disease (AD), and normal controls using samples from the parahippocampus/transentorhinal cortex. Compared to controls, DLB brains accumulate significantly greater amounts of sodium dodecyl sulfate (SDS)-soluble and SDS-insoluble ␣SN but levels of TBS-soluble ␣SN did not change. Levels of synaptophysin, a marker of synaptic integrity, were significantly lower in DLB cases than in normal aged controls regardless of whether concurrent changes of AD were present. This limbic synaptic dysfunction may contribute to cognitive impairment in DLB. Whether aggregated ␣SN is a cause or effect of the disease process in DLB and PD remains to be determined, but the presence of aggregated ␣SN is consistent with a pathogenesis similar to that associated with aggregates of A amyloid in AD.
Experimental Neurology, 2007
α-Synuclein (αSN) is implicated in Parkinson's disease (PD) and is the major component of Lewy bo... more α-Synuclein (αSN) is implicated in Parkinson's disease (PD) and is the major component of Lewy bodies (LBs). Although αSN is mainly expressed in neuronal cells and exists as a cytoplasmic protein, it has been found in body fluids including cerebrospinal fluid and blood. This study explored plasma αSN as a diagnostic marker for PD. Western blot analysis was used to characterize plasma αSN compared to brain αSN. Plasma αSN of 16 kDa migrates with the same mobility as its brain counterpart and recombinant αSN on denatured polyacrylamide gels and reacted with three different antibodies against the C-terminal and NAC regions of the αSN protein. The αSN levels in plasma from PD subjects are significantly lower than that in age-matched controls (p = 0.001), and the αSN levels in patients with early-onset PD are lower than that in both late-onset PD and controls. This initial study indicates that measurement of αSN in plasma can provide support for a clinical diagnosis of Parkinson's disease and warrants further study in a larger population.
The FASEB Journal, 2005
Dopamine (DA) and α-synuclein (α-SN) are two key molecules associated with Parkinson's disease (P... more Dopamine (DA) and α-synuclein (α-SN) are two key molecules associated with Parkinson's disease (PD). We have identified a novel action of DA in the initial phase of α-SN aggregation and demonstrate that DA induces α-SN to form soluble, SDS-resistant oligomers. The DA:α-SN oligomeric species are not amyloidogenic as they do not react with thioflavin T and lack the typical amyloid fibril structures as visualized with electron microscopy. Circular dichroism studies indicate that in the presence of lipid membranes DA interacts with α-SN, causing an alteration to the structure of the protein. Furthermore, DA inhibited the formation of ironinduced α-SN amyloidogenic aggregates, suggesting that DA acts as a dominant modulator of α-SN aggregation. These observations support the paradigm emerging for other neurodegenerative diseases that the toxic species is represented by a soluble oligomer and not the insoluble fibril.
Neuroreport, 2000
Presenilin (PS) mutations are associated with early-onset Alzheimer's disease and PS prot... more Presenilin (PS) mutations are associated with early-onset Alzheimer's disease and PS proteins are involved with gamma-secretase cleavage of the amyloid precursor protein, APP. We have shown previously that alpha-, beta- and gamma-secretase cleavages of APP are conserved in Pichia pastoris. Here, we report co-expression of APP and PSI in P. pastoris and show by immunoelectron microscopy colocalization of these two proteins in expanded endoplasmic reticulum. Western blot analysis indicates a drastic reduction of both alpha- and beta-secretase products. A relative increase in beta-secretase product derived from immature APP is also observed, pointing to a beta-secretase activity of P. pastoris associated with the early secretory pathway.