- Email: [email protected]
S.04.1 Analysis of cerebrospinal fluid of depressed patients
s102
I
S 03 05
S.04.
Protofibrils disease
L. Lauufelt. Health/Geriatrics,
in the
Hot
pathogenesis
topic
session
in preclinical
of Alzheimer’s
Uppsala University, Department Uppsala, Sweden
of Public
We have recently ideutified au APP nmtatiou (E693G) in a family from uorthem Sweden, named the “Arctic” family. Clinical examination, ueuropsychological testing, braiu imaging aud EEG were used in evaluating patieuts according to DSM-IV criteria. Clinical history was typical for AD, with a slow insidious progression aud decline in memory for recent eveuts as the first preseutiug symptom. Signs of strokes or vascular lesions were uot found ou braiu imaging in seveu investigated patients. Carriers of this “Arctic” mutation showed decreased Al342 aud Al340 levels in plasma. This fiudiug was corroborated in vitro, where the Al342 couceutratiou was low in couditioued media from cells trausfected with APP E693G. Fibrillizatiou studies demonstrated uo difference in fibrillizatiou rate, but Al3 peptides with the Arctic mutation (AnArc) formed protofibrils at a much higher rate aud in larger quantities thau wild-type (wt) Al3. We have seeu a shift in APP processing with the Arctic mutation, favouriug the l3secretase pathway, which should result in elevated Al3 levels. Thus, it is importaut to study the intracellular levels of Al3 to solve this puzzle. The unique fiudiug of decreased Al3 plasma levels aud iucreased protofibril formation in the Arctic AD is likely to reflect a novel pathogenic mechanism for AD iuvolviug a rapid Al3 protofibril formation leading to accelerated build-up of iusoluble Al3 i&aand/or extracellularly.
S.04. Hot topic session in preclinical neuropsychopharmacology IS
04 01
Analysis patients
M. Roseuhageu, C. Turck. Max Germany
of
cerebrospinal
D. Milfay, G. Maccarroue, Planck Institute, Proteomics
fluid
of depressed
M. Uhr, Group,
F. Holsboer, Munich,
Objectives: Comparative analysis of the proteome of cerebrospiual fluid (CSF) of depressed aud healthy subjects with the aim to ideutify disease specific proteins that may be targets for novel treatments in psychiatric aud neurological diseases. Background: Analysis of protein patterus of cerebrospiual fluid (CSF) is of great diagnostic importance because it reflects the metabolic state of the braiu in several diseases as well as uuder healthy conditions. The majority of CSF proteins are serum proteins, while ouly a small number are derived from the CNS. The ideutificatiou of CSF proteins is also importaut for the development of new, clinically useful ueuroual markers aud for studying the pathogeuesis of meutal disorders. Method: Cases were in-patients with the diagnosis of a severe major depressive episode. Controls consisted of patients without a history of psychiatric ilhless that were admitted to the neurology ward for routiue diagnosis aud the exclusion of severe neurological disorders. CSF proteins were separated by two dimeusioual (2D) gel electrophoresis. They are separated first by charge aud theu by size. Differences in spot patterus were analyzed by the computer assisted program PDQUEST. Ideutificatiou of proteins were doue by mass spectrometry.
neuropsychopharmacology Results: Preliminary evaluatiou of the data of our 2D analysis has showu both modest qualitative aud quantitative differences in the protein patterns betweeu cases aud controls. Several proteins were ideutified by mass spectrometry. Further analysis is on-going, focussiug ou the evaluatiou of the results.
I S 04 02
Biochemical function
modulation by antidepressants
of
AMPA
P. Svemliugssou’ , E. Tzavara2, G. Nomikos2, B. P. Greeugard4. ‘Karolinska Institute, Department and Pharmacology, Stockholm, Sweden; 2Eli Lilly Indianapolis, US.A.; 3Rockefeller University, New 4Rockefeller University, Laboratory of Molecular Neuroscience, New York, US.A.
receptor
McEweu3, of Physiology and Company, York, US.A.; and Cellular
Little is knowu about the molecular basis uuderlyiug the clinical efficacy of autidepressauts. The primary action of the autidepressauts, fluoxetiue (Prozac) aud tiaueptiue (Stablou) is markedly differeut. Whereas fluoxetiue iucreases the syuaptic availability of serotouiu, tiaueptiue decreases the levels of this ueurotrausmitter. Despite this, the ouset of therapeutic action aud the efficacy of these autidepressauts is similar. It has recently beeu described that phosphorylation of two specific sites of the GluRl subunit of AMPA receptor complex euhauces the fuuctiou of the AMPA receptor. Since it is possible that alteratious in syuaptic plasticity may underlie the efficacy of autidepressauts, we studied the effects of fluoxetiue aud tiaueptiue ou the phosphorylation state at these sites, a CanXIIIPKC site, Ser83 l-GluRl, aud a PKA site, Ser845GluRl. It was found that acute adminstration of fluoxetiue upregulates the phosphorylation at both Ser83 l- aud Ser845-GluRl, in froutal cortex aud hippocampus. Followiug chrouic admiuistratiou, fluoxetiue iucreases Ser845-GluRl, but uot Ser83 l-GluRl. In contrast, acute administration of tiaueptiue did uot siguificautly affect the phosphorylation state of Ser83 l- or Ser845-GluRl. However, chrouic admiustratiou of tiaueptiue iucreased the phosphorylatiou of Ser831-GluRl in froutal cortex. Thus, iucreased phosphorylation of AMPA receptors is a connnou cousequeuce of chrouic administration of fluoxetiue aud tiaueptiue, but this modulatiou occurs at distinct phosphorylation sites. A more detailed characterization of the mechanism uuderlyiug the phosphorylation of Ser845-GluRl by fluoxetiue has also beeu undertaken Previous work has established that protein phosphatase-1 (PP-1), a major seriueithreouiue protein phosphatase in the brain, regulates AMPA receptor phosphorylatiou aud function It has also beeu showu that dopamiue utilize the PKADARPP-32/PP-1 signaling cascade to AMPA receptor phosphorylatiou aud fuuctiou. Administration of fluoxetiue iucreases phosphorylation of DARPP-32 at the PKA site, Thr34, aud at the (X-1 site, Ser137, aud decreases phosphorylation at the Cdk5 site, Thr75. Pharmacological studies showed that stimulation of 5-HT4 aud 5-HT6 receptors causes au iucreased phosphorylation state at Thr34-DARPP-32, the protein kiuase (PKA) site, aud a decreased phosphorylation state at Thr75DARPP-32, the cyclic-depeudeut kiuase 5 (Cdk5) site, whereas stimulation of 5-HT2 receptors iucreases the phosphorylation state of Ser137-DARPP-32, the caseiu kiuase-1 ((X-1) site. Each of these chauges contributes, through distinct sigualiug pathways, to iucreased iuhibitiou of protein phosphatase-1 (PP-1). Indeed, the ability of fluoxetiue to iucrease Ser845-GluRl phosphorylation is strongly atteuuated in DARPP-32 KO mice. In agreement with this biochemical observation, it was also found that the ability of
I
S 03 05
S.04.
Protofibrils disease
L. Lauufelt. Health/Geriatrics,
in the
Hot
pathogenesis
topic
session
in preclinical
of Alzheimer’s
Uppsala University, Department Uppsala, Sweden
of Public
We have recently ideutified au APP nmtatiou (E693G) in a family from uorthem Sweden, named the “Arctic” family. Clinical examination, ueuropsychological testing, braiu imaging aud EEG were used in evaluating patieuts according to DSM-IV criteria. Clinical history was typical for AD, with a slow insidious progression aud decline in memory for recent eveuts as the first preseutiug symptom. Signs of strokes or vascular lesions were uot found ou braiu imaging in seveu investigated patients. Carriers of this “Arctic” mutation showed decreased Al342 aud Al340 levels in plasma. This fiudiug was corroborated in vitro, where the Al342 couceutratiou was low in couditioued media from cells trausfected with APP E693G. Fibrillizatiou studies demonstrated uo difference in fibrillizatiou rate, but Al3 peptides with the Arctic mutation (AnArc) formed protofibrils at a much higher rate aud in larger quantities thau wild-type (wt) Al3. We have seeu a shift in APP processing with the Arctic mutation, favouriug the l3secretase pathway, which should result in elevated Al3 levels. Thus, it is importaut to study the intracellular levels of Al3 to solve this puzzle. The unique fiudiug of decreased Al3 plasma levels aud iucreased protofibril formation in the Arctic AD is likely to reflect a novel pathogenic mechanism for AD iuvolviug a rapid Al3 protofibril formation leading to accelerated build-up of iusoluble Al3 i&aand/or extracellularly.
S.04. Hot topic session in preclinical neuropsychopharmacology IS
04 01
Analysis patients
M. Roseuhageu, C. Turck. Max Germany
of
cerebrospinal
D. Milfay, G. Maccarroue, Planck Institute, Proteomics
fluid
of depressed
M. Uhr, Group,
F. Holsboer, Munich,
Objectives: Comparative analysis of the proteome of cerebrospiual fluid (CSF) of depressed aud healthy subjects with the aim to ideutify disease specific proteins that may be targets for novel treatments in psychiatric aud neurological diseases. Background: Analysis of protein patterus of cerebrospiual fluid (CSF) is of great diagnostic importance because it reflects the metabolic state of the braiu in several diseases as well as uuder healthy conditions. The majority of CSF proteins are serum proteins, while ouly a small number are derived from the CNS. The ideutificatiou of CSF proteins is also importaut for the development of new, clinically useful ueuroual markers aud for studying the pathogeuesis of meutal disorders. Method: Cases were in-patients with the diagnosis of a severe major depressive episode. Controls consisted of patients without a history of psychiatric ilhless that were admitted to the neurology ward for routiue diagnosis aud the exclusion of severe neurological disorders. CSF proteins were separated by two dimeusioual (2D) gel electrophoresis. They are separated first by charge aud theu by size. Differences in spot patterus were analyzed by the computer assisted program PDQUEST. Ideutificatiou of proteins were doue by mass spectrometry.
neuropsychopharmacology Results: Preliminary evaluatiou of the data of our 2D analysis has showu both modest qualitative aud quantitative differences in the protein patterns betweeu cases aud controls. Several proteins were ideutified by mass spectrometry. Further analysis is on-going, focussiug ou the evaluatiou of the results.
I S 04 02
Biochemical function
modulation by antidepressants
of
AMPA
P. Svemliugssou’ , E. Tzavara2, G. Nomikos2, B. P. Greeugard4. ‘Karolinska Institute, Department and Pharmacology, Stockholm, Sweden; 2Eli Lilly Indianapolis, US.A.; 3Rockefeller University, New 4Rockefeller University, Laboratory of Molecular Neuroscience, New York, US.A.
receptor
McEweu3, of Physiology and Company, York, US.A.; and Cellular
Little is knowu about the molecular basis uuderlyiug the clinical efficacy of autidepressauts. The primary action of the autidepressauts, fluoxetiue (Prozac) aud tiaueptiue (Stablou) is markedly differeut. Whereas fluoxetiue iucreases the syuaptic availability of serotouiu, tiaueptiue decreases the levels of this ueurotrausmitter. Despite this, the ouset of therapeutic action aud the efficacy of these autidepressauts is similar. It has recently beeu described that phosphorylation of two specific sites of the GluRl subunit of AMPA receptor complex euhauces the fuuctiou of the AMPA receptor. Since it is possible that alteratious in syuaptic plasticity may underlie the efficacy of autidepressauts, we studied the effects of fluoxetiue aud tiaueptiue ou the phosphorylation state at these sites, a CanXIIIPKC site, Ser83 l-GluRl, aud a PKA site, Ser845GluRl. It was found that acute adminstration of fluoxetiue upregulates the phosphorylation at both Ser83 l- aud Ser845-GluRl, in froutal cortex aud hippocampus. Followiug chrouic admiuistratiou, fluoxetiue iucreases Ser845-GluRl, but uot Ser83 l-GluRl. In contrast, acute administration of tiaueptiue did uot siguificautly affect the phosphorylation state of Ser83 l- or Ser845-GluRl. However, chrouic admiustratiou of tiaueptiue iucreased the phosphorylatiou of Ser831-GluRl in froutal cortex. Thus, iucreased phosphorylation of AMPA receptors is a connnou cousequeuce of chrouic administration of fluoxetiue aud tiaueptiue, but this modulatiou occurs at distinct phosphorylation sites. A more detailed characterization of the mechanism uuderlyiug the phosphorylation of Ser845-GluRl by fluoxetiue has also beeu undertaken Previous work has established that protein phosphatase-1 (PP-1), a major seriueithreouiue protein phosphatase in the brain, regulates AMPA receptor phosphorylatiou aud function It has also beeu showu that dopamiue utilize the PKADARPP-32/PP-1 signaling cascade to AMPA receptor phosphorylatiou aud fuuctiou. Administration of fluoxetiue iucreases phosphorylation of DARPP-32 at the PKA site, Thr34, aud at the (X-1 site, Ser137, aud decreases phosphorylation at the Cdk5 site, Thr75. Pharmacological studies showed that stimulation of 5-HT4 aud 5-HT6 receptors causes au iucreased phosphorylation state at Thr34-DARPP-32, the protein kiuase (PKA) site, aud a decreased phosphorylation state at Thr75DARPP-32, the cyclic-depeudeut kiuase 5 (Cdk5) site, whereas stimulation of 5-HT2 receptors iucreases the phosphorylation state of Ser137-DARPP-32, the caseiu kiuase-1 ((X-1) site. Each of these chauges contributes, through distinct sigualiug pathways, to iucreased iuhibitiou of protein phosphatase-1 (PP-1). Indeed, the ability of fluoxetiue to iucrease Ser845-GluRl phosphorylation is strongly atteuuated in DARPP-32 KO mice. In agreement with this biochemical observation, it was also found that the ability of