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Integrative proteomic analysis of the NMDA NR1 knockdown mouse model reveals effects on central and peripheral pathways associated with schizophrenia and autism spectrum disorders.

Wesseling H, Guest PC, Lee CM, Wong EH, Rahmoune H, Bahn S - Mol Autism (2014)

Bottom Line: The highest magnitude changes were found for neurotrophic factors (VEGFA, EGF, IGF-1), apolipoprotein A1, and fibrinogen.We also found decreased levels of several chemokines.In contrast, increased levels of proteins involved in neurotransmitter metabolism and release were found only in the frontal cortex and abnormalities of proteins involved in the purinergic system were found exclusively in the hippocampus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, UK.

ABSTRACT

Background: Over the last decade, the transgenic N-methyl-D-aspartate receptor (NMDAR) NR1-knockdown mouse (NR1(neo-/-)) has been investigated as a glutamate hypofunction model for schizophrenia. Recent research has now revealed that the model also recapitulates cognitive and negative symptoms in the continuum of other psychiatric diseases, particularly autism spectrum disorders (ASD). As previous studies have mostly focussed on behavioural readouts, a molecular characterisation of this model will help to identify novel biomarkers or potential drug targets.

Methods: Here, we have used multiplex immunoassay analyses to investigate peripheral analyte alterations in serum of NR1(neo-/-) mice, as well as a combination of shotgun label-free liquid chromatography mass spectrometry, bioinformatic pathway analyses, and a shotgun-based 40-plex selected reaction monitoring (SRM) assay to investigate altered molecular pathways in the frontal cortex and hippocampus. All findings were cross compared to identify translatable findings between the brain and periphery.

Results: Multiplex immunoassay profiling led to identification of 29 analytes that were significantly altered in sera of NR1(neo-/-) mice. The highest magnitude changes were found for neurotrophic factors (VEGFA, EGF, IGF-1), apolipoprotein A1, and fibrinogen. We also found decreased levels of several chemokines. Following this, LC-MS(E) profiling led to identification of 48 significantly changed proteins in the frontal cortex and 41 in the hippocampus. In particular, MARCS, the mitochondrial pyruvate kinase, and CamKII-alpha were affected. Based on the combination of protein set enrichment and bioinformatic pathway analysis, we designed orthogonal SRM-assays which validated the abnormalities of proteins involved in synaptic long-term potentiation, myelination, and the ERK-signalling pathway in both brain regions. In contrast, increased levels of proteins involved in neurotransmitter metabolism and release were found only in the frontal cortex and abnormalities of proteins involved in the purinergic system were found exclusively in the hippocampus.

Conclusions: Taken together, this multi-platform profiling study has identified peripheral changes which are potentially linked to central alterations in synaptic plasticity and neuronal function associated with NMDAR-NR1 hypofunction. Therefore, the reported proteomic changes may be useful as translational biomarkers in human and rodent model drug discovery efforts.

No MeSH data available.


Related in: MedlinePlus

Computational pathway analysis of brain proteomic profiling. (A) IPA showing decreased and increased biological functions in NR1neo−/− mouse brain regions. Depicted are functions with an activation score (z-score) >1 (increased activation) or < −1 (decreased activation) with their corresponding log2 (P) (right graph, the dotted line represents the 0.05 P value threshold) as well as the five most-significantly dysregulated non-directionally (z-score > −1, <1, or not predicted) affected pathways (in the middle of the bar plots). (B) Identified IPA networks in the data set by global pathway analysis using the Ingenuity Pathways Knowledge Database (IPKB) software. All significantly altered proteins in the frontal cortex and hippocampus from the model were used for the network analysis based on criteria annotated in the IPKB database, which contains molecular information available in the scientific literature. Networks were generated algorithmically on the basis of the connectivity derived from molecular interaction information, scored according to the significant number of focus proteins and assigned associated biological functions (see network descriptions) by overlaying the network molecules onto predefined maps of functional or pathway information in the IPKB database. Proteins are indicated by their gene names. Red and green symbols/text indicate increased and decreased proteins, respectively. Blue symbols indicate predicted inhibition, orange lines indicate predicted activation. Yellow lines indicate inconsistencies with the states of the downstream molecules. Lines ending with arrows indicate activation, lines without arrows indicate interaction/binding.
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Figure 2: Computational pathway analysis of brain proteomic profiling. (A) IPA showing decreased and increased biological functions in NR1neo−/− mouse brain regions. Depicted are functions with an activation score (z-score) >1 (increased activation) or < −1 (decreased activation) with their corresponding log2 (P) (right graph, the dotted line represents the 0.05 P value threshold) as well as the five most-significantly dysregulated non-directionally (z-score > −1, <1, or not predicted) affected pathways (in the middle of the bar plots). (B) Identified IPA networks in the data set by global pathway analysis using the Ingenuity Pathways Knowledge Database (IPKB) software. All significantly altered proteins in the frontal cortex and hippocampus from the model were used for the network analysis based on criteria annotated in the IPKB database, which contains molecular information available in the scientific literature. Networks were generated algorithmically on the basis of the connectivity derived from molecular interaction information, scored according to the significant number of focus proteins and assigned associated biological functions (see network descriptions) by overlaying the network molecules onto predefined maps of functional or pathway information in the IPKB database. Proteins are indicated by their gene names. Red and green symbols/text indicate increased and decreased proteins, respectively. Blue symbols indicate predicted inhibition, orange lines indicate predicted activation. Yellow lines indicate inconsistencies with the states of the downstream molecules. Lines ending with arrows indicate activation, lines without arrows indicate interaction/binding.

Mentions: Ingenuity pathway analysis (IPA) was performed using all significantly changed proteins (P* <0.05) in the frontal cortex (142 proteins) and hippocampus (227 proteins), regardless of the magnitude of change. This assumed that even slight variations in the levels of multiple proteins can result in pathway alterations. Using IPA, the protein changes were assigned to groups of biological functions in the Ingenuity knowledge base and z-scores were calculated as a prediction of whether a biological function was either up- or down-regulated. The biological functions underlying the identified molecular changes in the NR1neo−/− mouse are shown in Figure 2A. The frontal cortex showed a decrease in “coordination”, “long-term potentiation”, and “quantity of filaments”. To a lesser extent, the behavioural domains of cognition, learning, and memory were decreased and hyperactive behaviour was increased. In the hippocampus a broader range of functions appeared to be affected. The most prominent finding here was an upregulation in “formation of cellular protrusions”. Full information including proteins underlying these functions can be found in (Additional file 4: Table S4). Furthermore, we generated functional networks using IPA. Both networks suggested an involvement of the ERK pathway in the two regions. Functional annotation using the ingenuity upstream analysis tool revealed an inhibition of this pathway (Figure 2B).


Integrative proteomic analysis of the NMDA NR1 knockdown mouse model reveals effects on central and peripheral pathways associated with schizophrenia and autism spectrum disorders.

Wesseling H, Guest PC, Lee CM, Wong EH, Rahmoune H, Bahn S - Mol Autism (2014)

Computational pathway analysis of brain proteomic profiling. (A) IPA showing decreased and increased biological functions in NR1neo−/− mouse brain regions. Depicted are functions with an activation score (z-score) >1 (increased activation) or < −1 (decreased activation) with their corresponding log2 (P) (right graph, the dotted line represents the 0.05 P value threshold) as well as the five most-significantly dysregulated non-directionally (z-score > −1, <1, or not predicted) affected pathways (in the middle of the bar plots). (B) Identified IPA networks in the data set by global pathway analysis using the Ingenuity Pathways Knowledge Database (IPKB) software. All significantly altered proteins in the frontal cortex and hippocampus from the model were used for the network analysis based on criteria annotated in the IPKB database, which contains molecular information available in the scientific literature. Networks were generated algorithmically on the basis of the connectivity derived from molecular interaction information, scored according to the significant number of focus proteins and assigned associated biological functions (see network descriptions) by overlaying the network molecules onto predefined maps of functional or pathway information in the IPKB database. Proteins are indicated by their gene names. Red and green symbols/text indicate increased and decreased proteins, respectively. Blue symbols indicate predicted inhibition, orange lines indicate predicted activation. Yellow lines indicate inconsistencies with the states of the downstream molecules. Lines ending with arrows indicate activation, lines without arrows indicate interaction/binding.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4109791&req=5

Figure 2: Computational pathway analysis of brain proteomic profiling. (A) IPA showing decreased and increased biological functions in NR1neo−/− mouse brain regions. Depicted are functions with an activation score (z-score) >1 (increased activation) or < −1 (decreased activation) with their corresponding log2 (P) (right graph, the dotted line represents the 0.05 P value threshold) as well as the five most-significantly dysregulated non-directionally (z-score > −1, <1, or not predicted) affected pathways (in the middle of the bar plots). (B) Identified IPA networks in the data set by global pathway analysis using the Ingenuity Pathways Knowledge Database (IPKB) software. All significantly altered proteins in the frontal cortex and hippocampus from the model were used for the network analysis based on criteria annotated in the IPKB database, which contains molecular information available in the scientific literature. Networks were generated algorithmically on the basis of the connectivity derived from molecular interaction information, scored according to the significant number of focus proteins and assigned associated biological functions (see network descriptions) by overlaying the network molecules onto predefined maps of functional or pathway information in the IPKB database. Proteins are indicated by their gene names. Red and green symbols/text indicate increased and decreased proteins, respectively. Blue symbols indicate predicted inhibition, orange lines indicate predicted activation. Yellow lines indicate inconsistencies with the states of the downstream molecules. Lines ending with arrows indicate activation, lines without arrows indicate interaction/binding.
Mentions: Ingenuity pathway analysis (IPA) was performed using all significantly changed proteins (P* <0.05) in the frontal cortex (142 proteins) and hippocampus (227 proteins), regardless of the magnitude of change. This assumed that even slight variations in the levels of multiple proteins can result in pathway alterations. Using IPA, the protein changes were assigned to groups of biological functions in the Ingenuity knowledge base and z-scores were calculated as a prediction of whether a biological function was either up- or down-regulated. The biological functions underlying the identified molecular changes in the NR1neo−/− mouse are shown in Figure 2A. The frontal cortex showed a decrease in “coordination”, “long-term potentiation”, and “quantity of filaments”. To a lesser extent, the behavioural domains of cognition, learning, and memory were decreased and hyperactive behaviour was increased. In the hippocampus a broader range of functions appeared to be affected. The most prominent finding here was an upregulation in “formation of cellular protrusions”. Full information including proteins underlying these functions can be found in (Additional file 4: Table S4). Furthermore, we generated functional networks using IPA. Both networks suggested an involvement of the ERK pathway in the two regions. Functional annotation using the ingenuity upstream analysis tool revealed an inhibition of this pathway (Figure 2B).

Bottom Line: The highest magnitude changes were found for neurotrophic factors (VEGFA, EGF, IGF-1), apolipoprotein A1, and fibrinogen.We also found decreased levels of several chemokines.In contrast, increased levels of proteins involved in neurotransmitter metabolism and release were found only in the frontal cortex and abnormalities of proteins involved in the purinergic system were found exclusively in the hippocampus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, UK.

ABSTRACT

Background: Over the last decade, the transgenic N-methyl-D-aspartate receptor (NMDAR) NR1-knockdown mouse (NR1(neo-/-)) has been investigated as a glutamate hypofunction model for schizophrenia. Recent research has now revealed that the model also recapitulates cognitive and negative symptoms in the continuum of other psychiatric diseases, particularly autism spectrum disorders (ASD). As previous studies have mostly focussed on behavioural readouts, a molecular characterisation of this model will help to identify novel biomarkers or potential drug targets.

Methods: Here, we have used multiplex immunoassay analyses to investigate peripheral analyte alterations in serum of NR1(neo-/-) mice, as well as a combination of shotgun label-free liquid chromatography mass spectrometry, bioinformatic pathway analyses, and a shotgun-based 40-plex selected reaction monitoring (SRM) assay to investigate altered molecular pathways in the frontal cortex and hippocampus. All findings were cross compared to identify translatable findings between the brain and periphery.

Results: Multiplex immunoassay profiling led to identification of 29 analytes that were significantly altered in sera of NR1(neo-/-) mice. The highest magnitude changes were found for neurotrophic factors (VEGFA, EGF, IGF-1), apolipoprotein A1, and fibrinogen. We also found decreased levels of several chemokines. Following this, LC-MS(E) profiling led to identification of 48 significantly changed proteins in the frontal cortex and 41 in the hippocampus. In particular, MARCS, the mitochondrial pyruvate kinase, and CamKII-alpha were affected. Based on the combination of protein set enrichment and bioinformatic pathway analysis, we designed orthogonal SRM-assays which validated the abnormalities of proteins involved in synaptic long-term potentiation, myelination, and the ERK-signalling pathway in both brain regions. In contrast, increased levels of proteins involved in neurotransmitter metabolism and release were found only in the frontal cortex and abnormalities of proteins involved in the purinergic system were found exclusively in the hippocampus.

Conclusions: Taken together, this multi-platform profiling study has identified peripheral changes which are potentially linked to central alterations in synaptic plasticity and neuronal function associated with NMDAR-NR1 hypofunction. Therefore, the reported proteomic changes may be useful as translational biomarkers in human and rodent model drug discovery efforts.

No MeSH data available.


Related in: MedlinePlus