Final Report Summary - TINTIN (Training in neurodegeneration, therapeutics intervention and neurorepair)
Summary description of the project objectives
Dopamine producing neurons play a central role in anxiety, mood disorders, schizophrenia, autism-spectrum disorders, Parkinson’s disease, epilepsy, and dementia. The overall objective of the TINTIN network (www.tintin-itn.nwt) was ‘To train ESRs and ERs in multidisciplinary aspects of research related to normal and abnormal function of the dopaminergic neuron so that new research discoveries will lead to improved therapies for cohorts of European patients in which dopaminergic systems are at the basis of their disorder.’ The scientific and technological objectives used interdisciplinary approaches to: (i) Discover how autophagy in the dopamine neuron is related to lysosomal and mitochondrial dysfunction. (ii) Discover how novel genetic mutations in glycolipid and ganglioside metabolism relate to dopamine neuron degeneration. (iii) Utilize pluripotent stem cell technologies for studying dopaminergic neurodegeneration. (iv) Utilize computational and molecular design techniques to identify novel aspects of the neurodegenerative processes that may be selected as therapeutic targets. (v) Identify and validate novel glycan-based biomarkers for use in clinical trials.
Description of the work performed and main results since the beginning of the project:
ESR1 – Modulation of antioxidant status in neurons and astrocytes: The (i) design and generation of a novel genetically modified mouse model that expressed mCAT and capable of decreasing physiological endogenous mROS in brain cells and (ii) physiological endogenous mROS modulation of the Nrf2 (Nuclear Factor (erythroid-derived 2)-like 2) pathway in astrocytes and (iii) assessment of how physiological astrocytic mROS regulates neuronal metabolism, redox status and function, were all completed.
ESR2 - Characterisation of the redox status inside dopamine neurons using conditional transgenic mouse models: The following were completed (i) To evaluate the oxidative metabolism under physiological oxygen conditions in comparison with the over-oxygenation (21% O2) that is normally used in cell culture incubators (ii) To study the effect of co-culturing astrocytes and neurons on neuronal-astrocytic metabolic and redox interactions (iii) To contribute to understand the main molecular factors lost in neurons, relevant to some neurodegenerative diseases, such as Parkinson’s disease.
ESR3 - The effect of glutathione on mitochondrial dynamics & function in the dopamine neuron: The following were completed (i) investigation of mitochondrial function in synaptic and non-synaptic mitochondria under different conditions (ii) investigation of the neurotoxic/neuroprotective effects of various compounds in the nerve terminal and in cell lines (iii) characterisation of the dopaminergic phenotype in synuclein cell lines.
ESR4 - How does the cytochrome P450 system control drug metabolism in the neuron?: The following were completed (i) effects of neurotoxins on cell lines (ii) the role of CYP expression on neurotoxin sensitivity. (iii) the identification of neuroprotective compounds (iv) the identification of CYP metabolism metabolites (v) assay of the enzymatic activity of CYP in microsomes and mitochondria.
ESR5 – Crosstalk between mitochondrial & lysosomal dysfunction in the dopamine neuron: The following were completed (i) elucidation of glucocerebrosidase activity in SH-SY5Y cells and the effect on mitochondrial function (ii) identification of signals that regulate mitochondrial-lysosomal interactions in PC12 cells and cortical neurons.
ESR6 – How does glucocerebrosidase control autophagy in the dopamine neuron?: The following were completed (i) neuronal cell model of GBA1 deficiency revealed effects on biogenic amine turnover (ii) Knock down of GBA1 in SH-SY5Y cells produced mitochondrial defects (iii) Glycolipid profiles in CSF from parkinson’s patients with low HVA demonstrated biomarker differences..
ESR7 - Human wild type and parkinsonian iPS cell lines: The following were completed (i) Removal of chemically damaged mitochondria does not involve the autophagy nucleation and initiation machinery, but does depend on the ubiquitin proteasome system. (ii) A stable PD iPS cell line was used to identify autophagy and mitophagy mechanisms in PD neurons.
ESR8 - Neurotoxin models of Parkinson’s disease: The following were completed (i) MPP+ treatment of dopamine and serotonergic stem cell derived neurons resulted in a misbalance between fusion and fission dynamics in mitochondria. (ii) MPP+ acts differentially on dopaminergic and serotonergic neurons derived from mESC.
ESR9 - In silico reconstruction of the dopamine transporter and design of new drugs with neuroprotective properties: The following were completed (i) By performing homology modeling and molecular dynamic (MD) simulations of human dopamine transporter (hDAT) 3D structure models of hDAT in complex with dopamine (natural substrate), amphetamine (substrate – psychostimulant), cocaine (inhibitor) and modafinil (atypical inhibitor) were obtained. (ii) Building pharmacophore models of DAT substrates and inhibitors demonstrated the chemical features that a compound needs to have in order to be substrate/inhibitor of the DAT.
ESR10 - Development of new drugs to inhibit the neurodegenerative process in dopamine neurons: The following were completed (i) A method was being developed for use of SHSY5Y cells in detection of lipid variance in PD and related diseases. The cell lipid profile was obtained using a newly developed lipid extraction method and Q-TOF LC/MS analysis. Preliminary trials suggest that the new lipid extraction method provides satisfactory recovery of lipids of cell samples compared to the gold standard methods. (ii) The toxicity of various neurotoxins on undifferentiated and differentiated cells displayed differential lipid profiles (iii) neuro-protective compounds were tested, allowing identification of lipid biomarkers for the disease and development of neuro-protective drugs.
ESR11 - Characterisation of the cell surface glycome on the dopamine neuron using glycochip technology: The following were completed (i) Development of MALDI-ToF-based platform to study relevant interactions between (neo)glycolipids and trimming enzymes. (ii) Development of a high-throughput computer aided processing method to compare proteomics data.
ESR12 - Glycans, glycolipids and gangliosides related to dopaminergic cell death?: The following were completed (i) MS based method for glycan quantitation was developed. These methods were used to characterize serum and cerebrospinal fluid N-glycans and reveal their biomarker potential in Parkinson’s disease. (ii) Correlations between glycosylation changes and disease progression in Parkinson’s disease using in vitro neuronal models were identified.
ER1 - The mechanisms that control mitochondrial dynamics and function in the dopamine neuron: The following were completed (i) This project focussed on the characterization of the role of monoamine oxidases (MAO) in the mitochondrial dynamics of Parkinson's disease-derived dopaminergic neurons. (ii) MAO activity levels in differentiated PC12 cells and iPS (wt iPS and alpha synuclein triplication iPS) neurons were characterized. The overall results show that inhibitors of MAO are able to protect against mitochondrial damage caused by parkinsonian neurotoxins.
ER2 - What mechanisms control autophagy in the dopamine neuron?: The following were completed (i) Interference RNAs (shRNA) were designed and developed to silence the human GBA1 gene through lentiviral transduction of several human cell lines. (ii) The results show GBA reduction induces complex I dysfunction in mitochondria, providing further evidence for GBA being an important enzyme in PD progression.
Final results and their potential impact and use: There has been considerable success at the individual research project level, resulting in novel mechanistic aspects of brain cells being uncovered. These initial findings were being extended into neurons of the dopaminergic phenotype as well as into the biomarker arena for conditions involving dopaminergic dysfunction. A number of high-impact scientific publications were produced as well as the generation of new putative anti-neurodegenerative therapeutics and intellectual property. All 12 TINTIN ESR and 2 ER projects were successfully completed and many of these reserchers have gone on to postdoctoral research positions in the EU and also directly into the pharmaceutical industry.
Dopamine producing neurons play a central role in anxiety, mood disorders, schizophrenia, autism-spectrum disorders, Parkinson’s disease, epilepsy, and dementia. The overall objective of the TINTIN network (www.tintin-itn.nwt) was ‘To train ESRs and ERs in multidisciplinary aspects of research related to normal and abnormal function of the dopaminergic neuron so that new research discoveries will lead to improved therapies for cohorts of European patients in which dopaminergic systems are at the basis of their disorder.’ The scientific and technological objectives used interdisciplinary approaches to: (i) Discover how autophagy in the dopamine neuron is related to lysosomal and mitochondrial dysfunction. (ii) Discover how novel genetic mutations in glycolipid and ganglioside metabolism relate to dopamine neuron degeneration. (iii) Utilize pluripotent stem cell technologies for studying dopaminergic neurodegeneration. (iv) Utilize computational and molecular design techniques to identify novel aspects of the neurodegenerative processes that may be selected as therapeutic targets. (v) Identify and validate novel glycan-based biomarkers for use in clinical trials.
Description of the work performed and main results since the beginning of the project:
ESR1 – Modulation of antioxidant status in neurons and astrocytes: The (i) design and generation of a novel genetically modified mouse model that expressed mCAT and capable of decreasing physiological endogenous mROS in brain cells and (ii) physiological endogenous mROS modulation of the Nrf2 (Nuclear Factor (erythroid-derived 2)-like 2) pathway in astrocytes and (iii) assessment of how physiological astrocytic mROS regulates neuronal metabolism, redox status and function, were all completed.
ESR2 - Characterisation of the redox status inside dopamine neurons using conditional transgenic mouse models: The following were completed (i) To evaluate the oxidative metabolism under physiological oxygen conditions in comparison with the over-oxygenation (21% O2) that is normally used in cell culture incubators (ii) To study the effect of co-culturing astrocytes and neurons on neuronal-astrocytic metabolic and redox interactions (iii) To contribute to understand the main molecular factors lost in neurons, relevant to some neurodegenerative diseases, such as Parkinson’s disease.
ESR3 - The effect of glutathione on mitochondrial dynamics & function in the dopamine neuron: The following were completed (i) investigation of mitochondrial function in synaptic and non-synaptic mitochondria under different conditions (ii) investigation of the neurotoxic/neuroprotective effects of various compounds in the nerve terminal and in cell lines (iii) characterisation of the dopaminergic phenotype in synuclein cell lines.
ESR4 - How does the cytochrome P450 system control drug metabolism in the neuron?: The following were completed (i) effects of neurotoxins on cell lines (ii) the role of CYP expression on neurotoxin sensitivity. (iii) the identification of neuroprotective compounds (iv) the identification of CYP metabolism metabolites (v) assay of the enzymatic activity of CYP in microsomes and mitochondria.
ESR5 – Crosstalk between mitochondrial & lysosomal dysfunction in the dopamine neuron: The following were completed (i) elucidation of glucocerebrosidase activity in SH-SY5Y cells and the effect on mitochondrial function (ii) identification of signals that regulate mitochondrial-lysosomal interactions in PC12 cells and cortical neurons.
ESR6 – How does glucocerebrosidase control autophagy in the dopamine neuron?: The following were completed (i) neuronal cell model of GBA1 deficiency revealed effects on biogenic amine turnover (ii) Knock down of GBA1 in SH-SY5Y cells produced mitochondrial defects (iii) Glycolipid profiles in CSF from parkinson’s patients with low HVA demonstrated biomarker differences..
ESR7 - Human wild type and parkinsonian iPS cell lines: The following were completed (i) Removal of chemically damaged mitochondria does not involve the autophagy nucleation and initiation machinery, but does depend on the ubiquitin proteasome system. (ii) A stable PD iPS cell line was used to identify autophagy and mitophagy mechanisms in PD neurons.
ESR8 - Neurotoxin models of Parkinson’s disease: The following were completed (i) MPP+ treatment of dopamine and serotonergic stem cell derived neurons resulted in a misbalance between fusion and fission dynamics in mitochondria. (ii) MPP+ acts differentially on dopaminergic and serotonergic neurons derived from mESC.
ESR9 - In silico reconstruction of the dopamine transporter and design of new drugs with neuroprotective properties: The following were completed (i) By performing homology modeling and molecular dynamic (MD) simulations of human dopamine transporter (hDAT) 3D structure models of hDAT in complex with dopamine (natural substrate), amphetamine (substrate – psychostimulant), cocaine (inhibitor) and modafinil (atypical inhibitor) were obtained. (ii) Building pharmacophore models of DAT substrates and inhibitors demonstrated the chemical features that a compound needs to have in order to be substrate/inhibitor of the DAT.
ESR10 - Development of new drugs to inhibit the neurodegenerative process in dopamine neurons: The following were completed (i) A method was being developed for use of SHSY5Y cells in detection of lipid variance in PD and related diseases. The cell lipid profile was obtained using a newly developed lipid extraction method and Q-TOF LC/MS analysis. Preliminary trials suggest that the new lipid extraction method provides satisfactory recovery of lipids of cell samples compared to the gold standard methods. (ii) The toxicity of various neurotoxins on undifferentiated and differentiated cells displayed differential lipid profiles (iii) neuro-protective compounds were tested, allowing identification of lipid biomarkers for the disease and development of neuro-protective drugs.
ESR11 - Characterisation of the cell surface glycome on the dopamine neuron using glycochip technology: The following were completed (i) Development of MALDI-ToF-based platform to study relevant interactions between (neo)glycolipids and trimming enzymes. (ii) Development of a high-throughput computer aided processing method to compare proteomics data.
ESR12 - Glycans, glycolipids and gangliosides related to dopaminergic cell death?: The following were completed (i) MS based method for glycan quantitation was developed. These methods were used to characterize serum and cerebrospinal fluid N-glycans and reveal their biomarker potential in Parkinson’s disease. (ii) Correlations between glycosylation changes and disease progression in Parkinson’s disease using in vitro neuronal models were identified.
ER1 - The mechanisms that control mitochondrial dynamics and function in the dopamine neuron: The following were completed (i) This project focussed on the characterization of the role of monoamine oxidases (MAO) in the mitochondrial dynamics of Parkinson's disease-derived dopaminergic neurons. (ii) MAO activity levels in differentiated PC12 cells and iPS (wt iPS and alpha synuclein triplication iPS) neurons were characterized. The overall results show that inhibitors of MAO are able to protect against mitochondrial damage caused by parkinsonian neurotoxins.
ER2 - What mechanisms control autophagy in the dopamine neuron?: The following were completed (i) Interference RNAs (shRNA) were designed and developed to silence the human GBA1 gene through lentiviral transduction of several human cell lines. (ii) The results show GBA reduction induces complex I dysfunction in mitochondria, providing further evidence for GBA being an important enzyme in PD progression.
Final results and their potential impact and use: There has been considerable success at the individual research project level, resulting in novel mechanistic aspects of brain cells being uncovered. These initial findings were being extended into neurons of the dopaminergic phenotype as well as into the biomarker arena for conditions involving dopaminergic dysfunction. A number of high-impact scientific publications were produced as well as the generation of new putative anti-neurodegenerative therapeutics and intellectual property. All 12 TINTIN ESR and 2 ER projects were successfully completed and many of these reserchers have gone on to postdoctoral research positions in the EU and also directly into the pharmaceutical industry.