Data_Sheet_3_Proteomics analysis of the p.G849D variant in neurexin 2 alpha may reveal insight into Parkinson’s disease pathobiology.docx

Parkinson’s disease (PD), the fastest-growing neurological disorder globally, has a complex etiology. A previous study by our group identified the p.G849D variant in neurexin 2 (NRXN2), encoding the synaptic protein, NRXN2α, as a possible causal variant of PD. Therefore, we aimed to perform functional studies using proteomics in an attempt to understand the biological pathways affected by the variant. We hypothesized that this may reveal insight into the pathobiology of PD. Wild-type and mutant NRXN2α plasmids were transfected into SH-SY5Y cells. Thereafter, total protein was extracted and prepared for mass spectrometry using a Thermo Scientific Fusion mass spectrometer equipped with a Nanospray Flex ionization source. The data were then interrogated against the UniProt H. sapiens database and afterward, pathway and enrichment analyses were performed using in silico tools. Overexpression of the wild-type protein led to the enrichment of proteins involved in neurodegenerative diseases, while overexpression of the mutant protein led to the decline of proteins involved in ribosomal functioning. Thus, we concluded that the wild-type NRXN2α may be involved in pathways related to the development of neurodegenerative disorders, and that biological processes related to the ribosome, transcription, and tRNA, specifically at the synapse, could be an important mechanism in PD. Future studies targeting translation at the synapse in PD could therefore provide further information on the pathobiology of the disease.</p

Data_Sheet_2_Proteomics analysis of the p.G849D variant in neurexin 2 alpha may reveal insight into Parkinson’s disease pathobiology.docx

Parkinson’s disease (PD), the fastest-growing neurological disorder globally, has a complex etiology. A previous study by our group identified the p.G849D variant in neurexin 2 (NRXN2), encoding the synaptic protein, NRXN2α, as a possible causal variant of PD. Therefore, we aimed to perform functional studies using proteomics in an attempt to understand the biological pathways affected by the variant. We hypothesized that this may reveal insight into the pathobiology of PD. Wild-type and mutant NRXN2α plasmids were transfected into SH-SY5Y cells. Thereafter, total protein was extracted and prepared for mass spectrometry using a Thermo Scientific Fusion mass spectrometer equipped with a Nanospray Flex ionization source. The data were then interrogated against the UniProt H. sapiens database and afterward, pathway and enrichment analyses were performed using in silico tools. Overexpression of the wild-type protein led to the enrichment of proteins involved in neurodegenerative diseases, while overexpression of the mutant protein led to the decline of proteins involved in ribosomal functioning. Thus, we concluded that the wild-type NRXN2α may be involved in pathways related to the development of neurodegenerative disorders, and that biological processes related to the ribosome, transcription, and tRNA, specifically at the synapse, could be an important mechanism in PD. Future studies targeting translation at the synapse in PD could therefore provide further information on the pathobiology of the disease.</p

Data_Sheet_1_Proteomics analysis of the p.G849D variant in neurexin 2 alpha may reveal insight into Parkinson’s disease pathobiology.docx

Parkinson’s disease (PD), the fastest-growing neurological disorder globally, has a complex etiology. A previous study by our group identified the p.G849D variant in neurexin 2 (NRXN2), encoding the synaptic protein, NRXN2α, as a possible causal variant of PD. Therefore, we aimed to perform functional studies using proteomics in an attempt to understand the biological pathways affected by the variant. We hypothesized that this may reveal insight into the pathobiology of PD. Wild-type and mutant NRXN2α plasmids were transfected into SH-SY5Y cells. Thereafter, total protein was extracted and prepared for mass spectrometry using a Thermo Scientific Fusion mass spectrometer equipped with a Nanospray Flex ionization source. The data were then interrogated against the UniProt H. sapiens database and afterward, pathway and enrichment analyses were performed using in silico tools. Overexpression of the wild-type protein led to the enrichment of proteins involved in neurodegenerative diseases, while overexpression of the mutant protein led to the decline of proteins involved in ribosomal functioning. Thus, we concluded that the wild-type NRXN2α may be involved in pathways related to the development of neurodegenerative disorders, and that biological processes related to the ribosome, transcription, and tRNA, specifically at the synapse, could be an important mechanism in PD. Future studies targeting translation at the synapse in PD could therefore provide further information on the pathobiology of the disease.</p

Application of an analysis technique to characterise impulse response of grounding systems

Transient response plays a key role in the evaluation of the performance of grounding systems and for the protection of electrical installations under lightning strikes. The frequency spectrum of the lightning impulse contains harmonics components up to the megahertz range. The measured transient response of grounding systems under test may be distorted by spurious high frequency interference in the acquired signals, which presents challenges for the accurate analysis of high frequency performance of such systems. In this paper, the high frequency performance of a rod electrode is investigated based on measurements of its transient response under impulse energisation. A practical method is implemented to eliminate the high frequency noise in the measured voltage and current shapes, which allows a frequency domain analysis based Fast Fourier Transforms

Regulation of AR-regulated proteins by DHT, 11KDHT, T and 11KT in VCaP cells.

<p>Cells were incubated with CS-FCS supplemented media for 48 hours prior to treatment with 1 nM steroid. Proteins were subsequently identified using mass spectrometry. Fold changes were calculated relative to the vehicle control. Statistically significant changes are indicated (P<0.05). Results are representative of three independent experiments.</p

Induction of cell proliferation in LNCaP and VCaP cells by DHT, 11KDHT, T and 11KT.

<p>Cells were incubated with media supplemented with CS-FCS for 24 hours prior to treatment with 0.1, 1 or 10 nM steroids. Resazurin assays were carried out on day 7 (LNCaP) or day 10 (VCaP) after treatment. Results are shown as means ± SEM of three independent experiments with eight replicates each.</p

Binding of DHT, T, 11KDHT and 11KT to the human AR (A) and transactivation via an ARE (B and C).

<p>Binding affinities, agonist potencies and efficacies of DHT, 11KDHT, T and 11KT relative to the synthetic AR agonist mibolerone are summarised in (D). Whole cell binding assays (A) were conducted in COS-1 cells transiently transfected with pSVARo. Cells were incubated with 0.2 nM [³H]-Mib in the absence and presence of increasing concentrations of either unlabelled Mib, DHT, 11KDHT, T and 11KT for 16 hours. Results are plotted as % specific binding where the total specific binding of [³H]-Mib only is set to 100% and binding of unlabelled steroid is set as a % binding relative to that. Whole cell binding results are shown as means ± SEM of three independent experiments performed in triplicate. Transactivation assays (B and C) where performed in COS-1 cells transiently transfected with the pSVARo expression vector and the 4xSC ARE1.2-luc reporter. Agonist activity was measured by incubating cells in the presence of increasing concentrations of either Mib, DHT, T, 11KDHT or 11KT for 24 h. Induction is shown as % luciferase activity expressed in relative light units (rlu’s), with the maximal response of Mib (10⁻⁵ M) set to 100%. Luciferase assays are shown as means ± SEM of six independent experiments performed in quadruplicate.</p

Metabolism of DHT, 11KDHT, T and 11KT by LNCaP and VCaP cells.

<p>Steroids were analysed by ultra-performance convergence chromatography-mass spectrometry (UPC²-MS/MS). Results are representative of two independent experiments performed in triplicate.</p

Biosynthesis of 11KT and 11KDHT from the adrenal androgen precursor 11OHA4.

<p>Enzymes: 11βHSD2, 11β-hydroxysteroid dehydrogenase; 17βHSD2, 17β-hydroxysteroid dehydrogenase; SRD5A1, steroid 5α-reductase type 1; 3αHSD2, 3α-hydroxysteroid dehydrogenase. Steroids: 11OHA4, 11β-hydroxyandrostenedione; 11KA4, 11-ketoandrostenedione; 11KT, 11-ketotestosterone; 11OH-5α-dione, 11OH-5α-androstanedione; 11K-5α-dione, 11-keto-5α-androstanedione; 11KDHT, 11-ketodihydrotestosterone; 11OHAST, 11β-hydroxyandrosterone; 11KAST, 11-ketoadrenosterone; 11K-3α-adiol, 11-keto-5α-androstane-3α,17β-diol.</p