Hereditary sensory neuropathy type I is an autosomal dominant disorder that affects the sensory neurons. Three missense mutations in serine palmitoyltransferase long chain subunit 1 cause hereditary sensory neuropathy type I. The endoplasmic reticulum, where the serine palmitoyltransferase long chain subunit 1 protein resides, and mitochondria are both altered in hereditary sensory neuropathy type I mutant cells. Employing a transfected neuronal cell line (ND15) , we have identified and confirmed altered protein expression levels of ubiquinol cytochrome C, Hypoxia Up regulated Protein 1, Chloride Intracellular Channel Protein 1, Ubiqutin-40s Ribosomal Protein S27a, and Coactosin. Additionally, further 14 new proteins that exhibited altered expression within V144D, C133W and C133Y mutants were identified. These data have shown that mutations in SPTLC1 alter the expression of a set of proteins that may help to establish a causal link between the mitochondria and ER and the “ dying back ” process of dorsal root ganglion neurons that occurs in HSN-I.
Hereditary Sensory Neuropathy type I (HSN-I) is an autosomal dominant inherited neurodegenerative disorder. It is caused by missense mutations in the open reading frame of serine palmitoyltransferase (SPT) long chain subunit 1 (SPTLC1) [
In previous studies, we investigated altered protein profile changes in the mitochondria and ER of HSN-I patient cells (SPTLC1 V144D mutation) [
Based on these earlier findings, this investigation employed ND15 cell line (hybrid of rat dorsal root ganglion neuron and a mouse neuroblastoma) which had been transiently transfected (TT) to overexpress the three SPTLC1 missense mutations: V144D, C133W and C133Y. The data obtained from this ND15 neuronal cell model confirmed previous results from the HSN-I patient lymphoblast, while notably identifying changes exhibited in the C133W and C133Y mutations. We have also identified an additional 14 proteins that are altered in abundance within the transfected ND15 cells. Together these findings offer a greater insight into the molecular mechanisms occurring in the three known mutations causing HSN-I.
All cell culture stock solutions, including DMEM, Foetal Bovine Serum (FBS), Penicillin (100 U/mL), Streptomycin (100 µg/mL), L-glutamine (2 M), NEAA (1 M), and phosphate buffered saline (PBS) were purchased from GIBCO Invitrogen (Australia). Cell culture consumables were purchased from BD Falcon (Greiner, USA). ORP-150, CLIC1, RPS27a, Calnexin, ubiquinol cytochrome C, MTCO2, and GAPDH primary antibodies were purchased from Abcam (USA); SPTLC1 primary antibody was purchased from Santa Cruz Biotechnology (USA). COTL1 primary antibody was purchased from Protein SciTech (USA). Kif2A and GFP primary antibodies were purchased from Merck Millipore (USA). Secondary horse radish peroxidase (HRP) mouse antibodies and DAPI stains were purchased from Sigma-Aldrich (Australia). ND15 cell lines were cultured in DMEM media, supplemented with FBS (10% v/v), Penicillin (1 U/mL), Streptomycin (1 µg/mL), L-glutamine (2 mM), and NEAA (1 mM) at 37˚C in a humidified atmosphere of 5% CO2, in T75 cm2 culture flasks (Greiner, Interpath). Prior to use in biochemical assays, ND15s were collected by centrifugation at 1500 ×g (5 min at RT) and washed in PBS. Cell counts were obtained using the Countess Automated Cell Counter (Invitrogen, Australia).
ND15 cells were transiently transfected (TT) with plasmid constructs (GFP-tagged SPTLC Wild type, V144D, C133W and C133Y mutants) using Lipofectamine 2000 (L2K; Invitrogen, USA). Cells were plated at a density of 2 × 105 per well in 6 well plates. Transfections were carried out when cells were 90% - 95% confluent (approximately 24 hours after plating, at 37˚C and 5% CO2), according to manufacturer instructions. Briefly, plasmid constructs and L2K reagent were diluted in 250 µL of Opti-MEM I Reduced Serum Media (Invitrogen, USA), to yield 16 μg/ml DNA and 40 μL/ml L2K. Within 5 min of each dilution, the DNA construct diluents and L2K diluents were combined and incubated for 30 min at 25˚C. After incubation, DNA-L2K complexes (500 μL) were added into each well, as required. The cells were then incubated at 37˚C in a humidified atmosphere of 5% CO2 for 6 h. Cells then had media replaced with fresh media and were left to incubate for 48 h. Post transfection, cells were harvested and cell lysate was used for further analyses.
Determination of total cellular protein was carried out using the EZQ Protein Estimation Assay (Invitrogen, Australia) as previously described [
Nontransfected, wild type and mutant protein fractions (25 µg total protein) were subjected to SDS-PAGE on 12.5% resolving gels and transferred to PVDF membrane. The membranes were blocked with 5% skim milk in TBS buffer containing 0.1% Tween-20. Whole membranes were blocked and incubated with anti-SPTLC1, anti-GAPDH, anti- ORP-150, anti-CLIC1, anti-RPS27a, anti-COTL1, anti-MTCO2, anti-GFP and anti- Kif2A antibodies respectively, at 1:1000 dilution, for 16 h. Membranes were washed and incubated with secondary HRP antibody (1:2000 dilution) for 1 h at RT. Blots were washed and developed using an enhanced chemiluminescence (ECL) detection kit (Pierce Thermo Scientific, USA). Membranes were developed on CL-Xposure Film (Thermo Fisher Scientific, USA) using an AGFA X-ray developer.
Immunofluorescence was carried out as previously described [
FACS analyses were carried out as previously described [
2DE was carried out as previously described [
The treated samples were added to 7 cm Non-Linear pH 3 - 10 IPG strips (Bio-Rad ReadyStrip), and rehydrated for 16 h at RT. Isoelectric focusing (IEF) was then carried out at 20˚C using the Protean IEF Cell (Bio-Rad, USA). After IEF, IPG strips were resolved in the second dimension using a 12.5% T, 2.6% C polyacrylamide gel buffered with 375 mM Tris buffer (pH 8.8), 0.1% (w/v) sodium dodecyl sulphate and polymerised with 0.05% (w/v) ammonium persulphate and 0.05% (v/v) tetramethylethylenediamine (TEMED). A stacking gel containing 5% T, 2.6% C polyacrylamide buffered with 375 mM Tris buffer (pH 6.8), 0.1% (w/v) SDS and 0.1% bromophenol blue was added to the resolving gel. The IPG strips were placed onto the stacking gel and overlaid with 0.5% (w/v) low melting agarose dissolved in 375 mM Tris (pH 8.8), with 0.1% (w/v) SDS. Electrophoresis was carried out at 4˚C; 150V initially for 10 min then reduced to 90 V for 2.5 h.
Following electrophoresis, the gels were placed in fixative containing 10% methanol and 7% acetic acid for 1 h. The gels were then washed with distilled water for 20 min, 3 times and subsequently stained with colloidal coomassie blue (0.1% (w/v) CCB G-250, 2% (v/v) phosphoric acid, 10% (w/v) ammonium sulphate and 20% (v/v) methanol) for 20 h, with constant shaking at RT [
The following selection criteria were applied for spot inclusion. Changes in mean normalised spot volume (the abundance of resolved protein species) had to be greater than a 1.0 fold difference between samples from wild type versus V144D, C133W and C133Y mutants and be present in all replicate gels [
ND15 cells were grown for 24 h in 35 mm glass bottom size 0 dish (MatTek, USA), and transfected as previously described. Molecular Probes Rhod-3 calcium imaging kit (Molecular Probes, USA) was used to stain the cells. Briefly, cells were incubated at RT in the dark for 1 h in 10 µM Rohd-3 AM, 2.5 mM probenecid and 1× power load. Cells were briefly washed in calcium-free PBS and incubated for a further 1 h at RT with 2.5 mM probenecid. To obtain low and high intracellular calcium images, NT ND15 cells were infused with PBS without calcium, containing 5 mM EGTA and 2 µM ionomycin to allow intracellular calcium to efflux from the cell. High intracellular calcium images were obtained by infusing the cells in PBS containing calcium and 2 µM ionomycin. Cells were ready for imaging after a further two washes in calcium-free PBS and imaged on the LSM 5 confocal microscope comprising the LSM 5 exciter laser scanning microscope with Axiovert 200M inverted optical microscope (Carl Zeiss, Jena, Germany).
In order to assess the level of expression of proteins previously reported as altered, total cellular protein fractions from nontransfected, wild type and mutant HSN-I TT ND15 cells were isolated and quantitative immunoblot analyses were carried out (
Quantitation of the immunoblot data (Figures 2(a)-(j)), confirmed that there were statistically significant (p < 0.05) changes in expression of COTL1 (
The intracellular localisation and abundance of the proteins SPTLC1, Kif2A, Cytochrome C, RPS27a, CLIC1, ORP-150, COTL1 and MTCO2 were established using immunostained nontransfected, wild type and mutant TT ND15 cells. There were no apparent changes in intracellular localisation of the SPTLC1 when transfected with GFP labelled SPTLC1, which was found localised to the perinuclear region where the ER resides (
cell in nontransfected, wild type and mutants (
Cytochrome C is typically located within the mitochondrial inner membrane [
ORP-150, a chaperon protein localised throughout the cell [
tingly, there appeared to be a more perinuclear clustering (as indicated by arrows,
Fluorescence assisted cell sorting (FACS) was used to determine the total fluorescence per cell of TT ND15 immunostained cells for the proteins SPTLC1, Kif2A, Cytochrome C, RPS27a, CLIC1, ORP-150, COTL1, MTCO2 and GFP (
cells compared to that of wild type. There were no changes to SPTLC1 (
Total isolated wild type and mutant ND15 proteins were resolved and quantitatively assessed using refined two dimensional gel electrophoresis (2DE) [
Wild type and mutant ND15 cells were analysed for total intracellular calcium using the
Rhod-3 Am calcium stain (
Mutations in the SPTLC1 subunit are known to be causal in HSN-I. Molecular and cellular studies of cells over-expressing the SPTLC1 mutations have identified potential dysfunction in sphingolipid biosynthesis and metabolic activity [
Ubiquinol cytochrome C reductase core protein 1 is a central component of the
electron transport chain, catalysing the oxidation of ubiquinol and reduction of cytochrome C [
Further quantitative analyses were carried out which confirmed that the protein expression of RPS27a (
Proteins Identified | Accession Number Matched | Unique Peptides | Sequence Coverage | Mascot Protein Score | Predicted pI | Predicted Mw (kDa) | Mascot pI | Mascot Mw (kDa) | Fold Increase or Decrease within the Mutant | |
---|---|---|---|---|---|---|---|---|---|---|
1. | Protein Disulphide Isomerase | P09103 | 35 | 42% | 1468 | 4.2 | 90 | 4.77 | 57.01 | V144D-1.3 Fold â |
2. | Alpha-Enolase | P17182 | 52 | 67% | 1901 | 6.5 | 65 | 6.37 | 47.11 | V144D-1.6 Fold á |
3. | Long-chain specific acyl-CoA dehydrogenase | P51174 | 20 | 50% | 1188 | 8.3 | 69 | 8.53 | 48.2 | V144D-1.7 Fold á |
4. | 26s protease regulatory subunit 8 | P62196 | 44 | 63% | 1287 | 7.0 | 60 | 7.11 | 45.2 | V144D-1.7 Fold á |
5. | RPS27a | P62983 | 18 | 42% | 193 | 9.2 | 16 | 8.00 | 9.68 | C133W-2.6 Fold á |
6. | Peptidyl-prolyl cis-trans isomerase | P30416 | 39 | 41% | 576 | 5.4 | 90 | 5.54 | 57.6 | C133W-1.7 Fold á |
7. | Stress-70 protein, Mitochondrial | P38647 | 33 | 38% | 884 | 4.3 | 75 | 5.81 | 73.70 | C133Y-2.3 Fold á |
8. | 10 kDa heat shock protein, Mitochondrial | Q64433 | 3 | 37% | 90 | 7.6 | 15 | 7.93 | 10.96 | C133Y-3.5 Fold â |
9. | Voltage-dependent anion-selective channel protein 2 | Q60930 | 7 | 29% | 229 | 7.9 | 30 | 7.44 | 32.34 | C133Y-1.6 Fold á |
10. | Long-chain specific acyl-CoA dehydrogenase, Mitochondrial | P51174 | 45 | 43% | 1117 | 8.2 | 50 | 8.53 | 48.37 | C133Y-2.1 Fold á |
11. | 26s Proteasome non-ATPase regulatory subunit 14 | O35593 | 25 | 39% | 580 | 5.8 | 30 | 6.06 | 34.77 | C133Y-1.5 Fold á |
12. | STIP1 homology and U box-containing protein 1 | Q9WUD1 | 27 | 58% | 366 | 5.9 | 35 | 5.71 | 35.34 | C133Y-2.1 Fold â |
13. | Eukaryotic Translation Initiator Factor 2 Subunit 1 | Q6ZWX6 | 47 | 64% | 944 | 4.5 | 37 | 5.02 | 36.37 | C133Y-2.4 Fold â |
14. | Triosephosphate Isomerase | P48500 | 34 | 79% | 1162 | 8.5 | 26 | 6.89 | 27.4 | C133Y-2.7 Fold â |
were significantly increased in the V144D TT ND15 cells. These findings correlated with results previously identified in the lymphoblast model [
The novel findings in this study indicate links to dysfunction in oxidative phosphorylation, viaubiquinol cytochrome C Reductase Core Protein 1 in all three mutations causing HSN-I. The increased expression of Cytochrome C results in the interference of energy production and oxidative stress upon the ER, eventually causing axonal retraction, a characteristic hallmark of HSN-I. Additionally, Stress-70 mitochondrial protein levels were identified in the C133Y mutant as being increased 2.3 fold relative to the wild type (
It is evident that there is an increase in oxidative and ER stress within the cells containing HSN-I mutations. This is demonstrated by the increased expression of ORP- 150, CLIC1, COTL1 and RPS27a. ORP-150 is an important molecular chaperone of the ER during stress [
Further strengthening the connection between ER stress and HSN-I, peptidyl-prolyl cis-trans isomerase was found to be increased in abundance by 1.7 fold in the C133W mutant. This protein ensures newly synthesised proteins are folded into their correct conformation [
Calcium is an important signaling molecule involved in the regulation of many cellular functions. Mitochondrial calcium uptake has been shown to lead to free radical production, with a delicate balance existing between moderate ROS production to modulate physiological signaling and overproduction of ROS which can ultimately lead to oxidative and ER stress [
This investigation has shown a correlation between previous studies, revealing an increase in a mitochondrial electron transport chain protein, increases in proteins induced by oxidative stress and changes in the intracellular calcium levels in all three SPTLC1 mutations causing HSN-I. These findings provide further evidence for mitochondrial and ER dysfunction occurring as a result of mutations in SPTLC1. These novel findings provide critical new directions in understanding the underlying molecular and cellular alterations broadly applicable (and specific) to all mutations causing HSN-I and neurodegenerations as a whole.
Stimpson, S.E., Shanu, A., Coorssen, J.R. and Myers, S.J. (2016) Identifying Unique Protein Alterations Caused by SPTLC1 Mutations in a Transfected Neuronal Cell Model. World Journal of Neuroscience, 6, 325-347. http://dx.doi.org/10.4236/wjns.2016.64035