Simon J. Myers

Protective effect of a synthetic anti-oxidant on neuronal cell apoptosis resulting from experimental hypoxia re-oxygenation injury

Oxidative stress is associated with the pathology of acute and chronic neurodegenerative disease. Cultured neuronal cells exposed to hypoxia‐reoxygenation (H/R) injury, as a model for stroke, yield a burst of reactive oxygen species (ROS) as measured with electron paramagnetic resonance (EPR) spectroscopy in combination with spin trapping. Added superoxide dismutase inhibited spin‐adduct formation verifying that superoxide radical anion was formed in neuronal cells following H/R injury. The intracellular ADP/ATP ratio increased rapidly over the first 5 h following injury and this was due primarily to the decreased cellular pools of ATP, consistent with the notion that H/R promotes mitochondrial dysfunction leading to decreased ATP reserve and increased ROS formation. As an early response to the enhanced oxidative stress, genes encoding for hypoxia‐inducible factor 1‐α (HIF1‐α), inducible haemoxygenase‐1 (HO‐1), and the oxygen‐sensor neuroglobin increased significantly. Up‐regulation of the HO‐1 gene was paralleled by increased HO protein expression and activity. Despite this cellular response, apoptosis increased significantly following H/R injury indicating that the endogenous anti‐oxidant defenses were unable to protect the cells. In contrast, addition of a phenolic anti‐oxidant, bisphenol (BP), prior to H/R injury, inhibited ROS production and gene regulation and significantly decreased neuronal cell apoptosis. Added BP was converted stoichiometrically to the corresponding diphenoquinone indicating the synthetic anti‐oxidant effectively decreased oxidative stress through a radical scavenging mechanism. Together, these data indicate that BP has the potential to act as a neuro‐protective drug.

Intermediate forms of Charcot-Marie-Tooth neuropathy

The Charcot-Marie-Tooth (CMT) neuropathies divide into two main electrophysiological groups with slow and near normal conduction velocities corresponding to Schwann cell and axonal pathology. An intermediate group also exists with nerve conduction velocities, which overlaps the two main groups. Families with intermediate CMT can be recognized in which different affected individuals in the same family have motor conduction velocities in both the CMT type 1 and 2 ranges (i.e., above and below 38 m/s). The intermediate group is caused by a limited number of distinct gene mutations in dynamin 2 (DNM2), gap-junction protein 1 (GJB1), neurofilament light polypeptide (NF-L) genes, and a rare mutation and as yet unknown genes on chromosome 1 and 10 loci. Intermediate forms of CMT may be associated with unique disease mechanisms affecting both Schwann cells and axons. It is useful to recognize this unique group of neuropathies for diagnostic and management purposes.

Intermediate forms of Charcot-Marie-Tooth neuropathy: a review.

The Charcot-Marie-Tooth (CMT) neuropathies divide into two main electrophysiological groups with slow and near normal conduction velocities corresponding to Schwann cell and axonal pathology. An intermediate group also exists with nerve conduction velocities, which overlaps the two main groups. Families with intermediate CMT can be recognized in which different affected individuals in the same family have motor conduction velocities in both the CMT type 1 and 2 ranges (i.e., above and below 38 m/s). The intermediate group is caused by a limited number of distinct gene mutations in dynamin2 (DNM2), gap-junction protein 1 (GJB1), neurofilament light polypeptide (NF-L) genes, and a rare mutation and as yet unknown genes on chromosome 1 and 10 loci. Intermediate forms of CMT may be associated with unique disease mechanisms affecting both Schwann cells and axons. It is useful to recognize this unique group of neuropathies for diagnostic and management purposes.

Cytotoxic platinum(II) intercalators that incorporate 1R,2R-diaminocyclopentane

Twelve metallointercalators of the type [Pt(I(L))(A(L))](2+), where A(L) is either the R,R or S,S enantiomer of 1,2-diaminocyclopentane (DACP) and I(L) is either 1,10-phenathroline, 4-methyl-1,10-phenanthroline, 5-methyl-1,10-phenanthroline, 4,7-dimethyl-1,10-phenanthroline, 5,6-dimethyl-1,10-phenanthroline or 3,4,7,8-tetramethyl-1,10-phenanthroline, were synthesised, characterised and the cytotoxicity to the L1210 cell line was determined. The crystal structures of PHENRRDACP and PHENSS were obtained as monoclinic with a space group of P2(1) (a/Å = 11.4966, b/Å = 6.6983, c/Å = 12.0235) and P2(1) (a/Å = 11.5777, b/Å = 7.0009, c/Å = 12.5079), respectively. The R,R enantiomer of 1,2-diaminocyclopentane (RRDACP) produced the most cytotoxic metallointercalators. The most cytotoxic metallointercalators were 56MERRDACP and 47MERRDACP with IC(50) values of 0.16 and 0.17 μM, respectively, in comparison to cisplatin (1 μM).

The effects of 56MESS on mitochondrial and cytoskeletal proteins and the cell cycle in MDCK cells

BACKGROUND 56MESS has been shown to be cytotoxic but the mode of this action is unclear. In order to probe the mechanism of action for 56MESS, MDCK cells were utilised to investigate the effect on treated cells. RESULTS IC50 values for 56MESS and cisplatin in the MDCK cell line, determined by a SRB assay, were 0.25 ± 0.03 and 18 ± 1.2 μM respectively. In a preliminary study, cells treated with 56MESS displayed no caspase-3/7 activity, suggesting that the mechanism of action is caspase independent. Protein expression studies revealed an increase the expression in the MTC02 protein associated with mitochondria in cells treated with 56MESS and cisplatin. Non-synchronised 56MESS-treated cells caused an arrest in the G2/M phase of the cell cycle, in comparison to the S phase arrest of cisplatin. In G0/G1 synchronised cells, both 56MESS and cisplatin both appeared to arrest within the S phase. CONCLUSIONS these results suggest that 56MESS is capable of causing cell-cycle arrest, and that mitochondrial and cell cycle proteins may be involved in the mode of action of cytotoxicity of 56MESS.

Increased lipid droplet accumulation associated with a peripheral sensory neuropathy.

Hereditary sensory neuropathy type 1 (HSN-1) is an autosomal dominant neurodegenerative disease caused by missense mutations in the SPTLC1 gene. The SPTLC1 protein is part of the SPT enzyme which is a ubiquitously expressed, critical and thus highly regulated endoplasmic reticulum bound membrane enzyme that maintains sphingolipid concentrations and thus contributes to lipid metabolism, signalling, and membrane structural functions. Lipid droplets are dynamic organelles containing sphingolipids and membrane bound proteins surrounding a core of neutral lipids, and thus mediate the intracellular transport of these specific molecules. Current literature suggests that there are increased numbers of lipid droplets and alterations of lipid metabolism in a variety of other autosomal dominant neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease. This study establishes for the first time, a significant increase in the presence of lipid droplets in HSN-1 patient-derived lymphoblasts, indicating a potential connection between lipid droplets and the pathomechanism of HSN-1. However, the expression of adipophilin (ADFP), which has been implicated in the regulation of lipid metabolism, was not altered in lipid droplets from the HSN-1 patient-derived lymphoblasts. This appears to be the first report of increased lipid body accumulation in a peripheral neuropathy, suggesting a fundamental molecular linkage between a number of neurodegenerative diseases.

An initial top-down proteomic analysis of the standard cuprizone mouse model of multiple sclerosis

An initial proteomic analysis of the cuprizone mouse model to characterise the breadth of toxicity by assessing cortex, skeletal muscle, spleen and peripheral blood mononuclear cells. Cuprizone treated vs. control mice for an initial characterisation. Select tissues from each group were pooled, analysed in triplicate using two-dimensional gel electrophoresis (2DE) and deep imaging and altered protein species identified using liquid chromatography tandem mass spectrometry (LC/MS/MS). Forty-three proteins were found to be uniquely detectable or undetectable in the cuprizone treatment group across the tissues analysed. Protein species identified in the cortex may potentially be linked to axonal damage in this model, and those in the spleen and peripheral blood mononuclear cells to the minimal peripheral immune cell infiltration into the central nervous system during cuprizone mediated demyelination. Primary oligodendrocytosis has been observed in type III lesions in multiple sclerosis. However, the underlying mechanisms are poorly understood. Cuprizone treatment results in oligodendrocyte apoptosis and secondary demyelination. This initial analysis identified proteins likely related to axonal damage; these may link primary oligodendrocytosis and secondary axonal damage. Furthermore, this appears to be the first study of the cuprizone model to also identify alterations in the proteomes of skeletal muscle, spleen and peripheral blood mononuclear cells. Notably, protein disulphide isomerase was not detected in the cuprizone cohort; its absence has been linked to reduced major histocompatibility class I assembly and reduced antigen presentation. Overall, the results suggest that, like experimental autoimmune encephalomyelitis, results from the standard cuprizone model should be carefully considered relative to clinical multiple sclerosis.

Mutations in the SPTLC1 protein cause mitochondrial structural abnormalities and endoplasmic reticulum stress in lymphoblasts.

Mutations in serine palmitoyltransferase long chain subunit 1 (SPTLC1) cause the typical length-dependent axonal degeneration hereditary sensory neuropathy type 1 (HSN1). Transmission electron microscopy studies on SPTLC1 mutant lymphoblasts derived from patients revealed specific structural abnormalities of mitochondria. Swollen mitochondria with abnormal cristae were clustered around the nucleus, with some mitochondria being wrapped in rough endoplasmic reticulum (ER) membranes. Total mitochondrial counts revealed a significant change in mitochondrial numbers between healthy and diseased lymphocytes but did not reveal any change in length to width ratios nor were there any changes to cellular function. However, there was a notable change in ER homeostasis, as assessed using key ER stress markers, BiP and ERO1-Lα, displaying reduced protein expression. The observations suggest that SPTLC1 mutations cause mitochondrial abnormalities and ER stress in HSN1 cells.

Mitochondrial protein alterations in a familial peripheral neuropathy caused by the V144D amino acid mutation in the sphingolipid protein, SPTLC1.

Axonal degeneration is the final common path in many neurological disorders. Subsets of neuropathies involving the sensory neuron are known as hereditary sensory neuropathies (HSNs). Hereditary sensory neuropathy type I (HSN-I) is the most common subtype of HSN with autosomal dominant inheritance. It is characterized by the progressive degeneration of the dorsal root ganglion (DRG) with clinical symptom onset between the second or third decade of life. Heterozygous mutations in the serine palmitoyltransferase (SPT) long chain subunit 1 (SPTLC1) gene were identified as the pathogenic cause of HSN-I. Ultrastructural analysis of mitochondria from HSN-I patient cells has displayed unique morphological abnormalities that are clustered to the perinucleus where they are wrapped by the endoplasmic reticulum (ER). This investigation defines a small subset of proteins with major alterations in abundance in mitochondria harvested from HSN-I mutant SPTLC1 cells. Using mitochondrial protein isolates from control and patient lymphoblasts, and a combination of 2D gel electrophoresis, immunoblotting and mass spectrometry, we have shown the increased abundance of ubiquinol-cytochrome c reductase core protein 1, an electron transport chain protein, as well as the immunoglobulin, Ig kappa chain C. The regulation of these proteins may provide a new route to understanding the cellular and molecular mechanisms underlying HSN-I.

Isolation and Identification of ER Associated Proteins with Unique ExpressionChanges Specific to the V144D SPTLC1 Mutations in HSN-I

Axonal degeneration is the final common path in many neurological disorders. Hereditary sensory neuropathies (HSN) are a group of neuropathies involving the sensory neurons. The most common subtype is autosomal dominant hereditary sensory neuropathy type I (HSN-I). Progressive degeneration of the dorsal root ganglion (DRG) neuron with an onset of clinical symptoms between the second or third decade of life characterises HSN-I. Mutations in the serine palmitoyltransferase (SPT) long chain subunit 1 (SPTLC1) gene cause HSN-I. The endoplasmic reticulum (ER) is a dynamic organelle that houses the SPTLC1 protein. Ultra structural analysis has shown the ER in the HSN-I mutant cells to wrap around dysfunctional mitochondria and tethers them to the perinucleus. This investigation establishes that the V144D mutant of SPTLC1 alters the expression of and potentially interacts with a set of proteins within the ER. Using ER protein lysates from HSN-I patient and control lymphoblasts: we have identified a change in regulation of five proteins; Hypoxia Up regulated Protein 1: Chloride intracellular channel protein 1: Ubiqutin-40s Ribosomal protein S27a: Coactosin and Ig Kappa chain C. The expression and regulation of these proteins may help to establish a link between the ER and the ‘dying back’ process of the DRG neuron.