Isolation and identification of ER associated proteins with unique expression changes 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

Suppression of the peripheral immune system limits the central immune response following cuprizone-feeding : relevance to modelling Multiple Sclerosis

Cuprizone (CPZ) preferentially affects oligodendrocytes (OLG), resulting in demyelination. To investigate whether central oligodendrocytosis and gliosis triggered an adaptive immune response, the impact of combining a standard (0.2%) or low (0.1%) dose of ingested CPZ with disruption of the blood brain barrier (BBB), using pertussis toxin (PT), was assessed in mice. 0.2% CPZ(±PT) for 5 weeks produced oligodendrocytosis, demyelination and gliosis plus marked splenic atrophy (37%) and reduced levels of CD4 (44%) and CD8 (61%). Conversely, 0.1% CPZ(±PT) produced a similar oligodendrocytosis, demyelination and gliosis but a smaller reduction in splenic CD4 (11%) and CD8 (14%) levels and no splenic atrophy. Long-term feeding of 0.1% CPZ(±PT) for 12 weeks produced similar reductions in CD4 (27%) and CD8 (43%), as well as splenic atrophy (33%), as seen with 0.2% CPZ(±PT) for 5 weeks. Collectively, these results suggest that 0.1% CPZ for 5 weeks may be a more promising model to study the ‘insideout’ theory of Multiple Sclerosis (MS). However, neither CD4 nor CD8 were detected in the brain in CPZ±PT groups, indicating that CPZ-mediated suppression of peripheral immune organs is a major impediment to studying the ‘inside-out’ role of the adaptive immune system in this model over long time periods. Notably, CPZ(±PT)-feeding induced changes in the brain proteome related to the suppression of immune function, cellular metabolism, synaptic function and cellular structure/organization, indicating that demyelinating conditions, such as MS, can be initiated in the absence of adaptive immune system involvement

The role of human coactosin-like protein in neurodegenerative disorders

Coactosin is one of the numerous actin-binding proteins which regulate the actin cytoskeleton. Coactosin binds F-actin, and also interacts with 5-lipoxygenase, which is the first committed enzyme in leukotriene biosynthesis. Coactosin and human coactosin like protein 1 (COTL1) have the potential to play a role in the degradation or impairment of neuronal cells and their functioning. Its homology to other proteins that affect neuronal cells also contributes to this notion. The objective of this review is to explore its structural novelty, regulation and its significance in neurodegenerative diseases

Proteome alterations associated with the V144D SPTLC1 mutation that causes hereditary sensory neuropathy-I

Background: Hereditary sensory neuropathy type is the most common subtype and presents with clinical onset in the second to third decade of life with progressive degeneration of the dorsal root ganglion neurons. Three different missense mutations in the gene encoding for serine palmitoyltransferase long chain subunit 1 have been linked to HSN-I. Here we quantitatively assess the proteomes and identify marked protein alterations in both mitochondria and endoplasmic reticulum from HSN-I patient lymphoblasts which harbour the V144D mutation. Methods: Mitochondria and endoplasmic reticulum were fractionated and lysed from control and patient-derived lymphoblasts. Protein samples were separated into total soluble and total membrane fractions and analysed using a well-established topdown proteomic protocol. Altered protein species were identified by LC MS/MS. Results: Using a detailed proteomic approach, we identified 36 proteins that were completely altered in abundance in cells harbouring the V144D SPTLC1 mutation relative to normal controls. Conclusion: The data establish that major protein alterations occur in both the endoplasmic reticulum, where the SPTLC1 protein resides, and in the mitochondria from V144D patient lymphoblasts. These proteins potentially play a major role in disease pathogenesis and may thus help to further elucidate the molecular mechanism(s) underlying hereditary sensory neuropathy type I and might also prove to be potential therapeutic targets

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

Optimal isolation of mitochondria for proteomic analyses

Considering the key role of mitochondria in cellular (dys)functions, we compared a standard isolation protocol, followed by lysis in urea/detergent buffer, with a commercially available isolation buffer that rapidly yields a mitochondrial protein fraction. The standard protocol yielded significantly better overall resolution and coverage of both the soluble and membrane mitochondrial proteomes; although the kit was faster, it resulted in recovery of only approximately 56% of the detectable proteome. The quality of ‘‘omic’’ analysis depends on sample handling; for large-scale protein studies, well-resolved proteomes are highly dependent on the purity of starting material and the rigor of the extraction protocol

Chloride intracellular channel protein 1 and its role in neurodegenerative disorders and cancerous tumors

Chloride intracellular channel protein 1 (CLIC1) is a highly conserved intracellular anion channel protein, thought to perform significant roles in maintaining cellular homeostasis. The novelty of its properties by which it can exist in soluble globular form and as integral membrane protein have earned it much interest. While the absolute functional role of CLIC1 is still debated, it is undoubtedly established that activation of CLIC1 increases membrane chloride ion permeability. The versatility of its redox regulated structural transitions has led to its addition to the rare category of metamorphic proteins. Although the exact functions of CLIC1 in maintaining cellular homeostasis still remain to be elucidated, several studies strongly indicate the possible involvement of CLIC1 up regulation in various disease states including cancer and neurodegenerative disorders, implying its significance as a potent drug target. The objective of this review is to explore its structural novelty, regulation and roles in pathologies delineating its significance in 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

Identifying Unique Protein Alterations Caused by SPTLC1 Mutations in a Transfected Neuronal Cell Model

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

Autophagy is up regulated in a neuronal model of Charcot-Marie-Tooth disease that overexpresses dynamin 2 mutant

Dominant-Intermediate Charcot-Marie-Tooth disease is one of the most common inherited disorders affecting the peripheral nervous system. Pleckstrin homology domain mutations in dynamin 2 cause dominant-intermediate Charcot Marie Tooth Syndrome. Autophagy in normal cells helps to maintain homeostasis and degrade damaged or old organelles and proteins. Here we link the pleckstrin homology domain mutants and the disease state to autophagy. Cells over-expressing the K558E mutation in the pleckstrin homology domain of dynamin 2 have shown an increase in expression of ER stress and autophagy markers. Although the exact link between autophagy and peripheral neurodegeneration has yet to be fully elucidated, these results set the foundation for further research into the interactions between dynamin 2 mutations, autophagy, and Dominant-Intermediate Charcot-Marie-Tooth