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1.  Biochemical characterization of protein quality control mechanisms during disease progression in the C22 mouse model of CMT1A 
ASN NEURO  2013;5(5):e00128.
Charcot–Marie–Tooth disease type 1A (CMT1A) is a hereditary demyelinating neuropathy linked with duplication of the peripheral myelin protein 22 (PMP22) gene. Transgenic C22 mice, a model of CMT1A, display many features of the human disease, including slowed nerve conduction velocity and demyelination of peripheral nerves. How overproduction of PMP22 leads to compromised myelin and axonal pathology is not fully understood, but likely involves subcellular alterations in protein homoeostatic mechanisms within affected Schwann cells. The subcellular response to abnormally localized PMP22 includes the recruitment of the ubiquitin–proteasome system (UPS), autophagosomes and heat-shock proteins (HSPs). Here we assessed biochemical markers of these protein homoeostatic pathways in nerves from PMP22-overexpressing neuropathic mice between the ages of 2 and 12 months to ascertain their potential contribution to disease progression. In nerves of 3-week-old mice, using endoglycosidases and Western blotting, we found altered processing of the exogenous human PMP22, an abnormality that becomes more prevalent with age. Along with the ongoing accrual of misfolded PMP22, the activity of the proteasome becomes compromised and proteins required for autophagy induction and lysosome biogenesis are up-regulated. Moreover, cytosolic chaperones are consistently elevated in nerves from neuropathic mice, with the most prominent change in HSP70. The gradual alterations in protein homoeostatic response are accompanied by Schwann cell de-differentiation and macrophage infiltration. Together, these results show that while subcellular protein quality control mechanisms respond appropriately to the presence of the overproduced PMP22, with aging they are unable to prevent the accrual of misfolded proteins.
In peripheral nerves of neuropathic C22 mice the frequency of cytosolic PMP22 aggregates increases with age, which elicits a response from protein quality control mechanisms. The combined effects of aging and neuropathic genotype exacerbate disease progression leading to nerve defects.
doi:10.1042/AN20130024
PMCID: PMC3848555  PMID: 24175617
autophagy; chaperone; ubiquitin; myelin; protein aggregation; Schwann cell.; AMC, amino-methyl coumarin; CathD, Cathepsin D; CMT1A, Charcot–Marie–Tooth disease type 1A; di-8-ANEPPS, 4-[2-(6-dibutylamino)-2-naphthalenyl)ethenyl]-1-(3-sulfopropyl) hydroxide; Egr2, early growth response 2; endoH, endoglycosidase H; ER, endoplasmic reticulum; HRP, horseradish peroxidase; HSF1, heat-shock factor 1; Hsp, heat-shock protein; IgG, immunoglobulin; LAMP1, lysosomal membrane-associated protein 1; LC3, light chain 3; MCP-1, monocyte chemoattractant protein 1; MS, multiple sclerosis; Oct6, octamer-binding transcription factor 6; PMP22, peripheral myelin protein 22; PNGaseF, N-glycosidase F; pUb, polyubiquitinated; TFEB, transcription factor EB; UPS, ubiquitin–proteasome system; Wt, wild-type
2.  Liposomes to Target Peripheral Neurons and Schwann Cells 
PLoS ONE  2013;8(11):e78724.
While a wealth of literature for tissue-specific liposomes is emerging, optimal formulations to target the cells of the peripheral nervous system (PNS) are lacking. In this study, we asked whether a novel formulation of phospholipid-based liposomes could be optimized for preferential uptake by microvascular endothelia, peripheral neurons and Schwann cells. Here, we report a unique formulation consisting of a phospholipid, a polymer surfactant and cholesterol that result in enhanced uptake by targeted cells. Using fluorescently labeled liposomes, we followed particle internalization and trafficking through a distinct route from dextran and escape from degradative compartments, such as lysosomes. In cultures of non-myelinating Schwann cells, liposomes associate with the lipid raft marker Cholera toxin, and their internalization is inhibited by disruption of lipid rafts or actin polymerization. In contrast, pharmacological inhibition of clathrin-mediated endocytosis does not significantly impact liposome entry. To evaluate the efficacy of liposome targeting in tissues, we utilized myelinating explant cultures of dorsal root ganglia and isolated diaphragm preparations, both of which contain peripheral neurons and myelinating Schwann cells. In these models, we detected preferential liposome uptake into neurons and glial cells in comparison to surrounding muscle tissue. Furthermore, in vivo liposome administration by intramuscular or intravenous injection confirmed that the particles were delivered to myelinated peripheral nerves. Within the CNS, we detected the liposomes in choroid epithelium, but not in myelinated white matter regions or in brain parenchyma. The described nanoparticles represent a novel neurophilic delivery vehicle for targeting small therapeutic compounds, biological molecules, or imaging reagents into peripheral neurons and Schwann cells, and provide a major advancement toward developing effective therapies for peripheral neuropathies.
doi:10.1371/journal.pone.0078724
PMCID: PMC3823803  PMID: 24244347
3.  The palmitoylation state of PMP22 modulates epithelial cell morphology and migration 
ASN NEURO  2012;4(6):e00101.
PMP22 (peripheral myelin protein 22), also known as GAS 3 (growth-arrest-specific protein 3), is a disease-linked tetraspan glycoprotein of peripheral nerve myelin and constituent of intercellular junctions in epithelia. To date, our knowledge of the post-translational modification of PMP22 is limited. Using the CSS-Palm 2.0 software we predicted that C85 (cysteine 85), a highly conserved amino acid located between the second and third transmembrane domains, is a potential site for palmitoylation. To test this, we mutated C85S (C85 to serine) and established stable cells lines expressing the WT (wild-type) or the C85S-PMP22. In Schwann and MDCK (Madin–Darby canine kidney) cells mutating C85 blocked the palmitoylation of PMP22, which we monitored using 17-ODYA (17-octadecynoic acid). While palmitoylation was not necessary for processing the newly synthesized PMP22 through the secretory pathway, overexpression of C85S-PMP22 led to pronounced cell spreading and uneven monolayer thinning. To further investigate the functional significance of palmitoylated PMP22, we evaluated MDCK cell migration in a wound-healing assay. While WT-PMP22 expressing cells were resistant to migration, C85S cells displayed lamellipodial protrusions and migrated at a similar rate to vector control. These findings indicate that palmitoylation of PMP22 at C85 is critical for the role of the protein in modulating epithelial cell shape and motility.
doi:10.1042/AN20120045
PMCID: PMC3563111  PMID: 23127255
lipid modification; myelin; protein trafficking; Schwann cell; tetraspan; Con A, concavalin A; C85, cysteine 85; C85S, C85 to serine; Endo H, endoglycosidase H; ER, endoplasmic reticulum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GAS3, growth-arrest-specific protein 3; GFP, green fluorescent protein; HA, haemagglutinin; HN, hydroxylamine; HRP, horseradish peroxidase; MDCK, Madin–Darby canine kidney; ODYA, octadecynoic acid; PLP, proteolipid protein; Palm-YFP, palmitoylatable yellow fluorescent protein; PMP22, peripheral myelin protein 22; PNGaseF, peptide N-glycosidase F; RIPA, radioimmunoprecipitation assay; TX-100, Triton X-100; VVL, Viciavillosa lectin; WT, wild-type
4.  Rapamycin activates autophagy and improves myelination in explant cultures from neuropathic mice 
Misexpression and cytosolic retention of peripheral myelin protein 22 (PMP22) within Schwann cells (SCs) is associated with a genetically heterogeneous group of demyelinating peripheral neuropathies. PMP22 overproducer C22 and spontaneous mutant Trembler J (TrJ) mice display neuropathic phenotypes and affected nerves contain abnormally localized PMP22. Nutrient deprivation-induced autophagy is able to suppress the formation of PMP22 aggregates in a toxin-induced cellular model, and improve locomotor performance and myelination in TrJ mice. As a step toward therapies, we assessed whether pharmacological activation of autophagy by rapamycin (RM) could facilitate the processing of PMP22 within neuropathic SCs and enhance their capacity to myelinate peripheral axons. Exposure of mouse SCs to RM induced autophagy in a dose- and time-dependent manner and decreased the accumulation of poly-ubiquitinated substrates. The treatment of myelinating dorsal root ganglion (DRG) explant cultures from neuropathic mice with RM (25 nM) improved the processing of PMP22 and increased the abundance and length of myelin internodes, as well as the expression of myelin proteins. Notably, RM is similarly effective in both the C22 and TrJ model, signifying that the benefit overlaps among distinct genetic models of PMP22 neuropathies. Furthermore, lentivirus-mediated shRNA knockdown of the autophagy-related gene 12 (Atg12) abolished the activation of autophagy and the increase in myelin proteins, demonstrating that autophagy is critical for the observed improvement. Together, these results support the potential use of RM and other autophagy-enhancing compounds as therapeutic agents for PMP22-associated demyelinating neuropathies.
doi:10.1523/JNEUROSCI.1356-10.2010
PMCID: PMC3478092  PMID: 20739560
CMT1A neuropathy; demyelination; Schwann cells; autophagy; rapamycin; myelin; peripheral myelin protein 22
5.  MicroRNAs in Oligodendrocyte and Schwann Cell Differentiation 
Developmental Neuroscience  2011;33(1):14-20.
MicroRNAs (miRNAs) are a class of small (approx. 22 nt) noncoding RNAs that are capable of post-transcriptionally silencing mRNAs that contain sequences complementary to the miRNAs’ 7- to 8-bp ‘seed’ sequence. As single miRNAs are often predicted to target up to hundreds of individual transcripts, miRNAs are able to broadly affect the overall protein expression state of the cell. This can translate into global effects on cellular health and differentiation state. Recently, several reports have identified crucial roles for miRNAs in controlling the production, differentiation, and health of myelinating cells of the mammalian nervous system. In this review, we will discuss how individual miRNAs regulate these various processes, and also how miRNA production in general is required for several stages of myelin generation and maintenance.
doi:10.1159/000323919
PMCID: PMC3080780  PMID: 21346322
miRNA; Oligodendrocyte; Schwann cell; Myelin
6.  Autophagy aids membrane expansion by neuropathic Schwann cells 
Autophagy  2011;7(2):238-239.
Demyelinating peripheral neuropathies associated with abnormal expression of peripheral myelin protein 22 (PMP22) involve the formation of cytosolic protein aggregates within Schwann cells. Towards developing a therapy for these progressive neurodegenerative diseases, we assessed whether pharmacological activation of autophagy by rapamycin (RM) could prevent protein aggregation and enhance Schwann cell myelination. Indeed, we found that glial cells from neuropathic mice activate autophagy in response to RM and produce abundant myelin internodes. Lentivirus-mediated shRNA shutdown of Atg12 abrogates the improvements in myelin production, demonstrating that autophagy is critical for the observed benefits.
doi:10.4161/auto.7.2.14278
PMCID: PMC3359469  PMID: 21135575
myelination; protein aggregation; neuropathy; neurodegeneration; Schwann cell
7.  Gene Expression in the Hippocampus: Regionally Specific Effects of Aging and Caloric Restriction 
We measured changes in gene expression, induced by aging and caloric restriction (CR), in three hippocampal subregions. When analysis included all regions, aging was associated with expression of genes linked to mitochondrial dysfunction, inflammation, and stress responses, and in some cases, expression was reversed by CR. An age-related increase in ubiquintination was observed, including increased expression of ubiquitin conjugating enzyme genes and cytosolic ubiquitin immunoreactivity. CR decreased cytosolic ubiquitin and upregulated deubiquitinating genes. Region specific analyses indicated that CA1 was more susceptible to aging stress, exhibiting a greater number of altered genes relative to CA3 and the dentate gyrus (DG), and an enrichment of genes related to the immune response and apoptosis. CA3 and the DG were more responsive to CR, exhibiting marked changes in the total number of genes across diet conditions, reversal of age-related changes in p53 signaling, glucocorticoid receptor signaling, and enrichment of genes related to cell survival and neurotrophic signaling. Finally, CR differentially influenced genes for synaptic plasticity in CA1 and CA3. It is concluded that regional disparity in response to aging and CR relates to differences in vulnerability to stressors, the availability of neurotrophic, and cell survival mechanisms, and differences in cell function.
doi:10.1016/j.mad.2010.10.006
PMCID: PMC3032012  PMID: 21055414
aging; diet; hippocampus; transcription; proteosome; ubiquitin
8.  Rewiring Integrin-Mediated Signaling and Cellular Response with the Peripheral Myelin Protein 22 and Epithelial Membrane Protein 2 Components of the Tetraspan Web 
Relative expression of EMP2 or PMP22 within the tetraspan web drives a cellular response toward a FAK or AKT dependent pathway, respectively. The functional consequence is differential regulation of collagen gel contraction in the ARPE-19 cell line.
Purpose.
Integrin-mediated collagen gel contraction by ARPE-19 is an in vitro model for proliferative vitreoretinopathy (PVR), an aberrant wound healing response after retinal detachment or ocular trauma. Expression of the tetraspan protein epithelial membrane protein 2 (EMP2) controls gel contraction through FAK activation. Peripheral myelin protein 22 (PMP22), another member of the tetraspan web, is closely related to EMP2. The purpose of this study was to determine whether PMP22 also controls the contractile phase associated with PVR.
Methods.
Integrin expression, adhesion, and protein expression were assessed, respectively, through flow cytometry, binding to collagen types I and IV, and Western blot analysis. Collagen gel contraction was assessed using an in vitro assay.
Results.
Overexpression of PMP22 in ARPE-19 cells (ARPE-19/PMP22) resulted in increased collagen adhesion. Gel contraction, however, was reduced by greater than 50% in ARPE-19/PMP22 cells (P < 0.001). In contrast to the FAK activation observed by increasing EMP2 expression, PMP22 overexpression led to increased AKT activation. The decrease in gel contraction by the ARPE-19/PMP22 cells was partially reversed through either PMP22 siRNA or by blockade of AKT.
Conclusions.
Relative expression of EMP2 or PMP22 within the tetraspan web drives a cellular response toward a FAK- or AKT-dependent pathway, respectively. EMP2 and PMP22 differentially regulate collagen gel contraction in the ARPE-19 cell line. The implication of this finding adds a new dimension to the concept of the tetraspan web, in which the abundance of individual tetraspan family members differentially regulates signal transduction and the downstream cellular response.
doi:10.1167/iovs.10-6139
PMCID: PMC3262552  PMID: 21421883
9.  Reduction of Dicer Impairs Schwann Cell Differentiation and Myelination 
Journal of neuroscience research  2010;88(12):2558-2568.
The process of Schwann cell myelination requires precisely coordinated gene expression. At the onset of myelination, there is an increase in the expression of differentiation-promoting transcription factors that regulate key Schwann cell genes. Further control of myelin gene expression occurs at the posttranscriptional level and, in part, is mediated by RNA binding proteins and micro-RNAs (miRNAs). miRNAs are small, endogenously derived RNA molecules that repress gene expression by specifically binding to their mRNA targets. In the experiments described here, we tested whether miRNAs were essential in controlling myelination by reducing the levels of Dicer, an essential endoribonuclease in miRNA biogenesis. We decreased the expression of Dicer by about 60% within Schwann cells using a lentiviral vector expressing an shRNA against Dicer. The reduced levels of Dicer led to a decrease in the steady-state expression of selected miRNAs and of the transcription factors Oct6 and Egr2/Krox20, both of which are critical for Schwann cells differentiation and myelination. In contrast, the levels of c-jun and Sox2 were up-regulated by the reduction in Dicer and were associated with an increase in Schwann cell proliferation. In dorsal root ganglion cocultures, Schwann cells transduced with Dicer shRNA synthesized less myelin, which was accompanied by significant reductions in the levels of myelin basic protein and protein zero. These findings support a critical role for Dicer and miRNAs in Schwann cell differentiation and myelination.
doi:10.1002/jnr.22418
PMCID: PMC3124220  PMID: 20648646
Schwann cell; myelin; micro-RNA; Dicer; myelination
10.  Pharmacological induction of the heat shock response improves myelination in a neuropathic model 
Neurobiology of disease  2008;32(1):105-115.
Misexpression and intracellular retention of peripheral myelin protein 22 (PMP22) is associated with hereditary neuropathies in humans, including Charcot-Marie-Tooth disease type 1A (CMT1A). Mice expressing extra copies of the human PMP22, termed C22, display morphologic and behavioral characteristics of CMT1A. In neuropathic Schwann cells, the turnover of the newly-synthesized PMP22 is decreased, leading to the formation of cytosolic protein aggregates. To aid the processing of PMP22 and alleviate the associated myelin defects, we pharmacologically stimulated the expression of protein chaperones by synthetic small-molecule inhibitors of heat shock protein 90 (HSP90). The exposure of Schwann cells to these compounds enhanced the levels of cytosolic chaperones in a time- and dose-dependent manner, with minimal cytotoxicity. Treatment of dorsal root ganglion (DRG) explants from neuropathic mice improved myelin formation and the processing of PMP22. These results warrant further studies with HSP90 inhibitors as potential therapeutic candidates for hereditary demyelinating neuropathies.
doi:10.1016/j.nbd.2008.06.015
PMCID: PMC3120161  PMID: 18655835
CMT1A neuropathy; demyelination; Schwann cells; protein misfolding; heat shock response; heat shock proteins; HSP90 inhibitors; myelination; myelin; peripheral myelin protein 22
11.  PERIPHERAL MYELIN PROTEIN-22 IS EXPRESSED IN CNS MYELIN 
Translational neuroscience  2010;1(4):282-285.
Myelin abnormalities exist in schizophrenia leading to the hypothesis that oligodendrocyte dysfunction plays a role in the pathophysiology of the disease. The expression of the mRNA for the peripheral myelin protein-22 (PMP-22) is decreased in schizophrenia and recent genetic evidence suggests a link between PMP-22 and schizophrenia. While PMP-22 mRNA is found in both rodent and human brain it has been generally thought that no protein expression occurs. Here we show that PMP-22 protein is present in myelin isolated from adult mouse and human brain. These results suggest that PMP-22 protein likely plays a role in the maintenance and function of central nervous system (CNS) myelin and provide an explanation for why altered PMP-22 expression may be pathophysiologically relevant in a CNS disorder such as schizophrenia.
doi:10.2478/v10134-010-0038-3
PMCID: PMC3093192  PMID: 21572910
Myelin; Peripheral myelin protein-22; Schizophrenia
12.  Peripheral myelin protein-22 (PMP22) modulates alpha 6 integrin expression in the human endometrium 
Background
PMP22, a member of the GAS3 family of tetraspan proteins, is associated with a variety of neurological diseases. Previous studies have shown that PMP22 is expressed in proliferative endometrium, but its function within this tissue is poorly understood. In this study, we first characterized the expression of PMP22 in the human menstrual cycle and began to characterize its function in the endometrium.
Methods
Using a combination of immunohistochemistry and quantitative PCR, we characterized the expression of PMP22 in both proliferative and secretory endometrium. Differences in PMP22 expression between proliferative and secretory endometrium were determined using a Mann-Whitney U test. In order to investigate the influence of PMP22 on α6 integrin expression, cells were created that ectopically overexpressed PMP22 or expressed a siRNA to inhibit its expression. These cells were analyzed for changes in integrins and binding to extracellular matrices.
Results
In this study, we show that PMP22 expression is higher in proliferative phase than secretory phase. Functionally, we have begun to characterize the functional significance of this expression. Previous studies have suggested a link between PMP22 and α6 integrin, and therefore we asked whether PMP22 could associate or potentially modulate the expression of α6 integrin. Expression of both PMP22 and α6 integrin were detectable in endometrial epithelial and stromal cells, and we show that both proteins can associate and colocalize with each other. To understand if PMP22 directly altered the expression of a6 integrin, we examined cell lines with modulated levels of the protein. Overexpression of PMP22 was sufficient to increase α6 integrin surface expression with a concominant increase in binding to the extracellular matrix laminin, while a reduction in PMP22 suppressed α6 integrin surface expression.
Conclusion
These findings suggest a physiologic role for PMP22 on the expression of α6 integrin. We predict that this may be important for the maintainence of endometrial integrity and to the disease biology associated with altered levels of α6 integrin expression in the endometrium.
doi:10.1186/1477-7827-9-56
PMCID: PMC3110552  PMID: 21518455
13.  Lifelong Calorie Restriction Alleviates Age-Related Oxidative Damage in Peripheral Nerves 
Rejuvenation Research  2010;13(1):65-74.
Abstract
Aging is associated with protein damage and imbalance in redox status in a variety of cells and tissues, yet little is known about the extent of age-related oxidative stress in the peripheral nervous system. Previously, we showed a drastic decline in the expression of glial and neuronal proteins in myelinated peripheral nerves with age, which is significantly ameliorated by lifelong calorie restriction. The age-related decline in functional molecules is associated with alterations in cellular protein homeostatic mechanisms, which could lead to a buildup of damaged, aggregated proteins. To determine the extent of oxidative damage within myelinated peripheral nerves, we studied sciatic nerves from rats of four different ages (8, 18, 29, and 38 months) maintained on an ad libitum or a 40% calorie-restricted diet. We found a prominent accumulation of polyubiquitinated substrates with age, which are associated with the conglomeration of distended lysosomes and lipofuscin adducts. The occurrence of these structures is notably less frequent within nerves of age-matched rodents kept on a lifelong reduced calorie diet. Markers for lipid peroxidation, inflammation, and immune cell infiltration are all elevated in nerves of ad libitum–fed rats, whereas food restriction is able to attenuate such deleterious processes with age. Together these results show that dietary restriction is an efficient means of defying age-related oxidative damage and maintaining a younger state in peripheral nerves.
doi:10.1089/rej.2009.0892
PMCID: PMC2877262  PMID: 20230280
14.  Peripheral Myelin Protein 22 is Regulated Post-Transcriptionally by miRNA-29a 
Glia  2009;57(12):1265-1279.
Peripheral myelin protein 22 (PMP22) is a dose-sensitive, disease-associated protein primarily expressed in myelinating Schwann cells. Either reduction or overproduction of PMP22 can result in hereditary neuropathy, suggesting a requirement for correct protein expression for peripheral nerve biology. PMP22 is post-transcriptionally regulated and the 3′untranslated region (3′UTR) of the gene exerts a negative effect on translation. MicroRNAs (miRNAs) are small regulatory molecules that function at a post-transcriptional level by targeting the 3′UTR in a reverse complementary manner. We used cultured Schwann cells to demonstrate that alterations in the miRNA biogenesis pathway affect PMP22 levels, and endogenous PMP22 is subjected to miRNA regulation. GW-body formation, the proposed cytoplasmic site for miRNA-mediated repression, and Dicer expression, an RNase III family ribonuclease involved in miRNA biogenesis, are co-regulated with the differentiation state of Schwann cells. Furthermore, the levels of Dicer inversely correlate with PMP22, while the inhibition of Dicer leads to elevated PMP22. Microarray analysis of actively-proliferating and differentiated Schwann cells, in conjunction with bioinformatics programs, identified several candidate PMP22-targeting miRNAs. Here we demonstrate that miR-29a binds and inhibits PMP22 reporter expression through a specific miRNA seed binding region. Over-expression of miR-29a enhances the association of PMP22 RNA with Argonaute 2, a protein involved in miRNA function, and reduces the steady-state levels of PMP22. In contrast, inhibition of endogenous miR-29a relieves the miRNA-mediated repression of PMP22. Correlation analyses of miR-29 and PMP22 in sciatic nerves reveal an inverse relationship, both developmentally and in post-crush injury. These results identify PMP22 as a target of miRNAs and suggest that myelin gene expression by Schwann cells is regulated by miRNAs.
doi:10.1002/glia.20846
PMCID: PMC2713384  PMID: 19170179
Schwann cell; myelin; microRNA; gene regulation; myelination
15.  Lifelong Calorie Restriction Alleviates Age-Related Oxidative Damage in Peripheral Nerves 
Rejuvenation research  2010;13(1):65-74.
Aging is associated with protein damage and imbalance in redox status in a variety of cells and tissues, yet little is known about the extent of age-related oxidative stress in the peripheral nervous system. Previously, we showed a drastic decline in the expression of glial and neuronal proteins in myelinated peripheral nerves with age, which is significantly ameliorated by lifelong calorie restriction. The age-related decline in functional molecules is associated with alterations in cellular protein homeostatic mechanisms, which could lead to a buildup of damaged, aggregated proteins. To determine the extent of oxidative damage within myelinated peripheral nerves, we studied sciatic nerves from rats of four different ages (8, 18, 29, and 38 months) maintained on an ad libitum or a 40% calorie-restricted diet. We found a prominent accumulation of polyubiquitinated substrates with age, which are associated with the conglomeration of distended lysosomes and lipofuscin adducts. The occurrence of these structures is notably less frequent within nerves of age-matched rodents kept on a lifelong reduced calorie diet. Markers for lipid peroxidation, inflammation, and immune cell infiltration are all elevated in nerves of ad libitum–fed rats, whereas food restriction is able to attenuate such deleterious processes with age. Together these results show that dietary restriction is an efficient means of defying age-related oxidative damage and maintaining a younger state in peripheral nerves.
doi:10.1089/rej.2009.0892
PMCID: PMC2877262  PMID: 20230280
16.  Intermittent fasting alleviates the neuropathic phenotype in a mouse model of Charcot-Marie-Tooth disease 
Neurobiology of disease  2009;34(1):146-154.
Charcot-Marie-Tooth type 1A (CMT1A) neuropathies linked to the misexpression of peripheral myelin protein 22 (PMP22) are progressive demyelinating disorders of the peripheral nervous system. In this study we asked whether dietary restriction by intermittent fasting (IF) could alleviate the neuropathic phenotype in the Trembler J (TrJ) mouse model of CMT1A. Our results show that neuropathic mice kept on a five month long IF regimen had improved locomotor performance compared to ad libitum (AL) fed littermates. The functional benefits of this dietary intervention are associated with an increased expression of myelin proteins combined with a thicker myelin sheath, less redundant basal lamina, and a reduction in aberrant Schwann cell proliferation. These morphological improvements are accompanied by a decrease in PMP22 protein aggregates, and enhanced expression of cytosolic chaperones and constituents of the autophagy-lysosomal pathway. These results indicate that dietary restriction is beneficial for peripheral nerve function in TrJ neuropathic mice, as it promotes the maintenance of locomotor performance.
doi:10.1016/j.nbd.2009.01.002
PMCID: PMC2757933  PMID: 19320048
neuropathy; myelin; dietary regimen; autophagy; chaperones; Schwann cells
17.  The heme precursor delta-aminolevulinate blocks peripheral myelin formation 
Journal of neurochemistry  2008;106(5):2068-2079.
Delta-aminolevulinic acid (δ-ALA) is a heme precursor implicated in neurological complications associated with porphyria and tyrosinemia type I. Delta-ALA is also elevated in the urine of animals and patients treated with the investigational drug dichloroacetate (DCA). We postulated that δ-ALA may be responsible, in part, for the peripheral neuropathy observed in subjects receiving DCA. To test this hypothesis, myelinating cocultures of Schwann cells and sensory neurons were exposed to δ-ALA (0.1–1 mM) and analyzed for the expression of neural proteins and lipids and markers of oxidative stress. Exposure of myelinating samples to δ-ALA is associated with a pronounced reduction in the levels of myelin-associated lipids and proteins, including myelin protein zero and peripheral myelin protein 22. We also observed an increase in protein carbonylation and the formation of hydroxynonenal and malondialdehyde after treatment with δ-ALA. Studies of isolated Schwann cells and neurons indicate that glial cells are more vulnerable to this pro-oxidant than neurons, based on a selective decrease in the expression of mitochondrial respiratory chain proteins in glial, but not in neuronal, cells. These results suggest that the neuropathic effects of δ-ALA are attributable, at least in part, to its pro-oxidant properties which damage myelinating Schwann cells.
doi:10.1111/j.1471-4159.2008.05552.x
PMCID: PMC2574579  PMID: 18665889
Schwann cells; demyelination; delta-aminolevulinate; peripheral neuropathy; oxidative stress; dichloroacetate
18.  The molecular architecture of myelinated peripheral nerves is supported by calorie restriction with aging 
Aging cell  2009;8(2):178-191.
Summary
Peripheral nerves from aged animals exhibit features of degeneration, including marked fiber loss, morphological irregularities in myelinated axons and notable reduction in the expression of myelin proteins. To investigate how protein homeostatic mechanisms change with age within the peripheral nervous system, we isolated Schwann cells from the sciatic nerves of young and old rats. The responsiveness of cells from aged nerves to stress stimuli is weakened, which in part may account for the observed age-associated alterations in glial and axonal proteins in vivo. While calorie restriction (CR) is known to slow the aging process in the central nervous system, its influence on peripheral nerves has not been investigated in detail. To determine if dietary restriction is beneficial for peripheral nerve health and glial function, we studied sciatic nerves from rats of four distinct ages (8-, 18-, 29- and 38-months) kept on an ad libitum (AL) or a 40% CR diet. Age-associated reduction in the expression of the major myelin proteins and widening of the nodes of Ranvier are attenuated by the dietary intervention, which is paralleled with the maintenance of a differentiated Schwann cell phenotype. The improvements in nerve architecture with diet restriction, in part, are underlined by sustained expression of protein chaperones and markers of the autophagy-lysosomal pathway. Together, the in vitro and in vivo results suggest that there might be an age-limit by which dietary intervention needs to be initiated to elicit a beneficial response on peripheral nerve health.
doi:10.1111/j.1474-9726.2009.00460.x
PMCID: PMC2715941  PMID: 19239416
peripheral nerve; Schwann cell; heat shock protein; autophagy; myelin; chaperones
19.  Identification of Dynamically Regulated microRNA and mRNA Networks in Developing Oligodendrocytes 
MicroRNAs (miRNAs) play important roles in modulating gene expression at the post-transcriptional level. In postnatal oligodendrocyte lineage cells, the miRNA expression profile -“microRNAome” - contains 43 miRNAs whose expression dynamically changes during the transition from A2B5+ oligodendrocyte progenitor cells to premyelinating GalC+ cells. The combination of microRNAome profiling with analyses of the oligodendrocyte transcriptome reveals a target bias for a class of miRNAs which includes miR-9. We show that miR-9 is down-regulated during oligodendrocyte differentiation. In addition, miR-9 expression level inversely correlates with the expression of its predicted targets, among which is the peripheral myelin protein PMP22. We found that PMP22 mRNA but not protein is detectable in oligodendrocytes, while Schwann cells producing PMP22 protein lack miR-9. We demonstrate that miR-9 interacts with the 3’ untranslated region of PMP22 and down-regulates its expression. Our results support models in which miRNAs can act as guardians of the transcriptome.
doi:10.1523/JNEUROSCI.1932-08.2008
PMCID: PMC2646797  PMID: 18987208
microRNA; post-transcriptional regulation; oligodendrocyte; PMP22; myelin; glia
20.  The formation of peripheral myelin protein 22 aggregates is hindered by the enhancement of autophagy and expression of cytoplasmic chaperones 
Neurobiology of disease  2006;25(2):252-265.
The accumulation of misfolded proteins is associated with various neurodegenerative conditions. Peripheral myelin protein 22 (PMP22) is a hereditary neuropathy-linked, short-lived molecule that forms aggresomes when the proteasome is inhibited or the protein is mutated. We previously showed that the removal of pre-existing PMP22 aggregates is assisted by autophagy. Here we examined whether the accumulation of such aggregates could be suppressed by experimental induction of autophagy and/or chaperones. Enhancement of autophagy during proteasome inhibition hinders protein aggregate formation and correlates with a reduction in accumulated proteasome substrates. Conversely, simultaneous inhibition of autophagy and the proteasome augments the formation of aggregates. An increase of heat shock protein levels by geldanamycin treatment or heat shock preconditioning similarly hampers aggresome formation. The beneficial effects of autophagy and chaperones in preventing the accumulation of misfolded PMP22 are additive and provide a potential avenue for therapeutic approaches in hereditary neuropathies linked to PMP22 mutations.
doi:10.1016/j.nbd.2006.09.018
PMCID: PMC1857308  PMID: 17174099
autophagosome; proteasome; neuropathy; Schwann cells; protein misfolding; aggresomes; heat shock proteins; heat shock
21.  Modulation of Epithelial Morphology, Monolayer Permeability, and Cell Migration by Growth Arrest Specific 3/Peripheral Myelin Protein 22 
Molecular Biology of the Cell  2005;16(3):1142-1151.
Peripheral myelin protein 22 (PMP22) is associated with a subset of hereditary peripheral neuropathies. Although predominantly recognized as a transmembrane constituent of peripheral nerve myelin, PMP22 is localized to epithelial and endothelial cell-cell junctions, where its function remains unknown. In this report, we investigated the role of PMP22 in epithelial biology. Expression of human PMP22 (hPMP22) slows cell growth and induces a flattened morphology in Madin-Darby canine kidney (MDCK) cells. The transepithelial electrical resistance (TER) and paracellular flux of MDCK monolayers are elevated by hPMP22 expression. After calcium switch, peptides corresponding to the second, but not the first, extracellular loop of PMP22 perturb the recovery of TER and paracellular flux. Finally, subsequent to wounding, epithelial monolayers expressing hPMP22 fail to migrate normally. These results indicate that PMP22 is capable of modulating several aspects of epithelial cell biology, including junctional permeability and wound closure.
doi:10.1091/mbc.E04-07-0551
PMCID: PMC551480  PMID: 15635102

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