Caveolin-1 and Altered Neuregulin Signaling Contribute to the Pathophysiological Progression of Diabetic Peripheral Neuropathy

Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.OBJECTIVE Evaluate if Erb B2 activation and the loss of caveolin-1 (Cav1) contribute to the pathophysiological progression of diabetic peripheral neuropathy (DPN). RESEARCH DESIGN AND METHODS Cav1 knockout and wild-type C57BL/6 mice were rendered diabetic with streptozotocin, and changes in motor nerve conduction velocity (MNCV), mechanical and thermal hypoalgesia, Erb B2 phosphorylation (pErb B2), and epidermal nerve fiber density were assessed. The contribution of Erb B2 to DPN was assessed using the Erb B2 inhibitors PKI 166 and erlotinib and a conditional bitransgenic mouse that expressed a constitutively active form of Erb B2 in myelinated Schwann cells (SCs). RESULTS Diabetic mice exhibited decreased MNCV and mechanical and thermal sensitivity, but the extent of these deficits was more severe in diabetic Cav1 knockout mice. Diabetes increased pErb B2 levels in both genotypes, but the absence of Cav1 correlated with a greater increase in pErb B2. Erb B2 activation contributed to the mechanical hypoalgesia and MNCV deficits in both diabetic genotypes because treatment with erlotinib or PKI 166 improved these indexes of DPN. Similarly, induction of a constitutively active Erb B2 in myelinated SCs was sufficient to decrease MNCV and induce a mechanical hypoalgesia in the absence of diabetes. CONCLUSIONS Increased Erb B2 activity contributes to specific indexes of DPN, and Cav1 may be an endogenous regulator of Erb B2 signaling. Altered Erb B2 signaling is a novel mechanism that contributes to SC dysfunction in diabetes, and inhibiting Erb B2 may ameliorate deficits of tactile sensitivity in DPN. Diabetic peripheral neuropathy (DPN) is a common complication of diabetes (1). Although hyperglycemia is the definitive cause of DPN (2), the vascular, glial, and neuronal damage that underlies the progressive axonopathy in DPN has a complex biochemical etiology involving oxidative stress (3,4), protein glycation (5), protein kinase C activation (6), polyol synthesis (7), and the hexosamine pathway (8). Altered neurotrophic support also contributes to sensory neuron dysfunction in DPN (9), but whether diabetes may alter growth factor signaling in Schwann cells (SCs), which also undergo substantial degeneration in diabetes, is poorly defined. Neuregulins are growth factors that control SC growth, survival, and differentiation via their interaction with Erb B receptors (10). Although Erb B2 signaling promotes developmental myelination and is clearly trophic for SCs, pharmacological evidence supports that pathologic activation of Erb B2 after axotomy (11) or infection with leprosy bacilli (12) is sufficient to induce SC dedifferentiation and demyelination. Additionally, genetic evidence supports that Erb B2 can promote the development of sensory neuropathies independent of diabetes because expression of a dominant-negative Erb B4 in nonmyelinating (13) or myelinating (14) SCs induced a temperature or mechanical sensory neuropathy, respectively. Given the contribution of Erb B2 to the degeneration of SCs, endogenous proteins that regulate Erb B2 activity may influence the development of certain aspects of sensory neuropathies. The interaction of Erb B2 with the protein caveolin-1 (Cav1) inhibits the intrinsic tyrosine kinase activity of the receptor (15). Cav1 is highly expressed in mature, myelinated SCs (16), and we have shown that prolonged hyperglycemia promoted the downregulation of Cav1 in SCs of sciatic nerve (17). Cav1 may regulate Erb B2 signaling in SCs because its forced downregulation was sufficient to enhance neuregulin-induced demyelination of SC–dorsal root ganglion (DRG) neuron cocultures (18). However, it is unknown whether an increase in Erb B2 activity may contribute to the pathophysiological development of DPN and if changes in Cav1 expression may alter Erb B2 activation in diabetic nerve. In the current study, we demonstrate that diabetic Cav1 knockout mice showed an increased activation of Erb B2 and developed greater motor nerve conduction velocity (MNCV) deficits relative to their wild-type counterparts. Inhibition of Erb B2 with two structurally diverse inhibitors corrected the MNCV deficits and mechanical hypoalgesia evident after 6 or 15 weeks of diabetes. Also, induction of a constitutively active Erb B2 in myelinated SCs of adult mice was sufficient to recapitulate the MNCV and mechanical sensitivity deficits observed in the diabetic mice. These studies provide the first evidence that activation of Erb B2 contributes to deficits associated with myelinated fiber function in diabetic nerve and suggest that Cav1 may serve as an endogenous regulator of Erb B2.This work was supported by grants from the Juvenile Diabetes Research Foundation and the National Institutes of Health (NS-054847 and DK-073594)

Miz1 Is a Critical Repressor of cdkn1a during Skin Tumorigenesis

The transcription factor Miz1 forms repressive DNA-binding complexes with the Myc, Gfi-1 and Bcl-6 oncoproteins. Known target genes of these complexes encode the cyclin-dependent kinase inhibitors (CKIs) cdkn2b (p15Ink4), cdkn1a (p21Cip1), and cdkn1c (p57Kip2). Whether Miz1-mediated repression is important for control of cell proliferation in vivo and for tumor formation is unknown. Here we show that deletion of the Miz1 POZ domain, which is critical for Miz1 function, restrains the development of skin tumors in a model of chemically-induced, Ras-dependent tumorigenesis. While the stem cell compartment appears unaffected, interfollicular keratinocytes lacking functional Miz1 exhibit a reduced proliferation and an accelerated differentiation of the epidermis in response to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Tumorigenesis, proliferation and normal differentiation are restored in animals lacking cdkn1a, but not in those lacking cdkn2b. Our data demonstrate that Miz1-mediated attenuation of cell cycle arrest pathways via repression of cdkn1a has a critical role during tumorigenesis in the skin

Exclusion of mutations in the PRNP, JPH3, TBP, ATN1, CREBBP, POU3F2 and FTL genes as a cause of disease in Portuguese patients with a Huntington-like phenotype

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder characterised by chorea, cognitive impairment, dementia and personality changes, caused by the expansion of a CAG repeat in the HD gene. Often, patients with a similar clinical presentation do not carry expansions of the CAG repeat in this gene [Huntington disease-like (HDL) patients]. We report the genetic analysis of 107 Portuguese patients with an HDL phenotype. The HDL genes PRNP and JPH3, encoding the prion protein and junctophilin-3, respectively, were screened for repeat expansions in these patients. Given the partial clinical overlap of SCA17, DRPLA and neuroferritinopathy with HD, their causative genes (TBP, ATN1, and FTL, respectively) were also analysed. Finally, repeat expansions in two candidate genes, CREBBP and POU3F2, which encode the nuclear transcriptional coactivator CREB-binding protein and the CNS-specific transcription factor N-Oct-3, respectively, were also studied. Expansions of the repetitive tracts of the PRNP, JPH3, TBP, ATN1, CREBBP and POU3F2 genes were excluded in all patients, as were sequence alterations in the FTL gene. Since none of the genes already included in the differential diagnosis of HD was responsible for the disease in our sample, the genetic heterogeneity of the HDL phenotype is still open for investigation.Fundação para a Ciência e a Tecnologia (FCT) and FEDER (grant CBO/33485/99). BIC included in grant CBO/33485/99, respectivel

Expression and localization of Ski determine cell type-specific TGFbeta signaling effects on the cell cycle

Transforming growth factor beta (TGFbeta) promotes epithelial cell differentiation but induces Schwann cell proliferation. We show that the protooncogene Ski (Sloan-Kettering viral oncogene homologue) is an important regulator of these effects. TGFbeta down-regulates Ski in epithelial cells but not in Schwann cells. In Schwann cells but not in epithelial cells, retinoblastoma protein (Rb) is up-regulated by TGFbeta. Additionally, both Ski and Rb move to the cytoplasm, where they partially colocalize. In vivo, Ski and phospho-Rb (pRb) appear to interact in the Schwann cell cytoplasm of developing sciatic nerves. Ski overexpression induces Rb hyperphosphorylation, proliferation, and colocalization of both proteins in Schwann cell and epithelial cell cytoplasms independently of TGFbeta treatment. Conversely, Ski knockdown in Schwann cells blocks TGFbeta-induced proliferation and pRb cytoplasmic relocalization. Our findings reveal a critical function of fine-tuned Ski levels in the control of TGFbeta effects on the cell cycle and suggest that at least a part of Ski regulatory effects on TGFbeta-induced proliferation of Schwann cells is caused by its concerted action with Rb

ErbB2 signaling in Schwann cells is mostly dispensable for maintenance of myelinated peripheral nerves and proliferation of adult Schwann cells after injury

Neuregulin/erbB signaling is critically required for survival and proliferation of Schwann cells as well as for establishing correct myelin thickness of peripheral nerves during development. In this study, we investigated whether erbB2 signaling in Schwann cells is also essential for the maintenance of myelinated peripheral nerves and for Schwann cell proliferation and survival after nerve injury. To this end, we used inducible Cre-loxP technology using a PLP-CreERT2 allele to ablate erbB2 in adult Schwann cells. ErbB2 expression was markedly reduced after induction of erbB2 gene disruption with no apparent effect on the maintenance of already established myelinated peripheral nerves. In contrast to development, Schwann cell proliferation and survival were not impaired in mutant animals after nerve injury, despite reduced levels of MAPK-P (phosphorylated mitogen-activated protein kinase) and cyclin D1. ErbB1 and erbB4 do not compensate for the loss of erbB2. We conclude that adult Schwann cells do not require major neuregulin signaling through erbB2 for proliferation and survival after nerve injury, in contrast to development and in cell culture

N-Oct 5 is generated by in vitro proteolysis of the neural POU-domain protein N-Oct 3

The neural POU-domain proteins N-Oct 3 and N-Oct 5 were first identified in electrophoretic mobility retardation assays through their ability to bind to the octamer sequence ATGCAAAT. These two N-Oct factors are detected in extracts from tumor-derived and normal neural cells. They are present differentially, however, in extracts from melanocytes and melanoma cells: N-Oct 3 is present in extracts from both melanocytes and melanoma cells, whereas N-Oct 5 is more evident in extracts from metastatic melanoma cells. We show here that a cDNA encoding N-Oct 3 directs synthesis of both the N-Oct 3 and N-Oct 5 proteins and that the N-Oct 5 protein in neural and melanoma-cell extracts is also related to N-Oct 3. N-Oct 5, however, is apparently not expressed in vivo: It is not detected if cells are rapidly lysed in SDS or if extracts are prepared with a cocktail of protease inhibitors that includes the serine-protease inhibitor 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). These data suggest that N-Oct 5 is a specific in vitro proteolytic cleavage product of N-Oct 3 and is not directly related to melanocyte malignancy

Differential cyclin D1 requirements of proliferating Schwann cells during development and after injury

Neurons regulate Schwann cell proliferation, but little is known about the molecular basis of this interaction. We have examined the possibility that cyclin D1 is a key regulator of the cell cycle in Schwann cells. Myelinating Schwann cells express cyclin D1 in the perinuclear region, but after axons are severed, cyclin D1 is strongly upregulated in parallel with Schwann cell proliferation and translocates into Schwann cell nuclei. During development, cyclin D1 expression is confined to the perinuclear region of proliferating Schwann cells and the analysis of cyclin D1-null mice indicates that cyclin D1 is not required for this type of Schwann cell proliferation. As in the adult, injury to immature peripheral nerves leads to translocation of cyclin D1 to Schwann cell nuclei and injury-induced proliferation is impaired in both immature and mature cyclin D1-deficient Schwann cells. Thus, our data indicate that the molecular mechanisms regulating proliferation of Schwann cells during development or activated by axonal damage are fundamentally different

Isolation of the human genomic brain-2/N-Oct 3 gene (POUF3) and assignment to chromosome 6q16

N-Oct 3 is a human POU domain transcription factor that binds to the octamer sequence ATGCAAAT. The protein is expressed in the central nervous system during development and in adult brain. We have isolated and characterized genomic clones encoding the human N-Oct 3 gene (HGMW-approved symbol POUF3). Comparison of the structure of these clones with the N-Oct 3 cDNA revealed that POUF3 is an intronless gene. Sequencing of 650 bp of the promoter region showed 84% sequence identity of POUF3 with its murine homologue, the brain-2 (designated brn-2) gene. Whereas both POUF3 and brn-2 lack a TATA box, consensus sequences for AP-2, GCF, and SP1 transcription factors were identified within the highly conserved 5'-flanking region. These sequences may play a crucial role for the tissue-specific transcription activation of the POUF3 gene. Southern blotting and in situ hybridization localized the human POUF3 gene to chromosome 6q16

Proliferation of Schwann cells and regulation of cyclin D1 expression in an animal model of Charcot-Marie-Tooth disease type 1A

Overexpression of PMP22 is responsible for the most common form of inherited neuropathy, Charcot-Marie-Tooth disease (CMT) type 1A. The PMP22-transgenic rat (CMT rat) is an animal model of CMT1A, and its peripheral nerves show the characteristic features of ongoing demyelination and remyelination that is also seen in CMT1A patients. Since Schwann cell proliferation is a prominent feature of peripheral nerves in inherited peripheral neuropathies, we examined proliferation and the expression of cyclin D1 in CMT rats. D-type cyclins are required for the initial steps in cell division and nuclear import is crucial for the function of cyclin D1 in promoting cell proliferation. Like normal myelinating Schwann cells in wild-type rats, remyelinating Schwann cells in CMT rats show perinuclear cyclin D1 expression. Schwann cells with nuclear cyclin D1 expression, as well as proliferating Schwann cells, were both associated with demyelinated axonal segments. Supernumerary onion bulb Schwann cells, however, do not express cyclin D1 and were not proliferating. Thus, cyclin D1 expression and its subcellular localization correlate directly with distinct physiological states of Schwann cells in this animal model of CMT1A

Fetal Actometer Based on Optical Fibre Gratings

Monitoring of fetal activity has been used to check wellbeing of fetus both clinically and in homecare. Clinically, fetal movements are monitored by an ultrasound transducer [1], simultaneously with the fetal heart rate and the uterine contractions measured by a tococardiograph (CTG) [2]. The absence of correlation between the fetal activity and the changes in its heart rate signals potential problems, the heaviest being the stillbirth [3]. In homecare, fetal movement is monitored by pregnant women directly, detection fully relying on mother's perception. It has been shown that mother's perception agrees with the fetal movement registered by an ultrasound actometer in 79% of cases in 34-40 weeks gestation period and in 72% of cases in 31-34 weeks gestation period [4]. The partial reliability of such a measurement indicates that a more accurate, objective and convenient monitoring method is needed in homecare. To further take a load off mother, who is supposed to perform measurements several times a day, the new sensor should be wearable and the readout of fetal movements automatic. We propose a fetal actometer based on optical fibre grating sensors that can satisfy the above requirements. Long-period gratings are sensitive to changes in pressure, tension and curvature, hence to external changes of mother's abdomen caused by fetal movement. Detection was performed by monochromatic lateral filtering technique that relies on the transmitted power measurement at a specified wavelength [5]. An additional reference sensor was used to register mother's perception. To extract the useful information and produce a user-friendly output, an algorithm for counting fetal movements and their demarcation in the signal was developed. Traces of the maternal heart beat and breathing as well as fetal hiccups were eliminated by spectral filtering. The signal processing software is sensor independent and can be used with other actographs. Results of the study performed on healthy women in the third trimester of pregnancy show 75% agreement between the number of movements registered by the LPG sensors and mothers. A systematic difference between the signals came from the movements that the sensors registered but mothers did not perceive. The number of false positives, however, can be established only in comparison with the ultrasound detection. The good agreement with the maternal perception and the absence of cross-talk with the fetal heart rate measurement, along with the sensors' light weight, robustness and wearability, indicates their potential for use in hospitals and homecare and encourages further clinical studies.VI International School and Conference on Photonics and COST actions: MP1406 and MP1402 : PHOTONICA2017 : August 23 - September 1, 2017; Belgrade