Regulation of L-periaxin by the Ubiquitin/Proteasome pathway

The morphological changes required for the ensheathment of peripheral nerve axons by Schwann cells are believed to be regulated by the cell cytoskeleton and its associated proteins. Control of the levels of these proteins is likely to be necessary for the assembly of compact myelin and the stability of the sheath.L-periaxin was initially identified as a putative cytoskeleton-associated protein expressed by myelin-forming Schwann cells based upon its insolubility in non-ionic detergent. The pattern of developmental expression of L-periaxin and its shift in localisation from the adaxonal to abaxonal membranes of myelinating Schwann cells following their association with axons, implied a role in the stabilisation of the myelin sheath. In this work, an F-box containing protein termed Fbxl6, was found to associate with the C-terminal acidic region of L-periaxin, in a search for binding partners of Lperiaxin using the yeast two-hybrid method. The observed interaction was verified by in vitro pull down assays using mouse sciatic nerve homogenate and L-periaxin generated by in vitro transcription/ translation.F-box proteins have been identified as components of a multi-enzyme complex termed SCF (Skp 1 / Cullin 1/F-box), which is responsible for the recruitment of substrates for ubiquitination and subsequent destruction. Fbxl6 belongs to the leucinerich repeat (LRR)-containing subfamily of F-box proteins. The C-terminal LRR region of the protein serves as the binding site for L-periaxin. whereas the F-box motif permits association with the core SCF complex. L-periaxin was detected as a ubiquitin conjugate in sciatic nerve explant cultures. Ubiquitination of the protein acts as a signal for degradation by the 26S proteasome, as revealed by stabilisation of L-periaxin upon inhibition of the proteasome by epoxomicin.The participation of L-periaxin in a recently identified dystroglycan- dystrophinrelated protein 2 (DRP2) complex, suggests an indirect role for Fbxl6 in the structural and signalling functions of the cortical cytoskeleton. Regulation of the levels of Lperiaxin by the ubiquitin/ proteasome pathway, mediated by Fbxl6, is likely to be important for the stabilisation of the Schwann cell-axon unit

Induction of Tachykinin Production in Airway Epithelia in Response to Viral Infection

The tachykinins are implicated in neurogenic inflammation and the neuropeptide substance P in particular has been shown to be a proinflammatory mediator. A role for the tachykinins in host response to lung challenge has been previously demonstrated but has been focused predominantly on the release of the tachykinins from nerves innervating the lung. We have previously demonstrated the most dramatic phenotype described for the substance P encoding gene preprotachykinin-A (PPT-A) to date in controlling the host immune response to the murine gammaherpesvirus 68, in the lung.In this study we have utilised transgenic mice engineered to co-ordinately express the beta-galactosidase marker gene along with PPT-A to facilitate the tracking of PPT-A expression. Using a combination of these mice and conventional immunohistology we now demonstrate that PPT-A gene expression and substance P peptide are induced in cells of the respiratory tract including tracheal, bronchiolar and alveolar epithelial cells and macrophages after viral infection. This induction was observed 24h post infection, prior to observable inflammation and the expression of pro-inflammatory chemokines in this model. Induced expression of the PPT-A gene and peptide persisted in the lower respiratory tract through day 7 post infection.Non-neuronal PPT-A expression early after infection may have important clinical implications for the progression or management of lung disease or infection aside from the well characterised later involvement of the tachykinins during the inflammatory response

The SLC6A4 VNTR genotype determines transcription factor binding and epigenetic variation of this gene in response to cocaine in vitro.

We demonstrated that the genotype of the variable number tandem repeats (VNTRs) in the linked polymorphic region (LPR) of the 5' promoter and in the intron 2 (Stin2) transcriptional regulatory domains of the serotonin transporter SLC6A4 gene determined its promoter interactions with transcription factors and co-activators in response to cocaine in the JAr cell line. The LPR variants contain 14 (short, s) or 16 (long, l) copies of a 22-23 bp repeat element, whereas the Stin2 VNTR exists as three variants containing 9, 10 or 12 copies of a 16-17 bp repeat. We observed a differential effect of cocaine on the association of the promoter with the transcription factor CTCF, which bound to both LPR alleles prior to cocaine exposure but only to the l-allele following exposure. Significantly, this differential effect of cocaine was correlated with the binding of the transcriptional regulator MeCP2 specifically to the s-allele and recruiting the histone deacetylase complex (HDAC). Concurrently, cocaine increased the association of positive histone marks over the SLC6A4 gene locus. At the Stin2 domain, we lost binding of the transcription factor YB-1, while CTCF remained bound. Our biochemical data are consistent with differential reporter gene activity directed by the individual or dual domains in response to cocaine in an Epstein-Barr virus-based episome model of stable transfections. These observations suggest that exposure of JAr cells to cocaine may result in differential binding of transcription factors and activators based on a specific genotype that might alter epigenetic parameters affecting gene expression after the initial challenge

Distinct gene expression profiles directed by the isoforms of the transcription factor neuron-restrictive silencer factor in human SK-N-AS neuroblastoma cells

Neuron-restrictive silencer factor (NRSF) and its isoforms are differentially regulated in rodent models of self-sustaining status epilepticus (SSSE). NRSF isoforms regulate genes associated with SSSE, including the proconvulsant tachykinins, brain-derived neurotrophic factor and multiple ion channels. NRSF isoforms may direct distinct gene expression patterns during SSSE, and the ratio of each isoform may be a causative factor in traumatic damage to the central nervous system. Here, we analysed global gene expression changes by microarray in human SK-N-AS neuroblastoma cells following the over-expression of NRSF and a truncated isoform, HZ4. We used bioinformatics software to analyse the microarray dataset and correlated these data with epilepsy candidate gene pathways. Findings were validated by reverse transcriptase-polymerase chain reaction. We demonstrated that NRSF and HZ4 direct overlapping as well as distinct gene expression patterns, and that they differentially modulated gene expression patterns associated with epilepsy. Finally, we revealed that NRSF gene expression may be modulated by the anticonvulsant, phenytoin. We have interpreted our data to reflect altered gene expression directed by NRSF that might be relevant for SSSE

Immunohistological detection of MHV-68 antigen in respiratory tissue.

<p>Groups of BALB/c mice were infected with MHV-68 and euthanized at various time-points post-infection. Micrographs show airway and lung tissue sections stained for the presence of MHV-68 antigen ((brown; paraffin-embedded tissue sections, peroxidase anti-peroxidase method, anti-MHV-68, Papanicolaou's haematoxylin counterstain). A. BALB/c mouse, day 3 post infection. MHV-68 antigen is expressed by an individual type II alveolar epithelial cells (arrow). Bar = 20 µm. B. BALB/c mouse, day 7 post infection. MHV-68 antigen is expressed by several individual and small groups of alveolar epithelial cells (arrows). Bar = 20 µm. C. BALB/c mouse, day 7 post infection. No reaction is seen in the negative control section, where the primary antibody was replaced by tris-buffered saline. Bar = 20 µm.</p

LacZ expression in lungs from infected and uninfected 143-YAChPPT-ALacZ mice.

<p>Groups of 143-YAChPPT-ALacZ mice were infected or mock-infected with MHV-68 and euthanized at various time-points post-infection. Micrographs show whole lung blocks from mock-infected (Panel A) and infected (Panel B) mice at 1 day p.i. stained for the presence of the LacZ marker (blue). Staining for LacZ is observed within trachea and bronchi (arrows) of the infected mouse only</p

Histological detection of PPT-A expression in respiratory tissue.

<p>Groups of 143-YAChPPT-ALacZ mice were infected or mock-infected with MHV-68 and euthanized at various time-points post-infection. Micrographs show trachea and lung tissue sections stained for the presence of the LacZ marker (blue; frozen sections). A. Mock-infected 143-YAChPPT-ALacZ mouse. LacZ expression is observed in individual bronchial (arrow) and alveolar (arrowheads) epithelial cells. C: bronchial cartilage. Bar = 40 µm. B. 143-YAChPPT-ALacZ mouse, day 1 post infection. A large patch of tracheal epithelial cells exhibit LacZ staining (arrows). Bar = 20 µm. C. 143-YAChPPT-ALacZ mouse, day 1 post infection. Several alveoli exhibit LacZ staining of all epithelial cells (arrows), in others only occasional cells, often with the morphology of type II alveolar epithelial cells (arrowhead) are positive. Bar = 40 µm. D. 143-YAChPPT-ALacZ mouse, day 1 post infection. Higher magnification of Panel C, showing staining for LacZ in numerous alveolar epithelial cells. A positive syncytial cell is also seen (arrow). Bar = 10 µm.</p

Immunohistological detection of SP expression in respiratory tissue.

<p>Groups of 143-YAChPPT-ALacZ mice (Panels A-E) and BALB/c mice (Panels F-H) were infected with MHV-68 or mock-infected and euthanized at various time-points post-infection. Micrographs show airway and lung tissue sections stained for the presence of SP (brown; frozen or paraffin-embedded tissue sections, peroxidase anti-peroxidase method, anti-SP, Papanicolaou's haematoxylin counterstain). A, B. Mock-infected 143-YAChPPT-ALacZ mouse. SP expression is seen as very weak cytoplasmic staining of occasional bronchial epithelial cells (arrow) or alveolar epithelial cells (arrowheads). Bars = 20 µm. C, D. 143-YAChPPT-ALacZ mouse, day 1 post infection. Numerous alveolar epithelial cells stain positive for SP (arrows); some of these can be identified as type II pneumocytes (arrowheads). C: PFA-fixed and paraffin wax-embedded tissue section, D: Frozen section. Bars = 20 µm. E. 143-YAChPPT-ALacZ mouse, day 1 post infection. No reaction is seen in the negative control section, where the primary antibody was replaced by tris-buffered saline. Bar = 20 µm. F. Mock-infected BALB/c mouse. SP expression is seen as cytoplasmic staining of occasional alveolar epithelial cells (arrowheads). Bar = 20 µm. G. BALB/c mouse, day 2 post infection. Numerous bronchial respiratory epithelial cells exhibit moderate to intense staining for SP (arrows). Bar = 20 µm. H. BALB/c mouse, day 1 post infection. Numerous alveolar epithelial cells stain positive for SP; many of these can be identified as type II pneumocytes (arrowheads). Bar = 20 µm.</p

Combinatorial interaction between two human serotonin transporter gene variable number tandem repeats and their regulation by CTCF

Two distinct variable number tandem repeats (VNTRs) within the human serotonin transporter gene (SLC6A4) have been implicated as predisposing factors for CNS disorders. The linked polymorphic region in the 5′-promoter exists as short (s) and long (l) alleles of a 22 or 23 bp elements. The second within intron 2 (Stin2) exists as three variants containing 9, 10 or 12 copies of a 16 or 17 bp element. These VNTRs, individually or in combination, supported differential reporter gene expression in rat neonate prefrontal cortical cultures. The level of reporter gene activity from the dual VNTR constructs indicated combinatorial action between the two domains. Chromatin immunoprecipitation demonstrated that both these VNTR domains can bind the CCCTC-binding factor and this correlated with the ability of exogenously supplied CCCTC-binding factor to modulate the expression supported by these reporter gene constructs. We suggest that the potential for interaction between multiple polymorphic domains should be incorporated into genetic association studies. © 2009 International Society for Neurochemistry