Mark R. Howard

Regulation and role of REST and REST4 variants in modulation of gene expression in in vivo and in vitro in epilepsy models.

Repressor element-1 silencing transcription factor (REST) is a candidate modulator of gene expression during status epilepticus in the rodent. In such models, full-length REST and the truncated REST4 variant are induced and can potentially direct differential gene expression patterns. We have addressed the regulation of these REST variants in rodent hippocampal seizure models and correlated this with expression of the proconvulsant, substance P encoding, PPT-A gene. REST and REST4 were differentially regulated following kainic acid stimulus both in in vitro and in vivo models. REST4 was more tightly regulated than REST in both models and its transient expression correlated with that of the differential regulation of PPT-A. Consistent with this, overexpression of a truncated REST protein (HZ4, lacking the C-terminal repression domain) increased expression of the endogenous PPT-A gene. Similarly the proximal PPT-A promoter reporter gene construct was differentially regulated by the distinct REST isoforms in hippocampal cells with HZ4 being the major inducer of increased reporter expression. Furthermore, REST and REST4 proteins were differentially expressed and compartmentalized within rat hippocampal cells in vitro following noxious stimuli. This differential localization of the REST isoforms was confirmed in the CA1 region following perforant path and kainic acid induction of status epilepticus in vivo. We propose that the interplay between REST and REST4 alter the expression of proconvulsant genes, as exemplified by the PPT-A gene, and may therefore regulate the progression of epileptogenesis.

Differential Regulation of the Serotonin Transporter Gene by Lithium Is Mediated by Transcription Factors, CCCTC Binding Protein and Y-Box Binding Protein 1, through the Polymorphic Intron 2 Variable Number Tandem Repeat

The serotoninergic pathways are possible targets for the action of lithium, a therapeutic agent for treatment of bipolar affective disorders. This study aimed to investigate the molecular mechanisms regulating human serotonin transporter gene (SLC6A4) expression by lithium and, specifically, the role of the variable number tandem repeat (VNTR) polymorphic region in intron 2, which is potentially a predisposing genetic factor for bipolar affective disorders. We demonstrated that addition of lithium to human JAr cells led to changes in the levels of SLC6A4 mRNA and protein. Additional investigations revealed that the intron 2 VNTR domain was a potential target for mediation of a transcriptional response to lithium. Properties of two transcription factors, CCCTC binding protein (CTCF) and Y-box binding protein 1 (YB-1), previously shown to be involved in the regulation of SLC6A4 VNTR, were found to be modulated by LiCl. Thus, levels of CTCF and YB-1 mRNA and protein were altered in vivo in response to LiCl. Furthermore, CTCF and YB-1 showed differential binding to the polymorphic alleles of the VNTR on exposure to LiCl. Our data suggest a model in which differential binding of CTCF and YB-1 to the allelic variants of the intron 2 VNTR can be regulated by lithium and in part result in differential and even aberrant expression of SLC6A4. Our study of the regulation of the SLC6A4 VNTR by lithium may improve the understanding of psychiatric disorders and enable the development of novel therapies for conditions such as bipolar affective disorder to target only the at-risk allele.

Co-localisation, heterophilic interactions and regulated expression of IgLON family proteins in the chick nervous system.

The chick glycoprotein GP55 has been shown to inhibit the growth and adhesion of DRG and forebrain neurons. GP55 consists of several members of the IgLON family, a group of glycoproteins including LAMP, OBCAM, CEPU-1 (chick)/neurotrimin (rat) and neurotractin (chick)/kilon (rat) thought to play a role in the guidance of growing axons. IgLONs belong to the Ig superfamily and have three C2 domains and a glycosyl phosphatidylinositol anchor which tethers them to the neuronal plasma membrane. We have now completed the deduced amino acid sequence for two isoforms of chicken OBCAM and used recombinant LAMP, OBCAM and CEPU-1 to raise antisera specific to these three IgLONs. LAMP and CEPU-1 are co-expressed on DRG and sympathetic neurons, while both overlapping and distinct expression patterns for LAMP, OBCAM and CEPU-1 are observed in retina. Analysis of IgLON mRNA expression reveals that alternatively spliced forms of LAMP and CEPU-1 are developmentally regulated. In an attempt to understand how the IgLONs function, we have begun to characterise their molecular interactions. LAMP and CEPU-1 have already been shown to interact homophilically. We now confirm that OBCAM will bind homophilically and also that LAMP, OBCAM and CEPU-1 will interact heterophilically with each other. We propose that IgLON activity will depend on the complement of IgLONs expressed by each neuron.

Promotion of neuronal cell adhesion by members of the IgLON family occurs in the absence of either support or modification of neurite outgrowth

The IgLONs are a family of glycosyl phosphatidyl inositol‐linked cell adhesion molecules which are thought to modify neurite outgrowth and may play a role in cell–cell recognition. The family consists of LAMP, OBCAM, neurotrimin/CEPU‐1 and neurotractin/kilon. In this paper we report the effect of recombinant LAMP, CEPU‐1 and OBCAM, and transfected cell lines expressing these molecules, on the adhesion and outgrowth of dorsal root ganglion (DRG) and sympathetic neurones. CHO cells transfected with cDNA for CEPU‐1 adhered to a recombinant CEPU‐1‐Fc substrate. However, DRG or sympathetic neurones only adhered to CEPU‐1‐Fc when presented on protein A. Although DRG and sympathetic neurones express IgLONs on their surface, both types of neurones exhibited differential adhesion to CEPU‐1‐Fc, LAMP‐Fc and OBCAM‐Fc. Neither DRG nor sympathetic neurones extended neurites on a protein A/IgLON‐Fc substrate and overexpression of CEPU‐1‐GFP in DRG neurones also failed to stimulate neurite outgrowth on an IgLON‐Fc substrate. DRG neurones adhered to and extended neurites equally on transfected and non‐transfected cell lines and the recombinant proteins did not modulate the outgrowth of neurones on laminin. In contrast to previous reports we suggest that IgLONs may not have a primary role in axon guidance but may be more important for cell–cell adhesion and recognition.

NO‐cGMP mediated galanin expression in NGF‐deprived or axotomized sensory neurons

Leukaemia inhibitory factor (LIF) and nerve growth factor (NGF) are well characterized regulators of galanin expression. However, LIF knockout mice containing the rat galanin 5′ proximal promoter fragment (− 2546 to + 15 bp) driving luciferase responded to axotomy in the same way as control mice. Also, LIF had no effect on reporter gene expression in vitro, neither in the presence or absence of NGF, suggesting that other factors mediate an axotomy response from the galanin promoter. We then addressed the role of nitric oxide (NO) using NGF‐deprived rat dorsal root ganglion (DRG) neuron cultures infected with viral vectors containing the above‐mentioned construct, and also studied endogenous galanin expression in axotomized DRG in vivo. Blocking endogenous NO in NGF‐deprived DRG cultures suppressed galanin promoter activity. Consistent with this, axotomized/NGF‐deprived DRG neurons expressed high levels of neuronal NO synthase (nNOS) and galanin. Further, using pharmacological NOS blockers, or adenoviral vectors expressing dominant‐negative either for nNOS or soluble guanylate cyclase in vivo and in vitro, we show that the NO‐cGMP pathway induces endogenous galanin in DRG neurons. We propose that both LIF and NO, acting at different promoter regions, are important for the up‐regulation of galanin, and for DRG neuron survival and regeneration after axotomy.

Identification and Characterization of CEPU-Se—A Secreted Isoform of the IgLON Family Protein, CEPU-1

CEPU-1/Neurotrimin is a neuronal glycoprotein thought to play a role in axon guidance and cell-cell recognition. It is a member of the IgLON family, has three C2 domains, and is attached to the plasma membrane by a GPI-anchor. We report here the characterisation of an alternatively-spliced isoform of CEPU-1 that is secreted. This isoform, termed CEPU-Se, is coexpressed with CEPU-1 in retina, cerebellum, and DRG neurons. In the cerebellum CEPU-1/CEPU-Se is expressed predominantly on granule cells and in the molecular layer. Divalent but not monovalent CEPU-Se interacts with CEPU-1 and other IgLONs, suggesting that the ability of CEPU-Se to modify the activity of the IgLON family may require an additional cofactor. CEPU-Se does not support the outgrowth of DRG neurons or the extension of established growth cones; however, neurite outgrowth on laminin is unaffected by CEPU-Se. Our data suggest that CEPU-Se may act to modulate the ability of CEPU-1, LAMP, and OBCAM to influence neurite outgrowth.

Evidence of postnatal neurogenesis in dorsal root ganglion : Role of nitric oxide and neuronal restrictive silencer transcription factor

The various mechanisms underlying postnatal neurogenesis from discrete CNS regions have emerged recently. However, little is known about postnatal neurogenesis in dorsal root ganglion (DRG). BrdU incorporation and subsequent immunostaining for BrdU, neural stem cell marker, nestin and neuronal marker, PGP 9.5 have provided evidence for postnatal neurogenesis in DRG. We further demonstrate, in vivo and in vitro, that nitric oxide (NO) regulates neural stem cells (nestin+) proliferation and, possibly, differentiation into neurons. Surprisingly, nerve growth factor (NGF) had no effect on nestin+ cells proliferation. Axotomy or NGF-deprivation of DRG neurons-satellite glia co-culture increases NO production by neurons and treating with a NO synthase (NOS) inhibitor, NG-nitro-L-arginine methylester (L-NAME) in vitro or 7-nitroindazole (7NI) in vivo, causes a significant increase in nestin+ cell numbers. However, a soluble guanylyl cyclase (sGC) blocker, 1H-[1, 2, 4] oxadiazolo [4, 3-a] quinoxalin-1-one (ODQ) treatment of NGF-deprived DRG neurons-satellite glia co-culture had no significant effect on nestin+ cell numbers. This implies NO regulates nestin+ cell proliferation independent of cGMP. We hypothesised that the neuronal-restrictive silencer transcription factor (NRSF, also termed REST), a master regulator of neuronal genes in non-neuronal cells, may be modulated by NO in satellite glia cultures. A NO donor, dimethyl-triamino-benzidine (DETA)-NO treatment of satellite glia cell cultures results in a significant increase in the NRSF/REST mRNA expression. The majority of cultured satellite glia cells express nestin, and also show increased levels of NOS, thus L-NAME treatment of these cultures causes a dramatic reduction in NRSF/REST mRNA. Overall these results suggest that NO inhibits neurogenesis in DRG and this is correlated with modulation of NRSF, a known modulator of differentiation.

Mechanical stimulation induces preprotachykinin gene expression in osteoarthritic chondrocytes which is correlated with modulation of the transcription factor neuron restrictive silence factor.

We have previously demonstrated that the transcription factor termed neuron restrictive silencer factor (NRSF) and the truncated splice variant, NRSF short form (sNRSF) are major modulators of preprotachykinin A (TAC1) gene expression. In this communication we addressed whether TAC1 gene expression would be effected in response to mechanical stimulation of both normal and osteoarthritic (OA) chondrocytes. Chondrocytes were mechanically stimulated for 20 min, and then incubated under normal tissue culture conditions for 1 or 3h. RT-PCR and quantitative PCR (qPCR) were used to investigate expression of TAC1, NRSF and sNRSF mRNA at these time points. Western blotting was used to validate and confirm expression of sNRSF protein in chondrocytes in response to mechanical stimulation. We observed that TAC1 was expressed in normal chondrocytes, with no evidence of NRSF or sNRSF expression. TAC1 mRNA expression did not significantly change following mechanical stimulation in normal cells. OA chondrocytes expressed TAC1 and sNRSF mRNA, though not NRSF, and following mechanical stimulation there was a significant upregulation of both TAC1 and sNRSF mRNA, which returned to baseline levels 3h post-stimulation. sNRSF protein was upregulated at 1 and 2h following stimulation of OA chondrocytes. In summary, differential expression of TAC1 and sNRSF in OA chondrocytes associates their expression with the disease. The change in expression of sNRSF and TAC1 mRNA following mechanical stimulation in OA but not normal chondrocytes suggests that sNRSF may be involved in the regulation of SP production in OA cartilage. These differences between normal and OA mechanotransduction responses may be important in the production of phenotypic changes present in diseased cartilage.

Nitric Oxide-NGF Mediated PPTA/SP, ADNP, and VIP Expression in the Peripheral Nervous System

Nerve growth factor (NGF)-deprivation or axotomy of dorsal root ganglion (DRG) neurons causes stress, which they cope by triggering various mechanisms. Among several molecular changes, in the present study, we demonstrate preprotachykinin-A–substance P (PPTA–SP) and activity-dependent neuroprotective protein–vasoactive intestinal peptide (ADNP–VIP) expression pattern using DRG neurons–Schwann cells coculture and axotomy model. In the presence of NGF, DRG cultures showed high levels of PPTA and ADNP mRNA expression, which were significantly suppressed in the absence of NGF and/or nitric oxide synthase (NOS) inhibition by NG-nitro-l-arginine methyl ester (l-NAME), suggesting that both NGF and nitric oxide (NO) can regulate PPTA and ADNP expression. However, treating coculture with NO donor, diethylenetriamine nitric oxide (DETA–NO) did not increase PPTA and ADNP expression in the presence or absence of NGF, although there was a marginal increase in ADNP expression in the absence of NGF. NGF-deprivation increases endogenous NO; thus, DETA–NO had no further effect on PPTA and ADNP expression. Alternatively, NGF produced from NO-stimulated Schwann cells influence gene expression. In addition, interestingly, DETA–NO treatment of Schwann cells alone suppresses both PPTA and ADNP, suggesting differential response of DRG neurons–Schwann cells coculture to DETA–NO. SP and ADNP immunostaining of axotomized DRGs revealed significant reduction in SP and ADNP compared to intact DRG, which was partially recovered in neuronal NOS blocker, 7-nitroindazole (7-NI)-treated DRGs, particularly intense ADNP staining in satellite glia. As ADNP is VIP-responsive gene, we further explored VIP expression in DRGs. Axotomy increased VIP in DRG neurons, but 7-NI treatment caused intense VIP staining in satellite glia. These observations suggest a complex interaction of NO–NGF with PPTA/SP and ADNP–VIP in neuron–glial communication when neurons are stressed.

A proximal E-box modulates NGF effects on rat PPT-A promoter activity in cultured dorsal root ganglia neurones.

The rat preprotachykinin A (rtPPTA) promoter fragment spanning -865+92, relative to the major transcriptional start, has previously been demonstrated to be nerve growth factor (NGF) responsive in primary cultures of rat dorsal root ganglion (DRG) neurones [Harrison, P.T., Dalziel, R.G., Ditchfield, N.A., Quinn, J.P., 1999. Neuronal-specific and nerve growth factor-inducible expression directed by the preprotachykinin-A promoter delivered by an adeno-associated virus vector. Neuroscience 94, 997-1003]. In this communication, we demonstrate that an E box element at -60, in part, regulates the activity of this rtPPT-A promoter fragment in DRG neurones in response to NGF. Differential regulation of the promoter is observed in the presence or absence of NGF when the E Box site is present. Under basal conditions binding of proteins to this -60 element may antagonise promoter activity. Hence, in the absence of NGF, mutation of the -60 E box increased reporter gene expression. Further, comparison of levels of reporter gene expression supported by both WT and mutated promoter indicate that in the presence of NGF the -60 E box element also plays a role as an activator domain. This represents a novel mechanism for NGF regulation of rtPPT-A. Similarly, an important role for this signalling pathway was observed in neonate rat DRG neuronal cultures, which require NGF for their survival, namely mutation of the -60 element resulted in higher levels of reporter gene expression.