Nigel G. F. Cooper

Increased chondroitin sulfate proteoglycan expression in denervated brainstem targets following spinal cord injury creates a barrier to axonal regeneration overcome by chondroitinase ABC and neurotrophin-3.

Increased chondroitin sulfate proteoglycan (CSPG) expression in the vicinity of a spinal cord injury (SCI) is a primary participant in axonal regeneration failure. However, the presence of similar increases of CSPG expression in denervated synaptic targets well away from the primary lesion and the subsequent impact on regenerating axons attempting to approach deafferented neurons have not been studied. Constitutively expressed CSPGs within the extracellular matrix and perineuronal nets of the adult rat dorsal column nuclei (DCN) were characterized using real-time PCR, Western blot analysis and immunohistochemistry. We show for the first time that by 2 days and through 3 weeks following SCI, the levels of NG2, neurocan and brevican associated with reactive glia throughout the DCN were dramatically increased throughout the DCN despite being well beyond areas of trauma-induced blood brain barrier breakdown. Importantly, regenerating axons from adult sensory neurons microtransplanted 2 weeks following SCI between the injury site and the DCN were able to regenerate rapidly within white matter (as shown previously by Davies et al. [Davies, S.J., Goucher, D.R., Doller, C., Silver, J., 1999. Robust regeneration of adult sensory axons in degenerating white matter of the adult rat spinal cord. J. Neurosci. 19, 5810-5822]) but were unable to enter the denervated DCN. Application of chondroitinase ABC or neurotrophin-3-expressing lentivirus in the DCN partially overcame this inhibition. When the treatments were combined, entrance by regenerating axons into the DCN was significantly augmented. These results demonstrate both an additional challenge and potential treatment strategy for successful functional pathway reconstruction after SCI.

Genomic Profiling of Messenger RNAs and MicroRNAs Reveals Potential Mechanisms of TWEAK-Induced Skeletal Muscle Wasting in Mice

Background Skeletal muscle wasting is a devastating complication of several physiological and pathophysiological conditions. Inflammatory cytokines play an important role in the loss of skeletal muscle mass in various chronic diseases. We have recently reported that proinflammatory cytokine TWEAK is a major muscle-wasting cytokine. Emerging evidence suggests that gene expression is regulated not only at transcriptional level but also at post-transcriptional level through the expression of specific non-coding microRNAs (miRs) which can affect the stability and/or translation of target mRNA. However, the role of miRs in skeletal muscle wasting is unknown. Methodology/Principal Findings To understand the mechanism of action of TWEAK in skeletal muscle, we performed mRNA and miRs expression profile of control and TWEAK-treated myotubes. TWEAK increased the expression of a number of genes involved in inflammatory response and fibrosis and reduced the expression of few cytoskeletal gene (e.g. Myh4, Ankrd2, and TCap) and metabolic enzymes (e.g. Pgam2). Low density miR array demonstrated that TWEAK inhibits the expression of several miRs including muscle-specific miR-1-1, miR-1-2, miR-133a, miR-133b and miR-206. The expression of a few miRs including miR-146a and miR-455 was found to be significantly increased in response to TWEAK treatment. Ingenuity pathway analysis showed that several genes affected by TWEAK are known/putative targets of miRs. Our cDNA microarray data are consistent with miRs profiling. The levels of specific mRNAs and miRs were also found to be similarly regulated in atrophying skeletal muscle of transgenic mice (Tg) mice expressing TWEAK. Conclusions/Significance Our results suggest that TWEAK affects the expression of several genes and microRNAs involved in inflammatory response, fibrosis, extracellular matrix remodeling, and proteolytic degradation which might be responsible for TWEAK-induced skeletal muscle loss.

The bovine rod outer segment guanylate cyclase, ROS-GC, is present in both outer segment and synaptic layers of the retina

Cyclic-GMP, which plays a pivotal role in visual transduction in the vertebrate retina, is synthesized by guanylate cyclase. The purpose of this study was to localize a rod outer segment-derived particulate guanylate cyclase (ROS-GC) to the retina of several species that have different populations of rods and cones. A rabbit antibody was raised against a synthetic peptide, corresponding to the sequence A107-L125 of bovine ROS-GC.Western blot analysis showed a single immunoreactive band at about 115 kDa with bovine rod outer segments but not with human rod outer segments. Light microscopic immunocytochemistry of tissue sections revealed immunoreactivity in the outer segment layer and in the outer and inner plexiform layers. The rod-rich rat retina showed uniform immunolabeling of outer segments; the cone-containing cat retina showed heavily labeled cone outer segments and lighter labeling of rod outer segments; the cone-rich chicken retina showed a uniformly and intensely labeled outer segment layer. Preincubation of the primary antibody with the peptide completely blocked antibody binding.Electron microscopic immunocytochemistry of the cat retina confirmed the presence of guanylate cyclase in photoreceptor outer segments and demonstrated its association with disk and plasma membranes. These data support a concept in which guanylate cyclase is much more concentrated in the outer segments of cones than rods. The immunolabeling of the plexiform layers suggests that the particulate guanylate cyclase is not unique to the photoreceptor outer segments, and may also play a role in transduction processes of retinal synapses.

Expression of two forms of glutamic acid decarboxylase (GAD67 and GAD65) during postnatal development of rat somatosensory barrel cortex.

The postnatal development of glutamic acid decarboxylase (GAD; GAD67 and GAD65) expression was studied in the rat somatosensory cortex. Delineation of barrels in layer IV by GAD67 immunoreactivity occurred between postnatal days P3 and P6 and remained evident into adulthood. At birth, a band of GAD67‐positive elements was already present in superficial layer V. This band was prominent until P6 and gradually disappeared after P9. In parallel, there was a gradual appearance of GAD67‐immunoreactive cells neuropil and puncta, which began in layer VI/subplate at P1 and achieved the adult laminar pattern by about P13. This later GAD67 immunoreactivity was responsible for the demarcation of barrels in layer IV. Development of GAD65 immunoreactivity was delayed relative to GAD67. GAD65 immunoreactivity, which was in little evidence before P6, increased markedly in density and in delineation of cell bodies over the next several weeks. During this prolonged developmental process, GAD65 first showed a negative image of the barrels compared with the septae and the surrounding cortex. Subsequently, there was a filling in of the barrels resulting in rather uniform GAD65 immunoreactivity across the barrel field and surrounding cortex. These results suggest that the development of the γ‐aminobutyric acid (GABA) synthetic system in the barrel cortex involves several processes: the disappearance of a precocious GAD67 system in layer V, the temporally overlapping maturation of the mature GAD67 system in an inside‐outside manner, and the delayed and prolonged development of the GAD65 system. J. Comp. Neurol. 402:62–74, 1998.

Expression of two forms of glutamic acid decarboxylase (GAD67 and GAD65) during postnatal development of cat visual cortex

The postnatal development of GAD67 and GAD65 protein expression and of GAD67 positive neurons and GAD65 containing axon terminals in cat visual cortex was studied. Western blot analysis showed that the expression of both GAD67 and GAD65 increased to approximately two-thirds of the adult level during the first 5 postnatal weeks and gradually increased thereafter. In adult cats, immunohistochemistry showed that GABA and GAD67 containing neurons were found in all cortical layers. Faint cell body staining was seen with the antibody to GAD65, but it densely labeled puncta. In neonates, GABA and GAD67 immunoreactivity was most intense in two distinct bands, one superficial (Layer 1/Marginal zone), another deep (Layer VI/Subplate). Unlike in adults, GAD65 positive cell bodies were clearly evident in neonates and distributed similarly to, but less frequently than, GABA and GAD67. These GAD65 positive cells frequently had morphologies suggestive of embryonic cells and largely disappeared in older animals. During postnatal development, the neurochemical differentiation of GAD67 positive neurons and GAD65 positive axon terminals across visual cortical laminae followed an inside-outside developmental pattern, which reached adult levels after 10 weeks of age. These results suggest that postnatal development of the visual cortical GABA system involves three distinct processes: (A) a dying off of embryonic GABA cells which could play a role in formation of the cortical plate; (B) a period of relative quiescence of the VC GABA system in the first 5 postnatal weeks which could maximize excitatory NMDA effects during the rising phase of the critical period; (C) the prolonged postnatal maturation of the adult GABA system which could be involved in the crystallization of adult physiological properties and the disappearance of neural plasticity.

Modeling microRNA-mRNA Interactions Using PLS Regression in Human Colon Cancer

BackgroundChanges in microRNA (miRNA) expression patterns have been extensively characterized in several cancers, including human colon cancer. However, how these miRNAs and their putative mRNA targets contribute to the etiology of cancer is poorly understood. In this work, a bioinformatics computational approach with miRNA and mRNA expression data was used to identify the putative targets of miRNAs and to construct association networks between miRNAs and mRNAs to gain some insights into the underlined molecular mechanisms of human colon cancer.MethodThe miRNA and mRNA microarray expression profiles from the same tissues including 7 human colon tumor tissues and 4 normal tissues, collected by the Broad Institute, were used to identify significant associations between miRNA and mRNA. We applied the partial least square (PLS) regression method and bootstrap based statistical tests to the joint expression profiles of differentially expressed miRNAs and mRNAs. From this analysis, we predicted putative miRNA targets and association networks between miRNAs and mRNAs. Pathway analysis was employed to identify biological processes related to these miRNAs and their associated predicted mRNA targets.ResultsMost significantly associated up-regulated mRNAs with a down-regulated miRNA identified by the proposed methodology were considered to be the miRNA targets. On average, approximately 16.5% and 11.0% of targets predicted by this approach were also predicted as targets by the common prediction algorithms TargetScan and miRanda, respectively. We demonstrated that our method detects more targets than a simple correlation based association. Integrative mRNA:miRNA predictive networks from our analysis were constructed with the aid of Cytoscape software. Pathway analysis validated the miRNAs through their predicted targets that may be involved in cancer-associated biological networks.ConclusionWe have identified an alternative bioinformatics approach for predicting miRNA targets in human colon cancer and for reverse engineering the miRNA:mRNA network using inversely related mRNA and miRNA joint expression profiles. We demonstrated the superiority of our predictive method compared to the correlation based target prediction algorithm through a simulation study. We anticipate that the unique miRNA targets predicted by the proposed method will advance the understanding of the molecular mechanism of colon cancer and will suggest novel therapeutic targets after further experimental validations.

Characterization of apoptosis-genes associated with NMDA mediated cell death in the adult rat retina

Calcium/calmodulin-dependent protein kinase II containing a nuclear localizing signal (CaMKII-alphaB) is altered in retinal neurons exposed to N-methyl-D-aspartate (NMDA). AIP (myristoylated autocamtide-2-related inhibitory peptide), a specific inhibitor of CaMKII provides neuroprotection against NMDA-mediated neurotoxicity. In this study, gene-arrays were used to investigate which apoptosis-associated genes are altered after exposure to NMDA. The data indicate an increased expression (2-7-fold) of five such genes encoding proteins that could be involved in NMDA induced cell death. The up-regulated genes are: FasL; GADD45; GADD153; Nur77 and TNF-R1. Treatment with AIP blocked their altered expression. The results suggest that multiples genes are involved in NMDA-induced excitotoxicity and that AIP, a specific inhibitor for CaMKII, regulates the expression of these apoptosis-associated genes in the retina.

Neuroprotective effect of AIP on N-methyl-d-aspartate-induced cell death in retinal neurons

Excessive activation of glutamate receptors mediates neuronal death, but the intracellular signaling pathways that mediate this type of neuronal death are only partly understood. Previously, we have demonstrated that calcium/calmodulin-dependent protein kinase II-alpha(B) (CaMKII-alpha(B)) containing a nuclear localizing signal but not CaMKII-alpha is altered in retinal neurons exposed to N-methyl-D-aspartate (NMDA). The present study describes a prospective function of CaMKII-alpha(B) in signal transduction leading to apoptosis. The terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL) method was used to detect fragmented DNA in fixed tissue sections of rat retina. The TUNEL assay confirmed that cell death occurs in the inner nuclear and ganglion cell layers following injection of 4 mM NMDA. A specific AIP (myristoylated autocamtide-2-related inhibitory peptide) with proven cell permeability inhibits CaMKII activity in vivo. Neuroprotection achieved by 500 microM AIP was complete when administered 2 h before and coincident with the NMDA application. Additionally, 100 microM of AIP protects only partially against the NMDA-induced excitotoxicity. The conformationally active fragment of caspase-3 (17 kDa), known to be involved in neuronal apoptosis was apparent within 30 min and at 2 h postinjection with NMDA. This activation was inhibited by 500 microM AIP when administered 2 h before and coincident with the NMDA application. The results suggest that CaMKII-alpha(B) isoform plays a role in excitotoxicity-induced neuronal apoptosis.

The role of CaMKII in BDNF-mediated neuroprotection of retinal ganglion cells (RGC-5)

The purpose of the study is to determine if expression or secretion of brain-derived neurotrophic factor (BDNF) in retinal ganglion cells (RGC-5) is mediated by NFkappaB or Ca2+/calmodulin-dependent protein kinase II (CaMKII). RGC-5 cells were exposed to 1 mM glutamate for various periods of time, in the presence or absence of prospective regulatory molecules. BDNF mRNA and protein expression were assessed with the aid of real-time PCR and immunoblots, respectively, and BDNF secretion was determined by ELISA. The NFkappaB inhibitor (TLCK and PTD-p65), or a specific CaMKII inhibitor (m-AIP), was used to study association of NFkappaB or CaMKII with BDNF expression/secretion in RGC-5 cells. Glutamate stimulated a transient increase in BDNF mRNA and protein in RGC-5 cells, and also stimulated an early release of BDNF into the culture media. Neutralizing the BDNF or blocking the TrkB receptor enhanced the glutamate-induced cytotoxicity. NFkappaB nuclear translocation was revealed in response to glutamate treatment. Application of TLCK or PTD-p65 inhibited the glutamate-induced BDNF expression and secretion. Inhibition of CaMKII by m-AIP did not affect expression but significantly enhanced the release of BDNF from glutamate challenged cells. Our data suggest that glutamate treatment may stimulate expression of BDNF in RGC-5 cells through NFkappaB activation. A novel mechanism for neuroprotection is proposed for the CaMKII inhibitor, AIP, which appears to protect RGC-5 cells from cytotoxicity by enhancing the release of BDNF from glutamate challenged cells.

Human adult olfactory neuroepithelial derived progenitors retain telomerase activity and lack apoptotic activity.

Olfactory epithelium (OE) contains a population of progenitors responsible for its life-long regenerative capacity. Procedures for the isolation of these progenitors have been established [F.J. Roisen, K.M. Klueber, C.L. Lu, L.M. Hatcher, A. Dozier, C.B. Shields, Adult human olfactory stem cells, Brain Res., 890 (2001) 11-12.] and over 40 patient-specific cell lines from adult postmortem OE and endoscopic biopsy from patients undergoing nasal sinus surgery have been obtained. As these cells emerged in primary cultures, they formed neurospheres (NSFCs). The purpose of the present study was to further characterize these adult human olfactory-derived progenitors. Subcultures of the NSFCs have been passaged nearly 200 times, with a mitotic cycle of 18-20 h. Telomerase activity remains in stem cells; therefore, ELISA was employed to determine the telomerase activity of different lines and passages. Since progenitors undergo low levels of apoptosis, the levels of apoptosis were also examined in these populations. The levels of telomerase and apoptotic activity in 12 NSFC lines remained relatively constant irrespective of donor age, culture duration, or sex. To further study the apoptotic characteristics of the NSFCs, nine different caspases (cysteine proteases) known to be critical in apoptosis were evaluated using gene-microarrays comparing cells from a single line at passages 14, 88, and 183. No increases were found in caspase activity in all passages studied. ELISA confirmed the absence of caspase activity over the entire range of passages. This study further suggests that NSFCs can be obtained and used from patients, irrespective of age, sex, or time in culture without altered viability expanding the potential utility of these cells for autologous transplantation and possible diagnostic testing.