Yee-Kong Ng

F3/Contactin Acts as a Functional Ligand for Notch during Oligodendrocyte Maturation

Axon-derived molecules are temporally and spatially required as positive or negative signals to coordinate oligodendrocyte differentiation. Increasing evidence suggests that, in addition to the inhibitory Jagged1/Notch1 signaling cascade, other pathways act via Notch to mediate oligodendrocyte differentiation. The GPI-linked neural cell recognition molecule F3/contactin is clustered during development at the paranodal region, a vital site for axoglial interaction. Here, we show that F3/contactin acts as a functional ligand of Notch. This trans-extracellular interaction triggers gamma-secretase-dependent nuclear translocation of the Notch intracellular domain. F3/Notch signaling promotes oligodendrocyte precursor cell differentiation and upregulates the myelin-related protein MAG in OLN-93 cells. This can be blocked by dominant negative Notch1, Notch2, and two Deltex1 mutants lacking the RING-H2 finger motif, but not by dominant-negative RBP-J or Hes1 antisense oligonucleotides. Expression of constitutively active Notch1 or Notch2 does not upregulate MAG. Thus, F3/contactin specifically initiates a Notch/Deltex1 signaling pathway that promotes oligodendrocyte maturation and myelination.

Neuronal and microglial response in the retina of streptozotocin-induced diabetic rats

This study reports Müller cell and neuronal changes and microglial reaction in streptozotocin-induced diabetic rats. Glial fibrillary acidic protein (GFAP) immunoreactivity was largely confined to astrocytes in the nerve fiber layer (NFL) and ganglion cell layer (GCL) in control rats. In diabetic rats especially those killed after 12 months, GFAP immunostaining could be traced along the entire length of Müller cell processes, extending from the inner to the outer limiting membrane. With the antibody neuronal nuclei, immunopositive cells were located in the GCL and the inner part of the inner nuclear layer (INL) in both diabetic and age-matched control rats. In diabetic rats, labelled cells were reduced in both layers being more marked in the INL. In age-matched control rats, OX42-immunoreactive microglial cells were distributed mainly in the NFL and GCL; some cells were localized in the inner plexiform layer, but rarely in the outer plexiform layer (OPL). Beginning 1 month after diabetes, the microglial cells appeared hypertrophic. Furthermore, microglial number as estimated from cell counts in different layers of the retina was significantly increased, with the occurrence of some cells in the OPL at 4 months. At 14 and 16 months, reactive microglial cells were detected in the outer nuclear layer and photoreceptor layer. Present results suggest that microglial reaction in induced diabetes was elicited by neuronal cell loss in both GCL and INL as well as by some pathologic changes affecting the photoreceptors.

Neuroprotection associated with running: is it a result of increased endogenous neurotrophic factors?

The possible neuroprotective effect of physical exercise was investigated in rats after middle cerebral artery occlusion (MCAO), a focal stroke model. It was found that physical exercise in the form of a 12-week treadmill running programme reduced the volume of infarction caused by MCAO. At the molecular level, reverse transcription polymerase chain reaction revealed that the runner had increased gene expression for nerve growth factor (NGF) over the nonrunner with or without MCAO. Expression of the NGF receptors, p75, was increased only in the absence of MCAO. In addition, runners showed a significantly higher number of cholinergic neurons, which constitutively expressed p75, in the horizontal diagonal band of Broca. The present findings suggest that neuroprotection after physical exercise may be a result of an increase in an endogenous neurotrophic factor nerve growth factor and the proliferation of its receptive cholinergic neurons.

Alterations in spatial learning and memory after forced exercise

Exercise has been shown to influence learning and memory. Most studies were performed with a voluntary running paradigm (e.g. running wheel) in mice. However, such effects of exercise on learning and memory are less well demonstrated using a forced running paradigm (e.g. treadmill). The present study was designed to examine the effects of 12 weeks of forced treadmill running on learning and memory performance in rats. We have previously shown that forced running resulted in qualitative and quantitative changes in the cholinergic neurons of the horizontal diagonal band of Broca (HDB) in the septum. This study was conducted in order to determine whether or not these changes occur simultaneously with enhanced learning and memory. The one-day version of the Morris water maze (MWM) test [Frick, K.M., Stillner, E.T., Berger-Sweeney, J., 2000. Mice are not little rats: species differences in a one-day water maze task. NeuroReport 11, 3461-3465] was used to test spatial learning and memory after the exercise period. Our data showed that runners displayed better spatial learning and memory when compared to nonrunners. This was evidently shown by a reduction in the time required for spatial acquisition (p<0.05) and superior probe trial performance (p<0.05). A shorter distance swam by the runners also suggested improved learning over the nonrunners (p<0.05). In an attempt to revalidate our earlier quantitative results, we used design-based stereology (DBS) to estimate the number of cholinergic neuronal profile population in the medial septum and diagonal band (MSDB). We confirmed that forced running increased the cholinergic neuronal profile subpopulation in the HDB (Coefficient of Error<0.2). Taken together, these results indicate that forced exercise could influence learning and memory with a concomitant increase in the number of cholinergic neurons in the HDB.

Cocaine- and amphetamine-regulated transcript peptide-immunoreactivity in adrenergic C1 neurons projecting to the intermediolateral cell column of the rat.

Cocaine- and amphetamine-regulated transcript (CART) peptide-immunoreactivity was detected in neurons of the rostral ventrolateral medulla (RVLM), but few in the caudal ventrolateral medulla (CVLM). Double-labeling the medullary sections with sheep polyclonal phenylethanolamine N-methyltransferase-antiserum (PNMT) or monoclonal tyrosine hydroxylase-antibody and rabbit polyclonal CART peptide-antiserum revealed that nearly all adrenergic cells in the C1 area were CART peptide-positive and vice versa; tyrosine hydroxylase-positive cells in the A1 area were not. In the thoracolumbar spinal cord, neurons in the intermediolateral cell column (IML) and other sympathetic autonomic nuclei were CART peptide-positive; some of these were contacted by immunoreactive fibers arising from the lateral funiculus. By immuno-electron microscopy, axon terminals containing closely packed agranular CART peptide-immunoreactive vesicles appeared to make synaptic contacts with immunoreactive dendrites and soma in the IML, albeit the incidence of such contacts was low. Microinjection of the retrograde tracer Fluorogold into the lateral horn area of the T1-T3 spinal segments labeled a population of neurons in the C1 area, many of which were also CART peptide-positive. The results indicate that CART peptide-immunoreactivity is expressed in C1 adrenergic neurons, some of which project to the thoracolumbar spinal cord. The presence of this novel peptide in C1 adrenergic neurons underscores the multiplicity of putative transmitters that may be involved in signaling between putative cardiovascular neurons in the medulla oblongata and sympathetic preganglionic neurons (SPNs) in the spinal cord.

Nogo‐A at CNS paranodes is a ligand of Caspr: possible regulation of K+ channel localization

We report Nogo‐A as an oligodendroglial component congregating and interacting with the Caspr–F3 complex at paranodes. However, its receptor Nogo‐66 receptor (NgR) does not segregate to specific axonal domains. CHO cells cotransfected with Caspr and F3, but not with F3 alone, bound specifically to substrates coated with Nogo‐66 peptide and GST–Nogo‐66. Binding persisted even after phosphatidylinositol‐ specific phospholipase C (PI‐PLC) removal of GPI‐linked F3 from the cell surface, suggesting a direct interaction between Nogo‐66 and Caspr. Both Nogo‐A and Caspr co‐immunoprecipitated with Kv1.1 and Kv1.2, and the developmental expression pattern of both paralleled compared with Kv1.1, implicating a transient interaction between Nogo‐A–Caspr and K+ channels at early stages of myelination. In pathological models that display paranodal junctional defects (EAE rats, and Shiverer and CGT−/− mice), distances between the paired labeling of K+ channels were shortened significantly and their localization shifted toward paranodes, while paranodal Nogo‐A congregation was markedly reduced. Our results demonstrate that Nogo‐A interacts in trans with axonal Caspr at CNS paranodes, an interaction that may have a role in modulating axon–glial junction architecture and possibly K+‐channel localization during development.

Factors contributing to neuronal degeneration in retinas of experimental glaucomatous rats

After our studies on ganglion cell degeneration in the glaucomatous retina, the current work further confirmed the reduction of amacrine cells in the retina after the onset of glaucoma. Present study also tried to understand the possible mechanisms underlying neuronal degeneration in the glaucomatous retina. Changes of expressions in immediate early genes (IEGs), glutamate receptors (GluRs), calcium‐binding proteins (CaBPs), 8‐hydroxy‐deoxyguanosine (8‐OH‐dG) and nitric oxide synthase (NOS), as well as apoptotic‐related factors including caspase 3, bax, and bcl‐2 were examined. IEGs such as c‐fos and c‐jun were induced in the retina of the glaucomatous rat as early as 2 hr after the onset of glaucoma and lasted up to 2 weeks. Expressions of GluRs and CaBPs (i.e., parvalbumin and calbindin D‐28k) were observed to be increased in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) at 3 days and 1 week after the onset of glaucoma. The increase occurred well before and during the phase where significant neuronal death was observed in the GCL and INL of the glaucomatous retinae. Induction of 8‐OH‐dG was present in both the GCL and INL of the glaucomatous retina at 3 days after the onset of glaucoma before significant neuronal death was observed. Furthermore, confocal microscopy study showed the complete colocalization of immunohistochemical expression of caspase 3 with glial fibrillary acidic protein (GFAP), but not with neuronal nuclei (NeuN). It indicates that astrocytes and Müller cells are involved in the pathological processes of neuronal death. The relationship between the linked factors and neuronal degeneration is also discussed.

Nitric oxide, microglial activities and neuronal cell death in the lateral geniculate nucleus of glaucomatous rats.

The present study was initiated to investigate neuronal degeneration, microglial reactivity and possible roles of NO in the lateral geniculate nucleus (LGN) of glaucomatous rats. An experimental one-eye glaucoma model was created by cauterization of the limbal-derived veins. Neuronal cell viability was studied by immunostaining with antibody against neuronal nuclei. Changes of expressions of nitric oxide synthase I (NOS I), NOS II, ED 1, OX6 and OX42 in the LGN were studied by immunohistochemistry. NADPH-d histochemistry was also employed. In the experimental glaucomatous rats, the number of NeuN labelled neurons was significantly decreased in both the ipsi- and contra-lateral sides of the ventral LGN (vLGN) but not the dorsal LGN (dLGN) at 1 month post-operation and beyond. Expressions of NOS I and NADPH-d were notably increased from 1 week post-operation in the ipsilateral vLGN. In the contralateral side of the vLGN, however, this change was only observed from 1 month post-operation. No NOS II immunoreaction was observed in LGN of both the normal control and glaucomatous rats. Increased microglial reactivity as indicated by OX-42 immunoreactivity was first observed in both sides of the LGN at 1 week post-operation, and this was most significant especially at 1 and 2 months post-operation. The present results suggest that NO and microglial cells may play some important roles in the pathologic processes of neuronal degeneration in the LGN of glaucomatous rats.

High plasma cyst(e)ine level may indicate poor clinical outcome in patients with acute stroke: possible involvement of hydrogen sulfide.

Abstract Cysteine is known to cause neuronal cell death and has been reported to be elevated in brain ischemia, but it has not been studied in clinical stroke. In this study, we correlated plasma levels of cyst(e)ine with long-term clinical outcome at 3 months in acute stroke. Patients were classified into 3 groups at 3 months as follows: good outcome (Rankin 0-1, n = 11), poor outcome (Rankin 2-5, n = 20), and dead (n = 5). Their plasma cyst(e)ine levels within 24 hours of stroke onset were 61 ± 12, 67 ± 9, and 82 ± 14 μmol/L (standard deviation), respectively. The correlation between early plasma cyst(e)ine levels and long-term clinical outcome assessed at 3 months is significant with p < 0.001. None of the other 4 amino acids studied showed any significant correlation. Cyst(e)ine was also significantly elevated in patients who had early stroke deterioration (p < 0.02). Dose-dependent administration of cysteine increased the infarct volume by approximately 30% in a rat stroke model. This effect of cysteine was abolished by aminooxyacetic acid, an inhibitor of the enzyme cystathionine β-synthase that converts cysteine to hydrogen sulfide (H2S), indicating that this novel neuromodulator may be acting as a mediator of ischemic brain damage. Raised plasma cyst(e)ine in patients with stroke may reflect increased production of H2S in the brain and thus predispose to poor outcome in clinical stroke. Inhibition of H2S formation may therefore be a novel approach in acute stroke therapy.

Cortical expression of endothelin receptor subtypes A and B following middle cerebral artery occlusion in rats.

This work aimed to define the spatial expression of endothelin A (ET(A)) and B (ET(B)) receptors in the cerebral cortex after permanent middle cerebral artery occlusion (MCAO) and to identify the phenotype of cells expressing ET(A) and ET(B) receptors. Cortical expression of ET(A) and ET(B) receptors was determined at the mRNA level by semi-quantitative reverse transcription-polymerase chain reaction and at the protein level by immunofluorescence staining, 12, 24 and 72 h after MCAO. Cells expressing endothelin receptors were phenotyped by double labelling with antibodies, anti-protein gene product (PGP9.5) and anti-ED1, towards neurons and activated microglia/macrophages, respectively. Both ET(A) and ET(B) receptor mRNA expressions increased significantly in the ipsilateral cortex in a time-dependent manner after MCAO. Robust expression of ET(A) receptors was noted in most neurons of the ischemic core and in several neurons in laminae 3 and 4 of the peri-infarct region 24 and 72 h after MCAO. ET(B) receptor immunoreactivity was observed in activated microglia/macrophages, beginning 24 h after MCAO. These results provide the first evidence that the action of endothelin during ischemia may be mediated by neuronal ET(A) receptors and activated microglia/macrophage ET(B) receptors. This differential localization of ET(A) and ET(B) receptors suggests that endothelin is involved in some complex neuron-glial interactions in addition to its vascular modulatory activity during ischemia.