Eve E. Kelland

The geometric and spatial constraints of the microenvironment induce oligodendrocyte differentiation.

The oligodendrocyte precursor cell (OPC) arises from the subventricular zone (SVZ) during early vertebrate development to migrate and proliferate along axon tracts before differentiating into the myelin-forming oligodendrocyte. We demonstrate that the spatial and temporal regulation of oligodendrocyte differentiation depends intimately on the axonal microenvironment and the density of precursor cells along a specified axonal area. Differentiation does not require dynamic axonal signaling, but instead is induced by packing constraints resulting from intercellular interactions. Schwann cells and even artificial beads bound to the axonal surface can mimic these constraints and promote differentiation. Together, these results describe the coordinately controlled biophysical interaction of oligodendrocyte precursors within an axonal niche leading to self-renewal and differentiation.

Characterisation of UBP296: A novel, potent and selective kainate receptor antagonist

Willardiine derivatives with an N3-benzyl substituent bearing an acidic group have been synthesized with the aim of producing selective antagonists for GLUK5-containing kainate receptors. UBP296 was found to be a potent and selective antagonist of native GLUK5-containing kainate receptors in the spinal cord, with activity residing in the S enantiomer (UBP302). In cells expressing human kainate receptor subunits, UBP296 selectively depressed glutamate-induced calcium influx in cells containing GLUK5 in homomeric or heteromeric forms. In radioligand displacement binding studies, the willardiine analogues displaced [3H]kainate binding with IC50 values >100 microM at rat GLUK6, GLUK2 or GLUK6/GLUK2. An explanation of the GLUK5 selectivity of UBP296 was obtained using homology models of the antagonist bound forms of GLUK5 and GLUK6. In rat hippocampal slices, UBP296 reversibly blocked ATPA-induced depressions of synaptic transmission at concentrations subthreshold for affecting AMPA receptor-mediated synaptic transmission directly. UBP296 also completely blocked the induction of mossy fibre LTP, in medium containing 2 mM (but not 4 mM) Ca2+. These data provide further evidence for a role for GLUK5-containing kainate receptors in mossy fibre LTP. In conclusion, UBP296 is the most potent and selective antagonist of GLUK5-containing kainate receptors so far described.

Group I metabotropic glutamate receptors limit AMPA receptor-mediated oligodendrocyte progenitor cell death.

Oligodendrocyte progenitor cells were found to be vulnerable to kainate excitotoxic insults, an effect inhibited by either the selective alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist (1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466)) or the selective group I metabotropic glutamate (mGlu) receptor agonist, (S)-3,5-dihydroxyphenylglycine. The protective effects of (S)-3,5-dihydroxyphenylglycine were reversed by the selective mGlu receptor antagonist, (S)-alpha-methyl-4-carboxyphenylglycine. These data suggest that group I mGlu receptors may limit oligodendrocyte progenitor cell degeneration during acute brain insults.

Regional mapping of low-affinity kainate receptors in mouse brain using [3H](2S,4R)-4-methylglutamate autoradiography

Recent data indicate that (2S,4R)-4-methylglutamate is a selective agonist for low affinity (GluR5 and GluR6) kainate receptor subunits. In the present study, we have employed [(3)H](2S,4R)-4-methylglutamate to examine low affinity kainate receptor distribution in mouse brain. [(3)H](2S,4R)-4-Methylglutamate labelled a single site in murine cerebrocortical membranes (K(d)=9.9+/-2.7 nM, B(max)=296.3+/-27.1 fmol mg protein(-1)). The binding of 8 nM [(3)H](2S,4R)-4-methylglutamate was displaced by several non-NMDA receptor ligands (K(i)+/-S.E.M.): domoate (1.1+/-0.2 nM)>kainate (7.1+/-1.1 nM) >> L-glutamate (187.6+/-31.9 nM) >> (S)-alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) (>50 microM). [(3)H](2S,4R)-4-Methylglutamate autoradiography revealed a widespread regional distribution of low affinity kainate receptors. Highest binding densities occurred within deep layers of the cerebral cortex, olfactory bulb, basolateral amygdala and hippocampal CA3 subregion. Moderate labelling was also evident in the nucleus accumbens, dentate gyrus, caudate putamen, hypothalamus and cerebellar granule cell layer. These data show that [(3)H](2S,4R)-4-methylglutamate is a useful radioligand for selectively labelling low affinity kainate receptors.

Characterisation of zinc uptake into rat cultured cerebrocortical oligodendrocyte progenitor cells

In the mammalian brain, extracellular Zn(2+) is reported to play a neuromodulatory role and, during acute CNS injury, increased Zn(2+) release may be neurotoxic. Although several recent studies have examined possible mechanisms of neuronal Zn(2+) accumulation, little is known about oligodendroglial Zn(2+) uptake, the focus of the present study. 65Zn(2+) uptake was time-dependent and saturable (K(m)=3.2+/-1.0 microM, V(max)=697.2+/-67.3 pmoles mg protein(-1) 15 min(-1)). Neither kainate (an AMPA/kainate receptor agonist) nor nicardipine (an L-type Ca(2+) channel inhibitor) influenced 65Zn(2+) uptake, in contrast with pyrithione (a Zn(2+) ionophore). Either increasing extracellular H(+) concentration (pH 5.5) or co-application of either 100 microM Co(2+) or 100 microM Cu(2+) reduced (65)Zn(2+) uptake. However, 100 microM Fe(2+) failed to influence 65Zn(2+) uptake and 100 microM La(3+) increased 65Zn(2+) accumulation. These data are consistent with oligodendrocyte progenitor cells possessing a high-affinity Zn(2+) uptake mechanism similar to that described for the Zrt, Irt-like protein (ZIP) transporter family.

The dual role of CXCL8 in human CNS stem cell function: Multipotent neural stem cell death and oligodendrocyte progenitor cell chemotaxis

Research into multiple sclerosis (MS) has shown that cells purportedly important to myelin repair within the CNS, namely neural stem cells (NSC) and oligodendrocyte progenitor cells (OPC), are recruited to active lesion sites during the course of the disease. However, over time these cells appear to become depleted or functionally blocked in and around lesions, accompanied by a failure of repair mechanisms. We have previously demonstrated elevated CXCL8 in patients with MS, and hypothesized that this chemokine may play a role in the pathology of this disease. Using NSC and OPC derived in vitro from human embryonic stem cells (hESC) we demonstrate here that CXCL8 has a dual role on stem cell biology in vitro. CXCL8 caused CXCR1‐mediated death of NSC, but not OPC, whilst also acting as a potent chemoattractant for both cell types. These data support a context‐dependent role for CXCL8 expression in the CNS in which it may drive recruitment of NSC and OPC to sites of inflammation, but as a side‐effect could also contribute to the failure of myelin repair in MS.

Pyruvate limits zinc-induced rat oligodendrocyte progenitor cell death.

A growing body of evidence suggests that excessive Zn2+ release plays a key role in inducing neuronal death during central nervous system injury. However, the possible cytotoxicity of extracellular Zn2+ to oligodendrocyte lineage cells remains unknown. Employing cultures of rat oligodendrocyte progenitor cells (OPC), we report here that OPC are vulnerable to increased extracellular Zn2+ levels and that pyruvate limits Zn2+‐induced OPC death. Zn2+‐induced concentration‐dependent (pEC50 = −4.1 ± 0.1) OPC death, which was insensitive to both α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (Evans Blue) and l‐type Ca2+ channel (nicardipine) inhibition. Neither kainate nor nicardipine influenced OPC 65Zn2+ accumulation, in contrast with the Zn2+ ionophore, pyrithione. Cytotoxic extracellular Zn2+ concentrations failed to increase OPC reactive oxygen species production and the antioxidant reagents, trolox, N,N′‐diphenyl‐1,4‐phenylenediamine and N‐tert‐butyl‐α‐phenylnitrone did not afford significant protection from Zn2+ insults. The apoptotic inducer staurosporine induced the appearance of known apoptotic markers [pyknotic nuclei and caspase‐3 specific (120 kDa) α‐fodrin cleavage fragment], events not reproduced with Zn2+ insults. Zn2+ insults were also insensitive to the pan‐caspase inhibitor Z‐VAD‐fmk. However, pyruvate afforded significant OPC protection from lethal Zn2+ insults. We conclude that cultured OPC are vulnerable to Zn2+ insults, via a nonoxidative stress and noncaspase‐3‐based mechanism, involving Zn2+ inhibition of OPC glycolysis.

Assessment of changes in immune measures of multiple sclerosis patients treated with laquinimod

Laquinimod is a novel orally active agent with immunomodulatory properties that was shown to be effective in suppressing disease activity in relapsing-remitting multiple sclerosis patients. Though many mechanisms of action of laquinimod have been described, little is known about the in vivo effects of laquinimod on the functionality of circulating human peripheral blood mononuclear cell populations. We assessed both phenotypical and functional measures of PBMC in a prospective longitudinal analysis comparing laquinimod and placebo treated cohorts. We determined that there were no significant changes in the relative proportion of T-cells, B-cells, monocytes & macrophages, NK-cells, dendritic cells or FoxP3(+) CD25(hi) T-regs in laquinimod treated patients. There were also no significant differences in the proliferative response to PHA or tetanus antigen, or in the inflammatory cytokine bias of these responses. These data demonstrated that there were no significant changes in immune function of PBMC in patients receiving two years of continuous laquinimod therapy who retained a full complement of the major populations of circulating PBMC and retained their capacity to respond to immunologic stimuli.

Measuring apoptosis in neural stem cells.

In trauma to, and diseases of, the central nervous system (CNS), apoptotic events are frequently observed in and around areas of damage. Human embryonic stem cells (hESCs) and their progeny have been suggested as possible therapeutic agents in the treatment of CNS diseases. The success of stem cell transplantation not only depends on the capacity of these cells to retain their functionality after transplant into the CNS but also on their ability to resist the in situ environmental cues that may lead to apoptosis. Although there are many methods used to detect apoptosis, the assessment of apoptosis in adherent cultures of primary stem cells and their progeny is more limited. We describe a series of protocols we have used to assess apoptosis in these cells.

In vitro assessment of the direct effect of laquinimod on basic functions of human neural stem cells and oligodendrocyte progenitor cells

Laquinimod is an orally active immunomodulatory small molecule that has shown clear clinical benefit in trials for relapsing-remitting multiple sclerosis and in experimental rodent models that emulate multiple sclerosis (MS). Studies in healthy mice, and in mice with experimental autoimmune encephalomyelitis, have demonstrated that laquinimod is capable of entering the central nervous system. It is therefore important to determine if laquinimod is capable of a direct influence on basic functions of neural stem cells (NSC) or oligodendrocyte progenitor cells (OPC)--cells critical for myelin repair in MS. In order to address this question, a series of experiments was conducted to determine the effect of exogenous laquinimod on viability, proliferation, migration and differentiation of human NSC and OPC in vitro. These data show, for the first time in cells of human origin, that direct, short-term interaction between laquinimod and NSC or OPC, in an isolated in vitro setting, is not detrimental to the basic cellular function of these cells.