Hadas Schori

Vaccination for protection of retinal ganglion cells against death from glutamate cytotoxicity and ocular hypertension: Implications for glaucoma

Our group recently demonstrated that autoimmune T cells directed against central nervous system-associated myelin antigens protect neurons from secondary degeneration. We further showed that the synthetic peptide copolymer 1 (Cop-1), known to suppress experimental autoimmune encephalomyelitis, can be safely substituted for the natural myelin antigen in both passive and active immunization for neuroprotection of the injured optic nerve. Here we attempted to determine whether similar immunizations are protective from retinal ganglion cell loss resulting from a direct biochemical insult caused, for example, by glutamate (a major mediator of degeneration in acute and chronic optic nerve insults) and in a rat model of ocular hypertension. Passive immunization with T cells reactive to myelin basic protein or active immunization with myelin oligodendrocyte glycoprotein-derived peptide, although neuroprotective after optic nerve injury, was ineffective against glutamate toxicity in mice and rats. In contrast, the number of surviving retinal ganglion cells per square millimeter in glutamate-injected retinas was significantly larger in mice immunized 10 days previously with Cop-1 emulsified in complete Freunds adjuvant than in mice injected with PBS in the same adjuvant (2,133 ± 270 and 1,329 ± 121, respectively, mean ± SEM; P < 0.02). A similar pattern was observed when mice were immunized on the day of glutamate injection (1,777 ± 101 compared with 1,414 ± 36; P < 0.05), but not when they were immunized 48 h later. These findings suggest that protection from glutamate toxicity requires reinforcement of the immune system by antigens that are different from those associated with myelin. The use of Cop-1 apparently circumvents this antigen specificity barrier. In the rat ocular hypertension model, which simulates glaucoma, immunization with Cop-1 significantly reduced the retinal ganglion cell loss from 27.8% ± 6.8% to 4.3% ± 1.6%, without affecting the intraocular pressure. This study may point the way to a therapy for glaucoma, a neurodegenerative disease of the optic nerve often associated with increased intraocular pressure, as well as for acute and chronic degenerative disorders in which glutamate is a prominent participant.

T-cell-based immunity counteracts the potential toxicity of glutamate in the central nervous system

Injuries to the central nervous system (CNS) evoke self-destructive processes, which eventually lead to a much greater loss of tissue than that caused by the trauma itself. The agents of self-destruction include physiological compounds, such as glutamate, which are essential for the proper functioning of the CNS, but become cytotoxic when their normal concentrations are exceeded. The CNS is equipped with buffering mechanisms that are specific for each compound. Here we show, using Balb/c mice (a strain resistant to induction of experimental autoimmune encephalomyelitis), that after intravitreal injection of any concentration of glutamate (a neurotransmitter that becomes toxic when in excess) or ammonium-ferrous sulfate hexahydrate (which increases the formation of toxic oxygen species), the loss of retinal ganglion cells in mice devoid of mature T cells (nude mice) is significantly greater than in matched wild-type controls. We further show that this outcome can be partially reversed by supplying the T cell-defective mice with splenocytes derived from the wild-type mice. The results suggest that potentially toxic physiological compounds, when present in excessive amounts, can recruit and activate a T-cell-dependent self-protective immune mechanism. This may represent a prototype mechanism for the physiological regulation of potentially destructive CNS events by T-cell-mediated immune activity, when the local buffering mechanisms cannot adequately cope with them.

A disaccharide derived from chondroitin sulphate proteoglycan promotes central nervous system repair in rats and mice

Chondroitin sulphate proteoglycan (CSPG) inhibits axonal regeneration in the central nervous system (CNS) and its local degradation promotes repair. We postulated that the enzymatic degradation of CSPG generates reparative products. Here we show that an enzymatic degradation product of CSPG, a specific disaccharide (CSPG‐DS), promoted CNS recovery by modulating both neuronal and microglial behaviour. In neurons, acting via a mechanism that involves the PKCα and PYK2 intracellular signalling pathways, CSPG‐DS induced neurite outgrowth and protected against neuronal toxicity and axonal collapse in vitro. In microglia, via a mechanism that involves ERK1/2 and PYK2, CSPG‐DS evoked a response that allowed these cells to manifest a neuroprotective phenotype ex vivo. In vivo, systemically or locally injected CSPG‐DS protected neurons in mice subjected to glutamate or aggregated β‐amyloid intoxication. Our results suggest that treatment with CSPG‐DS might provide a way to promote post‐traumatic recovery, via multiple cellular targets.

Immune‐related mechanisms participating in resistance and susceptibility to glutamate toxicity

Glutamate is an essential neurotransmitter in the CNS. However, at abnormally high concentrations it becomes cytotoxic. Recent studies in our laboratory showed that glutamate evokes T cell‐mediated protective mechanisms. The aim of the present study was to examine the nature of the glutamate receptors and signalling pathways that participate in immune protection against glutamate toxicity. We show, using the mouse visual system, that glutamate‐induced toxicity is strain dependent, not only with respect to the amount of neuronal loss it causes, but also in the pathways it activates. In strains that are genetically endowed with the ability to manifest a T cell‐dependent neuroprotective response to glutamate insult, neuronal losses due to glutamate toxicity were relatively small, and treatment with NMDA‐receptor antagonist worsened the outcome of exposure to glutamate. In contrast, in mice devoid of T cell‐dependent endogenous protection, NMDA receptor antagonist reduced the glutamate‐induced neuronal loss. In all strains, blockage of the AMPA/KA receptor was beneficial. Pharmacological (with α2‐adrenoceptor agonist) or molecular intervention (using either mice overexpressing Bcl‐2, or DAP‐kinase knockout mice) protected retinal ganglion cells from glutamate toxicity but not from the toxicity of NMDA. The results suggest that glutamate‐induced neuronal toxicity involves multiple glutamate receptors, the types and relative contributions of which, vary among strains. We suggest that a multifactorial protection, based on an immune mechanism independent of the specific pathway through which glutamate exerts its toxicity, is likely to be a safer, more comprehensive, and hence more effective strategy for neuroprotection. It might suggest that, because of individual differences, the pharmacological use of NMDA‐antagonist for neuroprotective purposes might have an adverse effect, even if the affinity is low.

Increased post-traumatic survival of neurons in IL-6-knockout mice on a background of EAE susceptibility

Axonal injury initiates a process of neuronal degeneration, with resulting death of neuronal cell bodies. We show here that in C57BL/6J mice, previously shown to have a limited ability to manifest a post-traumatic protective immunity, the rate of neuronal survival is increased if IL-6 is deficient during the first 24 hours after optic nerve injury. Immunocytochemical staining preformed 7 days after the injury revealed an increased number of activated microglia in the IL-6-deficient mice compared to the wild-type mice. In addition, IL-6-deficient mice showed an increased resistance to glutamate toxicity. These findings suggest that the presence of IL-6 during the early post-traumatic phase, at least in mice that are susceptible to autoimmune disease development, has a negative effect on neuronal survival. This further substantiates the contention that whether immune-derived factors are beneficial or harmful for nerve recovery after injury depends on the phenotype of the immune cells and the timing and nature of their dialog with the damaged neural tissue.

The mesenchyme expresses T cell receptor mRNAs: Relevance to cell growth control

The mesenchyme plays a crucial regulatory role in organ formation and maintenance. However, comprehensive molecular characterization of these cells is lacking. We found unexpectedly that primary mesenchyme, as well as mesenchymal cell clones, express T cell receptor (TCR)αβ mRNAs, lacking the variable region. Immunological and genetic evidence support the expression of a corresponding TCRβ protein. Additionally, mRNAs encoding TCR complex components including CD3 and ζ chain are present. A relatively higher expression of the mesenchymal TCRβ mRNA by cultured mesenchymal cell clones correlates with fast growth, whereas poorly expressing cells are slow growers and are contact inhibited. The clones that express relatively higher amount of the TCR mRNA exhibit an increased capacity to form tumors in nude mice. However, the expression of this mRNA in the mesenchyme is not per se leading to tumorigenesis, as demonstrated by primary mesenchyme that does not form tumors in mice while expressing moderate amounts of the TCR transcripts. The expression of mesencymal TCRβ was confined to the G2/M phases of the cell cycle in the MBA-13 mesenchymal cell line. This cell cycle dependent expression, considered together with the correlation between growth properties and the level of TCR expression by cell clones, implies association of mesenchymal TCR with cell growth control.

Reorganization of nuclear factors during myeloid differentiation

Differentiation in several stem cell systems is associated with major morphological changes in global nuclear shape. We studied the fate of inner‐nuclear structures, splicing factor‐rich foci and Cajal (coiled) bodies in differentiating hemopoietic, testis and skin tissues. Using antibodies to the splicing factors PSF, U2AF65 and snRNPs we find that these proteins localize in foci throughout the nuclei of immature bone marrow cells. Yet, when granulocytic cells differentiate and their nuclei condense and become segmented, the staining localizes in a unique compact and thread‐like structure. The splicing factor‐rich foci concentrate in the interior of these nuclei while the nuclear periphery and areas of highly compact chromatin remain devoid of these molecules. Differentiated myeloid cells do not stain for p80 coilin, the marker for Cajal bodies. Immature myeloid cells contain Cajal bodies although these usually do not coloclaize with PSF‐rich foci. Following complete inhibition of transcription in myeloid cells, the threaded PSF pattern becomes localized in several foci in the different lobes of mature granulocytes while in human HL‐60 immature myeloid leukemia cells PSF is found in the perinucleolar compartment. Studies of other differentiating stem cell systems show that PSF staining disappears completely in differentiated, transcriptionally inactive sperm cells, is scarce as cells migrate from the inner skin layers outward and is lost as cells of the hair follicle mature. We conclude that the formation and distribution of splicing factor‐rich foci in the nucleus during differentiation of various cell lineages is dependent on the levels of chromatin condensation and the differentiation status of the cell. J. Cell. Biochem. 81:379–392, 2001.

Severe Immunodeficiency Has Opposite Effects on Neuronal Survival in Glutamate-Susceptible and -Resistant Mice: Adverse Effect of B Cells

The resistance of rats or mice to glutamate-induced toxicity depends on their ability to spontaneously manifest a T cell-dependent response to the insult. Survival of retinal ganglion cells (RGCs) exposed to glutamate in BALB/c SCID mice (a strain relatively resistant to glutamate toxicity) was significantly worse than in the wild type. In the susceptible C57BL/6J mouse strain, however, significantly more RGCs survived among SCID mutants than in the matched wild type. RGC survival in the SCID mutants of the two strains was similar. These results suggest 1) that immunodeficiency might be an advantage in strains incapable of spontaneously manifesting protective T cell-dependent immunity and 2) that B cells might be destructive in such cases. After exposure of RGCs to toxic glutamate concentrations in three variants of B cell-deficient C57BL/6J mice, namely muMT−/− (B cell knockout mice) and Ii−/− mice reconstituted with transgenically expressed low levels of Ii p31 isoforms (p31 mice) or Ii p41 isoforms (p41 mice), significantly more RGCs survived in these mice than in the wild type. The improved survival was diminished by replenishment of the B cell-deficient mice with B cells derived from the wild type. It thus seems that B cells have an adverse effect on neuronal recovery after injury, at least in a strain that is unable to spontaneously manifest a T cell-dependent protective mechanism. These findings have clear implications for the design of immune-based therapies for CNS injury.

Genetic Manipulation of CD74 in Mouse Strains of Different Backgrounds Can Result in Opposite Responses to Central Nervous System Injury

The ability to recover from CNS injuries is strain dependent. Transgenic mice that weakly express the p41 CD74 isoform (an integral membrane protein functioning as a MHC class II chaperone) on an I-Ab genetic background have normal CD4+ T cell populations and normal surface expression of MHC class II, but their B cell development is arrested while the cells are still immature. After a CNS injury, these mice recover better than their matched wild-type controls. We generated p41-transgenic mice on an I-Ad background (p41-I-Ad mice), and found that their recovery from CNS injuries was worse than that of controls. A correlative inverse effect was seen with respect to the kinetics of T cell and B cell recruitment to the injured CNS and the expression of insulin-like growth factor at the lesion site. These results, besides verifying previous findings that B cells function in the damaged CNS, demonstrate that the outcome of a particular genetic manipulation may be strain dependent.