Harnessing the immune system for neuroprotection: therapeutic vaccines for acute and chronic neurodegenerative disorders

Nerve injury causes degeneration of directly injured neurons and the damage spreads to neighboring neurons. Research on containing the damage has been mainly pharmacological, and has not recruited the immune system. We recently discovered that after traumatic injury to the central nervous system (spinal cord or optic nerve), the immune system apparently recognizes certain injury-associated self-compounds as potentially destructive and comes to the rescue with a protective antiself response mediated by a T-cell subpopulation that can recognize self-antigens. We further showed that individuals differ in their ability to manifest this protective autoimmunity, which is correlated with their ability to resist the development of autoimmune diseases. This finding led us to suggest that the antiself response must be tightly regulated to be expressed in a beneficial rather than a destructive way. In seeking to develop a neuroprotective therapy by boosting the beneficial autoimmune response to injury-associated self-antigens, we looked for an antigen that would not induce an autoimmune disease. Candidate vaccines were the safe synthetic copolymer Cop-1, known to cross-react with self-antigens, or altered myelin-derived peptides. Using these compounds as vaccines, we could safely boost the protective autoimmune response in animal models of acute and chronic insults of mechanical or biochemical origin. Since this vaccination is effective even when given after the insult, and because it protects against the toxicity of glutamate (the most common mediator of secondary degeneration), it can be used to treat chronic neurodegenerative disorders such as glaucoma, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis.     

Dual action of glatiramer acetate (Cop-1) in the treatment of CNS autoimmune and neurodegenerative disorders

Protective autoimmunity is the body's defense mechanism against destructive self-compounds such as those commonly associated with neurodegenerative disorders. Autoimmune disease and neurodegenerative disorders can thus be viewed as two extreme manifestations of the same process. Therefore, when designing therapy, it is important to avoid an approach that will cure the one by invoking the other. One way to stop, or at least slow down, the progression of neurodegeneration without risking development of an autoimmune disease is by boosting protective autoimmunity in a well-controlled way. Copolymer 1 (Cop-1), an approved drug for the treatment of multiple sclerosis, can be used as a treatment for autoimmune diseases and as a therapeutic vaccine for neurodegenerative diseases. We propose that the protective effect of Cop-1 vaccination is obtained through a well-controlled inflammatory reaction, and that the activity of Cop-1 in driving this reaction derives from its ability to serve as a 'universal antigen' by weakly activating a wide spectrum of self-reactive T cells.     

Self-antigen maintains the innate antibacterial function of self-specific CD8 T cells in vivo

Self-specific CD8 T cells, which are selected by high-affinity interactions with self-Ags, develop into a lineage distinct from conventional CD8 T cells. We have previously shown that these self-specific cells acquire phenotypic and functional similarities to cells of the innate immune system including the expression of functional receptors associated with NK cells. In this study, we show that these self-specific cells have the ability to produce large amounts of IFN-gamma in response to infection with Listeria monocytogenes in a bystander fashion. The rapid production of IFN-gamma is associated with a dramatic reduction in the number of viable bacteria at the peak of infection. Self-specific CD8 T cells provide only marginal innate protection in the absence of self-Ag; however, the presence of self-Ag dramatically increases their protective ability. Exposure to self-Ag is necessary for the maintenance of the memory phenotype and responsiveness to inflammatory cytokines such as IL-15. Significantly, self-specific CD8 T cells are also more efficient in the production of IFN-gamma and TNF-alpha, thus providing more cytokine-dependent protection against bacterial infection when compared with NK cells. These findings illustrate that self-reactive CD8 T cells can play an important innate function in the early defense against bacterial infection.     

Protective autoimmunity: regulation and prospects for vaccination after brain and spinal cord injuries

Neuronal degeneration after traumatic injury to the central nervous system (CNS) can be reduced by active immunization or passive transfer of T cells against CNS-associated myelin antigens. We propose that a protective autoimmunity is evoked by CNS insult when non-immunological local protective mechanisms cannot adequately buffer the injury-induced toxicity. The ability of a particular strain to develop a protective autoimmune response appears to be inversely related to its susceptibility to autoimmune disease. We also propose that vaccination with specific CNS-derived'safe' (non-pathogenic) peptides after traumatic CNS insult, and possibly at any stage of chronic neurodegenerative disease, can be used to boost the protective autoimmunity and thereby to reduce further injury-induced damage. Such therapeutic vaccination ensures that the augmented beneficial autoimmunity will be free of accompanying disease.     

The stress protein response in cultured neurons: characterization and evidence for a protective role in excitotoxicity.

We used purified cultures of cerebellar granule cells to investigate the possible protective role of stress proteins in an in vitro model of excitotoxicity. Initial experiments used one- and two-dimensional polyacrylamide gel electrophoresis to confirm the induction of typical stress protein size classes by heat shock, sodium arsenite, and the calcium ionophore A23187. Immunoblot analysis and immunocytochemistry verified the expression of the highly inducible 72 kd heat shock protein (HSP72). Granule cell cultures exposed to glutamate showed evidence of cellular injury that was prevented by the noncompetitive NMDA antagonist MK-801, yet glutamate did not induce a detectable stress protein response. Nonetheless, preinduction of heat shock proteins was associated with protection from toxic concentrations of glutamate. These results imply that the HSP72 expression observed in in vivo models of excitotoxicity may not be directly related to the effects of excitatory amino acids. However, the ability of stress protein induction to protect against injury from glutamate may offer a novel approach toward ameliorating damage from excitotoxins.     

Redirection of T cell effector function in vivo and enhanced collagen-induced arthritis mediated by an IL-2R beta/IL-4R alpha chimeric cytokine receptor transgene

Chronic inflammatory autoimmune diseases such as diabetes, experimental autoimmune encephalomyelitis, and collagen-induced arthritis (CIA) are associated with type 1 (Th1, Tc1) T cell-dependent responses against autoantigens. Immune deviation toward type 2 (Th2, Tc2) response has been proposed as a potential means of gene therapy or immunomodulation to treat autoimmune diseases based on evidence that type 2 cytokines can prevent or alleviate these conditions. In this report we assessed the effects of elevated type 2 responses on CIA using transgenic mice expressing an IL-2R beta/IL-4R alpha chimeric cytokine receptor transgene specifically in T cells. In response to IL-2 binding, this chimeric receptor transduces IL-4-specific signals and dramatically enhances type 2 responses. In contrast to published reports of Th2-mediated protection, CIA was exacerbated in IL-2R beta/IL-4R alpha chimeric receptor transgenic mice, with increased disease incidence, severity, and earlier disease onset. The aggravated disease in transgenic mice was associated with an increase in type 2 cytokines (IL-4, IL-5, IL-10) and an increase in collagen-specific IgG1 levels. However, IFN-gamma production is not affected significantly in the induction phase of the disease. There is also an extensive eosinophilic infiltration in the arthritic joints of the transgenic animal, suggesting a direct contribution of type 2 response to joint inflammation. Taken together, our findings provide novel evidence that enhancement of a polyclonal type 2 response in immunocompetent hosts may exacerbate an autoimmune disease such as CIA, rather than serving a protective role. This finding raises significant caution with regard to the potential use of therapeutic approaches based on immune deviation toward type 2 responses.     

Insights into the pathogenesis of type 1 diabetes: a hint for novel immunospecific therapies

Type 1 diabetes is an organ-specific autoimmune disease whose incidence is increasing worldwide. At present, there is no effective therapy to prevent or cure this disease. The genetic background (MHC and non-MHC genes) and environmental factors (pathogens, drugs, and diet) are critical for the initiation of the autoimmune response against the pancreatic beta-cells. Recognition of the pancreatic autoantigens by T cells in a predetermined environment of antigen-presenting cells, costimulation, and cytokines is crucial for the selective activation of diabetogenic or protective/regulatory T cells. Once the autoimmune process is triggered, epitope spreading and sustaining the autoimmune responses by continuous antigen stimulation leads to expansion of effector cells, which launch the attack on the beta-cells. Despite of some controversy, most of the studies in humans and animal models suggest that CD4 (Th1) T cells are directly involved in the autoimmune attack by secretion of pro-inflammatory cytokines and recruitment of cytotoxic CD8 T cells. Secretion of anti-inflammatory cytokines by Th2 cells is protective against the disease. Therapy with peptides derived from major target antigens, such as glutamic acid decarboxylase 65 or proinsulin, can prevent the disease in animal models by rising protective Th2 cells. Herein, we review the recent progress in the immunopathogenesis of Type 1 diabetes and insights into the development of new diagnostic tools and antigen-specific immunomodulators, such as MHC-peptide chimeras.     

Conditioning of Langerhans cells induced by a primary CD8 T cell response to self-antigen in vivo

Using a previously described model of autoimmune skin disease, we addressed the question of how CD8 T cell responsiveness to self-Ag is regulated during chronic inflammation. In this model, CD8 T cells expand and induce tissue pathology directed at an epidermal self-Ag. However, we show here that this primary CD8 T cell response prevented subsequent expansion of a second CD8 T cell population with the same specificity. This lack of T cell accumulation was not due to Ag elimination, nor was it due to competition between the two T cell populations. However, skin-specific dendritic cells that present Ag in this model--Langerhans cells--underwent significant phenotypic changes associated with a compromised ability to stimulate naive T cells. Our study suggests that conditioning of dendritic cells may play a role in maintaining unresponsiveness to self-Ag during chronic inflammation.     

Inhibition of Drp1 provides neuroprotection in vitro and in vivo

Impaired regulation of mitochondrial dynamics, which shifts the balance towards fission, is associated with neuronal death in age-related neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. A role for mitochondrial dynamics in acute brain injury, however, has not been elucidated to date. Here, we investigated the role of dynamin-related protein 1 (Drp1), one of the key regulators of mitochondrial fission, in neuronal cell death induced by glutamate toxicity or oxygen-glucose deprivation (OGD) in vitro, and after ischemic brain damage in vivo. Drp1 siRNA and small molecule inhibitors of Drp1 prevented mitochondrial fission, loss of mitochondrial membrane potential (MMP), and cell death induced by glutamate or tBid overexpression in immortalized hippocampal HT-22 neuronal cells. Further, Drp1 inhibitors protected primary neurons against glutamate excitotoxicity and OGD, and reduced the infarct volume in a mouse model of transient focal ischemia. Our data indicate that Drp1 translocation and associated mitochondrial fission are key features preceding the loss of MMP and neuronal cell death. Thus, inhibition of Drp1 is proposed as an efficient strategy of neuroprotection against glutamate toxicity and OGD in vitro and ischemic brain damage in vivo.     

Apolipoprotein E protects against oxidative stress in mixed neuronal-glial cell cultures by reducing glutamate toxicity

Apolipoprotein E (ApoE) deficiency has been shown to adversely affect outcome after transient cerebral ischemia and head trauma. Since oxidative stress contributes to these injuries, the ability of ApoE to reduce irreversible oxidative damage was studied in primary mixed neuronal-glial cell cultures. Cells (13-16 days in vitro) were exposed to 50 microM hydrogen peroxide (H2O2) for 30 min, and toxicity was determined by the release of lactate dehydrogenase (LDH) 24 h after exposure. The presence of recombinant human ApoE2 (100, 300, or 1000 nM) in the culture media partially protected against oxidative injury. This protection was not reversed by pre-treatment with receptor associated protein. The NMDA receptor antagonist, MK-801, also provided partial protection against H2O2 toxicity. The degree of protection was similar to that conferred by ApoE treatment. The protective effects of ApoE and MK-801 were not additive; no ApoE protection was observed in cultures treated with MK-801 prior to H2O2 exposure. ApoE treatment had no effect on H2O2 stimulated glutamate release, but did increase the rate of glutamate uptake via the high affinity glutamate transporter in H2O2 treated cultures. Pre-treatment with ApoE also conferred partial protection against glutamate-induced LDH release. Taken together, these findings suggest that ApoE protects mixed neuronal-glial cell cultures against irreversible oxidative injury from H2O2 by reducing secondary glutamate excitotoxicity.     

l-Deprenyl Protects Mesencephalic Dopamine Neurons from Glutamate Receptor-Mediated Toxicity In Vitro

Abstract: l -Deprenyl is a relatively selective inhibitor of monoamine oxidase (MAO)-B that delays the emergence of disability and the progression of signs and symptoms of Parkinson's disease. Experimentally, deprenyl has also been shown to prevent neuronal cell death in various models through a mechanism that is independent of MAO-B inhibition. We examined the effect of deprenyl on cultured mesencephalic dopamine neurons subjected to daily changes of feeding medium, an experimental paradigm that causes neuronal death associated with activation of the NMDA subtype of glutamate receptors. Both deprenyl (0.5–50 µ M ) and the NMDA receptor blocker MK-801 (10 µ M ) protected dopamine neurons from damage caused by medium changes. The nonselective MAO inhibitor pargyline (0.5–50 µ M ) was not protective, indicating that protection by deprenyl was not due to MAO inhibition. Deprenyl (50 µ M ) also protected dopamine neurons from delayed neurotoxicity caused by exposure to NMDA. Because deprenyl had no inhibitory effect on NMDA receptor binding, it is likely that deprenyl protects from events occurring downstream from activation of glutamate receptors. As excitotoxic injury has been implicated in neurodegeneration, it is possible that deprenyl exerts its beneficial effects in Parkinson's disease by suppressing excitotoxic damage.     

Estradiol protects against ischemic brain injury in middle-aged rats

Several clinical studies suggest that estradiol acts as a potent growth and protective factor in the adult brain. Postmenopausal women experience permanent hypoestrogenicity and suffer from increased risk of brain injury associated with neurodegenerative diseases such as stroke and Alzheimer's disease. Estrogen replacement therapy appears to decrease the risk and severity of these neurodegenerative conditions. Studies using animal models have shown that estradiol exerts similar effects in rodents and can enhance cell survival and induce synaptic plasticity. Therefore, we undertook studies to assess whether estradiol treatment can decrease brain injury and cell death induced by an experimental model of ischemia and whether aging animals remain responsive to the protective effects of estradiol. We will review results from recent studies that demonstrate that 1) in young animals, estrogens exert profound protective effects against ischemic brain injury induced by cerebral artery occlusion and 2) the response of aging animals has been tested with varying results. We will discuss and compare our experimental findings that utilize a permanent cerebral artery occlusion model and physiological levels of estradiol replacement therapy in young and middle-aged rats with those of previous studies. These observations provide important insights into the potential protective actions of estrogen replacement therapy on age- and disease-related processes in the brain.     

Propofol hemisuccinate protects neuronal cells from oxidative injury

Oxidative stress contributes to the neuronal death observed in neurodegenerative disorders and neurotrauma. Some antioxidants for CNS injuries, however, have yet to show mitigating effects in clinical trials, possibly due to the impermeability of antioxidants across the blood-brain barrier (BBB). Propofol (2,6-diisopropylphenol), the active ingredient of a commonly used anesthetic, acts as an antioxidant, but it is insoluble in water. Therefore, we synthesized its water-soluble prodrug, propofol hemisuccinate sodium salt (PHS), and tested for its protective efficacy in neuronal death caused by non-receptor-mediated, oxidative glutamate toxicity. Glutamate induces apoptotic death in rat cortical neurons and the mouse hippocampal cell line HT-22 by blocking cystine uptake and causing the depletion of intracellular glutathione, resulting in the accumulation of reactive oxygen species (ROS). PHS has minimal toxicity and protects both cortical neurons and HT-22 cells from glutamate. The mechanism of protection is attributable to the antioxidative property of PHS because PHS decreases the ROS accumulation caused by glutamate. Furthermore, PHS protects HT-22 cells from oxidative injury induced by homocysteic acid, buthionine sulfoximine, and hydrogen peroxide. For comparison, we also tested alpha-tocopherol succinate (TS) and methylprednisolone succinate (MPS) in the glutamate assay. Although TS is protective against glutamate at lower concentrations than PHS, TS is toxic to HT-22 cells. In contrast, MPS is nontoxic but also nonprotective against glutamate. Taken together, PHS, a water-soluble prodrug of propofol, is a candidate drug to treat CNS injuries owing to its antioxidative properties, low toxicity, and permeability across the BBB.     

Glutamate induces oxidative stress and apoptosis in cerebral vascular endothelial cells: contributions of HO-1 and HO-2 to cytoprotection

In cerebral circulation, epileptic seizures associated with excessive release of the excitatory neurotransmitter glutamate cause endothelial injury. Heme oxygenase (HO), which metabolizes heme to a vasodilator, carbon monoxide (CO), and antioxidants, biliverdin/bilirubin, is highly expressed in cerebral microvessels as a constitutive isoform, HO-2, whereas the inducible form, HO-1, is not detectable. Using cerebral vascular endothelial cells from newborn pigs and HO-2-knockout mice, we addressed the hypotheses that 1) glutamate induces oxidative stress-related endothelial death by apoptosis, and 2) HO-1 and HO-2 are protective against glutamate cytotoxicity. In cerebral endothelial cells, glutamate (0.1–2.0 mM) increased formation of reactive oxygen species, including superoxide radicals, and induced major keystone events of apoptosis, such as NF-B nuclear translocation, caspase-3 activation, DNA fragmentation, and cell detachment. Glutamate-induced apoptosis was greatly exacerbated in HO-2 gene-deleted murine cerebrovascular endothelial cells and in porcine cells with pharmacologically inhibited HO-2 activity. Glutamate toxicity was prevented by superoxide dismutase, suggesting apoptotic changes are oxidative stress related. When HO-1 was pharmacologically upregulated by cobalt protoporphyrin, apoptotic effects of glutamate in cerebral endothelial cells were completely prevented. Glutamate-induced reactive oxygen species production and apoptosis were blocked by a CO-releasing compound, CORM-A1 (50 µM), and by bilirubin (1 µM), consistent with the antioxidant and cytoprotective roles of the end products of HO activity. We conclude that both HO-1 and HO-2 have anti-apoptotic effects against oxidative stress-related glutamate toxicity in cerebral vascular endothelium. Although HO-1, when induced, provides powerful protection, HO-2 is an essential endogenous anti-apoptotic factor against glutamate toxicity in the cerebral vascular endothelium. endothelium; carbon monoxide; bilirubin; injury; reactive oxygen species; heme oxygenase     

Estriol generates tolerogenic dendritic cells in vivo that protect against autoimmunity

Chronic inflammation contributes to numerous diseases, and regulation of inflammation is crucial for disease control and resolution. Sex hormones have potent immunoregulatory abilities. Specifically, estrogen influences immune cells and inflammation, which contributes to the sexual dimorphism of autoimmunity and protection against disease seen during pregnancy in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Although long thought to act primarily on T cells, recent evidence demonstrated that myeloid cells, such as dendritic cells (DCs), are essential in mediating estrogen's protective effects. Estriol (E3), a pregnancy-specific estrogen, has therapeutic efficacy in MS and EAE, and we evaluated whether E3 could act exclusively through DCs to protect against the inflammatory autoimmune disease EAE. Levels of activation markers (CD80 and CD86) and inhibitory costimulatory markers (PD-L1, PD-L2, B7-H3, and B7-H4) were increased in E3 DCs. E3 DCs had decreased proinflammatory IL-12, IL-23, and IL-6 mRNA expression, increased immunoregulatory IL-10 and TGF-β mRNA expression, and a decreased ratio of IL-12/IL-10 protein production. Importantly, transfer of E3 DCs to mice prior to active induction of EAE protected them from developing EAE through immune deviation to a Th2 response. This protection was apparent, even in the face of in vitro and in vivo inflammatory challenge. In summary, our results showed that E3 generates tolerogenic DCs, which protect against the inflammatory autoimmune disease EAE. Targeted generation of tolerogenic DCs with immunomodulatory therapeutics, such as E3, has potential applications in the treatment of numerous autoimmune and chronic inflammatory diseases.     

Iduna protects the brain from glutamate excitotoxicity and stroke by interfering with poly(ADP-ribose) polymer-induced cell death

Glutamate acting on N-methyl-D-aspartate (NMDA) receptors induces neuronal injury following stroke, through activation of poly(ADP-ribose) polymerase-1 (PARP-1) and generation of the death molecule poly(ADP-ribose) (PAR) polymer. Here we identify Iduna, a previously undescribed NMDA receptor-induced survival protein that is neuroprotective against glutamate NMDA receptor-mediated excitotoxicity both in vitro and in vivo and against stroke through interfering with PAR polymer-induced cell death (parthanatos). Iduna's protective effects are independent and downstream of PARP-1 activity. Iduna is a PAR polymer-binding protein, and mutation at the PAR polymer binding site abolishes the PAR binding activity of Iduna and attenuates its protective actions. Iduna is protective in vivo against NMDA-induced excitotoxicity and middle cerebral artery occlusion-induced stroke in mice. To our knowledge, these results define Iduna as the first known endogenous inhibitor of parthanatos. Interfering with PAR polymer signaling could be a new therapeutic strategy for the treatment of neurologic disorders.