Eric J. Benner

Nitrated α–Synuclein Immunity Accelerates Degeneration of Nigral Dopaminergic Neurons

Background The neuropathology of Parkinsons disease (PD) includes loss of dopaminergic neurons in the substantia nigra, nitrated α-synuclein (N-α-Syn) enriched intraneuronal inclusions or Lewy bodies and neuroinflammation. While the contribution of innate microglial inflammatory activities to disease are known, evidence for how adaptive immune mechanisms may affect the course of PD remains obscure. We reasoned that PD-associated oxidative protein modifications create novel antigenic epitopes capable of peripheral adaptive T cell responses that could affect nigrostriatal degeneration. Methods and Findings Nitrotyrosine (NT)-modified α-Syn was detected readily in cervical lymph nodes (CLN) from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxicated mice. Antigen-presenting cells within the CLN showed increased surface expression of major histocompatibility complex class II, initiating the molecular machinery necessary for efficient antigen presentation. MPTP-treated mice produced antibodies to native and nitrated α-Syn. Mice immunized with the NT-modified C-terminal tail fragment of α-Syn, but not native protein, generated robust T cell proliferative and pro-inflammatory secretory responses specific only for the modified antigen. T cells generated against the nitrated epitope do not respond to the unmodified protein. Mice deficient in T and B lymphocytes were resistant to MPTP-induced neurodegeneration. Transfer of T cells from mice immunized with N-α-Syn led to a robust neuroinflammatory response with accelerated dopaminergic cell loss. Conclusions These data show that NT modifications within α-Syn, can bypass or break immunological tolerance and activate peripheral leukocytes in draining lymphoid tissue. A novel mechanism for disease is made in that NT modifications in α-Syn induce adaptive immune responses that exacerbate PD pathobiology. These results have implications for both the pathogenesis and treatment of this disabling neurodegenerative disease.

Neuroinflammation, oxidative stress, and the pathogenesis of Parkinson's disease

Neuroinflammatory processes play a significant role in the pathogenesis of Parkinsons disease (PD). Epidemiologic, animal, human, and therapeutic studies all support the presence of an neuroinflammatory cascade in disease. This is highlighted by the neurotoxic potential of microglia . In steady state, microglia serve to protect the nervous system by acting as debris scavengers, killers of microbial pathogens, and regulators of innate and adaptive immune responses. In neurodegenerative diseases, activated microglia affect neuronal injury and death through production of glutamate, pro-inflammatory factors, reactive oxygen species, quinolinic acid amongst others and by mobilization of adaptive immune responses and cell chemotaxis leading to transendothelial migration of immunocytes across the blood-brain barrier and perpetuation of neural damage. As disease progresses, inflammatory secretions engage neighboring glial cells, including astrocytes and endothelial cells, resulting in a vicious cycle of autocrine and paracrine amplification of inflammation perpetuating tissue injury. Such pathogenic processes contribute to neurodegeneration in PD. Research from others and our own laboratories seek to harness such inflammatory processes with the singular goal of developing therapeutic interventions that positively affect the tempo and progression of human disease.

Regulatory T Cells Attenuate Th17 Cell-Mediated Nigrostriatal Dopaminergic Neurodegeneration in a Model of Parkinson’s Disease

Nitrated α-synuclein (N–α-syn) immunization elicits adaptive immune responses to novel antigenic epitopes that exacerbate neuroinflammation and nigrostriatal degeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson’s disease. We show that such neuroimmune degenerative activities, in significant measure, are Th17 cell-mediated, with CD4+CD25+ regulatory T cell (Treg) dysfunction seen among populations of N–α-syn–induced T cells. In contrast, purified vasoactive intestinal peptide induced and natural Tregs reversed N–α-syn T cell nigrostriatal degeneration. Combinations of adoptively transferred N–α-syn and vasoactive intestinal peptide immunocytes or natural Tregs administered to MPTP mice attenuated microglial inflammatory responses and led to robust nigrostriatal protection. Taken together, these results demonstrate Treg control of N–α-syn–induced neurodestructive immunity and, as such, provide a sound rationale for future Parkinson’s disease immunization strategies.

Protective astrogenesis from the SVZ niche after injury is controlled by Notch modulator Thbs4

Postnatal/adult neural stem cells (NSCs) within the rodent subventricular/subependymal zone (SVZ/SEZ) generate Doublecortin (DCX)+ neuroblasts that migrate and integrate into olfactory bulb circuitry1,2. Continuous production of neuroblasts is controlled by SVZ microenvironmental niche3,4. It is generally believed that enhancing neurogenic activities of endogenous NSCs may provide needed therapeutic options for disease states and after brain injury. However, SVZ NSCs can also differentiate into astrocytes. It remains unclear if there are conditions that favor astrogenesis over neurogenesis in the SVZ niche, and if astrocytes produced there exhibit different properties from others in the brain. We have uncovered that SVZ-generated astrocytes express high levels of Thrombospondin-4 (Thbs4)5,6, a secreted homopentameric glycoprotein, in contrast to cortical astrocytes which are Thbs4low. We found that localized photothrombotic/ischemic cortical injury initiates a marked increase in Thbs4hi astrocyte production from the postnatal SVZ niche. Tamoxifen-inducible nestin-CreERtm4 lineage-tracing demonstrated that it is these SVZ-generated Thbs4hi astrocytes, and not DCX+ neuroblasts, that home-in on the injured cortex. This robust post-injury astrogenic response required SVZ Notch activation, modulated by Thbs4 via direct Notch1 receptor binding and endocytosis to activate downstream signals, including increased Nfia transcription factor expression important for glia production7. Consequently, Thbs4KO/KO animals showed severe defects in cortical injury-induced SVZ astrogenesis, instead producing cells expressing DCX from SVZ to the injury sites. These alterations in cellular responses resulted in abnormal glial scar formation after injury, and significantly increased microvascular hemorrhage into the brain parenchyma of Thbs4KO/KO animals. Taken together, these findings have significant implications for post-injury applications of endogenous and transplanted NSCs in the therapeutic setting, as well as disease states where Thbs family members play important roles8,9.Postnatal/adult neural stem cells (NSCs) within the rodent subventricular zone (SVZ; also called subependymal zone) generate doublecortin (Dcx)+ neuroblasts that migrate and integrate into olfactory bulb circuitry. Continuous production of neuroblasts is controlled by the SVZ microenvironmental niche. It is generally thought that enhancing the neurogenic activities of endogenous NSCs may provide needed therapeutic options for disease states and after brain injury. However, SVZ NSCs can also differentiate into astrocytes. It remains unclear whether there are conditions that favour astrogenesis over neurogenesis in the SVZ niche, and whether astrocytes produced there have different properties compared with astrocytes produced elsewhere in the brain. Here we show in mice that SVZ-generated astrocytes express high levels of thrombospondin 4 (Thbs4), a secreted homopentameric glycoprotein, in contrast to cortical astrocytes, which express low levels of Thbs4. We found that localized photothrombotic/ischaemic cortical injury initiates a marked increase in Thbs4hi astrocyte production from the postnatal SVZ niche. Tamoxifen-inducible nestin-creERtm4 lineage tracing demonstrated that it is these SVZ-generated Thbs4hi astrocytes, and not Dcx+ neuroblasts, that home-in on the injured cortex. This robust post-injury astrogenic response required SVZ Notch activation modulated by Thbs4 via direct Notch1 receptor binding and endocytosis to activate downstream signals, including increased Nfia transcription factor expression important for glia production. Consequently, Thbs4 homozygous knockout mice (Thbs4KO/KO) showed severe defects in cortical-injury-induced SVZ astrogenesis, instead producing cells expressing Dcx migrating from SVZ to the injury sites. These alterations in cellular responses resulted in abnormal glial scar formation after injury, and significantly increased microvascular haemorrhage into the brain parenchyma of Thbs4KO/KO mice. Taken together, these findings have important implications for post-injury applications of endogenous and transplanted NSCs in the therapeutic setting, as well as disease states where Thbs family members have important roles.

Cadmium- and chromium-induced oxidative stress, DNA damage, and apoptotic cell death in cultured human chronic myelogenous leukemic K562 cells, promyelocytic leukemic HL-60 cells, and normal human peripheral blood mononuclear cells.

Sodium dichromate [Cr(VI)] and cadmium chloride [Cd(II)] are both cytotoxic and mutagenic. This study examined the toxic and apoptotic potentials of these two cations on three cell types in vitro, namely, human chronic myelogenous leukemic (CML) K562 cells, promyelocytic leukemic HL‐60 cells, and normal human peripheral blood mononuclear cells. The cells were incubated with 0–100 μM concentrations of the two cations for 0, 24, or 48 hours at 37°C. Both Cr(VI) and Cd(II) induced changes in intracellular oxidized states of cells, which were detected using laser scanning confocal microscopy. Cell cycle modulation and apoptosis of the K562 cells by Cr(VI) and Cd(II) were determined by flow cytometry. Significant decreases in the G2/M phase were observed in the Cr(VI) and Cd(II) treated CML cells compared with untreated cells. At 12.5 μM, Cr(VI) induced greater apoptosis in K562 cells as compared with Cd(II). In the K562 cells, 2.2‐ and 3.0‐fold increases in DNA fragmentation were observed following incubation with 12.5 and 25 μM Cr(VI), respectively, and 1.2‐ and 1.7‐fold increases in DNA fragmentation were observed with Cd(II). Furthermore, approximately 2.7‐ and 4.9‐fold increases in cytochrome c reduction were observed following incubation with 12.5 and 25 μM Cr(VI), respectively, and 1.6‐ and 3.3‐fold increases in cytochrome c reduction were observed with Cd(II), demonstrating enhanced production of superoxide anion. Approximately 3.1 to 6‐fold increases in hydroxyl radical production were observed following incubation of the K562 cells with these cations at 12.5 and 25 μM concentrations. These results in K562 cells were compared with promyelocytic leukemic HL‐60 cells and normal human peripheral blood mononuclear cells. More pronounced effects were observed on K562 and HL‐60 cells, and much lesser effects were observed on normal human peripheral blood mononuclear cells. The results demonstrate that both cations are toxic, producing oxidative tissue damage and apoptosis. Furthermore, more drastic effects were observed on K562 and HL‐60 cells as compared with normal human peripheral blood mononuclear cells.

Quantitative 1H magnetic resonance spectroscopic imaging determines therapeutic immunization efficacy in an animal model of Parkinson's disease.

Nigrostriatal degeneration, the pathological hallmark of Parkinsons disease (PD), is mirrored by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication. MPTP-treated animals show the common behavioral, motor, and pathological features of human disease. We demonstrated previously that adoptive transfer of Copaxone (Cop-1) immune cells protected the nigrostriatal dopaminergic pathway in MPTP-intoxicated mice. Herein, we evaluated this protection by quantitative proton magnetic resonance spectroscopic imaging (1H MRSI). 1H MRSI performed in MPTP-treated mice demonstrated that N-acetyl aspartate (NAA) was significantly diminished in the substantia nigra pars compacta (SNpc) and striatum, regions most affected in human disease. When the same regions were coregistered with immunohistochemical stains for tyrosine hydroxylase, numbers of neuronal bodies and termini were similarly diminished. MPTP-intoxicated animals that received Cop-1 immune cells showed NAA levels, in the SNpc and striatum, nearly equivalent to PBS-treated animals. Moreover, adoptive transfer of immune cells from ovalbumin-immunized to MPTP-treated mice failed to alter NAA levels or protect dopaminergic neurons and their projections. These results demonstrate that 1H MRSI can evaluate dopaminergic degeneration and its protection by Cop-1 immunization strategies. Most importantly, the results provide a monitoring system to assess therapeutic outcomes for PD.

Disrupted iron homeostasis causes dopaminergic neurodegeneration in mice

Significance The brain requires iron for mitochondrial respiration and synthesis of myelin, neurotransmitters, and monoamine oxidases. Iron accumulates in distinct parts of the brain in patients with neurodegenerative diseases, and some have proposed that neurons die because they contain too much iron. Neuronal iron handling is not well understood. We focused on dopaminergic neurons, affected in Parkinson’s disease, and manipulated molecules involve in iron uptake and release. We showed that loss of ferroportin, which exports cellular iron, had no apparent effect. In contrast, loss of transferrin receptor, involved in iron uptake, caused neuronal iron deficiency and neurodegeneration with features similar to Parkinson’s disease. We propose that neuronal iron deficiency may contribute to neurodegeneration in human disease. Disrupted brain iron homeostasis is a common feature of neurodegenerative disease. To begin to understand how neuronal iron handling might be involved, we focused on dopaminergic neurons and asked how inactivation of transport proteins affected iron homeostasis in vivo in mice. Loss of the cellular iron exporter, ferroportin, had no apparent consequences. However, loss of transferrin receptor 1, involved in iron uptake, caused neuronal iron deficiency, age-progressive degeneration of a subset of dopaminergic neurons, and motor deficits. There was gradual depletion of dopaminergic projections in the striatum followed by death of dopaminergic neurons in the substantia nigra. Damaged mitochondria accumulated, and gene expression signatures indicated attempted axonal regeneration, a metabolic switch to glycolysis, oxidative stress, and the unfolded protein response. We demonstrate that loss of transferrin receptor 1, but not loss of ferroportin, can cause neurodegeneration in a subset of dopaminergic neurons in mice.

Combination of antisense oligonucleotide and low-dose chemotherapy in hematological malignancies

Current conventional chemotherapy for the treatment of hematological malignancies, although quite effective, has associated toxicities to normal tissue and organs, which is still a major dose limiting factor. In addition, high dose chemotherapy followed by autologous stem cell transplantation is limited by tumor cell contamination in the stem cell harvest. The use of conventional chemotherapy alone to purge these tumor cell contaminants is known to damage normal hematopoietic progenitor cells, resulting in delayed engraftment. The combination of antisense oligodeoxynucleotides (ODN) and low doses of chemotherapy offer a potential regiment which may lower the doses of conventional therapeutics required to effectively combat disease, thus lowering cytotoxicity experienced by normal cells. Transient downregulation of genes by ODN treatment, which are involved in the transformation or perpetuation of the cancerous disease state, can remove the growth and survival advantages exploited by tumor cells. Many groups are currently investigating this combination and have produced intriguing results. This review article discusses the current research investigating the combination of antisense ODN therapy with conventional chemotherapy in the treatment of hematological malignancies. Although further improvements in this strategy are required, the results thus far support a future for this strategy in clinical management of hematological malignancy.

An interferon-[beta]-resistant and NLRP3 inflammasome-independent subtype of EAE with neuronal damage

Inflammation induced by innate immunity influences the development of T cell–mediated autoimmunity in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). We found that strong activation of innate immunity induced Nod-like receptor protein 3 (NLRP3) inflammasome–independent and interferon-β (IFNβ)-resistant EAE (termed type B EAE), whereas EAE induced by weak activation of innate immunity requires the NLRP3 inflammasome and is sensitive to IFNβ treatment. Instead, an alternative inflammatory mechanism, including membrane-bound lymphotoxin-β receptor (LTβR) and CXC chemokine receptor 2 (CXCR2), is involved in type B EAE development, and type B EAE is ameliorated by antagonizing these receptors. Relative expression of Ltbr and Cxcr2 genes was indeed enhanced in patients with IFNβ-resistant multiple sclerosis. Remission was minimal in type B EAE due to neuronal damages induced by semaphorin 6B upregulation on CD4+ T cells. Our data reveal a new inflammatory mechanism by which an IFNβ-resistant EAE subtype develops.

Oligonucleotide Enhanced Cytotoxicity of Idarubicin for Lymphoma Cells

Oligonucleotides offer the potential to manipulate gene expression in targeted cells which might be exploitable for therapeutic benefit. The effects of combining a phosphorothioate oligonucleotide OL(1) p53, which transiently down-regulates p53 levels, with an anthracycline, Idarubicin, on the growth of wild-type p53 WMN gene-expressing lymphoma cells was evaluated. Fluorescent OL(1) p53, was used to demonstrate oligonucleotide uptake and retention by the WMN cells. Uptake was maximal at 24 hours and compared to baseline (0 hours) increasing apoptotic cells were evident in WMN cells treated with OL(1) (1 μM) alone and in combination with Idarubicin (0.2 nM) for 24 to 48 hours. In cells treated with OL(1) p53 and Idarubicin, truncated p53 message of a predicted 201 base pair length based on RNAase H cleavage of the OL(1) p53-p53 mRNA heteroduplex was detected after 7 hours of incubation. The message for p53 was transiently downregulated as detected by RT-PCR analysis at 24 hours, and protein levels transiently reduced at 36 hours, as shown by a quantitative Western blot. Corresponding to these events, the growth of WMN cells ceased after 48 hours in the concurrent presence of OL(1) p53 and Idarubicin and, the lymphoma cells were dead after 72 hours. No reduction in hematopoietic colony forming cell capacity of similarly treated hematopoietic progenitor cells harvested from cytokine-mobilized blood by apheresis was observed. Therefore, synergistic cytotoxicity of Idarubicin for lymphoma cells treated with an oligonucleotide targeting p53 message was demonstrated at oligonucleotide and Idarubicin concentrations which were minimally toxic to hematopoietic progenitor cells. This approach offers new opportunities for purging of lymphoma cells from hematopoietic harvests and systemic lymphoma therapy.