Minocycline causes widespread cell death and increases microglial labeling in the neonatal mouse brain

Minocycline, an antibiotic of the tetracycline family, inhibits microglia in many paradigms and is among the most commonly used tools for examining the role of microglia in physiological processes. Microglia may play an active role in triggering developmental neuronal cell death, although findings have been contradictory. To determine whether microglia influence developmental cell death, we treated perinatal mice with minocycline (45 mg/kg) and quantified effects on dying cells and microglial labeling using immunohistochemistry for activated caspase‐3 (AC3) and ionized calcium‐binding adapter molecule 1 (Iba1), respectively. Contrary to our expectations, minocycline treatment from embryonic day 18 to postnatal day (P)1 caused a > tenfold increase in cell death 8 h after the last injection in all brain regions examined, including the primary sensory cortex, septum, hippocampus and hypothalamus. Iba1 labeling was also increased in most regions. Similar effects, although of smaller magnitude, were seen when treatment was delayed to P3–P5. Minocycline treatment from P3 to P5 also decreased overall cell number in the septum at weaning, suggesting lasting effects of the neonatal exposure. When administered at lower doses (4.5 or 22.5 mg/kg), or at the same dose 1 week later (P10‐P12), minocycline no longer increased microglial markers or cell death. Taken together, the most commonly used microglial “inhibitor” increases cell death and Iba1 labeling in the neonatal mouse brain. Minocycline is used clinically in infant and pediatric populations; caution is warrented when using minocycline in developing animals, or extrapolating the effects of this drug across ages. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 753–766, 2017     

Upregulated expression of Iba1 molecules in the central nervous system of mice in response to neurovirulent influenza A virus infection

The present study deals with the expression of Iba1 molecules, a novel EF-hand Ca2+-binding protein, in the brain after stereotaxic introduction of the neurovirulent WSN strain of influenza A virus into the olfactory bulb of C57BL/6 mice. The virus selectively targeted the paraventricular and anterior olfactory nuclei. Infected neurons appeared as early as at day 3 post infection and degenerated and vanished by day 12. The Iba1 molecule was normally expressed in resting microglia. The overexpression of the Iba1 in microglial cells was detected at day 3 post infection, culminating at day 7 with a morphological activation. Iba1-immunopositive macrophages outnumbered microglia in the paraventricular and anterior olfactory nuclei, where the infected neurons had degenerated. Macrophages totally disappeared by day 12, and the Iba1-expression in microglia was reduced to a normal level by day 35. Lack of perforin predisposed the mice to long-term virus infection of the brain, leading to the prolonged Iba1-overexpression. These results show that the Iba1 is upregulated in the mouse brain in response to influenza virus infection and may play significant roles in the regulation of some immunological and pathophysiological functions of microglia during virus infection.     

Alterations of Glial Cells in the Mouse Hippocampus During Postnatal Development

We investigated the postnatal alterations of neurons, astrocyte, oligodendrocyte, and microglia in the mouse hippocampal CA1 sector and dentate gyrus under the same conditions using immunohistochemistry. Neuronal nuclei (NeuN), Glial fibrillary acidic protein (GFAP), 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), and ionized calcium binding adaptor molecule 1 (Iba 1) immunoreactivity were measured in 1-, 2-, 4-, and 8-week-old mice. Total number of NeuN-positive neurons was unchanged in the mouse hippocampal CA1 sector and dentate gyrus from 1 to 8 weeks of birth. In contrast, a significant increase in the number of GFAP-positive astrocytes was observed only in the hippocampal CA1 sector of 1-week-old mice when compared with 8-week-old animals. Thereafter, total number of GFAP-positive astrocytes was unchanged in the hippocampal CA1 sector and dentate gyrus from 2 to 8 weeks of birth. For microglia, a significant increase in the number of Iba 1-positive microglia was observed in the hippocampal CA1 sector and dentate gyrus of 1-, 2-, and 4-week-old mice as compared with 8-week-old animals. On the other hand, a significant decrease in the area of expression of CNPase-positive fibers was observed in the hippocampal CA1 sector of 1- and 2-week-old mice as compared with 8-week-old animals. In dentate gyrus, a significant decrease in the area of expression of CNPase-positive fibers was found in 1-, 2-, and 4-week-old mice. Furthermore, our double-labeled immunostaining showed that brain-derived neurotrophic factor (BDNF) immunoreactivity was observed in GFAP-positive astrocytes and Iba 1-positive microglia in the hippocampal CA1 sector and dentate gyrus of 1- and 2-week-old mice. These results show that glial cells may play some role in the maintenance and neuronal functions of hippocampal CA1 pyramidal neurons and granule cells of dentate gyrus during postnatal development. Furthermore, our results demonstrate that glial BDNF may play an important role in the maturation of oligodendrocyte in the hippocampal CA1 sector and dentate gyrus during postnatal development. Thus, our findings provide valuable information on the developmental processes.     

Iba1 is an actin-cross-linking protein in macrophages/microglia

Iba1 is a 17-kDa EF hand protein that is specifically expressed in macrophages/microglia and is upregulated during the activation of these cells. When exposed to macrophage colony-stimulating factor (M-CSF), microglia cell line MG5 immediately produces intense membrane ruffles in which Iba1 accumulates together with filamentous actin. In this study, we investigated the physical interaction between Iba1 and actin by centrifugation assay and electron microscopic examination and showed that Iba1 possesses actin-binding and -cross-linking activities. Inhibitory mutant Iba1 that suppresses M-CSF-induced membrane ruffling had lost the actin-cross-linking activity, and it inhibited the cross-linking activity of intact Iba1. These results indicate that Iba1 is a macrophage/microglia-specific actin-cross-linking protein essential for M-CSF-induced membrane ruffling.     

Protein 4.1 G localizes in rodent microglia

Although it was reported that protein 4.1 G, a cytoskeletal protein characterized by its general expression in the body, interacts with some signal transduction molecules in the central nervous system (CNS), its distribution and significance in vivo remained to be elucidated. In the present study, we have identified 4.1 G-positive cells in the rodent CNS, and demonstrated its immunolocalization in the developing mouse CNS. In the rodent CNS, 4.1 G was colocalized with markers for microglia, such as CD45, OX-42 and ionized calcium-binding adapter molecule 1 (Iba1), but not with markers for neuronal or other glial cells. Additionally, colocalization of 4.1 G and A1 adenosine receptor was observed in the mouse cerebrum. In a mixed glial culture, most OX-42-positive microglia were positive for 4.1 G, and 4.1 G isoforms of the same molecular weight as in the rat brain were expressed in cultured microglia, where 4.1 G mRNA was detected by RT-PCR. In the developing mouse cerebral cortex, 4.1 G was detected in immature microglia, which were positive for Iba1. These results indicate that 4.1 G in the CNS is mainly distributed in microglia in vivo. Considering the interactions between 4.1 G and the signal transduction molecules, putative roles have been propsed for 4.1 G in microglial functions in the CNS.     

Microglia/macrophage-specific protein Iba1 binds to fimbrin and enhances its actin-bundling activity

Ionized calcium binding adaptor molecule 1 (Iba1) is a microglia/macrophage-specific calcium-binding protein. Iba1 has the actin-bundling activity and participates in membrane ruffling and phagocytosis in activated microglia. In order to understand the Iba1-related intracellular signalling pathway in greater detail, we employed a yeast two-hybrid screen to isolate an Iba1-interacting molecule and identified another actin-bundling protein, L-fimbrin. In response to stimulation, L-fimbrin accumulated and co-localized with Iba1 in membrane ruffles induced by M-CSF-stimulation and phagocytic cups formed by IgG-opsonized beads in microglial cell line MG5. L-fimbrin was shown to associate with Iba1 in cell lysate of COS-7 expressing L-fimbrin and Iba1. By using purified proteins, direct binding of Iba1 to L-fimbrin was demonstrated by immunoprecipitation, glutathione S-transferase pull-down assays and ligand overlay assays. The binding of Iba1 was also found to increase the actin-bundling activity of L-fimbrin. These results indicate that Iba1 forms complexes with L-fimbrin in membrane ruffles and phagocytic cups, and suggest that Iba1 co-operates with L-fimbrin in modulating actin reorganization to facilitate cell migration and phagocytosis by microglia.     

Direct evidence for spinal cord microglia in the development of a neuropathic pain-like state in mice

The present study was undertaken to further investigate the role of glial cells in the development of the neuropathic pain-like state induced by sciatic nerve ligation in mice. At 7 days after sciatic nerve ligation, the immunoreactivities (IRs) of the specific astrocyte marker glial fibrillary acidic protein (GFAP) and the specific microglial marker OX-42, but not the specific oligodendrocyte marker O4, were increased on the ipsilateral side of the spinal cord dorsal horn in nerve-ligated mice compared with that on the contralateral side. Furthermore, a single intrathecal injection of activated spinal cord microglia, but not astrocytes, caused thermal hyperalgesia in naive mice. Furthermore, 5-bromo-2'-deoxyuridine (BrdU)-positive cells on the ipsilateral dorsal horn of the spinal cord were significantly increased at 7 days after nerve ligation and were highly co-localized with another microglia marker, ionized calcium-binding adaptor molecule 1 (Iba1), but neither with GFAP nor a specific neural nuclei marker, NeuN, in the spinal dorsal horn of nerve-ligated mice. The present data strongly support the idea that spinal cord astrocytes and microglia are activated under the neuropathic pain-like state, and that the proliferated and activated microglia directly contribute to the development of a neuropathic pain-like state in mice.     

Remarkable increases of α1-antichymotrypsin in brain tissues of rodents during prion infection

α1-Antichymotrypsin (α1-ACT) belongs to a kind of acute-phase inflammatory protein. Recently, such protein has been proved exist in the amyloid deposits which is the hallmark of Alzheimer's disease, but limitedly reported in prion disease. To estimate the change of α1-ACT during prion infection, the levels of α1-ACT in the brain tissues of scrapie agents 263K-, 139A- and ME7-infected rodents were analyzed, respectively. Results shown that α1-ACT levels were significantly increased in the brain tissues of the three kinds of scrapie-infected rodents, displaying a time-dependent manner during prion infection. Immunohistochemistry assays revealed the increased α1-ACT mainly accumulated in some cerebral regions of rodents infected with prion, such as cortex, thalamus and cerebellum. Immunofluorescent assays illustrated ubiquitously localization of α1-ACT with GFAP positive astrocytes, Iba1-positive microglia and NeuN-positive neurons. Moreover, double-stained immunofluorescent assays and immunohistochemistry assays using series of brain slices demonstrated close morphological colocalization of α1-ACT signals with that of PrP and PrP^Sc in the brain slices of 263K-infected hamster. However, co-immunoprecipitation does not identify any detectable molecular interaction between the endogenous α1-ACT and PrP either in the brain homogenates of 263K-infected hamsters or in the lysates of prion-infected cultured cells. Our data here imply that brain α1-ACT is increased abnormally in various scrapie-infected rodent models. Direct molecular interaction between α1-ACT and PrP seems not to be essential for the morphological colocalization of those two proteins in the brain tissues of prion infection.     

Pax6 is misexpressed in Sox1 null lens fiber cells

Sox1 null lens fiber cells fail to elongate and have disrupted expression of gamma-crystallin. We have evaluated the expression of Sox1 and Pax6 proteins during critical stages of lens morphogenesis, with particular focus on fiber cell differentiation. While Pax6 and Sox1 are co-expressed during early stages of fiber cell differentiation, Sox1 up-regulation coincides temporally with the down-regulation of Pax6, and these proteins therefore display a striking inverse expression pattern in the lens fiber cell compartment. Furthermore, Pax6 is inappropriately expressed in the fiber cells of Sox1 null mice and the Pax6 target, alpha5 integrin, is simultaneously misexpressed. Finally, we demonstrate a genetic interaction between Sox1 and Pax6, as Sox1 heterozygosity partially rescues the diameter of Pax6(Sey) lenses by increasing the number of cells in the fiber cell compartment.     

Peripheral inflammatory mechanisms modulate microglial activation in response to mild impairment of oxidative metabolism

Thiamine deficiency (TD) models the selective neurodegeneration that accompanies the mild impairment of oxidative metabolism, which is observed in a variety of neurodegenerative diseases. Several markers of inflammation accompany neuronal death in TD and in these diseases. Studies in the submedial thalamic nucleus (SmTN), the region most sensitive to TD, have begun to define the temporal response of inflammation, immune response and neurodegeneration. Our previous studies show that the immune response is involved in TD-induced neurodegeneration. The current experiments tested the roles of other inflammatory cascades in TD-induced neuronal death. Deletion of genes for CD4, or CD8 (the co-receptors for T-cells), IFN-gamma (the cytokine produced by T-cell), or NADPH oxidase (the inflammation related oxidase) were tested. None protected against neuronal death in late stages of TD. On the other hand, deletion of the genes for CD4, CD8 and IFN-gamma increased the microglial activation, and deletion of the gene for NADPH oxidase decreased microglial activation when compared to control mice. In wild type mice, TD caused hypertrophy of CD68 positive microglia without increasing the number of microglia. However, TD induced hypertrophy and proliferation of CD68-positive microglia in the CD4 (97%), CD8 (57%) or IFN-gamma (96%) genetic knockout mice. In the genetic knockout mice for NADPH oxidase, the microglial activation was 65% less than the wild type mice. The results demonstrate that mice deficient in specific T cells (CD4-/-, CD8-/-) or activated T cell product, (IFN-gamma-/-) have increased microglia activation, but mice deficient in NADPH oxidase have decreased microglial activation. However, at the time point tested, the deletions were not neuroprotective. The results suggest that inflammatory responses play a role in TD-induced pathological changes in the brain, and the inflammation appears to be a late event that reflects a response to neuronal damage, which may spread the damage to other brain regions.     

Characteristics of the alternative phenotype of microglia/macrophages and its modulation in experimental gliomas

Microglia (brain resident macrophages) accumulate in malignant gliomas and instead of initiating the anti-tumor response, they switch to a pro-invasive phenotype, support tumor growth, invasion, angiogenesis and immunosuppression by release of cytokines/chemokines and extracellular matrix proteases. Using immunofluorescence and flow cytometry, we demonstrate an early accumulation of activated microglia followed by accumulation of macrophages in experimental murine EGFP-GL261 gliomas. Those cells acquire the alternative phenotype, as evidenced by evaluation of the production of ten pro/anti-inflammatory cytokines and expression profiling of 28 genes in magnetically-sorted CD11b(+) cells from tumor tissues. Furthermore, we show that infiltration of implanted gliomas by amoeboid, Iba1-positive cells can be reduced by a systematically injected cyclosporine A (CsA) two or eight days after cell inoculation. The up-regulated levels of IL-10 and GM-CSF, increased expression of genes characteristic for the alternative and pro-invasive phenotype (arg-1, mt1-mmp, cxcl14) in glioma-derived CD11b(+) cells as well as enhanced angiogenesis and tumor growth were reduced in CsA-treated mice. Our findings define for the first time kinetics and biochemical characteristics of glioma-infiltrating microglia/macrophages. Inhibition of the alternative activation of tumor-infiltrating macrophages significantly reduced tumor growth. Thus, blockade of microglia/macrophage infiltration and their pro-invasive functions could be a novel therapeutic strategy in malignant gliomas.     

Microglia of medicinal leech (Hirudo medicinalis) express a specific activation marker homologous to vertebrate ionized calcium‐binding adapter molecule 1 (Iba1/alias aif‐1)

The Ionized calcium‐Binding Adapter molecule 1 (Iba1), also known as Allograft Inflammatory Factor 1 (AIF‐1), is a 17 kDa cytokine‐inducible protein, produced by activated macrophages during chronic transplant rejection and inflammatory reactions in Vertebrates. In mammalian central nervous system (CNS), Iba1 is a sensitive marker associated with activated macrophages/microglia and is upregulated following neuronal death or brain lesions. The medicinal leech Hirudo medicinalis is able to regenerate its CNS after injury, leading to a complete functional repair. Similar to Vertebrates, leech neuroinflammatory processes are linked to microglia activation and recruitment at the lesion site. We identified a gene, named Hmiba1, coding a 17.8 kDa protein showing high similarity with Vertebrate AIF‐1. The present work constitutes the first report on an Iba1 protein in the nervous system of an invertebrate. Immunochemistry and gene expression analyses showed that HmIba1, like its mammalian counterpart, is modulated in leech CNS by mechanical injury or chemical stimuli (ATP). We presently demonstrate that most of leech microglial cells migrating and accumulating at the lesion site specifically expressed the activation marker HmIba1. While the functional role of Iba1, whatever species, is still unclear in reactive microglia, this molecule appeared as a good selective marker of activated cells in leech and presents an interesting tool to investigate the functions of these cells during nerve repair events. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 987–1001, 2014     

Involvement of TRPM2 in Peripheral Nerve Injury-Induced Infiltration of Peripheral Immune Cells into the Spinal Cord in Mouse Neuropathic Pain Model

Recent evidence suggests that transient receptor potential melastatin 2 (TRPM2) expressed in immune cells plays an important role in immune and inflammatory responses. We recently reported that TRPM2 expressed in macrophages and spinal microglia contributes to the pathogenesis of inflammatory and neuropathic pain aggravating peripheral and central pronociceptive inflammatory responses in mice. To further elucidate the contribution of TRPM2 expressed by peripheral immune cells to neuropathic pain, we examined the development of peripheral nerve injury-induced neuropathic pain and the infiltration of immune cells (particularly macrophages) into the injured nerve and spinal cord by using bone marrow (BM) chimeric mice by crossing wildtype (WT) and TRPM2-knockout (TRPM2-KO) mice. Four types of BM chimeric mice were prepared, in which irradiated WT or TRPM2-KO recipient mice were transplanted with either WT-or TRPM2-KO donor mouse-derived green fluorescence protein-positive (GFP⁺) BM cells (TRPM2^BM+/Rec+, TRPM2^BM–/Rec+, TRPM2^BM+/Rec–, and TRPM2^BM–/Rec– mice). Mechanical allodynia induced by partial sciatic nerve ligation observed in TRPM2^BM+/Rec+ mice was attenuated in TRPM2^BM–/Rec+, TRPM2^BM+/Rec–, and TRPM2^BM–/Rec– mice. The numbers of GFP⁺ BM-derived cells and Iba1/GFP double-positive macrophages in the injured sciatic nerve did not differ among chimeric mice 14 days after the nerve injury. In the spinal cord, the number of GFP⁺ BM-derived cells, particularly GFP/Iba1 double-positive macrophages, was significantly decreased in the three TRPM2-KO chimeric mouse groups compared with TRPM2^BM+/Rec+ mice. However, the numbers of GFP^–/Iba1⁺ resident microglia did not differ among chimeric mice. These results suggest that TRPM2 plays an important role in the infiltration of peripheral immune cells, particularly macrophages, into the spinal cord, rather than the infiltration of peripheral immune cells into the injured nerves and activation of spinal-resident microglia. The spinal infiltration of macrophages mediated by TRPM2 may contribute to the pathogenesis of neuropathic pain.     

Multi-Session Transcranial Direct Current Stimulation (tDCS) Elicits Inflammatory and Regenerative Processes in the Rat Brain

Transcranial direct current stimulation (tDCS) is increasingly being used in human studies as an adjuvant tool to promote recovery of function after stroke. However, its neurobiological effects are still largely unknown. Electric fields are known to influence the migration of various cell types in vitro, but effects in vivo remain to be shown. Hypothesizing that tDCS might elicit the recruitment of cells to the cortex, we here studied the effects of tDCS in the rat brain in vivo. Adult Wistar rats (n = 16) were randomized to either anodal or cathodal stimulation for either 5 or 10 consecutive days (500 µA, 15 min). Bromodeoxyuridine (BrdU) was given systemically to label dividing cells throughout the experiment. Immunohistochemical analyses ex vivo included stainings for activated microglia and endogenous neural stem cells (NSC). Multi-session tDCS with the chosen parameters did not cause a cortical lesion. An innate immune response with early upregulation of Iba1-positive activated microglia occurred after both cathodal and anodal tDCS. The involvement of adaptive immunity as assessed by ICAM1-immunoreactivity was less pronounced. Most interestingly, only cathodal tDCS increased the number of endogenous NSC in the stimulated cortex. After 10 days of cathodal stimulation, proliferating NSC increased by ∼60%, with a significant effect of both polarity and number of tDCS sessions on the recruitment of NSC. We demonstrate a pro-inflammatory effect of both cathodal and anodal tDCS, and a polarity-specific migratory effect on endogenous NSC in vivo. Our data suggest that tDCS in human stroke patients might also elicit NSC activation and modulate neuroinflammation.     

Deregulation of the p57-E2F1-p53 axis results in nonobstructive hydrocephalus and cerebellar malformation in mice

The cyclin-dependent kinase inhibitor (CKI) p57(Kip2) plays a pivotal role in cell cycle arrest during development, in particular, in the regulation of the entry of proliferating progenitors into quiescence. The gene encoding p57 undergoes genomic imprinting, and impairment of the regulation of p57 expression results in various developmental anomalies in humans and mice. We now show that p57 is expressed predominantly in the subcommissural organ and cerebellar interneurons in the mouse brain and that mice with brain-specific deletion of the p57 gene (Kip2) manifest prominent nonobstructive hydrocephalus as well as cerebellar malformation associated with the loss of Pax2-positive interneuron precursors and their descendants, including Golgi cells and γ-aminobutyric acid-containing neurons of the deep cerebellar nuclei. These abnormalities were found to be attributable to massive apoptosis of precursor cells in the developing brain. The morphological defects of the p57-deficient mice were corrected by knock-in of the gene for the related CKI p27(Kip1) at the Kip2 locus. The abnormalities were also prevented by additional genetic ablation of p53 or E2F1. Our results thus implicate p57 in cell cycle arrest in the subcommissural organ and Pax2-positive interneuron precursors, with the lack of p57 resulting in induction of p53-dependent apoptosis due to hyperactivation of E2F1.     

Role of age and neuroinflammation in the mechanism of cognitive deficits in sickle cell disease

This study aims to determine whether sickle cell mice could recapitulate features of cognitive and neurobehavioral impairment observed in sickle cell patients and whether neuroinflammation could be a potential therapeutic target as in other non-sickle cell disease-related cognitive dysfunction. Cognitive (learning and memory) and behavioral (anxiety) deficits in 13- and later 6-month-old male Townes humanized sickle cell (SS) and matched control (AA) mice were evaluated using novel object recognition (NOR) and fear conditioning tests. Immunohistochemistry was performed to quantify peripheral immune cell (CD45⁺) and activated microglia (Iba1⁺) as markers of neuroinflammation in the dentate and peri-dentate gyrus areas. We evaluated cell fate by measuring 5''-bromodeoxyuridine and doublecortin fluorescence and phenotyped proliferating cells using either glial fibrillary acid protein (GFAP⁺), neuronal nuclei (NeuN⁺), CD45⁺, and Iba1⁺. In addition, Golgi-Cox staining was used to assess markers of neuroplasticity (dendritic spine density and morphology and density of dendrite arbors) on cortical and hippocampal pyramidal neurons. Compared to matched AA controls, 13-month-old SS mice showed significant evidence of cognitive and behavioral deficit on NOR and fear conditioning tests. Also, SS mice had significantly higher density of CD45⁺ and activated microglia cells (i.e. more evidence of neuroinflammation) in the dentate and peri-dentate gyrus area. Additionally, SS mice had significantly lower dendritic spine density, but a higher proportion of immature dendritic spines. Treatment of 13-month-old SS mice with minocycline resulted in improvement of cognitive and behavioral deficit compared to matched vehicle-treated SS mice. Also, treated SS mice had significantly fewer CD45⁺ and activated microglia cells (i.e. less evidence of neuroinflammation) in the dentate and peri-dentate gyrus, as well as a significant improvement in markers of neuroplasticity.Impact statementThis study provides crucial information that could be helpful in the development of new or repurposing of existing therapies for the treatment of cognitive deficit in individuals with sickle cell disease (SCD). Its impact is in demonstrating for the first time that neuroinflammation and along with abnormal neuroplasticity are among the underlying mechanism of cognitive and behavioral deficits in SCD and that drugs such as minocycline which targets these pathophysiological mechanisms could be repurposed for the treatment of this life altering complication of SCD.     

Inhibition of repulsive guidance molecule-a protects dopaminergic neurons in a mouse model of Parkinson’s disease

Repulsive guidance molecule-a (RGMa), a glycosylphosphatidylinositol-anchored membrane protein, has diverse functions in axon guidance, cell patterning, and cell survival. Inhibition of RGMa attenuates pathological dysfunction in animal models of central nervous system (CNS) diseases including spinal cord injury, multiple sclerosis, and neuromyelitis optica. Here, we examined whether antibody-based inhibition of RGMa had therapeutic effects in a mouse model of Parkinson’s disease (PD). We treated mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and found increased RGMa expression in the substantia nigra (SN). Intraventricular, as well as intravenous, administration of anti-RGMa antibodies reduced the loss of tyrosine hydroxylase (TH)-positive neurons and accumulation of Iba1-positive microglia/macrophages in the SN of MPTP-treated mice. Selective expression of RGMa in TH-positive neurons in the SN-induced neuronal loss/degeneration and inflammation, resulting in a progressive movement disorder. The pathogenic effects of RGMa overexpression were attenuated by treatment with minocycline, which inhibits microglia and macrophage activation. Increased RGMa expression upregulated pro-inflammatory cytokine expression in microglia. Our observations suggest that the upregulation of RGMa is associated with the PD pathology; furthermore, inhibitory RGMa antibodies are a potential therapeutic option.Subject terms: Cell death in the nervous system, Parkinson''s disease     

Specific induction of macrophage inflammatory protein 1-alpha in glial cells of Sandhoff disease model mice associated with accumulation of N-acetylhexosaminyl glycoconjugates

Sandhoff disease is a lysosomal storage disease caused by simultaneous deficiencies of beta-hexosaminidase A (HexA; alphabeta) and B (HexB; betabeta), due to a primary defect of the beta-subunit gene (HEXB) associated with excessive accumulation of GM2 ganglioside (GM2) and oligosaccharides with N-acetylhexosamine residues at their non-reducing termini, and with neurosomatic manifestations. To elucidate the neuroinflammatory mechanisms involved in its pathogenesis, we analyzed the expression of chemokines in Sandhoff disease model mice (SD mice) produced by disruption of the murine Hex beta-subunit gene allele (Hexb-/-). We demonstrated that chemokine macrophage inflammatory protein-1 alpha (MIP-1alpha) was induced in brain regions, including the cerebral cortex, brain stem and cerebellum, of SD mice from an early stage of the pathogenesis but not in other systemic organs. On the other hand, little changes in other chemokine mRNAs, including those of RANTES (regulated upon activation, normal T expressed and secreted), MCP-1 (monocyte chemotactic protein-1), SLC (secondary lymphoid-tissue chemokine), fractalkine and SDF-1 (stromal derived factor-1), were detected. Significant up-regulation of MIP-1alpha mRNA and protein in the above-mentioned brain regions was observed in parallel with the accumulation of natural substrates of HexA and HexB. Immunohistochemical analysis revealed that MIP-1alpha-immunoreactivity (IR) in the above-mentioned brain regions of SD mice was co-localized in Iba1-IR-positive microglial cells and partly in glial fibrillary acidic protein (GFAP)-IR-positive astrocytes, in which marked accumulation of N-acetylglucosaminyl (GlcNAc)-oligosaccharides was observed from the presymptomatic stage of the disease. In contrast, little MIP-1alpha-IR was observed in neurons in which GM2 accumulated predominantly. These results suggest that specific induction of MIP-1alpha might coincide with the accumulation of GlcNAc-oligosaccharides due to a HexB deficiency in resident microglia and astrocytes in the brains of SD mice causing their activation and acceleration of the progressive neurodegeneration in SD mice.     

Possible role of Pax-6 in promoting breast cancer cell proliferation and tumorigenesis

Pax 6, a member of the paired box (Pax) family, has been implicated in oncogenesis. However, its therapeutic potential has been never examined in breast cancer. To explore the role of Pax6 in breast cancer development, a lentivirus based short hairpin RNA (shRNA) delivery system was used to knockdown Pax6 expression in estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) breast cancer cells. Effect of Pax6 silencing on breast cancer cell proliferation and tumorigenesis was analyzed. Pax6-RNAi-lentivirus infection remarkably downregulated the expression levels of Pax6 mRNA and protein in MCF-7 and MDA-MB-231 cells. Accordingly, the cell viability, DNA synthesis, and colony formation were strongly suppressed, and the tumorigenesis in xenograft nude mice was significantly inhibited. Moreover, tumor cells were arrested at G0/G1 phase after Pax6 was knocked down. Pax6 facilitates important regulatory roles in breast cancer cell proliferation and tumor progression, and could serve as a diagnostic marker for clinical investigation.