Sonja Johann

17β-Estradiol and progesterone prevent cuprizone provoked demyelination of corpus callosum in male mice

Sex hormones, for example, estrogen and progesterone, are thought to affect and delay progression of multiple sclerosis (MS) in pregnant women. Although both steroid hormones are neuroprotective in the brain and elevated during pregnancy, only estrogen was tested in clinical trials. To evaluate the role of 17β‐estradiol (E) and progesterone (P) in prevention demyelination, young adult male mice were fed with cuprizone for a defined time interval and simultaneously treated with steroids by repeated injections into the neck region. The status of myelination was analyzed by magnetic resonance imaging and conventional histological staining. The individual application of E and P resulted only in a moderate prevention of demyelination in the corpus callosum (CC). The combined treatment with both steroid hormones counteracted the process of demyelination. Expression of the mature (PLP and MBP) and premature (PDGF‐α‐R) oligodendrocyte markers were significantly increased after hormone application in the affected CC. In addition, both hormones stimulated astrogliosis and the expression of IGF‐1. Microglial invasion in demyelinated CC was pronounced and additionally localized in the midline of CC after hormone treatment. These data show that sex steroids can protect the brain from demyelination and stimulate remyelination. It appears that only the administration of both hormones is fully effective. The beneficial steroid effect requires interactions with oligodendrocytes possibly by preventing their degeneration or recruitment from precursor cells which are stimulated to remyelinated fibers. The positive hormonal influence on myelination in the CNS may be a future therapeutically strategy for the treatment of MS.

Altered localization, abnormal modification and loss of function of Sigma receptor-1 in amyotrophic lateral sclerosis

Intracellular accumulations of mutant, misfolded proteins are major pathological hallmarks of amyotrophic lateral sclerosis (ALS) and related disorders. Recently, mutations in Sigma receptor 1 (SigR1) have been found to cause a form of ALS and frontotemporal lobar degeneration (FTLD). Our goal was to pinpoint alterations and modifications of SigR1 in ALS and to determine how these changes contribute to the pathogenesis of ALS. In the present study, we found that levels of the SigR1 protein were reduced in lumbar ALS patient spinal cord. SigR1 was abnormally accumulated in enlarged C-terminals and endoplasmic reticulum (ER) structures of alpha motor neurons. These accumulations co-localized with the 20s proteasome subunit. SigR1 accumulations were also observed in SOD1 transgenic mice, cultured ALS-8 patients fibroblasts with the P56S-VAPB mutation and in neuronal cell culture models. Along with the accumulation of SigR1 and several other proteins involved in protein quality control, severe disturbances in the unfolded protein response and impairment of protein degradation pathways were detected in the above-mentioned cell culture systems. Furthermore, shRNA knockdown of SigR1 lead to deranged calcium signaling and caused abnormalities in ER and Golgi structures in cultured NSC-34 cells. Finally, pharmacological activation of SigR1 induced the clearance of mutant protein aggregates in these cells. Our results support the notion that SigR1 is abnormally modified and contributes to the pathogenesis of ALS.

Oestrogen and progesterone reduce lipopolysaccharide-induced expression of tumour necrosis factor-alpha and interleukin-18 in midbrain astrocytes.

Besides microglia, astrocytes exert an important regulatory function in the initiation and control of neuro‐inflammatory processes in the central nervous system. Clinical and experimental data suggest that sex steroids are neuroprotective and that neurological/neurodegenerative disorders display sex‐specific characteristics. Astroglia is known to respond to toxic stimuli by secretion of distinct pro‐inflammatory/apoptotic cytokines. In the present study, we investigated the influence of oestrogen and progesterone on the expression of the cytokines tumour necrosis factor (TNF)‐α and interleukin (IL)‐18 in primary astrocytes obtained from neonatal mouse midbrain and cerebral cortex after the stimulation with lipopolysaccharides (LPS). LPS strongly induced the expression of TNF‐α in astrocytes from both brain regions and IL‐18 in those from midbrain. Oestrogen significantly attenuated LPS‐induced TNF‐α expression in the midbrain glia but not in the cortex glia. Combined treatment with oestrogen and progesterone together diminished LPS‐induced IL‐18 expression in the midbrain completely. Both steroid effects could be specifically antagonised by the steroid hormone receptor antagonists ICI 182 780 and mifepristone. We conclude that neuroprotective oestrogen and progesterone effects in the midbrain might be in part the consequence of a reduced pro‐inflammatory response of astroglia.

Cuprizone treatment induces demyelination and astrocytosis in the mouse hippocampus

Memory impairment is outstanding within the spectrum of cognitive deficits in multiple sclerosis (MS) patients. Demyelination has been reported in the hippocampus formation of MS patients. The degree of hippocampus lesions in MS strongly correlates with progression of cognitive dysfunction. Because no appropriate animal model for the study of hippocampus demyelination has been established, we used the cuprizone mouse model to investigated demyelination in young adult and aged mice. The myelin status was analyzed by classical histological staining, immunocytochemistry for proteolipoprotein, and electron microscopy. Oligodendrocyte, astroglial, and microglia markers were studied. Cuprizone intoxication induced an almost complete demyelination of distinct hippocampus subregions to a similar extent in young adult and aged male mice. Demyelination was pronounced in a subset of white and gray matter areas, i.e., the stratum lacunosum moleculare containing the perforant path, medial alveus, stratum pyramidale in the cornu ammonis 2/3 region, and hilus region. Besides demyelination, affected areas displayed hypertrophic and hyperplastic astrocytosis. No significant effect on microglia invasion was detected at any investigated time point (0, 3, 5, and 7 weeks). We conclude that cuprizone‐induced demyelination provides an adequate animal model to investigate appropriate therapy strategies for the prevention of hippocampus demyelination.

Brain-Region-Specific Astroglial Responses In Vitro After LPS Exposure

Astroglia is well-known to be integrated in the complex regulation of neuroinflammation in the central nervous system. Astrocytes become activated and synthesize cytokines, chemokines, and prostanoids during degenerative and vulnerable processes and interact with other immune-competent cells. Degenerative disorders often occur in a brain-region-specific fashion suggesting differences in the activity and reactivity of innate immune cells. We have investigated the potency of lipopolysaccharides (LPS) to differently stimulate astrocytes from the cortex and midbrain. Astroglial cultures were prepared from Bagg albino/c mice and exposed to LPS. Astrocytes from both brain areas already differed in their capacity and profile of cytokine expression under basal unstimulated conditions. In response to LPS, we observed both a region-specific pattern of up-regulation of distinct cytokines and differences in the extent and time-course of activation. Our data demonstrate that astrocytes reveal a region-specific basal profile of cytokine expression and a selective area-specific regulation of cytokines upon LPS-induced inflammation. This makes astrocytes likely candidates to be responsible for region-specific incidence rates of neurological and neurodegenerative disorders.

Inflammatory cytokine release of astrocytes in vitro is reduced by all-trans retinoic acid

In the central nervous system inflammation is mediated by microglia and astrocytes. To investigate its regulation, murine astrocyte cultures were treated with bacterial lipopolysaccharides (LPS) and analyzed with Affymetrix gene array, qRT-PCR and ELISA. Cells responded to LPS with a strong upregulation of pro-inflammatory cytokines and chemokines. Treatment with the transcriptional activator retinoic acid (RA) suppressed mRNA expression and protein release of several important cytokines (IL-1β 4%, IL-6 21%, TNFα 30%, IL-12p40 42%, and IL-12p35/p40 27%; p<0.01). The data are consistent with the hypothesis that all-trans RA takes part in endogenous anti-inflammatory feedback loops in the CNS.

Expression of Enzymes Involved in the Prostanoid Metabolism by Cortical Astrocytes after LPS- induced Inflammation

Neuroinflammatory processes are a common epiphenomenon for a number of neurological and neurodegenerative diseases. Besides microglia, astrocytes are implicated in brain inflammation in response to harmful stimuli and pathological processes. Bacterial endotoxins can induce the synthesis and release of proinflammatory mediators, i.e., cytokines and chemokines, by astroglia. In this study, we have investigated the effect of lipopolysaccharide (LPS) treatment on the expression of enzymes of prostanoid synthesis and degradation in cultured mouse cortical astrocytes using an Affymetrix Gene Chip array, quantitative reverse transcriptase polymerase chain reaction (RT-PCR), and an enzyme-immunosorbent assay. LPS treatment induced an upregulation of enzymes responsible for prostaglandin E2 synthesis, a downregulation of enzymes that catalyzes prostaglandin E2 (PGE2) degradation and production of proinflammatory leukotrienes. Changes in enzyme expression were accompanied by a highly significant increase in extracellular PGE2. Our data demonstrate that astrocytes are directly involved in the complex regulation of proinflammatory prostanoids in the CNS under pathological processes, thus being of potential interest as targets for therapeutical interventions. Further studies are required to unravel the different roles and interactions between astroglia and other cells of the brain-intrinsic innate immune system during inflammation.

NLRP3 inflammasome is expressed by astrocytes in the SOD1 mouse model of ALS and in human sporadic ALS patients.

Amyotrophic lateral sclerosis (ALS) is characterized by the degeneration of motoneurons in the cerebral cortex, brainstem and spinal cord. Neuroinflammation plays an important role in the pathogenesis of ALS and involves the activation of microglia and astrocytes. Intracellular inflammasome complexes are part of the innate immunity as they sense and execute host inflammatory responses. The best characterized component is the NLRP3 inflammasome comprised of the NLR protein NLRP3, the adaptor ASC and pro‐caspase 1. The NLRP3 inflammasome is critical for the activation of caspase 1 and the processing and release of IL1β and IL18. In this study, we investigated the expression, activation and co‐localization of the NLRP3 inflammasome in the spinal cord of male SOD1(G93A) mice carrying a mutant human superoxide dismutase 1 (SOD1) variant and regarded as an animal model for ALS as well as in post‐mortem tissue of ALS patients. NLRP3 and its molecular components as well as IL1β were already detectable in SOD1 mice at a pre‐symptomatic stage after 9 weeks and further increased in 14 week old animals. Spinal cord astrocytes were identified as the major cell type expressing NLRP3 components. In human ALS tissue, we also found increased NLRP3, ASC, IL18 and active caspase 1 levels compared to control patients. Our findings suggest that astroglial NLRP3 inflammasome complexes are critically involved in neuroinflammation in ALS. GLIA 2015;63:2260–2273

Selective regulation of growth factor expression in cultured cortical astrocytes by neuro-pathological toxins

Astrocytes are integrated in the complex regulation of neurodegeneration and neuronal damage in the CNS. It is well-known that astroglia produces a plethora of growth factors which might be protective for neurons. Growth factors prevent neurons from cell death and promote proliferation and differentiation of precursor cells. Previous data suggest that astrocytes may respond to toxic stimuli by a selective mobilization of guarding molecules. In the present study, we have investigated the potency of different pathological stimuli such as lipopolysaccharides, tumor necrosis factor alpha, glutamate, and hydrogen peroxide to activate cultured cortical astroglia and stimulate growth factor expression. Astroglial cultures were exposed to the above factors for 24h at non-toxic concentrations for astrocytes. Growth factor expression was analyzed by real-time PCR, oligo-microarray technique, and ELISA. Insulin-like growth factor-1 was selectively down-regulated by lipopolysaccharides and tumor necrosis factor alpha, bone morphogenetic protein 6 by all stimuli. In contrast, lipopolysaccharides, tumor necrosis factor alpha, and glutamate increased leukemia inhibitory factor. Fibroblast growth factor 2 was up-regulated by lipopolysaccharides and tumor necrosis factor alpha and down-regulated by hydrogen peroxide. Besides hydrogen peroxide, all other stimuli promoted vascular epithelial growth factor A mRNA and protein expression. It appears that lipopolysaccharides but not tumor necrosis factor alpha effects on vascular epithelial growth factor A depend on the classic NFkappaB pathway. Our data clearly demonstrate that astroglia actively responses to diverse pathological compounds by a selective expression pattern of growth factors. These findings make astrocytes likely candidates to participate in disease-specific characteristics of neuronal support or damage.

Regulation of Hypoxia-Induced Inflammatory Responses and M1-M2 Phenotype Switch of Primary Rat Microglia by Sex Steroids

Microglia cells are the primary mediators of the CNS immune defense system and crucial for the outcome of shaping inflammatory responses. They are highly dynamic, moving constantly, and become activated by neuronal signaling under pathological conditions. They fulfill a dual role by not only regulating local neuroinflammation but also conferring neuronal protection. Gonadal steroids are known to exert anti-inflammatory effects in the CNS. Recently, we have shown that the microglial-like cell line BV-2 is hypoxia-sensitive and regulated by gonadal steroids. The present study used primary rat cerebral cortex-derived microglia to analyze whether this cell type directly perceive and respond to acute hypoxia. Second, we investigated whether 17β-estradiol (E2) and progesterone (P) interfere with hypoxia-induced changes. Short-term hypoxia increased the expression of a subset of pro-inflammatory (TNFa, IL1b) and oxidative stress-related (Hif1a) genes. The induction of TNFa and IL1b was counteracted by P. Hypoxia shifted the primary microglia to the pro-inflammatory M1 phenotype. The administration of E2 and P favored the neuroprotective M2 phenotype. Our findings extend previous data obtained with BV-2 cells and show that the primary microglia directly perceive hypoxia which increase their inflammatory activity. Both steroid hormones directly and indirectly interact with the microglia cells by reducing the inflammatory scenario and stimulating neuroprotection.