6-Methyl flavone inhibits Nogo-B expression and improves high fructose diet-induced liver injury in mice.

Excessive fructose consumption increases hepatic de novo lipogenesis, resulting in cellular stress, inflammation and liver injury. Nogo-B is a resident protein of the endoplasmic reticulum that regulates its structure and function. Hepatic Nogo-B is a key protein in glycolipid metabolism, and inhibition of Nogo-B has protective effects against metabolic syndrome, thus small molecules that inhibit Nogo-B have therapeutic benefits for glycolipid metabolism disorders. In this study we tested 14 flavones/isoflavones in hepatocytes using dual luciferase reporter system based on the Nogo-B transcriptional response system, and found that 6-methyl flavone (6-MF) exerted the strongest inhibition on Nogo-B expression in hepatocytes with an IC50 value of 15.85 µM. Administration of 6-MF (50 mg· kg-1 ·d-1, i.g. for 3 weeks) significantly improved insulin resistance along with ameliorated liver injury and hypertriglyceridemia in high fructose diet-fed mice. In HepG2 cells cultured in a media containing an FA-fructose mixture, 6-MF (15 µM) significantly inhibited lipid synthesis, oxidative stress and inflammatory responses. Furthermore, we revealed that 6-MF inhibited Nogo-B/ChREBP-mediated fatty acid synthesis and reduced lipid accumulation in hepatocytes by restoring cellular autophagy and promoting fatty acid oxidation via the AMPKα-mTOR pathway. Thus, 6-MF may serve as a potential Nogo-B inhibitor to treat metabolic syndrome caused by glycolipid metabolism dysregulation.

Loss of parkin causes endoplasmic reticulum calcium dyshomeostasis by upregulation of reticulocalbin 1.

Increasing evidence suggests that astrocytes play an important role in the progression of Parkinson's disease (PD). Previous studies on our parkin knockout mouse demonstrated a higher accumulation of damaged mitochondria in astrocytes than in surrounding dopaminergic (DA) neurons, suggesting that Parkin plays a crucial role regarding their interaction during PD pathogenesis. In the current study, we examined primary mesencephalic astrocytes and neurons in a direct co-culture system and discovered that the parkin deletion causes an impaired differentiation of mesencephalic neurons. This effect required the parkin mutation in astrocytes as well as in neurons. In Valinomycin-treated parkin-deficient astrocytes, ubiquitination of Mitofusin 2 was abolished, whereas there was no significant degradation of the outer mitochondrial membrane protein Tom70. This result may explain the accumulation of damaged mitochondria in parkin-deficient astrocytes. We examined differential gene expression in the substantia nigra region of our parkin-KO mouse by RNA sequencing and identified an upregulation of the endoplasmic reticulum (ER) Ca2+ -binding protein reticulocalbin 1 (RCN1) expression, which was validated using qPCR. Immunostaining of the SN brain region revealed RCN1 expression mainly in astrocytes. Our subcellular fractionation of brain extract has shown that RCN1 is located in the ER and in mitochondria-associated membranes (MAM). Moreover, a loss of Parkin function reduced ATP-stimulated calcium-release in ER mesencephalic astrocytes that could be attenuated by siRNA-mediated RCN1 knockdown. Our results indicate that RCN1 plays an important role in ER-associated calcium dyshomeostasis caused by the loss of Parkin function in mesencephalic astrocytes, thereby highlighting the relevance of astrocyte function in PD pathomechanisms.

BF-1--a novel selective 5-HT2B receptor antagonist blocking neurogenic dural plasma protein extravasation in guinea pigs.

Serotonin 5-HT2B receptor antagonists have been proposed as migraine prophylactic drugs, but previously available 5-HT2B receptor antagonists displayed multiple monoaminergic side effects and had to be withdrawn from the market. Here, we set out to identify a novel antagonist with high affinity and selectivity towards 5-HT2B receptors. To test the affinity of new compounds towards various receptors, we generated a broad series of cells functionally coupling human monoaminergic receptors to luciferase. Using the cell lines we revealed pimethixene (1-methyl-4-(9H-thioxanthen-9-ylidene)piperidine) as highly potent, albeit non-selective 5-HT2B receptor antagonist and optimized its chemical structure to create highly potent and selective 5-HT2B receptor antagonists. We selected the methoxythioxanthene BF-1 for further analysis. In comparison to pimethixene, it lacked high affinities to 5-HT1A, 5-HT2A, 5-HT2C, histamine H1, dopamine D1 and D2 as well as muscarinic M1 and M2 receptors. BF-1 was tested as potential migraine prophylactic drug by blocking meta-chlorophenylpiperazine, (mCPP) or BW723C86 (5-((thiophen-2-yl)methoxy)-α-methyltryptamine) induced neurogenic dural plasma protein extravasation in a guinea pig model that may resemble a migraine attack. BF-1 was significantly more potent in this assay compared to the well know non-selective 5-HT2B antagonists, methysergide ((6aR,9R)-N-[(2S)-1-Hydroxybutan-2-yl]-4,7-dimethyl-6,6a,8,9-tetrahydroindolo[4,3-fg]quinoline-9-carboxamide) or pizotifen (4-(1-methyl-4-piperidylidine)-9,10-dihydro-4H-benzo-[4,5]cyclohepta[1,2]-thiophene). Therefore, we propose BF-1 as a new compound that may be developed for prophylactic migraine treatment without the typical monoaminergic side effects.

GABAA receptors involved in sleep and anaesthesia: ß1- versus ß3-containing assemblies.

The histaminergic neurons of the posterior hypothalamus (tuberomamillary nucleus-TMN) control wakefulness, and their silencing through activation of GABA(A) receptors (GABA(A)R) induces sleep and is thought to mediate sedation under propofol anaesthesia. We have previously shown that the ß1 subunit preferring fragrant dioxane derivatives (FDD) are highly potent modulators of GABA(A)R in TMN neurons. In recombinant receptors containing the ß3N265M subunit, FDD action is abolished and GABA potency is reduced. Using rat, wild-type and ß3N265M mice, FDD and propofol, we explored the relative contributions of ß1- and ß3-containing GABA(A)R to synaptic transmission from the GABAergic sleep-on ventrolateral preoptic area neurons to TMN. In ß3N265M mice, GABA potency remained unchanged in TMN neurons, but it was decreased in cultured posterior hypothalamic neurons with impaired modulation of GABA(A)R by propofol. Spontaneous and evoked GABAergic synaptic currents (IPSC) showed ß1-type pharmacology, with the same effects achieved by 3 µM propofol and 10 µM PI24513. Propofol and the FDD PI24513 suppressed neuronal firing in the majority of neurons at 5 and 100 µM, and in all cells at 10 and 250 µM, respectively. FDD given systemically in mice induced sedation but not anaesthesia. Propofol-induced currents were abolished (1-6 µM) or significantly reduced (12 µM) in ß3N265M mice, whereas gating and modulation of GABA(A)R by PI24513 as well as modulation by propofol were unchanged. In conclusion, ß1-containing (FDD-sensitive) GABA(A)R represent the major receptor pool in TMN neurons responding to GABA, while ß3-containing (FDD-insensitive) receptors are gated by low micromolar doses of propofol. Thus, sleep and anaesthesia depend on different GABA(A)R types.

Spinal cord pathology in alpha-synuclein transgenic mice.

Accumulation of α-synuclein is observed in neurodegenerative diseases like Parkinson's disease and Multiple System Atrophy. In previous studies with transgenic C57BL/6 mice overexpressing α-synuclein carrying the mutations A53T and A30P found in Parkinson's disease or with a parkin-null background, we reported severe mitochondrial impairments in neurons and to a larger extent in glial cells of the mesencephalon. Neuron death was not observed in the brain. Here we show that the mice show severe motor impairments in behavioral tests. In addition, these mice exhibit astrocytic cell death in the spinal cord, accompanied by extensive gliosis and microglial activation. This is shown by cell death staining and immunohistochemistry. Ultrastructural analyses revealed severe mitochondrial impairments not only in astrocytes, but also in oligodendrocytes and, to a small extent, in neurons. Thus, the transgenic mice show a profound pathology in glial cells of the spinal cord.

Fragrant dioxane derivatives identify beta1-subunit-containing GABAA receptors.

Nineteen GABA(A) receptor (GABA(A)R) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of beta1-subunit-containing GABA(A)Rs is unknown. Here we report the discovery of a new structural class of GABA(A)R positive modulators with unique beta1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed alpha1betaxgamma2L (x-for 1,2,3) GABA(A)R FDD were 6 times more potent at beta1- versus beta2- and beta3-containing receptors. Serine at position 265 was essential for the high sensitivity of the beta1-subunit to FDD and the beta1N286W mutation nearly abolished modulation; vice versa the mutation beta3N265S shifted FDD sensitivity toward the beta1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to beta1-negative cerebellar Purkinje neurons. Immunostaining for the beta1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by beta1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of beta1-containing GABA(A)Rs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABA(A)Rs.

The neuronal RhoA GEF, Tech, interacts with the synaptic multi-PDZ-domain-containing protein, MUPP1.

Tech is a RhoA guanine nucleotide exchange factor (GEF) that is highly enriched in hippocampal and cortical neurons. To help define its function, we have conducted studies aimed at identifying partner proteins that bind to its C-terminal PDZ ligand motif. Yeast two hybrid studies using the Tech C-terminal segment as bait identified MUPP1, a protein that contains 13 PDZ domains and has been localized to the post-synaptic compartment, as a candidate partner protein for Tech. Co-transfection of Tech and MUPP1 in human embryonic kidney 293 cells confirmed that these full-length proteins interact in a PDZ-dependent fashion. Furthermore, we confirmed that endogenous Tech co-precipitates with MUPP1, but not PSD-95, from hippocampal and cortical extracts prepared from rat brain. In addition, immunostaining of primary cortical cultures revealed co-localization of MUPP1 and Tech puncta in the vicinity of synapses. In assessing which PDZ domains of MUPP1 mediate binding to Tech, we found that Tech can bind to either PDZ domain 10 or 13 of MUPP1 as mutation of both these domains is needed to disrupt their interaction. Taken together, these findings demonstrate that Tech binds to MUPP1 and suggest that it regulates RhoA signaling pathways in the vicinity of synapses.

Non-motor behavioural impairments in parkin-deficient mice.

Mutations in the parkin gene are the major cause of early-onset familial Parkinson's disease (PD). We previously reported the generation and analysis of a knockout mouse carrying a deletion of exon 3 in the parkin gene. F1 hybrid pa+/- mice were backcrossed to wild-type C57Bl/6 for three more generations to establish a pa-/-(F4) mouse line. The appearance of tyrosine hydroxylase-positive neurons was normal in young and aged pa-/- (F4) animals. Loss of parkin function in mice did not enhance vulnerability of dopaminergic neurons to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity. However, the pa-/- (F4) mice displayed impaired exploration and habituation to a new environment and exhibited thigmotaxis behaviour in the open field and Morris water maze. Abnormal anxiety-related behaviour of pa-/- (F4) mice was also observed in the light/dark exploration test paradigm. Dopamine metabolism was enhanced in the striatum of pa-/- (F4) mice, as revealed by increased homovanillic acid (HVA) content and a reduced ratio of dihydroxyphenylacetic acid (DOPAC)/HVA. The alterations found in the dopaminergic system could be responsible for the behavioural impairments of pa-/- (F4) mice. Consistent with a recent observation of cognitive dysfunction in parkin-linked patients with PD, our findings provide evidence of a physiological role of parkin in non-motor behaviour, possibly representing a disease stage that precedes dopaminergic neuron loss.

Mono- and double-mutant mouse models of Parkinson's disease display severe mitochondrial damage.

Mutations in the gene encoding alpha-synuclein (asyn) causes autosomal-dominant, in the parkin gene autosomal-recessive forms of Parkinson's disease (PD). The pathophysiology of PD is poorly understood, even though published evidence suggests a role for mitochondria in the pathogenesis. To gain insight into the influence of asyn and parkin on mitochondrial integrity and function, we have generated several mono-mutant mouse lines expressing doubly mutated human asyn (hm(2)asyn) under the control of two different promoters, or a targeted deletion of Parkin (Parkin-Exon3-knockout). Both mouse lines were crossed to generate the double-mutant. Here we compare the ultrastructure and functional properties of mitochondria in the substantia nigra (SN), the striatum, the cerebral cortex (Cx) and skeletal muscle of young (2-3 months) and aged (12-14 months) mono- and double-mutants mice. We observed severe genotype-, age- and region-dependent morphological alterations of mitochondria in neuronal somata. The number of structurally altered mitochondria was significantly increased in the SN of both double-mutants and in the Cx of one mono- and one double-mutant line. These alterations coincided with a reduced complex I capacity in the SN, but were neither accompanied by alterations in the number or the size of the mitochondria nor by leakage of cytochrome c, Smac/DIABLO or Omi/HtrA2. None of the transgenic animals developed any gross histopathological abnormalities or overt motor disabilities. Together our results provide compelling evidence that (i) both, asyn and parkin are relevant for mitochondrial integrity, (ii) the influence of these proteins on mitochondria are age- and tissue-specific and (iii) changes of mitochondrial morphology do not inevitably cause functional impairments.

Differential expression of cathepsin X in aging and pathological central nervous system of mice.

Increasing evidence of a fundamental influence of cathepsins on inflammation has drawn interest in a thorough understanding of their role in physiological and pathological processes. Even though the number of identified cathepsins has more than doubled in the last years, information about their expression, regulation and function in the brain is still incomplete. In the present study we analyzed the regional, cellular and subcellular localization and the activity of the recently discovered cathepsin X in the normal, developing and pathological mouse brain. Our results show that CATX is: (i) is expressed in almost all cells in the mouse brain with a preference for glial cells; (ii) already widely expressed early in development and age-dependently upregulated in amount and activity; (iii) prominently localized in the lysosomal system but also scattered in the somal cytoplasm in the aged brain; (iv) upregulated in numerous glial cells of degenerating brain regions in a transgenic mouse model of amyotrophic lateral sclerosis; and (v) associated with plaques in a transgenic mouse model and in Alzheimer patients. These results strongly suggest that cathepsin X is an important player in degenerative processes during normal aging and in pathological conditions.

Ascending neuropathology in the CNS of a mutant SOD1 mouse model of amyotrophic lateral sclerosis.

Transgenic mice expressing a mutated human Cu/Zn superoxide dismutase (SOD1) gene develop a motor neuron disease similar to familial amyotrophic lateral sclerosis (FALS). While the histopathology and the inflammatory reactions in the spinal cord of these mice are well described, their spatiotemporal extension into brain areas and the relationship between degenerative and inflammatory events remain obscure. In the present study, we investigated the time course and extent of degenerative changes and inflammatory reactions in the CNS during progression of the disease in a transgenic FALS model, the SOD1-G93A mouse with histological and immunohistochemical methods. Compared to non-transgenic littermates, the SOD1-G93A transgenics developed widespread degeneration in both motor and extra-motor regions up to telencephalic regions, including the cerebral cortex but sparing distinct regions like the striatum and hippocampus. We provide evidence that these degenerative processes are accompanied by intense inflammatory reactions in the brain, which spatiotemporally correlate with degeneration and comprise besides strong astro- and microgliotic reactions also an influx of peripheral immune cells such as T-lymphocytes and dendritic cells. Both degeneration and inflammatory reactions spread caudocranially, starting at 2 months in the spinal cord and reaching the telencephalon at 5 months of age. Since the corticospinal tract lacked any signs of degeneration, we conclude that the upper and the lower motor neurons degenerate independently of each other.

Sgk1, a cell survival response in neurodegenerative diseases.

Serum and glucocorticoid-regulated kinase 1 (sgk1) belongs to a family of serine/threonine kinases that is under acute transcriptional control by serum and glucocorticoids. An expanding set of receptors and cellular stress pathways has been shown to enhance sgk1 expression, which is implicated in the regulation of ion channel conductance, cell volume, cell cycle progression, and apoptosis. Recent evidence for the involvement of sgk1 in the early pathogenesis of MPTP-induced Parkinson's disease (PD) prompted us to investigate in more detail its expression and role in animal models of different neurodegenerative diseases. Here, we show that transcription of sgk1 is increased in several animal models of PD and a transgenic model of amyotrophic lateral sclerosis (ALS). The upregulation of sgk1 strongly correlates with the occurrence of cell death. Furthermore, we provide evidence that the Forkhead transcription factor FKHRL1 and some of the voltage-gated potassium channels are physiological substrates of sgk1 in vivo. Using a small interfering RNA approach to silence sgk1 transcripts in vitro, we give evidence that sgk1 exerts a protective role in oxidative stress situations. These findings underline a key role for sgk1 in the molecular pathway of cell death, in which sgk1 seems to exert a protective role.

sgk1, a member of an RNA cluster associated with cell death in a model of Parkinson's disease.

In an effort to gain deeper insight into the molecular processes underlying neurodegeneration in Parkinson's disease, we performed gene expression profiling at several early time points after MPTP-injection into old (1-year) mice. We used a PCR-based gene expression profiling method, digital expression pattern display (DEPD), a method of very high sensitivity and reproducibility, which displays almost all transcripts of a tissue. To identify cell death-associated genes, we defined clusters of differentially expressed transcripts with expression behaviour that correlated with the temporal profile of cell death progression and characterized one of these cell death clusters further. We selected one of the strongest regulated genes, the serum and glucocorticoid-regulated kinase 1 (sgk1), and validated its differential expression by Northern blot analysis, semiquantitative PCR and in situ hybridization. Up-regulation of sgk1 (i) coincides with the onset of dopaminergic cell death in both the 8-week acute and 1-year subacute MPTP models, (ii) spans the entire brain, (iii) is attenuated by the l-deprenyl-mediated inhibition of the MPTP conversion to its active metabolite MPP+ and (iv) is not induced by dehydration. This study demonstrated that the combination of the DEPD technology, clustering analysis and a detailed histopathology is a useful tool for elucidating molecular pathways in neurodegenerative diseases.

Parkin expression in the developing mouse.

Parkin is an E3 ubiquitin ligase causally involved in the pathogenesis of autosomal recessive juvenile parkinsonism. In this paper, we analysed the formation of alternative splice products and the spatio-temporal expression pattern of parkin during pre- and postnatal mouse development. Using RT-PCR, Northern blot, in situ hybridization, Western blot analysis, and immunohistochemistry we found (i) alternative splice forms of parkin; (ii) an early and widespread expression of parkin mRNA and protein in the CNS and several organs, already at E10/12; (iii) a marked increase in expression level during midgestational development (E15-18) in the CNS, followed by a steady increase until adulthood; (iv) an ubiquitous distribution throughout CNS ontogeny. Our results show that parkin expression is correlated with cell maturation and suggests an important physiological role of parkin in neurons that is at no time limited to the dopaminergic system.