2010

Tissue Distribution of 5-Hydroxymethylcytosine and Search for Active Demethylation Intermediates

PLoS ONE, 2010, doi:10.1371/journal.pone.0015367, published on 23.12.2010
PLoS ONE, online article
5–Hydroxymethylcytosine (hmC) was recently detected as the sixth base in mammalian tissue at so far controversial levels. The function of the modified base is currently unknown, but it is certain that the base is generated from 5-methylcytosine (mC). This fuels the hypothesis that it represents an intermediate of an active demethylation process, which could involve further oxidation of the hydroxymethyl group to a formyl or carboxyl group followed by either deformylation or decarboxylation. Here, we use an ultra-sensitive and accurate isotope based LC-MS method to precisely determine the levels of hmC in various mouse tissues and we searched for 5–formylcytosine (fC), 5-carboxylcytosine (caC), and 5–hydroxymethyluracil (hmU) as putative active demethylation intermediates. Our data suggest that an active oxidative mC demethylation pathway is unlikely to occur. Additionally, we show using HPLC-MS analysis and immunohistochemistry that hmC is present in all tissues and cell types with highest concentrations in neuronal cells of the CNS.

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Planar Patch Clamp Approach to Characterize Ionic Currents from Intact Lysosomes

Science Signaling, 2010, doi:10.1126/scisignal.3151pl3, published on 07.12.2010
Science Signaling, online article
Since its launch in the early 1980s, the patch clamp method has been extensively used to study ion channels in the plasma membrane, but its application to the study of intracellular ion channels has been limited. Unlike the plasma membrane, intracellular membranes are usually not stable enough to withstand mechanical manipulation by glass electrodes during seal formation and rupturing of the membrane. To circumvent these problems, we developed a method involving the immobilization of isolated organelles on a solid matrix planar glass chip. This glass chip contains a microstructured hole that supports the formation of gigaseals and subsequent electrophysiological recordings despite the high fragility of intracellular membranes. Here, we report the experimental details of this method using lysosomes, which are the smallest cellular organelles, as a model system. We demonstrate that we can record endogenous ionic currents from wild-type lysosomes, as well as from lysosomes overexpressing ion channels, and expect that this method will provide electrophysiological access to a broad range of intracellular ion channels.

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Restoration of Cone Vision in the CNGA3−/− Mouse Model of Congenital Complete Lack of Cone Photoreceptor Function

Molecular Therapy, 2010, doi:10.1038/mt.2010.149, published on 05.12.2010
Molecular Therapy, online article
Congenital absence of cone photoreceptor function is associated with strongly impaired daylight vision and loss of color discrimination in human achromatopsia. Here, we introduce viral gene replacement therapy as a potential treatment for this disease in the CNGA3(-/-) mouse model. We show that such therapy can restore cone-specific visual processing in the central nervous system even if cone photoreceptors had been nonfunctional from birth. The restoration of cone vision was assessed at different stages along the visual pathway. Treated CNGA3(-/-) mice were able to generate cone photoreceptor responses and to transfer these signals to bipolar cells. In support, we found morphologically that treated cones expressed regular cyclic nucleotide-gated (CNG) channel complexes and opsins in outer segments, which previously they did not. Moreover, expression of CNGA3 normalized cyclic guanosine monophosphate (cGMP) levels in cones, delayed cone cell death and reduced the inflammatory response of Müller glia cells that is typical of retinal degenerations. Furthermore, ganglion cells from treated, but not from untreated, CNGA3(-/-) mice displayed cone-driven, light-evoked, spiking activity, indicating that signals generated in the outer retina are transmitted to the brain. Finally, we demonstrate that this newly acquired sensory information was translated into cone-mediated, vision-guided behavior.

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Inhibition of mitochondrial fusion by Alpha-synuclein is rescued by PINK1 Parkin and DJ-1

The EMBO Journal, 2010, 1-19, doi:10.1038/emboj.2010.223 published on 20.10.2010

The Embo Journal, online article

Aggregation of alpha-synuclein (alphaS) is involved in the pathogenesis of Parkinson’s disease (PD) and a variety of related neurodegenerative disorders. The physiological function of alphaS is largely unknown. We demonstrate with in vitro vesicle fusion experiments that alphaS has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, alphaS binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age-dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous alphaS. In contrast, siRNA-mediated downregulation of alphaS results in elongated mitochondria in cell culture. AlphaS can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, alphaS prevents fusion of differently labelled mitochondrial populations. Thus, alphaS inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of alphaS is rescued by coexpression of PINK1, parkin or DJ-1 but not the PD-associated mutations PINK1 G309D and parkin D1–79 or by DJ-1 C106A.

 

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Altered synaptic plasticity and behavioral abnormalities in CNGA3-deficient mice

Genes, Brain and Behavior, 2010, doi:10.1111/j.1601-183X.2010.00646.x, published on 24.08.2010
Genes, Brain and Behavior, online article
The role of the cyclic nucleotide-gated (CNG) channel CNGA3 is well established in cone photoreceptors and guanylyl cyclase-D-expressing olfactory neurons. To assess a potential function of CNGA3 in the mouse amygdala and hippocampus, we examined synaptic plasticity and performed a comparative analysis of spatial learning, fear conditioning and step-down avoidance in wild-type mice and CNGA3 null mutants (CNGA3(-/-) ). CNGA3(-/-) mice showed normal basal synaptic transmission in the amygdala and the hippocampus. However, cornu Ammonis (CA1) hippocampal long-term potentiation (LTP) induced by a strong tetanus was significantly enhanced in CNGA3(-/-) mice as compared with their wild-type littermates. Unlike in the hippocampus, LTP was not significantly altered in the amygdala of CNGA3(-/-) mice. Enhanced hippocampal LTP did not coincide with changes in hippocampus-dependent learning, as both wild-type and mutant mice showed a similar performance in water maze tasks and contextual fear conditioning, except for a trend toward higher step-down latencies in a passive avoidance task. In contrast, CNGA3(-/-) mice showed markedly reduced freezing to the conditioned tone in the amygdala-dependent cued fear conditioning task. In conclusion, our study adds a new entry on the list of physiological functions of the CNGA3 channel. Despite the dissociation between physiological and behavioral parameters, our data describe a so far unrecognized role of CNGA3 in modulation of hippocampal plasticity and amygdala-dependent fear memory.

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Transmembrane Protein 147 (TMEM147) Is a Novel Component of the Nicalin-NOMO Protein Complex

JBC, 2010, 285, 26174-81 published on 20.08.2010
JBC, online article
Nicastrin and its relative Nicalin (Nicastrin-like protein) are both members of larger protein complexes, namely gamma-secretase and the Nicalin-NOMO (Nodal modulator) complex. The gamma- secretase complex, which contains Presenilin, APH-1 and PEN-2 in addition to Nicastrin, catalyzes the proteolytic cleavage of the transmembrane domain of various proteins including the beta-Amyloid Precursor Protein (APP) and Notch. Nicalin and its binding partner NOMO form a complex which was shown to modulate Nodal signaling in developing zebrafish embryos. Since its experimentally determined native size (200-220 kDa) could not be satisfyingly explained by the molecular weights of Nicalin (60 kDa) and NOMO (130 kDa), we searched in affinitypurified complex preparations for additional components in the low-molecular-weight range. A 22-kDa protein was isolated and identified by mass spectrometry as transmembrane protein 147 (TMEM147), a novel, highly conserved membrane protein with a putative topology similar to APH-1. Like Nicalin and NOMO it localizes to the endoplasmic reticulum and is expressed during early zebrafish development. Overexpression and knockdown experiments in cultured cells demonstrate a close relationship between the three proteins and suggest that they are components of the same complex. We present evidence that, similar to γ-secretase, its assembly is hierarchical starting with the formation of a Nicalin-NOMO intermediate. Nicalin appears to represent the limiting factor regulating the assembly rate by stabilizing the other two components. We conclude that TMEM147 is a novel core component of the Nicalin-NOMO complex further emphasizing its similarity with gamma-secretase.

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Quantification of the Sixth DNA Base Hydroxymethylcytosine in the Brain

Angewandte Chemie, 2010, 49, 31, 5375 - 77 published on 19.07.2010

Angewandte Chemie, online article

LC-MS has allowed the amount of the post-replicatively formed DNA base 5-hydroxymethylcytosine (see picture; left) to be quantified in brain tissue. The nucleoside is most abundant in areas that are associated with higher cognitive functions, and its content in mouse hippocampi seems to increase with age. The new method enables hydroxymethylcytosine to be quantified with unprecedented accuracy.

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An Olfactory Subsystem that Detects Carbon Disulfide and Mediates Food-Related Social Learning

Current Biology, 2010, doi:10.1016/j.cub.2010.06.021, published on 15.07.2010
Current Biology, online article
Olfactory signals influence social interactions in a variety of species. In mammals, pheromones and other social cues can promote mating or aggression behaviors; can communicate information about social hierarchies, genetic identity and health status; and can contribute to associative learning [1,2,3,4,5]. However, the molecular, cellular, and neural mechanisms underlying many olfactory-mediated social interactions remain poorly understood. Here, we report that a specialized olfactory subsystem that includes olfactory sensory neurons (OSNs) expressing the receptor guanylyl cyclase GC-D, the cyclic nucleotide-gated channel subunit CNGA3, and the carbonic anhydrase isoform CAII (GC-D+ OSNs) [6,7,8,9,10,11] is required for the acquisition of socially transmitted food preferences (STFPs) in mice. Using electrophysiological recordings from gene-targeted mice, we show that GC-D+ OSNs are highly sensitive to the volatile semiochemical carbon disulfide (CS2), a component of rodent breath and a known social signal mediating the acquisition of STFPs [12,13,14]. Olfactory responses to CS2 are drastically reduced in mice lacking GC-D, CNGA3, or CAII. Disruption of this sensory transduction cascade also results in a failure to acquire STFPs from either live or surrogate demonstrator mice or to exhibit hippocampal correlates of STFP retrieval [15]. Our findings indicate that GC-D+ OSNs detect chemosignals that facilitate food-related social interactions.

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ALS-associated fused in sarcoma (FUS) mutations disrupt Transportin-mediated nuclear import

The EMBO Journal, 2010, 29, 2841 - 57 published on 06.07.2010

The EMBO Journal, online article

Mutations in fused in sarcoma (FUS) are a cause of familial amyotrophic lateral sclerosis (fALS). Patients carrying point mutations in the C-terminus of FUS show neuronal cytoplasmic FUS-positive inclusions, whereas in healthy controls, FUS is predominantly nuclear. Cytoplasmic FUS inclusions have also been identified in a subset of frontotemporal lobar degeneration (FTLD-FUS). We show that a non-classical PY nuclear localization signal (NLS) in the C-terminus of FUS is necessary for nuclear import. The majority of fALS-associated mutations occur within the NLS and impair nuclear import to a degree that correlates with the age of disease onset. This presents the first case of disease-causing mutations within a PY-NLS. Nuclear import of FUS is dependent on Transportin, and interference with this transport pathway leads to cytoplasmic redistribution and recruitment of FUS into stress granules. Moreover, proteins known to be stress granule markers co-deposit with inclusions in fALS and FTLD-FUS patients, implicating stress granule formation in the pathogenesis of these diseases. We propose that two pathological hits, namely nuclear import defects and cellular stress, are involved in the pathogenesis of FUS-opathies.

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ALS-associated fused in sarcoma (FUS) mutations disrupt Transportin-mediated nuclear import

The EMBO Journal, 2010, 29, 2841 - 57 published on 06.07.2010

The EMBO Journal, online article

Mutations in fused in sarcoma (FUS) are a cause of familial amyotrophic lateral sclerosis (fALS). Patients carrying point mutations in the C-terminus of FUS show neuronal cytoplasmic FUS-positive inclusions, whereas in healthy controls, FUS is predominantly nuclear. Cytoplasmic FUS inclusions have also been identified in a subset of frontotemporal lobar degeneration (FTLD-FUS). We show that a non-classical PY nuclear localization signal (NLS) in the C-terminus of FUS is necessary for nuclear import. The majority of fALS-associated mutations occur within the NLS and impair nuclear import to a degree that correlates with the age of disease onset. This presents the first case of disease-causing mutations within a PY-NLS. Nuclear import of FUS is dependent on Transportin, and interference with this transport pathway leads to cytoplasmic redistribution and recruitment of FUS into stress granules. Moreover, proteins known to be stress granule markers co-deposit with inclusions in fALS and FTLD-FUS patients, implicating stress granule formation in the pathogenesis of these diseases. We propose that two pathological hits, namely nuclear import defects and cellular stress, are involved in the pathogenesis of FUS-opathies.

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Disruption of the olivo-cerebellar circuit by Purkinje neuron-specific ablation of BK channels

PNAS, 2010, 107, 27, 12323 - 28 published on 06.07.2010

PNAS, online article

The large-conductance voltage- and calcium-activated potassium (BK) channels are ubiquitously expressed in the brain and play an important role in the regulation of neuronal excitation. Previous work has shown that the total deletion of these channels causes an impaired motor behavior, consistent with a cerebellar dysfunction. Cellular analyses showed that a decrease in spike firing rate occurred in at least two types of cerebellar neurons, namely in Purkinje neurons (PNs) and in Golgi cells. To determine the relative role of PNs, we developed a cell-selective mouse mutant, which lacked functional BK channels exclusively in PNs. The behavioral analysis of these mice revealed clear symptoms of ataxia, indicating that the BK channels of PNs are of major importance for normal motor coordination. By using combined two-photon imaging and patch-clamp recordings in these mutant mice, we observed a unique type of synaptic dysfunction in vivo, namely a severe silencing of the climbing fiber–evoked complex spike activity. By performing targeted pharmacological manipulations combined with simultaneous patch-clamp recordings in PNs, we obtained direct evidence that this silencing of climbing fiber activity is due to a malfunction of the tripartite olivo-cerebellar feedback loop, consisting of the inhibitory synaptic connection of PNs to the deep cerebellar nuclei (DCN), followed by a projection of inhibitory DCN afferents to the inferior olive, the origin of climbing fibers. Taken together, our results establish an essential role of BK channels of PNs for both cerebellar motor coordination and feedback regulation in the olivo-cerebellar loop.

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Bepridil and Amiodarone Simultaneously Target the Alzheimer's Disease beta - and gamma-Secretase via Distinct Mechanisms

The Journal of Neuroscience, 2010, 30 (26), 8973-83, DOI:10.1523/JNEUROSCI.1199-10.2010 published on 30.06.2010

The Journal of Neuroscience, online article

The two proteases beta-secretase and gamma-secretase generate the amyloid beta peptide and are drug targets for Alzheimer’s disease. Here we tested the possibility of targeting the cellular environment of beta-secretase cleavage instead of the beta-secretase enzyme itself. beta-Secretase has an acidic pH optimum and cleaves the amyloid precursor protein in the acidic endosomes. We identified two drugs, bepridil and amiodarone, that are weak bases and are in clinical use as calcium antagonists. Independently of their calcium-blocking activity, both compounds mildly raised the membrane-proximal, endosomal pH and inhibited beta-secretase cleavage at therapeutically achievable concentrations in cultured cells, in primary neurons, and in vivo in guinea pigs. This shows that an alkalinization of the cellular environment could be a novel therapeutic strategy to inhibit beta-secretase. Surprisingly, bepridil and amiodarone also modulated gamma-secretase cleavage independently of endosomal alkalinization. Thus, both compounds act as dual modulators that simultaneously target beta- and gamma-secretase through distinct molecular mechanisms. In addition to Alzheimer’s disease, compounds with dual properties may also be useful for drug development targeting other membrane proteins.

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Genetic Reactivation of Cone Photoreceptors Restores Visual Responses in Retinitis Pigmentosa

Science, 2010, doi:10.1126/science.1190897, published on 24.06.2010
Science, online article
Retinitis pigmentosa refers to a diverse group of hereditary diseases that lead to incurable blindness, affecting two million people worldwide. As a common pathology, rod photoreceptors die early, whereas light-insensitive, morphologically altered cone photoreceptors persist longer. It is unknown if these cones are accessible for therapeutic intervention. Here, we show that expression of archaebacterial halorhodopsin in light-insensitive cones can substitute for the native phototransduction cascade and restore light sensitivity in mouse models of retinitis pigmentosa. Resensitized photoreceptors activate all retinal cone pathways, drive sophisticated retinal circuit functions (including directional selectivity), activate cortical circuits, and mediate visually guided behaviors. Using human ex vivo retinas, we show that halorhodopsin can reactivate light-insensitive human photoreceptors. Finally, we identified blind patients with persisting, light-insensitive cones for potential halorhodopsin-based therapy.

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Three-Amino Acid Spacing of Presenilin Endoproteolysis Suggests a General Stepwise Cleavage of gamma-Secretase-Mediated Intramembrane Proteolysis

The Journal of Neuroscience, 2010, 30 Issue 23, 7853-62 published on 09.06.2010
The Journal of Neuroscience, online article
Presenilin (PS1 or PS2) is the catalytic component of the gamma-secretase complex, which mediates the final proteolytic processing step leading to the Alzheimer's disease (AD)-characterizing amyloid β-peptide. PS is cleaved during complex assembly into its characteristic N- and C-terminal fragments. Both fragments are integral components of physiologically active gamma-secretase and harbor the two critical aspartyl residues of the active site domain. While the minimal subunit composition of gamma-secretase has been defined and numerous substrates were identified, the cellular mechanism of the endoproteolytic cleavage of PS is still unclear. We addressed this pivotal question by investigating whether familial AD (FAD)-associated PS1 mutations affect the precision of PS endoproteolysis in a manner similar to the way that such mutations shift the intramembrane cleavage of gamma-secretase substrates. We demonstrate that all FAD mutations investigated still allow endoproteolysis to occur. However, the precision of PS1 endoproteolysis is affected by PS1 mutations. Comparing the cleavage products generated by a variety of PS1 mutants revealed that specifically cleavages at positions 293 and 296 of PS1 are selectively affected. Systematic mutagenesis around the cleavage sites revealed a stepwise three amino acid spaced cleavage mechanism of PS endoproteolysis reminiscent to the epsilon-, zeta-, and gamma-cleavages described for typical gamma-secretase substrates, such as the beta-amyloid precursor protein. Our findings therefore suggest that intramembranous cleavage by gamma-secretase and related intramembrane-cleaving proteases may generally occur via stepwise endoproteolysis.

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Grueneberg Ganglion Neurons Are Finely Tuned Cold Sensors

The Journal of Neuroscience, 2010, doi:0.1523/​JNEUROSCI.0608-10.2010, published on 02.06.2010
The Journal of Neuroscience, online article
The Grueneberg ganglion is a newly appreciated nasal subsystem with neural connections to the olfactory forebrain, but its functional role has not been well defined. Here, we assess whether Grueneberg ganglion neurons (GGNs) function as thermosensors. By investigating the effect of acute temperature changes on the cytosolic Ca2+ concentration of genetically labeled mouse GGNs (either gender), we demonstrate that GGNs are thermosensory neurons specialized to detect a temperature decline within a given temperature window. Furthermore, GGNs comprise a relatively homogeneous cell population with respect to temperature sensitivity. GGNs do not respond to ligands of the temperature-sensitive TRP channels TRPM8 and TRPA1, suggesting a novel mechanism for temperature sensing. One possibility is a cGMP-mediated mechanism, as GGNs express the receptor guanylyl cyclase GC-G, the cGMP-sensitive phosphodiesterase PDE2 and the cGMP-sensitive channel CNGA3. Surprisingly, Cnga3-null mice show normal cooling-induced Ca2+ responses although cGMP-dependent Ca2+ increases are absent in these mice. Rather, the cooling-induced Ca2+ response of GGNs depends critically on the activity of a tetrodotoxin-sensitive voltage-gated sodium channel whereas the cGMP-dependent Ca2+ signal does not. These findings establish the Grueneberg ganglion as a sensory organ mediating cold-evoked neural responses, possibly in conjunction with the sensing of other stress- or fear-related chemical social cues.

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Induction of STAT3-related genes in fast degenerating cone photoreceptors of cpfl1 mice

Cellular and Molecular Life Sciences, 2010, doi:10.1007/s00018-010-0376-9, published on 14.05.2010
Cell. Mol. Life Sci., online article
Cone dystrophies are genetic diseases characterized by loss of cone photoreceptor function and severe impairment of daylight vision. Loss of function is accompanied by a progressive degeneration of cones limiting potential therapeutic interventions. In this study we combined microarray-based gene-expression analysis with electroretinography and immunohistochemistry to characterize the pathological processes in the cone photoreceptor function loss 1 (cpfl1) mouse model. The cpfl1-mouse is a naturally arising mouse mutant with a loss-of-function mutation in the cone-specific Pde6c gene. Cpfl1-mice displayed normal rod-specific light responses while cone-specific responses were strongly diminished. Despite the lack of a general retinal degeneration, the cone-specific functional defect resulted in a marked activation of GFAP, a hallmark of Müller-cell gliosis. Microarray-based network-analysis confirmed activation of Müller-glia-specific transcripts. Unexpectedly, we found up-regulation of the cytokine LIF and the anti-apoptotic transcription factor STAT3 in cpfl1 cone photoreceptors. We postulate that STAT3-related pathways are induced in cpfl1 cone photoreceptors to counteract degeneration.

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Christian Haass received an honorary doctorate from the University of Zurich

2010, published on 03.05.2010
Press release of the University of Zurich
CIPSM's Christian Haass received an honorary doctorate from the University of Zurich for his contributions to the characterisation of the intramembrane proteolysis and its importance in neurodegeneration. Congratulation Christian, well done!

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Dendritic organization of sensory input to cortical neurons in vivo

Nature, 2010, 464, 1307-1312 published on 29.04.2010
Nature, online article
In sensory cortex regions, neurons are tuned to specific stimulus features. For example, in the visual cortex, many neurons fire predominantly in response to moving objects of a preferred orientation. However, the characteristics of the synaptic input that cortical neurons receive to generate their output firing pattern remain unclear. Here we report a novel approach for the visualization and functional mapping of sensory inputs to the dendrites of cortical neurons in vivo. By combining high-speed two-photon imaging with electrophysiological recordings, we identify local subthreshold calcium signals that correspond to orientation-specific synaptic inputs. We find that even inputs that share the same orientation preference are widely distributed throughout the dendritic tree. At the same time, inputs of different orientation preference are interspersed, so that adjacent dendritic segments are tuned to distinct orientations. Thus, orientation-tuned neurons can compute their characteristic firing pattern by integrating spatially distributed synaptic inputs coding for multiple stimulus orientations.

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Methylene blue fails to inhibit Tau and polyglutamine protein-dependent toxicity in zebrafish

Neurobiol. Dis., 2010, doi:10.1016/j.nbd.2010.03.023 published on 08.04.2010
Neurobiology of Disease, online article
Methylene blue is an FDA approved compound with a variety of pharmacologic activities. It inhibits aggregation of several amyloidogenic proteins known to be deposited in neurodegenerative diseases. Recently, it has been proposed that methylene blue shows significant beneficial effects in a phase 2 clinical trial by slowing cognitive decline in Alzheimer's disease patients. To analyze its therapeutic potential, we investigated the effect of methylene blue on neurotoxicity in a zebrafish model for tauopathies. Transgenic expression of the frontotemporal dementia associated Tau-P301L mutation recapitulates a number of the pathological features observed in humans including abnormal phosphorylation and folding of Tau, tangle formation and Tau-dependent neuronal loss. Upon incubation of zebrafish larvae with methylene blue, neither abnormal phosphorylation nor neuronal cell loss, reduced neurite outgrowth or a swimming defect were rescued. Methylene blue is biologically active in zebrafish since it reduced aggregation of a huntingtin variant containing a stretch of 102 glutamine residues. However, although huntingtin aggregation was largely prevented by methylene blue, huntingtin-dependent toxicity was unaffected. Our findings are consistent with the hypothesis that toxicity is not necessarily associated with deposition of insoluble amyloid proteins.

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Expression of the anti-amyloidogenic secretase ADAM10 is suppressed by its 5′-untranslated region

J. Biol. Chem., 2010, doi: 10.1074/jbc.M110.110742 published on 26.03.2010
The Journal of Biological Chemistry, online article
Proteolytic processing of the amyloid precursor protein (APP) by α-secretase prevents formation of the amyloid β-peptide (Aβ), which is the main constituent of amyloid plaques in brains of Alzheimer′s disease (AD) patients. α-Secretase activity is decreased in AD and overexpression of the α-secretase ADAM10 (a disintegrin and metalloprotease) in an AD animal model prevents amyloid pathology. ADAM10 has a 444 nucleotides long,very GC-rich 5′ untranslated region (5′UTR) with two upstream open reading frames. Since similar properties of 5′UTRs are found in transcripts of many genes, which are regulated by translational control mechanisms, we asked whether ADAM10 expression is translationally controlled by its 5′UTR. We demonstrate that the 5′UTR of ADAM10 represses the rate of ADAM10 translation. In the absence of the 5′UTR we observed a significant increase of ADAM10 protein levels in HEK293 cells while mRNA levels were not changed. Moreover, the 5′UTR of ADAM10 inhibits translation of a luciferase reporter in an in vitro transcription / translation assay. Successive deletion of the first half of the ADAM10 5′UTR revealed a striking increase in ADAM10 protein expression in HEK293 cells, suggesting that this part of the 5′UTR contains inhibitory elements for translation. Moreover, we detect an enhanced α-secretase activity and consequently reduced Aβ levels in conditioned media of HEK293-APP cells expressing a 5′UTR-ADAM10 deletion construct lacking the first half of the 5′UTR. Thus, we provide evidence that the 5′UTR of ADAM10 may have an important role for posttranscriptional regulation of ADAM10 expression and consequently Aβ production.

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Beta-amyloid precursor protein mutants respond to gamma-secretase modulators

JBC, 2010, doi: 10.1074/jbc.M110.103283, published on 26.03.2010
JBC, online article
Pathogenic generation of the 42 amino acid variant of the amyloid beta-peptide (Abeta42) by beta- and gamma-secretase cleavage of the beta-amyloid precursor protein (APP) is believed to be causative for Alzheimer 's disease (AD). Lowering of Abeta42 production by gamma-secretase modulators (GSMs) is a hopeful approach towards AD treatment. The mechanism of GSM action is not fully understood. Moreover, whether GSMs target the Abeta domain is controversial. To further our understanding of the mode of action of GSMs and the cleavage mechanism of gamma-secretase, we analyzed mutations located at different positions of the APP TMD around or within the Abeta domain regarding their response to GSMs. We found that Abeta42-increasing familial AD (FAD) mutations of the gamma-secretase cleavage site domain responded robustly to Abeta42-lowering GSMs, especially to the potent compound GSM-1, irrespective of the amount of Abeta42 produced. We thus expect that FAD patients carrying mutations at the gamma-secretase cleavage sites of APP should respond to GSM-based therapeutic approaches. Systematic phenylalanine scanning mutagenesis of this region revealed a high permissiveness to GSM-1 and demonstrated a complex mechanism of GSM action as also other Abeta species (Abeta41, Abeta39) could be lowered besides Abeta42. Moreover, certain mutations simultaneously increased Abeta42 and the shorter peptide Abeta38 arguing that the proposed precursor-product relationship of these Abeta species is not general. Finally, mutations of residues in the proposed GSM-binding site implicated in Abeta42 generation (G29, G33) and potentially in GSM-binding (K28) were also responsive to GSMs, a finding that may question APP substrate targeting of GSMs.

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Transgenic Zebrafish as a Novel Animal Model to Study Tauopathies and Other Neurodegenerative Disorders in vivo

Neurodegenerative Diseases, 2010, DOI: 10.1159/000285515, Vol. 7, No. 1-3, published on 18.02.2010
Neurodegenerative Diseases online article
Our ageing society is confronted with a dramatic increase in patients suffering from tauopathies such as Alzheimer’s disease, frontotemporal dementia and others. Typical neuropathological lesions including tangles composed of hyperphosphorylated tau protein as well as severe neuronal cell death characterize these disorders. No mechanism-based cures are available at present. Genetically modified animals are invaluable models to understand the molecular disease mechanisms and to screen for modifying compounds. We recently introduced tau-transgenic zebrafish as a novel model for tauopathies. Our model allows recapitulating key pathological features of tauopathies within an extremely short time. Moreover, life imaging of tau-dependent neuronal cell death was performed for the very first time. This demonstrated tau-dependent neuronal cell loss independent of tangle formation. Finally, we exemplified that the zebrafish frontotemporal dementia model can be used to screen for drugs that prevent abnormal tau phosphorylation and neuronal cell death.

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The cyclic nucleotide-gated ion channel CNGA3 contributes to coolness-induced responses of Grueneberg ganglion neurons

Cellular and Molecular Life Sciences, 2010, doi:10.1007/s00018-010-0296-8, published on 18.02.2010
Cell. Mol. Life Sci., online article
Localized to the vestibule of the nasal cavity, neurons of the Grueneberg ganglion (GG) respond to cool ambient temperatures. The molecular mechanisms underlying this thermal response are still elusive. Recently, it has been suggested that cool temperatures may activate a cyclic guanosine monophosphate (cGMP) pathway in the GG, which would be reminiscent of thermosensory neurons in Caenorhabditis elegans. In search for other elements of such a cascade, we have found that the cyclic nucleotide-gated ion channel CNGA3 was strongly expressed in the GG and that expression of CNGA3 was confined to those cells that are responsive to coolness. Further experiments revealed that the response of GG neurons to cool temperatures was significantly reduced in CNGA3-deficient mice compared to wild-type conspecifics. The observation that a cGMP-activated non-selective cation channel significantly contributes to the coolness-evoked response in GG neurons strongly suggests that a cGMP cascade is part of the transduction process.

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Rapid time course of action potentials in spines and remote dendrites of mouse visual cortex neurons

The Journal of Physiology, 2010, doi: 10.1113/jphysiol.2009.184960 published on 15.02.2010
The Journal of Physiology, online article
Axonally initiated action potentials back-propagate into spiny dendrites of central mammalian neurons and thereby regulate plasticity at excitatory synapses on individual spines as well as linear and supralinear integration of synaptic inputs along dendritic branches. Thus, the electrical behavior of individual dendritic spines and terminal dendritic branches is critical for the integrative function of nerve cells. The actual dynamics of action potentials in spines and terminal branches, however, are not entirely clear, mostly because electrode recording from such small structures is not feasible. Additionally, the available membrane potential imaging techniques are limited in their sensitivity and require substantial signal averaging for the detection of electrical events at the spatial scale of individual spines. We made a critical improvement in voltage-sensitive dye imaging technique to achieve multisite recordings of backpropagating action potentials from individual dendritic spines at a high frame rate. With this approach, we obtained direct evidence that in layer 5 pyramidal neurons from the visual cortex of juvenile mice the rapid time-course of somatic action potentials is preserved throughout all cellular compartments, including dendritic spines and terminal branches of basal and apical dendrites. The rapid time-course of the action potential in spines may be a critical determinant for the precise regulation of spike-timing dependent synaptic plasticity within a narrow time window.

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The Retinitis Pigmentosa Mutation c.3444+1G>A in CNGB1 Results in Skipping of Exon 32

PLoS ONE, 2010, doi:10.1371/journal.pone.0008969, published on 29.01.2010
PLoS ONE, online article
Retinitis pigmentosa (RP) is a severe hereditary eye disorder characterized by progressive degeneration of photoreceptors and subsequent loss of vision. Two of the RP associated mutations were found in the CNGB1 gene that encodes the B subunit of the rod cyclic nucleotide-gated channel (CNGB1a). One of them (c.3444+1G>A) is located at the donor site of exon 32 and has been proposed to result in a frameshift and truncation of the last 28 aa of the corresponding protein. However, this ambiguous conclusion was not verified by experimental data. Recently, another study reported that the last 28 aa of CNGB1a harbor a motif required for the proper targeting of this subunit to rod photoreceptor outer segments. This suggests that defective targeting is the major cause for the RP phenotype in affected patients. Here, we investigated the splicing of c.3444+1G>A by exon trapping experiments and could demonstrate that instead of the proposed truncation of the last 28 aa this mutation leads to replacement of the last 170 aa of CNGB1a by 68 unrelated amino acids. The 170 aa deletion covers the complete distal C-terminus including the last 10 aa of an important alpha (αC) helix within the ligand-binding domain of CNGB1a. When expressed in a heterologous expression system the corresponding mutant full-length CNGB1a subunit was more susceptible to proteosomal degradation compared to the wild-type counterpart. In conclusion, our experimental data do not support the hypothesis proposed by the original study on the c.3444+1G>A mutation. Based on this, we suggest that apart from the defective targeting other mechanisms may be responsible for the RP phenotype in affected individuals.

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In Vivo Analysis of Cone Survival in Mice

IOVS, 2010, doi:10.1167/iovs.09-4003, published on 05.01.2010
IOVS, online article
Purpose. To identify individual cone photoreceptors in a transgenic mouse line in vivo based on selective expression of green fluorescent protein (GFP) using cSLO (confocal scanning laser ophthalmoscopy) and to use this approach to monitor cone cell fate in mouse models of retinal degeneration. Methods. Transgenic mice expressing GFP under the control of a red-green opsin promoter (RG-GFP mice) were analyzed in vivo with respect to GFP expression in cone cells using cSLO and functional integrity using electroretinography (ERG). Histology was performed to correlate the pattern of GFP expression with light microscopic data. Longitudinal monitoring of cone survival was evaluated in crossbreds of RG-GFP mice with cpfl1 and Rpe65−/− mutant mice, respectively. Results. The authors found that RG-GFP transgenic mice had a stable GFP expression that did not interfere with retinal function up to at least 3 months of age. Thus, a longitudinal analysis of cone degeneration in individual RG cpfl1 and RG Rpe65−/− cross-bred mice in vivo was successfully performed and demonstrated distinct time frames of cone survival in the particular mouse model. Conclusions. Monitoring GFP expression in cone photoreceptor cells, such as in the RG-GFP mouse, is a promising in vivo approach for the analysis of cone survival in mice.

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LMUexcellent
TU München
MPG
Helmholz Muenchen
MPI of Neurobiology
MPI of Biochemistry