Content
2012
Characterization of neurite outgrowth and ectopic synaptogenesis in response to photoreceptor dysfunction
03.12.2012
Cellular and Molecular Life Sciences,
2012,
10.1007/s00018-012-1230-z,
published on 03.12.2012
Cellular and Molecular Life Sciences, online article
Cellular and Molecular Life Sciences, online article
In the mammalian retina, light signals generated in photoreceptors are passed to bipolar and horizontal cells via synaptic contacts. In various pathological conditions, these second-order neurons extend neurites into the outer nuclear layer (ONL). However, the molecular events associated with this neurite outgrowth are not known. Here, we characterized the morphological synaptic changes in the CNGA3/CNGB1 double-knockout (A3B1) mouse, a model of retinitis pigmentosa. In these mice, horizontal cells looked normal until postnatal day (p) 11, but started growing neurites into the ONL 1 day later. At p28, the number of sprouting processes decreased, but the remaining sprouts developed synapse-like contacts at rod cell bodies, with an ultrastructural appearance reminiscent of ribbon synapses. Hence, neurite outgrowth and ectopic synaptogenesis in the A3B1 retina were precisely timed events starting at p12 and p28, respectively. We therefore performed microarray analysis of retinal gene expression in A3B1 and wild-type mice at those ages to evaluate the genomic response underlying these two events. This analysis identified 163 differentially regulated genes in the A3B1 retina related to neurite outgrowth or plasticity of synapses. The global changes in gene expression in the A3B1 retina were consistent with activation of signaling pathways related to Tp53, Smad, and Stat3. Moreover, key molecules of these signaling pathways could be localized at or in close proximity to outgrowing neurites. We therefore propose that Tp53, Smad, and Stat3 signaling pathways contribute to the synaptic plasticity in the A3B1 retina.
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In Vivo Imaging of Disease-Related Mitochondrial Dynamics in a Vertebrate Model System
14.11.2012
The Journal of Neuroscience,
2012,
doi: 10.1523/JNEUROSCI.1327-12.2012.,
32(46):16203-12
published on 14.11.2012
The Journal of Neuroscience, online article
The Journal of Neuroscience, online article
Mitochondria provide ATP, maintain calcium homeostasis, and regulate apoptosis. Neurons, due to their size and complex geometry, are particularly dependent on the proper functioning and distribution of mitochondria. Thus disruptions of these organelles and their transport play a central role in a broad range of neurodegenerative diseases. While in vitro studies have greatly expanded our knowledge of mitochondrial dynamics, our understanding in vivo remains limited. To address this shortcoming, we developed tools to study mitochondrial dynamics in vivo in optically accessible zebrafish. We demonstrate here that our newly generated tools, including transgenic "MitoFish," can be used to study the in vivo "life cycle" of mitochondria and allows identifying pharmacological and genetic modulators of mitochondrial dynamics. Furthermore we observed profound mitochondrial transport deficits in real time in a zebrafish tauopathy model. By rescuing this phenotype using MARK2 (microtubule-affinity regulating kinase 2), we provide direct in vivo evidence that this kinase regulates axonal transport in a Tau-dependent manner. Thus, our approach allows detailed studies of the dynamics of mitochondria in their natural environment under normal and disease conditions.
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Isotope-Based Analysis of Modified tRNA Nucleosides Correlates Modification Density with Translational Efficiency
04.10.2012
Angew. Chem.,
2012,
DOI: 10.1002/anie.201203769,
Volume 51, Issue 44, pages 11162–11165,
published on 04.10.2012
Transfer RNAs (tRNAs) are adapter molecules needed to translate genetic information into a peptide sequence. At the ribosome, the anticodon of each tRNA reads the corresponding codon of the messenger RNA. This anticodon– codon interaction allows the ribosome_s large subunit to catalyze amide-bond formation between the cognate amino acids present at the 3’ terminus of aminoacyl-tRNAs and the growing peptide chain. The tRNA adapters required for this process display a surprisingly large chemical diversity. Aside from the four canonical nucleosides A, C,G, and U, more than 100 modified nucleosides are key constituents. The most diverse and complex chemical structures are found in the anticodon stem-loop either in the anticodon at the wobble position or directly adjacent to the 3’ position of the anticodon, suggesting that here the chemical complexity is necessary to establish translational fidelity. The ribosome seems to need the modified anticodon region to better distinguish correctly base-paired tRNA from mispaired interactions in order to prohibit, for example, codon-slippage processes that would lead to frameshifts.
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LOTOS-based two-photon calcium imaging of dendritic spines in vivo
13.09.2012
Nature Protocols,
2012,
doi:10.1038/nprot.2012.106,
7, 1818–1829
published on 13.09.2012
Nature Protocols, online article
Nature Protocols, online article
Neurons in the mammalian brain receive thousands of synaptic inputs on their dendrites. In many types of neurons, such as cortical pyramidal neurons, excitatory synapses are formed on fine dendritic protrusions called spines. Usually, an individual spine forms a single synaptic contact with an afferent axon. In this protocol, we describe a recently established experimental procedure for measuring intracellular calcium signals from dendritic spines in cortical neurons in vivo by using a combination of two-photon microscopy and whole-cell patch-clamp recordings. We have used mice as an experimental model system, but the protocol may be readily adapted to other species. This method involves data acquisition at high frame rates and low-excitation laser power, and is termed low-power temporal oversampling (LOTOS). Because of its high sensitivity of fluorescence detection and reduced phototoxicity, LOTOS allows for prolonged and stable calcium imaging in vivo. Key aspects of the protocol, which can be completed in 5–6 h, include the use of a variant of high-speed two-photon imaging, refined surgery procedures and optimized tissue stabilization.
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Complex Regulation of Voltage-dependent Activation and Inactivation Properties of Retinal Voltage-gated Cav1.4 L-type Ca2+ Channels by Ca2+-binding Protein 4 (CaBP4)
30.08.2012
The Journal of Biological Chemistry,
2012,
doi: 10.1074/jbc.M112.392811,
VOL. 287, NO. 43, pp. 36312–36321
published on 30.08.2012
The Journal of Biological Chemistry, online article
The Journal of Biological Chemistry, online article
Cav1.4 L-type Ca2+ channels are crucial for synaptic transmission in retinal photoreceptors and bipolar neurons. Recent studies suggest that the activity of this channel is regulated by the Ca2+-binding protein 4 (CaBP4). In the present study, we explored this issue by examining functional effects of CaBP4 on heterologously expressed Cav1.4. We show that CaBP4 dramatically increases Cav1.4 channel availability. This effect crucially depends on the presence of the C-terminal ICDI (inhibitor of Ca2+-dependent inactivation) domain of Cav1.4 and is absent in a Cav1.4 mutant lacking the ICDI. Using FRET experiments, we demonstrate that CaBP4 interacts with the IQ motif of Cav1.4 and that it interferes with the binding of the ICDI domain. Based on these findings, we suggest that CaBP4 increases Cav1.4 channel availability by relieving the inhibitory effects of the ICDI domain on voltage-dependent Cav1.4 channel gating. We also functionally characterized two CaBP4 mutants that are associated with a congenital variant of human night blindness and other closely related nonstationary retinal diseases. Although both mutants interact with Cav1.4 channels, the functional effects of CaBP4 mutants are only partially preserved, leading to a reduction of Cav1.4 channel availability and loss of function. In conclusion, our study sheds new light on the functional interaction between CaBP4 and Cav1.4. Moreover, it provides insights into the mechanism by which CaBP4 mutants lead to loss of Cav1.4 function and to retinal disease.
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Biocompatibility of a genetically encoded calcium indicator in a transgenic mouse model
28.08.2012
Nature Communications,
2012,
doi:10.1038/ncomms2035,
3, Article number: 1031
published on 28.08.2012
Nature Communications, online article
Nature Communications, online article
Engineering efforts of genetically encoded calcium indicators predominantly focused on enhancing fluorescence changes, but how indicator expression affects the physiology of host organisms is often overlooked. Here, we demonstrate biocompatibility and widespread functional expression of the genetically encoded calcium indicator TN-XXL in a transgenic mouse model. To validate the model and characterize potential effects of indicator expression we assessed both indicator function and a variety of host parameters, such as anatomy, physiology, behaviour and gene expression profiles in these mice. We also demonstrate the usefulness of primary cells and organ explants prepared from these mice for imaging applications. Although we find mild signatures of indicator expression that may be further reduced in future sensor generations, the 'green' indicator mice generated provide a well-characterized resource of primary cells and tissues for in vitro and in vivo calcium imaging applications.
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Gene Therapy Restores Vision and Delays Degeneration in the CNGB1-/- Mouse Model of Retinitis Pigmentosa
16.07.2012
Hum. Mol. Genet.,
2012,
doi: 10.1093/hmg/dds290,
published on 16.07.2012
Retinitis pigmentosa (RP) is a group of genetically heterogeneous severe retinal diseases commonly leading to legal blindness. Mutations in the CNGB1a subunit of the rod cyclic nucleotide-gated (CNG) channel have been found to cause RP in patients. Here, we demonstrate the efficacy of gene therapy as a potential treatment for RP by means of recombinant adeno-associated viral (AAV) vectors in the CNGB1 knockout (CNGB1-/-) mouse model. To enable efficient packaging and rod-specific expression of the relatively large CNGB1a cDNA (approx. 4 kb), we used an AAV expression cassette with a short rod-specific promoter and short regulatory elements. After injection of therapeutic AAVs into the subretinal space of two-week-old CNGB1-/- mice, we assessed the restoration of the visual system by analyzing i) CNG channel expression and localization, ii) retinal function and morphology and iii) vision-guided behavior. We found that the treatment not only led to expression of full-length CNGB1a, but also restored normal levels of the previously degraded CNGA1 subunit of the rod CNG channel. Both proteins co-localized in rod outer segments and formed regular CNG channel complexes within the treated area of the CNGB1-/- retina, leading to significant morphological preservation and a delay of retinal degeneration. In the electroretinographic analysis (ERG), we also observed restoration of rod-driven light responses. Finally, treated CNGB1-/- mice performed significantly better than untreated mice in a rod-dependent vision-guided behavior test. In summary, this work provides a proof-of-concept for the treatment of rod channelopathy-associated retinitis pigmentosa by AAV-mediated gene replacement.
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Dendritic spines: from structure to in vivo function
13.07.2012
EMBO reports,
2012,
doi:10.1038/embor.2012.102,
13, 699 - 708
published on 13.07.2012
EMBO reports, online article
EMBO reports, online article
Dendritic spines arise as small protrusions from the dendritic shaft of various types of neuron and receive inputs from excitatory axons. Ever since dendritic spines were first described in the nineteenth century, questions about their function have spawned many hypotheses. In this review, we introduce understanding of the structural and biochemical properties of dendritic spines with emphasis on components studied with imaging methods. We then explore advances in in vivo imaging methods that are allowing spine activity to be studied in living tissue, from super-resolution techniques to calcium imaging. Finally, we review studies on spine structure and function in vivo. These new results shed light on the development, integration properties and plasticity of spines.
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Regulation of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) Channel Activity by cCMP
19.06.2012
The Journal of Biological Chemistry,
2012,
doi: 10.1074/jbc.M112.357129,
VOL. 287, NO. 32, pp. 26506–26512,
published on 19.06.2012
The Journal of Biological Chemistry, online article
The Journal of Biological Chemistry, online article
Activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated in vivo by direct binding of the second messenger cAMP. This process plays a fundamental role in the fine-tuning of HCN channel activity and is critical for the modulation of cardiac and neuronal rhythmicity. Here, we identify the pyrimidine cyclic nucleotide cCMP as another regulator of HCN channels. We demonstrate that cCMP shifts the activation curves of two members of the HCN channel family, HCN2 and HCN4, to more depolarized voltages. Moreover, cCMP speeds up activation and slows down deactivation kinetics of these channels. The two other members of the HCN channel family, HCN1 and HCN3, are not sensitive to cCMP. The modulatory effect of cCMP is reversible and requires the presence of a functional cyclic nucleotide-binding domain. We determined an EC50 value of ∼30 μm for cCMP compared with 1 μm for cAMP. Notably, cCMP is a partial agonist of HCN channels, displaying an efficacy of ∼0.6. cCMP increases the frequency of pacemaker potentials from isolated sinoatrial pacemaker cells in the presence of endogenous cAMP concentrations. Electrophysiological recordings indicated that this increase is caused by a depolarizing shift in the activation curve of the native HCN current, which in turn leads to an enhancement of the slope of the diastolic depolarization of sinoatrial node cells. In conclusion, our findings establish cCMP as a gating regulator of HCN channels and indicate that this cyclic nucleotide has to be considered in HCN channel-regulated processes.
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Critical role of soluble amyloid-β for early hippocampal hyperactivity in a mouse model of Alzheimer’s disease
16.05.2012
PNAS,
2012,
doi: 10.1073/pnas.1206171109,
8740–8745 , vol. 109 , no. 22
published on 16.05.2012
PNAS, online article
PNAS, online article
Alzheimer’s disease (AD) is characterized by a progressive dysfunction of central neurons. Recent experimental evidence indicates that in the cortex, in addition to the silencing of a fraction of neurons, other neurons are hyperactive in amyloid-β (Aβ) plaque-enriched regions. However, it has remained unknown what comes first, neuronal silencing or hyperactivity, and what mechanisms might underlie the primary neuronal dysfunction. Here we examine the activity patterns of hippocampal CA1 neurons in a mouse model of AD in vivo using two-photon Ca2+ imaging. We found that neuronal activity in the plaque-bearing CA1 region of older mice is profoundly altered. There was a marked increase in the fractions of both silent and hyperactive neurons, as previously also found in the cortex. Remarkably, in the hippocampus of young mice, we observed a selective increase in hyperactive neurons already before the formation of plaques, suggesting that soluble species of Aβ may underlie this impairment. Indeed, we found that acute treatment with the γ-secretase inhibitor LY-411575 reduces soluble Aβ levels and rescues the neuronal dysfunction. Furthermore, we demonstrate that direct application of soluble Aβ can induce neuronal hyperactivity in wild-type mice. Thus, our study identifies hippocampal hyperactivity as a very early functional impairment in AD transgenic mice and provides direct evidence that soluble Aβ is crucial for hippocampal hyperactivity.
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Role of TRPML and Two-Pore Channels in Endolysosomal Cation Homeostasis
19.04.2012
The Journal of Pharmacology and Experimental Therapeutics,
2012,
doi: 10.1124/jpet.112.192880,
Vol. 342, No. 2
published on 19.04.2012
The Journal of Pharmacology and Experimental Therapeutics, online article
The Journal of Pharmacology and Experimental Therapeutics, online article
The transient receptor potential (TRP) channels TRPML1, TRPML2, and TRPML3 (also called mucolipins 1–3 or MCOLN1–3) are nonselective cation channels. Mutations in the Trpml1 gene cause mucolipidosis type IV in humans with clinical features including psychomotor retardation, corneal clouding, and retinal degeneration, whereas mutations in the Trpml3 gene cause deafness, circling behavior, and coat color dilution in mice. No disease-causing mutations are reported for the Trpml2 gene. Like TRPML channels, which are expressed in the endolysosomal pathway, two-pore channels (TPCs), namely TPC1, TPC2, and TPC3, are found in intracellular organelles, in particular in endosomes and lysosomes. Both TRPML channels and TPCs may function as calcium/cation release channels in endosomes, lysosomes, and lysosome-related organelles with TRPMLs being activated by phosphatidylinositol 3,5-bisphosphate and regulated by pH and TPCs being activated by nicotinic acid adenine dinucleotide phosphate in a calcium- and pH-dependent manner. They may also be involved in endolysosomal transport and fusion processes, e.g., as intracellular calcium sources. Currently, however, the exact physiological roles of TRPML channels and TPCs remain quite elusive, and whether TRPML channels are purely endolysosomal ion channels or whether they may also be functionally active at the plasma membrane in vivo remains to be determined.
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Staged decline of neuronal function in vivo in an animal model of Alzheimer's disease
10.04.2012
Nature Communications,
2012,
doi:10.1038/ncomms1783,
3; Article number: 774
published on 10.04.2012
Nature Communications, online article
Nature Communications, online article
The accumulation of amyloid-β in the brain is an essential feature of Alzheimer’s disease. However, the impact of amyloid-β-accumulation on neuronal dysfunction on the single cell level in vivo is poorly understood. Here we investigate the progression of amyloid-β load in relation to neuronal dysfunction in the visual system of the APP23×PS45 mouse model of Alzheimer’s disease. Using in vivo two-photon calcium imaging in the visual cortex, we demonstrate that a progressive deterioration of neuronal tuning for the orientation of visual stimuli occurs in parallel with the age-dependent increase of the amyloid-β load. Importantly, we find this deterioration only in neurons that are hyperactive during spontaneous activity. This impairment of visual cortical circuit function also correlates with pronounced deficits in visual-pattern discrimination. Together, our results identify distinct stages of decline in sensory cortical performance in vivo as a function of the increased amyloid-β-load.
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Endoplasmic Reticulum Stress-associated Cone Photoreceptor Degeneration in Cyclic Nucleotide-gated Channel Deficiency
09.04.2012
JBC,
2012,
doi: 10.1074/jbc.M112.342220,
Vol 287, Number 22
published on 09.04.2012
JBC, online article
JBC, online article
Cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 account for >70% of all known cases of achromatopsia. Cones degenerate in achromatopsia patients and in CNGA3−/− and CNGB3−/− mice. This work investigates the molecular basis of cone degeneration in CNG channel deficiency. As cones comprise only 2–3% of the total photoreceptor population in the wild-type mouse retina, we generated mouse lines with CNG channel deficiency on a cone-dominant background, i.e. CNGA3−/−/Nrl−/− and CNGB3−/−/Nrl−/− mice. The retinal phenotype and potential cell death pathways were examined by functional, biochemical, and immunohistochemical approaches. CNGA3−/−/Nrl−/− and CNGB3−/−/Nrl−/− mice showed impaired cone function, opsin mislocalization, and cone degeneration similar to that in the single knock-out mice. The endoplasmic reticulum stress marker proteins, including Grp78/Bip, phospho-eIF2α, phospho-IP3R, and CCAAT/enhancer-binding protein homologous protein, were elevated significantly in CNGA3−/−/Nrl−/− and CNGB3−/−/Nrl−/− retinas, compared with the age-matched (postnatal 30 days) Nrl−/− controls. Along with these, up-regulation of the cysteine protease calpains and cleavage of caspase-12 and caspase-7 were found in the channel-deficient retinas, suggesting an endoplasmic reticulum stress-associated apoptosis. In addition, we observed a nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G in CNGA3−/−/Nrl−/− and CNGB3−/−/Nrl−/− retinas, implying a mitochondrial insult in the endoplasmic reticulum stress-activated cell death process. Taken together, our findings suggest a crucial role of endoplasmic reticulum stress in cone degeneration associated with CNG channel deficiency.
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Imaging Calcium in Neurons
08.03.2012
Neuron,
2012,
doi:10.1016/j.neuron.2012.02.011,
Volume 73, Issue 5, 862-885
published on 08.03.2012
Neuron, online article
Neuron, online article
Calcium ions generate versatile intracellular signals that control key functions in all types of neurons. Imaging calcium in neurons is particularly important because calcium signals exert their highly specific functions in well-defined cellular subcompartments. In this Primer, we briefly review the general mechanisms of neuronal calcium signaling. We then introduce the calcium imaging devices, including confocal and two-photon microscopy as well as miniaturized devices that are used in freely moving animals. We provide an overview of the classical chemical fluorescent calcium indicators and of the protein-based genetically encoded calcium indicators. Using application examples, we introduce new developments in the field, such as calcium imaging in awake, behaving animals and the use of calcium imaging for mapping single spine sensory inputs in cortical neurons in vivo. We conclude by providing an outlook on the prospects of calcium imaging for the analysis of neuronal signaling and plasticity in various animal models.
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Axonal transport deficits and degeneration can evolve independently in mouse models of amyotrophic lateral sclerosis
27.02.2012
Axonal transport deficits have been reported in many neurodegenerative conditions and are widely assumed to be an immediate causative step of axon and synapse loss. By imaging changes in axonal morphology and organelle transport over time in several animal models of amyotrophic lateral sclerosis (ALS), we now find that deficits in axonal transport of organelles (mitochondria, endosomes) and axon degeneration can evolve independently. This conclusion rests on the following results: (i) Axons can survive despite long-lasting transport deficits: In the SODG93A model of ALS, transport deficits are detected soon after birth, months before the onset of axon degeneration. (ii) Transport deficits are not necessary for axon degeneration: In the SODG85R model of ALS, motor axons degenerate, but transport is unaffected. (iii) Axon transport deficits are not sufficient to cause immediate degeneration: In mice that overexpress wild-type superoxide dismutase-1 (SODWT), axons show chronic transport deficits, but survive. These data suggest that disturbances of organelle transport are not a necessary step in the emergence of motor neuron degeneration.
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CNGA3 Deficiency Affects Cone Synaptic Terminal Structure and Function and Leads to Secondary Rod Dysfunction and Degeneration
12.01.2012
Investigative Ophthalmology & Visual Science,
2012,
doi: 10.1167/iovs.11-8168,
Vol. 53, No. 3
published on 12.01.2012
Investigative Ophthalmology & Visual Science, online article
Investigative Ophthalmology & Visual Science, online article
Purpose. To investigate rod function and survival after cone dysfunction and degeneration in a mouse model of cone cyclic nucleotide-gated (CNG) channel deficiency.
Methods. Rod function and survival in mice with cone CNG channel subunit CNGA3 deficiency (CNGA3−/− mice) were evaluated by electroretinographic (ERG), morphometric, and Western blot analyses. The arrangement, integrity, and ultrastructure of photoreceptor terminals were investigated by immunohistochemistry and electron microscopy.
Results. The authors found loss of cone function and cone death accompanied by impairment of rods and rod-driven signaling in CNGA3−/− mice. Scotopic ERG b-wave amplitudes were reduced by 15% at 1 month, 30% at 6 months, and 40% at 9 months and older, while scotopic a-wave amplitudes were decreased by 20% at 9 months, compared with ERGs of age-matched wild-type mice. Outer nuclear layer thickness in CNGA3−/− retina was reduced by 15% at 12 months compared with age-matched wild-type controls. This was accompanied by a 30%–40% reduction in expression of rod-specific proteins, including rhodopsin, rod transducin α-subunit, and glutamic acid-rich protein (GARP). Cone terminals in the CNGA3−/− retina showed a progressive loss of neurochemical and ultrastructural integrity. Abnormalities were observed as early as 1 month. Disorganized rod terminal ultrastructure was noted by 12 months.
Conclusions. These findings demonstrate secondary rod impairment and degeneration after cone degeneration in mice with cone CNG channel deficiency. Loss of cone phototransduction accompanies the compromised integrity of cone terminals. With time, rod synaptic structure, function, and viability also become compromised.
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