Human autoantibodies to amphiphysin induce defective presynaptic vesicle dynamics and composition.

Stiff-person syndrome is the prototype of a central nervous system disorder with autoantibodies targeting presynaptic antigens. Patients with paraneoplastic stiff-person syndrome may harbour autoantibodies to the BAR (Bin/Amphiphysin/Rvs) domain protein amphiphysin, which target its SH3 domain. These patients have neurophysiological signs of compromised central inhibition and respond to symptomatic treatment with medication enhancing GABAergic transmission. High frequency neurotransmission as observed in tonic GABAergic interneurons relies on fast exocytosis of neurotransmitters based on compensatory endocytosis. As amphiphysin is involved in clathrin-mediated endocytosis, patient autoantibodies are supposed to interfere with this function, leading to disinhibition by reduction of GABAergic neurotransmission. We here investigated the effects of human anti-amphiphysin autoantibodies on structural components of presynaptic boutons ex vivo and in vitro using electron microscopy and super-resolution direct stochastic optical reconstruction microscopy. Ultrastructural analysis of spinal cord presynaptic boutons was performed after in vivo intrathecal passive transfer of affinity-purified human anti-amphiphysin autoantibodies in rats and revealed signs of markedly disabled clathrin-mediated endocytosis. This was unmasked at high synaptic activity and characterized by a reduction of the presynaptic vesicle pool, clathrin coated intermediates, and endosome-like structures. Super-resolution microscopy of inhibitory GABAergic presynaptic boutons in primary neurons revealed that specific human anti-amphiphysin immunoglobulin G induced an increase of the essential vesicular protein synaptobrevin 2 and a reduction of synaptobrevin 7. This constellation suggests depletion of resting pool vesicles and trapping of releasable pool vesicular proteins at the plasma membrane. Similar effects were found in amphiphysin-deficient neurons from knockout mice. Application of specific patient antibodies did not show additional effects. Blocking alternative pathways of clathrin-independent endocytosis with brefeldin A reversed the autoantibody induced effects on molecular vesicle composition. Endophilin as an interaction partner of amphiphysin showed reduced clustering within presynaptic terminals. Collectively, these results point towards an autoantibody-induced structural disorganization in GABAergic synapses with profound changes in presynaptic vesicle pools, activation of alternative endocytic pathways, and potentially compensatory rearrangement of proteins involved in clathrin-mediated endocytosis. Our findings provide novel insights into synaptic pathomechanisms in a prototypic antibody-mediated central nervous system disease, which may serve as a proof-of-principle example in this evolving group of autoimmune disorders associated with autoantibodies to synaptic antigens.

Functional and structural characterization of axonal opioid receptors as targets for analgesia.

BACKGROUND: Opioids are the gold standard for the treatment of acute pain despite serious side effects in the central and enteric nervous system. µ-opioid receptors (MOPs) are expressed and functional at the terminals of sensory axons, when activated by exogenous or endogenous ligands. However, the presence and function of MOP along nociceptive axons remains controversial particularly in naïve animals. Here, we characterized axonal MOPs by immunofluorescence, ultrastructural, and functional analyses. Furthermore, we evaluated hypertonic saline as a possible enhancer of opioid receptor function. RESULTS: Comparative immunolabeling showed that, among several tested antibodies, which all provided specific MOP detection in the rat central nervous system (CNS), only one monoclonal MOP-antibody yielded specificity and reproducibility for MOP detection in the rat peripheral nervous system including the sciatic nerve. Double immunolabeling documented that MOP immunoreactivity was confined to calcitonin gene-related peptide (CGRP) positive fibers and fiber bundles. Almost identical labeling and double labeling patterns were found using mcherry-immunolabeling on sciatic nerves of mice producing a MOP-mcherry fusion protein (MOP-mcherry knock-in mice). Preembedding immunogold electron microscopy on MOP-mcherry knock-in sciatic nerves indicated presence of MOP in cytoplasm and at membranes of unmyelinated axons. Application of [D-Ala(2), N-MePhe(4), Gly-ol]-enkephalin (DAMGO) or fentanyl dose-dependently inhibited depolarization-induced CGRP release from rat sciatic nerve axons ex vivo, which was blocked by naloxone. When the lipophilic opioid fentanyl was applied perisciatically in naïve Wistar rats, mechanical nociceptive thresholds increased. Subthreshold doses of fentanyl or the hydrophilic opioid DAMGO were only effective if injected together with hypertonic saline. In vitro, using ß-arrestin-2/MOP double-transfected human embryonic kidney cells, DAMGO as well as fentanyl lead to a recruitment of ß-arrestin-2 to the membrane followed by a ß-arrestin-2 reappearance in the cytosol and MOP internalization. Pretreatment with hypertonic saline prevented MOP internalization. CONCLUSION: MOPs are present and functional in the axonal membrane from naïve animals. Hypertonic saline acutely decreases ligand-induced internalization of MOP and thereby might improve MOP function. Further studies should explore potential clinical applications of opioids together with enhancers for regional analgesia.

Initial characterization of a Syap1 knock-out mouse and distribution of Syap1 in mouse brain and cultured motoneurons.

Synapse-associated protein 1 (Syap1/BSTA) is the mammalian homologue of Sap47 (synapse-associated protein of 47 kDa) in Drosophila. Sap47 null mutant larvae show reduced short-term synaptic plasticity and a defect in associative behavioral plasticity. In cultured adipocytes, Syap1 functions as part of a complex that phosphorylates protein kinase Bα/Akt1 (Akt1) at Ser(473) and promotes differentiation. The role of Syap1 in the vertebrate nervous system is unknown. Here, we generated a Syap1 knock-out mouse and show that lack of Syap1 is compatible with viability and fertility. Adult knock-out mice show no overt defects in brain morphology. In wild-type brain, Syap1 is found widely distributed in synaptic neuropil, notably in regions rich in glutamatergic synapses, but also in perinuclear structures associated with the Golgi apparatus of specific groups of neuronal cell bodies. In cultured motoneurons, Syap1 is located in axons and growth cones and is enriched in a perinuclear region partially overlapping with Golgi markers. We studied in detail the influence of Syap1 knockdown and knockout on structure and development of these cells. Importantly, Syap1 knockout does not affect motoneuron survival or axon growth. Unexpectedly, neither knockdown nor knockout of Syap1 in cultured motoneurons is associated with reduced Ser(473) or Thr(308) phosphorylation of Akt. Our findings demonstrate a widespread expression of Syap1 in the mouse central nervous system with regionally specific distribution patterns as illustrated in particular for olfactory bulb, hippocampus, and cerebellum.

Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling.

In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3-stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features.

Disruption of tumor cell adhesion promotes angiogenic switch and progression to micrometastasis in RAF-driven murine lung cancer.

Progression of non-small-cell lung cancer (NSCLC) to metastasis is poorly understood. Two genetic approaches were used to evaluate the role of adherens junctions in a C-RAF driven mouse model for NSCLC: conditional ablation of the cdh1 gene and expression of dominant-negative (dn) E-cadherin. Disruption of E-cadherin caused massive formation of intratumoral vessels that was reversible in the early phase of induction. Vascularized tumors grew more rapidly, developed invasive fronts, and gave rise to micrometastasis. beta-catenin was identified as a critical effector of E-cadherin disruption leading to upregulation of VEGF-A and VEGF-C. In vivo, lung tumor cells with disrupted E-cadherin expressed beta-catenin target genes normally found in other endodermal lineages suggesting that reprogramming may be involved in metastatic progression.

Mechanisms for axon maintenance and plasticity in motoneurons: alterations in motoneuron disease.

In motoneuron disease and other neurodegenerative disorders, the loss of synapses and axon branches occurs early but is compensated by sprouting of neighboring axon terminals. Defective local axonal signaling for maintenance and dynamics of the axonal microtubule and actin cytoskeleton plays a central role in this context. The molecular mechanisms that lead to defective cytoskeleton architecture in two mouse models of motoneuron disease are summarized and discussed in this manuscript. In the progressive motor neuropathy (pmn) mouse model of motoneuron disease that is caused by a mutation in the tubulin-specific chaperone E gene, death of motoneuron cell bodies appears as a consequence of axonal degeneration. Treatment with bcl-2 overexpression or with glial-derived neurotrophic factor prevents loss of motoneuron cell bodies but does not influence the course of disease. In contrast, treatment with ciliary neurotrophic factor (CNTF) significantly delays disease onset and prolongs survival of pmn mice. This difference is due to the activation of Stat-3 via the CNTF receptor complex in axons of pmn mutant motoneurons. Most of the activated Stat-3 protein is not transported to the nucleus to activate transcription, but interacts locally in axons with stathmin, a protein that destabilizes microtubules. This interaction plays a major role in CNTF signaling for microtubule dynamics in axons. In Smn-deficient mice, a model of spinal muscular atrophy, defects in axonal translocation of ß-actin mRNA and possibly other mRNA species have been observed. Moreover, the regulation of local protein synthesis in response to signals from neurotrophic factors and extracellular matrix proteins is altered in motoneurons from this model of motoneuron disease. These findings indicate that local signals are important for maintenance and plasticity of axonal branches and neuromuscular endplates, and that disturbances in these signaling mechanisms could contribute to the pathophysiology of motoneuron diseases.

Fast motor axon loss in SMARD1 does not correspond to morphological and functional alterations of the NMJ.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a childhood motoneuron disease caused by mutations in the gene encoding for IGHMBP2, an ATPase/Helicase. Paralysis of the diaphragm is an early and prominent clinical sign resulting both from denervation and myopathy. In skeletal muscles, muscle atrophy mainly results from loss of motoneuron cell bodies and axonal degeneration. Although it is well known that loss of motoneurons at the lumbar spinal cord is an early event in the pathogenesis of the disease, it is not clear whether the corresponding proximal axons and NMJs are also early affected. In order to address this question, we have investigated the time course of the disease progression at the level of the motoneuron cell body, proximal axon (ventral root), distal axon (sciatic nerve), NMJ, and muscle fiber in Nmd(2J) mice, a mouse model for SMARD1. Our results show an early and apparently parallel loss of motoneurons, proximal axons, and NMJs. In affected muscles, however, denervated fibers coexist with NMJs with normal morphology and unaltered neurotransmission. Furthermore, unaffected axons are able to sprout and reinnervate muscle fibers, suggesting selective vulnerability of neurons to Ighmbp2 deficiency. The preservation of the NMJ morphology and neurotransmission in the Nmd(2J) mouse until motor axon loss takes place, differs from that observed in SMA mouse models in which NMJ impairment is an early and more general phenomenon in affected muscles.

Pathologic endothelial response and impaired function of circulating angiogenic cells in patients with Fabry disease.

Fabry disease is an X-chromosomal recessive deficiency of the lysosomal hydrolase alpha-galactosidase A (alpha-Gal). This results in an accumulation of globotriaosylceramide (GL-3) in a variety of cells often with subsequent functional impairment. Here, the impact of Fabry disease on the biology of circulating angiogenic cells (CACs) and the endothelial response to transient ischemia was investigated. Untreated patients with Fabry disease (n = 26), patients after initiation of alpha-Gal enzyme replacement therapy (ERT) (n = 16) and healthy controls (n = 26) were investigated. Endothelial function was assessed by the EndoPAT2000 device. CAC numbers were assessed by flow-cytometry, CAC function by a modified Boyden chamber assay. Fabry patients showed a pathologic endothelial response, which normalized after ERT. CACs were increased in number, but functionally impaired. Immunofluorescence and electron microscopy identified an accumulation of GL-3 in Fabry CACs. ERT attenuated CAC dysfunction and improved markers of oxidative stress response in Fabry patients via a reduction in GL-3 accumulation in vitro and in vivo. Silencing of alpha-Gal in healthy CACs impaired their migratory capacity underlining a key role of this enzyme for CAC function. CAC supernatant as well as CACs from Fabry patients impaired angiogenesis and migratory capacity of HUVECs providing a mechanistic link between CAC and endothelial dysfunction. CAC adhesion to TNF-α pre-stimulated HUVECs and tube formation was impaired by alpha-Gal knockdown. Fabry patients show a dysfunction of CAC and a pathologic endothelial response. ERT improves CAC and endothelial function and thus may attenuate development of cardiovascular disease in the long term in this patient population.

Serotonergic innervation of the amygdala: targets, receptors, and implications for stress and anxiety.

The amygdala is a core component of neural circuits that mediate processing of emotional, particularly anxiety and fear-related stimuli across species. In addition, the nuclear complex plays a key role in the central nervous system stress response, and alterations in amygdala responsivity are found in neuropsychiatric disorders, especially those precipitated or sustained by stressors. Serotonin has been shown to shape and fine-tune neural plasticity in development and adulthood, thereby allowing for network flexibility and adaptive capacity in response to environmental challenges, and is implicated in the modulation of stimulus processing and stress sensitivity in the amygdala. The fact that altered amygdala activity patterns are observed upon pharmacological manipulations of serotonergic transmission, as well as in carriers of genetic variations in serotonin pathway-associated signaling molecules representing risk factors for neuropsychiatric disorders, underlines the importance of understanding the role and mode of action of serotonergic transmission in the amygdala for human psychopathology. Here, we present a short overview over organizational principles of the amygdala in rodents, non-human primates and humans, and review findings on the origin, morphology, and targets of serotonergic innervation, the distribution patterns and cellular expression of serotonin receptors, and the consequences of stress and pharmacological manipulations of serotonergic transmission in the amygdala, focusing particularly on the extensively studied basolateral complex and central nucleus.

GABA concentration and GABAergic neuron populations in limbic areas are differentially altered by brain serotonin deficiency in Tph2 knockout mice.

While tryptophan hydroxylase-2 (Tph2) null mutant (Tph2(-/-)) mice are completely deficient in brain serotonin (5-HT) synthesis, the formation of serotonergic neurons and pathfinding of their projections are not impaired. However, 5-HT deficiency, during development and in the adult, might affect morphological and functional parameters of other neural systems. To assess the influence of 5-HT deficiency on γ-amino butyric acid (GABA) systems, we carried out measurements of GABA concentrations in limbic brain regions of adult male wildtype (wt), heterozygous (Tph2(+/-)) and Tph2(-/-) mice. In addition, unbiased stereological estimation of GABAergic interneuron numbers and density was performed in subregions of amygdala and hippocampus. Amygdala and prefrontal cortex displayed significantly increased and decreased GABA concentrations, respectively, exclusively in Tph2(+/-) mice while no changes were detected between Tph2(-/-) and wt mice. In contrast, in the hippocampus, increased GABA concentrations were found in Tph2(-/-) mice. While total cell density in the anterior basolateral amygdala did not differ between genotypes, the number and density of the GABAergic interneurons were significantly decreased in Tph2(-/-) mice, with the group of parvalbumin (PV)-immunoreactive (ir) interneurons contributing somewhat less to the decrease than that of non-PV-ir GABAergic interneurons. Major morphological changes were also absent in the dorsal hippocampus, and only a trend toward reduced density of PV-ir cells was observed in the CA3 region of Tph2(-/-) mice. Our findings are the first to document that life-long reduction or complete lack of brain 5-HT transmission causes differential changes of GABA systems in limbic regions which are key players in emotional learning and memory processes. The changes likely reflect a combination of developmental alterations and functional adaptations of emotion circuits to balance the lack of 5-HT, and may underlie altered emotional behavior in 5-HT-deficient mice. Taken together, our findings provide further insight into the mechanisms how life-long 5-HT deficiency impacts the pathogenesis of anxiety- and fear-related disorders.

Brain Noradrenergic Innervation Supports the Development of Parkinson's Tremor: A Study in a Reserpinized Rat Model.

The pathophysiology of tremor in Parkinson's disease (PD) is evolving towards a complex alteration to monoaminergic innervation, and increasing evidence suggests a key role of the locus coeruleus noradrenergic system (LC-NA). However, the difficulties in imaging LC-NA in patients challenge its direct investigation. To this end, we studied the development of tremor in a reserpinized rat model of PD, with or without a selective lesioning of LC-NA innervation with the neurotoxin DSP-4. Eight male rats (Sprague Dawley) received DSP-4 (50 mg/kg) two weeks prior to reserpine injection (10 mg/kg) (DR-group), while seven male animals received only reserpine treatment (R-group). Tremor, rigidity, hypokinesia, postural flexion and postural immobility were scored before and after 20, 40, 60, 80, 120 and 180 min of reserpine injection. Tremor was assessed visually and with accelerometers. The injection of DSP-4 induced a severe reduction in LC-NA terminal axons (DR-group: 0.024 ± 0.01 vs. R-group: 0.27 ± 0.04 axons/um2, p < 0.001) and was associated with significantly less tremor, as compared to the R-group (peak tremor score, DR-group: 0.5 ± 0.8 vs. R-group: 1.6 ± 0.5; p < 0.01). Kinematic measurement confirmed the clinical data (tremor consistency (% of tremor during 180 s recording), DR-group: 37.9 ± 35.8 vs. R-group: 69.3 ± 29.6; p < 0.05). Akinetic-rigid symptoms did not differ between the DR- and R-groups. Our results provide preliminary causal evidence for a critical role of LC-NA innervation in the development of PD tremor and foster the development of targeted therapies for PD patients.

Double and triple in situ hybridization for coexpression studies: combined fluorescent and chromogenic detection of neuropeptide Y (NPY) and serotonin receptor subtype mRNAs expressed at different abundance levels.

Multiple fluorescence in situ hybridization is the method of choice for studies aimed at determining simultaneous production of signal transduction molecules and neuromodulators in neurons. In our analyses of the monoamine receptor mRNA expression of peptidergic neurons in the rat telencephalon, double tyramide-signal-amplified fluorescence in situ hybridization delivered satisfactory results for coexpression analysis of neuropeptide Y (NPY) and serotonin receptor 2C (5-HT2C) mRNA, a receptor subtype expressed at high-to-moderate abundance in the regions analyzed. However, expression of 5-HT1A mRNA, which is expressed at comparatively low abundance in many telencephalic areas, could not be unequivocally identified in NPY mRNA-reactive neurons due to high background and poor signal-to-noise ratio in fluorescent receptor mRNA detections. Parallel chromogenic in situ hybridization provided clear labeling for 5-HT1A mRNA and additionally offered the possibility to monitor the chromogen deposition at regular time intervals to determine the optimal signal-to-noise ratio. We first developed a double labeling protocol combining fluorescence and chromogenic in situ hybridization and subsequently expanded this variation to combine double fluorescence and chromogenic in situ hybridization for triple labelings. With this method, we documented expression of 5-HT2C and/or 5-HT1A in subpopulations of telencephalic NPY-producing neurons. The method developed in the present study appears suitable for conventional light and fluorescence microscopy, combines advantages of fluorescence and chromogenic in situ hybridization protocols and thus provides a reliable non-radioactive alternative to previously published multiple labeling methods for coexpression analyses in which one mRNA species requires highly sensitive detection.

Ultrastructural analysis reveals cAMP-dependent enhancement of microvascular endothelial barrier functions via Rac1-mediated reorganization of intercellular junctions.

Evidence exists that cAMP stabilizes the endothelial barrier, in part via activation of the small GTPase Rac1. However, despite the high medical relevance of this signaling pathway, the mechanistic effects on intercellular contacts on the ultrastructural level are largely unknown. In microvascular endothelial cell monolayers, in which increased cAMP strengthened barrier properties, similar to intact microvessels in vivo, both forskolin and rolipram (F/R) to increase cAMP and 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphorothioate (O-Me-cAMP) to stimulate exchange protein directly activated by cAMP/Ras proximate-1 (EPac/Rap 1) signaling enhanced transendothelial electrical resistance and induced activation of Rac1. Concurrently, augmented immunofluorescence intensity and linearization of signals at cell borders were observed for intercellular junction proteins VE-cadherin and claudin 5. Ultrastructural analysis of the intercellular contact zone architecture documented that exposure to F/R or O-Me-cAMP led to a significant increase in the proportion of contact sites displaying complex interdigitations of cell borders, in which membranes of neighboring cells were closely apposed over comparatively long distances; in addition, they were stabilized by numerous intercellular junctions. Interference with Rac1 activation by NSC-23766 completely abolished both barrier stabilization and contact zone reorganization in response to O-Me-cAMP, whereas F/R-mediated Rac1 activation and barrier enhancement were not affected by NSC-23766. In parallel experiments using macrovascular endothelium, increased cAMP failed to induce Rac1 activation, barrier enhancement, and contact zone reorganization. These results indicate that, in microvascular endothelium, Rac1-mediated alterations in contact zone architecture contribute to cAMP-induced barrier stabilization.

Bruchpilot in ribbon-like axonal agglomerates, behavioral defects, and early death in SRPK79D kinase mutants of Drosophila.

Defining the molecular structure and function of synapses is a central theme in brain research. In Drosophila the Bruchpilot (BRP) protein is associated with T-shaped ribbons ("T-bars") at presynaptic active zones (AZs). BRP is required for intact AZ structure and normal evoked neurotransmitter release. By screening for mutations that affect the tissue distribution of Bruchpilot, we have identified a P-transposon insertion in gene CG11489 (location 79D) which shows high homology to mammalian genes for SR protein kinases (SRPKs). SRPKs phosphorylate serine-arginine rich splicing factors (SR proteins). Since proteins expressed from CG11489 cDNAs phosphorylate a peptide from a human SR protein in vitro, we name CG11489 the Drosophila Srpk79D gene. We have characterized Srpk79D transcripts and generated a null mutant. Mutation of the Srpk79D gene causes conspicuous accumulations of BRP in larval and adult nerves. At the ultrastructural level, these correspond to extensive axonal agglomerates of electron-dense ribbons surrounded by clear vesicles. Basic synaptic structure and function at larval neuromuscular junctions appears normal, whereas life expectancy and locomotor behavior of adult mutants are significantly impaired. All phenotypes of the mutant can be largely or completely rescued by panneural expression of SRPK79D isoforms. Isoform-specific antibodies recognize panneurally overexpressed GFP-tagged SRPK79D-PC isoform co-localized with BRP at presynaptic active zones while the tagged -PB isoform is found in spots within neuronal perikarya. SRPK79D concentrations in wild type apparently are too low to be revealed by these antisera. We propose that the Drosophila Srpk79D gene characterized here may be expressed at low levels throughout the nervous system to prevent the assembly of BRP containing agglomerates in axons and maintain intact brain function. The discovery of an SR protein kinase required for normal BRP distribution calls for the identification of its substrate and the detailed analysis of SRPK function for the maintenance of nervous system integrity.

Identification of L-ferritin in neuromelanin granules of the human substantia nigra: a targeted proteomics approach.

In the pigmented dopaminergic neurons of the human substantia nigra pars compacta the system relevant in iron storage is the polymer neuromelanin (NM). Although in most cells this function is mainly accomplished by ferritin, this protein complex appears not to be expressed in NM-containing neurons. Nevertheless the conceivable presence of iron-storing proteins as part of the NM granules has recently been discussed on the basis of Mössbauer spectroscopy and synchrotron x-ray microspectroscopy. Intriguingly by combining subcellular fractionation of NM granules, peptide sequencing via tandem mass spectrometry, and the additional confirmation by multiple reaction monitoring and immunogold labeling for electron microscopy, L-ferritin could now be unambiguously identified and localized in NM granules for the first time. This finding not only supports direct evidence for a regulatory role of L-ferritin in neuroectodermal cell pigmentation but also integrates a new player within a complicated network governing iron homeostasis in the dopamine neurons of the human substantia nigra. Thus our finding entails far reaching implications especially when considering etiopathogenetic aspects of Parkinson disease.

Serotonin transporter genotype modulates resting state and predator stress-induced amygdala perfusion in mice in a sex-dependent manner.

The serotonin transporter (5-HTT) is a key molecule of serotoninergic neurotransmission and target of many anxiolytics and antidepressants. In humans, 5-HTT gene variants resulting in lower expression levels are associated with behavioral traits of anxiety. Furthermore, functional magnetic resonance imaging (fMRI) studies reported increased cerebral blood flow (CBF) during resting state (RS) and amygdala hyperreactivity. 5-HTT deficient mice as an established animal model for anxiety disorders seem to be well suited for investigating amygdala (re-)activity in an fMRI study. We investigated wildtype (5-HTT+/+), heterozygous (5-HTT+/-), and homozygous 5-HTT-knockout mice (5-HTT-/-) of both sexes in an ultra-high-field 17.6 Tesla magnetic resonance scanner. CBF was measured with continuous arterial spin labeling during RS, stimulation state (SS; with odor of rats as aversive stimulus), and post-stimulation state (PS). Subsequently, post mortem c-Fos immunohistochemistry elucidated neural activation on cellular level. The results showed that in reaction to the aversive odor CBF in total brain and amygdala of all mice significantly increased. In male 5-HTT+/+ mice amygdala RS CBF levels were found to be significantly lower than in 5-HTT+/- mice. From RS to SS 5-HTT+/+ amygdala perfusion significantly increased compared to both 5-HTT+/- and 5-HTT-/- mice. Perfusion level changes of male mice correlated with the density of c-Fos-immunoreactive cells in the amygdaloid nuclei. In female mice the perfusion was not modulated by the 5-Htt-genotype, but by estrous cycle stages. We conclude that amygdala reactivity is modulated by the 5-Htt genotype in males. In females, gonadal hormones have an impact which might have obscured genotype effects. Furthermore, our results demonstrate experimental support for the tonic model of 5-HTTLPR function.

Bruchpilot, a protein with homology to ELKS/CAST, is required for structural integrity and function of synaptic active zones in Drosophila.

Neurotransmitters are released at presynaptic active zones (AZs). In the fly Drosophila, monoclonal antibody (MAB) nc82 specifically labels AZs. We employ nc82 to identify Bruchpilot protein (BRP) as a previously unknown AZ component. BRP shows homology to human AZ protein ELKS/CAST/ERC, which binds RIM1 in a complex with Bassoon and Munc13-1. The C terminus of BRP displays structural similarities to multifunctional cytoskeletal proteins. During development, transcription of the bruchpilot locus (brp) coincides with neuronal differentiation. Panneural reduction of BRP expression by RNAi constructs permits a first functional characterization of this large AZ protein: larvae show reduced evoked but normal spontaneous transmission at neuromuscular junctions. In adults, we observe loss of T bars at active zones, absence of synaptic components in electroretinogram, locomotor inactivity, and unstable flight (hence "bruchpilot"-crash pilot). We propose that BRP is critical for intact AZ structure and normal-evoked neurotransmitter release at chemical synapses of Drosophila.

Proteomic analysis of cathepsin B- and L-deficient mouse brain lysosomes.

Cathepsins B and L are lysosomal cysteine proteases which have been implicated in a variety of pathological processes such as cancer, tumor angiogenesis, and neurodegeneration. However, only a few protein substrates have thus far been described and the mechanisms by which cathepsins B and L regulate cell proliferation, invasion, and apoptosis are poorly understood. Combined deficiency of both cathepsins results in early-onset neurodegeneration in mice reminiscent of neuronal ceroid lipofuscinoses in humans. Therefore, we intended to quantify accumulated proteins in brain lysosomes of double deficient mice. A combination of subcellular fractionation and LC-MS/MS using isobaric tagging for relative and absolute quantitation (iTRAQ) allowed us to simultaneously assess wildtype and cathepsin B(-/-)L(-/-) cerebral lysosomes. Altogether, 19 different proteins were significantly increased in cathepsin B(-/-)L(-/-) lysosomes. Most elevated proteins had previously been localized to neuronal biosynthetic, recycling/endocytic or lysosomal compartments. A more than 10-fold increase was observed for Rab14, the Delta/Notch-like epidermal growth factor-related receptor (DNER), calcyon, and carboxypeptidase E. Intriguingly, immunohistochemistry demonstrated that Rab14 and DNER specifically stain swollen axons in double deficient brains. Since dense accumulations of expanded axons are the earliest phenotypic and pathognomonic feature of cathepsin B(-/-)L(-/-) brains, our data suggest a role for cathepsins B and L in recycling processes during axon outgrowth and synapse formation in the developing postnatal central nervous system.

Single-dose application of CNTF and BDNF improves remyelination of regenerating nerve fibers after C7 ventral root avulsion and replantation.

Although axonal regeneration has been observed after replantation of avulsed ventral roots (VR) into the spinal cord, the functional outcome of this treatment in terms of motor reinnervation is unsatisfactory. In the present study, effects of single-dose ciliary and/or brain-derived neurotrophic factor (CNTF, BDNF) application on axon regeneration after C7 VR avulsion and replantation in adult rabbits were morphologically assessed by analysis of numbers, calibers, and myelination of axons in replanted VRs. Electromyography (EMG) was carried out to document the time course of de- and reinnervation in individual animals. After 3 weeks, replanted C7 VRs were almost devoid of myelinated axons. At week 8, active EMG-denervation was confirmed in affected muscles, but was less pronounced in neurotrophic factor (NF)-treated animals than in controls. Reinnervation potentials were identified in paraspinal muscles in more NF-treated animals than in controls. After 6 months, the number of myelinated axons in replanted VRs was approximately 45% of that in unlesioned roots in all groups, with small-sized axons constituting the majority of axons. At this time, more NF-treated animals than controls featured reinnervation. Moreover, myelination deficits of regenerated axons in controls were less pronounced in NF-treated animals. Especially in CNTF + BDNF-treated animals, myelination of regenerated axons of specific sizes was significantly increased compared to regenerated controls. In summary, NFs stimulated reinnervation early after the lesion and, for the first time, our morphological data quantitatively indicate positive effects of CNTF + BDNF on remyelination.