[Show abstract][Hide abstract] ABSTRACT: Background:
Afamin is a human plasma vitamin E-binding glycoprotein primarily expressed in the liver and secreted into the bloodstream. Because little is known about (patho)-physiological functions of afamin, we decided to identify phenotypes associated with afamin by investigating transgenic mice overexpressing the human afamin gene and performing large-scale human epidemiological studies.
Methods and results:
Transgenic mice overexpressing afamin revealed increased body weight and serum concentrations of lipids and glucose. We applied a random-effects meta-analysis using age- and sex-adjusted baseline and follow-up investigations in the population-based Bruneck (n=826), Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR; n=1499), and KOoperative Gesundheitsforschung in der Region Augsburg (KORA) F4 studies (n=3060). Mean afamin concentrations were 62.5±15.3, 66.2±14.3, and 70.6±17.2 mg/L in Bruneck, SAPHIR, and KORA F4, respectively. Per 10 mg/L increment in afamin measured at baseline, the number of metabolic syndrome components increased by 19% (incidence rate ratio=1.19; 95% confidence interval [CI], 1.16-1.21; P=5.62×10(-64)). With the same afamin increment used at baseline, we observed an 8% gain in metabolic syndrome components between baseline and follow-up (incidence rate ratio=1.08; 95% CI, 1.06-1.10; P=8.87×10(-16)). Afamin concentrations at baseline were highly significantly related to all individual metabolic syndrome components at baseline and at follow-up. This observation was most pronounced for elevated waist circumference (odds ratio, 1.79; 95% CI, 1.54-2.09; P=4.15×10(-14) at baseline and odds ratio, 1.46; 95% CI, 1.31-1.63; P=2.84×10(-11) for change during follow-up) and for elevated fasting glucose concentrations (odds ratio, 1.46; 95% CI, 1.40-1.52; P=1.87×10(-69) and odds ratio, 1.46; 95% CI, 1.24-1.71; P=5.13×10(-6), respectively).
This study in transgenic mice and >5000 participants in epidemiological studies shows that afamin is strongly associated with the prevalence and development of metabolic syndrome and all its components.
Preview · Article · Aug 2014 · Circulation Cardiovascular Genetics
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Jul 2010 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Cumulative evidence indicates that amyloid-beta peptides exert some of their neurodegenerative effects through modulation of L-type voltage gated calcium channels, which play key roles in a diverse range of CNS functions. In this study we examined the expression of CaV1.2 L-type voltage gated calcium channels in transgenic mice overexpressing human AbetaPP751 with the London (V717I) and Swedish (K670M/N671L) mutations by immunohistochemistry in light and electron microscopy. In hippocampal layers of wild type and transgenic mice, CaV1.2 channels were predominantly localized to somato-dendritic domains of neurons, and to astrocytic profiles with an age-dependent increase in labeling density. In transgenic animals, CaV1.2-like immunoreactive clusters were found in neuronal profiles in association with amyloid-beta plaques. Both the number and density of these clusters depended upon age of animals and number of plaques. The most striking difference between wild type and transgenic mice was the age-dependent expression of CaV1.2 channels in reactive astrocytes. At the age of 6 month, CaV1.2 channels were rarely detected in reactive astrocytes of transgenic mice, but an incremental number of CaV1.2 expressing reactive astrocytes was found with increasing age of animals and number of amyloid-beta plaques. This study demonstrates that CaV1.2 channels are highly expressed in reactive astrocytes of 12-months of age transgenic mice, which might be a consequence of the increasing amyloid burden. Further studies should clarify which functional implications are associated with the higher availability of CaV1.2 channels in late stage Alzheimer's disease.
[Show abstract][Hide abstract] ABSTRACT: Calcium-activated potassium channels have been shown to be critically involved in neuronal function, but an elucidation of their detailed roles awaits identification of the microdomains where they are located. This study was undertaken to unravel the precise subcellular distribution of the large-conductance calcium-activated potassium channels (called BK, KCa1.1, or Slo1) in the somatodendritic compartment of cerebellar Purkinje cells by means of postembedding immunogold cytochemistry and SDS-digested freeze-fracture replica labeling (SDS-FRL). We found BK channels to be unevenly distributed over the Purkinje cell plasma membrane. At distal dendritic compartments, BK channels were scattered over the plasma membrane of dendritic shafts and spines but absent from postsynaptic densities. At the soma and proximal dendrites, BK channels formed two distinct pools. One pool was scattered over the plasma membrane, whereas the other pool was clustered in plasma membrane domains overlying subsurface cisterns. The labeling density ratio of clustered to scattered channels was about 60:1, established in SDS-FRL. Subsurface cisterns, also called hypolemmal cisterns, are subcompartments of the endoplasmic reticulum likely representing calciosomes that unload and refill Ca2+ independently. Purkinje cell subsurface cisterns are enriched in inositol 1,4,5-triphosphate receptors that mediate the effects of several neurotransmitters, hormones, and growth factors by releasing Ca2+ into the cytosol, generating local Ca2+ sparks. Such increases in cytosolic [Ca2+] may be sufficient for BK channel activation. Clustered BK channels in the plasma membrane may thus participate in building a functional unit (plasmerosome) with the underlying calciosome that contributes significantly to local signaling in Purkinje cells.
No preview · Article · Jul 2009 · The Journal of Comparative Neurology
[Show abstract][Hide abstract] ABSTRACT: α-Tocopherol (αTocH), a member of the vitamin E family, is essential for normal neurological function. Despite the importance of αTocH transport into the CNS, transfer mechanisms across the blood–brain barrier (BBB) are not entirely clear. We here investigate whether afamin, a known αTocH-binding protein, contributes to αTocH transport across an in vitro model of the BBB consisting of primary porcine brain capillary endothelial cells (BCEC) and basolaterally cultured astrocytoma cells. Exogenously added afamin had no adverse effects on BCEC viability or barrier function and was transported across BCEC Transwell cultures. Furthermore, αTocH transport across polarized BCEC cultures to astrocytoma cells is facilitated by afamin, though to a lesser extent than by high-density lipoprotein-mediated transport, an essential and in vivo operating αTocH import pathway at the cerebrovasculature. We also demonstrate that porcine BCEC endogenously synthesize afamin. In line with these in vitro findings, afamin was detected by immunohistochemistry in porcine, human postmortem, and mouse brain, where prominent staining was observed almost exclusively in the cerebrovasculature. The demonstration of afamin mRNA expression in isolated brain capillaries suggests that afamin might be a new family member of binding/transport proteins contributing to αTocH homeostasis at the BBB in vivo.
Full-text · Article · Nov 2008 · Journal of Neurochemistry
[Show abstract][Hide abstract] ABSTRACT: Chromogranin B and secretogranin II are major soluble constituents of large dense core vesicles of presynaptic structures and have been found in neuritic plaques of Alzheimer patients. We examined the distribution and expression of these peptides in both transgenic mice over expressing human amyloid-beta protein precursor APP751 with the London (V717I) and Swedish (K670M/N671L) mutations and in human post-mortem brain. In transgenic mice, the number of amyloid-beta plaques and chromogranin immunopositive plaques increased from 6 to 12 months. About 60% of amyloid-beta plaques were associated with chromogranin B and about 40% with secretogranin II. Chromogranin immunoreactivity appeared mainly as swollen dystrophic neurites. Neither synaptophysin- nor glial fibrillary acidic protein- immunoreactivity was expressed in chromogranin immunoreactive structures at any timepoint. Density of chromogranin peptides in hippocampal structures did not change in transgenic animals at any timepoint, even though animals had a poorer performance in the Morris water maze task. In conclusion, our findings in transgenic animals partly resembled findings in Alzheimer patients. Chromogranin peptides were associated with amyloid-beta plaques, but were not reduced in specific brain areas as previously reported by our group. Therefore specific changes of chromogranin peptides observed in Alzheimer patients can be related to amyloid-beta pathology only.
[Show abstract][Hide abstract] ABSTRACT: Stress activates the hypothalamic-pituitary-adrenal (HPA) axis, releasing ACTH from the anterior pituitary gland and glucocorticoids from the adrenal cortex. Stress also activates the sympathetic nervous system, evoking adrenaline release from the adrenal medulla. Large-conductance calcium- and voltage-activated potassium (BK) channels have been implicated in regulation of cellular excitability in these systems. Here, we examine the functional role of BK channels in HPA axis regulation in vivo using female mice genetically deficient (BK(-/-)) for the pore-forming subunits of BK channels. BK(-/-) phenotype in the HPA was confirmed by immunohistochemistry, Western blot analysis, and corticotrope patch-clamp recording. Restraint stress-induced plasma concentrations of ACTH and corticosterone were significantly blunted in BK(-/-) mice compared with wild type (WT) controls. This stress hyporesponsiveness was associated with reduced activation of hypothalamic paraventricular nucleus (PVN) neurons. Basal expression of CRH, but not arginine vasopressin mRNA in the PVN was significantly lower in BK(-/-) mice compared with WT controls. Total anterior pituitary ACTH peptide content, but not proopiomelanocortin mRNA expression or corticotrope number, was significantly reduced in BK(-/-) mice compared with WT. However, anterior pituitary corticotropes from BK(-/-) mice fully supported ACTH output, releasing a significantly greater proportion of stored ACTH in response to secretagogue in vitro compared with WT. These results support an important role for BK channels in both the neural circuitry and endocrine output of the HPA axis and indicate that the stress hyporesponsiveness in BK(-/-) mice primarily results from reduced activation of hypothalamic PVN neurosecretory neurons.
[Show abstract][Hide abstract] ABSTRACT: Substance P-like immunoreactivity (-LI) is found in neuritic plaques, and is reduced in patients suffering from Alzheimer disease (AD). In this study, we examined the distribution and expression of substance P in transgenic mice overexpressing human amyloid precursor protein (hAPP) APP751 with the London (V717I) and Swedish (K670M/N671L) mutations. Immunohistochemistry was performed to localize substance P- and glial fibrillary acidic protein-LI by confocal microscopy. In hAPP transgenic mice, the number of beta-amyloid plaques significantly increased from 6 to 12 months. About 5% of beta-amyloid plaques were substance P-immunoreactive. In transgenic mice, the morphology of substance P-immunoreactive structures changed by consisting of swollen and dystrophic neurites mostly associated with beta-amyloid plaques. The overall localization and the relative substance P densities were not different between wild type and transgenic mice at 6 and 12 months. At month 12, a dramatic change in the distribution pattern of substance P-LI was observed as it was now expressed in a high number of reactive astrocytes. This expression of substance P in astrocytes was mainly found in the hippocampal formation and thalamic nuclei with a preferential association with beta-amyloid plaques, whereas in cortical regions only faintly substance P-immunoreactive astrocytes were observed. This study indicates that substance P undergoes complex changes in this animal Alzheimer disease model. Future experiments including substance P antagonists are necessary to further explore the interaction between beta-amyloid deposits and substance P.
[Show abstract][Hide abstract] ABSTRACT: Cardiac L-type calcium channels are formed by two α-subunits, Cav1.2 (α1C) and Cav1.3 (α1D). In contrast to the uniform expression pattern of Cav1.2, Cav1.3 is highly expressed in sino-atrial node (SAN) and atrial tissue, but not in the ventricle. Accordingly, knockout of Cav1.3 (Cav1.3−/−) in mice was shown to lead to a cardiac phenotype characterised by severe bradycardia in vivo and in isolated SAN cells. Cav1.3 may therefore constitute a novel pharmacological target for specific bradycardic agents.
RNAse protection assays of murine wild type hearts revealed rather high Cav1.3 levels comparable to Cav1.2, suggesting functional relevance of Cav1.3 outside specialised tissues such as SAN. Due to the lack of specific Cav1.3 blockers, we directly examined the functional role of Cav1.3 using isolated working hearts from adult wild type (WT) and Cav1.3−/− mice. Histological analysis of hearts revealed no pathological changes. Ventricular contractility and inotropic effects of isoproterenol were unaltered in Cav1.3−/− hearts. Severe sinus bradycardia already noted in vivo was accompanied by ventricular extrasystoles. This phenotype was restored to nearly normal values by the cumulative addition of isoproterenol. Electrocardiograms of Cav1.3−/− hearts revealed delayed atrio-ventricular (AV) conduction and a decoupling of heart rate and PR interval duration. Isoproterenol did not improve disturbance of AV conduction.
In conclusion, suppression of Cav1.3 does not alter ventricular contractile function, and the decrease in sinus node frequency is counterbalanced by adrenergic stimulation. Importantly, bradyarrhythmia is partly due to an intrinsic AV node dysfunction, which is resistant to adrenergic counterbalance. These findings help to predict the clinical pattern of selective Cav1.3 blockade.
No preview · Article · Jul 2004 · Archiv für Experimentelle Pathologie und Pharmakologie
[Show abstract][Hide abstract] ABSTRACT: Ca(v)1.2 and Ca(v)1.3 L-type Ca(2+) channels (LTCCs) are believed to underlie Ca(2+) currents in brain, pancreatic beta cells, and the cardiovascular system. In the CNS, neuronal LTCCs control excitation-transcription coupling and neuronal plasticity. However, the pharmacotherapeutic implications of CNS LTCC modulation are difficult to study because LTCC modulators cause cardiovascular (activators and blockers) and neurotoxic (activators) effects. We selectively eliminated high dihydropyridine (DHP) sensitivity from Ca(v)1.2 alpha 1 subunits (Ca(v)1.2DHP-/-) without affecting function and expression. This allowed separation of the DHP effects of Ca(v)1.2 from those of Ca(v)1.3 and other LTCCs. DHP effects on pancreatic beta cell LTCC currents, insulin secretion, cardiac inotropy, and arterial smooth muscle contractility were lost in Ca(v)1.2DHP-/- mice, which rules out a direct role of Ca(v)1.3 for these physiological processes. Using Ca(v)1.2DHP-/- mice, we established DHPs as mood-modifying agents: LTCC activator-induced neurotoxicity was abolished and disclosed a depression-like behavioral effect without affecting spontaneous locomotor activity. LTCC activator BayK 8644 (BayK) activated only a specific set of brain areas. In the ventral striatum, BayK-induced release of glutamate and 5-HT, but not dopamine and noradrenaline, was abolished. This animal model provides a useful tool to elucidate whether Ca(v)1.3-selective channel modulation represents a novel pharmacological approach to modify CNS function without major peripheral effects.
Full-text · Article · Jun 2004 · Journal of Clinical Investigation
[Show abstract][Hide abstract] ABSTRACT: EBP (emopamil-binding protein) is a high-affinity binding protein for [3H]emopamil and belongs to the family of so-called sigma receptors. Mutations that disrupt EBP's 3beta-hydroxysteroid sterol delta8-delta7 isomerase activity (EC 126.96.36.199) impair cholesterol biosynthesis and cause X-chromosomal dominant chondrodysplasia punctata. We identified a human cDNA for a novel EBPL (EBP-like protein) with a calculated mass of 23.2 kDa. Amino acid sequence alignments and phylogenetic analysis revealed that EBPL is distantly related to EBP (31% identity and 52% similarity) and found in animals but not in plants. EBPL is encoded by four exons on human chromosome 13q14.2 covering 30.7 kb, and a partially processed EBPL pseudogene was found on 16q21. The EBPL mRNA was expressed ubiquitously and most abundant in liver, lung and kidney. Upon heterologous expression in yeast EBPL had no detectable 3beta-hydroxysteroid sterol delta8-delta7 isomerase and sigma-ligand-binding activity. Nine out of ten amino acid residues essential for catalytic activity of EBP were conserved in EBPL. Replacement of the only differing residue (EBP-Y111W) reduced catalytic activity of EBP. Transfer of the divergent residue from EBP to EBPL (EBPL-W91Y) and chimaerization of EBP and EBPL at various positions failed to restore catalytic activity of EBPL. Chemical cross-linking induced homodimerization of EBPL and EBP. Whereas mevinolin increased the mRNA for EBP and DHCR7 (delta7-sterol reductase) in HepG2 cells, it had no effect on mRNAs for EBPL and sigma1 receptor, indicating that EBP and EBPL expression are not co-ordinated. We propose that EBPL has a yet-to-be-discovered function other than cholesterol biosynthesis.
[Show abstract][Hide abstract] ABSTRACT: Voltage-gated Ca(2+) channels formed by subunits (class D Ca(2+) channels) tightly regulate neurotransmitter release from cochlear inner hair cells (IHCs) by controlling the majority of depolarisation-induced Ca(2+) entry. We have recently shown that the absence of these channels can cause deafness and degeneration of outer hair cells (OHCs) and IHCs in alpha1D-deficient mice (alpha1D(-/-)) (Platzer et al., 2000. Cell 102, 89-97). We investigated the time-dependent patterns of degeneration during postnatal development in the alpha1D(-/-) mouse cochlea using light and electron microscopy. At postnatal day 3 (P3), electron microscopy revealed no morphological aberrations in sensory cells, in afferent as well as in efferent nerve endings. But at P7 we observed a beginning degeneration of afferent nerve fibres by electron microscopy. By P15, we found a loss of OHCs in apical turns but electron microscopy revealed no ultrastructural changes in IHCs and efferent axons as compared to C57 black control animals (C57BL). We demonstrated by serial ultrathin sectioning of 15 days old alpha1D(-/-) mice that intact efferent nerve fibres formed direct contacts with IHCs as the degeneration of afferent nerve fibres progressed. We also saw a notable degeneration of spiral ganglion cells at P15. By 8 months, nearly all spiral ganglion and sensory cells of the organ of Corti were absent. Random ultrathin sectioning gave the impression that synaptic bodies abundant in wild-type animals were absent in nearly all alpha1D(-/-) mice investigated. We conclude that besides presumably reduced synaptic bodies the absence of class D L-type Ca(2+) channels does not prevent morphological development of the cochlea until P3 but may cause cochlear degeneration thereafter. The observed pattern of degeneration involves afferent nerve fibres (P7) followed by cell bodies in the spiral ganglion (P15), OHCs (P15) and IHCs (after P15).
No preview · Article · May 2003 · Hearing Research
[Show abstract][Hide abstract] ABSTRACT: Neuronal L-type calcium channels have been implicated in pain perception and neuronal synaptic plasticity. To investigate this we have examined the effect of disrupting the gene encoding the CaV1.3 (alpha 1D) alpha subunit of L-type Ca2+ channels on neurological function, acute nociceptive behavior, and hippocampal synaptic function in mice. CaV1.3 alpha 1 subunit knockout (CaV1.3 alpha 1(-/-)) mice had relatively normal neurological function with the exception of reduced auditory evoked behavioral responses and lower body weight. Baseline thermal and mechanical thresholds were unaltered in these animals. CaV1.3 alpha 1(-/-) mice were also examined for differences in N-methyl-D-aspartate (NMDA) receptor-dependent (100 Hz tetanization for 1 s) and NMDA receptor-independent (200 Hz in 100 microM DL-2-amino-5-phosphopentanoic acid) long-term potentiation within the CA1 region of the hippocampus. Both NMDA receptor-dependent and NMDA receptor-independent forms of long-term potentiation were expressed normally. Radioligand binding studies revealed that the density of (+)[3H]isradipine binding sites in brain homogenates was reduced by 20-25% in CaV1.3 alpha 1(-/-) mice, without any detectable change in CaV1.2 (alpha 1C) protein levels as detected using Western blot analysis. Taken together these data indicate that following loss of CaV1.3 alpha 1 subunit expression there is sufficient residual activity of other Ca2+ channel subtypes to support NMDA receptor-independent long-term potentiation and some forms of sensory behavior/function.