Synaptic coupling of inner ear sensory cells is controlled by brevican-based extracellular matrix baskets resembling perineuronal nets.

BACKGROUND: Perineuronal nets (PNNs) are specialized aggregations of extracellular matrix (ECM) molecules surrounding specific neurons in the central nervous system (CNS). PNNs are supposed to control synaptic transmission and are frequently associated with neurons firing at high rates, including principal neurons of auditory brainstem nuclei. The origin of high-frequency activity of auditory brainstem neurons is the indefatigable sound-driven transmitter release of inner hair cells (IHCs) in the cochlea. RESULTS: Here, we show that synaptic poles of IHCs are ensheathed by basket-like ECM complexes formed by the same molecules that constitute PNNs of neurons in the CNS, including brevican, aggreccan, neurocan, hyaluronan, and proteoglycan link proteins 1 and 4 and tenascin-R. Genetic deletion of brevican, one of the main components, resulted in a massive degradation of ECM baskets at IHCs, a significant impairment in spatial coupling of pre- and postsynaptic elements and mild impairment of hearing. CONCLUSIONS: These ECM baskets potentially contribute to control of synaptic transmission at IHCs and might be functionally related to PNNs of neurons in the CNS.

Tau phosphorylation-associated spine regression does not impair hippocampal-dependent memory in hibernating golden hamsters.

The microtubule-associated protein tau, in its hyperphosphorylated form, is the major component of paired helical filaments and other aggregates in neurodegenerative disorders commonly referred to as "tauopathies". Recent evidence, however, indicates that mislocalization of hyperphosphorylated tau to subsynaptic sites leads to synaptic impairment and cognitive decline even long before formation of tau aggregates and neurodegeneration occur. A similar, but reversible hyperphosphorylation of tau occurs under physiologically controlled conditions during hibernation. Here, we study the hibernating Golden hamster (Syrian hamster, Mesocricetus auratus). A transient spine reduction was observed in the hippocampus, especially on apical dendrites of hippocampal CA3 pyramidal cells, but not on their basal dendrites. This distribution of structural synaptic regression was correlated to the distribution of phosphorylated tau, which was highly abundant in apical dendrites but hardly detectable in basal dendrites. Surprisingly, hippocampal memory assessed by a labyrinth maze was not affected by hibernation. The present study suggests a role for soluble hyperphosphorylated tau in the process of reversible synaptic regression, which does not lead to memory impairment during hibernation. We hypothesize that tau phosphorylation associated spine regression might mainly affect unstable/dynamic spines while sparing established/stable spines.

Functional changes of the gastric bypass microbiota reactivate thermogenic adipose tissue and systemic glucose control via intestinal FXR-TGR5 crosstalk in diet-induced obesity.

BACKGROUND: Bariatric surgery remains the most effective therapy for adiposity reduction and remission of type 2 diabetes. Although different bariatric procedures associate with pronounced shifts in the gut microbiota, their functional role in the regulation of energetic and metabolic benefits achieved with the surgery are not clear. METHODS: To evaluate the causal as well as the inherent therapeutic character of the surgery-altered gut microbiome in improved energy and metabolic control in diet-induced obesity, an antibiotic cocktail was used to eliminate the gut microbiota in diet-induced obese rats after gastric bypass surgery, and gastric bypass-shaped gut microbiota was transplanted into obese littermates. Thorough metabolic profiling was combined with omics technologies on samples collected from cecum and plasma to identify adaptions in gut microbiota-host signaling, which control improved energy balance and metabolic profile after surgery. RESULTS: In this study, we first demonstrate that depletion of the gut microbiota largely reversed the beneficial effects of gastric bypass surgery on negative energy balance and improved glucolipid metabolism. Further, we show that the gastric bypass-shaped gut microbiota reduces adiposity in diet-induced obese recipients by re-activating energy expenditure from metabolic active brown adipose tissue. These beneficial effects were linked to improved glucose homeostasis, lipid control, and improved fatty liver disease. Mechanistically, these effects were triggered by modulation of taurine metabolism by the gastric bypass gut microbiota, fostering an increased abundance of intestinal and circulating taurine-conjugated bile acid species. In turn, these bile acids activated gut-restricted FXR and systemic TGR5 signaling to stimulate adaptive thermogenesis. CONCLUSION: Our results establish the role of the gut microbiome in the weight loss and metabolic success of gastric bypass surgery. We here identify a signaling cascade that entails altered bile acid receptor signaling resulting from a collective, hitherto undescribed change in the metabolic activity of a cluster of bacteria, thereby readjusting energy imbalance and metabolic disease in the obese host. These findings strengthen the rationale for microbiota-targeted strategies to improve and refine current therapies of obesity and metabolic syndrome. Video Abstract Bariatric Surgery (i.e. RYGB) or the repeated fecal microbiota transfer (FMT) from RYGB donors into DIO (diet-induced obesity) animals induces shifts in the intestinal microbiome, an effect that can be impaired by oral application of antibiotics (ABx). Our current study shows that RYGB-dependent alterations in the intestinal microbiome result in an increase in the luminal and systemic pool of Taurine-conjugated Bile acids (TCBAs) by various cellular mechanisms acting in the intestine and the liver. TCBAs induce signaling via two different receptors, farnesoid X receptor (FXR, specifically in the intestines) and the G-protein-coupled bile acid receptor TGR5 (systemically), finally resulting in metabolic improvement and advanced weight management. BSH, bile salt hydrolase; BAT brown adipose tissue.

FAD-mutation of APP is associated with a loss of its synaptotrophic activity.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder associated with extracellular accumulation of Abeta peptide that derives from the amyloid precursor protein (APP). While amyloidogenic processing of APP has received most attention, the physiological function of APP and the sequelae of potentially impaired APP function are less understood. APP is a transmembrane glycoprotein being widely expressed in neurons in both central and peripheral nervous system. Its physiological function has been associated with neuronal survival, neurite outgrowth and neuronal plasticity. The aim of the present study was to determine whether FAD-linked mutations of APP, known to be associated with early onset of the disease, might impair its synaptotrophic function, potentially contributing to synaptic deficiencies seen in AD. We performed a quantitative electron microscopy study on synapses in well characterized expression-matched transgenic mice lines expressing either wildtype or FAD-mutated hAPP. Using serial electron microscopic sections, we comparatively analyzed by stereological methods the number and sizes of synaptic contacts and the number of synaptic vesicles in the neocortex. We could clearly show a synaptotrophic effect in mice overexpressing wildtype hAPP evidenced by a significant increase in the number of synapses and the number of vesicles per synapse. This effect was abolished when FAD-mutated APP(Sw,Ind) was expressed instead of wildtype APP. The present study demonstrates a synaptotrophic effect of APP which is lost in the presence of a FAD-mutation. This failure could either be due to a synaptotoxic effect of Abeta potentially counteracting the synaptotrophic effect of APP. Alternatively, the FAD-mutation might impair the physiological function of the extracellular domain of APP and its fragments which might be required for the synaptotrophic effect. This suggests that not only "too much Abeta" but also "too less functional intact APP" might be relevant for synaptic pathology and degeneration in AD.

Effects of wild-type and mutant human amyloid precursor protein on cortical afferent network.

Alzheimer's disease is characterized by severe neuronal disintegration supposed to be partly associated with amyloid pathology. Recently, we described morphological alterations of pyramidal cell structure in transgenic mice expressing wild-type or mutant human amyloid precursor protein (hAPP) (strains B6-Py8.9 and Tg2576), which are unrelated to direct plaque-associated changes. In this study, we focused on the pattern of cortical afferent connections in these transgenic mice. The quantity of cholinergic afferents is increased in both transgenic lines. Glutamatergic intra- and interhemispheric afferents are augmented in B6-Py8.9 mice but decreased in Tg2576 mice. Furthermore, perisomatic inhibition of pyramidal neurons was found to be reduced in Tg2576 mice. Findings suggest different effects of wild-type and mutant hAPP on neuronal connectivity.

Different dendrite and dendritic spine alterations in basal and apical arbors in mutant human amyloid precursor protein transgenic mice.

The extracellular deposition of amyloid-beta peptide (Abeta) in brain parenchyma is one of the characteristic features of Alzheimer's disease and is suggested to induce reactive and degenerative changes in neuronal cell bodies, axons and dendritic processes. In particular, within and in close proximity to amyloid plaques, distinctive morphological alterations have been observed, including changes in neurite trajectory and decreases in dendritic diameter and in spine density. Apart from these plaque-associated focal aberrations, little is known regarding modifications of the global dendritic morphology including the detailed and comparative quantitative analysis of apical and basal arbors. The objective of the present study was to investigate the effects of amyloid plaque deposition and elevated soluble Abeta on neuronal morphology in mutant human amyloid precursor protein (hAPP) transgenic mice (line Tg2576; [K. Hsiao, P. Chapman, S. Nilsen, C. Eckman, Y. Harigaya, S. Younkin, F. Yang, G. Cole, Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice, Science 274 (1996) 99-102]). Retrogradelly labeled callosal-projecting pyramidal cells in the primary somatosensory cortex were three-dimensionally analyzed. Although basal dendrites remained unaffected, analysis of apical trees revealed a number of unambiguous morphological changes. Thus, in TG2576 mice, the apical arbors were shortened in total length and less branched. Furthermore, the diameter of proximal dendritic segments was increased whereas that of distal segments was reduced. Analysis of spine numbers and distribution on basal and apical trees demonstrated a significant reduction in spine densities along the whole course of dendrites. The findings suggest that Abeta-related pathology induces morphological aberrations in basal and apical arbors to different degrees which are unrelated to direct plaque-associated changes.

Activation of Ras in neurons modifies synaptic vesicle docking and release.

We have recently demonstrated that constitutive activation of Ha-Ras in differentiated neurons induces structural remodeling of both axons and dendrites in a transgenic mouse model (referred as synRas mice). Here we show that this activation of neuronal Ras enhances docking of synaptic vesicles to active zones, thereby leading to an increase in the size of the readily releasable pool of vesicles, while the size of the total pool of synaptic vesicles remained unchanged. The morphological phenotype was associated with corresponding increases in the probability of glutamate release as revealed by changes in short-term synaptic plasticity. We, therefore, conclude that neuronal Ras activity contributes to the regulation of synaptic plasticity in adult mammalian brain at the presynaptic level.

Enhanced Ras activity in pyramidal neurons induces cellular hypertrophy and changes in afferent and intrinsic connectivity in synRas mice.

Neurotrophic actions are critically controlled and transmitted to cellular responses by the small G protein Ras which is therefore essential for normal functioning and plasticity of the nervous system. The present study summarises findings of recent studies on morphological changes in the neocortex of synRas mice expressing Val12-Ha-Ras in vivo under the control of the rat synapsin I promoter. In the here reported model (introduced by Heumann et al. [J. Cell Biol. 151 (2000) 1537]), transgenic Val12-Ha-Ras expression is confined to the pyramidal cell population and starts postnatally at a time, when neurons are postmitotic and their developmental maturation has been basically completed. Expression of Val12-Ha-Ras results in a significant enlargement of pyramidal neurons. Size, complexity and spine density of dendritic trees are increased, which leads, finally, to cortical expansion. However, the main morphological design principles of 'transgenic' pyramidal cells remain preserved. In addition to somato-dendritic changes, expression of Val12-Ha-Ras in pyramidal cells induces augmented axon calibres and upregulates the establishment of efferent boutons. Despite the enlargement of cortical size, the overall density of terminals representing intra- or interhemispheric, specific and non-specific afferents is unchanged or even higher in transgenic mice suggesting a significant increase in the total afferent input to the neocortex. Although interneurons do not express the transgene and are therefore excluded from direct, intrinsic Val12-Ha-Ras effects, they respond with morphological adaptations to structural changes. Thus, dendritic arbours of interneurons are extended to follow the cortical expansion and basket cells establish a denser inhibitory innervation of 'transgenic' pyramidal cells perikarya. It is concluded that expression of Val12-Ha-Ras in pyramidal neurons results in remodelling of neocortical structuring which strongly implicates a crucial involvement of Ras in cortical plasticity.

Adaptive morphological changes of neocortical interneurons in response to enlarged and more complex pyramidal cells in p21H-Ras(Val12) transgenic mice.

Morphological features of interneuronal adaptation to an altered, more complex neuronal architecture have been investigated in p21H-Ras(Val12) transgenic mice. This transgenic strain serves as a model for studying the morphogenetic role of the G-protein p21Ras on cortical principal neurons. We have recently demonstrated that postmitotic expression of constitutively active p21H-Ras(Val12) in the neocortical pyramidal cell population results in increased size and dendritic complexity of the affected neurons, leading to an enlarged cortical volume. Interneurons do not express the transgene and are therefore excluded from direct, intrinsic p21H-Ras(Val12) effects. In the present study, immunolabelling of gamma-amino-butyric-acid (GABA), and of the calcium-binding proteins parvalbumin, calbindin and calretinin revealed that in the transgenic mice local circuit neurons are not increased in either somal size or number and their main morphological characteristics are preserved. However, the dendritic arbour of interneurons was found to be extended, at least in the vertical dimension, to follow the cortical expansion. Immunostaining for the vesicular GABA transporter revealed a denser inhibitory innervation of p21H-Ras(Val12)-expressing pyramidal cell perikarya than in those of wild-type animals, while the overall density of inhibitory axon terminals within the cortex was decreased in the transgenic animals as a consequence of cortical expansion. The findings of the present study demonstrate the morphogenetic capacity of interneurons for adapting to morphological alterations of principal neurons in the cerebral cortex.

Impact of 5-lipoxygenase inhibitors on the spatiotemporal distribution of inflammatory cells and neuronal COX-2 expression following experimental traumatic brain injury in rats.

The inflammatory response following traumatic brain injury (TBI) contributes to neuronal death with poor outcome. Although anti-inflammatory strategies were beneficial in the experimental TBI, clinical translations mostly failed, probably caused by the complexity of involved cells and mediators. We recently showed in a rat model of controlled cortical impact (CCI) that leukotriene inhibitors (LIs) attenuate contusion growth and improve neuronal survival. This study focuses on spatiotemporal characteristics of macrophages and granulocytes, typically involved in inflammatory processes, and neuronal COX-2 expression. Effects of treatment with LIs (Boscari/MK-886), started prior trauma, were evaluated by quantifying CD68(+), CD43(+) and COX-2(+) cells 24h and 72 h post-CCI in the parietal cortex (PC), CA3 region, dentate gyrus (DG) and visual/auditory cortex (v/aC). Correlations were applied to identify intercellular relationships. At 24h, untreated animals showed granulocyte invasion in all regions, decreasing towards 72 h. Macrophages increased from 24h to 72 h post-CCI in PC and v/aC. COX-2(+) neurones showed no temporal changes, except of an increase in the CA3 region at 72 h. Treatment reduced granulocytes at 24h in the pericontusional zone and hippocampus, and macrophages at 72 h in the PC and v/aC. COX-2 expression remained unaffected by LIs, except of time-specific changes in the DG (increase/decrease at 24/72 h). Interrelations confirmed concomitant cellular reactions beyond the initial trauma site. In conclusion, LIs attenuated the cellular inflammatory response following CCI. Future studies have to clarify region-specific effects and explore the potential of a clinically more relevant therapeutic approach applying LIs after CCI.

Involvement of perineuronal and perisynaptic extracellular matrix in Alzheimer's disease neuropathology.

Brain extracellular matrix (ECM) is organized in specific patterns assumed to mirror local features of neuronal activity and synaptic plasticity. Aggrecan-based perineuronal nets (PNs) and brevican-based perisynaptic axonal coats (ACs) form major structural phenotypes of ECM contributing to the laminar characteristics of cortical areas. In Alzheimer's disease (AD), the deposition of amyloid proteins and processes related to neurofibrillary degeneration may affect the integrity of the ECM scaffold. In this study we investigate ECM organization in primary sensory, secondary and associative areas of the temporal and occipital lobe. By detecting all major PN components we show that the distribution, structure and molecular properties of PNs remain unchanged in AD. Intact PNs occurred in close proximity to amyloid plaques and were even located within their territory. Counting of PNs revealed no significant alteration in AD. Moreover, neurofibrillary tangles never occurred in neurons associated with PNs. ACs were only lost in the core of amyloid plaques in parallel with the loss of synaptic profiles. In contrast, hyaluronan was enriched in the majority of plaques. We conclude that the loss of brevican is associated with the loss of synapses, whereas PNs and related matrix components resist disintegration and may protect neurons from degeneration.

Perineuronal nets are largely unaffected in Alzheimer model Tg2576 mice.

Changes in the molecular organization of the extracellular matrix are key factors in neuropathology. We investigated aggrecan-based perineuronal nets (PNs) in relation to neurodegeneration and activation of glial cells in a transgenic mouse (Tg2576) model of Alzheimer's disease. The formation of amyloid plaques in the cerebral cortex occurred independently of the area-specific distribution of PNs. Matrix components were only affected in the core of plaques in advanced stages of pathology. PNs remained unchanged in the large marginal zone occupied by reactive astrocytic processes. We conclude that the aggrecan-based extracellular matrix of PNs is not enzymatically altered in peripheral plaque territories and is only removed after neuronal death.

Inducible neuronal expression of transgenic TGF-beta1 in vivo: dissection of short-term and long-term effects.

Various chronic neurological diseases are associated with increased expression of transforming growth factor-beta1 (TGF-beta1) in the brain. TGF-beta1 has both neuroprotective and neurodegenerative functions, depending on conditions such as duration and the local and temporal pattern of its expression. Previous transgenic approaches did not enable control for these dynamic aspects. To overcome these limitations, we established a transgenic mouse model with inducible neuron-specific expression of TGF-beta1 based on the tetracycline-regulated gene expression system. TGF-beta1 expression was restricted to the brain where it was particularly pronounced in the neocortex, hippocampus and striatum. Transgene expression was highly sensitive to the presence of doxycycline and completely silenced within 6 days after doxycycline application. After long-term expression, perivascular thioflavin-positive depositions, formed by amyloid fibrils, developed. These depositions persisted even after prolonged silencing of the transgene, indicating an irreversible process. Similarly, strong perivascular apolipoprotein E (ApoE) depositions were found after TGF-beta1 expression and these remained despite TGF-beta1 removal. These in vivo observations suggests that the continuous presence of TGF-beta1 as initial trigger is not necessary for the persistence and development of chronic lesions. Neuroprotective effects were observed after short-term expression of TGF-beta1. Death of striatal neurons induced by 3-nitropropionic acid was markedly reduced after induced TGF-beta1 expression.

Constitutive expression of p21H-Ras(Val12) in neurons induces increased axonal size and dendritic microtubule density in vivo.

The small G protein p21Ras is a key signal transducer mediating cellular growth and proliferation responses to extracellular stimuli. We investigated by electron microscopy the effects of augmented p21Ras activity on neuronal processes and microtubule arrangement in vivo. We used transgenic mice with a neuron-specific overexpression of p21H-RasVal12, which starts postnatally around Day 15. Axonal and dendritic diameters and the numerical density of dendritic microtubules were analyzed at postnatal Day 12 before the onset of transgene expression and in adult mice. In adult transgenic mice, calibers of both axons (corpus callosum) and dendrites (layers II/III of somatosensory cortex) were enlarged by about 57% and 79%, respectively. The increase in dendritic calibers was associated with an increment in the amount of microtubules. Even in dendrites of equivalent diameters, the number of microtubules was higher in transgenic mice compared to that in wild-type mice suggesting an elevated microtubule density. Changes in process diameters or microtubule density were not observed at postnatal Day 12 before relevant transcription of transgenic p21H-RasVal12. The present results extend previous findings on neuronal hypertrophy as a consequence of p21H-RasVal12 expression and suggest a profound influence on the dendritic microtubule network.

Constitutive Ras activity induces hippocampal hypertrophy and remodeling of pyramidal neurons in synRas mice.

The small G protein Ras, which is involved critically in neurotrophic signal transduction, has been implicated in neuronal plasticity of both the developing and the adult nervous systems. In the present study, the cumulative effects of constitutive Ras activity from early in postnatal development into the adult upon the morphology of hippocampal pyramidal neurons were investigated in synRas mice overexpressing Val12-Ha-Ras postmitotically under the control of the rat synapsin I promoter. In synRas mice, stereologic investigations revealed hypertrophy of the hippocampus associated with an increase in perikaryal size of pyramidal neurons within the CA2/CA3 region and the gyrus dentatus. Morphometric analyses of Lucifer Yellow-filled CA1 pyramidal neurons, in addition, demonstrated considerable expansion of dendritic arbors. The increase in basal dendritic size was caused primarily by alterations of intermediate and distal segments and was associated with an enlarged dendritic surface. Apical dendrites showed similar but more moderate changes, which were attributed mainly to elongation of terminal segments. Sholl analyses illustrated higher complexity of both basal and apical trees. Despite significant morphologic alterations, dendritic arbors preserve their major design principles. The synaptic density within the stratum radiatum of CA1 remained unchanged; however, increases in the total hippocampal volume and in apical dendritic size imply an increment in the absolute number of synaptic contacts. The data presented here suggest a critical involvement of Ras dependent signaling in morphoregulatory processes during the maturation and in the maintenance of hippocampal pyramidal neurons.

Constitutive expression of p21H-ras(Val12) in pyramidal neurons results in reorganization of mouse neocortical afferents.

The effect of constitutive expression of p21H-ras(Val12) in pyramidal neurons upon the establishment of afferent input has been investigated in the primary somatosensory cortex of transgenic mice. In these animals, relevant transgene expression is confined to cortical pyramidal neurons and starts postnatally at a period when neuronal morphogenesis has been largely completed. We have shown recently that overexpression of p21H-ras(Val12) in these cells results in considerable enlargement of their size and consequently in expansion of the cortex. In the present study we demonstrate that the density of terminals representing intra- or interhemispheric afferents within cortical layers II/III, however, is only slightly decreased. The density of thalamocortical boutons within layer IV is even higher and the number of afferent contacts to transgenic pyramidal neurons is significantly increased compared to the wild-type. The number of catecholaminergic and cholinergic terminals is augmented proportionally to cortical size or even overproportionally, respectively. Along intercortical and striatal fibers arising from p21H-ras(Val12)-expressing pyramidal neurons, frequency of varicosities is significantly increased, but remains unchanged on cortical cholinergic and catecholaminergic axons originating from "nontransgenic" neurons. Additionally, a higher number of multiple synaptic bodies are found in transgenic mice, suggesting subtle effects on synaptic plasticity. It is concluded that the enlargement of pyramidal neurons due to transgenic expression of p21H-ras(Val12) is paralleled by significant changes in the quantity and pattern of afferent connections. Moreover, expression of p21H-ras(Val12) in pyramidal cells induces an enhanced establishment of efferent boutons.

Neuronal activation of Ras regulates synaptic connectivity.

A synRas mouse model was used expressing constitutively activated Ha-Ras (Val12 mutation) in neurons to investigate the role of Ras-MAPkinase signalling for neuronal connectivity in adult brain. Expression of the transgene in the cortex of these mice starts after neuronal differentiation is completed and allows to directly investigate the effects of enhanced Ras activity in differentiated neurons. Activation of Ha-Ras induced an increase in soma size which was sensitive to MEK inhibitor in postnatal organotypic cultures. Adult cortical pyramidal neurons showed complex structural rearrangements associated with an increased size and ramification of dendritic arborization. Dendritic spine density was elevated and correlated with a twofold increase in number of synapses. In acute brain slices of the somatosensory and of the visual cortex, extracellular field potentials were recorded from layer II/III neurons. The input-output relation of synaptically evoked field potentials revealed a significantly higher basal excitability of the transgenic mice cortex compared to wild-type animals. In whole cell patch clamp preparations, the frequency of AMPA receptor-mediated spontaneous excitatory postsynaptic currents was increased while the ratio between NMDA and AMPA-receptor mediated signal amplitude was unchanged. A pronounced depression of paired pulse facilitation indicated that Ras contributes to changes at the presynaptic site. Furthermore, synRas mice showed an increased synaptic long-term potentiation, which was sensitive to blockers of NMDA-receptors and of MEK. We conclude that neuronal Ras is a common switch of plasticity in adult mammalian brain sculpturing neuronal architecture and synaptic connectivity in concert with tuning synaptic efficacy.