Systemic inhibition of the membrane attack complex impedes neuroinflammation in chronic relapsing experimental autoimmune encephalomyelitis.

The complement system is a key driver of neuroinflammation. Activation of complement by all pathways, results in the formation of the anaphylatoxin C5a and the membrane attack complex (MAC). Both initiate pro-inflammatory responses which can contribute to neurological disease. In this study, we delineate the specific roles of C5a receptor signaling and MAC formation during the progression of experimental autoimmune encephalomyelitis (EAE)-mediated neuroinflammation. MAC inhibition was achieved by subcutaneous administration of an antisense oligonucleotide specifically targeting murine C6 mRNA (5 mg/kg). The C5a receptor 1 (C5aR1) was inhibited with the C5a receptor antagonist PMX205 (1.5 mg/kg). Both treatments were administered systemically and started after disease onset, at the symptomatic phase when lymphocytes are activated. We found that antisense-mediated knockdown of C6 expression outside the central nervous system prevented relapse of disease by impeding the activation of parenchymal neuroinflammatory responses, including the Nod-like receptor protein 3 (NLRP3) inflammasome. Furthermore, C6 antisense-mediated MAC inhibition protected from relapse-induced axonal and synaptic damage. In contrast, inhibition of C5aR1-mediated inflammation diminished expression of major pro-inflammatory mediators, but unlike C6 inhibition, it did not stop progression of neurological disability completely. Our study suggests that MAC is a key driver of neuroinflammation in this model, thereby MAC inhibition might be a relevant treatment for chronic neuroinflammatory diseases.

Architecture of the Synaptophysin/Synaptobrevin Complex: Structural Evidence for an Entropic Clustering Function at the Synapse.

We have purified the mammalian synaptophysin/synaptobrevin (SYP/VAMP2) complex to homogeneity in the presence of cholesterol and determined the 3D EM structure by single particle reconstruction. The structure reveals that SYP and VAMP2 assemble into a hexameric ring wherein 6 SYP molecules bind 6 VAMP2 dimers. Using the EM map as a constraint, a three dimensional atomic model was built and refined using known atomic structures and homology modeling. The overall architecture of the model suggests a simple mechanism to ensure cooperativity of synaptic vesicle fusion by organizing multiple VAMP2 molecules such that they are directionally oriented towards the target membrane. This is the first three dimensional architectural data for the SYP/VAMP2 complex and provides a structural foundation for understanding the role of this complex in synaptic transmission.

[A synaptic marker synaptophysin].

The review summarizes the current data on synaptophysin (SYP), its functional role in the cell and the use of SYP immunocytochemistry for labeling the synaptic contacts. SYP is a transmembrane glycoprotein found in small presynaptic vesicles of the nerve cells and in microvesicles of the neuroendocrine cells. Literature data and the authors' own experience suggest that currently SYP is an important synaptic marker, which allows, with the use of light and confocal laser microscopy, to obtain the reliable data on the morphological organization of the synaptic structures in the central nervous system. It is also indispensable in the study of the efferent innervation of the internal organs. Applicatioin of the quantitative analysis of SYP-immunopositive structures using light and confocal laser microscopy allows to solve some problems that previously could be solved only by using electron microscopy.

Stereological and allometric studies on neurons and axo-dendritic synapses in the superior cervical ganglia of rats, capybaras and horses.

The superior cervical ganglion (SCG) in mammals varies in structure according to developmental age, body size, gender, lateral asymmetry, the size and nuclear content of neurons and the complexity and synaptic coverage of their dendritic trees. In small and medium-sized mammals, neuron number and size increase from birth to adulthood and, in phylogenetic studies, vary with body size. However, recent studies on larger animals suggest that body weight does not, in general, accurately predict neuron number. We have applied design-based stereological tools at the light-microscopic level to assess the volumetric composition of ganglia and to estimate the numbers and sizes of neurons in SCGs from rats, capybaras and horses. Using transmission electron microscopy, we have obtained design-based estimates of the surface coverage of dendrites by postsynaptic apposition zones and model-based estimates of the numbers and sizes of synaptophysin-labelled axo-dendritic synaptic disks. Linear regression analysis of log-transformed data has been undertaken in order to establish the nature of the relationships between numbers and SCG volume (V(scg)). For SCGs (five per species), the allometric relationship for neuron number (N) is N=35,067xV (scg) (0.781) and that for synapses is N=20,095,000xV (scg) (1.328) , the former being a good predictor and the latter a poor predictor of synapse number. Our findings thus reveal the nature of SCG growth in terms of its main ingredients (neurons, neuropil, blood vessels) and show that larger mammals have SCG neurons exhibiting more complex arborizations and greater numbers of axo-dendritic synapses.

Structure of synaptophysin: a hexameric MARVEL-domain channel protein.

Synaptophysin I (SypI) is an archetypal member of the MARVEL-domain family of integral membrane proteins and one of the first synaptic vesicle proteins to be identified and cloned. Most all MARVEL-domain proteins are involved in membrane apposition and vesicle-trafficking events, but their precise role in these processes is unclear. We have purified mammalian SypI and determined its three-dimensional (3D) structure by using electron microscopy and single-particle 3D reconstruction. The hexameric structure resembles an open basket with a large pore and tenuous interactions within the cytosolic domain. The structure suggests a model for Synaptophysin's role in fusion and recycling that is regulated by known interactions with the SNARE machinery. This 3D structure of a MARVEL-domain protein provides a structural foundation for understanding the role of these important proteins in a variety of biological processes.

Macromolecular-scale resolution in biological fluorescence microscopy.

We demonstrate far-field fluorescence microscopy with a focal-plane resolution of 15-20 nm in biological samples. The 10- to 12-fold multilateral increase in resolution below the diffraction barrier has been enabled by the elimination of molecular triplet state excitation as a major source of photobleaching of a number of dyes in stimulated emission depletion microscopy. Allowing for relaxation of the triplet state between subsequent excitation-depletion cycles yields an up to 30-fold increase in total fluorescence signal as compared with reported stimulated emission depletion illumination schemes. Moreover, it enables the reduction of the effective focal spot area by up to approximately 140-fold below that given by diffraction. Triplet-state relaxation can be realized either by reducing the repetition rate of pulsed lasers or by increasing the scanning speed such that the build-up of the triplet state is effectively prevented. This resolution in immunofluorescence imaging is evidenced by revealing nanoscale protein patterns on endosomes, the punctuated structures of intermediate filaments in neurons, and nuclear protein speckles in mammalian cells with conventional optics. The reported performance of diffraction-unlimited fluorescence microscopy opens up a pathway for addressing fundamental problems in the life sciences.

On the atrophy of the internal carotid artery in capybara.

Capybara might be a useful model for studying changes in cerebral circulation as the natural atrophy of the internal carotid artery (ICA) occurs in this animal at maturation. In this study, confocal and electron microscopy combined with immunohistochemical techniques were applied in order to reveal the changes in morphology and innervation to the proximal part of ICA in young (6-month-old) and mature (12-month-old) capybaras. Some features of the basilar artery (BA) were also revealed. The ICA of young animals degenerated to a ligamentous cord in mature animals. Immunolabelling positive for pan-neuronal marker protein gene product 9.5 but negative for tyrosine hydroxylase was observed in the proximal part of ICA at both ages examined. Axon varicosities positive for synaptophysin were present in the adventitia of ICA of young animals but were absent in the ligamentous cord of mature animals. In the ICA of young animals, adventitial connective tissue invaded the media suggesting that the process of regression of this artery began within the first 6 months of life. An increase in size of the BA was found in mature animals indicating increased blood flow in the vertebro-basilar system, possibly making capybara susceptible to cerebrovascular pathology (e.g. stroke). Capybara may therefore provide a natural model for studying adaptive responses to ICA regression/occlusion.

Demyelinating disease simulating brain tumours: a histopathologic assessment of seven cases.

BACKGROUND: Demyelinating diseases can present as space occupying lesions with in the brain. It is clinically and radiologically difficult to differentiate them from primary neoplasms. Histopathologically they mimic astrocytic neoplasms closely and identifying these lesions correctly has a profound impact in treatment and prognosis of these patients. AIMS AND OBJECTIVES: The objective was to determine the histopathologic features of such acute focal demyelinating disease that clinically presented as brain tumors. MATERIAL AND METHODS: Seven cases were included for the study. Detailed histopathological examination including stains for myelin and axon were performed. The histopathological keys in arriving at the right diagnoses included a well demarcated lesion that contains uniform distribution of foamy macrophages in the absence of any associated coagulative necrosis, sheets of gemistocytic astrocytes in the white matter that show well-formed processes, perivascular chronic inflammatory cell infiltration and total absence of myelin with relative preservation of axons within these areas. CONCLUSION: The degree of suspicion (clinical, radiological and histopathological) should be high to diagnose these group of lesions. The above-mentioned diagnostic keys should help in arriving at the correct histopathological diagnoses of such cases.

Neural membrane protein 35/Lifeguard is localized at postsynaptic sites and in dendrites.

We have previously identified and characterized a cDNA coding for neural membrane protein 35 (NMP35). We showed that NMP35 mRNA is predominantly expressed in the adult CNS with a neuronal expression pattern. Functional analysis indicates that the human homologue of NMP35, Lifeguard, plays a role in Fas-mediated cell death. In this study we used affinity-purified antibodies raised against the putative cytoplasmic N-terminal domain of NMP35 to determine its precise subcellular localization in the adult CNS. NMP35 protein is widely expressed throughout the brain and spinal cord, most prominently in dendrites of several neuronal cell types and in the surrounding neuropil. Immunofluorescence confocal microscopy reveals colocalization of NMP35 with the glutamate receptor GluR2 and adjacent localization to the presynaptic vesicle protein synaptophysin. These data suggest that NMP35 may be localized to the postsynaptic membrane. Immunoelectron microscopy with NMP35 antibodies confirms the expression of the protein in dendritic processes and in a subset of synapses at the postsynaptic membrane and density. These findings suggest a role for NMP35 in synapses of the adult central nervous system.

Synaptic vesicle protein synaptoporin is differently expressed by subpopulations of mouse hippocampal neurons.

In the hippocampus, the synaptic vesicle protein synaptoporin (SPO) has been reported to be exclusively enriched in the granule cell axons, the mossy fibers. In this study, we show that in adult rats and mice SPO immunoreactivity (IR) is also detectable in strata oriens, radiatum, and lacunosum-moleculare of CA1-CA3, as well as perisomatically in the hippocampus proper and fascia dentata. In situ hybridization confirmed that SPO mRNA was present in granule cells and CA3 pyramidal cells but not in CA1 pyramidal cells. Importantly, cells scattered throughout the hippocampal layers resembling the distribution of interneurons were found to synthesize high amounts of SPO mRNA, too. Thus, these findings indicate that SPO expression in the hippocampus was underestimated until now. Moreover, double-labeling immunohistochemistry and confocal microscopy revealed selective colocalization of SPO and glutamate decarboxylase (GAD 65), a marker for gamma-aminobutyric acid (GABA)ergic terminals. To identify SPO expressing interneurons, in situ hybridization was combined with immunocytochemistry against parvalbumin (PV), calbindin (CB), calretinin (CR), cholecystokinin (CCK), and vasoactive intestinal polypeptide (VIP). We found that SPO transcripts were differentially expressed by various interneuron subpopulations in the hippocampus of C57Bl/6 mice (PV 44.2%, CB 46.3%, CR 19.3%, CCK 38.6%, VIP 59.9%). Immunoelectron microscopy for SPO labeled synaptic vesicle profiles in distinct symmetric and asymmetric synapses. In conclusion, our data demonstrate that hippocampal principal cells and interneurons display a variety of synaptic vesicles that are likely to contribute to the functional characteristics of their output synapses.

[Adrenocortical tumors with neuroendocrine differentiation]. / Adrenokortikal'nye opukholi s neiroéndokrinnoi differentsirovkoi.

18 adrenocortical tumours and 5 pheochromocytomas were studied immunohistochemically. Expression of synaptophysin and chromogranin A was found in cells of cortical adenomas, "frontier" neoplasms and in 20-75% of carcinoma cells, this ultramicroscopically was confirmed by observation of typical neuroendocrine granules. Some groups of cells of cortico-medullary tumours also expressed proteins of neural differentiation (protein S-100).

[Central neurocytoma]. / Neurocitoma central.

INTRODUCTION: A central neurocytoma (CN) is a rare tumor, of neuronal origin, well-differentiated and found intraventricularly. It mainly affects young adults. Firm diagnosis is made on immunohistochemical (IHQ) and ultrastructural studies, since on optic microscopy it is similar in appearance to an oligodendroglioma or to an ependymoma. PATIENTS AND METHODS: We studied 4 cases, three after surgical resection and one on autopsy. The average age was 29, ranging from 3 to 63. Both sexes were equally affected. In all cases IHQ techniques were used (GFAP, neurofilament, synaptophysin and specific neuronal enolase) and they were studied by electron microscopy. RESULTS: IHQ was negative for GFAP and neurofilament, but intensely positive for synaptophysin and specific neuronal enolase. On ultrastructural study there were few neurofilaments, microtubules and dense central granules typical of neural differentiation. CONCLUSIONS: The findings in our cases lead to diagnosis of NC and confirm that this tumor is a distinct clinicopathological entity.

Immunoelectron-microscopic localization of synaptophysin in the organ of Corti of the guinea pig.

Synaptophysin has been localized previously in the mammalian cochlea at the light-microscopic level and in few reports by electron microscopy using either a preincubation procedure or the avidin-biotin reaction. Here we present results of the electron-microscopic analysis for postembedding immunoreactivity of synaptophysin in the cochlea of the guinea pig of LR-White-embedded samples. Strong synaptophysin immunoreactivity is located in the cytoplasm of the efferent nerve endings at the base of inner and outer hair cells. Besides this, some antibodies to synaptophysin were also identified in the cytoplasm of outer hair cells. To get more information about the cellular content of synaptophysin, the density of the immunoreaction per square micrometer was determined for the outer hair cells of the second turn of the cochlea counting the gold particles in numerous ultrathin sections. The role of synaptophysin identified in the cytoplasm of outer hair cells is discussed.

Spatial periodicity of NADPH-diaphorase and synaptophysin, but not SNAP-25, reactivity in the monkey cerebellar cortex.

We recently described a parasagittal patchy organisation of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity in the granular layer of the rat cerebellum. We now report the pattern of NADPH-d distribution in the primate cerebellum and its relationship to two synaptic proteins, synaptophysin and synaptosomal associated protein 25 kDa (SNAP-25), using histochemistry and immunocytochemistry. NADPH-d reactivity was localised in the molecular and granular layers (ML, GL) and a subset of infraganglionic plexuses (IGPs), but not in the Purkinje cell layer and the white matter. In ML, the histochemical reactivity was dense and relatively homogeneous in the neuropil, and moderate in the stellate cells. A patchy organisation of NADPH-d in GL was detected in both horizontal and parasagittal sections. In the IGPs staining for NADPH-d revealed modular positive zones alternating with negative ones. The positive and negative IGP zones were usually congruent with the high and low NADPH-d reactivity in GL, respectively. Both synaptic proteins were strongly expressed in the neuropil in ML and GL, and their patterns were relatively homogeneous. However, synaptophysin was present in a subpopulation of IGPs organised in modules which corresponded to those expressing NADPH-d. Our results indicate that the NADPH-d modular system is more complicated in the primate cerebellum than in the rat. In addition, we have provided suggestive evidence of a co-expression of NADPH-d and synaptophysin in selected IGP modules in primate cerebellum, which suggests that nitric oxide may be involved in the activity of the Purkinje cells by affecting the basket cell synaptic input.