Evaluation of intrathecal serum amyloid P (SAP) and C-reactive protein (CRP) synthesis in Alzheimer's disease with the use of index values

Serum amyloid P (SAP) and C-reactive protein (CRP) are proteins involved in innate immunity. The expression of SAP and CRP is increased in Alzheimer's disease (AD) brain tissue, compared to healthy controls. Although both proteins are found in cerebrospinal fluid (CSF), their origin is unclear. We investigated if increased local production of SAP and CRP in AD brain results in higher levels in CSF with the use of index values. To study this, SAP, CRP, and albumin levels were determined in CSF and serum samples of 30 control (65 ± 11 years; 57% female) and 140 AD subjects (65 ± 9 years; 53% female). To correct for inter-individual differences in protein diffusion from blood to CSF, quotients (Q =CSF/serum) of SAP, CRP, and albumin and index values (Qprotein/Qalb) were calculated. The results showed no significant differences in SAP and CRP index values between control and AD subjects, although eight percent of individual AD patients showed evidence of intrathecal SAP or CRP production using the Reiber hyperbolic model. Interestingly, the SAP index value was much lower than expected, based on its molecular size. In conclusion, these data suggest that local production of SAP and CRP in the AD brain does not substantially contribute to the CSF levels.     

A novel scaffold protein, TANC, possibly a rat homolog of Drosophila rolling pebbles (rols), forms a multiprotein complex with various postsynaptic density proteins

We cloned from the rat brain a novel gene, tanc (GenBank Accession No. AB098072), which encoded a protein containing three tetratricopeptide repeats (TPRs), ten ankyrin repeats and a coiled-coil region, and is possibly a rat homolog of Drosophila rolling pebbles (rols). The tanc gene was expressed widely in the adult rat brain. Subcellular distribution, immunohistochemical study of the brain and immunocytochemical studies of cultured neuronal cells indicated the postsynaptic localization of TANC protein of 200 kDa. Pull-down experiments showed that TANC protein bound PSD-95, SAP97, and Homer via its C-terminal PDZ-binding motif, -ESNV, and fodrin via both its ankyrin repeats and the TPRs together with the coiled-coil domain. TANC also bound the alpha subunit of Ca2+/calmodulin-dependent protein kinase II. An immunoprecipitation study showed TANC association with various postsynaptic proteins, including guanylate kinase-associated protein (GKAP), alpha-internexin, and N-methyl-D-aspartate (NMDA)-type glutamate receptor 2B and AMPA-type glutamate receptor (GluR1) subunits. These results suggest that TANC protein may work as a postsynaptic scaffold component by forming a multiprotein complex with various postsynaptic density proteins.     

Differential interaction of the tSXV motifs of the NR1 and NR2A NMDA receptor subunits with PSD-95 and SAP97.

The NR1 and NR2 subunits of the N-methyl-D-aspartate (NMDA) receptor are encoded by distinct genes. In the rat brain, four C-terminal variants of the NR1 subunit (NR1-1 to NR1-4) are encoded by a single gene, and are generated by alternative splicing of the C1 and C2 exon cassettes, while four different genes encode the NR2 subunits (NR2 A-D). Functional NMDA receptors result from the heteromultimeric assembly of NR1 variants with distinct NR2 subunits. The NR2B subunit interacts with post-synaptic density protein 95 (PSD-95), SAP97 and members of the membrane-associated guanylate-like kinase (MAGUK) family of proteins. This interaction occurs through the binding of the C-terminal tSXV intracellular motif of the NR2B subunit to the N-terminal PDZ (PSD-95, discs-large, ZO-1) domains of the PSD-95 and SAP97 proteins. Both NR1-3 and NR1-4 also display a consensus C-terminal tSXV motif. Using the two-hybrid genetic system in yeast and site-directed mutagenesis, we compared the binding of the NR2A, NR1-3 and NR1-4 tSXV motifs with the PDZ domains of PSD-95 and SAP97. The main conclusions of the present report are that: (i) while NR2A displays a strong interaction with PSD-95 and SAP97, the NR1-3 and NR1-4 NMDA receptor subunits do not display any interaction despite the presence of tSXV motifs; (ii) the C-terminal tSXV motif of the NR2A subunit is mandatory but not sufficient for efficient interaction with the PSD-95 and SAP97 proteins; (iii) as yet unidentified upstream sequences of the receptor subunits determine whether the tSXV motifs will bind to the PSD-95 and SAP97 PDZ domains; (iv) different tSXV motifs elicit interactions of variable strengths; and (v) residues in positions -3 and -4 modulate the binding affinity of the C-terminal tSXV motifs. Using immunohistochemistry, we also compared the distribution of the PSD-95, NR2A and SAP97 proteins in adult rat brain, and we show that in the cortex, hippocampus and cerebellum, there is evidence for colocalization of these proteins.     

p55 protein is a member of PSD scaffold proteins in the rat brain and interacts with various PSD proteins

p55 is a membrane-associated guanylate kinase (MAGuK) family member that consists of a single PDZ followed by SH3, HOOK and guanylate kinase (GuK or GK) domains. We investigated rat p55 (r-p55) in the brain. r-p55 mRNA was expressed widely in various tissues and in various regions of the brain. r-p55 protein was also expressed widely in various rat tissues, including brain and erythrocytes. The protein was enriched in the synaptic plasma membrane and postsynaptic density (PSD) fractions of the forebrain. An immunocytochemical study using cultured cortical neurons suggested postsynaptic localization of r-p55 protein. Pull-down assay showed that r-p55 protein interacted with r-p55 itself and various PSD proteins, such as PSD-95, SAP97, GKAP, CASK, GRIP, neuroligin, cadherin, tubulin, actin, alpha-internexin, neurofilament-L and Ca(2+)/calmodulin-dependent protein kinase II, through its PDZ, SH3, HOOK or GK domains. The interaction with PSD-95 was found to occur between the PDZ domains of PSD-95 and the HOOK and GK domains of r-p55 protein. These findings, together with the presence of r-p55 puncta in a period of early synaptogenesis, suggest that r-p55 protein functions as one of postsynaptic scaffold component in an early stage of synaptogenesis in the brain. r-p55 protein may form a basic structure, which interlinks diverse functional molecules of the PSD necessary for postsynaptic signaling and synaptic adhesion.     

Progesterone counteracts estrogen-induced increases in neurotrophins in the aged female rat brain

Neurotrophin alterations have been associated with normal aging and age-related neurodegenerative disease, as well as cognitive status. Estrogen influences expression of mRNA and protein of neurotrophins and their receptors, and affects cognitive performance in young ovariectomized (Ovx) rats. The current investigation evaluated whether estrogen or estrogen plus progesterone affects neurotrophin protein levels in cognitive brain regions in the aged Ovx rat. While estrogen treatment increased BDNF, NGF, and NT3 levels in entorhinal cortex, progesterone abated the effects of estrogen resulting in neurotrophin levels comparable to aged Ovx rats not given hormone. Our findings suggest that the aged female brain is responsive to estrogen in cognitive brain regions, and that progesterone can reverse these estrogen effects.     

Serum amyloid P immunoreactivity in hippocampal tangles, plaques and vessels: implications for leakage across the blood-brain barrier in Alzheimer''s disease

Serum amyloid P (SAP) has been shown to be consistently present in all types of amyloid deposits except cerebral lesions of neurofibrillary tangles and senile plaques. We used immunohistochemical methods to demonstrate SAP reactivity in both tangles and plaques, as well as vessels, in lightly fixed frozen tissue sections of hippocampus and parahippocampal gyrus from subjects with Alzheimer's disease (AD) and normal controls. As confirmed by thioflavin S staining, heavy deposition of immunoperoxidase reaction product was evident in Sommer's sector (CA1), the subiculum and entorhinal cortex with both the antisera to SAP used. Serial sections immunostained with antiserum to amyloid A or preimmune rabbit serum showed no evidence for staining in plaques or tangles. These observations provide evidence for extravasation of the protein across the blood-brain barrier (BBB) in disease although expression of it by cellular elements within or entering the brain through the BBB cannot be ruled out. Our results also implicate the use of lightly fixed tissue for localization of some antigens by immunohistochemistry in postmortem human brain.     

Heterogeneity and regulation of cellular prion protein glycoforms in neuronal cell lines

The normal cellular prion protein is a small sialoglycoprotein highly expressed in neurons, the physiological function of which is largely unknown. Due to extensive N-glycosylations with a wide range of oligosaccharides, the prion protein displays a complex glycosylation pattern that could be of relevance for its function. The cellular prion protein patterns in adult mouse and rat brain, and in neuronal cell lines, appeared highly heterogeneous, as distinct levels and glycoforms of cellular prion protein were revealed by immunoblotting of corresponding samples. Amongst neuronal cell lines, mouse N2a neuroblastoma cells expressed low levels of endogenous prion protein. Mouse hypothalamic GT1-7 cells and rat pheochromocytoma PC-12 cells expressed highly glycosylated forms of cellular prion protein that were found neither in adult mouse and rat brain, nor in mouse brain during development. In contrast, rat B104 neuroblastoma cells abundantly expressed N-glycosylated cellular prion protein forms similar to those observed in mouse and rat brain. In all these cell lines, the prion protein was normally exported to and expressed at the outer cell membrane. Our results suggest that B104 cells may represent an appropriate cell model to investigate the physiological role of cellular prion protein in further detail as they highly express the normal 'brain-like' cellular prion protein glycoforms. In addition, we observed that the various prion glycoforms in B104 cells were tightly regulated both as a function of cell density and during neuronal differentiation, implying a potential role of cellular prion protein in cell-cell interactions and differentiation.     

Developmental changes in the association of NMDA receptors with lipid rafts

Lipid rafts (LR) are lipid microdomains present in the cell surface membrane that are organizational platforms involved in protein trafficking and formation of cell signaling complexes. In the adult brain, NMDA receptors (NMDAR) and receptor-associated proteins such as membrane-associated guanylate kinases (PSD-95 and SAP102), are distributed between the postsynaptic density (PSD) and lipid rafts. However, the time course of the association of NMDAR with LR during neural development is not known. We therefore investigated the effect of development on the association of NMDAR with LR prepared from rat brains ranging in postnatal age from 1-35 days and compared this with their expression in PSDs. LR and PSD fractions were prepared by extraction of P2 membranes with Tx-100 followed by sucrose density gradient centrifugation. The yield of LR, as reflected by levels of protein, Thy-1, and flotillin-1 increased during postnatal development. NR2A was associated predominantly with the lipid raft fraction at all ages examined whereas NR2B underwent a gradual shift from PSDs to lipid rafts during the first 3 weeks after birth. These changes in the distribution of NR2A and NR2B were paralleled by changes in the distribution of PSD-95 and SAP102 respectively. Tyrosine-phosphorylated proteins, including NR2A and NR2B, were preferentially associated with lipid rafts in older, as compared to younger, animals. These results show that the association of NMDAR with LR is regulated developmentally.     

Regional brain glycogen stores and metabolism during complete global ischaemia

Microwave fixation in situ was used to assess regional glycogen and glucose stores in normal rat brain. Glycogen levels were highest in the cerebellum and pons/medulla (38.0 and 35.6 nmol/mg protein), and lowest in the striatum and cerebral cortex (17.4 and 23.6 nmol/mg protein respectively). Glucose concentrations paralleled glycogen, ranging from 5.9 to 10.8 nmol/mg protein. Glycogen, glucose, and lactate were measured during complete global ischaemia (decapitation) to assess regional differences in ischaemic metabolism. Those regions which in normal brain contain higher glycogen and glucose stores were found to maintain significantly higher levels of glycogen and glucose for at least 2 minutes of ischaemia. Lactate accumulated to highest levels after 30 minutes of ischaemia in those regions with highest glucose and glycogen stores. Lactate levels did not, however, rise above 90 nmol/mg protein in any brain region, a level well below that considered potentially neurotoxic. The data indicate considerable regional differences in normal and ischaemic glycogen metabolism that might contribute to known regional differences in vulnerability to global ischaemia.     

Serum amyloid P is not present in amyloid beta deposits of a transgenic animal model

Serum amyloid P component (SAP) is associated with amyloid beta (A beta) deposition in Alzheimer disease (AD). Since SAP is exclusively synthesized by peripheral organs, its presence in the brain of AD suggests impairment of the blood-brain barrier (BBB). We studied the association of SAP with A beta deposits in a transgenic mouse model overexpressing beta-protein precursor (betaPP). Both SAP and another extracellular matrix binding protein, basic fibroblastic growth factor bind to the heparinase sensitive sites of A beta deposits in this model. However, no endogenous SAP immunoreactivity was found in the transgenic mouse brain. These results suggest that SAP is not required for A beta deposition, and that this mouse model does not develop the same BBB abnormalities as those seen in AD.     

Serum amyloid P component induces neuronal apoptosis and beta-amyloid immunoreactivity

Previously we have reported serum amyloid P component (SAP) induced cell death in cerebro-cortical cultures of rat brain. In this paper we studied the types of target cells and the molecular mechanism of SAP-induced cell death. Immuno-electron and light microscopy revealed that SAP penetrates the plasma membrane and translocates selectively into the nuclei of neurons. Neuronal cells with SAP immunoreactivity exhibit the morphological hallmarks of apoptosis in vitro. The apoptotic mechanism of cell death is also supported by the increased Bax/Bcl-2 ratio. In addition to neurotoxic effects, we detected elevated beta-amyloid (Abeta) immunoreactivity following SAP treatment. This study supports the thesis that SAP plays an important role in the pathomechanism of neurodegenerative diseases, including Alzheimer's disease by inducing neuronal apoptosis.     

Immunocytochemical localization of the synapse-associated protein SAP102 in the rat retina

An elaborate network of transmitter receptors, synapse associated proteins (SAPs), and cytoskeletal elements, generally known as the postsynaptic density, is involved with efficient synaptic signaling. The localization of the synapse associated protein SAP102 was studied in the rat retina by using immunocytochemical methods. Immunofluorescence for SAP102 was most prominent in the inner plexiform layer (IPL). It had a punctate appearance, suggesting a synaptic clustering of SAP102 in the IPL. Electron microscopy by use of pre-embedding immunocytochemistry showed that SAP102 is concentrated in the IPL in processes which are postsynaptic at bipolar cell ribbon synapses (dyads). As a rule, only one of the two postsynaptic members of the dyad was labeled for SAP102. Double-labeling experiments were performed in order to find out whether SAP102 is involved with the clustering the N-methyl-D-aspartate (NMDA) receptor 2A subunit (NR2A). Only a fraction (approximately 23%) of the SAP102 clusters expressed NR2A, suggesting SAP102 is also associated with other subunits or receptors. Distinct SAP102 labeling was also present in horizontal cell processes in the outer plexiform layer (OPL), which are inserted as lateral elements into photoreceptor ribbon synapses (triads). The optic nerve fibre layer was also diffusely immunoreactive for SAP102. The postsynaptic aggregation of SAP102 at bipolar cell dyads and at photoreceptor triads suggests SAP102 is associated with the clustering of transmitter receptors. However, the labeling of the optic nerve fibre layer indicates additional functions of SAP102 in the retina. J. Comp. Neurol. 397:326–336, 1998. © 1998 Wiley-Liss, Inc.     

Identification of 11-HETE in the rat brain and its relevancy to ischemic cerebral edema

Eicosanoids are thought to be important in the pathogenetic mechanism of ischemic brain damage. But little is known about lipoxygenase products and their roles in brain. In the present study, lipoxygenase metabolism in the brain and its relevancy to ischemic brain edema were studied using high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). The rat middle cerebral artery (MCA) occlusion model was used because the time course of ischemic edema formation has been well known. The rat brain was fixed by in situ freezing 24 and 72 hours after MCA occlusion, and removed. Identification and quantitative analysis of hydroxyeicosatetraenoic acids (HETEs) in normal and ischemic brain homogenates was performed by HPLC and GC-MS. The rat brain was divided into the microvessel (MV) fraction and the rest. Then the same analysis was carried out in the both fractions obtained from the normal rat brain. Only 11-HETE was detected in the normal and ischemic brain. Quantitatively, normal brains contained 1288 +/- 66 (mean +/- SE) of 11-HETE ng/g wet weight, while the hemispheres rendered ischemic for 24 and 72 hours after MCA occlusion contained 1101 +/- 48, and 1663 +/- 147, respectively. The 11-HETE content was significantly increased 72 hours after MCA occlusion (p less than 0.05). The MV fraction of rat brain contained 11-HETE (653 ng/mg protein) ten times as much as the other fraction. The identification of 11-HETE in the rat brain is a new finding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuronal expression and regulation of rat inhibitor of apoptosis protein-2 by kainic acid in the rat brain

Inhibitors of apoptosis proteins (IAPs) define a protein family with the ability to counteract cell death by the inhibition of different caspases activated during apoptosis. These proteins are present in different cells, however, the function and roles of IAPs in brain tissue are not fully understood. We report here that RIAP-2, the rat homologue of human cIAP-1/HIAP-2, is expressed in different areas of rat brain as shown by in situ hybridization and immunohistochemistry. Brain regions with relatively high expression of RIAP-2 mRNA included cortex, cerebellum and different subregions of rat hippocampus. Double labelling using a specific anti-RIAP antibody and markers for neurons and glial cells, showed that RIAP-2 is predominantly expressed by nerve cells. Kainic acid treatment, which induces seizures, transiently up-regulated RIAP-2 mRNA levels in cerebral cortex, in the CA1 and dentate gyrus regions of hippocampus, which returned to normal levels at 24 h. However in the CA3 region, RIAP-2 mRNA was decreased at 6 h following an early up-regulation. This region contains neurons particularly vulnerable to kainic acid induced cell degeneration. The decrease in RIAP-2 following kainic acid was also observed using immunohistochemistry. RIAP-2 protein did not colocalize with TUNEL labelling present in cells undergoing cell death. The results show that in the adult rat brain RIAP-2 is expressed mainly by neurons, and that the levels are regulated by kainic acid, which activates glutamate receptors. The decrease in RIAP-2 in specific neuronal populations may contribute to cell degeneration in vulnerable brain regions observed after kainic acid treatment.     

A novel mutation in the DLG3 gene encoding the synapse-associated protein 102 (SAP102) causes non-syndromic mental retardation

We have identified a novel splice site mutation (IVS6-1G?>?A) in the disc-large homolog 3 (DLG3) gene, encoding the synapse-associated protein 102 (SAP102) in one out of 300 families with moderate to severe non-syndromic mental retardation. SAP102 is a member of the neuronal membrane-associated guanylate kinase protein subfamily comprising SAP97, postsynaptic density (PSD)95, and PSD93, which interacts with methyl-d-aspartate receptor and associated protein complexes at the postsynaptic density of excitatory synapses. DLG3 is the first mental retardation gene directly linked to glutamate receptor signalling and trafficking, increasingly recognised as a central mechanism in the regulation of synaptic formation and plasticity in brain and cognitive development.     

Improved immunohistochemical detection of postsynaptically located PSD-95/SAP90 protein family by protease section pretreatment: a study in the adult mouse brain

Postsynaptic density (PSD)-95, SAP102, and Chapsyn-110 are members of the PSD-95/SAP90 protein family, which interact with the C-terminus of N-methyl-D-aspartate (NMDA) receptor and shaker-type potassium channel subunits. Here we report that appropriate section pretreatment with pepsin has led to qualitative and quantitative changes in light microscopic immunohistochemical detection of the protein family. First, pepsin pretreatment lowered the concentration of affinity-purified primary antibodies, while it greatly increased the intensity of immunoreactions. Second, the resulting overall distributions of PSD-95, SAP102, and Chapsyn-110 in the adult mouse brain were consistent with their mRNA distributions. Third, instead of the reported patterns of somatodendritic labeling, tiny punctate staining in the neuropil became overwhelming. Fourth, many PSD-95-immunopositive puncta were apposed closely to synaptophysin-positive nerve terminals and overlapped with NMDA receptor subunits. By postembedding immunogold, the PSD-95 antibody was shown to label exclusively the postsynaptic density at asymmetrical synapses. Based on these results, we conclude that antibody access and binding to the postsynaptically located PSD-95/SAP90 protein family are hindered when conventional immunohistochemistry is adopted, and that pepsin pretreatment effectively unmasks the postsynaptic epitopes. On the other hand, PSD-95 in axon terminals of cerebellar basket cells, where high levels of potassium channels are present, was detectable irrespective of pepsin pretreatment, suggesting that PSD-95 antibody is readily accessible to the presynaptic epitopes. Consequently, the present immunohistochemical results have provided light microscopic evidence supporting the prevailing notion that the PSD-95/SAP90 protein family interacts with NMDA receptor subunits and potassium channel subunits.     

Multiple calmodulin genes exhibit systematically differential responses to chronic ethanol treatment and withdrawal in several regions of the rat brain

Ethanol induces profound alterations in the neuronal signaling systems, including the calcium (Ca(2+)) signaling. Prolonged exposure to ethanol evokes adaptive changes in the affected systems as they strive to restore the normal neuronal function. We investigated the involvement of calmodulin (CaM) genes, coding for the major mediator protein of intracellular Ca(2+) signals, in these adaptive processes at the mRNA level. The changes induced in the regional abundances of the CaM I, II, and III mRNA classes by chronic ethanol treatment and withdrawal were examined by means of quantitative in situ hybridization, employing gene-specific [35S]cRNA probes on rat brain cryostat sections. Regional analysis of the resulting changes in mRNA levels highlighted brain areas that belong in neuronal systems known to be especially sensitive to the action of ethanol. The results revealed systematically differential regulation for the three mRNA classes: the CaM I and CaM III mRNA levels displayed increases, and CaM II levels decreases in the affected brain regions, in both chronic ethanol- and withdrawal-treated animals. As regards the numbers of brain regions undergoing significant alterations in mRNA content, the CaM I mRNA levels exhibited changes in most brain areas, the CaM II levels did so in a lower number of brain regions, and the CaM III levels changed in only a few brain areas. These results suggest a differential regulation for the CaM genes in the rat brain and may help towards elucidation of the functional significance of the multiple CaM genes in the mammalian genome.     

SAP97 concentrates at the postsynaptic density in cerebral cortex

SAP97, a PDZ-containing protein, is reported to concentrate in axon terminals, where its function remains unknown. Using highly specific new antibodies, we show that SAP97 in rat cerebral cortex is associated with heteromeric AMPA receptors via a selective biochemical interaction between SAP97 and the GluR1 subunit. Using light and electron microscopic immunocytochemistry, we demonstrate cellular and synaptic colocalization of SAP97 and GluR1, and show that SAP97 concentrates at synapses that contain GluR1 but not necessarily GluR2 or GluR3. Using quantitative postembedding immunogold electron microscopy, we find that SAP97 is at highest concentration within the postsynaptic density of asymmetric synapses. These data suggest that SAP97 may help to anchor GluR1-containing AMPA receptors at the synapse. As a multifunctional scaffolding protein, SAP97 may organize components of AMPA-related intracellular signalling pathways, including those associated with calcium-permeable homomeric GluR1 channels.