Neuroplastin splice variants Np55 and Np65: Who is doing the job in macrophages?

Neuroplastin, a paralog of CD147/Basigin, is known as a neuronal cell adhesion molecule and as an auxiliary subunit of plasma membrane calcium ATPases in both neurons and adaptive immune cells. Recently, an interesting study by Ren et al. (2022) provided evidence for an important role of neuroplastin in macrophages during bacterial infection. Here, we critically discuss one aspect of this study, the assignment of this role to Np65 as one of two prominent splice variants of neuroplastin.

Ca2+ Homeostasis by Plasma Membrane Ca2+ ATPase (PMCA) 1 Is Essential for the Development of DP Thymocytes.

The strength of Ca2+ signaling is a hallmark of T cell activation, yet the role of Ca2+ homeostasis in developing T cells before expressing a mature T cell receptor is poorly understood. We aimed to unveil specific functions of the two plasma membrane Ca2+ ATPases expressed in T cells, PMCA1 and PMCA4. On a transcriptional and protein level we found that PMCA4 was expressed at low levels in CD4-CD8- double negative (DN) thymocytes and was even downregulated in subsequent stages while PMCA1 was present throughout development and upregulated in CD4+CD8+ double positive (DP) thymocytes. Mice with a targeted deletion of Pmca1 in DN3 thymocytes had an almost complete block of DP thymocyte development with an accumulation of DN4 thymocytes but severely reduced numbers of CD8+ immature single positive (ISP) thymocytes. The DN4 thymocytes of these mice showed strongly elevated basal cytosolic Ca2+ levels and a pre-mature CD5 expression, but in contrast to the DP thymocytes they were only mildly prone to apoptosis. Surprisingly, mice with a germline deletion of Pmca4 did not show any signs of altered progression through the developmental thymocyte stages, nor altered Ca2+ homeostasis throughout this process. PMCA1 is, therefore, non-redundant in keeping cellular Ca2+ levels low in the early thymocyte development required for the DN to DP transition.

Plasma membrane Ca2+ ATPase 1 (PMCA1) but not PMCA4 is critical for B-cell development and Ca2+ homeostasis in mice.

The amplitude and duration of Ca2+ signaling is crucial for B-cell development and self-tolerance; however, the mechanisms for terminating Ca2+ signals in B cells have not been determined. In lymphocytes, plasma membrane Ca2+ ATPase (PMCA) isoforms 1 and 4 (PMCA1 and PMCA4, aka ATP2B1 and ATP2B4) are the main candidates for expelling Ca2+ from the cell through the plasma membrane. We report here that Pmca4 (Atp2b4) KO mice had normal B-cell development, while mice with a conditional KO of Pmca1 (Atp2b1) had greatly reduced numbers of B cells, particularly splenic follicular B cells, marginal zone B cells, and peritoneal B-1a cells. Mouse and naïve human B cells showed only PMCA1 expression and no PMCA4 by western blot, in contrast to T cells, which did express PMCA4. Calcium handling was normal in Pmca4-/- B cells, but Pmca1 KO B cells had elevated basal levels of Ca2+ , elevated levels in ER stores, and reduced Ca2+ clearance. These findings show that the PMCA1 isoform alone is required to ensure normal B-cell Ca2+ signaling and development, which may have implications for therapeutic targeting of PMCAs and Ca2+ in B cells.

A complex of Neuroplastin and Plasma Membrane Ca2+ ATPase controls T cell activation.

The outcome of T cell activation is determined by mechanisms that balance Ca2+ influx and clearance. Here we report that murine CD4 T cells lacking Neuroplastin (Nptn -/-), an immunoglobulin superfamily protein, display elevated cytosolic Ca2+ and impaired post-stimulation Ca2+ clearance, along with increased nuclear levels of NFAT transcription factor and enhanced T cell receptor-induced cytokine production. On the molecular level, we identified plasma membrane Ca2+ ATPases (PMCAs) as the main interaction partners of Neuroplastin. PMCA levels were reduced by over 70% in Nptn -/- T cells, suggesting an explanation for altered Ca2+ handling. Supporting this, Ca2+ extrusion was impaired while Ca2+ levels in internal stores were increased. T cells heterozygous for PMCA1 mimicked the phenotype of Nptn -/- T cells. Consistent with sustained Ca2+ levels, differentiation of Nptn -/- T helper cells was biased towards the Th1 versus Th2 subset. Our study thus establishes Neuroplastin-PMCA modules as important regulators of T cell activation.

Vasopressin signaling at brain level controls stress hormone release: the vasopressin-deficient Brattleboro rat as a model.

The nonapeptide arginine vasopressin (AVP) has long been suggested to play an important role as a secretagogue for triggering the activity of the endocrine stress response. Most recent studies employed mutant mice for analyzing the importance of AVP for endocrine regulation under stress. However, it is difficult to compare and draw overall conclusions from all these studies as mixing the genetic material from different mouse strains has consequences on the individual's stress response. Moreover, mice are not ideal subjects for several experimental procedures. Therefore, to get more insight, we used a rather old mutant rat model: the AVP-deficient Brattleboro rat. The present short review is aimed at providing the most interesting results of these studies within the last 8 years that allowed gaining new insights in the potential signal function of AVP in stress and endocrine regulation.

Blunted HPA axis response in lactating, vasopressin-deficient Brattleboro rats.

Adaptation to stress is a basic phenomenon in mammalian life that is mandatorily associated with the activity of the hypothalamic-pituitary-adrenal (HPA) axis. An increased resting activity of the HPA axis can be measured during pregnancy and lactation, suggesting that these reproductive states lead to chronic load in females. In this study, we examined the consequences of the congenital lack of vasopressin on the activity of the HPA axis during lactation using vasopressin-deficient Brattleboro rats. Virgin and lactating, homozygous vasopressin-deficient rats were compared with control, heterozygous rats. In control dams compared with virgins, physiological changes similar to those observed in a chronic stress state (thymus involution, adrenal gland hyperplasia, elevation of proopiomelanocortin mRNA levels in the adenohypophysis, and resting plasma corticosterone levels) were observed. In vasopressin-deficient dams, adrenal gland hyperplasia and resting corticosterone level elevations were not observed. Corticotropin-releasing hormone (Crh) mRNA levels in the hypothalamic paraventricular nucleus were elevated in only the control dams, while oxytocin (OT) mRNA levels were higher in vasopressin-deficient virgins and lactation induced a further increase in both the genotypes. Suckling-induced ACTH and corticosterone level elevations were blunted in vasopressin-deficient dams. Anaphylactoid reaction (i.v. egg white) and insulin-induced hypoglycemia stimulated the HPA axis, which were blunted in lactating rats compared with the virgins and in vasopressin-deficient rats compared with the controls without interaction of the two factors. Vasopressin seems to contribute to the physiological changes observed during lactation mimicking a chronic stress state, but its role in acute HPA axis regulation during lactation seems to be similar to that observed in virgins. If vasopressin is congenitally absent, OT, but not the CRH, compensates for the missing vasopressin; however, the functional restitution remains incomplete.

Arginase and Arginine Decarboxylase - Where Do the Putative Gate Keepers of Polyamine Synthesis Reside in Rat Brain?

Polyamines are important regulators of basal cellular functions but also subserve highly specific tasks in the mammalian brain. With this respect, polyamines and the synthesizing and degrading enzymes are clearly differentially distributed in neurons versus glial cells and also in different brain areas. The synthesis of the diamine putrescine may be driven via two different pathways. In the "classical" pathway urea and carbon dioxide are removed from arginine by arginase and ornithine decarboxylase. The alternative pathway, first removing carbon dioxide by arginine decarboxlyase and then urea by agmatinase, may serve the same purpose. Furthermore, the intermediate product of the alternative pathway, agmatine, is an endogenous ligand for imidazoline receptors and may serve as a neurotransmitter. In order to evaluate and compare the expression patterns of the two gate keeper enzymes arginase and arginine decarboxylase, we generated polyclonal, monospecific antibodies against arginase-1 and arginine decarboxylase. Using these tools, we immunocytochemically screened the rat brain and compared the expression patterns of both enzymes in several brain areas on the regional, cellular and subcellular level. In contrast to other enzymes of the polyamine pathway, arginine decarboxylase and arginase are both constitutively and widely expressed in rat brain neurons. In cerebral cortex and hippocampus, principal neurons and putative interneurons were clearly labeled for both enzymes. Labeling, however, was strikingly different in these neurons with respect to the subcellular localization of the enzymes. While with antibodies against arginine decarboxylase the immunosignal was distributed throughout the cytoplasm, arginase-like immunoreactivity was preferentially localized to Golgi stacks. Given the apparent congruence of arginase and arginine decarboxylase distribution with respect to certain cell populations, it seems likely that the synthesis of agmatine rather than putrescine may be the main purpose of the alternative pathway of polyamine synthesis, while the classical pathway supplies putrescine and spermidine/spermine in these neurons.

Dysregulation of Rho GTPases in the αPix/Arhgef6 mouse model of X-linked intellectual disability is paralleled by impaired structural and synaptic plasticity and cognitive deficits.

Mutations in the ARHGEF6 gene, encoding the guanine nucleotide exchange factor αPIX/Cool-2 for the Rho GTPases Rac1 and Cdc42, cause X-linked intellectual disability (ID) in humans. We show here that αPix/Arhgef6 is primarily expressed in neuropil regions of the hippocampus. To study the role of αPix/Arhgef6 in neuronal development and plasticity and gain insight into the pathogenic mechanisms underlying ID, we generated αPix/Arhgef6-deficient mice. Gross brain structure in these mice appeared to be normal; however, analysis of Golgi-Cox-stained pyramidal neurons revealed an increase in both dendritic length and spine density in the hippocampus, accompanied by an overall loss in spine synapses. Early-phase long-term potentiation was reduced and long-term depression was increased in the CA1 hippocampal area of αPix/Arhgef6-deficient animals. Knockout animals exhibited impaired spatial and complex learning and less behavioral control in mildly stressful situations, suggesting that this model mimics the human ID phenotype. The structural and electrophysiological alterations in the hippocampus were accompanied by a significant reduction in active Rac1 and Cdc42, but not RhoA. In conclusion, we suggest that imbalance in activity of different Rho GTPases may underlie altered neuronal connectivity and impaired synaptic function and cognition in αPix/Arhgef6 knockout mice.

Phosphoprotein associated with glycosphingolipid-enriched microdomains differentially modulates SRC kinase activity in brain maturation.

Src family kinases (SFK) control multiple processes during brain development and function. We show here that the phosphoprotein associated with glycosphigolipid-enriched microdomains (PAG)/Csk binding protein (Cbp) modulates SFK activity in the brain. The timing and localization of PAG expression overlap with Fyn and Src, both of which we find associated to PAG. We demonstrate in newborn (P1) mice that PAG negatively regulates Src family kinases (SFK). P1 Pag1(-/-) mouse brains show decreased recruitment of Csk into lipid rafts, reduced phosphorylation of the inhibitory tyrosines within SFKs, and an increase in SFK activity of >/ = 50%. While in brain of P1 mice, PAG and Csk are highly and ubiquitously expressed, little Csk is found in adult brain suggesting altered modes of SFK regulation. In adult brain Pag1-deficiency has no effect upon Csk-distribution or inhibitory tyrosine phosphorylation, but kinase activity is now reduced (-20-30%), pointing to the development of a compensatory mechanism that may involve PSD93. The distribution of the Csk-homologous kinase CHK is not altered. Importantly, since the activities of Fyn and Src are decreased in adult Pag1(-/-) mice, thus presenting the reversed phenotype of P1, this provides the first in vivo evidence for a Csk-independent positive regulatory function for PAG in the brain.

Minocycline neuroprotection in a rat model of asphyxial cardiac arrest is limited.

OBJECTIVE: The study investigated a possible neuroprotective potency of minocycline in an experimental asphyxial cardiac arrest (ACA) rat model. Clinically important survival times were evaluated thus broadening common experimental approaches. METHODS: Adult rats were subjected to 5 min of ACA followed by resuscitation. There were two main treatment groups: ACA and sham operated. Relating to minocycline treatment each group consisted of three sub-groups: pre-, post-, and sans-mino, with three different survival times: 4, 7, and 21 days. Neurodegeneration and microgliosis were monitored by immunohistochemistry. Alterations of microglia-associated gene expression were analyzed by quantitative RT-PCR. RESULTS: ACA induced massive nerve cell loss and activation of microglia/macrophages in hippocampal CA1 cell layer intensifying with survival time. After 7 days, minocycline significantly decreased both, neuronal degeneration and microglia response in dependence on the application pattern; application post ACA was most effective. After 21 days, neuroprotective effects of minocycline were lost. ACA significantly induced expression of the microglia-associated factors Ccl2, CD45, Mac-1, F4-80, and Tnfa. Independent on survival time, minocycline affected these parameters not significantly. Expression of iNOS was unaffected by both, ACA and minocycline. CONCLUSIONS: In adult rat hippocampus microglia was significantly activated by ACA. Minocycline positive affected neuronal survival and microglial response temporary, even when applied up to 18 h after ACA, thus defining a therapeutically-relevant time window. As ACA-induced neuronal cell death involves acute and delayed events, longer minocycline intervention targeting also secondary injury cascades should manifest neuroprotective potency, a question to be answered by further experiments.

Expression of cAMP-dependent protein kinase isoforms in the human prostate: functional significance and relation to PDE4.

OBJECTIVES: To investigate the expression of isoforms of the cyclic AMP (cAMP)-dependent protein kinase (cAK) in the transition zone of the human prostate and the functional significance of the enzyme in the control of prostate smooth muscle. METHODS: Using Western blot analysis and immunohistochemistry, the expression and distribution in the prostate of cAKIalpha, cAKIbeta, cAKIIalpha, and cAKIIbeta in relation to alpha-actin and the phosphodiesterase PDE4 (types A and B) were investigated. The effects of the cAK inhibitor Rp-8-CPT-cAMPS on the reversion of the adrenergic tension of isolated prostate tissue induced by forskolin, rolipram, sodium nitroprusside (SNP), and tadalafil were examined by means of the organ bath technique. RESULTS: Immunosignals specific for cAKIalpha, cAKIIalpha, and cAKIIbeta were observed in the smooth musculature and glandular structures of the prostate. Double stainings revealed the colocalization of alpha-actin and PDE4 with the cAK isoforms. The expression of the cAK isoforms was confirmed by Western blot analysis. The relaxation of the tension induced by norepinephrine brought about by forskolin, rolipram, SNP, and tadalafil was significantly attenuated by Rp-8-CPT-cAMPS. CONCLUSIONS: The colocalization of smooth muscle alpha-actin and PDE4 with cAK, as well as the results from the organ bath experiments, provide further evidence for a pivotal role of the cAMP-dependent signaling in the regulation of prostate smooth muscle contractility. Compounds interacting with the cAMP/cAK pathway might represent a new therapeutic avenue to treat symptoms of benign prostatic hyperplasia and lower urinary tract symptomatology.

An intrinsic vasopressin system in the olfactory bulb is involved in social recognition.

Many peptides, when released as chemical messengers within the brain, have powerful influences on complex behaviours. Most strikingly, vasopressin and oxytocin, once thought of as circulating hormones whose actions were confined to peripheral organs, are now known to be released in the brain, where they have fundamentally important roles in social behaviours. In humans, disruptions of these peptide systems have been linked to several neurobehavioural disorders, including Prader-Willi syndrome, affective disorders and obsessive-compulsive disorder, and polymorphisms of V1a vasopressin receptor have been linked to autism. Here we report that the rat olfactory bulb contains a large population of interneurons which express vasopressin, that blocking the actions of vasopressin in the olfactory bulb impairs the social recognition abilities of rats and that vasopressin agonists and antagonists can modulate the processing of information by olfactory bulb neurons. The findings indicate that social information is processed in part by a vasopressin system intrinsic to the olfactory system.

Vasopressin administration into the paraventricular nucleus normalizes plasma oxytocin and corticosterone levels in Brattleboro rats.

Adult male rats of the Brattleboro strain were used to investigate the impact of the congenital absence of vasopressin on plasma adrenocorticotropin, corticosterone, and oxytocin concentrations as well as the release pattern of oxytocin within the hypothalamic paraventricular nucleus (PVN), in response to a 10-min forced swimming session. Measurement of adrenocorticotropin in plasma samples collected via chronically implanted jugular venous catheters revealed virtually identical stress responses for vasopressin-lacking Brattleboro (KO) and intact control animals. In contrast, plasma corticosterone and oxytocin levels were found to be significantly elevated 105 min after onset of the stressor in KO animals only. Microdialysis samples collected from the extracellular fluid of the PVN showed significantly higher levels of oxytocin both under basal conditions and in response to stressor exposure in KO vs. intact control animals accompanied by elevated oxytocin mRNA levels in the PVN of KO rats. These findings suggest that the increased oxytocin levels in the PVN caused by the congenital absence of vasopressin may contribute to normal adrenocorticotropin stress responses in KO animals. However, whereas the stressor-induced elevation of plasma oxytocin in KO rats may be responsible for their maintained corticosterone levels, oxytocin seems unable to fully compensate for the lack of vasopressin. This hypothesis was tested by retrodialyzing synthetic vasopressin into the PVN area concomitantly with blood sampling in KO animals. Indeed, this treatment normalized plasma oxytocin and corticosterone levels 105 min after forced swimming. Thus, endogenous vasopressin released within the PVN is likely to act as a paracrine signal to facilitate the return of plasma oxytocin and corticosterone to basal levels after acute stressor exposure.

Selection of reference genes for quantitative real-time PCR in a rat asphyxial cardiac arrest model.

BACKGROUND: Cardiac arrest, and the associated arrest of blood circulation, immediately leads to permanent brain damage because of the exhaustion of oxygen, glucose and energy resources in the brain. Most hippocampal CA1 neurons die during the first week post the insult. Molecular data concerning the recovery after resuscitation are sparse and limited to the early time period. Expression analysis of marker genes via quantitative real-time RT-PCR enables to follow up the remodeling process. However, proper validation of the applied normalization strategy is a crucial prerequisite for reliable conclusions.Therefore, the present study aimed to determine the expression stability of ten commonly used reference genes (Actb, actin, beta; B2m, beta-2 microglobulin;CypA, cyclophilin A; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; Hprt, hypoxanthine guanine phosphoribosyl transferase; Pgk1, phosphoglycerate kinase 1; Rpl13a, ribosomal protein L13A; Sdha, succinat dehydrogenase complex, subunit a, flavoprotein (Fp); Tbp, TATA box binding protein; Ywhaz, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) in the rat hippocampus four, seven and twenty-one days after cardiac arrest. Moreover, experimental groups treated with the anti-inflammatory and anti-apoptotic drug minocycline have been included in the study as well. RESULTS: The microglial marker Mac-1, used as a target gene to validate the experimental model, was found to be upregulated about 10- to 20-fold after cardiac arrest. Expression stability of candidate reference genes was analyzed using geNorm and NormFinder software tools. Several of these genes behave rather stable. CypA and Pgk1 were identified by geNorm as the two most stable genes 4 and 21 days after asphyxial cardiac arrest, CypA and Gapdh at 7 days post treatment. B2m turned out to be the most variable candidate reference gene, being about 2-fold upregulated in the cardiac arrest treatment groups. CONCLUSION: We have validated endogenous control genes for qRT-PCR analysis of gene expression in rat hippocampus after resuscitation from cardiac arrest. For normalization purposes in gene profiling studies a combination of CypA and Pgk1 should be considered 4 and 21 days post injury, whereas CypA and Gapdh is the best combination at 7 days. CypA is most favorable if restriction to a single reference gene for all time points is required.

Expression and distribution of cyclic GMP-dependent protein kinase-1 isoforms in human penile erectile tissue.

INTRODUCTION: Besides the bioavailability of nitric oxide (NO), downstream guanine monophosphate (cGMP) effector proteins are also considered to play a significant role in penile vascular disease. In animal studies, a downregulation of the cGMP-dependent protein kinase-1 (cGKI) alpha isoform has been linked to erectile dysfunction and diabetes mellitus. So far, the expression of cGKI alpha and beta isoforms has not been evaluated in human penile erectile tissue. AIM: To evaluate the expression of cGKI alpha and beta isoforms in relation to smooth muscle alpha-actin, cGMP, and endothelial NO synthase (eNOS) in human cavernous arteries (HCAs) and human corpus cavernosum (HCC). METHODS: Cryostat sections of HCA and HCC were incubated with primary antibodies directed against alpha-actin, cGMP, eNOS, cGKI, cGKI alpha, and cGKI beta. Visualization of double-labeled immunofluorescent stainings was achieved by laser microscopy. Western blot analysis was performed in order to confirm the expression of cGKI isoforms. MAIN OUTCOME MEASURES: Expression of cGKI alpha and beta isoforms in relation to smooth muscle alpha-actin, cGMP, and eNOS in human penile erectile tissue. RESULTS: Immunoreactivities specific for cGKI, cGKI alpha, and cGKI beta were observed within the smooth musculature and the endothelium of cavernous arteries and sinusoids. Double stainings revealed the colocalization of alpha-actin, cGMP, eNOS, and cGKI isoforms. The expression of cGKI isoforms was confirmed by Western blot analysis. CONCLUSIONS: Our results demonstrate, for the first time, the expression of both cGKI alpha and beta isoforms in the smooth musculature of HCA and HCC. Corresponding to recent findings from animal studies, the presence of cGKI alpha and beta provides further evidence for a significant role of these enzymes in the control of smooth muscle function in human penile erectile tissue.

Endothelial nitric oxide synthase is predominantly involved in angiotensin II modulation of renal vascular resistance and norepinephrine release.

Nitric oxide (NO) is mainly generated by endothelial NO synthase (eNOS) or neuronal NOS (nNOS). Recent studies indicate that angiotensin II generates NO release, which modulates renal vascular resistance and sympathetic neurotransmission. Experiments in wild-type [eNOS(+/+) and nNOS(+/+)], eNOS-deficient [eNOS(-/-)], and nNOS-deficient [nNOS(-/-)] mice were performed to determine which NOS isoform is involved. Isolated mice kidneys were perfused with Krebs-Henseleit solution. Endogenous norepinephrine release was measured by HPLC. Angiotensin II dose dependently increased renal vascular resistance in all mice species. EC(50) and maximal pressor responses to angiotensin II were greater in eNOS(-/-) than in nNOS(-/-) and smaller in wild-type mice. The nonselective NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 0.3 mM) enhanced angiotensin II-induced pressor responses in nNOS(-/-) and wild-type mice but not in eNOS(-/-) mice. In nNOS(+/+) mice, 7-nitroindazole monosodium salt (7-NINA; 0.3 mM), a selective nNOS inhibitor, enhanced angiotensin II-induced pressor responses slightly. Angiotensin II-enhanced renal nerve stimulation induced norepinephrine release in all species. L-NAME (0.3 mM) reduced angiotensin II-mediated facilitation of norepinephrine release in nNOS(-/-) and wild-type mice but not in eNOS(-/-) mice. 7-NINA failed to modulate norepinephrine release in nNOS(+/+) mice. (4-Chlorophrnylthio)guanosine-3', 5'-cyclic monophosphate (0.1 nM) increased norepinephrine release. mRNA expression of eNOS, nNOS, and inducible NOS did not differ between mice strains. In conclusion, angiotensin II-mediated effects on renal vascular resistance and sympathetic neurotransmission are modulated by NO in mice. These effects are mediated by eNOS and nNOS, but NO derived from eNOS dominates. Only NO derived from eNOS seems to modulate angiotensin II-mediated renal norepinephrine release.

Expression pattern of peptidylarginine deiminase in rat and human Schwann cells.

The peptidylarginine deiminase (PAD) family of enzymes are responsible for conversion of protein-bound arginine to citrulline in most tissues of the body and are garnering increased interest for their physiological and pathological roles. Although it has been shown that oligodendrocytes of the CNS express the PAD isoenzyme type 2, nothing is presently known about PAD expression in Schwann cells, the myelinating cells of the PNS. To evaluate PAD expression in the PNS, cultivated rat and human Schwann cells and slices of fetal, juvenile, and normal and regenerated adult sciatic nerves were examined with RT-PCR, Western blot, and immunohistochemical analysis. Samples from cerebellar cultures and skin served as positive controls. One of the principle findings was that cultivated Schwann cells expressed significant levels of mRNA and protein for the PAD isoenzymes 2 and 3. PAD1 and PAD4, however, were not expressed in any types of Schwann cells. Using double immunofluorescence, the majority of PAD2 staining was localized in immature cell stages. Moreover, increased amounts of PAD2, PAD3, and peptidyl-citrulline were also found in human fetal and rat juvenile and regenerated sciatic nerves as compared to similar normal adult specimens. Neuronal and inducible nitric oxide synthases, enzymes that convert free arginine to citrulline, were also expressed in Schwann cells; however, their massive induction by LPS/K(+), was not reflected in an enhanced peptidyl-citrulline immunosignal. These data suggest that, similar to the CNS, citrullination of proteins may also exert a specific role in thecourse of PNS development and repair.

Inhibiting effect of minocycline on the regeneration of peripheral nerves.

The effect of minocycline on nerve regeneration was studied in a rat model of acute sciatic nerve injury, in which the injury was caused by resection and reimplantation of the right sciatic nerve. Immunohistochemical and molecular biological methods, as well as morphometric and electron microscopic techniques, were used. Compared with uninjured and PBS-treated injured nerves, the minocycline-treated injured nerve showed: (i) a decrease in macrophage recruitment and activation, probably resulting from inhibition of blood-brain-barrier break-down via reduced MMP2 and MMP9 induction, inhibition of revascularization via additional reduction of VEGF induction, and inhibition of inducible NO synthase (iNOS) induction; (ii) reduced activation of phagocytic Schwann cells, probably by inhibition of iNOS, MMP2 and MMP9 expression; (iii) a slowed Wallerian degeneration; and subsequently, (iv) a diminished nerve regeneration. Macrophages, especially their function in the removal of cellular debris and formation of a microenvironment beneficial for nerve regeneration, are strongly implicated in constructive events after nerve injuries. Therefore, we suggest that additional research into optimizing minocycline intervention for treatment of neurodegenerative diseases is needed before further clinical trials are performed.

Splice-isoform specific immunolocalization of neuronal nitric oxide synthase in mouse and rat brain reveals that the PDZ-complex-building nNOSalpha beta-finger is largely exposed to antibodies.

Knock out mice deficient for the splice-isoform alphaalpha of neuronal nitric oxide synthase (nNOSalphaalpha) display residual nitric oxide synthase activity and immunosignal. To attribute this signal to the two minor neuronal nitric oxide synthase splice variants, betabeta and gammagamma, we generated isoform-specific anti-peptide antibodies against the nNOSalphaalpha specific betabeta-finger motif involved in PDZ domain scaffolding and the nNOSbetabeta specific N-terminus. The nNOSalphaalpha betabeta-finger-specific antibody clearly recognized the 160-kDa band of recombinant nNOSalphaalpha on Western blots. Using immunocytochemistry, this antibody displayed, in rats and wild-type mice, a labeling pattern similar to but not identical with that obtained using a commercial pan-nNOS antibody. This similarity indicates that the majority of immunocytochemically detectable nNOS is not likely to be complexed with PDZ-domain proteins via the betabeta-finger motif. This conclusion was confirmed by the inhibition of PSD-95/nNOS interaction by the nNOSalphaalpha betabeta-finger antibody in pull-down assays. By contrast, nNOSalphaalpha betabeta-finger labeling was clearly reduced in hippocampal and cortical neuropil areas enriched in NMDA receptor complex containing spine synapses. In nNOSalphaalpha knock out mice, nNOSalphaalpha was not detectable, whereas the pan-nNOS antibody showed a distinct labeling of cell bodies throughout the brain, most likely reflecting betabeta/gammagamma-isoforms in these cells. The nNOSbetabeta antibody clearly detected bacterial expressed nNOSbetabeta fusion protein and nNOSbetabeta in overexpressing HEK cells by Western blotting. Immunocytochemically, individual cell bodies in striatum, cerebral cortex, and in some brain stem nuclei were labeled in knock out but not in wild-type mice, indicating an upregulation of nNOSbetabeta in nNOSalphaalpha deficient animals.

New aspects of the location of neuronal nitric oxide synthase in the skeletal muscle: a light and electron microscopic study.

The action of nitric oxide (NO) synthesized by NO synthases (NOS) is spatially restricted. Hence, the intracellular location of NOS might play an important role for the functional interactions of NO with its target molecules. In the skeletal muscle the neuronal NOS (nNOS) is considered to be the predominant isoform expressed as a muscle specific elongated splice variant. There are only a few and highly discrepant reports of the subcellular distribution of nNOS, which prompted us to re-examine the distribution of nNOS in the skeletal muscle of rat and mouse applying immunocytochemistry and NADPH-diaphorase (NADPH-d) histochemistry. Light microscopically, the sarcolemma, areas beneath the sarcolemma, areas around the nuclei, and the cross striation were labeled by antibodies and by the NADPH-d reaction as well. Ultrastructurally, nNOS visualized immunocytochemically or by the histochemical BSPT-reaction, was associated discretely with extrajunctional portions of the sarcolemma. Both reaction products were additionally observed in the vicinity of endoplasmic reticulum and mitochondria, or associated with their outer membranes. In the neuromuscular junction (NMJ)-region NOS was localized to the cytoplasm of nerve terminals and terminal Schwann cells. In contrast to the commonly accepted assumption, the enzyme was found in association with the presynaptic, and not with the postsynaptic membrane. Cytosolic NADPH-d was exhibited especially between mitochondria accumulated in the postsynaptic region of the NMJ. Surprisingly, in nNOS-/--mice the skeletal muscle showed patterns of significant nNOS-immunoreactivity and NADPH-d activity possibly due to alternative nNOS-splice isoforms, which might be up-regulated to compensate for decreased NO formation.