Developmental vitamin D deficiency alters brain protein expression in the adult rat: implications for neuropsychiatric disorders

An increased risk for multiple sclerosis and schizophrenia is observed at increasing latitude and in patients born in winter or spring. To explore a possible link between maternal vitamin D deficiency and these brain disorders, we examined the impact of prenatal hypovitaminosis D on protein expression in the adult rat brain. Vitamin D-deficient female rats were mated with vitamin D normal males. Pregnant females were kept vitamin D-deficient until birth whereupon they were returned to a control diet. At week 10, protein expression in the progeny's prefrontal cortex and hippocampus was compared with control animals using silver staining 2-D gels associated with MS and newly devised data mining software. Developmental vitamin D (DVD) deficiency caused a dysregulation of 36 brain proteins involved in several biological pathways including oxidative phosphorylation, redox balance, cytoskeleton maintenance, calcium homeostasis, chaperoning, PTMs, synaptic plasticity and neurotransmission. A computational analysis of these data revealed that (i) nearly half of the molecules dysregulated in our animal model have also been shown to be misexpressed in either schizophrenia and/or multiple sclerosis and (ii) an impaired synaptic network may be a consequence of mitochondrial dysfunction.     

Developmental vitamin D deficiency and schizophrenia: the role of animal models

Schizophrenia is a debilitating neuropsychiatric disorder that affects 1% of the US population. Based on twin and genome‐wide association studies, it is clear that both genetics and environmental factors increase the risk for developing schizophrenia. Moreover, there is evidence that conditions in utero, either alone or in concert with genetic factors, may alter neurodevelopment and lead to an increased risk for schizophrenia. There has been progress in identifying genetic loci and environmental exposures that increase risk, but there are still considerable gaps in our knowledge. Furthermore, very little is known about the specific neurodevelopmental mechanisms upon which genetics and the environment act to increase disposition to developing schizophrenia in adulthood. Vitamin D deficiency during the perinatal period has been hypothesized to increase risk for schizophrenia in humans. The developmental vitamin D (DVD) deficiency hypothesis of schizophrenia arises from the observation that disease risk is increased in individuals who are born in winter or spring, live further from the equator or live in urban vs. rural settings. These environments result in less exposure to sunlight, thereby reducing the initial steps in the production of vitamin D. Rodent models have been developed to characterize the behavioral and developmental effects of DVD deficiency. This review focuses on these animal models and discusses the current knowledge of the role of DVD deficiency in altering behavior and neurobiology relevant to schizophrenia.     

Membrane proteins of synaptic vesicles and cytoskeletal specializations at the node of Ranvier in electric ray and rat

Summary Binding sites for antibodies against membrane proteins of synaptic vesicles have been shown to be enhanced at nodes of Ranvier in electromotor axons of the electric ray Torpedo marmorata and sciatic nerve axons of the rat, using indirect immunofluorescence and monoclonal antibodies against the synaptic vesicle transmembrane proteins SV2 and synaptophysin (rat) or SV2 (Torpedo). In the electric lobe of Torpedo, vesicle-membrane constituents occurred at higher density in the proximal axon segments covered by oligodendroglia cells than in the distal axon segments where myelin is formed by Schwann cells. Antibody binding sites were enhanced at nodes forming the borderline of the central and peripheral nervous systems. Filamentous actin was present in the Schwann-cell processes covering both the nodal and the paranodal axon segments as suggested by the pattern of phalloidin labelling. Furthermore, in rat sciatic nerve, Schmidt-Lanterman incisures were intensely labelled by phalloidin. A similar nodal distribution was found for binding sites of antibodies against actin and myosin. Binding of antibodies to tubulin was enhanced at nodes in Torpedo electromotor axons. The apparent nodal accumulation of constituents of synaptic vesicle membranes and the presence of filamentous actin and of myosin are discussed in relation to the substantial constriction of the axoplasm at nodes of Ranvier.     

Association of vitamin D,BMD and knee osteoarthritis in postmenopausal women

Objectives:The aim of this study was to analyze the association of knee OA with bone mineral density (BMD) and vitamin D serum levels in postmenopausal women.Methods:A cross-sectional study including 240 postmenopausal women with knee OA was conducted. Demographic data were recorded along with balance and functionality scores. Knee OA severity was assessed by the radiological Kellgren & Lawrence scale. BMD and T-scores were calculated in hips and lumbar spine. Serum levels of vitamin D were also measured.Results:High BMI (p<0.005), high number of children (p=0.022) and family history of hip fracture (p=0.011) are significantly associated with knee OA severity. Lumbar spine OP is negatively associated with knee OA (p<0.005). A significant difference was detected between vitamin D deficiency and severe knee OA, adjusted for BMD [OR (95%CI); 3.1 (1.6-6.1), p=0.001]. BMD does not affect the relationship of vitamin D levels in relation to OA and vitamin D levels do not affect the relationship of BMD with OA.Conclusions:Low BMD has a protective role against knee OA while vitamin D deficiency contributes significantly to knee OA severity. However, the association between OA and OP is not affected by vitamin D deficiency and the association of OA and vitamin D serum levels is not affected by BMD.     

High prevalence of vitamin D deficiency in HIV-infected individuals in comparison with the general population across Punjab province,Pakistan

The status of vitamin D in individuals infected with human immunodeficiency virus (HIV), particularly in naïve as well as treated patients, has never been reported in the Pakistani population. A cross-sectional study was performed to measure vitamin D in individuals infected with HIV living in various districts of the Punjab, Pakistan. 1000 persons attending various treatment centers of the Punjab were screened for HIV, hepatitis B virus (HBV), hepatitis C virus (HCV), and Syphilis. Total 398 patients met inclusion criteria and vitamin D level was measured in respective cases by using enzyme-linked immunosorbent assay (ELISA) technique. 232 samples from the healthy population were also included in present research. Demographic history and clinical parameters regarding HIV disease were evaluated. Comparison of variables was done to find out the link between vitamin D levels and characteristics of HIV infected persons and comparison to that of healthy individuals was performed. Among 398 HIV patients vitamin D deficiency and insufficiency was found among 15 % and 39 % while majority of the control participants had sufficient levels of vitamin D (78 %). Most of the HIV infected individuals were males (68.6 %) and had age between 24 and 47 years (67.8 %). A significant relationship was found for vitamin D level, lifestyle and CD4 count among HIV + ve non acquired immunodeficiency syndrome (AIDS) subjects (95 % CI; p < 0.001, p = 0.09). For HIV + ve AIDS patients vitamin D had a significant relationship with lifestyle along with HIV viral load and CD4 count. Hypovitaminosis D prevails among the HIV infected population of Punjab, Pakistan.     

Characteristics of the calcium-triggered mitochondrial permeability transition in nonsynaptic brain mitochondria: effect of cyclosporin A and ubiquinone O

The objective of the present study was to assess the capacity of nonsynaptic brain mitochondria to accumulate Ca2+ when subjected to repeated Ca2+ loads, and to explore under what conditions a mitochondrial permeability transition (MPT) pore is assembled. The effects of cyclosporin A (CsA) on Ca2+ accumulation and MPT pore assembly were compared with those obtained with ubiquinone 0 (Ubo), a quinone that is a stronger MPT blocker than CsA, when tested on muscle and liver mitochondria. When suspended in a solution containing phosphate (2 mM) and Mg2+ (1 mM), but no ATP or ADP, the brain mitochondria had a limited capacity to accumulate Ca2+ (210 nmol/mg of mitochondrial protein). Furthermore, when repeated Ca2+ pulses (40 nmol/mg of protein each) saturated the uptake system, the mitochondria failed to release the Ca2+ accumulated. However, in each instance, the first Ca2+ pulse was accompanied by a moderate release of Ca2+, a release that was not observed during the subsequent pulses. The initial release was accompanied by a relatively marked depolarization, and by swelling, as assessed by light-scattering measurements. However, as the swelling was <50% of that observed following addition of alamethicin, it is concluded that the first Ca2+ pulse gives rise to an MPT in a subfraction of the mitochondrial population. CsA, an avid blocker of the MPT pore, only marginally increased the Ca(2+)-sequestrating capacity of the mitochondria. However, CsA eliminated the Ca2+ release accompanying the first Ca2+ pulse. The effects of CsA were shared by Ubo, but when the concentration of Ubo exceeded 20 microM, it proved toxic. The results thus suggest that brain mitochondria are different from those derived from a variety of other sources. The major difference is that a fraction of the brain mitochondria, studied presently, depolarized and showed signs of an MPT. This fraction, but not the remaining ones, contributed to the chemically and electron microscopically verified mitochondrial swelling.     

An electron microscopic study of the normal synaptic relationships and early degenerative changes in the rat olfactory tubercle

Summary The olfactory tubercle of the rat was studied by electron microscopy both in the normal and after ipsilateral olfactory bulb ablation at survival times of from 14 hours to seven days. Particular emphasis was placed on synaptic structures and their changes following the lesion. Normal synapses are similar to those described in previous studies and presynaptic profiles are of at least three types. Types-A and -B contain round vesicles and form asymmetrical contacts and type-C profiles contain flattened vesicles and form symmetrical contacts.There appear to be two major types of degenerative changes. The electron-lucent type predominates at early survival times and is seen first at 14 hours. These profiles show an early reduction in numbers of vesicles with mitochondrial swelling followed by shrinkage of the profile. These profiles become increasingly electron-dense at later survival times. The second major type of degenerating profile is initially electron-dense. The earliest changes in these profiles are an increased axoplasmic density and increased microtubular density and clumping without apparent loss of vesicles. These profiles also become progressively more electron-dense at longer survival times. The observations are discussed in relation to previous reports.This study represents a part of a thesis (C. A. Anderson) for the Doctor of Medicine degree, University of Washington School of Medicine. It was supported by PHS grants NS 04053, NS 02896 and NS 09678 from the N.I.N.D.S., National Institutes of Health. The authors gratefully acknowledge this support.     

Distribution of anti-Leu-7, anti-Leu-11a and anti-Leu-M1 immunoreactivity in the brain of the adult rat

Summary This study reports a specific cross-reactivity of the three anti-human-hematopoetic-cell monoclonal antibodies, anti-Leu-7 (HNK-1), anti-Leu-11a (NKP-15), and anti-Leu-M1 (MMA), with different epitopes in the brain of the adult rat. The distribution of these epitopes in rat brain is determined by means of immunohistochemistry in paraffin-embedded frontal serial sections.The reaction pattern of anti-Leu-11a monoclonal antibody is very similar to that of polyclonal antibodies against the myelin basic protein. Both antisera give a specific reaction with myelinated fibers. Immunoreaction products with the anti-Leu-7 monoclonal antibody are found as diffuse, mostly punctiform material in the neuropil and even more evident as small granules coating the cell surface of many neurons. In the white matter anti-Leu-7 reveals a moderate reactivity, which occurs predominantly as spots and fine-stranded material within the myelinated fiber tracts.Anti-Leu-M1 immunoreactivity is present between myelinated fiber bundles of the white matter, where it has a reticulate appearance, and as fine-granulated material within the grey matter of the cortex and the nuclei. The characteristic feature in the grey matter is that of irregularly shaped immunopositive plaques, which are often located around small blood vessels. The cytoplasm of glial and neuronal cells appeared negative with this MAB.The exact topographical distribution of the Leu-7 and Leu-M1 epitopes throughout the rat brain is described. The present hypotheses concerning the nature of this shared antigenicity between hematopoetic cells and nervous tissue are discussed.Supported by the Deutsche Forschungsgemeinschaft, SFB 200     

Developmental changes and regional distribution of phospholipase D and base exchange enzyme activities in rat brain

Phospholipase D (PL-D) activity per mg protein of whole homogenate increased 5.1 fold between Embryonic (E) day 17 and Postpartum (P) day 14 and slightly decreased by P 30 days. This was due to the decrease of PL-D activity of the P₂ fraction. The PL-D activity of P₂ and P₃ fractions increased 11.2 and 6.1 fold respectively between E 17 and P 14. The 3 base exchange enzyme (BEE) activities per mg protein of whole homogenate increased up to P 14 or P 21 and then decreased. This decrease was greater in the P₂ fraction and the P₃ fraction increased after P14. Brains from 1 day to 25 month old rats were dissected into 7 separate regions and both PL-D and BEE activities were measured. In adult rats, the hippocampus and hypothalamus had the highest PL-D activities while medulla+pons and cerebellum had the lowest PL-D activities. The developmental patterns of 5 regions except for hippocampus and hypothalamus were similar. PL-D activity in the hippocampus was maximum at P 7 followed by a steep decrease till P30 suggesting that the PL-D activity of the hypothalamus develops later and that of the hippocampus develops earlier than any other region. The distributions of BEE activities were quite different from those of PL-D activities. In adult rats, the cerebellum had the highest activity while the striatum and medulla+pons had the lowest. The BEE activities of cerebellum were lowest at P 1 and showed steep increase during the next 2 weeks.To whom to address reprint request are to be sent.     

Expression of D2 dopamine receptor in the mouse brain

The neurotransmitter, dopamine, binds to dopamine receptor (DR), and is involved in several functions of the brain, such as initiation and execution of movement, emotion, prolactin secretion, etc. Of all the five DRs, D2 dopamine receptor has maximal affinity for dopamine. D2 has a short isoform, D2S, and a long isoform D2L. D2L is longer than D2S by 29 amino acid residues. We studied the expression of the gene and protein of D2 receptor in the cerebral and cerebellar cortices of the brain of new born, developing, adult, and old male mice to find out: (i) at what stage of development, expression of the gene peaks and (ii) if it undergoes any changes as the animal ages, which may account for the neurodegenerative changes and symptoms of Parkinson's and other diseases seen in old age. RT-PCR and Western blot studies show that peak expression of D2 gene occurs in the cerebral and cerebellar cortices around 15-day after birth. We speculate that the majority of dopaminergic synapses are established and possibly become functional in the brain around 15-day after birth. The expression of D2 receptor is upregulated in the cerebral cortex in old mice. However, it is down-regulated in the cerebellar cortex.     

Effect of vitamin D deficiency on the composition and in vitro labeling of rat kidney proteins

Densitometric analysis of single-dimension gels consistently demonstrated that, in addition to rat renal calcium binding protein (CaBP) (Mr 28,000), two other kidney proteins of Mr 16,500 and Mr 18,000 were significantly enriched in their contents in the vitamin D-replete rat. Partial characterization of the Mr 18,000 and 16,500 proteins revealed that these proteins were heat-stable and distinct from calmodulin, as determined by their inability to undergo the calcium-dependent mobility shift in sodium dodecyl sulfate gels which is characteristic of calmodulin. The Mr 16,500 and Mr 18,000 kidney proteins did not cross-react with rat renal or rat intestinal CaBP antisera, as assessed by radioimmunoassay and Western blot analysis. A comparison of peptide maps of tryptic digests of these proteins and purified rat renal CaBP, as analyzed by high-pressure liquid chromatography, revealed no apparent homology. Protein synthesis studies using [35S]methionine and short-term tissue culture of kidney cortex fragments indicated that the most pronounced effect of vitamin D or 1,25 dihydroxyvitamin D3 was increased synthesis of the Mr 28,000 protein (3.2- to 4.6-fold increase compared to -D rats, P less than 0.001). Synthesis of a Mr 54,500 protein increased by 1.3- to 1.5-fold (P less than 0.05) and [35S]methionine incorporation into a Mr 66,000 protein decreased by 1.2- to 1.3-fold (P less than 0.05) in +D rats. This study represents the first detailed characterization of the effects of vitamin D on the composition and synthesis of rat kidney proteins. The data indicate that the most significant effect of vitamin D on kidney proteins is increased synthesis of the Mr 28,000 CaBP, suggesting that a major role of vitamin D in renal function is regulation of calcium transport at the distal tubule. However, dietary vitamin D or 1,25(OH)2D3 can influence the expression as well as the suppression of other specific kidney proteins.     

异氟烷预处理对大鼠肝缺血再灌注时脑线粒体钙及线粒体转运通道的影响

目的：通过观察在体大鼠肝部分缺血再灌注损伤后脑线粒体游离钙、线粒体转运通道（ mitochondrial permeability transition pore ,MPTP）及外周血中S-100β蛋白含量的变化,明确异氟烷预处理对大鼠肝部分缺血再灌注时脑损伤是否具有保护作用及可能的机制。方法 SD大鼠75只随机分成假手术组（ S组）;缺血再灌注组（ I/R组）：肝缺血60 min,再灌注120 min;异氟烷预处理组（ ISO组）：肝I/R前60 min ISO预处理30 min,后用空气洗脱30 min：CsA＋ISO组,CsA50 mg/kg静脉内注射,30 min后同ISO组;CsA组,I/R前30 min CsA50 mg/kg静脉内注射。再灌注24 h迅速断头取前脑,分离线粒体进行线粒体游离钙、MPTP含量检测,各组分别于缺血前及再灌注120 min后抽取静脉血采用双抗体夹心-ELAISA 法测定 S-100β蛋白含量。结果 I/R组（287.32±26.17）线粒体游离Ca2＋浓度明显增加,高于S组（198.54±21.02）和ISO组（209.74±29.49）（P ＜0.05）;CsA＋ISO（267.31±37.52）明显高于ISO组（ P ＜0.05）;CsA（288.63±23.15）组与I/R组间比较差异无显著意义（ P ＜0.05）;I/R组（1.73±0.24）的ΔS与S组（2.36±0.35）和ISO 组（2.11±0.32）相比明显减少（P ＜0.05）,既MPTP大量开放,而后两组的差异无统计学意义（P ＜0.05）;I/R组与CsA＋ISO组（1.72±0.34）和CsA组（1.77±0.35）△S之间差异无统计学意义（P ＜0.05）;CsA＋ISO组的ΔS值与ISO组相比明显降低（P ＜0.05）。外周血液S-100β蛋白I/R组明显高于S组和ISO组（P ＜0.05）;CsA＋ISO组与ISO组比较显著升高（P ＜0.05）,I/R组,CsA＋ISO组和CsA组与缺血前比较明显升高（ P ＜0.05）,缺血前S-100β蛋白含量五组无显著性差异（ P ＜0.05）。结论大鼠肝部分缺血再灌注后对脑组织造成了一定程度损伤,而异氟烷预处理对此损伤具有一定保护作用;其作用的机制可能与异氟烷抑制MPTP开放,降低线粒体游离Ca2＋浓度,防止了线粒体Ca2＋超载有关。     

Developmental pattern of three vesicular glutamate transporters in the rat superior olivary complex

Vesicular glutamate transporters (VGLUTs) mediate the packaging of the excitatory neurotransmitter glutamate into synaptic vesicles. Three VGLUT subtypes have been identified so far, which are differentially expressed in the brain. Here, we have investigated the spatiotemporal distribution of the three VGLUTs in the rat superior olivary complex (SOC), a prominent processing center, which receives strong glutamatergic inputs and which lies within the auditory brainstem. Immunoreactivity (ir) against all three VGLUTs was found in the SOC nuclei throughout development (postnatal days P0–P60). It was predominantly seen in axon terminals, although cytoplasmic labeling also occurred. Each transporter displayed a characteristic expression pattern. In the adult SOC, VGLUT1 labeling varied from strong in the medial nucleus of the trapezoid body, lateral superior olive, and medial superior olive (MSO) to moderate (ventral and lateral nuclei of the trapezoid body) to faint (superior paraolivary nucleus). VGLUT2-ir was moderate to strong throughout the SOC, whereas VGLUT3 was only weakly expressed. These results extend previous reports on co-localization of VGLUTs in the auditory brainstem. As in the adult, specific features were seen during development for all three transporters. Intensity increases and decreases occurred with both VGLUT1 and VGLUT3, whereas VGLUT2-ir remained moderately high throughout development. A striking result was obtained with VGLUT3, which was only transiently expressed in the different SOC nuclei between P0 and P12. A transient occurrence of VGLUT1-immunoreactive terminals on somata of MSO neurons was another striking finding. Our results imply a considerable amount of synaptic reorganization in the glutamatergic inputs to the SOC and suggest differential roles of VGLUTs during maturation and in adulthood. This work was supported by the Graduate Research School Molecular, physiological and pharmacological analysis of cellular membrane transport, DFG GRK 845/1.     

Dysregulated mitochondrial genes and networks with drug targets in postmortem brain of patients with posttraumatic stress disorder (PTSD) revealed by human mitochondria-focused cDNA microarrays

Posttraumatic stress disorder (PTSD) is associated with decreased activity in the dorsolateral prefrontal cortex (DLPFC), the brain region that regulates working memory and preparation and selection of fear responses. We investigated gene expression profiles in DLPFC Brodmann area (BA) 46 of postmortem patients with (n=6) and without PTSD (n=6) using human mitochondria-focused cDNA microarrays. Our study revealed PTSD-specific expression fingerprints of 800 informative mitochondria-focused genes across all of these 12 BA46 samples, and 119 (+/->1.25, p<0.05) and 42 (+/->1.60, p<0.05) dysregulated genes between the PTSD and control samples. Quantitative RT-PCR validated the microarray results. These fingerprints can essentially distinguish the PTSD DLPFC BA46 brains from controls. Of the 119 dysregulated genes (+/-> or =125%, p<0.05), the highest percentages were associated with mitochondrial dysfunction (4.8%, p=6.61 x 10(-6)), oxidative phosphorylation (3.8%, p=9.04 x 10(-4)), cell survival-apoptosis (25.2%, p<0.05) and neurological diseases (23.5%, p<0.05). Fifty (50) dysregulated genes were present in the molecular networks that are known to be involved in neuronal function-survival and contain 7 targets for neuropsychiatric drugs. Thirty (30) of the dysregulated genes are associated with a number of neuropsychiatric disorders. Our results indicate mitochondrial dysfunction in the PTSD DLPFC BA46 and provide the expression fingerprints that may ultimately serve as biomarkers for PTSD diagnosis and the drugs and molecular targets that may prove useful for development of remedies for prevention and treatment of PTSD.