Succination is Increased on Select Proteins in the Brainstem of the NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) Knockout Mouse,a Model of Leigh Syndrome

Elevated fumarate concentrations as a result of Krebs cycle inhibition lead to increases in protein succination, an irreversible post-translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). Metabolic events that reduce NADH re-oxidation can block Krebs cycle activity; therefore we hypothesized that oxidative phosphorylation deficiencies, such as those observed in some mitochondrial diseases, would also lead to increased protein succination. Using the Ndufs4 knockout (Ndufs4 KO) mouse, a model of Leigh syndrome, we demonstrate for the first time that protein succination is increased in the brainstem (BS), particularly in the vestibular nucleus. Importantly, the brainstem is the most affected region exhibiting neurodegeneration and astrocyte and microglial proliferation, and these mice typically die of respiratory failure attributed to vestibular nucleus pathology. In contrast, no increases in protein succination were observed in the skeletal muscle, corresponding with the lack of muscle pathology observed in this model. 2D SDS-PAGE followed by immunoblotting for succinated proteins and MS/MS analysis of BS proteins allowed us to identify the voltage-dependent anion channels 1 and 2 as specific targets of succination in the Ndufs4 knockout. Using targeted mass spectrometry, Cys⁷⁷ and Cys⁴⁸ were identified as endogenous sites of succination in voltage-dependent anion channels 2. Given the important role of voltage-dependent anion channels isoforms in the exchange of ADP/ATP between the cytosol and the mitochondria, and the already decreased capacity for ATP synthesis in the Ndufs4 KO mice, we propose that the increased protein succination observed in the BS of these animals would further decrease the already compromised mitochondrial function. These data suggest that fumarate is a novel biochemical link that may contribute to the progression of the neuropathology in this mitochondrial disease model.We previously identified the formation of S-(2-succino)cysteine (2SC)¹ (protein succination) as a result of the irreversible reaction of fumarate with reactive cysteine thiols (1, 2). Fumarate concentrations are increased during adipogenesis and adipocyte maturation (2, 3), and the excess of glucose and insulin leads to augmented protein succination in the adipose tissue of type 2 diabetic mice (4, 5). Protein succination is also specifically increased in fumarate hydratase deficient hereditary leiomyomatosis and renal cell carcinoma (HLRCC), because of the decreased conversion of fumarate to malate (6, 7). In both cases, intracellular fumarate concentrations are elevated; in fumarate hydratase deficient cells, the fumarate concentration is about 5 mm (8), whereas fumarate levels increase up to fivefold in adipocytes grown in the presence of high (30 mm) versus normal (5 mm) glucose concentrations (2). In the adipocyte the increase in fumarate and succinated proteins develops as a direct result of mitochondrial stress induced by nutrient excess. Mechanistically, excess glucose without increased ATP demand inhibits the electron transport chain resulting in an elevated NADH/NAD⁺ ratio. This inhibits NAD⁺-dependent Krebs cycle enzymes and leads to an increase in fumarate and protein succination (9). In support of this we have also shown that low concentrations of chemical uncouplers of oxidative phosphorylation (OXPHOS) can decrease fumarate concentrations and protein succination (9). The physiological consequences of protein succination include a decrease in the functionality of the target protein (8, 10–12), for example succination of adiponectin prevents the formation of multimeric complexes and reduces plasma adiponectin levels in diabetes (4). Considering the impact of glucotoxicity driven mitochondrial stress in the adipocyte, we predicted that deficiencies in OXPHOS associated with NADH accumulation would also result in increased protein succination.Mitochondrial respiratory chain disorders encompass a broad range of encephalopathies and myopathies associated with the defective assembly, activity or maintenance of the OXPHOS machinery (13), and are estimated to occur in about 1 in 5,000 live births (14). A common feature in most mitochondrial diseases (MD) is a failure to thrive because of reduced mitochondrial energy production; both the brain and muscle are usually affected because of their high dependence on oxidative metabolism (13). Leigh syndrome is one of the most common manifestations of MD and is characterized by progressive neurodegeneration with bilateral necrotizing lesions of the brainstem and basal ganglia, resulting in lactic acidosis, ataxia, seizures, dystonia, and respiratory failure (15, 16). Mutations in genes encoding the five complexes of the OXPHOS machinery can lead to Leigh syndrome; however, the majority of these mutations affect subunits of complexes I and IV (17), and both mitochondrial and nuclear encoded proteins may be affected (17–19). Complex I is a large (980 kDa) l-shaped protein assembly consisting of 45 peptides, with one flavin mononucleotide and eight iron–sulfur clusters (20). One of the first identified mutations of complex I encoded Ndufs4, a small (18 kDa) assembly protein (21–23). Ndufs4 assists in the final stages of complex I assembly, and its absence results in the formation of a smaller ∼830 kDa subcomplex that lacks the NADH dehydrogenase module and has significantly less electron shuttling activity than the intact holoenzyme (24, 25). Ndufs4 mutations are associated with brainstem deterioration in humans (26), and a recently described Ndufs4 knockout mouse (Ndufs4 KO) exhibits many of the clinical and neurological symptoms observed in human Leigh syndrome (27, 28).One of the most common clinical features of MD is lactic acidosis, derived from the accumulation of pyruvate and elevated NADH. Increased lactate or lactate:pyruvate ratios have been measured in the blood, urine, and cerebrospinal fluid of a large number of Leigh syndrome patients (15, 16). Increases in other organic acids in urine have also been reported (16), indicating that metabolic acidosis is a prominent clinical feature. Interestingly, a study designed to find new diagnostic metabolites in MD demonstrated that within certain age ranges the measurement of urinary fumarate and malate was a more useful discriminator of MD than lactate or other organic acids (29). Barshop''s findings support the hypothesis that MD derived from OXPHOS deficiencies may exhibit increased protein succination because of the accumulation of NADH and subsequently fumarate. In this study we report for the first time that protein succination is present in the brain in an animal model of Leigh syndrome, the Ndufs4 KO mouse, suggesting that this modification may be an important biochemical link between the genetic defect and the onset of neuropathology observed in Leigh syndrome.     

Brain Region-Specific Glutathione Redox Imbalance in Autism

Autism is a heterogeneous, behaviorally defined neurodevelopmental disorder. Recently, we reported a brain region-specific increase in lipid peroxidation, and deficits in mitochondrial electron transport chain complexes in autism, suggesting the role of oxidative stress and mitochondrial dysfunction in the pathophysiology of autism. However, the antioxidant status of the brain is not known in autism. Glutathione is a major endogenous antioxidant that plays a crucial role in protecting cells from exogenous and endogenous toxins, particularly in the central nervous system. The present study examines the concentrations of glutathione (GSH, reduced form; and GSSG, oxidized form) and the redox ratio of GSH to GSSG (marker of oxidative stress) in different regions of brains from autistic subjects and age-matched control subjects. In the cerebellum and temporal cortex from subjects with autism, GSH levels were significantly decreased by 34.2 and 44.6 %, with a concomitant increase in the levels of GSSG by 38.2 and 45.5 %, respectively, as compared to the control group. There was also a significant decrease in the levels of total GSH (tGSH) by 32.9 % in the cerebellum, and by 43.1 % in the temporal cortex of subjects with autism. In contrast, there was no significant change in GSH, GSSG and tGSH levels in the frontal, parietal and occipital cortices in autism versus control group. The redox ratio of GSH to GSSG was also significantly decreased by 52.8 % in the cerebellum and by 60.8 % in the temporal cortex of subjects with autism, suggesting glutathione redox imbalance in the brain of individuals with autism. These findings indicate that autism is associated with deficits in glutathione antioxidant defense in selective regions of the brain. We suggest that disturbances in brain glutathione homeostasis may contribute to oxidative stress, immune dysfunction and apoptosis, particularly in the cerebellum and temporal lobe, and may lead to neurodevelopmental abnormalities in autism.     

Post-Translational Decrease in Respiratory Chain Proteins in the Polg Mutator Mouse Brain

Mitochondrial DNA damage is thought to be a causal contributor to aging as mice with inactivating mutations in polymerase gamma (Polg) develop a progeroid phenotype. To further understand the molecular mechanisms underlying this phenotype, we used iTRAQ and RNA-Seq to determine differences in protein and mRNA abundance respectively in the brains of one year old Polg mutator mice compared to control animals. We found that mitochondrial respiratory chain proteins are specifically decreased in abundance in the brains of the mutator mice, including several nuclear encoded mitochondrial components. However, we found no evidence that the changes we observed in protein levels were the result of decreases in mRNA expression. These results show that there are post-translational effects associated with mutations in Polg.     

Vinyl Chloride Monomer (VCM) Induces High Occurrence of Neural Tube Defects in Embryonic Mouse Brain During Neurulation

The aim of this study was to explore the direct embryonic teratogenicity of vinyl chloride monomer (VCM), especially the toxic effects on the early development of the nervous system and its underlying mechanisms. Pregnant mice at embryonic day 6.5 (E6.5) were injected with different doses of VCM (200, 400 and 600 mg/kg) and embryos were harvested at E10.5. Our results showed that doses higher than 400 mg/kg of VCM increased the incidence of malformed embryos, especially the neural tube defects (NTDs). In addition, high-dose of VCM decreased mitotic figure counts in the neuroepithelium and enhanced the percentage of cells in G0/G1 phase, while they were reduced in S phase. The more VCM was injected into mice, the fewer positive PCNA cells were seen and the more positive TUNEL cells were observed in the neuroepithelium. Moreover, significant increases in the levels of caspase-3 protein were observed in NTD embryos. Our results demonstrate that during early pregnancy, exposure to doses higher than 400 mg/kg of VCM increases the incidence of malformations and particularly the rate of NTDs. High-dose of VCM inhibits the proliferation of neural cells and induces cell apoptosis, leading to an imbalance in the ratio of proliferation and apoptosis. Meanwhile, the apoptosis of neuroepithelial cells might be accelerated by the activation of the caspase-3 pathway, and it might be a reason for NTDs.     

Complex I Deficiency Due to Selective Loss of Ndufs4 in the Mouse Heart Results in Severe Hypertrophic Cardiomyopathy

Mitochondrial complex I, the primary entry point for electrons into the mitochondrial respiratory chain, is both critical for aerobic respiration and a major source of reactive oxygen species. In the heart, chronic dysfunction driving cardiomyopathy is frequently associated with decreased complex I activity, from both genetic and environmental causes. To examine the functional relationship between complex I disruption and cardiac dysfunction we used an established mouse model of mild and chronic complex I inhibition through heart-specific Ndufs4 gene ablation. Heart-specific Ndufs4-null mice had a decrease of ∼50% in complex I activity within the heart, and developed severe hypertrophic cardiomyopathy as assessed by magnetic resonance imaging. The decrease in complex I activity, and associated cardiac dysfunction, occurred absent an increase in mitochondrial hydrogen peroxide levels in vivo, accumulation of markers of oxidative damage, induction of apoptosis, or tissue fibrosis. Taken together, these results indicate that diminished complex I activity in the heart alone is sufficient to drive hypertrophic cardiomyopathy independently of alterations in levels of mitochondrial hydrogen peroxide or oxidative damage.     

High Fat Diet Induced Developmental Defects in the Mouse: Oocyte Meiotic Aneuploidy and Fetal Growth Retardation/Brain Defects

Background

Maternal obesity is associated with poor outcomes across the reproductive spectrum including infertility, increased time to pregnancy, early pregnancy loss, fetal loss, congenital abnormalities and neonatal conditions. Furthermore, the proportion of reproductive-aged woman that are obese in the population is increasing sharply. From current studies it is not clear if the origin of the reproductive complications is attributable to problems that arise in the oocyte or the uterine environment.

Methodology/Principal Findings

We examined the developmental basis of the reproductive phenotypes in obese animals by employing a high fat diet mouse model of obesity. We analyzed very early embryonic and fetal phenotypes, which can be parsed into three abnormal developmental processes that occur in obese mothers. The first is oocyte meiotic aneuploidy that then leads to early embryonic loss. The second is an abnormal process distinct from meiotic aneuploidy that also leads to early embryonic loss. The third is fetal growth retardation and brain developmental abnormalities, which based on embryo transfer experiments are not due to the obese uterine environment but instead must be from a defect that arises prior to the blastocyst stage.

Conclusions/Significance

Our results suggest that reproductive complications in obese females are, at least in part, from oocyte maternal effects. This conclusion is consistent with IVF studies where the increased pregnancy failure rate in obese women returns to the normal rate if donor oocytes are used instead of autologous oocytes. We postulate that preconceptional weight gain adversely affects pregnancy outcomes and fetal development. In light of our findings, preconceptional counseling may be indicated as the preferable, earlier target for intervention in obese women desiring pregnancy and healthy outcomes.     

Poly(ADP-Ribose) Polymerase Inhibitor PJ34 Reduces Brain Damage after Stroke in the Neonatal Mouse Brain

The poly(ADP-ribose) polymerase inhibitor PJ34 has recently been reported to increase cerebral blood flow, via the endothelial NO synthase, in the naive mouse brain throughout life. We addressed here the benefits of PJ34 after neonatal ischemia on hemodynamics and components of the neurovascular unit including the blood-brain barrier (BBB), microglia, and astrocytes. Nine-day-old mice were subjected to permanent MCA occlusion (pMCAo), and treated with either PBS or PJ34 (10 mg/kg). Mean blood-flow velocities (mBFV) were measured in both internal carotid arteries (ICA) and basilar trunk (BT) using Doppler-ultrasonography. BBB opening was assessed through somatostatin-receptor type-2 internalization and immunohistochemistry at 24 and 48 h. Lesion areas were measured 8 days after ischemia. In PBS-treated mice, pMCAo involved a drop in mBFV in the left ICA (p < 0.001 vs. basal), whereas mBFV remained stable in both right ICA and BT. PJ34 prevented this drop in the left ICA (NS vs. basal) and increased mBFV in the right ICA (p = 0.0038 vs. basal). No modification was observed in the BT. In contrast to PBS, BBB disruption extent and astrocyte demise were reduced in PJ34 mice only in the rostral brain at 48 h and 8 days post-pMCAo, respectively. Accordingly, 8 days after pMCAo, affected areas were reduced in the rostral brain (Bregma +0.86 and +0.14 mm), whereas total tissue loss was not reduced after PJ34 (4.0 ± 3.1%) vs. PBS (5.8 ± 3.4%). These results show that PJ34 reduced BBB permeability, astrocyte demise, and tissue loss (particularly in the rostral territories), suggesting that collateral supply mainly proceeds from the anterior ICA’s branches in the ischemic neonatal mouse brain.     

Development of Reserve Capacities of the Pulmonary Respiratory Function in Schoolboys

New data on the developmental features of the forming of reserve respiratory capacities were recorded in studies of a high risk group of physiologically retarded children and adolescents. The data showed that negative effects of passive smoking on these capacities are especially harmful in adolescents. The monitoring of the respiratory functional capacities in schoolboys made it possible to determine certain highly vulnerable periods characterized by stronger individual manifestations of the functional development of the respiratory system.     

Cyclic AMP in Female Mouse Brain is Altered by the Adrenocorticotropic Hormone(4–9) Analogue Organon 2766

Abstract: Cyclic AMP content was determined in 12 brain regions of young adult female mice at 30 min and at 24 h following an intraperitoneal injection of the tri-substituted adrenocorticotropic hormone(4–9) [ACTH(4–9)] analogue Organon 2766 [ORG 2766]. Animals were killed by focused 3.5 kW microwave radiation applied for 350 ms. Unlike previously reported responses in male mice, at 30 min post-injection there were no detectable differences in cyclic AMP content between the placebo and ORG 2766-treated animals. By contrast, 24 h after injection, the content of cyclic AMP was changed significantly in 8 of the 12 brain regions examined: medulla-pons, septal area, thalamus, hypothalamus, hippocampus, olfactory bulb, and parietal and occipital cortices. In most of the regions examined, differences consisted of 50% or greater reductions of tissue cyclic AMP content. The changes were unrelated to the estrus cycle of these animals     

Region-Specific Interrelations between Apoptotic Proteins Expression and DNA Fragmentation in the Neonatal Rat Brain

DNA fragmentation, mRNA and protein levels of Bcl-XL, Bax and caspase-3 were determined to characterize interrelations between expression of these apoptotic markers in the neonatal brain regions. High DNA fragmentation intensity in the cortex was in consonance with the lowest Bcl-XL/Bax expression ratio, the highest procaspase-3 and active caspase-3 levels. Low and intermediate DNA fragmentation levels in the cerebellum and hippocampus respectively were also in a good agreement with apoptotic proteins expression in these structures. In the cortex, hippocampus and cerebellum DNA fragmentation intensity was proportional to the active caspase-3 level. In contrast to these structures, in the brainstem, the lowest level of this protease was accompanied by the highest intensity of DNA fragmentation among the brain regions studied. The data suggest that cell death normally occurring during early postnatal life could be realized in the developing brainstem via caspase-3-independent pathways in animals that express this protease.     

Schistosome-infected IL-4 receptor knockout (KO) mice, in contrast to IL-4 KO mice, fail to develop granulomatous pathology while maintaining the same lymphokine expression profile.

Th2 lymphocytes have been postulated to play a major role in the immunopathology induced by Schistosoma mansoni infection. Nevertheless, infected IL-4 knockout (KO) and wild-type (wt) mice develop egg granulomas comparable in size. To further investigate the function of the Th2 response in egg pathology we studied IL-4Ralpha-deficient mice, which are nonresponsive to both IL-4 and IL-13. In striking contrast to IL-4 KO animals, infected IL-4Ralpha KO mice developed only minimal hepatic granulomas and fibrosis despite the presence of CD3+ T cells in the residual egg lesions. Moreover, liver lymphokine mRNA levels in these animals and IL-4 KO mice were equivalent. In addition, infected IL-4Ralpha-deficient, IL-4-deficient, and wt animals developed similar egg Ag-specific IgG Ab titers, arguing that CD4-dependent Th activity is intact in KO mice. As expected, IFN-gamma secretion was strongly up-regulated in mesenteric lymph node cultures from both groups of deficient animals, a change reflected in increased serum IgG2a and IgG2b Ab levels. Surprisingly, Th2 cytokine production in infected IL-4Ralpha KO mice was not abolished but was only reduced and resembled that previously documented in IL-4 KO animals. This residual Th2 response is likely to explain the ability of IL-4 KO mice to generate egg granulomas, which cannot be formed in IL-4Ralpha-deficient animals because of their lack of responsiveness to the same cytokine ligands. Taken together, these findings argue that tissue pathology in schistosomiasis requires, in addition to egg-specific CD4+ lymphocytes, a previously unrecognized IL-4Ralpha+ non-T cell effector population.     

Rearrangement and Reactivation of the Myelin Basic Protein Locus in Myelin-Deficient (mld) Mouse Brain

Abstract: Myelin-deficient ( mld ) is a complex mutation affecting the myelin basic protein (MBP) locus of the mouse. It consists of duplication and partial inversion of the MBP gene and results in a dysfunctional MBP locus. The mutant phenotype is reversed, both in vivo and in vitro, in ∼5% of mld oligodendrocytes. One possible mechanism for the somatic reversion is recombination between homologous sequences of the duplicated gene copies to reconstitute a functional MBP locus. There are several possible recombination events that could reconstitute a functional MBP locus by DNA rearrangement. Two of these would result in reinversion and circularization of specific MBP gene sequences, respectively. In this work polymerase chain reaction analysis was used to detect both reinverted and circularized MBP gene sequences in mld mouse tissues, indicating that DNA rearrangement at the MBP locus does occur. Analysis of individually harvested cells showed that in revertant MBP-positive mld oligodendrocytes DNA rearrangement at the MBP locus was correlated with reactivation of the MBP gene. Fluctuation analysis showed that reactivation of the MBP locus is a stochastic event occurring with a frequency of ∼1.4 × 10⁻⁶ per cell per cell cycle during oligodendrocyte development. The frequency of rearrangement and reactivation of the MBP locus was comparable in double mutant ( mld/mld , scid/scid ) and single mutant ( mld/mld , + ^scid /+ ^scid ) mice, indicating that the scid factor is not required for MBP gene reactivation in mld . The significance of DNA rearrangement in mammalian development is discussed.     

Genetic Ablation of Type III Adenylyl Cyclase Exerts Region-Specific Effects on Cilia Architecture in the Mouse Nose

We recently reported that olfactory sensory neurons in the dorsal zone of the mouse olfactory epithelium exhibit drastic location-dependent differences in cilia length. Furthermore, genetic ablation of type III adenylyl cyclase (ACIII), a key olfactory signaling protein and ubiquitous marker for primary cilia, disrupts the cilia length pattern and results in considerably shorter cilia, independent of odor-induced activity. Given the significant impact of ACIII on cilia length in the dorsal zone, we sought to further investigate the relationship between cilia length and ACIII level in various regions throughout the mouse olfactory epithelium. We employed whole-mount immunohistochemical staining to examine olfactory cilia morphology in phosphodiesterase (PDE) 1C^-/-;PDE4A^-/- (simplified as PDEs^-/- hereafter) and ACIII^-/- mice in which ACIII levels are reduced and ablated, respectively. As expected, PDEs^-/- animals exhibit dramatically shorter cilia in the dorsal zone (i.e., where the cilia pattern is found), similar to our previous observation in ACIII^-/- mice. Remarkably, in a region not included in our previous study, ACIII^-/- animals (but not PDEs^-/- mice) have dramatically elongated, comet-shaped cilia, as opposed to characteristic star-shaped olfactory cilia. Here, we reveal that genetic ablation of ACIII has drastic, location-dependent effects on cilia architecture in the mouse nose. These results add a new dimension to our current understanding of olfactory cilia structure and regional organization of the olfactory epithelium. Together, these findings have significant implications for both cilia and sensory biology.     

Uptake of 1-Methyl-4-Phenylpyridinium and Dopamine in the Mouse Brain Cell Nuclei

Abstract: The neurotoxin N -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes, via its metabolite 1-methyl-4-phenylpyridinium (MPP⁺), parkinsonism in humans, monkeys, and mice but not in rats. When incubated with mouse brain homogenates, [³H]-MPP⁺ is recovered in relatively large concentrations in the brain cell nucleus. Although isolated cell nuclei from rat and mouse brain contain uptake systems for dopamine (DA), only brain cell nuclei from mice avidly take up [³H]MPP⁺. This nuclear uptake is ATP dependent and can be blocked by ouabain and Nethylmaleimide. It is not, however, affected by neuronal and vesicular blockers such as benztropine. mazindol, and reserpine. Selective uptake of MPP⁺ into brain cell nuclei may provide a new avenue for future investigation into the complex modes of action of the neurotoxin MPTP.     

Processing of the Matricellular Protein Hevin in Mouse Brain Is Dependent on ADAMTS4

The matricellular SPARC family member hevin (SPARC-like 1/SPARCL-1/SC1/Mast9) contributes to neural development and alters tumor progression in a range of mammalian models. The distribution of hevin in mouse tissues was reexamined with a novel monoclonal antibody that discriminates between hevin and its ortholog SPARC. We now report proteolysis of hevin in many tissues, with the most extensive processing in the brain. We demonstrate a cleavage site within the hevin sequence for the neural tissue proteinase ADAMTS4. Digestion of hevin by ADAMTS4 in vitro produced fragments similar to those present in brain lysates. Monoclonal antibodies revealed a SPARC-like fragment generated from hevin that was co-localized with ADAMTS4 in vivo. We show that proteolysis of hevin by ADAMTS4 in the mouse cerebellum is important for the normal development of this tissue. In conclusion, we have identified the fragmentation of hevin by ADAMTS4 in the mouse brain and propose that this specific proteolysis is integral to cell morphology and extracellular matrix deposition in the developing brain.     

The Vasculome of the Mouse Brain

The blood vessel is no longer viewed as passive plumbing for the brain. Increasingly, experimental and clinical findings suggest that cerebral endothelium may possess endocrine and paracrine properties – actively releasing signals into and receiving signals from the neuronal parenchyma. Hence, metabolically perturbed microvessels may contribute to central nervous system (CNS) injury and disease. Furthermore, cerebral endothelium can serve as sensors and integrators of CNS dysfunction, releasing measurable biomarkers into the circulating bloodstream. Here, we define and analyze the concept of a brain vasculome, i.e. a database of gene expression patterns in cerebral endothelium that can be linked to other databases and systems of CNS mediators and markers. Endothelial cells were purified from mouse brain, heart and kidney glomeruli. Total RNA were extracted and profiled on Affymetrix mouse 430 2.0 micro-arrays. Gene expression analysis confirmed that these brain, heart and glomerular preparations were not contaminated by brain cells (astrocytes, oligodendrocytes, or neurons), cardiomyocytes or kidney tubular cells respectively. Comparison of the vasculome between brain, heart and kidney glomeruli showed that endothelial gene expression patterns were highly organ-dependent. Analysis of the brain vasculome demonstrated that many functionally active networks were present, including cell adhesion, transporter activity, plasma membrane, leukocyte transmigration, Wnt signaling pathways and angiogenesis. Analysis of representative genome-wide-association-studies showed that genes linked with Alzheimer’s disease, Parkinson’s disease and stroke were detected in the brain vasculome. Finally, comparison of our mouse brain vasculome with representative plasma protein databases demonstrated significant overlap, suggesting that the vasculome may be an important source of circulating signals in blood. Perturbations in cerebral endothelial function may profoundly affect CNS homeostasis. Mapping and dissecting the vasculome of the brain in health and disease may provide a novel database for investigating disease mechanisms, assessing therapeutic targets and exploring new biomarkers for the CNS.     

Homeobox Genes in the Developing Mouse Brain

Abstract: Any list of past and recent findings on vertebrate brain prenatal development would have to include the fundamental roles of homeobox genes, the genes encoding the nuclear regulatory homeodomain proteins. The discovery of homeobox genes and their involvement as master regulatory elements in programing the development of an embryo into a complete adult organism has provided a key to our understanding of ontogenesis. Also, the correlation of mouse developmental mutants and their corresponding human syndromes with mutations in homeobox genes has provided further evidence for the fundamental role of homeobox genes during the vertebrate brain embryonic development. Here, we review the expression patterns and the phenotypes of gene mutations that implicate a large repertoire of mouse homeobox genes in the specification of neuronal functions during brain embryogenesis.