Impairment of an Endothelial NAD+-H2S Signaling Network Is a Reversible Cause of Vascular Aging.

A decline in capillary density and blood flow with age is a major cause of mortality and morbidity. Understanding why this occurs is key to future gains in human health. NAD precursors reverse aspects of aging, in part, by activating sirtuin deacylases (SIRT1-SIRT7) that mediate the benefits of exercise and dietary restriction (DR). We show that SIRT1 in endothelial cells is a key mediator of pro-angiogenic signals secreted from myocytes. Treatment of mice with the NAD+ booster nicotinamide mononucleotide (NMN) improves blood flow and increases endurance in elderly mice by promoting SIRT1-dependent increases in capillary density, an effect augmented by exercise or increasing the levels of hydrogen sulfide (H2S), a DR mimetic and regulator of endothelial NAD+ levels. These findings have implications for improving blood flow to organs and tissues, increasing human performance, and reestablishing a virtuous cycle of mobility in the elderly.

SirT1 is required in the male germ cell for differentiation and fecundity in mice.

Sirtuins are NAD(+)-dependent deacylases that regulate numerous biological processes in response to the environment. SirT1 is the mammalian ortholog of yeast Sir2, and is involved in many metabolic pathways in somatic tissues. Whole body deletion of SirT1 alters reproductive function in oocytes and the testes, in part caused by defects in central neuro-endocrine control. To study the function of SirT1 specifically in the male germ line, we deleted this sirtuin in male germ cells and found that mutant mice had smaller testes, a delay in differentiation of pre-meiotic germ cells, decreased spermatozoa number, an increased proportion of abnormal spermatozoa and reduced fertility. At the molecular level, mutants do not have the characteristic increase in acetylation of histone H4 at residues K5, K8 and K12 during spermiogenesis and demonstrate corresponding defects in the histone to protamine transition. Our findings thus reveal a germ cell-autonomous role of SirT1 in spermatogenesis.

Pcdh19 mediates olfactory sensory neuron coalescence during postnatal stages and regeneration.

The mouse olfactory system regenerates constantly throughout life. While genes critical for the initial projection of olfactory sensory neurons (OSNs) to the olfactory bulb have been identified, what genes are important for maintaining the olfactory map during regeneration are still unknown. Here we show a mutation in Protocadherin 19 (Pcdh19), a cell adhesion molecule and member of the cadherin superfamily, leads to defects in OSN coalescence during regeneration. Surprisingly, lateral glomeruli were more affected and males in particular showed a more severe phenotype. Single cell analysis unexpectedly showed OSNs expressing the MOR28 odorant receptor could be subdivided into two major clusters. We showed that at least one protocadherin is differentially expressed between OSNs coalescing on the medial and lateral glomeruli. Moreover, females expressed a slightly different complement of genes from males. These features may explain the differential effects of mutating Pcdh19 on medial and lateral glomeruli in males and females.

4-Phenylbutyrate restores localization and membrane repair to human dysferlin mutations.

Dysferlinopathies are muscular dystrophies caused by recessive loss-of-function mutations in dysferlin (DYSF), a membrane protein involved in skeletal muscle membrane repair. We describe a cell-based assay in which human DYSF proteins bearing missense mutations are quantitatively assayed for membrane localization by flow cytometry and identified 64 localization-defective DYSF mutations. Using this platform, we show that the clinically approved drug 4-phenylbutryric acid (4-PBA) partially restores membrane localization to 25 mutations, as well as membrane repair to cultured myotubes expressing 2 different mutations. Two-day oral administration of 4-PBA to mice homozygous for one of these mutations restored myofiber membrane repair. 4-PBA may hold therapeutic potential for treating a subset of humans with muscular dystrophy due to dysferlin deficiency.

Tuning of delta-protocadherin adhesion through combinatorial diversity.

The delta-protocadherins (δ-Pcdhs) play key roles in neural development, and expression studies suggest they are expressed in combination within neurons. The extent of this combinatorial diversity, and how these combinations influence cell adhesion, is poorly understood. We show that individual mouse olfactory sensory neurons express 0-7 δ-Pcdhs. Despite this apparent combinatorial complexity, K562 cell aggregation assays revealed simple principles that mediate tuning of δ-Pcdh adhesion. Cells can vary the number of δ-Pcdhs expressed, the level of surface expression, and which δ-Pcdhs are expressed, as different members possess distinct apparent adhesive affinities. These principles contrast with those identified previously for the clustered protocadherins (cPcdhs), where the particular combination of cPcdhs expressed does not appear to be a critical factor. Despite these differences, we show δ-Pcdhs can modify cPcdh adhesion. Our studies show how intra- and interfamily interactions can greatly amplify the impact of this small subfamily on neuronal function.

Novel subdomains of the mouse olfactory bulb defined by molecular heterogeneity in the nascent external plexiform and glomerular layers.

BACKGROUND: In the mouse olfactory system, the role of the olfactory bulb in guiding olfactory sensory neuron (OSN) axons to their targets is poorly understood. What cell types within the bulb are necessary for targeting is unknown. What genes are important for this process is also unknown. Although projection neurons are not required, other cell-types within the external plexiform and glomerular layers also form synapses with OSNs. We hypothesized that these cells are important for targeting, and express spatially differentially expressed guidance cues that act to guide OSN axons within the bulb. RESULTS: We used laser microdissection and microarray analysis to find genes that are differentially expressed along the dorsal-ventral, medial-lateral, and anterior-posterior axes of the bulb. The expression patterns of these genes divide the bulb into previously unrecognized subdomains. Interestingly, some genes are expressed in both the medial and lateral bulb, showing for the first time the existence of symmetric expression along this axis. We use a regeneration paradigm to show that several of these genes are altered in expression in response to deafferentation, consistent with the interpretation that they are expressed in cells that interact with OSNs. CONCLUSION: We demonstrate that the nascent external plexiform and glomerular layers of the bulb can be divided into multiple domains based on the expression of these genes, several of which are known to function in axon guidance, synaptogenesis, and angiogenesis. These genes represent candidate guidance cues that may act to guide OSN axons within the bulb during targeting.

Sirtuin 1 Promotes Deacetylation of Oct4 and Maintenance of Naive Pluripotency.

The enhancer landscape is dramatically restructured as naive preimplantation epiblasts transition to the post-implantation state of primed pluripotency. A key factor in this process is Otx2, which is upregulated during the early stages of this transition and ultimately recruits Oct4 to a different set of enhancers. In this study, we discover that the acetylation status of Oct4 regulates the induction of the primed pluripotency gene network. Maintenance of the naive state requires the NAD-dependent deacetylase, SirT1, which deacetylates Oct4. The activity of SirT1 is reduced during the naive-to-primed transition; Oct4 becomes hyper-acetylated and binds to an Otx2 enhancer to induce Otx2 expression. Induction of Otx2 causes the reorganization of acetylated Oct4 and results in the induction of the primed pluripotency gene network. Regulation of Oct4 by SirT1 may link stem cell development to environmental conditions, and it may provide strategies to manipulate epiblast cell state.

SIRT1 suppresses the epithelial-to-mesenchymal transition in cancer metastasis and organ fibrosis.

The epithelial-to-mesenchymal transition (EMT) is important for the development of cancer metastases and organ fibrosis, conditions prevalent in aging. Because sirtuins affect the pathology of aging, we tested the effect of SirT1 on EMT. Reduced SIRT1 levels in HMLER breast cancer cells led to increased metastases in nude mice, and the loss of SIRT1 in kidney tubular epithelial cells exacerbated injury-induced kidney fibrosis. SIRT1 reduces EMT in cancer and fibrosis by deacetylating Smad4 and repressing the effect of TGF-ß signaling on MMP7, a Smad4 target gene. Consequently, less E-cadherin is cleaved from the cell surface and ß-catenin remains bound to E-cadherin at the cell-cell junctions. Our findings suggest that the SIRT1/Smad4/ß-catenin axis may be a target for diseases driven by EMT.

Delta Protocadherin 10 is Regulated by Activity in the Mouse Main Olfactory System.

Olfactory sensory neurons (OSNs) are thought to use activity-dependent and independent mechanisms to regulate the expression of axon guidance genes. However, defining the molecular mechanisms that underlie activity-dependent OSN guidance has remained elusive. Only a handful of genes have been identified whose expression is regulated by activity. Interestingly, all of these genes have been shown to play a role in OSN axon guidance, underscoring the importance of identifying other genes regulated by activity. Furthermore, studies suggest that more than one downstream mechanism regulates target gene expression. Thus, both the number of genes regulated by activity and how many total mechanisms control this expression are not well understood. Here we identify delta protocadherin 10 (pcdh10) as a gene regulated by activity. Delta protocadherins are members of the cadherin superfamily, and pcdh10 is known to be important for axon guidance during development. We show pcdh10 is expressed in the nasal epithelium and olfactory bulb in patterns consistent with providing guidance information to OSNs. We use naris occlusion, genetic manipulations, and pharmacological assays to show pcdh10 can be regulated by activity, consistent with activation via the cyclic nucleotide-gated channel. Transgenic analysis confirms a potential role for pcdh10 in OSN axon guidance.

Nordihydroguaiaretic acid increases glutamate uptake in vitro and in vivo: therapeutic implications for amyotrophic lateral sclerosis.

Synaptic accumulation of glutamate causes neuronal death in many neurodegenerative pathologies including amyotrophic lateral sclerosis. Drugs capable of increasing glutamate uptake could therefore be therapeutically effective. We screened in a cell-based assay a library of 1040 FDA-approved drugs and nutrients for compounds that could enhance glutamate uptake. Nordihydroguaiaretic acid (NDGA), an anti-inflammatory drug that inhibits lipoxygensases, potently enhanced glutamate uptake in MN-1 cells. Given subcutaneously at 1 mg/day for 30 days in mice, NDGA increased glutamate uptake in spinal cord synaptosomes persistently throughout the treatment. However, when administered following the same regimen to the SOD1-G93A transgenic mouse model of ALS at disease onset, NDGA did not extend survival of these mice. We found that NDGA failed to sustain increased glutamate uptake in the SOD1-G93A mice despite an initial upregulation measured during the first 10 days of treatment. SOD1-G93A mice displayed a progressive increase in spinal cord expression levels of the efflux transporter P-glycoprotein beginning at disease onset. This increase was not mediated by the NDGA treatment because it was measured in untreated SOD1-G93A mice. Since P-glycoproteins control the extrusion of a broad range of toxins and xenobiotics and are responsible for drug resistance in many diseases including cancer and brain diseases such as epilepsy, we propose that the failure of NDGA in maintaining glutamate uptake upregulated in SOD1-G93A mice and its therapeutic inefficacy are due to acquired pharmacoresistance mediated by the increased expression of P-glycoprotein.

Controlling microarray spot morphology with polymer liftoff arrays.

Biological arrays are hindered by the lack of uniformity in the deposition of biomaterials. Efforts aimed at improving this deposition have focused on altering the composition of the solution or the tool used to deposit the material. However, little attention has been paid to controlling material deposition by constraining the physical and chemical topography of the surface. Here we present the use of a hybrid hydrophilic/hydrophobic micropatterned surface to direct the deposition of spotted DNA on microarrays. These polymer "liftoff" arrays combine the hydrophobic surface properties of di-p-xylylene (Parylene) with photolithographically etched hydrophilic openings within the polymer. We show that the flow pattern of solutes on these substrates favors the concentration of dissolved material into the mesoscopic openings underlying the printed spot, resulting in significantly improved uniformity of deposition. Moreover, the micropatterned surface allows for increased replication of spotted materials. Finally, these polymer liftoff arrays display reduced array-to-array variation, improving the reproducibility of data acquisition. We envision that these novel substrates can be generalized to produce more uniform arrays of other patterned biomaterials.

Caspase-3 cleaves and inactivates the glutamate transporter EAAT2.

EAAT2 is a high affinity, Na+-dependent glutamate transporter with predominant astroglial localization. It accounts for the clearance of the bulk of glutamate released at central nervous system synapses and therefore has a crucial role in shaping glutamatergic neurotransmission and limiting excitotoxicity. Caspase-3 activation and impairment in expression and activity of EAAT2 are two distinct molecular mechanisms occurring in human amyotrophic lateral sclerosis (ALS) and in the transgenic rodent model of the disease. Excitotoxicity caused by down-regulation of EAAT2 is thought to be a contributing factor to motor neuron death in ALS. In this study, we report the novel evidence that caspase-3 cleaves EAAT2 at a unique site located in the cytosolic C-terminal domain of the transporter, a finding that links excitotoxicity and activation of caspase-3 as converging mechanisms in the pathogenesis of ALS. Caspase-3 cleavage of EAAT2 leads to a drastic and selective inhibition of this transporter. Heterologous expression of mutant SOD1 proteins linked to the familial form of ALS leads to inhibition of EAAT2 through a mechanism that largely involves activation of caspase-3 and cleavage of the transporter. In addition, we found evidence in spinal cord homogenates of mutant SOD1 ALS mice of a truncated form of EAAT2, likely deriving from caspase-3-mediated proteolytic cleavage, which appeared concurrently to the loss of EAAT2 immunoreactivity and to increased expression of activated caspase-3. Taken together, our findings suggest that caspase-3 cleavage of EAAT2 is one mechanism responsible for the impairment of glutamate uptake in mutant SOD1-linked ALS.