Transcriptome profiling of hippocampal CA1 after early-life seizure-induced preconditioning may elucidate new genetic therapies for epilepsy.

Injury of the CA1 subregion induced by a single injection of kainic acid (1 × KA) in juvenile animals (P20) is attenuated in animals with two prior sustained neonatal seizures on P6 and P9. To identify gene candidates involved in the spatially protective effects produced by early-life conditioning seizures we profiled and compared the transcriptomes of CA1 subregions from control, 1 × KA- and 3 × KA-treated animals. More genes were regulated following 3 × KA (9.6%) than after 1 × KA (7.1%). Following 1 × KA, genes supporting oxidative stress, growth, development, inflammation and neurotransmission were upregulated (e.g. Cacng1, Nadsyn1, Kcng1, Aven, S100a4, GFAP, Vim, Hrsp12 and Grik1). After 3 × KA, protective genes were differentially over-expressed [e.g. Cat, Gpx7, Gad1, Hspa12A, Foxn1, adenosine A1 receptor, Ca(2+) adaptor and homeostasis proteins, Cacnb4, Atp2b2, anti-apoptotic Bcl-2 gene members, intracellular trafficking protein, Grasp and suppressor of cytokine signaling (Socs3)]. Distinct anti-inflammatory interleukins (ILs) not observed in adult tissues [e.g. IL-6 transducer, IL-23 and IL-33 or their receptors (IL-F2 )] were also over-expressed. Several transcripts were validated by real-time polymerase chain reaction (QPCR) and immunohistochemistry. QPCR showed that casp 6 was increased after 1 × KA but reduced after 3 × KA; the pro-inflammatory gene Cox1 was either upregulated or unchanged after 1 × KA but reduced by ~70% after 3 × KA. Enhanced GFAP immunostaining following 1 × KA was selectively attenuated in the CA1 subregion after 3 × KA. The observed differential transcriptional responses may contribute to early-life seizure-induced pre-conditioning and neuroprotection by reducing glutamate receptor-mediated Ca(2+) permeability of the hippocampus and redirecting inflammatory and apoptotic pathways. These changes could lead to new genetic therapies for epilepsy.

beta2-glycoprotein-I (apolipoprotein H) and beta2-glycoprotein-I-phospholipid complex harbor a recognition site for the endocytic receptor megalin.

Screening of serum by using a surface plasmon resonance analysis assay identified beta2-glycoprotein-I/apolipoprotein H as a plasma component binding to the renal epithelial endocytic receptor megalin. A calcium-dependent megalin-mediated beta2-glycoprotein-I endocytosis was subsequently demonstrated by ligand blotting of rabbit renal cortex and uptake analysis in megalin-expressing cells. Immunohistochemical and immunoelectron microscopic examination of kidneys and the presence of high concentrations of beta2-glycoprotein-I in urine of mice with disrupted megalin gene established that megalin is the renal clearance receptor for beta2-glycoprotein-I. A significant increase in functional affinity for purified megalin was observed when beta2-glycoprotein-I was bound to the acidic phospholipids, phosphatidylserine and cardiolipin. The binding of beta2-glycoprotein-I and beta2-glycoprotein-I- phospholipid complexes to megalin was completely blocked by receptor-associated protein. In conclusion, we have demonstrated a novel receptor recognition feature of beta2-glycoprotein-I. In addition to explaining the high urinary excretion of beta2-glycoprotein-I in patients with renal tubule failure, the data provide molecular evidence for the suggested function of beta2-glycoprotein-I as a linking molecule mediating cellular recognition of phosphatidylserine-exposing particles.

Immunocytochemical localization of DNA double-strand breaks in human and rat brains.

Post-mitotic neurons are particularly susceptible to DNA double-strand breaks during their relatively long lifespan. Here, we report the anatomical distribution and subcellular localization of a molecule first identified as a DNA damage checkpoint protein. Immunocytochemical analysis of 53BP1 showed that this nuclear molecule is widely expressed in adult human and rat brains. Further, we showed that 53BP1 routinely co-clusters with γ-aminobutyric acid neurons throughout the rat neuraxis. Notably, 53BP1 is only expressed in neuronal cells as the DNA damage checkpoint protein was virtually absent from glial cells. Finally, we found that human neural progenitors showed a differential index of DNA fragmentation at different stages of cellular differentiation. These data provide additional and important anatomical findings for the distribution and phenotype of DNA double-strand breaks in the mammalian brain, and suggest that DNA fragmentation is a spontaneous event routinely occurring in neural progenitors and adult neurons.

Inverse relationship of cannabimimetic (R+)WIN 55, 212 on behavior and seizure threshold during the juvenile period.

Cannabinoids have anti-convulsant effects in both in vivo and in vitro models of status epilepticus. Since the development of spontaneous seizures and neuronal vulnerability are age-dependent, we hypothesized that the anti-convulsant effects of cannabimimetics are also age-dependent. We administered a single injection of varied doses of (R+)WIN 55,212 (0.5, 1, 5 mg/kg) to postnatal (P) day 20 rats 90 min prior to induction of kainate (KA)-induced status epilepticus. The highest dose of (R+)WIN 55,212 (5 mg/kg) resulted in rapid onset of behavioral stupor, loss of balance, stiffening and immobility while standing on hind legs or laying flat in prone position; lower doses had minimal or no behavioral effect. After KA administration, seizure scores and electroencephalography (EEG) recordings were inversely related to (R+)WIN 55,212 dosage whereby higher doses were associated with high seizures scores and synchronous epileptiform activity and low doses with low seizure scores and diminished spiking in the EEG. Immunohistochemistry revealed a dose-dependent reduction in CB1 receptor expression with increasing concentrations of (R+)WIN 55,212 in presence or absence of KA seizures. Nissl and NeuN staining showed hippocampal injury was attenuated only when seizures were mild following low doses of WIN 55,212 (0.5, 1 mg/kg), consistent with the level of CB1 expression. Since low doses abolished seizures without psychotropic side-effects further study may facilitate a groundbreaking cannabamimetic therapeutic strategy to treat early-life seizures. Higher doses had adverse effects on behavior and failed to prevent seizures and protect CA1 neurons possibly due to inactivation or loss of CB1 receptors.

Distribution maps of D-dopachrome tautomerase in the mouse brain.

D-Dopachrome tautomerase is an enzyme related by amino acid sequence and catalytic activity to macrophage migration inhibitory factor. Both of these small molecules are pro-inflammatory cytokines mediating broad innate immune responses. Although it is well established that the gene product of D-dopachrome tautomerase is widely expressed in liver and kidney cells, no study has mapped the distribution pattern of this tautomeric enzyme in the mammalian nervous system. Here, we address this void by characterizing the cellular localization of D-dopachrome tautomerase in the adult mouse brain. Two well-characterized polyclonal antibodies were used for Western blotting and immunohistochemical localization of the endogenous tautomeric enzyme. Our results show that D-dopachrome tautomerase is present throughout the brain parenchyma with a large fraction of heterogeneous interneurons harboring a stable and robust expression of the enzyme. These data point to a potential involvement of D-dopachrome tautomerase activity in the mature mouse brain, and suggest some functional and evolutionary relationship between innate immunity and tautomerization of D-dopachrome in mammalian species.

Resveratrol protects neurons from cannulae implantation injury: implications for deep brain stimulation.

Brain-implantable electrodes such as those used in deep brain stimulation (DBS) have a promising future in end-stage Parkinson's disease therapy. However, there is considerable injury when electrodes penetrate brain tissue. For instance, broken blood vessels and glial scar formation may impede continual DBS or electrical recording from specific neurons. To begin addressing this key safety issue, we tested the therapeutic potential of resveratrol in reducing brain trauma caused by DBS-like surgery. Microinfusion of resveratrol (10 µM) directly applied to the sub-thalamic nucleus (STN) of the rat brain significantly minimized the formation of astrocytic gliosis in response to a 27-G precision-glide cannula implant. The therapeutic effects of resveratrol extended to the "kill zone", a boundary zone of about 100 µm comprising the cannula implant and surrounding neurons. We also found that resveratrol not only provided almost complete protection from mechanical injury to the brain, but that it also prevented undesirable motor deficits often seen in animals with lesions to the STN. Lastly, continuous infusion of resveratrol over a 4-week period led to the inhibition of pro-apoptotic, neurodegenerative and cell division cycle genes that may be associated with a reduction in astrocytic gliosis and glial scar formation within the STN. Taken together, these data suggest that application of resveratrol to the brain is an effective adjunct surgical procedure for minimizing acute neuronal injury when electrodes are implanted directly into the STN.

Silent information regulator 1 mediates hippocampal plasticity through presenilin1.

Silent information regulator 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase directly implicated in protecting a wide range of organisms against internal and external metabolic insults. However, the identification of SIRT1-specific DNA targets that confer such protection have remained elusive. Using human cells, we show that SIRT1 binds to, and transcriptionally regulates, a gene locus encoding presenilin1 (PSEN1), a protein intrinsically involved in the function of the γ-secretase protein complex. We also demonstrate that rats fed with resveratrol exhibit a significant increase in sirt1 and psen1 expression. Finally, dietary consumption of resveratrol also leads to an enhanced proliferative state of neuronal stem cells in the rat hippocampus. Our findings reveal a strong link between resveratrol-dependent SIRT1 signaling and hippocampal plasticity in the mammalian brain.

Two splice variants of the Wilms' tumor 1 gene have distinct functions during sex determination and nephron formation.

Alternative splicing of Wt1 results in the insertion or omission of the three amino acids KTS between zinc fingers 3 and 4. In vitro experiments suggest distinct molecular functions for + and -KTS isoforms. We have generated mouse strains in which specific isoforms have been removed. Heterozygous mice with a reduction of +KTS levels develop glomerulosclerosis and represent a model for Frasier syndrome. Homozygous mutants of both strains die after birth due to kidney defects. Strikingly, mice lacking +KTS isoforms show a complete XY sex reversal due to a dramatic reduction of Sry expression levels. Our data demonstrate distinct functions for the two splice variants and place the +KTS variants as important regulators for Sry in the sex determination pathway.

Normal blood pressure and plasma renin activity in mice lacking the renin-binding protein, a cellular renin inhibitor.

In renal extracts, some renin is present as "high molecular weight renin," a heterodimeric complex of renin with the 46-kDa renin-binding protein (RnBP), also known as N-acyl-D-glucosamine 2-epimerase. Because RnBP specifically inhibits renin activity, the protein was proposed to play an important role in the regulation of the renin-angiotensin system (RAS). Using gene targeting, we have generated mice lacking RnBP and tested this hypothesis in vivo. In particular, we analyzed biosynthesis, secretion, and activity of renin and other components of the RAS in mice lacking RnBP. Despite extensive investigations, we were unable to detect any major effects of RnBP deficiency on the plasma and renal RAS or on blood pressure regulation. Contrary to previous hypotheses, we conclude that RnBP does not play a significant role in the regulation of renin activity in plasma or kidney. However, RnBP knockout mice excrete an abnormal pattern of carbohydrates in the urine, indicating a role of the protein in renal carbohydrate metabolism.

Megalin knockout mice as an animal model of low molecular weight proteinuria.

Megalin is an endocytic receptor expressed on the luminal surface of the renal proximal tubules. The receptor is believed to play an important role in the tubular uptake of macromolecules filtered through the glomerulus. To elucidate the role of megalin in vivo and to identify its endogenous ligands, we analyzed the proximal tubular function in mice genetically deficient for the receptor. We demonstrate that megalin-deficient mice exhibit a tubular resorption deficiency and excrete low molecular weight plasma proteins in the urine (low molecular weight proteinuria). Proteins excreted include small plasma proteins that carry lipophilic compounds including vitamin D-binding protein, retinol-binding protein, alpha(1)-microglobulin and odorant-binding protein. Megalin binds these proteins and mediates their cellular uptake. Urinary loss of carrier proteins in megalin-deficient mice results in concomitant loss of lipophilic vitamins bound to the carriers. Similar to megalin knockout mice, patients with low molecular weight proteinuria as in Fanconi syndrome are also shown to excrete vitamin/carrier complexes. Thus, these results identify a crucial role of the proximal tubule in retrieval of filtered vitamin/carrier complexes and the central role played by megalin in this process.

Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D(3).

Steroid hormones are central regulators of a variety of biological processes. According to the free hormone hypothesis, steroids enter target cells by passive diffusion. However, recently we demonstrated that 25(OH) vitamin D(3) complexed to its plasma carrier, the vitamin D-binding protein, enters renal proximal tubules by receptor-mediated endocytosis. Knockout mice lacking the endocytic receptor megalin lose 25(OH) vitamin D(3) in the urine and develop bone disease. Here, we report that cubilin, a membrane-associated protein colocalizing with megalin, facilitates the endocytic process by sequestering steroid-carrier complexes on the cellular surface before megalin-mediated internalization of the cubilin-bound ligand. Dogs with an inherited disorder affecting cubilin biosynthesis exhibit abnormal vitamin D metabolism. Similarly, human patients with mutations causing cubilin dysfunction exhibit urinary excretion of 25(OH) vitamin D(3). This observation identifies spontaneous mutations in an endocytic receptor pathway affecting cellular uptake and metabolism of a steroid hormone.