Notch and Wnt Signaling Mediated Rod Photoreceptor Regeneration by Müller Cells in Adult Mammalian Retina

Background

Evidence emerging from a variety of approaches used in different species suggests that Müller cell function may extend beyond its role of maintaining retinal homeostasis to that of progenitors in the adult retina. Enriched Müller cells in vitro or those that re-enter cell cycle in response to neurotoxin-damage to retina in vivo display multipotential and self-renewing capacities, the cardinal features of stem cells.

Methodology/Principal Findings

We demonstrate that Notch and Wnt signaling activate Müller cells through their canonical pathways and that a rare subset of activated Müller cells differentiates along rod photoreceptor lineage in the outer nuclear layer. The differentiation of activated Müller cells along photoreceptor lineage is confirmed by multiple approaches that included Hoechst dye efflux analysis, genetic analysis using retina from Nrl-GFP mice, and lineage tracing using GS-GFP lentivirus in wild type and rd mice in vitro and S334ter rats in vivo. Examination of S334ter rats for head-neck tracking of visual stimuli, a behavioral measure of light perception, demonstrates a significant improvement in light perception in animals treated to activate Müller cells. The number of activated Müller cells with rod photoreceptor phenotype in treated animals correlates with the improvement in their light perception.

Conclusion/Significance

In summary, our results provide a proof of principle for non-neurotoxin-mediated activation of Müller cells through Notch and Wnt signaling toward the regeneration of rod photoreceptors.     

Differential sorting of the vesicular glutamate transporter 1 into a defined vesicular pool is regulated by light signaling involving the clock gene Period2

Synaptic strength depends on the amount of neurotransmitter stored in synaptic vesicles. The vesicular transmitter content has recently been shown to be directly dependent on the expression levels of vesicular neurotransmitter transporters indicating that the transport capacity of synaptic vesicles is a critical determinant for synaptic efficacy. Using synaptic vesicles prepared from whole brain at different times of the day we now show that the amount of vesicular glutamate transporter (VGLUT) 1 undergoes strong diurnal cycling. VGLUT1 protein levels are high before the start of the light period, decline at noon, increase again before start of the dark period, and decline again at midnight. Mice kept in complete darkness showed within a 24-h period only a single peak of VGLUT1 expression in the middle of the rest phase. In contrast, mice lacking the period gene Period 2, a core component of the circadian clock, did not show any light-cycle-dependent changes of VGLUT1 levels. No other of several synaptic vesicle proteins examined underwent circadian cycling. Circadian cycling of VGLUT1 was not seen when analyzing homogenate or synaptosomes, the starting fraction for vesicle preparation. Circadian cycling of VGLUT1 was also not reflected at the mRNA level. We conclude that nerve terminals are endowed with mechanisms that regulate quantal size by changing the copy number of transporters in synaptic vesicles. A reduced amount of VGLUT1 per vesicle is probably achieved by means of selective sorting controlled by clock genes.     

Role of cardiac isoform of alpha-2 macroglobulin in diabetic myocardium

Earlier studies from one of the investigator’s laboratory have demonstrated the presence of a high molecular weight protein (182 kDa) in the blood serum of laboratory animals subjected to pressure-induced cardiac hypertrophy and suggested that this protein may be involved in the development of cardiac hypertrophy. Studies have shown that this protein is also involved in earlier stages of cardiac complications associated with diabetes, but the role of this protein in diabetic heart is less understood. So we aimed to check whether this protein is having any protective role in diabetic heart. The protein was purified from serum of rats induced with cardiac hypertrophy and the purified protein was injected through tail vein of diabetic rats for further studies. The results of various antioxidant enzymes and the TBARS levels have indicated the antioxidant activity of this protein. Real-time PCR analysis of gene expression revealed the upregulation of certain muscle-specific genes like β-MHC, MLC-2, and skeletal α actin in diabetic group and also in presence of 182-kDa protein. The results further showed a down regulation of genes such as cardiac α-actin and α- MHC implicating the role of this protein in the development of cardiac hypertrophy in diabetes. Increased cardiac hypertrophy as revealed by the expression of various genes and improved antioxidant potential in presence of 182 kDa protein in diabetes at the earlier stages is beneficial for counteracting the myocardial damage associated with diabetes.     

Discovery of 4-aminomethylphenylacetic acids as γ-secretase modulators via a scaffold design approach

Starting from literature examples of nonsteroidal anti-inflammatory drugs (NSAIDs)-type carboxylic acid γ-secretase modulators (GSMs) and using a scaffold design approach, we identified 4-aminomethylphenylacetic acid 4 with a desirable γ-secretase modulation profile. Scaffold optimization led to the discovery of a novel chemical series, represented by 6b, having improved brain penetration. Further SAR studies provided analog 6q that exhibited a good pharmacological profile. Oral administration of 6q significantly reduced brain Aβ42 levels in mice and rats.     

An Autolytic Process for Recovery of Antioxidant Activity Rich Carotenoprotein from Shrimp Heads

Studies were carried out to utilize in situ proteases of shrimp heads to recover carotenoproteins possessing antioxidant activity. Highest protease activity of the buffer extract was found at pH 8.0 (9.85 ± 0.61 units). The protease activity increased with temperature up to 50°C and reduced thereafter with highest activity being 19.32 ± 2.0 units. Thus, the autolysis of shrimp heads for recovery of carotenoprotein was carried out at pH 8.0 and at 50°C. Waste to buffer ratio had a significant (p < 0.05) effect on recovery of carotenoids in carotenoprotein filtrate with a maximum of 58.5 ± 6.4% recovery with a waste to buffer ratio of 1:2.5 (w:v). The carotenoid recovery increased significantly to 63.4% ± 3.6% at the end of a 4-h autolysis. The studies on combined effect of waste to buffer ratio and autolysis time indicated increase in protein recovery with increase in waste to buffer ratio but not with autolysis time. DPPH scavenging activity of the carotenoprotein isolate increased with autolysis time up to 100 min, and thereafter, reduced above 160 min of autolysis time. With increase in waste to buffer ratio, the scavenging activity increased, reaching more than 12.5 mg TBHQ equivalent/mg protein at waste to buffer ratio of 1:5. The optimum autolysis condition for obtaining antioxidant activity rich carotenoprotein from shrimp heads was found to be waste to buffer (pH 8.0) ratio of 1:5 and an autolysis time of 2 h at 50°C. The isolated carotenoprotein was found to have antioxidant activity with respect to singlet oxygen quenching, reducing power and metal chelating activity.     

Somatic embryogenesis and plant regeneration through cell suspension in diploid (AB) banana cultivar Elakki Bale (syn Neypoovan)

An efficient protocol was developed using cell suspensions for somatic embryogenesis and plantlet regeneration in a most popular diploid AB banana (M.accuminata X M.bulbisiana hybrid) cv. Elakki Bale (syn Neypoovan) known for its taste and keeping quality in southern India. Floral primodia from position 8–16 of male inflorescence which were more responsive for embryogenesis were used as explants for the embryogenic callus production in MS media supplemented with different concentration of 2,4-D. A concentration of 18.1 μM 2, 4-D produced maximum embryogenic calli in 1 % of the explants inoculated. Embryogenic calli on repeated sub culturing on MA2 media produced good embryogenic cell suspensions (ECS). Microscopic examination of ECS showed globular, smaller with dense cytoplasm filled with starchy granules characteristic of embryogenic cells. Highest number of somatic embryos (189) was produced on modified MA3 media. A germination percentage of 31 % were observed in BAP 22.19 μM concentration. Regenerated plants with normal shoot and root were hardened in soilrite. Direct somatic embryogenesis and plant regeneration was also noticed in embryogenic calli which did not pass through the ECS stage. The protocol optimized for somatic embryogenesis through cell suspension and also direct embryogenesis leading to plantlet regeneration can be used for the micropropagation and genetic manipulation.     

Impact of nitrogen limitation on biomass,photosynthesis, and lipid accumulation in <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis>

Induction of oil accumulation in algae for biofuel production is often achieved by withholding nitrogen. However, withholding nitrogen often reduces total biomass yield. In this report, it is demonstrated that Chlorella sorokiniana will not only accumulate substantial quantities of neutral lipids when grown in the absence of nitrogen but will also exhibit unimpeded growth rates for up to 2 weeks. To determine the physiological basis for the observed increase in oil and biomass accumulation, we compared photosynthetic and respiration rates and chlorophyll, lipid, and total energy content under ammonia replete and deplete conditions. Under N-depleted growth conditions, there was a 64 % increase in total energy density and a ～20-fold increase in oil accumulation relative to N-replete growth leading to a 1.6-fold greater total energy yield in N-depleted than in N-replete cultures. We propose that the higher energy accumulation in N-depleted cultures is due to enhanced photosynthetic energy capture and conversion associated with reduced chlorophyll levels and reduced self-shading as well as a shift in metabolism leading to the accumulation of oils.     

Plasmodium falciparum adhesion domains linked to severe malaria differ in blockade of endothelial protein C receptor

Cytoadhesion of Plasmodium falciparum‐infected erythrocytes to endothelial protein C receptor (EPCR) is associated with severe malaria. It has been postulated that parasite binding could exacerbate microvascular coagulation and endothelial dysfunction in cerebral malaria by impairing the protein C–EPCR interaction, but the extent of binding inhibition has not been fully determined. Here we expressed the cysteine‐rich interdomain region (CIDRα1) domain from a variety of domain cassette (DC) 8 and DC13 P. falciparum erythrocyte membrane protein 1 proteins and show they interact in a distinct manner with EPCR resulting in weak, moderate and strong inhibition of the activated protein C (APC)–EPCR interaction. Overall, there was a positive correlation between CIDRα1–EPCR binding activity and APC blockade activity. In addition, our analysis from a combination of mutagenesis and blocking antibodies finds that an Arg81 (R81) in EPCR plays a pivotal role in CIDRα1 binding, but domains with weak and strong APC blockade activity were distinguished by their sensitivity to inhibition by anti‐EPCR mAb 1535, implying subtle differences in their binding footprints. These data reveal a previously unknown functional heterogeneity in the interaction between P. falciparum and EPCR and have major implications for understanding the distinct clinical pathologies of cerebral malaria and developing new treatment strategies.