Determining Rates of Molecular Secretion from Supernatant Concentration Measurements in a 3D-Bioprinted Human Liver Tissue Model.

The secretion rate of albumin is a key indicator of function in liver tissue models used for hepatotoxicity and pharmacokinetic testing. However, it is not generally clear how to determine molecular secretion rates from measurements of the molecular concentration in supernatant media. Here, we develop computational and analytical models of molecular transport in an experimental system that enable determination of albumin secretion rates based on measurements of albumin concentration in supernatant media. The experimental system is a 3D-bioprinted human liver tissue construct embedded in a 3D culture environment made from packed microgel particles swollen in liquid growth media. The mathematical models reveal that the range of albumin synthesis rates necessary to match experimentally measured albumin concentrations corresponds to reaction-limited conditions, where a steady state of albumin spatial distribution is rapidly reached between media exchanges. Our results show that temporally resolved synthesis rates can be inferred from serial concentration measurements of collected supernatant media. This link is critical to confidently assessing in vitro tissue performance in applications where critical quality attributes must be quantified, like in drug development and screening.

Tsc1 Regulates the Proliferation Capacity of Bone-Marrow Derived Mesenchymal Stem Cells.

TSC1 is a tumor suppressor that inhibits cell growth via negative regulation of the mammalian target of rapamycin complex (mTORC1). TSC1 mutations are associated with Tuberous Sclerosis Complex (TSC), characterized by multiple benign tumors of mesenchymal and epithelial origin. TSC1 modulates self-renewal and differentiation in hematopoietic stem cells; however, its effects on mesenchymal stem cells (MSCs) are unknown. We investigated the impact of Tsc1 inactivation in murine bone marrow (BM)-MSCs, using tissue-specific, transgelin (Tagln)-mediated cre-recombination, targeting both BM-MSCs and smooth muscle cells. Tsc1 mutants were viable, but homozygous inactivation led to a dwarfed appearance with TSC-like pathologies in multiple organs and reduced survival. In young (28 day old) mice, Tsc1 deficiency-induced significant cell expansion of non-hematopoietic BM in vivo, and MSC colony-forming potential in vitro, that was normalized upon treatment with the mTOR inhibitor, everolimus. The hyperproliferative BM-MSC phenotype was lost in aged (1.5 yr) mice, and Tsc1 inactivation was also accompanied by elevated ROS and increased senescence. ShRNA-mediated knockdown of Tsc1 in BM-MSCs replicated the hyperproliferative BM-MSC phenotype and led to impaired adipogenic and myogenic differentiation. Our data show that Tsc1 is a negative regulator of BM-MSC proliferation and support a pivotal role for the Tsc1-mTOR axis in the maintenance of the mesenchymal progenitor pool.

A Proteolytic Regulator Controlling Chalcone Synthase Stability and Flavonoid Biosynthesis in Arabidopsis.

Flavonoids represent a large family of specialized metabolites involved in plant growth, development, and adaptation. Chalcone synthase (CHS) catalyzes the first step of flavonoid biosynthesis by directing carbon flux from general phenylpropanoid metabolism to flavonoid pathway. Despite extensive characterization of its function and transcriptional regulation, the molecular basis governing its posttranslational modification is enigmatic. Here, we report the discovery of a proteolytic regulator of CHS, namely, KFBCHS, a Kelch domain-containing F-box protein in Arabidopsis thaliana KFBCHS physically interacts with CHS and specifically mediates its ubiquitination and degradation. KFBCHS exhibits developmental expression patterns in Arabidopsis leaves, stems, and siliques and strongly responds to the dark-to-light (or the light-to-dark) switch, the blue, red, and far-red light signals, and UV-B irradiation. Alteration of KFBCHS expression negatively correlates to the cellular concentration of CHS and the production of flavonoids. Our study suggests that KFBCHS serves as a crucial negative regulator, via mediating CHS degradation, coordinately controlling flavonoid biosynthesis in response to the developmental cues and environmental stimuli.

Light Quality Dependent Changes in Morphology, Antioxidant Capacity, and Volatile Production in Sweet Basil (Ocimum basilicum).

Narrow-bandwidth light treatments may be used to manipulate plant growth, development and metabolism. In this report LED-based light treatments were used to affect yield and metabolic content of sweet basil (Ocimum basilicum L. cv "Ceasar") grown in controlled environments. This culinary herb produces an aroma highly appreciated by consumers, primarily composed of terpenes/terpenoids, phenylpropanoids, and fatty-acid- derived volatile molecules. Basil plants were grown under narrow-bandwidth light conditions, and leaf area, height, mass, antioxidant capacity and volatile emissions were measured at various time points. The results indicate reproducible significant differences in specific volatiles, and in biochemical classes of volatiles, compared to greenhouse grown plants. For example, basil plants grown under blue/red/yellow or blue/red/green wavelengths emit higher levels of a subset of monoterpenoid volatiles, while a blue/red/far-red treatment leads to higher levels of most sesquiterpenoid volatile molecules. Specific light treatments increase volatile content, mass, and antioxidant capacity. The results show that narrow-bandwidth illumination can induce discrete suites of volatile classes that affect sensory quality in commercial herbs, and may be a useful tool in improving commercial production.

AAV-Mediated Clarin-1 Expression in the Mouse Retina: Implications for USH3A Gene Therapy.

Usher syndrome type III (USH3A) is an autosomal recessive disorder caused by mutations in clarin-1 (CLRN1) gene, leading to progressive retinal degeneration and sensorineural deafness. Efforts to develop therapies for preventing photoreceptor cell loss are hampered by the lack of a retinal phenotype in the existing USH3 mouse models and by conflicting reports regarding the endogenous retinal localization of clarin-1, a transmembrane protein of unknown function. In this study, we used an AAV-based approach to express CLRN1 in the mouse retina in order to determine the pattern of its subcellular localization in different cell types. We found that all major classes of retinal cells express AAV-delivered CLRN1 driven by the ubiquitous, constitutive small chicken ß-actin promoter, which has important implications for the design of future USH3 gene therapy studies. Within photoreceptor cells, AAV-expressed CLRN1 is mainly localized at the inner segment region and outer plexiform layer, similar to the endogenous expression of other usher proteins. Subretinal delivery using a full strength viral titer led to significant loss of retinal function as evidenced by ERG analysis, suggesting that there is a critical limit for CLRN1 expression in photoreceptor cells. Taken together, these results suggest that CLRN1 expression is potentially supported by a variety of retinal cells, and the right combination of AAV vector dose, promoter, and delivery method needs to be selected to develop safe therapies for USH3 disorder.

The Potential Use of PGC-1α and PGC-1ß to Protect the Retina by Stimulating Mitochondrial Repair.

Damage to mitochondria is a common mechanism of cell death in inherited neurodegenerative disorders. Therefore, mitochondrial protection and mitochondrial repair are promising strategies to induce retinal neuroprotection. Peroxisome proliferator-activated receptor γ coactivator-α (PGC-1α) and ß (PGC-1ß) are transcriptional coactivators that are the main regulators of mitochondrial biogenesis. We propose that PGC-1α and PGC-1ß could play a role in regulating retina cell survival, and may be important therapeutic targets to prevent retinal degeneration.

Müller glial cells induce stem cell properties in retinal progenitors in vitro and promote their further differentiation into photoreceptors.

Using stem cells to replace lost neurons is a promising strategy for treating retinal neurodegenerative diseases. Among their multiple functions, Müller glial cells are retina stem cells, with a robust regenerative potential in lower vertebrates, which is much more restricted in mammals. In rodents, most retina progenitors exit the cell cycle immediately after birth, differentiate as neurons, and then cannot reenter the cell cycle. Here we demonstrate that, in mixed cultures with Müller glial cells, rat retina progenitor cells expressed stem cell properties, maintained their proliferative potential, and were able to preserve these properties and remain mitotically active after several consecutive passages. Notably, these progenitors retained the capacity to differentiate as photoreceptors, even after successive reseedings. Müller glial cells markedly stimulated differentiation of retina progenitors; these cells initially expressed Crx and then developed as mature photoreceptors that expressed characteristic markers, such as opsin and peripherin. Moreover, they were light responsive, insofar as they decreased their cGMP levels when exposed to light, and they also showed high-affinity glutamate uptake, a characteristic of mature photoreceptors. Our present findings indicate that, in addition to giving rise to new photoreceptors, Müller glial cells might instruct a pool of undifferentiated cells to develop and preserve stem cell characteristics, even after successive reseedings, and then stimulate their differentiation as functional photoreceptors. This complementary mechanism might contribute to enlarge the limited regenerative capacity of mammalian Müller cells.

Ceramide-1-phosphate, a new mediator of development and survival in retina photoreceptors.

PURPOSE. Simple sphingolipids control crucial cellular processes in several cell types. Previous work demonstrated that sphingolipids, such as ceramide, sphingosine, and sphingosine-1-phosphate, are key mediators in the regulation of survival, differentiation, and proliferation of retina photoreceptors. Ceramide-1-phosphate (C1P) regulates growth and survival in several cell types; however, little is known concerning its functions in the retina. Whether C1P also participates in controlling photoreceptor development was also explored. METHODS. Rat retina neuronal cultures were supplemented with 1 to 10 µM C1P. Proliferation was determined by evaluating 5-bromo-2-deoxyuridine (BrdU) uptake and the number of mitotic figures and differentiation by evaluating opsin and peripherin expression by immunocytochemistry and Western blot. Apoptosis was inhibited with the pan caspase inhibitor ZVADFMK and evaluated by TUNEL assay, propidium iodide/annexin V, and DAPI labeling. Preservation of mitochondrial membrane potential was evaluated. RESULTS. C1P enhanced BrdU uptake and increased mitosis in retinal progenitors. C1P addition advanced photoreceptor differentiation, enhancing opsin and peripherin expression and stimulating development of the apical processes in which these proteins were concentrated. In the absence of these trophic factors, photoreceptors degenerated after 4 days in vitro, and at day 6, almost 50% of photoreceptors were apoptotic. C1P decreased photoreceptor apoptosis, reducing this percentage by half. Inhibiting caspase activity reduced photoreceptor apoptosis in the controls, but did not increase opsin expression, implying that C1P has separate effects on differentiation and survival. CONCLUSIONS. These results suggest for the first time that C1P is a novel mediator that has multiple functions in photoreceptors, initially regulating their proliferation and then promoting their survival and differentiation.

Regulating survival and development in the retina: key roles for simple sphingolipids.

Many sphingolipids have key functions in the regulation of crucial cellular processes. Ceramide (Cer) and sphingosine (Sph) induce growth arrest and cell death in multiple situations of cellular stress. On the contrary, sphingosine-1-phosphate (S1P), the product of Sph phosphorylation, promotes proliferation, differentiation, and survival in different cell systems. This review summarizes the roles of these simple sphingolipids in different tissues and then analyzes their possible functions in the retina. Alterations in proliferation, neovascularization, differentiation, and cell death are critical in major retina diseases and collective evidence points to a role for sphingolipids in these processes. Cer induces inflammation and apoptosis in endothelial and retinal pigmented epithelium cells, leading to several retinopathies. S1P can prevent this death but also promotes cell proliferation that might lead to neovascularization and fibrosis. Recent data support Cer and Sph as crucial mediators in the induction of photoreceptor apoptosis in diverse models of oxidative damage and neurodegeneration, and suggest that regulating their metabolism can prevent this death. New evidence proposes a central role for S1P controlling photoreceptor survival and differentiation. Finally, this review discusses the ability of trophic factors to regulate sphingolipid metabolism and transactivate S1P signaling pathways to control survival and development in retina photoreceptors.

Synthesis of sphingosine is essential for oxidative stress-induced apoptosis of photoreceptors.

PURPOSE: Oxidative stress is involved in inducing apoptosis of photoreceptors in many retinal neurodegenerative diseases. It has been shown that oxidative stress increases in photoreceptors the synthesis of ceramide, a sphingolipid precursor that then activates apoptosis. In several cell types, ceramide is converted by ceramidases to sphingosine (Sph), another apoptosis mediator; hence, this study was undertaken to determine whether Sph participates in triggering photoreceptor apoptosis. METHODS: Rat retina neurons were incubated with [(3)H]palmitic acid and treated with the oxidant paraquat (PQ) to evaluate Sph synthesis. Sph was added to cultures with or without docosahexaenoic acid (DHA), the major retina polyunsaturated fatty acid and a photoreceptor survival factor, to evaluate apoptosis. Synthesis of Sph and sphingosine-1-phosphate (S1P), a prosurvival signal, were inhibited with alkaline ceramidase or sphingosine kinase inhibitors, respectively, before adding PQ, C(2)-ceramide, or Sph. Apoptosis, mitochondrial membrane polarization, cytochrome c localization, and reactive oxygen species (ROS) production were determined. RESULTS: PQ increased [(3)H]Sph synthesis in photoreceptors and blocking this synthesis by inhibiting alkaline ceramidase decreased PQ-induced apoptosis. Addition of Sph induced photoreceptor apoptosis, increased ROS production, and promoted cytochrome c release from mitochondria. Although DHA prevented this apoptosis, inhibiting Sph conversion to S1P blocked DHA protection. CONCLUSIONS: These results suggest that oxidative stress enhances formation of ceramide and its subsequent breakdown to Sph; ceramide and/or Sph would then trigger photoreceptor apoptosis. Preventing Sph synthesis or promoting its phosphorylation to S1P rescued photoreceptors, suggesting that Sph is a mediator of their apoptosis and modulation of Sph metabolism may be crucial for promoting photoreceptor survival.

Sphingosine-1-phosphate is a key regulator of proliferation and differentiation in retina photoreceptors.

PURPOSE: Identifying the cues required for the survival and development of photoreceptors is essential for treating retinal neurodegeneration. The authors previously established that glial-derived neurotrophic factor (GDNF) stimulates proliferation and that docosahexaenoic acid (DHA) promotes photoreceptor survival and differentiation. Later findings that ceramide triggers photoreceptor apoptosis suggested sphingolipids might also control photoreceptor development. The present study investigated whether sphingosine-1-phophate (S1P), which promotes survival and differentiation in several cell types, regulates photoreceptor proliferation and differentiation and whether it is a mediator in GDNF and DHA effects. METHODS: Rat retina neuronal cultures were supplemented at day 0 or 1 with S1P, GDNF, or DHA and were treated with DL-threo-dihydrosphingosine to inhibit S1P synthesis or with brefeldin A (BFA) to block intracellular trafficking. Proliferation was quantified to determine bromodeoxyuridine uptake and number of mitotic figures. Opsin, peripherin, and sphingosine kinase (SphK), the enzyme required for S1P synthesis, were quantified by immunocytochemistry and Western blot analysis. RESULTS: S1P increased the proliferation of photoreceptor progenitors. It also stimulated the formation of apical processes, enhanced opsin and peripherin expression, and promoted their localization in these processes; DHA had similar effects. BFA prevented S1P and DHA enhancement of apical process formation without affecting opsin expression. GDNF and DHA enhanced SphK expression in photoreceptors, while inhibiting S1P synthesis blocked GDNF mitogenic effects and DHA effects on differentiation. CONCLUSIONS: The authors propose S1P as a key regulator in photoreceptor development. GDNF and DHA might upregulate SphK levels to promote S1P synthesis, which would initially promote proliferation and then advance photoreceptor differentiation.

Oxidative stress promotes proliferation and dedifferentiation of retina glial cells in vitro.

Oxidative damage is involved in triggering neuronal death in several retinal neurodegenerative diseases. The recent finding of stem cells in the retina suggests that both preventing neuronal death and replacing lost neurons might be useful strategies for treating these diseases. We have previously shown that oxidative stress induces apoptosis in cultured retinal neurons. We now investigated the response of Müller cells, proposed as retina stem cells, to this damage. Treatment of glial cell cultures prepared from rat retinas with the oxidant paraquat (PQ) did not induce glial cell apoptosis. Instead, PQ promoted their rapid dedifferentiation and proliferation. PQ decreased expression of a marker of differentiated glial cells, simultaneously increasing the expression of smooth muscle actin, shown to increase with glial dedifferentiation, the levels of cell-cycle markers, and the number of glial cells in the cultures. In addition, glial cells protected neurons in coculture from apoptosis induced by PQ and H(2)O(2). In pure neuronal cultures, PQ induced apoptosis of photoreceptors and amacrine neurons, simultaneously decreasing the percentage of neurons preserving mitochondrial membrane potential; coculturing neurons with glial cells completely prevented PQ-induced apoptosis and preserved mitochondrial potential in both neuronal types. These results demonstrate that oxidative damage activated different responses in Müller glial cells; they rapidly dedifferentiated and enhanced their proliferation, concurrently preventing neuronal apoptosis. Glial cells might not only preserve neuronal survival but also activate their cell cycle in order to provide a pool of new progenitor cells that might eventually be manipulated to preserve retinal functionality.

Ceramide is a mediator of apoptosis in retina photoreceptors.

PURPOSE: The precise mechanisms involved in photoreceptor apoptosis are still unclear. In the present study, the role of ceramide, a sphingolipid precursor that induces apoptosis on cellular stress, was investigated in relation to the activation of cell death in photoreceptors. METHODS: Rat retina neuronal cultures, with or without docosahexaenoic acid (DHA), were treated with the ceramide analogue acetylsphingosine (C2-ceramide), and with a glucosylceramide synthase inhibitor. Ceramide synthesis in cultures treated with the oxidant paraquat was evaluated with [3H]palmitate. The effect of inhibitors of ceramide de novo synthesis, fumonisin B1 and cycloserine, on photoreceptor apoptosis was investigated. Apoptosis, mitochondrial membrane potential, and Bcl-2 expression were determined. RESULTS: Addition of C2-ceramide induced photoreceptor apoptosis. Paraquat increased formation of [3H]ceramide in photoreceptors, compared with the control, whereas inhibition of ceramide synthesis, immediately before paraquat treatment, prevented paraquat-induced photoreceptor apoptosis. Fumonisin also reduced photoreceptor apoptosis during early development in vitro. DHA, the retina major polyunsaturated fatty acid, which protects photoreceptors from oxidative stress-induced apoptosis, completely blocked C2-ceramide-induced photoreceptor death, simultaneously increasing Bcl-2 expression. Inhibiting glucosylceramide synthase, which catalyzes ceramide glucosylation, before ceramide or paraquat treatment blocked DHA's protective effect. CONCLUSIONS: The results suggest that oxidative stress stimulated an increase in ceramide levels that induced photoreceptor apoptosis. DHA prevented oxidative stress and ceramide damage by upregulating Bcl-2 expression and glucosylating ceramide, thus decreasing its intracellular concentration. This shows for the first time that ceramide is a critical mediator for triggering photoreceptor apoptosis in mammalian retina and suggests that modulating ceramide levels may provide a therapeutic tool for preventing photoreceptor death in neurodegenerative diseases.