Identification of Penicillin Binding Protein 4 (PBP4) as a critical factor for Staphylococcus aureus bone invasion during osteomyelitis in mice.

Staphylococcus aureus infection of bone is challenging to treat because it colonizes the osteocyte lacuno-canalicular network (OLCN) of cortical bone. To elucidate factors involved in OLCN invasion and identify novel drug targets, we completed a hypothesis-driven screen of 24 S. aureus transposon insertion mutant strains for their ability to propagate through 0.5 µm-sized pores in the Microfluidic Silicon Membrane Canalicular Arrays (µSiM-CA), developed to model S. aureus invasion of the OLCN. This screen identified the uncanonical S. aureus transpeptidase, penicillin binding protein 4 (PBP4), as a necessary gene for S. aureus deformation and propagation through nanopores. In vivo studies revealed that Δpbp4 infected tibiae treated with vancomycin showed a significant 12-fold reduction in bacterial load compared to WT infected tibiae treated with vancomycin (p<0.05). Additionally, Δpbp4 infected tibiae displayed a remarkable decrease in pathogenic bone-loss at the implant site with and without vancomycin therapy. Most importantly, Δpbp4 S. aureus failed to invade and colonize the OLCN despite high bacterial loads on the implant and in adjacent tissues. Together, these results demonstrate that PBP4 is required for S. aureus colonization of the OLCN and suggest that inhibitors may be synergistic with standard of care antibiotics ineffective against bacteria within the OLCN.

Drusen in patient-derived hiPSC-RPE models of macular dystrophies.

Age-related macular degeneration (AMD) and related macular dystrophies (MDs) are a major cause of vision loss. However, the mechanisms underlying their progression remain ill-defined. This is partly due to the lack of disease models recapitulating the human pathology. Furthermore, in vivo studies have yielded limited understanding of the role of specific cell types in the eye vs. systemic influences (e.g., serum) on the disease pathology. Here, we use human induced pluripotent stem cell-retinal pigment epithelium (hiPSC-RPE) derived from patients with three dominant MDs, Sorsby's fundus dystrophy (SFD), Doyne honeycomb retinal dystrophy/malattia Leventinese (DHRD), and autosomal dominant radial drusen (ADRD), and demonstrate that dysfunction of RPE cells alone is sufficient for the initiation of sub-RPE lipoproteinaceous deposit (drusen) formation and extracellular matrix (ECM) alteration in these diseases. Consistent with clinical studies, sub-RPE basal deposits were present beneath both control (unaffected) and patient hiPSC-RPE cells. Importantly basal deposits in patient hiPSC-RPE cultures were more abundant and displayed a lipid- and protein-rich "drusen-like" composition. Furthermore, increased accumulation of COL4 was observed in ECM isolated from control vs. patient hiPSC-RPE cultures. Interestingly, RPE-specific up-regulation in the expression of several complement genes was also seen in patient hiPSC-RPE cultures of all three MDs (SFD, DHRD, and ADRD). Finally, although serum exposure was not necessary for drusen formation, COL4 accumulation in ECM, and complement pathway gene alteration, it impacted the composition of drusen-like deposits in patient hiPSC-RPE cultures. Together, the drusen model(s) of MDs described here provide fundamental insights into the unique biology of maculopathies affecting the RPE-ECM interface.

Pluripotent Stem Cells to Model Degenerative Retinal Diseases: The RPE Perspective.

Pluripotent stem cell technology, including human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), has provided a suitable platform to investigate molecular and pathological alterations in an individual cell type using patient's own cells. Importantly, hiPSCs/hESCs are amenable to genome editing providing unique access to isogenic controls. Specifically, the ability to introduce disease-causing mutations in control (unaffected) and conversely correct disease-causing mutations in patient-derived hiPSCs has provided a powerful approach to clearly link the disease phenotype with a specific gene mutation. In fact, utilizing hiPSC/hESC and CRISPR technology has provided significant insight into the pathomechanism of several diseases. With regard to the eye, the use of hiPSCs/hESCs to study human retinal diseases is especially relevant to retinal pigment epithelium (RPE)-based disorders. This is because several studies have now consistently shown that hiPSC-RPE in culture displays key physical, gene expression and functional attributes of human RPE in vivo. In this book chapter, we will discuss the current utility, limitations, and plausible future approaches of pluripotent stem cell technology for the study of retinal degenerative diseases. Of note, although we will broadly summarize the significant advances made in modeling and studying several retinal diseases utilizing hiPSCs/hESCs, our specific focus will be on the utility of patient-derived hiPSCs for (1) establishment of human cell models and (2) molecular and pharmacological studies on patient-derived cell models of retinal degenerative diseases where RPE cellular defects play a major pathogenic role in disease development and progression.

Decreasing mitochondrial fission alleviates hepatic steatosis in a murine model of nonalcoholic fatty liver disease.

Mitochondria produce the majority of cellular ATP through oxidative phosphorylation, and their capacity to do so is influenced by many factors. Mitochondrial morphology is recently suggested as an important contributor in controlling mitochondrial bioenergetics. Mitochondria divide and fuse continuously, which is affected by environmental factors, including metabolic alterations. Underscoring its bioenergetic influence, altered mitochondrial morphology is reported in tissues of patients and in animal models of metabolic dysfunction. In this study, we found that mitochondrial fission plays a vital role in the progression of nonalcoholic fatty liver disease (NAFLD). The development of hepatic steatosis, oxidative/nitrative stress, and hepatic tissue damage, induced by a high-fat diet, were alleviated in genetically manipulated mice suppressing mitochondrial fission. The alleviation of steatosis was recapitulated in primary hepatocytes with the inhibition of mitochondrial fission. Mechanistically, our study indicates that fission inhibition enhances proton leak under conditions of free fatty acid incubation, implicating bioenergetic change through manipulating mitochondrial fission. Taken together, our results suggest a mechanistic role for mitochondrial fission in the etiology of NAFLD. The efficacy of decreasing mitochondrial fission in the suppression of NAFLD suggests that mitochondrial fission represents a novel target for therapeutic treatment of NAFLD.

Arachnoid granulations are lymphatic conduits that communicate with bone marrow and dura-arachnoid stroma.

Arachnoid granulations (AG) are poorly investigated. Historical reports suggest that they regulate brain volume by passively transporting cerebrospinal fluid (CSF) into dural venous sinuses. Here, we studied the microstructure of cerebral AG in humans with the aim of understanding their roles in physiology. We discovered marked variations in AG size, lobation, location, content, and degree of surface encapsulation. High-resolution microscopy shows that AG consist of outer capsule and inner stromal core regions. The fine and porous framework suggests uncharacterized functions of AG in mechanical CSF filtration. Moreover, internal cytokine and immune cell enrichment imply unexplored neuroimmune properties of these structures that localize to the brain-meningeal lymphatic interface. Dramatic age-associated changes in AG structure are additionally identified. This study depicts for the first time microscopic networks of internal channels that communicate with perisinus spaces, suggesting that AG subserve important functions as transarachnoidal flow passageways. These data raise new theories regarding glymphatic-lymphatic coupling and mechanisms of CSF antigen clearance, homeostasis, and diseases.

The mitochondrial fusion protein OPA1 is dispensable in the liver and its absence induces mitohormesis to protect liver from drug-induced injury.

Mitochondria are critical for metabolic homeostasis of the liver, and their dysfunction is a major cause of liver diseases. Optic atrophy 1 (OPA1) is a mitochondrial fusion protein with a role in cristae shaping. Disruption of OPA1 causes mitochondrial dysfunction. However, the role of OPA1 in liver function is poorly understood. In this study, we delete OPA1 in the fully developed liver of male mice. Unexpectedly, OPA1 liver knockout (LKO) mice are healthy with unaffected mitochondrial respiration, despite disrupted cristae morphology. OPA1 LKO induces a stress response that establishes a new homeostatic state for sustained liver function. Our data show that OPA1 is required for proper complex V assembly and that OPA1 LKO protects the liver from drug toxicity. Mechanistically, OPA1 LKO decreases toxic drug metabolism and confers resistance to the mitochondrial permeability transition. This study demonstrates that OPA1 is dispensable in the liver, and that the mitohormesis induced by OPA1 LKO prevents liver injury and contributes to liver resiliency.

Efficacy of Bisphosphonate-Conjugated Sitafloxacin in a Murine Model of S. aureus Osteomyelitis: Evidence of "Target & Release" Kinetics and Killing of Bacteria Within Canaliculi.

S. aureus infection of bone is difficult to eradicate due to its ability to colonize the osteocyte-lacuno-canalicular network (OLCN), rendering it resistant to standard-of-care (SOC) antibiotics. To overcome this, we proposed two bone-targeted bisphosphonate-conjugated antibiotics (BCA): bisphosphonate-conjugated sitafloxacin (BCS) and hydroxybisphosphonate-conjugate sitafloxacin (HBCS). Initial studies demonstrated that the BCA kills S. aureus in vitro. Here we demonstrate the in vivo efficacy of BCS and HBCS versus bisphosphonate, sitafloxacin, and vancomycin in mice with implant-associated osteomyelitis. Longitudinal bioluminescent imaging (BLI) confirmed the hypothesized "target and release"-type kinetics of BCS and HBCS. Micro-CT of the infected tibiae demonstrated that HBCS significantly inhibited peri-implant osteolysis versus placebo and free sitafloxacin (p < 0.05), which was not seen with the corresponding non-antibiotic-conjugated bisphosphonate control. TRAP-stained histology confirmed that HBCS significantly reduced peri-implant osteoclast numbers versus placebo and free sitafloxacin controls (p < 0.05). To confirm S. aureus killing, we compared the morphology of S. aureus autolysis within in vitro biofilm and infected tibiae via transmission electron microscopy (TEM). Live bacteria in vitro and in vivo presented as dense cocci ~1 µm in diameter. In vitro evidence of autolysis presented remnant cell walls of dead bacteria or "ghosts" and degenerating (non-dense) bacteria. These features of autolyzed bacteria were also present among the colonizing S. aureus within OLCN of infected tibiae from placebo-, vancomycin-, and sitafloxacin-treated mice, similar to placebo. However, most of the bacteria within OLCN of infected tibiae from BCA-treated mice were less dense and contained small vacuoles and holes >100 nm. Histomorphometry of the bacteria within the OLCN demonstrated that BCA significantly increased their diameter versus placebo and free antibiotic controls (p < 0.05). As these abnormal features are consistent with antibiotic-induced vacuolization, bacterial swelling, and necrotic phenotype, we interpret these findings to be the initial evidence of BCA-induced killing of S. aureus within the OLCN of infected bone. Collectively, these results support the bone targeting strategy of BCA to overcome the biodistribution limits of SOC antibiotics and warrant future studies to confirm the novel TEM phenotypes of bacteria within OLCN of S. aureus-infected bone of animals treated with BCS and HBCS.

Periarteriolar spaces modulate cerebrospinal fluid transport into brain and demonstrate altered morphology in aging and Alzheimer's disease.

Perivascular spaces (PVS) drain brain waste metabolites, but their specific flow paths are debated. Meningeal pia mater reportedly forms the outermost boundary that confines flow around blood vessels. Yet, we show that pia is perforated and permissive to PVS fluid flow. Furthermore, we demonstrate that pia is comprised of vascular and cerebral layers that coalesce in variable patterns along leptomeningeal arteries, often merging around penetrating arterioles. Heterogeneous pial architectures form variable sieve-like structures that differentially influence cerebrospinal fluid (CSF) transport along PVS. The degree of pial coverage correlates with macrophage density and phagocytosis of CSF tracer. In vivo imaging confirms transpial influx of CSF tracer, suggesting a role of pia in CSF filtration, but not flow restriction. Additionally, pial layers atrophy with age. Old mice also exhibit areas of pial denudation that are not observed in young animals, but pia is unexpectedly hypertrophied in a mouse model of Alzheimer's disease. Moreover, pial thickness correlates with improved CSF flow and reduced ß-amyloid deposits in PVS of old mice. We show that PVS morphology in mice is variable and that the structure and function of pia suggests a previously unrecognized role in regulating CSF transport and amyloid clearance in aging and disease.

Metabolic regulation of APOBEC-1 complementation factor trafficking in mouse models of obesity and its positive correlation with the expression of ApoB protein in hepatocytes.

APOBEC-1 Complementation Factor (ACF) is an RNA-binding protein that interacts with apoB mRNA to support RNA editing. ACF traffics between the cytoplasm and nucleus. It is retained in the nucleus in response to elevated serum insulin levels where it supports enhanced apoB mRNA editing. In this report we tested whether ACF may have the ability to regulate nuclear export of apoB mRNA to the sites of translation in the cytoplasm. Using mouse models of obesity-induced insulin resistance and primary hepatocyte cultures we demonstrated that both nuclear retention of ACF and apoB mRNA editing were reduced in the livers of hyperinsulinemic obese mice relative to lean controls. Coincident with an increase in the recovery of ACF in the cytoplasm was an increase in the proportion of total cellular apoB mRNA recovered in cytoplasmic extracts. Cytoplasmic ACF from both lean controls and obese mouse livers was enriched in endosomal fractions associated with apoB mRNA translation and ApoB lipoprotein assembly. Inhibition of ACF export to the cytoplasm resulted in nuclear retention of apoB mRNA and reduced both intracellular and secreted ApoB protein in primary hepatocytes. The importance of ACF for modulating ApoB was supported by the finding that RNAi knockdown of ACF reduced ApoB secretion. An additional discovery from this study was the finding that leptin is a suppressor ACF expression. Dyslipidemia is a common pathology associated with insulin resistance that is in part due to the loss of insulin controlled secretion of lipid in ApoB-containing very low density lipoproteins. The data from animal models suggested that loss of insulin regulated ACF trafficking and leptin regulated ACF expression may make an early contribution to the overall pathology associated with very low density lipoprotein secretion from the liver in obese individuals.

Distinct vasculotropic versus osteotropic features of S. agalactiae versus S. aureus implant-associated bone infection in mice.

Osteomyelitis is a devastating complication of orthopaedic surgery and commonly caused by Staphylococcus aureus (S. aureus) and Group B Streptococcus (GBS, S. agalactiae). Clinically, S. aureus osteomyelitis is associated with local inflammation, abscesses, aggressive osteolysis, and septic implant loosening. In contrast, S. agalactiae orthopaedic infections generally involve soft tissue, with acute life-threatening vascular spread. While preclinical models that recapitulate the clinical features of S. aureus bone infection have proven useful for research, no animal models of S. agalactiae osteomyelitis exist. Here, we compared the pathology caused by these bacteria in an established murine model of implant-associated osteomyelitis. In vitro scanning electron microscopy and CFU quantification confirmed similar implant inocula for both pathogens (~105 CFU/pin). Assessment of mice at 14 days post-infection demonstrated increased S. aureus virulence, as S. agalactiae infected mice had significantly greater body weight, and fewer CFU on the implant and in bone and adjacent soft tissue (p < 0.05). X-ray, µCT, and histologic analyses showed that S. agalactiae induced significantly less osteolysis and implant loosening, and fewer large TRAP+ osteoclasts than S. aureus without inducing intraosseous abscess formation. Most notably, transmission electron microscopy revealed that although both bacteria are capable of digesting cortical bone, S. agalactiae have a predilection for colonizing blood vessels embedded within cortical bone while S. aureus primarily colonizes the osteocyte lacuno-canalicular network. This study establishes the first quantitative animal model of S. agalactiae osteomyelitis, and demonstrates a vasculotropic mode of S. agalactiae infection, in contrast to the osteotropic behavior of S. aureus osteomyelitis.

Emerging electron microscopy and 3D methodologies to interrogate Staphylococcus aureus osteomyelitis in murine models.

Recent breakthroughs in our understanding of orthopaedic infections have come from advances in transmission electron microscopy (TEM) imaging of murine models of bone infection, most notably Staphylococcus aureus invasion and colonization of osteocyte-lacuno canalicular networks of live cortical bone during the establishment of chronic osteomyelitis. To further elucidate this microbial pathogenesis and evaluate the mechanism of action of novel interventions, additional advances in TEM imaging are needed. Here we present detailed protocols for fixation, decalcification, and epoxy embedment of bone tissue for standard TEM imaging studies, as well as the application of immunoelectron microscopy to confirm S. aureus occupation within sub-micron canaliculi. We also describe the first application of the novel Automated-Tape-UltraMicrotome system with three-dimensional reconstruction and volumetric analyses to quantify S. aureus occupation within the osteocyte-lacuno canalicular networks. Reconstruction of the three-dimensional volume broadened our perspective of S. aureus colonization of the canalicular network and, surprisingly, revealed adjacent noninfected canaliculi. This observation has led us to hypothesize that viable osteocytes of the osteocyte-lacuno canalicular networks respond and resist infection, opening future research directions to explain the paradox of adjacent uninfected canaliculi and life-long deep bone infection in patients with chronic osteomyelitis.

Staphylococcus aureus Cell Wall Biosynthesis Modulates Bone Invasion and Osteomyelitis Pathogenesis.

Staphylococcus aureus invasion of the osteocyte lacuno-canalicular network (OLCN) is a novel mechanism of bacterial persistence and immune evasion in chronic osteomyelitis. Previous work highlighted S. aureus cell wall transpeptidase, penicillin binding protein 4 (PBP4), and surface adhesin, S. aureus surface protein C (SasC), as critical factors for bacterial deformation and propagation through nanopores in vitro, representative of the confined canaliculi in vivo. Given these findings, we hypothesized that cell wall synthesis machinery and surface adhesins enable durotaxis- and haptotaxis-guided invasion of the OLCN, respectively. Here, we investigated select S. aureus cell wall synthesis mutants (Δpbp3, Δatl, and ΔmreC) and surface adhesin mutants (ΔclfA and ΔsasC) for nanopore propagation in vitro and osteomyelitis pathogenesis in vivo. In vitro evaluation in the microfluidic silicon membrane-canalicular array (µSiM-CA) showed pbp3, atl, clfA, and sasC deletion reduced nanopore propagation. Using a murine model for implant-associated osteomyelitis, S. aureus cell wall synthesis proteins were found to be key modulators of S. aureus osteomyelitis pathogenesis, while surface adhesins had minimal effects. Specifically, deletion of pbp3 and atl decreased septic implant loosening and S. aureus abscess formation in the medullary cavity, while deletion of surface adhesins showed no significant differences. Further, peri-implant osteolysis, osteoclast activity, and receptor activator of nuclear factor kappa-B ligand (RANKL) production were decreased following pbp3 deletion. Most notably, transmission electron microscopy (TEM) imaging of infected bone showed that pbp3 was the only gene herein associated with decreased submicron invasion of canaliculi in vivo. Together, these results demonstrate that S. aureus cell wall synthesis enzymes are critical for OLCN invasion and osteomyelitis pathogenesis in vivo.

3D iPSC modeling of the retinal pigment epithelium-choriocapillaris complex identifies factors involved in the pathology of macular degeneration.

The retinal pigment epithelium (RPE)-choriocapillaris (CC) complex in the eye is compromised in age-related macular degeneration (AMD) and related macular dystrophies (MDs), yet in vitro models of RPE-CC complex that enable investigation of AMD/MD pathophysiology are lacking. By incorporating iPSC-derived cells into a hydrogel-based extracellular matrix, we developed a 3D RPE-CC model that recapitulates key features of both healthy and AMD/MD eyes and provides modular control over RPE and CC layers. Using this 3D RPE-CC model, we demonstrated that both RPE- and mesenchyme-secreted factors are necessary for the formation of fenestrated CC-like vasculature. Our data show that choroidal neovascularization (CNV) and CC atrophy occur in the absence of endothelial cell dysfunction and are not necessarily secondary to drusen deposits underneath RPE cells, and CC atrophy and/or CNV can be initiated systemically by patient serum or locally by mutant RPE-secreted factors. Finally, we identify FGF2 and matrix metalloproteinases as potential therapeutic targets for AMD/MDs.

A human model of Batten disease shows role of CLN3 in phagocytosis at the photoreceptor-RPE interface.

Mutations in CLN3 lead to photoreceptor cell loss in CLN3 disease, a lysosomal storage disorder characterized by childhood-onset vision loss, neurological impairment, and premature death. However, how CLN3 mutations cause photoreceptor cell death is not known. Here, we show that CLN3 is required for phagocytosis of photoreceptor outer segment (POS) by retinal pigment epithelium (RPE) cells, a cellular process essential for photoreceptor survival. Specifically, a proportion of CLN3 in human, mouse, and iPSC-RPE cells localized to RPE microvilli, the site of POS phagocytosis. Furthermore, patient-derived CLN3 disease iPSC-RPE cells showed decreased RPE microvilli density and reduced POS binding and ingestion. Notably, POS phagocytosis defect in CLN3 disease iPSC-RPE cells could be rescued by wild-type CLN3 gene supplementation. Altogether, these results illustrate a novel role of CLN3 in regulating POS phagocytosis and suggest a contribution of primary RPE dysfunction for photoreceptor cell loss in CLN3 disease that can be targeted by gene therapy.

The expression of apoB mRNA editing factors is not the sole determinant for the induction of editing in differentiating Caco-2 cells.

Apolipoprotein B mRNA is edited at cytidine 6666 in the enterocytes lining the small intestine of all mammals; converting a CAA codon to a UAA stop codon. The conversion is approximately 80% efficient in this tissue and leads to the expression of the truncated protein, ApoB48, essential for secretion of dietary lipid as chylomicrons. Caco-2 cell raft cultures have been used as an in vitro model for the induction of editing activity during human small intestinal cell differentiation. This induction of apoB mRNA editing has been ascribed to the expression of APOBEC-1. In agreement our data demonstrated differentiation-dependent induction of expression of the editing enzyme APOBEC-1 and in addition we show alternative splicing of the essential auxiliary factor ACF. However, transfection of these editing factors in undifferentiated proliferating Caco-2 cells was not sufficient to induce robust apoB mRNA editing activity. Only differentiation of Caco-2 cells could induce more physiological like levels of apoB mRNA editing. The data suggested that additional regulatory mechanism(s) were induced by differentiation that controlled the functional activity of editing factors.

Environmental stress impairs photoreceptor outer segment (POS) phagocytosis and degradation and induces autofluorescent material accumulation in hiPSC-RPE cells.

Retinal pigment epithelium (RPE) cell dysfunction is central to the pathogenesis of age-related macular degeneration (AMD), a leading cause of adult blindness. Aging, the single biggest risk factor for AMD development, favors increase in RPE autofluorescent material due to accumulation of POS-digestion by-products through lysosomal dysfunction and impaired POS degradation. Apart from aging, environmental agents affect lysosomal function in multiple model systems and are implicated in AMD. Iron (Fe) overload and cigarette smoke exposure are the two environmental factors that are known to affect the lysosomal pathway and impact RPE cell health. However, the impact of Fe and cigarette smoke, on POS processing and its consequence for autofluorescent material accumulation in human RPE cells are yet to be established. Human induced pluripotent stem cell (hiPSC)-derived RPE, which phagocytoses and degrades POS in culture and can be derived from control individuals (no history/susceptibility for retinal disease), provides a model system to investigate the singular effect of excess Fe and/or cigarette smoke on POS processing by RPE cells. Using at least three distinct control hiPSC lines, we show that, compared to untreated hiPSC-RPE cells, POS uptake is reduced in both Fe (ferric ammonium citrate or FAC) and FAC + CSE (cigarette smoke extract)-treated hiPSC-RPE cells. Furthermore, exposure of hiPSC-RPE cultures to FAC + CSE leads to reduced levels of active cathepsin-D (CTSD), a lysosomal enzyme involved in POS processing, and causes delayed degradation of POS. Notably, delayed degradation of POS over time (2 weeks) in hiPSC-RPE cells exposed to Fe and CSE was sufficient to increase autofluorescent material build-up in these cells. Given that inefficient POS processing-mediated autofluorescent material accumulation in RPE cells has already been linked to AMD development, our results implicate a causative role of environmental agents, like Fe and cigarette smoke, in AMD.

Functional characterization of APOBEC-1 complementation factor phosphorylation sites.

ApoB mRNA editing involves site-specific deamination of cytidine 6666 producing an in-frame translation stop codon. Editing minimally requires APOBEC-1 and APOBEC-1 complementation factor (ACF). Metabolic stimulation of apoB mRNA editing in hepatocytes is associated with serine phosphorylation of ACF localized to editing competent, nuclear 27S editosomes. We demonstrate that activation of protein kinase C (PKC) stimulated editing and enhanced ACF phosphorylation in rat primary hepatocytes. Conversely, activation of protein kinase A (PKA) had no effect on editing. Recombinant PKC efficiently phosphorylated purified ACF64 protein in vitro, whereas PKA did not. Mutagenesis of predicted PKC phosphorylation sites S154 and S368 to alanine inhibited ethanol-stimulated induction of editing suggesting that these sites function in the metabolic regulation of editing. Consistent with this interpretation, substitution of S154 and S368 with aspartic acid stimulated editing to levels comparable to ethanol treatment in control McArdle RH7777 cells. These data suggest that phosphorylation of ACF by PKC may be a key regulatory mechanism of apoB mRNA editing in rat hepatocytes.

Metabolic regulation of apoB mRNA editing is associated with phosphorylation of APOBEC-1 complementation factor.

Apolipoprotein B (apoB) mRNA editing is a nuclear event that minimally requires the RNA substrate, APOBEC-1 and APOBEC-1 Complementation Factor (ACF). The co-localization of these macro-molecules within the nucleus and the modulation of hepatic apoB mRNA editing activity have been described following a variety of metabolic perturbations, but the mechanism that regulates editosome assembly is unknown. APOBEC-1 was effectively co-immunoprecipitated with ACF from nuclear, but not cytoplasmic extracts. Moreover, alkaline phosphatase treatment of nuclear extracts reduced the amount of APOBEC-1 co-immunoprecipitated with ACF and inhibited in vitro editing activity. Ethanol stimulated apoB mRNA editing was associated with a 2- to 3-fold increase in ACF phosphorylation relative to that in control primary hepatocytes. Significantly, phosphorylated ACF was restricted to nuclear extracts where it co-sedimented with 27S editing competent complexes. Two-dimensional phosphoamino acid analysis of ACF immunopurified from hepatocyte nuclear extracts demonstrated phosphorylation of serine residues that was increased by ethanol treatment. Inhibition of protein phosphatase I, but not PPIIA or IIB, stimulated apoB mRNA editing activity coincident with enhanced ACF phosphorylation in vivo. These data demonstrate that ACF is a metabolically regulated phosphoprotein and suggest that this post-translational modification increases hepatic apoB mRNA editing activity by enhancing ACF nuclear localization/retention, facilitating the interaction of ACF with APOBEC-1 and thereby increasing the probability of editosome assembly and activity.

Characterization of Human iPSC-RPE on a Prosthetic Bruch's Membrane Manufactured From Silk Fibroin.

Purpose: RPE cell transplantation as a potential treatment for AMD has been extensively investigated; however, in AMD, ultrastructural damage affects both the RPE and its underlying matrix support, the Bruch's membrane (BrM). An RPE monolayer supported by a surrogate scaffold could thus provide a more effective approach to cell-based therapy for AMD. Toward this goal, we aimed to establish a functional human induced pluripotent stem cell-derived (hiPSC)-RPE monolayer on a Bombyx mori silk fibroin (BMSF) scaffold. Methods: RPE differentiated from five distinct hiPSC lines were cultured on BMSF membrane coated with extracellular matrix (ECM, COL1), and either regular tissue culture plastic or Transwell coated with ECM (LAM-TCP). Morphologic, gene and protein expression, and functional characteristics of the hiPSC-RPE cultured on different membranes were compared in longitudinal experiments spanning 1 day to ≥3 months. Results: The hiPSC-RPE monolayers on ECM-coated BMSF and TCP could be maintained in culture for ≥3 months and displayed RPE-characteristic morphology, pigmentation, polarity, and expression of RPE signature genes and proteins. Furthermore, hiPSC-RPE on both ECM-coated BMSF and TCP displayed robust expression and secretion of several basement membrane proteins. Importantly, hiPSC-RPE cells on COL1-BMSF and LAM-TCP showed similar efficacy in the phagocytosis and degradation of photoreceptor outer segments. Conclusions: A biomaterial scaffold manufactured from silk fibroin supports the maturation and long-term survival of a functional hiPSC-RPE monolayer. This has significant implications for both in vitro disease modeling and in vivo cell replacement therapy.

Hepatic very-low-density lipoprotein and apolipoprotein B production are increased following in vivo induction of betaine-homocysteine S-methyltransferase.

We have previously reported a positive correlation between the expression of BHMT (betaine-homocysteine S-methyltransferase) and ApoB (apolipoprotein B) in rat hepatoma McA (McArdle RH-7777) cells [Sowden, Collins, Smith, Garrow, Sparks and Sparks (1999) Biochem. J. 341, 639-645]. To examine whether a similar relationship occurs in vivo, hepatic BHMT expression was induced by feeding rats a Met (L-methionine)-restricted betaine-containing diet, and parameters of ApoB metabolism were evaluated. There were no generalized metabolic abnormalities associated with Met restriction for 7 days, as evidenced by control levels of serum glucose, ketones, alanine aminotransferase and L-homocysteine levels. Betaine plus the Met restriction resulted in lower serum insulin and non-esterified fatty acid levels. Betaine plus Met restriction induced hepatic BHMT 4-fold and ApoB mRNA 3-fold compared with Met restriction alone. No changes in percentage of edited ApoB mRNA were observed on the test diets. An increase in liver ApoB mRNA correlated with an 82% and 46% increase in ApoB and triacylglycerol production respectively using in vivo Triton WR 1339. Increased secretion of VLDL (very-low-density lipoprotein) with Met restriction plus betaine was associated with a 45% reduction in liver triacylglycerol compared with control. Nuclear run-off assays established that transcription of both bhmt and apob genes was also increased in Met-restricted plus betaine diets. No change in ApoB mRNA stability was detected in BHMT-transfected McA cells. Hepatic ApoB and BHMT mRNA levels were also increased by 1.8- and 3-fold respectively by betaine supplementation of Met-replete diets. Since dietary betaine increased ApoB mRNA, VLDL ApoB and triacylglycerol production and decreased hepatic triacylglycerol, results suggest that induction of apob transcription may provide a potential mechanism for mobilizing hepatic triacylglycerol by increasing ApoB available for VLDL assembly and secretion.