Impaired axonal transport at the optic nerve head contributes to neurodegeneration in a novel Cre-inducible mouse model of myocilin glaucoma.

Elevation of intraocular pressure (IOP) due to trabecular meshwork (TM) dysfunction, leading to neurodegeneration, is the pathological hallmark of primary open-angle glaucoma (POAG). Impaired axonal transport is an early and critical feature of glaucomatous neurodegeneration. However, a robust mouse model that replicates these human POAG features accurately has been lacking. We report the development and characterization of a novel Cre-inducible mouse model expressing a DsRed-tagged Y437H mutant of human myocilin (Tg.CreMYOCY437H). A single intravitreal injection of HAd5-Cre induced selective MYOC expression in the TM, causing TM dysfunction, reducing outflow facility, and progressively elevating IOP in Tg.CreMYOCY437H mice. Sustained IOP elevation resulted in significant retinal ganglion cell (RGC) loss and progressive axonal degeneration in Cre-induced Tg.CreMYOCY437H mice. Notably, impaired anterograde axonal transport was observed at the optic nerve head before RGC degeneration, independent of age, indicating that impaired axonal transport contributes to RGC degeneration in Tg.CreMYOCY437H mice. In contrast, axonal transport remained intact in ocular hypertensive mice injected with microbeads, despite significant RGC loss. Our findings indicate that Cre-inducible Tg.CreMYOCY437H mice replicate all glaucoma phenotypes, providing an ideal model for studying early events of TM dysfunction and neuronal loss in POAG.

Sigma-1R Protects Retinal Ganglion Cells in Optic Nerve Crush Model for Glaucoma.

Purpose: The purpose of this study was to determine the effects of the Sigma-1R (σ-1r) on retinal ganglion cell (RGC) survival following optic nerve crush (ONC) and the signaling mechanism involved in the σ-1r protection. Methods: The overall strategy was to induce injury by ONC and mitigate RGC death by increasing σ-1r expression and/or activate σ-1r activity in σ-1r K/O mice and wild type (WT) mice. AAV2-σ-1r vector was used to increase σ-1r expression and σ-1r agonist used to activate the σ-1r and RGCs were counted. Immunohistochemical and Western blot analysis determined phosphorylated (p)-c-Jun, c-Jun, and Caspase-3. Pattern electroretinography (PERG) determined RGC activity. Results: RGC counts and function were similar in pentazocine-treated WT mice when compared to untreated mice and in WT mice when compared with σ-1r K/O mice. Pentazocine-induced effects and the effects of σ-1r K/O were only observable after ONC. ONC resulted in decreased RGC counts and activity in both WT and σ-1r K/O mice, with σ-1r K/O mice experiencing significant decreases compared with WT mice. The σ-1r transgenic expression resulted in increased RGC counts and activity following ONC. In WT mice, treatment with σ-1r agonist pentazocine resulted in increased RGC counts and increased activity when compared with untreated WT mice. There were time-dependent increases in c-jun, p-c-jun, and caspase-3 expression in ONC mice that were mitigated with pentazocine-treatment. Conclusions: These findings suggest that the apoptotic pathway is involved in RGC losses seen in an ONC model. The σ-1r offers neuroprotection, as activation and/or transgenic expression of σ-1r attenuated the apoptotic pathway and restored RGCs number and function following ONC.

Hybrid Compound SA-2 is Neuroprotective in Animal Models of Retinal Ganglion Cell Death.

Purpose: Determine the toxicity, bioavailability in the retina, and neuroprotective effects of a hybrid antioxidant-nitric oxide donor compound SA-2 against oxidative stress-induced retinal ganglion cell (RGC) death in neurodegenerative animal models. Methods: Optic nerve crush (ONC) and ischemia reperfusion (I/R) injury models were used in 12-week-old C57BL/6J mice to mimic conditions of glaucomatous neurodegeneration. Mice were treated intravitreally with either vehicle or SA-2. Retinal thickness was measured by spectral-domain optical coherence tomography (SD-OCT). The electroretinogram and pattern ERG (PERG) were used to assess retinal function. RGC survival was determined by counting RBPMS-positive RGCs and immunohistochemical analysis of superoxide dismutase 1 (SOD1) levels was carried out in the retina sections. Concentrations of SA-2 in the retina and choroid were determined using HPLC and MS. In addition, the direct effect of SA-2 treatment on RGC survival was assessed in ex vivo rat retinal explants under hypoxic (0.5% O2) conditions. Results: Compound SA-2 did not induce any appreciable change in retinal thickness, or in a- or b-wave amplitude in naive animals. SA-2 was found to be bioavailable in both the retina and choroid after a single intravitreal injection (2% wt/vol). An increase in SOD1 levels in the retina of mice subjected to ONC and SA-2 treatment, suggests an enhancement in antioxidant activity. SA-2 provided significant (P < 0.05) RGC protection in all three of the tested RGC injury models in rodents. PERG amplitudes were significantly higher in both I/R and ONC mouse eyes following SA-2 treatment (P ≤ 0.001) in comparison with the vehicle and control groups. Conclusions: Compound SA-2 was effective in preventing RGC death and loss of function in three different rodent models of acute RGC injury: ONC, I/R, and hypoxia.

Sigma-1 Receptor Regulates Mitochondrial Function in Glucose- and Oxygen-Deprived Retinal Ganglion Cells.

Purpose: Understanding the role of mitochondria in retinal ganglion cells (RGCs) is relevant to human disease as studies have shown mitochondrial abnormalities in primary open-angle glaucoma patients. This study seeks to determine the effects of the sigma-1 receptor (σ-1r) and its agonists on mitochondrial function in oxygen- and glucose- deprived (OGD) purified neonatal RGCs. Methods: Retinal ganglion cells were isolated from rat pups and subjected to OGD in varying conditions in the presence or absence of σ-1r agonist and antagonist and following addition of an AAV2-σ-1r vector that was used to increase σ-1r expression. Western blots and immunofluorescence microscopy validated findings. Mitochondrial function was determined by measuring mitochondrial membrane potential (Δψm) using the dye, fluorescence tetraethylbenzimidazolylcarbocyanineiodide (JC-1), and determination of cytochrome c oxidase activity using a cytochrome c oxidase assay kit. Caspase 3 and 7 activities were also measured using a luminescent assay kit. Results: Oxygen and glucose deprivation in RGCs resulted in decreased mitochondrial membrane potential and cytochrome c oxidase activity when compared with normoxic RGCs. σ-1r agonists or overexpression of the σ-1r restored the mitochondrial membrane potential comparable to normoxic conditions, while σ-1r antagonists abolished these effects. Oxygen and glucose depreavtation induced decreases in cytochrome c activity were partially restored by overexpression or activation of σ-1r. Caspase activity was increased in response to OGD and was decreased by the addition of σ-1r agonist, pentazocine, and following σ-1r overexpression. Conclusions: These data suggest that activation and/or overexpression of σ-1r restores RGCs mitochondrial function following OGD and that mitochondrial function is vital to the function of RGCs.

Involvement of AMPA Receptor and Its Flip and Flop Isoforms in Retinal Ganglion Cell Death Following Oxygen/Glucose Deprivation.

PURPOSE: The α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptors (AMPAR) subunits can be posttranscriptionally modified by alternative splicing forming flip and flop isoforms. We determined if an ischemia-like insult to retinal ganglion cells (RGCs) increases AMPAR susceptibility to s-AMPA-mediated excitotoxicity through changes in posttranscriptional modified isoforms. METHODS: Purified neonatal rat RGCs were subjected to either glucose deprivation (GD) or oxygen/glucose deprivation (OGD) conditions followed by treatment with either 100 µM s-AMPA or Kainic acid. A live-dead assay and caspase 3 assay was used to assess cell viability and apoptotic changes, respectively. We used JC-1 dye and dihydroethidium to measure mitochondria depolarization and reactive oxygen species (ROS), respectively. Calcium imaging with fura-2AM was used to determine intracellular calcium, while the fluorescently-labeled probe, Nanoprobe1, was used to detect calcium-permeable AMPARs. Quantitative PCR (qPCR) analysis was done to determine RNA editing sites AMPAR isoforms. RESULTS: Glucose deprivation, as well as an OGD insult followed by AMPAR stimulation, produced a significant increase in RGC death. Retinal ganglion cell death was independent of caspase 3/7 activity, but was accompanied by increased mitochondrial depolarization and increased ROS production. This was associated with an elevated intracellular Ca(2+) and calcium permeable-AMPARs. The mRNA expression of GLUA2 and GLUA3 flop isoform decreased significantly, while no appreciable changes were found in the corresponding flip isoforms. There were no changes in the Q/R editing of GLUA2, while R/G editing of GLUA2 flop declined under these conditions. CONCLUSIONS: Following oxidative injury, RGCs become more susceptible to AMPAR-mediated excitotoxicity. RNA editing and changes in alternative spliced flip and flop isoforms of AMPAR subunits may contribute to increased RGC death.

Mitochondrial Dihydrolipoamide Dehydrogenase is Upregulated in Response to Intermittent Hypoxic Preconditioning.

Intermittent hypoxia preconditioning (IHP) has been shown to protect neurons against ischemic stroke injury. Studying how proteins respond to IHP may identify targets that can help fight stroke. The objective of the present study was to investigate whether mitochondrial dihydrolipoamide dehydrogenase (DLDH) would respond to IHP and if so, whether such a response could be linked to neuroprotection in ischemic stroke injury. To do this, we subjected male rats to IHP for 20 days and measured the content and activity of DLDH as well as the three α-keto acid dehydrogenase complexes that contain DLDH. We also measured mitochondrial electron transport chain enzyme activities. Results show that DLDH content was indeed upregulated by IHP and this upregulation did not alter the activities of the three α-keto acid dehydrogenase complexes. Results also show that the activities of the five mitochondrial complexes (I-V) were not altered either by IHP. To investigate whether IHP-induced DLDH upregulation is linked to neuroprotection against ischemic stroke injury, we subjected both DLDH deficient mouse and DLDH transgenic mouse to stroke surgery followed by measurement of brain infarction volume. Results indicate that while mouse deficient in DLDH had exacerbated brain injury after stroke, mouse overexpressing human DLDH also showed increased brain injury after stroke. Therefore, the physiological significance of IHP-induced DLDH upregulation remains to be further investigated.

Preclinical pharmacology, ocular tolerability and ocular hypotensive efficacy of a novel non-peptide bradykinin mimetic small molecule.

We sought to characterize the ocular pharmacology, tolerability and intraocular pressure (IOP)-lowering efficacy of FR-190997, a non-peptidic bradykinin (BK) B2-receptor agonist. FR-190997 possessed a relatively high receptor binding affinity (Ki = 27 nM) and a high in vitro potency (EC50 = 18.3 ± 4.4 nM) for inositol-1-phosphate generation via human cloned B2-receptors expressed in host cells with mimimal activity at B1-receptors. It also mobilized intracellular Ca2+ in isolated human trabecular meshwork (h-TM), ciliary muscle (h-CM), and in immortalized non-pigmented ciliary epithelial (h-iNPE) cells (EC50s = 167-384 nM; Emax = 32-86% of BK-induced response). HOE-140, a selective B2-receptor antagonist, potently blocked the latter effects of FR-190997 (e.g., IC50 = 7.3 ± 0.6 nM in h-CM cells). FR-190997 also stimulated the release of prostaglandins (PGs) from h-TM and h-CM cells (EC50s = 60-84 nM; Emax = 29-44% relative to max. BK-induced effects). FR-190997 (0.3-300 µg t.o.) did not activate cat corneal polymodal nociceptors and did not cause ocular discomfort in Dutch-Belted rabbits, but it was not well tolerated in New Zealand albino rabbits and Hartley guinea pigs. A single topical ocular (t.o.) dose of 1% FR-190997 in Dutch-Belted rabbits and mixed breed cats did not lower IOP. However, FR-190997 efficaciously lowered IOP of conscious ocular hypertensive cynomolgus monkey eyes (e.g., 34.5 ± 7.5% decrease; 6 h post-dose of 30 µg t.o.; n = 8). Thus, FR-190997 is an unexampled efficacious ocular hypotensive B2-receptor non-peptide BK agonist that activates multiple signaling pathways to cause IOP reduction.

FR-190997, a nonpeptide bradykinin B2-receptor partial agonist, is a potent and efficacious intraocular pressure lowering agent in ocular hypertensive cynomolgus monkeys.

Preclinical Research FR-190997 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl) cinnaminoacetyl]-N-methylamino]benzyloxy]-2-methyl-4- (2-pyridylmethoxy) quinoline), a nonpeptide bradykinin (BK) B2-receptor-selective agonist, represents a novel class of ocular hypotensive agents. FR-190997 exhibited a high affinity for the human cloned B2-receptor (Ki = 9.8 nM) and a relatively high potency (EC50 = 155 nM) for mobilizing intracellular Ca(2+) ([Ca(2+)]i) in human ocular cells from nonpigmented ciliary epithelium; trabecular meshwork [h-TM]; ciliary muscle [h-CM] that are involved in regulating intraocular pressure (IOP). Unlike BK, FR-190997 behaved as a partial agonist (Emax = 38-80%) in these cells and its [Ca(2+)]i-mobilizing effects were blocked by the B2-receptor-selective antagonists (HOE-140, Ki = 0.8-7 nM; WIN-64338, Ki = 157-425 nM). FR-190997 stimulated the production of prostaglandins (PGs) in h-CM and h-TM cells (EC50 = 15-19 nM; Emax = 27-33%); an effect that was reduced by the cyclooxygenase-2 inhibitor bromfenac, and by HOE-140. FR-190997 also induced pro-matrix metalloproteinase (MMP)-1 and MMP-3 release from h-CM cells. FR-190997 significantly lowered IOP (37% [P < 0.001] with 30 µg, 24 h post-topical ocular dosing) in ocular hypertensive eyes of conscious Cynomolgus monkeys. This effect was reduced by bromfenac and completely blocked by a B2-antagonist. FR-190997 primarily stimulated uveoslceral outflow (UVSO) of aqueous humor (2.6 to 3.9-fold above baseline). In conclusion, FR-190997 is a B2-receptor selective partial agonist that activates phospholipase C, mobilizes [Ca(2+)]; induces PG and pro-MMP production, and that profoundly lowers IOP by promoting UVSO in ocular hypertensive Cynomolgus monkey eyes.

Human non-pigmented ciliary epithelium bradykinin B2-receptors: receptor localization, pharmacological characterization of intracellular Ca(2+) mobilization, and prostaglandin secretion.

PURPOSE: To characterize the bradykinin (BK) receptor system in human non-pigmented ciliary epithelium (NPCE) using immunohistochemistry and functional cell-based techniques. METHODS: B2-receptor protein expression was studied in sections of human donor eyes and in Cynomolgus monkey eyes using immunohistochemical methods. The pharmacological characteristics of intracellular Ca(2+) ([Ca(2+)]i) mobilization in response to BK and related peptides, and blockade by two antagonists, was studied in primary human (p-h-NPCE) and in immortalized human NPCE (imh-NPCE) cells. Prostaglandins (PGs) release induced by BK was also studied in both cell-types using ELISA assays. Limited studies on primary human ciliary muscle (h-CM) cells and human trabecular meshwork (h-TM) cells and Chinese hamster ovary cells expressing human cloned B2-receptors (CHO-B2) were performed to compare with responses in both the NPCE cell-types. RESULTS: B2-receptor immunoreactivity was observed on human and Cynomolgus monkey NPCE cells on eye sections from both species. BK and related analog peptides differentially activated signaling mechanisms in NPCE cells by mobilizing [Ca(2+)]i, and the BK-evoked responses were blocked by B2-receptor-selective antagonists, HOE-140 and (S)-WIN-64338. Relative agonist potencies (EC50, nM) in p-h-NPCE cells [and in imh-NPCE cells] were: BK=3.4 ± 0.4 [6.3 nM]; Hyp(3)-BK EC50=1.7 ± 0.2 [6.0 nM], Lys-BK EC50=7.0 ± 0.3 [19.8 nM]; Met-Lys-BK EC50=106 ± 57.8 [125 nM]; Des-Arg(9)-BK EC50=>10,000 [16 µM]. The antagonist potencies for attenuating BK-induced mobilization of [Ca(2+)]i in these cells were: HOE-140 (Ki=7.9 ± 1.8 nM, n=4) and (S)-WIN-64338 (Ki=451 ± 44 nM, n=4). These NPCE cell data correlated well with those obtained for h-CM and h-TM cells, and with B2-receptor binding (r=0.99, p<0.0001). However, BK failed to stimulate total PGs production in both NPCE cell-types even though 10% bovine serum increased PG release (by 4.9-fold above baseline), and even though BK stimulated PG release from h-CM, h-TM and in CHO-B2 cells. BK (1 µM) also failed to increase nitric oxide (NO) levels in NPCE cells even though sodium nitropruside increased NO production by 3-fold. CONCLUSIONS: Human and monkey NPCE express immunoreactive B2-receptor proteins. These proteins were functionally active, since BK and related peptides potently stimulated mobilization of [Ca(2+)]i in p-h-NPCE and imNPCE cells that was blocked by two B2-selective antagonists. Down-stream signaling from B2-receptor activation did not appear to involve PG synthesis/release (or NO production) in NPCE cell-types under the present conditions, even though h-CM, h-TM and CHO-B2 cells exhibited robust PG synthesis and release in response to BK.

Trabecular meshwork bradykinin receptors: mRNA levels, immunohistochemical visualization, signaling processes pharmacology, and linkage to IOP reduction.

PURPOSE: To localize mRNA and protein of bradykinin (BK) receptors, BK precursor polypeptide (kininogen) mRNA, and to study functional biochemical pharmacology of the signal transduction processes mediated by B2-receptors in isolated human trabecular meshwork (h-TM) cells. Intraocular pressure (IOP) lowering effects of 2 kinins were also investigated. METHODS: Previously documented procedures were utilized throughout these studies. RESULTS: Kinninogen mRNA was most abundant in TM, ciliary body (CB), and optic nerve head and appeared elevated in glaucomatous h-TM tissue. High levels of B2-receptor mRNA were found in the sclera, iris, TM, and CB. B2-receptor subtype protein was localized in cells of the monkey and h-TM, and the treatment of isolated h-TM cells with transforming growth factor-ß2 (5 ng/mL) caused significant (P<0.04) downregulation of B2-receptor mRNA. In isolated primary h-TM cells, BK (EC50=0.8±0.2 nM; n=19) and Met-Lys-BK (EC50=6.5±1.5 nM) mobilized intracellular Ca(2+) and induced the release of prostaglandins (PGs) that was blocked by 2 B2-receptor antagonists [HOE-140; (S)-WIN-64338]. The cyclooxygenase inhibitor, bromfenac, abolished BK-induced PGs production. BK concentration dependently increased cell impedance, and it significantly (P<0.05) decreased h-TM cell volume in vitro. Intravitreal (ivt) administration of BK (50 µg), but not a B1-agonist (Sar-[D-Phe(9)]-Des-Arg(9)-BK; also at 50 µg), efficaciously lowered IOP (22.9% to 37% from baseline) of Dutch-Belted rabbits that naturally have high IOPs (27-28 mmHg). CONCLUSIONS: BK activates multiple signal transduction pathways in h-TM cells via B2-receptors that also mediate IOP reduction as observed in rabbits following ivt administration of BK. These ocular hypotensive effects of BK may be physiologically important and suggest a novel therapeutic potential of BK-related B2-agonists.

Protein expression, biochemical pharmacology of signal transduction, and relation to intraocular pressure modulation by bradykinin B2 receptors in ciliary muscle.

PURPOSE: To examine the bradykinin (BK) B2-receptor system in human and monkey ciliary muscle (CM) using immunohistochemical techniques, and to pharmacologically characterize the associated biochemical signal transduction systems in human CM (h-CM) cells. BK-induced modulation of intraocular pressure (IOP) in pigmented Dutch-Belt rabbits and cynomolgus monkeys was also studied. METHODS: Previously published procedures were used throughout these studies. RESULTS: The human and monkey ciliary bodies expressed high levels of B2-receptor protein immunoreactivity. Various kinins differentially stimulated [Ca²âº](i) mobilization in primary h-CM cells (BK EC50=2.4±0.2 nM > Hyp³,ß-(2-thienyl)-Ala5,Tyr(Me)8-(®)-Arg9-BK (RMP-7) > Des-Arg9-BK EC50=4.2 µM [n=3-6]), and this was blocked by B2-selective antagonists, HOE-140 (IC50=1.4±0.1 nM) and WIN-63448 (IC50=174 nM). A phospholipase C inhibitor (U73122; 10-30 µM) and ethylene glycol tetraacetic acid (1-2 mM) abolished the BK-induced [Ca²âº](i) mobilization. Total prostaglandin (primarily PGE2) secretion stimulated by BK and other kinins in h-CM cells was attenuated by the cyclooxygenase inhibitors bromfenac and flurbiprofen, and by the B2-antagonists. BK and RMP-7 (100 nM) induced a twofold increase in extracellular signal-regulated kinase-1/2 phosphorylation, and BK (0.1-1 µM; at 24 h) caused a 1.4-3.1-fold increase in promatrix metalloproteinases-1-3 release. Topical ocular BK (100 µg) failed to alter IOP in cynomolgus monkeys. However, intravitreal injection of 50 µg of BK, but not Des-Arg9-BK, lowered IOP in rabbit eyes (22.9±7.3% and 37.0±5.6% at 5 h and 8 h post-injection; n=7-10). CONCLUSIONS: These studies have provided evidence of a functional endogenously expressed B2-receptor system in the CM that appears to be involved in modulating IOP.

Katanin localization requires triplet microtubules in Chlamydomonas reinhardtii.

Centrioles and basal bodies are essential for a variety of cellular processes that include the recruitment of proteins to these structures for both centrosomal and ciliary function. This recruitment is compromised when centriole/basal body assembly is defective. Mutations that cause basal body assembly defects confer supersensitivity to Taxol. These include bld2, bld10, bld12, uni3, vfl1, vfl2, and vfl3. Flagellar motility mutants do not confer sensitivity with the exception of mutations in the p60 (pf19) and p80 (pf15) subunits of the microtubule severing protein katanin. We have identified additional pf15 and bld2 (ε-tubulin) alleles in screens for Taxol sensitivity. Null pf15 and bld2 alleles are viable and are not essential genes in Chlamydomonas. Analysis of double mutant strains with the pf15-3 and bld2-6 null alleles suggests that basal bodies in Chlamydomonas may recruit additional proteins beyond katanin that affect spindle microtubule stability. The bld2-5 allele is a hypomorphic allele and its phenotype is modulated by nutritional cues. Basal bodies in bld2-5 cells are missing proximal ends. The basal body mutants show aberrant localization of an epitope-tagged p80 subunit of katanin. Unlike IFT proteins, katanin p80 does not localize to the transition fibers of the basal bodies based on an analysis of the uni1 mutant as well as the lack of colocalization of katanin p80 with IFT74. We suggest that the triplet microtubules are likely to play a key role in katanin p80 recruitment to the basal body of Chlamydomonas rather than the transition fibers that are needed for IFT localization.

Whole-Genome Sequencing to Identify Mutants and Polymorphisms in Chlamydomonas reinhardtii.

Whole-genome sequencing (WGS) provides a new platform for the identification of mutations that produce a mutant phenotype. We used Illumina sequencing to identify the mutational profile of three Chlamydomonas reinhardtii mutant strains. The three strains have more than 38,000 changes from the reference genome. NG6 is aflagellate and maps to 269 kb with only one nonsynonymous change; the V(12)E mutation falls in the FLA8 gene. Evidence that NG6 is a fla8 allele comes from swimming revertants that are either true or pseudorevertants. NG30 is aflagellate and maps to 458 kb that has six nonsynonomous changes. Evidence that NG30 has a causative nonsense allele in IFT80 comes from rescue of the nonswimming phenotype with a fragment bearing only this gene. This gene has been implicated in Jeune asphyxiating thoracic dystrophy. Electron microscopy of ift80-1 (NG30) shows a novel basal body phenotype. A bar or cap is observed over the distal end of the transition zone, which may be an intermediate in preparing the basal body for flagellar assembly. In the acetate-requiring mutant ac17, we failed to find a nonsynonymous change in the 676 kb mapped region, which is incompletely assembled. In these strains, 43% of the changes occur on two of the 17 chromosomes. The excess on chromosome 6 surrounds the mating-type locus, which has numerous rearrangements and suppressed recombination, and the changes extend beyond the mating-type locus. Unexpectedly, chromosome 16 shows an unexplained excess of single nucleotide polymorphisms and indels. Overall, WGS in combination with limited mapping allows fast and accurate identification of point mutations in Chlamydomonas.

Retrograde intraflagellar transport mutants identify complex A proteins with multiple genetic interactions in Chlamydomonas reinhardtii.

The intraflagellar transport machinery is required for the assembly of cilia. It has been investigated by biochemical, genetic, and computational methods that have identified at least 21 proteins that assemble into two subcomplexes. It has been hypothesized that complex A is required for retrograde transport. Temperature-sensitive mutations in FLA15 and FLA17 show defects in retrograde intraflagellar transport (IFT) in Chlamydomonas. We show that IFT144 and IFT139, two complex A proteins, are encoded by FLA15 and FLA17, respectively. The fla15 allele is a missense mutation in a conserved cysteine and the fla17 allele is an in-frame deletion of three exons. The flagellar assembly defect of each mutant is rescued by the respective transgenes. In fla15 and fla17 mutants, bulges form in the distal one-third of the flagella at the permissive temperature and this phenotype is also rescued by the transgenes. These bulges contain the complex B component IFT74/72, but not alpha-tubulin or p28, a component of an inner dynein arm, which suggests specificity with respect to the proteins that accumulate in these bulges. IFT144 and IFT139 are likely to interact with each other and other proteins on the basis of three distinct genetic tests: (1) Double mutants display synthetic flagellar assembly defects at the permissive temperature, (2) heterozygous diploid strains exhibit second-site noncomplemention, and (3) transgenes confer two-copy suppression. Since these tests show different levels of phenotypic sensitivity, we propose they illustrate different gradations of gene interaction between complex A proteins themselves and with a complex B protein (IFT172).

Improving gene-finding in Chlamydomonas reinhardtii:GreenGenie2.

BACKGROUND: The availability of whole-genome sequences allows for the identification of the entire set of protein coding genes as well as their regulatory regions. This can be accomplished using multiple complementary methods that include ESTs, homology searches and ab initio gene predictions. Previously, the Genie gene-finding algorithm was trained on a small set of Chlamydomonas genes and shown to improve the accuracy of gene prediction in this species compared to other available programs. To improve ab initio gene finding in Chlamydomonas, we assemble a new training set consisting of over 2,300 cDNAs by assembling over 167,000 Chlamydomonas EST entries in GenBank using the EST assembly tool PASA. RESULTS: The prediction accuracy of our cDNA-trained gene-finder, GreenGenie2, attains 83% sensitivity and 83% specificity for exons on short-sequence predictions. We predict about 12,000 genes in the version v3 Chlamydomonas genome assembly, most of which (78%) are either identical to or significantly overlap the published catalog of Chlamydomonas genes 1. 22% of the published catalog is absent from the GreenGenie2 predictions; there is also a fraction (23%) of GreenGenie2 predictions that are absent from the published gene catalog. Randomly chosen gene models were tested by RT-PCR and most support the GreenGenie2 predictions. CONCLUSION: These data suggest that training with EST assemblies is highly effective and that GreenGenie2 is a valuable, complementary tool for predicting genes in Chlamydomonas reinhardtii.

Two flagellar genes, AGG2 and AGG3, mediate orientation to light in Chlamydomonas.

Ciliary membranes have a large repertoire of receptors and ion channels that act to transduce information from the environment to the cell. Chlamydomonas offers a tractable system for dissecting the transport and function of ciliary and flagellar membrane proteins. Isolation of ergosterol and sphingolipid-enriched Chlamydomonas flagellar membrane domains identified potential signaling molecules by mass spectroscopy. These include a membrane protein and a matrix flavodoxin protein that are encoded by the AGG2 and AGG3 genes, respectively. Agg2p localizes to the proximal flagellar membrane near the basal bodies. Agg3p is distributed throughout the flagellar matrix, with an increased concentration in the proximal regions where Agg2p is located. Chlamydomonas cells sense light by using a microbial-type rhodopsin , transduce a signal from the cell body to the flagella, and alter the waveform of the flagella to turn a cell toward the light. Protein depletion by RNA interference reveals that both AGG gene products play roles in the orientation of cells to a directional light source. The depleted strains mimic the phenotype of the previously identified agg1 mutant, which swims away from light. We propose that the localization of Agg2p and Agg3p to the proximal region of the flagella may be important for interpreting light signals.

Comparative genomics identifies a flagellar and basal body proteome that includes the BBS5 human disease gene.

Cilia and flagella are microtubule-based structures nucleated by modified centrioles termed basal bodies. These biochemically complex organelles have more than 250 and 150 polypeptides, respectively. To identify the proteins involved in ciliary and basal body biogenesis and function, we undertook a comparative genomics approach that subtracted the nonflagellated proteome of Arabidopsis from the shared proteome of the ciliated/flagellated organisms Chlamydomonas and human. We identified 688 genes that are present exclusively in organisms with flagella and basal bodies and validated these data through a series of in silico, in vitro, and in vivo studies. We then applied this resource to the study of human ciliation disorders and have identified BBS5, a novel gene for Bardet-Biedl syndrome. We show that this novel protein localizes to basal bodies in mouse and C. elegans, is under the regulatory control of daf-19, and is necessary for the generation of both cilia and flagella.