In vitro studies have involved AID in apoptosis and gene transcription. In vivo studies, which utilize transgenic mice expressing AID in the forebrain, only support a role for AID in apoptosis but not gene transcription.Regulated intramembranous proteolysis of the amyloid-β precursor protein by the γ-secretase yields amyloid-β, which is the major component of the amyloid plaques found in Alzheimer's disease (AD), and the APP intracellular domain (AID). Here, we have further characterized several lines of AID transgenic mice by crossing them with human Tau-bearing mice, to determine whether over-expression of AID in the forebrain provokes AD-like pathologic features in this background. We have found no evidence that AID overexpression induces AD-like characteristics, such as activation of GSK-3β, hyperphosphorylation of Tau and formation of neurofibrillary pathology.Overall, these data suggest that AID transgenic mice do not represent a model that reproduces the overt biochemical and anatomo-pathologic lesions observed in AD patients. They can still be a valuable tool to understand the role of AID in enhancing the cell sensitivity to apoptotic stimuli, whose pathways still need to be characterized. Because of these evidence, various APP-derived fragments, such as AID/AICD Alzheimer disease (AD) is characterized by the production of amyloidogenic peptides, neurofibrillary tangles (NFT) and neurodegeneration in vivo, we have made transgenic mice expressing AID-peptides in the CNS in vivo. However, a role of AID in gene transcription in vivoMouse models are critical to explore both pathogenesis and therapy of human diseases. All animal models used to study human neurodegenerative diseases consist of transgenic mice carrying mutant forms of genes shown to be involved in human dementia Mice were handled according to the Ethical Guidelines for Treatment of Laboratory Animals of Albert Einstein College of Medicine. The procedures were described and approved in animal protocol number 20040707.AID mice were generated as described before The following monoclonal antibodies have been produced as described earlier DA9 (IgG1): total Tau aa102–140; TG5 (IgG1): total Tau aa220–240 (murine and human); CP27 (Ig2B): total Tau aa130–170 (human specific); CP13 (IgG1): pSer202; PHF1 (IgG1): pSer396/Ser404; MC1 (IgG1): N-terminal conformational change, Exon 10; CP17 (IgG3): pThr231; CP9 (IgM): pThr231. Antibodies were used pure (hybridoma medium), purified or biotinylated. Biotinylation of antibodies was performed using EZ-Link NHS-PEO Solid Phase Biotinylation Kit .Mice were sacrificed by cervical dislocation after Isoflurane anesthesia. One of the two brain hemispheres was processed for protein and Tau extraction, the other for immunohistochemistry. Forebrains were homogenized in 400µL/100mg of Homogenizing Buffer  for 20″ with a Polytron homogenizer, on ice. For heat stable Tau, to a 500µL aliquot of homogenate, NaCl was added to 250mM, and βME was added to 5% final concentrations, and the mix vortexed. Homogenates were heated at 100°C for 15′, mixed again by vortexing and cooled in ice for 30′, vortexed briefly again and spun down at 20,000g for 10′ at 4°C. The supernatant, containing heat stable microtuble protein enriched for Tau, was retained and used for subsequent experiments. Protein concentration was measured in the original homogenate in order to load equal amounts of protein for western blot and ELISA.Clear, flat bottom, 96 well plates were coated overnight with an antibody specific for total Tau (DA9) 2µg/mL in Coating Buffer . Wells were blocked with 100% Starting Block (Pierce) for 1 hour at room temperature. Homogenates were then added: brain homogenates, prepared as described above, were mixed 1/1 with 0.1% SDS, briefly vortexed and spun for 10′ at 20,000g, at room temperature, and the supernatant loaded on the ELISA plate at 1/250 to 1/2000 dilution in 20% Superblock-TBS (Pierce) as needed, and left overnight, at 4°C, gently shaking. Purified primary antibodies we added to the plate, diluted in 20% Superblock-TBS, as follow: TG5-biotinylated 1/500000, CP27 (Ig2B) 1/20000, CP13 (IgG1) 1/20000, PHF1-biotinylated 1/10000, CP17-biotinylated 1/20000, MC1 (IgG1) 1/20000, and left for 2 hours at room temperature, gently shaking. Secondary antibodies, Streptavidin-HRP or HRP-Goat-anti mouse IgG2B/IgG1, 1/5000 in 20% Superblock-TBS (Pierce), were left on the plate for 1 hour as above. Fifty µL of TMB (Pierce) were added and left for 20′, room temperature, light shielded, gently shaking, and the reaction was stopped with same volume of 2M Sulfuric Acid, and read at 450nM with an automated Tecan plate reader. Every step was followed by 5 automated washes with washing buffer .For Tau, heat stable preparations were run on a 4–12% tris-HCl precast gel (Biorad), blotted on a 0.45µM Nitrocellulose membrane and probed with monoclonal antibodies specific for total Tau or site specific Tau phosphorylations as follows: DA9 (1/3000), CP13 (1/3000CP9 (1/3000), PHF1 (1/3000), MC1 (1/500). Antibodies were diluted in 5% milk in TBS. Western Blots were developed either with the ECL system on film or by means of 4-Chloro-Naftol reaction on the membranes. For GSK, brains were homogenized as described for ELISA, subjected to PAGE as described above, and the membranes were probed with antibodies against GSK3α/β , GSK3βpSer9 , GSK3α pTyrα279/β216 (Invitrogen #44604G), diluted in 20% Superblock-TBS, and anti β-Actin (Sigma-Aldrich A1978) 1/8000 in 5% Milk-TBS-0.1%Tween20.th section inward (i.e. 350µM apart from each other). Free-floating sections were conserved in TBS /0.02%NaN3, and the staining was performed on multiwell plates. Endogenous peroxidases were quenched with 3% H2O2/0.25% Triton X-100/TBS for 30′. Non-specific binding was blocked with 5% Milk-TBS for 1 hour at room temperature. Primary antibodies are as follows: NeuN , IbA1 , and non-purified anti Tau antibodies DA9 (1/5000), CP13 (1/5000), PHF1 (1/5000), MC1 (1/200), all diluted in 5% Milk-TBS, and left overnight at 4°C, shaking. Biotin-conjugated secondary antibodies directed against the specific isotypes were diluted 1/1000 in 20% Superblock, left for 2 hours at room temperature, and likely Streptavidin-HRP was incubated for 1 hour. Staining was visualized by 3,3′-Diaminobenzidine. Each step was followed by 5×5′ washes in TBS.Brains were fixed in 4% PFA overnight and cut sagittally with a vibratome the next day. Selection of corresponding sections from each mouse was performed as follows: brains were cut sagittally at 50µM thickness, from the lateral to the medial aspect, preserving the cerebellum. For each antibody, 3 sections were stained, starting at section #15 , and taking every 7All quantified data represent an average of at least triplicate samples. Error bars represent standard errors of the mean. Statistical analysis was done by 2-Way ANOVA and Bonferroni Post Test. Significance was determined by Student's t test and a p<0.05 was considered significant.Mice are presenting with normal growth and weaning, thrive at appropriate age. No significant differences were observed in breeding and litter size and survival compared to C57BL/6 mice. Up to 21 months of age, there is no prevalent pathology, susceptibility to infections, and animals appear active and alert.in vitroTau accumulation, phosphorylation and axonopathy can be subtle and precede neuronal degeneration, tangle formation and cognitive or behavioural deficits CP13 recognizes an early Tau phosphorylation epitope (pSer202). This phopshorylation was decreased in line 57.13.3 at 4 and 7 months on WB , and alsOur biochemical data does not reveal a net trend of Tau phosphorylation induced by AID overexpression. Nonetheless, we wanted to ascertain if subtle changes could be seen at the tissue level, in both the quantity and the distribution of Tau phosphorylation. Furthermore, we have searched if any neuronal loss would be evident and if any microglia activation would be induced by AID overexpression. In Although the data presented do not hint to a role for AID in promoting Tau accumulation or deranged phosphorylation, it is still possible that transgenic AID may cause hyperphosphorylation of Tau at later time, by activating kinases. Another model of AID59 overexpression in vivo, in the CNS, we generated transgenic mice expressing CaMKIIα-AID, to target AID expression to areas that are most relevant to Alzheimer's pathology To directly examine the effects of AID in vivo AID expression could initiate aspects of AD that precede and lead to neuronal loss.A previous analysis of these mice showed that overexpression of AID did not affect gene transcription. However, cultured neuronal cells derived from AID transgenic mice were more sensitive to selective apoptotic and toxic stimuli. This evidence suggests that AID overexpression may predispose to neuronal degeneration. Thus, we tested whether in vivo by processing of APP-CTFs. This is problematic, for the reasons explained above. Although some evidence pointed to a role of AID as a “nuclear regulator” per se Contrarily to previous reports however PSEN1/PSEN2 that cause Familial Alzheimer's disease are loss of function mutants, and that loss of PSEN1/PSEN2 function causes neurodegeneration in mice, supports this hypothesis Do our data exclude a role for AID in AD? A common view for mechanisms underlying early pathophysiology of Alzheimer's disease (AD) includes axonopathy in vivo physiopathological context in which it is generated. Given these premises, AID and its functions should be investigated not much as a source of AD pathology, but as a possible interesting intracellular signaling pathway, much similar to NOTCH and NICD, that is still obscure but highly likely to be effective in neurons, possibly under stressing conditions.In conclusion, our transgenic model shows that overexpression of several isoforms of AID in the CNS fails to reproduce obvious signs of AD-like pathology in mice. These data question whether a generalized overexpression of this APP fragment can address its role in both biology and disease if it is disjointed from the


Chrna9L9′T line of knockin mice with a threonine for leucine change (L9′T) at position 9′ of the second transmembrane domain of the α9 nicotinic cholinergic subunit, rendering α9-containing receptors that were hypersensitive to acetylcholine and had slower desensitization kinetics. The Chrna9L9′T allele produced a 3-fold prolongation of efferent synaptic currents in vitro. In vivo, Chrna9L9′T mice had baseline elevation of cochlear thresholds and efferent-mediated inhibition of cochlear responses was dramatically enhanced and lengthened: both effects were reversed by strychnine blockade of the α9α10 hair cell nicotinic receptor. Importantly, relative to their wild-type littermates, L9′T/L9′TChrna9 mice showed less permanent hearing loss following exposure to intense noise. Thus, a point mutation designed to alter α9α10 receptor gating has provided an animal model in which not only is efferent inhibition more powerful, but also one in which sound-induced hearing loss can be restrained, indicating the ability of efferent feedback to ameliorate sound trauma.The transduction of sound in the auditory periphery, the cochlea, is inhibited by efferent cholinergic neurons projecting from the brainstem and synapsing directly on mechanosensory hair cells. One fundamental question in auditory neuroscience is what role(s) this feedback plays in our ability to hear. In the present study, we have engineered a genetically modified mouse model in which the magnitude and duration of efferent cholinergic effects are increased, and we assess the consequences of this manipulation on cochlear function. We generated the  Nicotinic cholinergic receptors are essential to higher order brain function. Structurally, these operate through a myriad of ligand-gated pentameric arrangements of different homologous subunits. Here, we report progress in understanding the structural properties of a neuronal nicotinic receptor at the synapse. Receptors assembled from two nicotinic cholinergic subunits (α9 and α10) serve exclusively at the synapse between central nervous system descending fibers and cochlear hair cells. This enabled us to show direct causality between a point mutation of the α9 subunit, and predicted alterations in the synaptic strength in sensory hair cells of the cochlea of α9 point mutant mice. Furthermore, this single mutation results in profound enhancement of central nervous system feedback to the cochlea. And finally, as a consequence, mutant mice possessing this altered receptor have substantially improved resistance to traumatic sound. Thus, central neuronal feedback on cochlear hair cells provides an opportunity to define one specific role that nicotinic receptors can play in the nervous system, enabling study from biophysical to behavioral levels and promoting a target for the prevention of noise-induced hearing loss. A point mutation in the cochlear hair cell nicotinic cholinergic receptor leads to strengthened central nervous system feedback to the cochlea and enhances protection from noise-induced hearing loss. In bringing information about the world to an individual, sensory systems perform a series of common functions. Each system responds with some specificity to a stimulus, and each one employs some specialized receptor cells at the periphery to translate specific stimuli into electrical signals that all neurons can use. That initial electrical event begins the process by which the central nervous system constructs an orderly representation of, for example, sounds, odors, tastes, and objects. Thus, basic sound detection begins when sound waves strike the eardrum, which transmits that physical stimulus to the organ of Corti within the cochlea, the sensory epithelium of the mammalian inner ear. Here, the primary receptor cells known as inner hair cells (IHCs) transform the information into electrical signals that are sent to the central nervous system by the auditory nerve . HoweverThe medial olivocochlear (MOC) efferents A origina2(α10)3 stoichiometry [2+ and the subsequent opening of small conductance Ca2+-activated K+ SK2 channels, thus leading to hyperpolarization of hair cells [In addition to their significance in auditory processing, efferent cholinergic synapses provide a unique opportunity to assess the function of neuronal nAChRs. In contrast to nAChR-expressing cells in the central nervous system , hair cehiometry –15. Currir cells ,17,  of the second transmembrane domain of the α9 subunit and examined the neurotransmitter responses, synaptic efficiency, and cochlear function of wt/wtChrna9, wt/L9′TChrna9, and L9′T/L9′TChrna9 mice. We show that the L9′T mutation produced an increase in sensitivity to ACh, and decreased rates of ACh-induced desensitization. More significantly, synaptic currents were dramatically prolonged in the OHCs of L9′T/L9′TChnra9 mice. Consistent with these effects, L9′T/L9′TChnra9 mice had elevated acoustic thresholds, and shock-evoked MOC activation produced both enhanced and prolonged cochlear suppression. This enhanced efferent inhibitory drive attenuated sound-induced, permanent acoustic injury. Our results establish efferent feedback inhibition's function in preventing acoustic trauma, and provide preliminary characterization of a mouse model to explore additional physiological functions of MOC innervation. In addition, it motivates further exploration of the efferent synapse as a model system to study fast neurotransmission mediated by a neuronal nAChR.In the present study, we have generated a genetically modified mouse model in which the magnitude and duration of the MOC efferent effect is increased, and assessed the consequences of this manipulation on auditory thresholds and susceptibility to noise-induced hearing loss. We have substituted a threonine for a leucine at position 9′ .The targeting construct and generation of cochleae . As asse9′T mice . In addi9′T mice . Lastly,currents . Hair ceXenopus oocytes, α9α10 nAChRs assembled from mutant α9L9′T subunits exhibit an increased sensitivity to ACh, a reduced desensitization rate, and an increased channel mean open time [Chrna9L9′T mutant mice.In pen time . To detepen time ,20. Sincwt/L9′TChrna9 and L9′T/L9′TChrna9 mice responded to ACh at P6–P10. In each case, ACh dose-response curves of IHCs were left-shifted when compared to wild type, with a concomitant decrease in the half maximal effective concentration (EC50). The EC50 was significantly different (p < 0.01) between homozygous mutant and wild-type IHCs . During prolonged (>60 s) application of 1 mM ACh at −90 mV , 0.45 ± 0.05 in wt/L9′TChrna9 , and 0.48 ± 0.06 in  L9′T/L9′TChrna9 .As observed in t −90 mV B, the cuτdecay, no difference in τrise, and a 3-fold increase in the duration at half amplitude . In addition, the sIPSCs in L9′T/L9′TChrna9 IHCs had significantly smaller amplitudes than those of wild-type IHCs. Despite the amplitude reduction, overall charge transfer  of the “nAChR-only” synaptic current was more than doubled in L9′T/L9′TChrna9 when compared to wt/wtChrna9 IHCs.Following efferent transmitter release in vitro, spontaneous inhibitory postsynaptic currents (sIPSCs) can be measured in neonatal IHCs. As determined for MOC synapses located on OHCs, the IHC inhibitory postsynaptic currents (IPSCs) result from ion flux through α9α10 nAChRs and calcium-activated SK2 channels ,20, FigC. To onlChrna9L9′T mutant mice. At −90 mV, sIPSCs of L9′T/L9′TChrna9 mice were dramatically prolonged compared to wt/wtChrna9, with a 4- and 3-fold increase in the τdecay and halfwidth, respectively. At −40 mV, τdecay and halfwidth were 3-fold greater than in wild type, and a slight increase in τrise also was observed. The peak current at −40 mV, carried by SK2 channels, was equivalent in wt/wtChrna9 and L9′T/L9′TChrna9 IHCs. Nonetheless, the smaller amplitude nAChR-only current observed in IHCs of mutant mice activates SK2 channels as effectively as in wild-type IHCs. Indeed, the area under the L9′T/L9′TChrna9 SK2 current was nearly three times greater than that of the wt/wtChrna9 SK2 current. Presumably, this enhancement results from the increased total calcium ion flux during the prolonged activity of the nAChRs in mutant mice.When sIPSCs were recorded with KCl-EGTA in the pipette, currents were inward at −90 mV and outward at −40 mV B. As not+ in IHCs was reversibly blocked by the α9α10 blocker strychnine (300 nM) [L9′T/L9′TChrna9 was in fact mediated through mutant α9α10 nAChRs.As shown in (300 nM) , indicat+ plus EGTA, minus apamin) to depolarize the efferent terminals . Analysis of waveforms revealed a 4- and 3.5-fold increase in τdecay and halfwidth, respectively, and a 0.6-fold reduction in amplitude  was 2-fold larger in L9′T/L9′TChrna9 compared to wt/wtChrna9 OHCs.Since hair cell sIPSCs were infrequently observed, synaptic currents and the effects of exogenous ACh were measured in P10–P11 OHCs using high-potassium buffer  and distortion product otoacoustic emissions (DPOAEs). Wave 1 of the ABR, which is recorded from scalp electrodes in response to short tone pips, represents the summed activity of cochlear nerve fibers projecting from IHCs  = 4.30, p = 0.043) and L9′T/L9′TChrna9  = 7.87, p = 0.06) when compared to wt/wtChrna9 mice. DPOAE thresholds  = 14.82, p < 0.001) and L9′T/L9′TChrna9  = 9.20, p = 0.003). Threshold shifts were larger at frequencies above 16 kHz and were similar in magnitude whether measured by ABRs or DPOAEs, suggesting that all dysfunction can be explained by changes in OHC contributions to cochlear amplification.As shown in resholds B were alL9′T/L9′TChrna9 mice by almost 9 dB, thus restoring the baseline thresholds to that seen in the wild type.Since MOC activity in vivo decreases OHC-based amplification of cochlear responses , we askeChrna9L9′T mutant mice, suppression had a much slower onset , suppression in L9′T/L9′TChrna9 resembled the normal wt/wtChrna9 response  = 8.894, p = 0.018; wt/L9′TChrna9: F = 6.263, p = 0.041). With a lower intensity exposure designed to produce a TTS, there was no significant difference in vulnerability between wild-type and mutant mice, when examined 12 h after exposure  enhances nAChR function in cochlear hair cells , increases synaptic efficacy, and dramatically strengthens cochlear suppression in vivo. More importantly, it shows that the MOC efferent system inhibits cochlear sensitivity and protects the inner ear from acoustic injury. Thus, the efferent pathway provides a promising target for pharmacological prevention of inner ear pathologies derived from acoustic injury, such as hearing loss and tinnitus.The present study demonstrates that substitution of a single amino acid in transmembrane region 2 of the α9 subunit  are expressed in OHCs at high levels, similar to those found in skeletal muscle [Thus, the cholinergic synapse between MOC terminals and cochlear hair cells provides a valuable model for the study of fast neurotransmission mediated by nAChRs of known composition ,13. Targkinetics . Both thkinetics  and thatkinetics ,33. Morekinetics . Finally neurons  and apop neurons , respect to Ca2+ ,38 pointl muscle .L9′T mutants can be “rescued” via strychnine, the potent α9α10 nAChR blocker, suggests that it arises from an enhancement of cholinergic effects on OHCs. Therefore, compared to Chrna9 and Chrna10 knockouts, in which baseline cochlear thresholds were normal [L9′T mutant receptors in the absence of ACh [The main peripheral effect of the MOC activity is to inhibit cochlear responses by decreasing the gain of the cochlear amplifier . MOC neue normal ,41, the e of ACh . The appe of ACh , may alsL9′T mutant mice in the present study show (1) ACh-evoked currents with a greatly reduced desensitization rate, and (2) spontaneous miniature synaptic currents with slower activation and decay kinetics. These changes in ACh response kinetics provide likely explanations for some aspects of the electrically evoked suppression of DPOAEs. The doubling of onset time for the ACh-evoked SK2 current in mutant hair cells  is too large to be explained only by the 2- to 3-fold prolongation of synaptic currents observed in the mutant hair cells. Another contributing factor may be the observation that choline, the metabolite produced when acetylcholinesterase degrades ACh released at the synapse, is a full agonist of the mutant α9α10 receptor, and only a weak partial agonist of the wild-type receptor [2+ influx through mutated α9α10 receptors leads to changes in prestin structure or function resulting in prolonged MOC efferent effects.In addition to rising more slowly, MOC-mediated cochlear suppression was also larger and longer lasting in receptor . Thus, ireceptor , a low creceptor . Stiffnereceptor ,45. Thus+ conductance in neighboring SK2 channels and the effects of the resultant OHC hyperpolarization on the magnitude of electromotility and thus on cochlear vibration amplitude. The slow suppression, with an onset time course of approximately 10 s, may require a wave of calcium-induced calcium release along the OHC membrane and appears to reflect a change in OHC stiffness [The presence of mutant α9L9′T receptors, designed to increase the magnitude of MOC effects on OHCs, also increased the protective action of the MOC system in vivo. This is consistent with previous work showing that overexpression of wild-type α9 channels, which more modestly increased the magnitude of MOC-mediated DPOAE suppression, also increased the resistance of the ear to acoustic injury . Prior itiffness  that alstiffness , althougThere are two fundamentally different ways in which slow effects of α9 activation could reduce acoustic injury: (1) by reducing mechanical vibration of the sensory epithelium, or (2) via intracellular modifications of OHCs arising as other downstream effects of the calcium entry through α9α10 receptors. The further observation that in the α9L9′T mutants, protection was seen only for exposures intense enough to produce irreversible damage and permanent threshold shifts, and not in less traumatic exposures producing only reversible changes, argues against a general reduction of vibration amplitude as the underlying mechanism. Most of the permanent threshold elevation underlying this type of noise exposure arises from damage to the hair cell stereocilia bundles, including disarray loss and/or fusion of these modified microvilli , which hThis work shows that a point mutation in the hair cell's nAChR produces dramatic prolongation of efferent MOC inhibitory effects at both cellular and systems levels. This alteration provides enhanced protection from permanent acoustic trauma, indicating that cholinergic synaptic feedback is not only necessary, but sufficient for this effect. In addition, the enhanced activity of the modified nAChR revealed a novel tonic inhibitory effect, raising baseline acoustic thresholds over those of wild-type littermates, confirming the inhibitory nature of the MOC efferent system. Thus, this α9L9′T knockin presents new insights into the cellular mechanisms of cholinergic inhibition, as well as a promising model in which to probe the functional role of MOC efferents. In addition, the hair cell's efferent synapse, much like the neuromuscular junction, can provide insights into cholinergic signaling, and promises to be an equally informative model for studying activity-dependent synaptic function.Chrna9 exons 1–4 (see GenBank accession number NT_039305.7 and Chrna9L9′T mutation (*) introduced via site-directed mutagenesis using the QuickChange Site-Directed Mutagenesis kit (Stratagene) and amplimers A9sense (5′-CTCTGGGAGTGACCATCCTAacGGCCATGACTGTATTTCAGC-3′) and A9antisense (5′-GCTGAAATACAGTCATGGCCgtTAGGATGGTCACTCCCAGAG-3′). This targeting vector was used to electroporate 129S4/SvJae embryonic stem (ES) cells, and homologous recombinants were obtained following gentamicin (G418) selection and Southern blot hybridization analyses. Genomic DNA was purified from G418-resistant ES cell clones, digested with HindIII and KpnI, electrophoresed on 0.8% agarose, and hybridized to a 32P-labled DNA 962-bp SacII-KpnI fragment probe prepared from the DNA fragment shown in Chrna9 subunit gene (see GenBank accession number NT_039305.7), wild-type ES cell DNA yielded a fragment of 13,800 bp, whereas ES cells that have undergone homologous recombination yielded a 7,300-bp fragment C5379Lmgd/J, stock 003314; Jackson Laboratory) to remove the “floxed” neomycin resistance cassette. The excision of the neo cassette and subsequent segregation and loss of the cre transgene were monitored via PCR. The single loxP site footprint and flanking regions of the targeted allele in neo-deleted mice were confirmed by DNA sequencing.A HindIII-NotI (the latter derived from the construction of the library) restriction endonuclease fragment of approximately 9,500 bp encoding ment see B. TransfChrna9L9′T mutant allele has been maintained in congenic FVB.129P2-Pde6b+ Tyrc-ch/AntJ  strain. C57BL/6J mice develop a marked and progressive late-onset hearing loss characterized by cochlear degeneration. The FVB background lacks this hearing loss. Moreover, the wild-type Pde6b allele avoids blindness due to retinal degeneration typical of the FBV strain. All experiments reported in this paper were performed using neo-deleted wt/wtChrna9, wt/L9′TChrna9, or L9′T/L9′TChrna9 mutant mice backcrossed with congenic FVB.129P2-Pde6b+ Tyrc-ch/AntJ stock for four to five generations .The Chrna9 mice was performed using tail biopsy tissue DNA samples , amplimers A9LOXP.1 (5′-TAC TGG CTA TCC TCC AGA CAG AGC-3′) and A9LOXP.2 (5′-AGG AGC GAG CAG AGG TCC TAA ATT-3′)  and L9T3′ (5′-CCGCACACATACAGGGTTCGAT-3′) (Routine genotyping of -3′) see A, and Falele see C. In eacCGAT-3′) D.AVMA Guidelines on Euthanasia (June 2007). Apical turns of the organ of Corti were excised from mice at postnatal ages P6–P11 for IHCs and P10–P11 for OHCs, and used within 3 h. Day of birth was considered postnatal day 0 (P0). Cochlear preparations were mounted under a Leica DMLFS microscope (Leica Microsystems) and viewed with differential interference contrast (DIC) using a 40× water immersion objective and a Hamamatsu C7500–50 camera with contrast enhancement (Hamamatsu). Methods to record from IHCs and OHCs were essentially as described previously [Mice were sacrificed by decapitation. All experimental protocols were carried out in accordance with the American Veterinary Medical Associations' eviously ,19.+ and K+ currents [2, 0.9 MgCl2, 0.7 NaH2PO4, 5.6 d-glucose, and 10 Hepes buffer (pH 7.4). Two different pipette solutions were used. One contained (in mM): 150 KCl, 3.5 MgCl2, 0.1 CaCl2, 5 ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′- teraacetic acid (EGTA), 5 Hepes buffer, 2.5 Na2ATP, pH 7.2 . In the other, EGTA was replaced with 10 mM bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA) . The latter solution was used where indicated, in order to record the current through the ACh receptor in isolation from the coupled SK2 current (nAChR-only). In addition, in the latter condition, the SK blocker apamine (5 nM) was added to the recording solution.Briefly, hair cells were identified visually with the 40× objective and during recordings, by the size of their capacitance (7 to 12 pF), by their characteristic voltage-dependent Nacurrents . Some ce+ were applied by a gravity-fed multichannel glass pipette (∼150 μm tip diameter), positioned about 300 μm from the recorded cell. All working solutions containing either ACh or elevated K+ or both, were made up in a saline containing low Ca2+ (0.5 mM) and no Mg2+ so as to optimize the experimental conditions for measuring currents flowing through the α9α10 receptors [2+ and no Mg2+. All experiments designed to record synaptic currents, either spontaneous or evoked with high K+, were done using an extracellular solution containing 1.3 mM Ca2+ and no Mg2+. sIPSCs were recorded immediately after rupturing the cell.Solutions containing ACh or high Keceptors . sIPSCs τrise and τdecay were fit to a monoexponential.Glass pipettes, 1.2-mm I.D., had resistances of 5–8 MΩ. Currents in both IHCs and OHCs were recorded in the whole-cell patch-clamp mode with an Axopatch 200B amplifier, low-pass filtered at 2–10 kHz, and digitized at 5–20 kHz with a Digidata 1322A board (Molecular Devices). Recordings were made at room temperature (22–25 °C). Holding potentials were not corrected for liquid junction potentials or for the voltage drop across the uncompensated series resistance. IPSCs were analyzed with Minianalyis . IPSCs were identified using a search routine for event detection and confirmed by eye. The individual events of each cell were aligned and averaged using Minianalysis. Rise time was used as the criterion to align. For further analysis, Prism 4 (GraphPad Software) was used. The maxI/I = nA/(nA + EC50n), where I is the peak inward current evoked by agonist at concentration A; maxI is current evoked by the concentration of agonist eliciting a maximal response; EC50 is the concentration of agonist inducing half-maximal current response, and n is the Hill coefficient.Prolonged agonist application responses and concentration-response curves to ACh in IHCs were performed in the nAChR-only condition, normalized to the maximal agonist response, and iteratively fitted with the equation: 1–f2 were recorded with a custom acoustic assembly consisting of two electrostatic drivers  to generate primary tones (f1 and f2 with f2/f1 = 1.2 and f2 level 10 dB < f1 level) and a Knowles miniature microphone (EK3103) to record ear-canal sound pressure. Stimuli were generated digitally, while resultant ear-canal sound pressure was amplified and digitally sampled at 4 μs . Fast Fourier Transforms were computed and averaged over five consecutive waveform traces, and 2f1–f2 DPOAE amplitude and surrounding noise floor were extracted. Iso-response curves were interpolated from plots of amplitude versus sound level, performed in 5-dB steps of f1 level. “Threshold” is defined as the f1 level required to produce a DPOAE with amplitude = 0 dB sound pressure level (SPL). For measurement of ABRs, needle electrodes were inserted at vertex and pinna, with a ground near the tail. ABRs were evoked with 5-ms tone pips . The response was amplified , filtered (100 Hz–3 kHz), and averaged with an A-D board in a LabVIEW-driven data-acquisition system. Sound level was raised in 5-dB steps from 10 dB below threshold to 80 dB SPL. At each level, 1,024 responses were averaged , using an “artifact reject” whereby response waveforms were discarded when peak-to-peak amplitude exceeded 15 μV. Upon visual inspection of stacked waveforms, “threshold” was defined as the lowest SPL level at which any wave could be detected, usually the level step just below that at which the response amplitude exceeded the noise floor (∼0.25 μV). For amplitude versus level functions, wave-I peak was identified by visual inspection at each sound level and the peak-to-peak amplitude computed.Mice were anesthetized with xylazine (20 mg/kg i.p.) and ketamine (100 mg/kg i.p.). DPOAEs at 2f2 level was set to produce a DPOAE 10–15 dB or 20–25 dB greater than the noise floor. To measure MOC effects, repeated measures of baseline DPOAE amplitude were first obtained (n = 56), followed by a series of 70 contiguous periods in which DPOAE amplitudes were measured with simultaneous shocks to the MOC bundle and additional periods during which DPOAE measures continued after the termination of the shock train.Mice were anesthetized with urethane (1.20 g/kg i.p.) and xylazine (20 mg/kg i.p.). A posterior craniotomy and partial cerebellar aspiration were performed to expose the floor of the IVth ventricle. To stimulate the MOC bundle, shocks  were applied through fine silver wires (0.4-mm spacing) placed along the midline, spanning the olivocochlear decussation. Shock threshold for facial twitches was determined, muscle paralysis induced with α-d-tubocurarine (1.25 mg/kg i.p.), and the animal connected to a respirator via a tracheal cannula. Shock levels were raised to 6 dB above twitch threshold. During the MOC suppression assay, fAnimals were exposed free-field, in a small reverberant chamber. Acoustic trauma consisted of a 2-h exposure to an 8–16-kHz octave band noise presented at 100 dB SPL (for permanent injury) or a 15-min exposure to the same noise at 94 dB SPL (for temporary injury). For the higher level exposure, animals were anesthetized , because many mutant animals experienced audiogenic seizures as soon as the high-level noise was turned on. The exposure stimulus was generated by a custom white-noise source, filtered , amplified (Crown power amplifier), and delivered (JBL compression driver) through an exponential horn fitted securely to a hole in the top of a reverberant box. Sound exposure levels were measured at four positions within each cage using a 0.25'' Bruel and Kjaer condenser microphone: sound pressure was found to vary by less than 0.5 dB across these measurement positions.t-test in the case of pairwise comparisons or a one-way ANOVA followed by a Dunnet test for multiple comparisons. In the case of in vivo data, a two-way ANOVA was performed. A p < 0.05 was selected as the criterion for statistical significance. Mean values are quoted as means ± the standard error of the mean (S.E.M.).Statistical analyses of in vitro electrophysiology experiments were carried by the Student 2ATP, BAPTA, and all other reagents were from Sigma Chemical. EGTA and Na2ATP were dissolved at the moment of preparing the intracellular solutions.ACh chloride, strychnine HCl, NaAll experimental protocols were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals as well as Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI), Tufts University, and Massachusetts Eye and Ear Infirmary Institutional Animal Care and Use Committee (IACUC) guidelines, and best practice procedures.Figure S1L9′T/L9′TChrna9 cochleae showed no morphological changes when compared to wt/wt.Chrna9 Sections through the middle turn are shown. Black and grey arrows in wt/wtChrna9 indicate OHCs and IHCs, respectively. Histological procedures were performed as described in Gross histological assessment via plastic sections of osmium-stained (2.38 MB TIF)Click here for additional data file.Figure S2wt/wt; nChrna9 = 10 animals, 13 cells; wt/L9′TChrna9: n = 9 animals, 13 cells; and L9′T/L9′TChrna9: n = 14 animals, 17 cells); (B) same as in (A), but for P10–P14 OHCs .(A) Voltage-dependent currents obtained in P6–P10 IHCs by holding the cell at −80 mV and stepping the voltage to 80 mV in 10-mV increments (200-ms duration). Representative traces are shown for the three genotypes as well as for averaged steady state current-voltage curves ((225 KB TIF)Click here for additional data file.Figure S32 = 22.6 kHz). All data were collected 60 min after strychnine injection. Although thresholds in wild-type mice slightly rise with strychnine dosage, thresholds in knockin mice improve by as much as 8 dB.Each point shows data from different animals extracted from runs such as those shown in (117 KB TIF)Click here for additional data file.Table S1Chrna9: gene encoding the α9 nAChR subunit; Chrna10: gene encoding the α10 nAChR subunit; Kcnn2: gene encoding the SK2 channel; Ryr1: gene encoding the ryanodine receptor 1; Ryr2: gene encoding the ryanodine receptor 2; Ryr3: gene encoding the ryanodine receptor 3; Kcnmb1: gene encoding the calcium-activated potassium channel (BK) β-1subunit; Cacna1d: gene encoding the voltage-gated calcium channel alpha subunit CaV1.3; Myo7a: gene encoding myosin VIIa. CTs: threshold cycle of sample; CTm: threshold cycle of Myo7a. Results shown are the mean ± S.E.M of three experiments per group, each performed in triplicates. The protocol for quantitative PCR was as indicated in (88 KB DOC)Click here for additional data file.Text S1(24 KB DOC)Click here for additional data file.Text S2(27 KB DOC)Click here for additional data file.


Uridine has been advocated for the treatment of HIV-1/HAART-associated lipodystrophy (HALS), although its metabolism in HIV-1-infected patients is poorly understood.Plasma uridine concentrations were measured in 35 controls and 221 HIV-1-infected patients and fat uridine in 15 controls and 19 patients. The diagnosis of HALS was performed following the criteria of the Lipodystrophy Severity Grading Scale. Uridine was measured by a binary gradient-elution HPLC method. Analysis of genes encoding uridine metabolizing enzymes in fat was performed with TaqMan RT-PCR.Median plasma uridine concentrations for HIV-1-infected patients were 3.80 µmol/l (interquartile range: 1.60), and for controls 4.60 µmol/l (IQR: 1.8) (P = 0.0009). In fat, they were of 6.0 (3.67), and 2.8 (4.65) nmol/mg of protein, respectively (P = 0.0118). Patients with a mixed HALS form had a median plasma uridine level of 4.0 (IC95%: 3.40–4.80) whereas in those with isolated lipoatrophy it was 3.25 (2.55–4.15) µmol/l/l (P = 0.0066). The expression of uridine cytidine kinase and uridine phosphorylase genes was significantly decreased in all groups of patients with respect to controls. A higher expression of the mRNAs for concentrative nucleoside transporters was found in HIV-1-infected patients with respect to healthy controls.HIV-1 infection is associated with a decrease in plasma uridine and a shift of uridine to the adipose tissue compartment. Antiretroviral therapy was not associated with plasma uridine concentrations, but pure lipoatrophic HALS was associated with significantly lower plasma uridine concentrations. Specifically, we postulated that the toxic effects of thymidine analogues might result in pyrimidine depletion, detectable as a reduction of systemic and/or fat uridine concentrations. Therefore, the aim of the present study was to look for a relationship between plasma and fat uridine concentrations and HIV-1/highly active antiretroviral (HAART)-associated lipodystrophy syndrome (HALS).Hospital de la Santa Creu I Sant Pau, which attends a population of 1455 HIV-1-infected patients on active follow-up, and were consecutive patients with an established diagnosis of HIV-1 infection. Patients were eligible whether or not they had HALS, whether or not they were on antiretroviral therapy, and if they were not taking uridine or derivatives. Subjects who were hospitalized or had a frank cognitive impairment such as delirium or dementia on enrolment were not eligible. Patients with opportunistic infections, acute hepatitis, liver insufficiency, neoplasms or fever of undetermined origin were excluded from the study. At the time of study entry no patient used any other drug known to influence glucose metabolism or fat distribution. Informed consent was obtained from the patients at study entry. The diagnosis of AIDS was based on the 1993 revised case definition of the Centers for Disease Control and Prevention (CDC) Hospital de la Santa Creu i Sant Pau.All patients were recruited through the same clinic at the Body composition measurements, definitions of HALS and metabolic syndrome have been described elsewhere These were obtained from subcutaneous adipose tissue (SAT) depots through a small surgical biopsy performed by an 8 mm punch under local anesthesia with mepivacaine. One half of the SAT obtained was immediately frozen in liquid nitrogen and stored at −80°C until RNA extraction (see below). The remaining was used in fresh for determining fat uridine concentrations.2; plus Complete-Mini™ protease inhibitor cocktail, Roche), in an ice-cold bath. After centrifugation, the supernatant was saved and protein concentration was determined by the Bradford method For uridine determination, anticoagulated blood was centrifuged at 1,500× g and plasma was separated and kept at −20 C until analysis. Fat tissue was homogenated in the presence of 10 volumes  of homogenization buffer  to eliminate enzymatically the uridine peak. The area from residual peaks coeluting with uridine and still present after treatment with E. coli thymidine phosphorylase was substracted from the area of the uridine peak obtained in the untreated aliquot, to ensure no overestimation of uridine concentration. The quantitation of the nucleoside was based on peak areas using external aqueous standards. The method had a between-day imprecision of 2.3% (coefficient of variation) for a uridine concentration of 4.30 µmol/l. Uridine concentrations in plasma of ∼3 to 5 µmol/l are found in different species and individuals Uridine was measured by a binary gradient-elution HPLC method. 200 µl of plasma or tissue homogenate were deproteinized by the addition of 9 µl of cold perchloric acid 11.7 mol/l  and kept 5 min on ice. Precipitated proteins were eliminated by centrifugation and 5 µl of the supernatant were injected into an Acquity UPLC apparatus  and eluted at 0.5 ml/min with a saline buffer eluent A  and an organic eluent B (methanol gradient grade) according to the following gradient: 0 to 1.1 min, 100% eluent A; 1.1 to 6.1 min, 100% to 82.6% eluent A; 6.1 to 6.2 min, 82.6 to 100% eluent A; 6.2 to 7.2 min, 100% eluent A. The column used was an Acquity UPLC BEH C18 column 100×2.1 mm, 130 Å pore size, 1.7 µm particle size (Waters). Optical absorbance of the eluate was monitored at 267 nm and definitive identification of the uridine peak was based upon retention time and treatment of a second aliquot of the sample with a large excess of purified RNA extraction was performed using a column-affinity based methodology . On-column DNA digestion was performed during RNA purification . TaqMan Reverse Transcription and RT-PCR reagents were used for mRNA analysis . One microgram RNA was transcribed into cDNA random-hexamer primers and the real-time reverse transcriptase-polymerase chain reaction was performed on the ABI PRISM 7700HT sequence detection system (Applied Biosystems). The TaqMan RT-PCR reaction was performed in a final volume of 25 µl using TaqMan Universal PCR Master Mix, NoAmpErase UNG reagent and the specific gene expression primer probes. The TaqManGene Expression assays (Applied Biosystems) used were: uridine-monophosphate synthase, Hs00923516; uridine cytidine kinase-1, Hs00258815; uridine phosphorylase, Hs00427695; uridine 5′-monophosphate hydrolase , Hs00826433; concentrative nucleoside transport (CNT-1), Hs00984402; CNT-2, Hs01035852; CNT-3, Hs00223220. Controls with no RNA, primers, or reverse transcriptase were included in each set of experiments. Each sample was run in duplicate and the amount of the mRNA for the gene of interest in each sample was normalized to that of the reference housekeeping control , as already reported The serum composition of fatty acids was determined using the method by Lepage and Roy Samples were then heated for 60 minutes at 100°C in a heating block and shaking continuously at 600 rpm. After the tubes had been cooled to room temperature, five millilitres of 6% (w/v) potassium carbonate were then added. The samples were vortexed for 30 seconds and centrifuged at 2500 rpm for 20 minutes at 15°C. The fatty acid methyl esters contained in the upper benzene phase were transferred to gas chromatography vials and stored at 4°C until injection into the chromatograph.The analysis was performed on a Varian CP-3900 gas chromatograph equipped with a flame ionization detector, using a capillary column model CP9205-VF-WAXms (Varian), 30 m length ×0.25 mm internal diameter ×0.25 µm film thickness. Individual fatty acids were identified by order of elution and upon comparison with known commercially prepared fatty acid standards . Fatty acid methyl ester peaks were identified by comparison of retention times of standards and quantified in comparison to known commercially prepared reference standards. The percentage of each fatty acid class was expressed as percentage of total fatty acids.All analyses were performed with the Statistical Package for Social Sciences version 17.0 . Data are expressed as mean ± SD or median with interquartile range (IQR). Data that were not normally distributed, as determined using the Kolmogorov-Smirnov test, were logarithmically transformed before analysis. Student's t test was used for comparison between two groups, Pearson's correlations or one-way ANOVA and multiple testing were corrected using Bonferroni correction. Stepwise logistic regression analysis was used to examine the association of plasma uridine concentrations and other parameters with HALS. The variables selected to enter into stepwise regression were those that correlated significantly with plasma uridine concentrations (after Bonferroni correction for multiple testing). In all statistical tests, P values <0.05 were considered significant.Two-hundred and twenty one HIV-1-infected patients and 35 healthy controls were studied. The demographics, HIV-1 infection, and antiretroviral exposure parameters from patients and controls are shown in 10 copies/ml (IQR: 1.1 log10 copies/ml). The mean CD4 count was 589±320 cells/mm3 (median: 531 [IQR. 425] cells/mm3). Nadir CD4 cell count was <100 cells/mm3 in 57 patients (25.8%). The cumulated exposure to antiretroviral drugs is shown in Among treated patients, 59 (41.3%) had undetectable viral load at the moment of the study. The other 84 treated patients presented a median detectable viral load of 2.3 logHALS, based on 7 or more points on the Lipodystrophy Scale Grading Score (LSGS), was diagnosed in 84 treated patients (58.7%) whereas absence of HALS was found in 59 treated patients (41.3%), and in all the naives. All 84 patients presented with lipoatrophy, but in addition 44 52.4%) had a mixed lipoatrophic-lipohypertrophic form. Differences between HALS and non-HALS patients are shown in .4% had a3, did or did not have not clinical toxicity , HALS, HCV infection, and metabolic syndrome with respect to plasma uridine concentrations. Lean subjects had median plasma uridine concentrations of 3.7 [IQR: 1.5], overweight subjects 4.1 [IQR: 1.8], and obese subjects 3.9 [IQR: 1.67] µmol/l (P = 0.0152 for lean vs. obese). The correlations of plasma uridine concentrations with metabolic, infectious and treatment factors are shown in Plasma uridine concentrations for HIV-1-infected patients ranged from 1.60 to 7.4 µmol/l/l, with a mean value of 3.85±1.18 µmol/l . The corresponding values for controls were: range: 2.0–9.4 µmol/l, mean: 4.36±1.28 µmol/l, median: 4.60, IQR: 1.80 µmol/l (P = 0.0009). Plasma uridine concentrations were not significantly different between HALS and non-HALS patients (P = 0.3377). Patients with plasma uridine concentrations <3 µmol/l were 8.9% among controls, 16.7% among naïves, 19.0% among treated patients without HALS, and 29.6% among patients with HALS (P = 0.0460). There was no sex difference in plasma uridine concentrations for HIV-1-infected patients, but there were statistically significant differences between controls . In naïve patients, there was a negative correlation between plasma concentrations of uridine and viral load . Patients who were currently receiving treatment with AZT or d4T had a median plasma uridine level of 3.90 [IQR: 1.67] whereas in those who did not it was 3.70 , and in controls, 2.8 [4.65] nmol/mg protein (P = 0.0118). Median fat uridine level was 6.1 [0.50], 2.6 [1.85], and 6.3 [3.4] nmol/mg protein, for naïve, non-HALS patients, and HALS patients, respectively (P = 0.3767). There was a negative correlation between plasma and fat uridine concentrations in HIV-1-infected patients , whereas such a correlation was not found for controls . Median fat uridine level was 5.5 [IQR: 4.0] nmol/mg protein for those who were currently treated with AZT or d4T, whereas it was 2.6 [IQR: 1.27] nmol/mg protein for those not treated (P = 0.5369). There were no statistically significant differences between patients with mixed HALS and pure lipoatrophic HALS with respect to fat uridine concentrations 6.80 [IQR: 0] vs. 6.55 [IQR: 0.50] nmol/mg protein, respectively, P = 0.4028). The only correlations were for EFV exposure , and for PUFAs level .The expression of genes involved in uridine metabolism is depicted in Our study does not suggest an association of the development of features of HALS or metabolic syndrome with plasma and fat concentrations of uridine, except for lipohypertrophy, but suggests that HIV-1 infection is associated with a significant decrease in uridine plasma concentrations. However, the number of patients with uridine <3 µmol/l was higher in patients with than in non-HALS patients. Antiretroviral therapy that included thymidine analogues did not influence uridine plasma concentrations. On the other hand, fat uridine concentrations showed an inverse correlation with plasma concentrations. This finding may be explained by an over-expression of those genes that encode concentrative nucleoside transporters, and particularly CNT3 However, the present results have to be viewed in the light of their inherent limitations. The first one comes from the nature of the study; this is a cross-sectional study and therefore, no causal relationships can or should be drawn. Second, for the majority of patients, uridine was measured in plasma whereas the anatomical and biological processes giving rise to HALS occur in the adipose depots. We have some measurements of uridine in fat, and although such measurements are highly reproducible and with tight variability coefficients, their reliability is difficult to ascertain E. coli, which ensures that we only measured true uridine, with no interfering compounds eluting at the same retention time in the chromatogram. This may explain why plasma uridine values obtained in our patients with HALS are lower than those reported by Sutinen et al. The plasma uridine results are trustworthy since we were especially careful to avoid overestimations of the uridine peak, as we have observed that some unknown compounds sometimes coelute with uridine in our chromatographic separation. This fact could explain, at least in part, the values slightly higher for uridine plasma concentration reported by other groups We have found that HIV-1 infection is associated with significantly lower concentrations of uridine in plasma, whereas there were not differences between HIV-1-infected patients irrespective of their treatment status, but it was by the presence of lipoatrophic HALS. This may be explained by the fact that, unlike what happens in non-infected patients, there is a shift of uridine towards adipose depots and maybe other tissues. Additionally, among naïve patients, a negative correlation was found between uridine plasma concentrations and the amount of plasma HIV-1 RNA. This may suggest its consumption during the HIV-1 replicative cycle. On the other hand, alterations due to HIV-1-infection in adipose tissue from patients, i.e. reduction in the amounts of mitochondrial DNA Uridine supplementation has been advocated as a successful treatment for thymidine analogues-related lipoatrophy based on a short-term randomized, double-blind, placebo-controlled trial The concentrations of uridine in animal experimentation tissues are usually far in excess of the concentration of uridine in plasma In summary, our results suggest that uridine homeostasis is profoundly disturbed by HIV-1 infection. Although plasma concentrations in HIV-1-infected patients were lower than in controls, the only additional uridine disturbance that could be documented was unrelated to antiretroviral therapy but it was to the development of pure lipoatrophic syndrome. These results are in agreement with the lack of efficacy of uridine supplements in HALS reversal as shown by ACTG 5229.File S1(0.03 MB DOC)Click here for additional data file.


Wittig olefination–Claisen rearrangement protocol was applied to obtain 3-allyl oxindole. This oxindole was then converted to (±)-coerulescine and (±)-horsfiline. Coerulescine (1) was isolated from the blue canary grass, Phalaris coerulescens  ring system is a widely distributed structural framework present in a number of cytostatic alkaloids. For example, coerulescine  and horsrsfiline  represenrba Warb –5, elacorba Warb  and rychrba Warb –8, have rba Warb , found t framework for horsfiline and coerulescine –34, bothcinimide . Other acinimide , ring excinimide –16, 1,3-cinimide –19, intrcinimide –24, eleccinimide , asymmetcinimide , palladicinimide , palladicinimide , Pd-catacinimide –30, NHC-cinimide , dimethycinimide , and by cinimide .1) and horsfiline (2) (The Wittig olefination–Claisen rearrangement protocol  providesline (2) .o-nitrobenzaldehyde with allyloxymethylenetriphenylphosphorane under standard conditions [5 as an inseparable mixture of E and Z isomers. However, the NMR signals of the E and Z isomers in the olefinic region were well separated, which allowed us to estimate the ratio of these isomers as 1:2. The mixture of allyl vinyl ethers was heated in refluxing xylene to effect the Claisen rearrangement to obtain 4-pentenal 6 in 85% yield. Aldehyde 6 was transformed into acid 7 by Jones oxidation, which was immediately converted to the ethyl ester 8. Subsequently, reduction of the compound 8 with Zn and NH4Cl resulted in clean cyclization leading to oxindole 9.The Wittig olefination of nditions  furnishe10 was treated with NaH, followed by ethyl chloroformate at 0 °C to give 11 in 80% yield. Oxidative cleavage of the allyl group was accomplished by catalytic osmium tetroxide and N-methylmorpholine N-oxide (NMO), followed by cleavage of the diol with sodium metaperiodate on silica in methylene chloride. Reductive amination of the aldehyde 12 was conducted using methylamine hydrochloride and NaBH3CN and gave spiro-oxindole 13. The Boc group of 13 was removed by treatment with 2.5 M HCl to give 14. Finally, chemoselective reduction of amide 14 with n-BuLi and LAH  gave coIn summary, we have described a new and efficient synthesis of (±)-coerulescine and (±)-horsfiline.File 1Experimental and spectral data.


M. tuberculosis is central to its success as a pathogen. Mycolic acids are key components of this cell wall. The genes involved in joining the α and mero mycolates are located in a cluster, beginning with Rv3799c and extending at least until Rv3804c. The role of each enzyme encoded by these five genes is fairly well understood, except for Rv3802c. Rv3802 is one of seven putative cutinases encoded by the genome of M. tuberculosis. In phytopathogens, cutinases hydrolyze the waxy layer of plants, cutin. In a strictly mammalian pathogen, such as M. tuberculosis, it is likely that these proteins perform a different function. Of the seven, we chose to focus on Rv3802c because of its location in a mycolic acid synthesis gene cluster, its putative essentiality, its ubiquitous presence in actinomycetes, and its conservation in the minimal genome of Mycobacterium leprae. We expressed Rv3802 in Escherichia coli and purified the enzymatically active form. We probed its activities and inhibitors characterizing those relevant to its possible role in mycolic acid biosynthesis. In addition to its reported phospholipase A activity, Rv3802 has significant thioesterase activity, and it is inhibited by tetrahydrolipstatin (THL). THL is a described anti-tuberculous compound with an unknown mechanism, but it reportedly targets cell wall synthesis. Taken together, these data circumstantially support a role for Rv3802 in mycolic acid synthesis and, as the cell wall is integral to M. tuberculosis pathogenesis, identification of a novel cell wall enzyme and its inhibition has therapeutic and diagnostic implications.The cell wall of  M. tuberculosis infects one third of the world, makes over 8 million ill each year, and kills 1.8 million, 450,000 of whom are children M. tuberculosis, they contribute to pathogenesis through a variety of mechanisms from cell signaling and host evasion to granuloma formation. The genes involved in joining the mero and α mycolate into a mature mycolic acid and transferring it to trehalose or arabinogalactan are located in a gene cluster from Rv3799c to at least Rv3804c  and AccD4 (Rv3799c), along with the other acyl-CoA carboxylases, AccA3 and AccD5, participate in deriving meroacyl-AMP (from FAS-II) and 2-carboxyl-C26-S-CoA (from FAS-I), respectively. These are attached via thioester bonds to phosphopentathienes on the acyl carrier domains of Pks13 , a multidomain protein which, via Claisen condensation, creates a mature mycolic acid. A reduction step is needed and one enzyme so far, CmrA, has been identified for this role Mycobacterium smegmatisRv3804c) Rv3802c gene product to the mycolic acid synthesis pathway. Our data demonstrate that the products encoded by Rv3802c and the non-orthologous but homologue MSMEG_1403 possess both phospholipase A (PLA) activity and thioesterase activity. These activities are consistent with a role in mycolic acid biosynthesis as this pathway involves multiple ester and thioester bonds. In addition, the Rv3802c gene product, but not that of the distant homologue MSMEG_1403 in M. smegmatis, is inhibited by the known human fatty acid synthase thioesterase (FASTE) domain inhibitor, THL. THL has been demonstrated to decrease mycolic acid synthesis, leading to defects in the mycobacterial cell wall, though the targets of its antimycobacterial action were unknown. Though these lines of evidence are circumstantial, taken together with the genetic data, these findings support a role for Rv3802 in mycobacterial mycolic acid synthesis. Rv3804c . The rolRv3802c in M. tuberculosis H37Rv and compared it to that of other mycobacteria and corynebacteria. Rv3802c is located in a mycolic acid synthesis gene cluster from Rv3799c to 3804c, which encode AccD4, Pks13, FadD32, Rv3802, Ag85D and Ag85A, respectively  M. tuberculosis and M. smegmatisC. glutamicum; it shares only 0–40% similarity with the other annotated cutinases in M. tuberculosis. Rv3802 is a 337 amino acid protein with a predicted secretion signal and an altered, but recognizable, cutinase motif We analyzed a 30-kb chromosomal region surrounding ectively . The cluRv3802c and MSMEG_1403 were PCR-amplified from their respective genomes without their secretion signals and placed under the control of the IPTG inducible T7 promoter in the expression vector pET23a. The vector derived 6× His tag was not used in lieu of an engineered streptavidin tag, and expression in each recombinant strain was verified using an anti-streptavidin antibody  on phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, but not sphingomyelin  was investigated as a surrogate for cutinase activity. This is essentially an esterase activity with colorimetric detection of the nitrophenyl group freed from NPB. Both enzymes were active esterases, though the turnover rates were relatively slow .14C-trehalose was used as the acceptor, and cell wall lipids or crude cell wall extract (with lipid and protein) were used for donation; no transacylation was detected except with the positive control, Ag85C (data not shown). T6P is not currently commercially available, thus a similar assay was done with the acceptors mannose-6-phosphate and glycerol-6-phosphate, with no observed acylated product. These same acceptors were used but the donor changed to 14C-P-CoA (thioester bond) or 14C-DPPC (ester bond), and again no acylation occurred, though hydrolysis of the donors did occur. 14C-P-CoA and 14C-DPPC were again used as donors, with crude cell wall extract  used for the acceptor; no new acylated products emerged. These 14C-P-CoA and 14C-DPPC assays were repeated with the addition of crude cell wall extract alone and with cytosolic extract, with the hope that native donor or acceptor would be present, and again, no transacylation was observed. Transacylation assays were repeated several times with different amounts of donor, acceptor and enzyme, and different amounts of time from 1 to 72 H. The only result for Rv3802 and MSMEG_1403 in all assays was the release of fatty acid (not shown).Though Rv3802 may function for hydrolysis only, evaluation of the mycolic acid synthesis pathway suggests a possible role as a transferase . Rv3802 M. smegmatis mc2 155 cell wall was radiolabeled with 14C-acetate, and the outer cell wall was extracted as in 14C-palmitic acid resulted. Mass spectrometry of the apparent substrate revealed it as phosphatidylinositol mannoside 2 (PIM2); this was purified by normal phase chromatography on a silica column, and hydrolysis by Rv3802 and MSMEG_1403 confirmed .For the kinetic analysis of Rv3802, the time-dependent hydrolysis of palmitoyl CoA (P-CoA) and decanoyl CoA (D-CoA) was measured for thioesterase activity, and of NPB for esterase activity see . These kM. tuberculosis Rv3802. Inhibition of PLA activity is demonstrated in 50 for THL against Rv3802 are 5 and 250 nm, respectively, which are lower than against human FASTE, for which the apparent Ki and IC50 are reported at 100 nm and 1.35±0.34 µM, respectively THL is reported to inhibit the thioesterase domain of human FAS, thus we investigated the ability of THL to inhibit M. tuberculosis cutinases, including Rv3802, have been immunologically characterized, their function in organisms that do not encounter cutin has not been elucidated M. tuberculosis, it may be they serve a variety of functions. The function of Rv3802c and its orthologs is suggested by its location in a gene cluster for mycolic acid synthesis. In addition, the importance of Rv3802c is supported by its putative essentiality, its ubiquitous presence in actinomycetes, and its unique conservation in the minimal genome of M. leprae. To date, all of the genes surrounding Rv3802c are demonstrated to serve vital functions in mycolic acid synthesis. This genetic data implying a role in mycolic acid synthesis is further supported by Rv3802's: (i) membership in the α/β-hydrolase fold family with known PLAs, mycolyltransferases and thioesterases; (ii) possession of thioesterase activity; and (iii) enzymatic inhibition by a known mycolic acid synthesis inhibitor, THL.In this report we described the heterologous expression, purification and enzymatic characterization of an annotated cutinase, Rv3802, from a mammalian pathogen. Though the proteins encoded by 50 for THL against human FASTE are 100 nm and 1.35±0.34 µM, respectivelyM. tuberculosis growth at <30 µg/mL, decreasing the production of mycolic acids with concomitant defects in the cell wall, though the target(s) were undefined M. tuberculosis.The compound THL is sold over the counter as the weight loss agent Alli™. It inhibits pancreatic lipase so that long chain fatty acids cannot be absorbed. During activity based screening, THL was noted to be a selective inhibitor of the TE domain on human FAS Though further investigation will be required to pinpoint the exact function of Rv3802, examination of the mycolic acid synthetic pathway suggests a role as a Pks associated thioesterase or as a mycolyltransferase. Though we could not demonstrate mycolyltransferase activity, our data is limited by the need to use surrogate substrates and acceptors, thus this remains a distinct possibility. There are multiple unknown mycolyltransferases required in the pathway, and similarities between the known mycolyltranferase Ag85 proteins and Rv3802 are hard to ignore. However, the unusual ability to hydrolyze thioesters strongly suggests some type of interaction with nascent mycolic acids while docked via thioester bonds on Pks13.M. tuberculosis has all the necessary modules to produce a mature mycolic acid: one acyltranferase domain for loading and transfer of selected acyl chains to the phosphopentatheine (PP) binding motif of the acyl-S-carrier protein (ACP) domain; two ACPs for thioester binding, via a PP, of the meroacyl chain from FadD32 and the 2 carboxyl-C26 chain from AccD4, AccA3 and AccD5; a ketoacyl synthase domain for Claisen-type condensation; and one TE1 domain for chain termination and hydrolysis. The reduction event to form the mature mycolic acid may be catalyzed by more than one protein, but to date CmrA has been identifiedInteraction with Pks13 could be in the form of an exogenous type 1 thioesterase, (TE1) or as an exogenous type 2 thiosterase (TE2) An alternative role for this protein is that of the precedented TE2. TE2s are well described in association with Pks and NRPS modules in bacteria, fungi and plants, and serve to proofread the nascent product on the synthetic module Rv3802c and the decrease in mycolic acids observed with THL. Significant amino acid similarity between TE1s or TE2s and cutinases is not found by search programs, yet the TE1s, TE2s and cutinases are all α/β-hydrolase fold enzymes with SDH motifs. In Rv3802, which otherwise shares little amino acid similarity to the cutinases (0–40%), the spacing of the SDH is more similar to thioesterases than to cutinases; structure prediction models find structural similarity of Rv3802 with known cutinases and thioesterases at 100% and 75% precision, respectively  was purchased from ATCC. M. smegmatis was grown at 37°C in Luria-Bertani (LB) broth with 0.05% Tween80®. Escherichia coli strains DH5α and Rosetta 2 (DE3)pLysS (Merck) were grown at 37°C in LB broth. Antibiotics (Sigma) were added to media at the following concentrations: Ampicillan 100 µg/mL, Kanamycin 25–50 µg/mL, Chloramphenicol 34 µg/mL.M. tuberculosis Rv3802c and M. smegmatis M_SMEG 1403 genes. PCR amplifications consisted of one cycle of denaturation , followed by 30 cycles of amplification that included denaturation , annealing , and primer extension . The primers used were as follows: catatgggatgccctgacctgcactggatcggc-3′5′- and gagctctcatttttcgaactgcgggtggctccaagcgctgcctaacgtcggagcgggctc-3′5′- for M. smegmatis MSMEG_1403 gene, and catatgggcgaatcgccgcccagcgcgg-3′5′- and gagctctcatttttcgaactgcgggtggctccaagcgctcctatgtttggggtggggcgc-3′5′- for M. tuberculosis Rv3802c gene. Primers were designed to provide PCR amplified fragments containing a C-terminal streptavidin tag (bold) and flanking NdeI and SacI restriction sites (underline). Fragments were cloned into PCR2.1 (Invitrogen) and colonies for subsequent cloning were confirmed with sequencing. Correct clones were sub cloned into pET23a (Novagen). This vector was used as a template to make three mutants in M. tuberculosis Rv3802c via the QuickChange® II method . Primers used were 5′-ctgatcgggtttgagcagggcgcggtg-3′ and 5′-caccgcgccctgctcaaacccgatcag-3′ for Rv3802 ΔS175E, 5′-ggaacctgggaggaatcgccgcagcag-3′ and 5′-ctgctgcggcgattcctcccaggttcc-3′ for Rv3802 ΔS86E, and 5′-cctgggagtcagagccgcagcagaaccc-3′ and 5′-gggttctcgtgcggctctgactcccagg-3′ for Rv3802 ΔS87E.Standard PCR strategies with Taq DNA polymerase (Invitrogen) were used to amplify the 2PO4 and 300 mM NaCl, pH 8.0 with 1 mg/mL lysozyme and 5 µg/mL DNase I) per 200 mL original induction culture, and stored at −20°C overnight. Thawed resuspensions with 2% (w/v) n-lauryl sarcosine was mixed for ≥60 min at room temperature until lysate was clear of large clumps. After centrifugation, target protein was purified from lysate using strep-tactin Superflow resin  per company protocol. Protein concentration and absorbance spectra of these aliquots were evaluated at 280 nm on a Nanodrop™ system. All eluted protein samples, if not used immediately, were stored at −70°C. Selected fractions were also analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and either stained or immunoblotted using an anti-streptavidin tag antibody (IBA) at 1∶1500 and secondary horseradish peroxidase goat anti-mouse (1∶20 K), and visualized with chemiluminescence. Fractions were estimated to be at least 95% pure using SYPRO orange staining  with phosphoimager detection.The constructs in pET23a described above were transformed into electrocompetent Rosetta 2 (DE3)pLysS for expression using the T7 promoter. Cultures were induced with 1 mM isopropyl-B-D-thiogalactopyranoside (IPTG) for 3 H at 37°C. Following induction, cell pellets were collected and re-suspended in 5 mL Strep-tactin lysis buffer  was added at 1 µCi/mL and cells were grown for four additional H. Cells were then pelleted, and extracted with 30 mL CHCl3∶CH3OH  per gram of wet cells. Debris was again pelleted, extract removed, and re-extracted with the same volume of CHCl3∶CH3OH . Extracts were combined and Folch washed. Organic layer was aliquoted and dried down in pre-weighed vials. For mass spectrometry, a parallel experiment was done without 14C-acetate. To isolate PIM species, 400 µg was resuspended in CHCl3 and applied to a silica column  and eluted with CHCl3∶CH3OH, in percentages (v/v) of 90∶10, 80∶20, 70∶30, 60∶40 and 50∶50. PIMs were collected from the 60∶40 and 50∶50 fractions.For radiolabeling of whole 3∶CH3OH∶NH4OH . Individual bands of lipid were scraped from the plate into columns made from Pasteur pipettes that were fitted with 0.2 µm, Type FG Fluoropore membrane filters (Millipore). The silica gel was rinsed with 1 mL of 2∶1 i-C3H7OH/H2O and lipid species were eluted with 1 mL of 2∶1 (v/v) CHCl3/CH3OH. Samples were evaporated to dryness, re-dissolved in 2 µL of the same solvent and 0.5 µL was deposited onto a sample stage that had been pre-spotted with 2,5-dihydroxybenzoic acid (DHB) for analysis by matrix assisted laser desorption ionization (MALDI) mass spectrometry. Mass spectra using a laser wavelength of 337 nm were obtained from m/z 100 to 2400 using a QSTAR XL tandem mass spectrometer . The mass analyzer configuration of this instrument, mass-resolving quadrupole – collision quadrupole – orthogonal time-of-flight (Q-q-oTOF), allowed both full and collision induced dissociation (CID) spectra to be obtained. PIM species were identified based on molecular weight, interpretation of CID spectra, and comparison of spectra with data found in the literature Cell wall lipids were isolated as above. Lipids were applied to Silica gel 60Å plates , and developed in CHCl14C-radiolabeled cell wall or purified PIM were resuspended at 2 mg/mL in 50 mM Bis-tris, pH 7, with 0.25% Triton®-X 100. 50 µL of this was added to 10–50 µg of enzyme in 50 µL, for a total reaction volume of 100 µL. After 2–5 H, the reaction was quenched with 200 µL of CHCl3∶CH3OH∶HCl , and the organic phase removed and plated under a nitrogen stream on Silica gel 60Å plates . Plates were developed in CHCl3∶CH3OH∶NH4OH  and visualized with a phosphoimager (radiolabel) and/or cupric sulfate or Dittmer-Lester stains (radiolabeled and unlabeled). For mass spectrometric analysis, a parallel experiment was performed without radiolabel and lipid species were recovered as described above.Aliquots of 2  were used per reaction for a PLA activity control. Lyophilized F. solani cutinase  was resuspended at 3 mg/mL in H2O and then further diluted depending on assay.6 units of snake venom PLA14C] , bovine sphingomyelin [choline-methyl-14C] , and phosphatidylethanolamine, L-a-palmitoyl-2-arachidonyl [arachidonyl-1-14C] , all from NEN Life Science Products, and 3-phosphatidyl-[3-14C]serine 1,2-dioleoyl  from Amersham Biosciences. Cold DPPC with 0.1 µL of 14C-radiolabeled phospholipid per reaction were dried down and then resuspended with 50 µL of 50 mM bis-tris pH 7.4 buffer with 0.25% Triton®-X 100 per reaction to make mixed micelles. This solution was sonicated 30 s×3, vortexing in between. 50 µL of this substrate was then added to 50 µL of enzyme solution (10–50 µg), and incubated at 37°C three H. The reaction was extracted with 100 µL of chloroform/methanol/HCl , vortexed and centrifuged at 13,000 rpms for two min. The organic phase was then plated on 60 Å silica plates (Whatman 4807-425) and then developed in CHCl3∶CH3OH∶H2O  solvent. Lipids were visualized with a phosphoimager.Unlabeled dipalmitoyl phosphatidylcholine (DPPC) was resuspended at 23 mM in chloroform, and used as a micellar backbone in all reactions. Radiolabeled phospholipids were as follows: phosphatidylcholine L-a-dipalmitoyl [dipalmitoyl-1-F. solani cutinase, the general esterase Thermomyces lanuginosus (Sigma L-0777) and M. tuberculosis Ag85C .Control enzymes included M. smegmatis MSMEG_1403 and M. tuberculosis Rv3802 were active in this assay, kinetics was done with only Rv3802. For kinetic calculations, each 200 µL reaction contained 0.01 mg of enzyme (1.53 µM), the path length was 0.8 cm and the extinction coefficient was 15250 M−1 cm−1m and Vmax and these were used to determine the kcat and specificity constant for the esterase activity of Rv3802 and mutants. Up to 10 units of snake venom PLA was also tested and was inactive.hydrolysis of NPB  was used as a surrogate for cutinase activity. Though both 2−). For kinetic calculations, each 200 µL reaction contained 3 µg of enzyme (461 nm), the path length was 0.8 cm and the extinction coefficient was 14150 M−1 cm−1 for TNB2− (Sigma). The P-CoA 20 mM stock was diluted in 50 mM bis-tris (pH 7.0) for use in the assays. The DTNB 40 mM stock was diluted in EtOH to give a molar equivalent amount of DTNB to acyl-CoA for the reaction . The final P-CoA concentration ranged from 4.875 µM – 2.5 mM, and the final D-CoA concentration ranged from 0.078 to 5.0 mM. Assays were done in 96 well plates, in triplicate, at 37°C for three H, with proper controls. There were four replicate experiments. Absorbance was read by a plate spectrophotometer at 415 nm at various time points. Prizm 5.0 software was used to determine Km and Vmax and these were used to determine the kcat and specificity constant for the thioesterase activity of Rv3802. For radiolabel 14C-P-CoA assays, protocol was followed as in 14C-phospholipids assays above.hydrolysis of P-CoA  and D-CoA,  were used to measure thioesterase activity. When the thioester bond is hydrolyzed, the free sulfur on CoA is attacked by 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), which releases a measurable nitrophenyl group, 5-thio-2-nitrobenzoate  were determined using Prizm 5.0 software, as were apparent Ki and IC50 values. For inhibition of M. smegmatis MSMEG_1403, each 200 µL reaction contained 0.3 µg (0.495 µM). THL concentration was increased to a maximum of 25 µM (a molar excess compared to enzyme of 505) in an attempt to detect inhibition.four concentrations of THL  were selected in order to determine K14C-trehalose (0.25 µCi) with crude cell wall lipids (50 µg) from M. tuberculosis; 2) 14C-P-CoA (0.25 µCi) as donor with mannose-6-phosphate (1–10 mM) or glycerol-6-phosphate (1 mM) as acceptor with crude cell wall extract ; 5) same as two and three with 14C-DPPC instead of 14C-P-CoA. As a positive control for the mycolyltranferase assays using 14C-trehalose as an acceptor, Ag85C was used at 10–50 µg per assay.donors and recipients were paired for various transferase assays to assess whether the purified enzymes could hydrolyze a known substrate and transfer it to an acceptor. Assays included 10–50 µg of either Rv3802 or MSMEG_1403 protein, and were incubated for 1–72 H. Pairings included: 1)