MeCP2 gene therapy ameliorates disease phenotype in mouse model for Pitt Hopkins syndrome.

The neurodevelopmental disorder Pitt Hopkins syndrome (PTHS) causes clinical symptoms similar to Rett syndrome (RTT) patients. However, RTT is caused by MECP2 mutations whereas mutations in the TCF4 gene lead to PTHS. The mechanistic commonalities underling these two disorders are unknown, but their shared symptomology suggest that convergent pathway-level disruption likely exists. We reprogrammed patient skin derived fibroblasts into induced neuronal progenitor cells. Interestingly, we discovered that MeCP2 levels were decreased in PTHS patient iNPCs relative to healthy controls and that both iNPCs and iAstrocytes displayed defects in function and differentiation in a mutation-specific manner. When Tcf4+/- mice were genetically crossed with mice overexpressing MeCP2, molecular and phenotypic defects were significantly ameliorated, underlining and important role of MeCP2 in PTHS pathology. Importantly, post-natal intracerebroventricular gene replacement therapy with adeno-associated viral vector serotype 9 (AAV9)-expressing MeCP2 (AAV9.P546.MeCP2) significantly improved iNPC and iAstrocyte function and effectively ameliorated histological and behavioral defects in Tcf4+/- mice. Combined, our data suggest a previously unknown role of MeCP2 in PTHS pathology and common pathways that might be affected in multiple neurodevelopmental disorders. Our work highlights potential novel therapeutic targets for PTHS, including upregulation of MeCP2 expression or its downstream targets or, potentially, MeCP2-based gene therapy.

Tartrate Dehydrogenase in Bacillus Species: Deciphering Unique Catalytic Diversity Through Kinetic, Structural and Molecular Docking Analysis.

Divergently evolved Tartrate dehydrogenase (TDH) exhibits multiple catalytic activities at a single active site; the enzyme from P. putida (pTDH) being structurally and biochemically well-characterized. Occurrence of TDH-associated ability to aerobically metabolize L-tartrate in Bacillus isolates and limited resemblance of ycsA-encoded protein sequences with pTDH rendered Bacillus TDH as an intriguing enzyme with possible catalytic diversity as well as evolutionary significance. The present study explores substrate interactions of TDHs from B. subtilis 168 (168bTDH) and B. licheniformis DSM-13 (429bTDH) through kinetic, structural and molecular docking-based analysis. Heterologously expressed bTDHs, purified from insoluble fractions of E. coli BL21(DE3) cells, could significantly catalyze L-tartrate and meso-tartrate as substrates in forward reaction. Unlike pTDH, bTDHs distinctly and more efficiently catalyzed the reverse reaction using dihydroxyfumarate substrate following sigmoidal kinetics; the ability being ~ 4 fold higher in 168bTDH. Their binding energies predicted from molecular docking, further substantiated the relative substrate specificities, while revealing major residues involved in protein-ligand interactions at active site. The kinetic analysis and homology modelling validated using Ramachandran Plot analysis predicted a dimeric nature for bTDH. Collectively, the results highlight unique catalytic potential of phylogenetically recent bTDHs, offering an important protein engineering target to mediate efficient enantioselective enzymatic biotransformations.

Unusual concurrence of P-solubilizing and biocontrol traits under P-limitation in plant-beneficial Pseudomonas aeruginosa P4: insights from in vitro metabolic and gene expression analysis.

Plant-beneficial fluorescent Pseudomonas species with concurrent P-solubilizing and biocontrol traits could have improved rhizospheric survival and efficacy; this rare ability being subject to diverse environmental and endogenous regulations. This study correlates growth patterns, time-course analysis of selected metabolites, non-targeted metabolomics of exometabolites and selected gene expression analysis to elucidate P-limitation-induced physiological shifts enabling co-production of metabolites implied in P-solubilization and biocontrol by P. aeruginosa P4 (P4). P-limited culture supernatants showed enhanced production of selected biocontrol metabolites such as pyocyanin, pyoverdine and pyochelin and IAA while maintaining biomass yield despite reduced growth rate and glucose consumption. Non-targeted exometabolomics further indicated that P-limitation positively impacted pentose phosphate pathway as well as pyruvate, C5-branched dibasic acid and amino acid metabolism. Its correlation with unusually reduced aroC expression and growth phase-dependent changes in the expression of key biosynthetic genes pchA, pchE, pchG, pvdQ and phzM implied a probable regulation of biosynthesis of chorismate-derived secondary metabolites, not neglecting the possibility of multiple factors influencing the gene expression profiles. Similar increase in biocontrol metabolite production was also observed in Artificial Root Exudates (ARE)-grown P4 cultures. While such metabolic flexibility could impart physiological advantage in sustaining P-starvation stress, it manifests as unique coexistence of P-solubilizing and biocontrol abilities.

Exploration of group II metabotropic glutamate receptor modulation in mouse models of Rett syndrome and MECP2 Duplication syndrome.

Rett syndrome (RTT) and MECP2 Duplication syndrome (MDS) have opposing molecular origins in relation to expression and function of the transcriptional regulator Methyl-CpG-binding protein 2 (MeCP2). Several clinical and preclinical phenotypes, however, are shared between these disorders. Modulation of MeCP2 levels has recently emerged as a potential treatment option for both of these diseases. However, toxicity concerns remain with these approaches. Here, we focus on pharmacologically modulating the group II metabotropic glutamate receptors (mGlu), mGlu2 and mGlu3, which are two downstream targets of MeCP2 that are bidirectionally affected in expression in RTT patients and mice (Mecp2Null/+) versus an MDS mouse model (MECP2Tg1/o). Mecp2Null/+ and MECP2Tg1/o animals also exhibit contrasting phenotypes in trace fear acquisition, a form of temporal associative learning and memory, with trace fear deficiency observed in Mecp2Null/+ mice and abnormally enhanced trace fear acquisition in MECP2Tg1/o animals. In Mecp2Null/+ mice, treatment with the mGlu2/3 agonist LY379268 reverses the deficit in trace fear acquisition, and mGlu2/3 antagonism with LY341495 normalizes the abnormal trace fear learning and memory phenotype in MECP2Tg1/o mice. Altogether, these data highlight the role of group II mGlu receptors in RTT and MDS and demonstrate that both mGlu2 and mGlu3 may be potential therapeutic targets for these disorders.

Profiling beneficial and potential adverse effects of MeCP2 overexpression in a hypomorphic Rett syndrome mouse model.

De novo loss-of-function mutations in methyl-CpG-binding protein 2 (MeCP2) lead to the neurodevelopmental disorder Rett syndrome (RTT). Despite promising results from strategies aimed at increasing MeCP2 levels, additional studies exploring how hypomorphic MeCP2 mutations impact the therapeutic window are needed. Here, we investigated the consequences of genetically introducing a wild-type MECP2 transgene in the Mecp2 R133C mouse model of RTT. The MECP2 transgene reversed the majority of RTT-like phenotypes exhibited by male and female Mecp2 R133C mice. However, three core symptom domains were adversely affected in female Mecp2R133C/+ animals; these phenotypes resemble those observed in disease contexts of excess MeCP2. Parallel control experiments in Mecp2Null/+ mice linked these adverse effects to the hypomorphic R133C mutation. Collectively, these data provide evidence regarding the safety and efficacy of genetically overexpressing functional MeCP2 in Mecp2 R133C mice and suggest that personalized approaches may warrant consideration for the clinical assessment of MeCP2-targeted therapies.

A GRM7 mutation associated with developmental delay reduces mGlu7 expression and produces neurological phenotypes.

The metabotropic glutamate receptor 7 (mGlu7) is a G protein-coupled receptor that has been recently linked to neurodevelopmental disorders. This association is supported by the identification of GRM7 variants in patients with autism spectrum disorder, attention deficit hyperactivity disorder, and severe developmental delay. One GRM7 mutation previously reported in 2 patients results in a single amino acid change, I154T, within the mGlu7 ligand-binding domain. Here, we report 2 new patients with this mutation who present with severe developmental delay and epilepsy. Functional studies of the mGlu7-I154T mutant reveal that this substitution resulted in significant loss of mGlu7 protein expression in HEK293A cells and in mice. We show that this occurred posttranscriptionally at the level of protein expression and trafficking. Similar to mGlu7-global KO mice, mGlu7-I154T animals exhibited reduced motor coordination, deficits in contextual fear learning, and seizures. This provides functional evidence that a disease-associated mutation affecting the mGlu7 receptor was sufficient to cause neurological dysfunction in mice and further validates GRM7 as a disease-causing gene in the human population.

Phenotypic profiling of mGlu7 knockout mice reveals new implications for neurodevelopmental disorders.

Neurodevelopmental disorders are characterized by deficits in communication, cognition, attention, social behavior and/or motor control. Previous studies have pointed to the involvement of genes that regulate synaptic structure and function in the pathogenesis of these disorders. One such gene, GRM7, encodes the metabotropic glutamate receptor 7 (mGlu7 ), a G protein-coupled receptor that regulates presynaptic neurotransmitter release. Mutations and polymorphisms in GRM7 have been associated with neurodevelopmental disorders in clinical populations; however, limited preclinical studies have evaluated mGlu7 in the context of this specific disease class. Here, we show that the absence of mGlu7 in mice is sufficient to alter phenotypes within the domains of social behavior, associative learning, motor function, epilepsy and sleep. Moreover, Grm7 knockout mice exhibit an attenuated response to amphetamine. These findings provide rationale for further investigation of mGlu7 as a potential therapeutic target for neurodevelopmental disorders such as idiopathic autism, attention deficit hyperactivity disorder and Rett syndrome.

Colonization by multi-potential Pseudomonas aeruginosa P4 stimulates peanut (Arachis hypogaea L.) growth, defence physiology and root system functioning to benefit the root-rhizobacterial interface.

The beneficial associations between Arachis hypogaea L. (peanut) and fluorescent Pseudomonas species have been poorly explored despite their predominance in the peanut rhizosphere. The present study explores the mutually beneficial interactions between peanut roots and P. aeruginosa P4 (P4) in terms of their impact on plant growth, defence physiology and the root-rhizobacterial interface. The efficient phosphate solubilizer P4 exhibited biocontrol abilities, including the production of siderophores, pyocyanin, indole-3-acetic acid and hydrogen cyanide. The bacterization of peanut seeds with multi-potential P4 significantly enhanced in vitro seed germination and seedling vigour. Under sand-based gnotobiotic (10 days post-inoculation) and sterile soil-based cultivation systems (30 days post-inoculation), sustained P4 colonization enhanced the peanut root length and dry plant biomass. The subsequent increase in catalase, polyphenol oxidase and phenylalanine ammonia lyase activities with increased phenolic contents in the peanut roots and shoots suggested the systemic priming of defences. Consequently, the altered root exudate composition caused enhanced chemo-attraction towards P4 itself and the symbiotic N2-fixing Bradyrhizobium strain. Co-inoculating peanuts with P4 and Bradyrhizobium confirmed the improved total bacterial colonization (∼2 fold) of the root tip, with the successful co-localization of both, as substantiated by scanning electron microscopy. Collectively, the peanut-P4 association could potentially model the beneficial Pseudomonas-driven multi-trophic rhizosphere benefits, emphasizing the plausible role of non-rhizobium PGPR in promoting N2 fixation.

Improvisation of a spectrophotometric method to quantify hydroxycitric acid.

Existing spectrophotometric method to quantify hydroxycitric acid (HCA), although is specific and sensitive; finds limited use owing to poor stability of HCA-metavanadate complex. Present study describes improvisation of this method with respect to source of HCA standard and assay parameters. Assay system consisting of HCA and metavanadate reagent was modified to include 1 M NaOH to neutralize excess acidity. Resulting complex showed λmax at 485 nm, obeying Beer-Lambert's law within concentration range of 33-677 µg/ml, with linear calibration curve showing a good coefficient of determination (R2 = 0.998). Moreover, HCA-metavanadate complex showed enhanced stability retaining up to 70% absorbance even after 60 min of its formation. Similar consistency in scaled-down assay system renders the method suitable for high-throughput screening of HCA-producing microbes. Of the tested metabolites and media components, only tartrate interfered with the spectrophotometric estimation of HCA; a correction factor to eliminate which was also established. Accordingly measured HCA level in the culture supernatant of a bacterial isolate IT6 was comparable to that determined using the standardized HPLC method. The proposed procedure therefore is a convenient, sensitive, accurate and high-throughput method suitable for primary screening of HCA producing microbes; the only ecofriendly alternative source of optically pure HCA.

Broad-host-range plasmid-mediated metabolic perturbations in Pseudomonas fluorescens 13525.

Genetic engineering of fluorescent pseudomonads for various industrially, agriculturally and environmentally important bioprocesses often involves the use of suitable plasmids. Plasmid-mediated alterations in host physiology and metabolism are poorly understood for this group of organisms. Thus, we investigated the metabolic perturbations in Pseudomonas fluorescens 13525 due to the independent and combined presence of broad-host-range plasmids, pBBR1MCS-2 (copy number 30) and pUCPM18 derived pAB4 and pAB8 (copy number 14-16). Presence of pAB4 and pAB8 not only significantly increased the growth rate and glucose utilization of P. fluorescens 13525, but also increased glucose dehydrogenase activity and gluconic acid production indicating enhanced direct oxidative pathway for glucose catabolism. Additionally, increased secretion of pyruvic, acetic, and citric acids caused faster media acidification in presence of pAB4 and pAB8. Simultaneous presence of pAB4/pAB8 in Pf (pAB48) and pAB4/pBBR1MCS-2 in Pf (pAB4BBR1MCS-2) reduced their respective copy numbers to nearly half. Pf (pAB48) demonstrated further increase in direct oxidation pathway without altering growth and glucose depletion rates, as compared with single transformants. Conversely, pBBR1MCS-2 plasmid did not greatly alter P. fluorescens 13525 metabolism when present independently but masked the effects imposed by pAB4 when present in its combination. In conclusion, P. fluorescens 13525 redesigns its metabolism in response to the presence of plasmids irrespective of their nature, by enhancing anaplerosis with a simultaneous reduction in catabolism as indicated by increased pyruvate carboxylase and decreased citrate synthase activities, respectively. Such information will be helpful for vector designing during genetic engineering of fluorescent pseudomonads.

Heterologous expression of phosphoenolpyruvate carboxylase enhances the phosphate solubilizing ability of fluorescent pseudomonads by altering the glucose catabolism to improve biomass yield.

The Synechococcus elongatus PCC 6301 phosphoenolpyruvate carboxylase (ppc) gene was constitutively overexpressed in fluorescent pseudomonads, to increase the supply of oxaloacetate, a crucial anabolic precursor and an intermediate in biosynthesis of organic acids implicated in phosphate (P) solubilization. Pseudomonas fluorescens ATCC 13525, transformed with pAB3 plasmid containing the ppc gene showed a 14-fold increase in PPC activity under P-sufficiency resulting in increased carbon flow through the direct oxidative pathway and reduced metabolic overflow. Under P-limitation, contribution of the direct oxidative pathway significantly increased in P. fluorescens ATCC 13525; however, ppc overexpression enhanced glucose catabolism through intracellular phosphorylative pathway. These results correlated with gluconic, pyruvic and acetic acid levels as well as the activities of key glucose catabolic enzymes. Irrespective of the P-status, ppc overexpression improved biomass yield without altering growth rate, resulting in improved P- solubilizing abilities of P. fluorescens ATCC 13525 as well as of the wheat rhizosphere fluorescent pseudomonads isolates Fp585, P109 and Fp315. Collectively, ppc overexpression reversed the P-status dependent glucose distribution between the direct oxidative and phosphorylative pathways of glucose catabolism in P. fluorescens ATCC 13525 and presents a feasible genetic engineering approach for developing efficient P-solubilizing bacteria.

Enhanced citric acid biosynthesis in Pseudomonas fluorescens ATCC 13525 by overexpression of the Escherichia coli citrate synthase gene.

Citric acid secretion by fluorescent pseudomonads has a distinct significance in microbial phosphate solubilization. The role of citrate synthase in citric acid biosynthesis and glucose catabolism in pseudomonads was investigated by overexpressing the Escherichia coli citrate synthase (gltA) gene in Pseudomonas fluorescens ATCC 13525. The resultant approximately 2-fold increase in citrate synthase activity in the gltA-overexpressing strain Pf(pAB7) enhanced the intracellular and extracellular citric acid yields during the stationary phase, by about 2- and 26-fold, respectively, as compared to the control, without affecting the growth rate, glucose depletion rate or biomass yield. Decreased glucose consumption was paralleled by increased gluconic acid production due to an increase in glucose dehydrogenase activity. While the extracellular acetic acid yield increased in Pf(pAB7), pyruvic acid secretion decreased, correlating with an increase in pyruvate carboxylase activity and suggesting an increased demand for the anabolic precursor oxaloacetate. Activities of two other key enzymes, glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase, remained unaltered, and the contribution of phosphoenolpyruvate carboxylase and isocitrate lyase to glucose catabolism was negligible. Strain Pf(pAB7) demonstrated an enhanced phosphate-solubilizing ability compared to the control. Co-expression of the Synechococcus elongatus PCC 6301 phosphoenolpyruvate carboxylase and E. coli gltA genes in P. fluorescens ATCC 13525, so as to supplement oxaloacetate for citrate biosynthesis, neither significantly affected citrate biosynthesis nor caused any change in the other physiological and biochemical parameters measured, despite approximately 1.3- and 5-fold increases in citrate synthase and phosphoenolpyruvate carboxylase activities, respectively. Thus, our results demonstrate that citrate synthase is rate-limiting in enhancing citrate biosynthesis in P. fluorescens ATCC 13525. Significantly low extracellular citrate levels as compared to the intracellular levels in Pf(pAB7) suggested a probable limitation of efficient citrate transport.

Metabolic channeling of glucose towards gluconate in phosphate-solubilizing Pseudomonas aeruginosa P4 under phosphorus deficiency.

Most phosphate-solubilizing bacteria (PSB), including the Pseudomonas species, release P from sparingly soluble mineral phosphates by producing high levels of gluconic acid from extracellular glucose, in a reaction catalyzed by periplasmic glucose dehydrogenase, which is an integral component of glucose catabolism of pseudomonads. To investigate the differences in the glucose metabolism of gluconic acid-producing PSB pseudomonads and low gluconic acid-producing/non-PSB strains, several parameters pertaining to growth and glucose utilization under P-sufficient and P-deficient conditions were monitored for the PSB isolate Pseudomonas aeruginosa P4 (producing approximately 46 mM gluconic acid releasing 437 microM P) and non-PSB P. fluorescens 13525. Our results show interesting differences in the channeling of glucose towards gluconate and other catabolic end-products like pyruvate and acetate with respect to P status for both strains. However, PSB strain P. aeruginosa P4, apart from exhibiting better growth under both low and high Pi conditions, differed from P. fluorescens 13525 in its ability to accumulate gluconate under P-solubilizing conditions. These alterations in growth, glucose utilization and acid secretion are correlated with glucose dehydrogenase, glucose-6-phosphate dehydrogenase and pyruvate carboxylase activities. The ability to shift glucose towards a direct oxidative pathway under P deficiency is speculated to underlie the differential gluconic acid-mediated P-solubilizing ability observed amongst pseudomonads.