mTOR drives cerebrovascular,synaptic, and cognitive dysfunction in normative aging

Cerebrovascular dysfunction and cognitive decline are highly prevalent in aging, but the mechanisms underlying these impairments are unclear. Cerebral blood flow decreases with aging and is one of the earliest events in the pathogenesis of Alzheimer's disease (AD). We have previously shown that the mechanistic/mammalian target of rapamycin (mTOR) drives disease progression in mouse models of AD and in models of cognitive impairment associated with atherosclerosis, closely recapitulating vascular cognitive impairment. In the present studies, we sought to determine whether mTOR plays a role in cerebrovascular dysfunction and cognitive decline during normative aging in rats. Using behavioral tools and MRI‐based functional imaging, together with biochemical and immunohistochemical approaches, we demonstrate that chronic mTOR attenuation with rapamycin ameliorates deficits in learning and memory, prevents neurovascular uncoupling, and restores cerebral perfusion in aged rats. Additionally, morphometric and biochemical analyses of hippocampus and cortex revealed that mTOR drives age‐related declines in synaptic and vascular density during aging. These data indicate that in addition to mediating AD‐like cognitive and cerebrovascular deficits in models of AD and atherosclerosis, mTOR drives cerebrovascular, neuronal, and cognitive deficits associated with normative aging. Thus, inhibitors of mTOR may have potential to treat age‐related cerebrovascular dysfunction and cognitive decline. Since treatment of age‐related cerebrovascular dysfunction in older adults is expected to prevent further deterioration of cerebral perfusion, recently identified as a biomarker for the very early (preclinical) stages of AD, mTOR attenuation may potentially block the initiation and progression of AD.     

Host specificity testing and examination for plant pathogens reveals that the gall‐inducing psyllid Calophya latiforceps is safe to release for biological control of Brazilian peppertree

Brazilian peppertree, Schinus terebinthifolia Raddi (Anacardiaceae), is one of the worst upland exotic weeds in Florida, USA. Foreign exploration for natural enemies led to the discovery of a pit‐galling psyllid, Calophya latiforceps Burckhardt (Hemiptera: Calophyidae), in the state of Bahia, Brazil, in 2010. Crawlers of C. latiforceps stimulate the formation of galls on the leaves of S. terebinthifolia resulting in leaf discoloration and in some cases leaf abscission. To determine whether C. latiforceps is a safe candidate for biological control of S. terebinthifolia, host specificity and the presence of selected plant pathogens were examined. Adult oviposition, gall formation, and adult survival of C. latiforceps were examined on 89 plant species under no‐choice and choice conditions. We found that C. latiforceps laid eggs on plants in seven families; however, crawlers stimulated gall formation and completed development to adult only on S. terebinthifolia. All crawlers on non‐target plants died, likely due to starvation caused either by the absence of a feeding stimulus or by a hypersensitive plant response. Under no‐choice conditions, 10% of adults lived for 19 days on the target weed, but adult survival was reduced to <3 days on non‐target plants. Choice testing revealed that females preferred to oviposit on S. terebinthifolia compared to non‐target plants. Molecular methods and indicator host inoculations did not detect the presence of ‘Candidatus Liberibacter solanacearum’, ‘Ca. L. asiaticus’, ‘Ca. L. americanus’, ‘Ca. L. africanus’, or plant viruses in adult C. latiforceps. We conclude that releasing C. latiforceps in the USA will have extremely low risk to non‐target plants, and provides another tool for the management of S. terebinthifolia.     

Cryptic diversity and morphological convergence in threatened species of fossorial skinks in the genus Scelotes (Squamata: Scincidae) from the Western Cape Coast of South Africa: implications for species boundaries, digit reduction and conservation

We investigated the evolutionary relationships among populations of two threatened Red Data Book fossorial skinks, Scelotes gronovii and Scelotes kasneri, along the Western Cape Coast of South Africa. The genus Scelotes shows considerable variation in limb and digit reduction. We sampled four localities purported to contain S. gronovii and seven of S. kasneri, encompassing all of each species' limited distribution. Each of these species lack forelimbs, and differ by the number of digits on the hind limbs, among other morphological characters; S. gronovii bears a single digit and S. kasneri bears two digits on the hind limbs. Sequence data obtained from three mtDNA (16S ribosomal RNA, cytochrome b, and nicotinamide adenine dinucleotide dehydrogenase 1 unit; 2035 bp ttl.) and two nuclear (dynein axonemal heavy chain 3 and the natural killer tumor recognition; 1848 bp ttl.) gene regions were used to reconstruct the evolutionary relationships among the two focal species and several other co-distributed species (Scelotes bipes, Scelotes montispectus, and Scelotes sexlineatus). Phylogenetic results (Bayesian and parsimony) revealed that several populations previously considered S. kasneri actually belong to other species, and others are paraphyletic with respect to one another. Additionally, populations of S. gronovii were also found to be paraphyletic, with populations south of the Berg River supported as sister to S. bipes, and populations north of the Berg River sister the remaining sampled species. Our results require a redefinition of S. sexlineatus to encompass populations morphologically convergent with S. kasneri and restrict the ranges of the already threatened S. kasneri and S. gronovii even further. The paraphyly of S. gronovii and the placement of each clade as sister to clades of species bearing two digits on the hind limbs suggests that digit loss has occurred at least twice in this group.     

Inferred Paternity and Male Reproductive Success in a Killer Whale (Orcinus orca) Population

We used data from 78 individuals at 26 microsatellite loci to infer parental and sibling relationships within a community of fish-eating ("resident") eastern North Pacific killer whales (Orcinus orca). Paternity analysis involving 15 mother/calf pairs and 8 potential fathers and whole-pedigree analysis of the entire sample produced consistent results. The variance in male reproductive success was greater than expected by chance and similar to that of other aquatic mammals. Although the number of confirmed paternities was small, reproductive success appeared to increase with male age and size. We found no evidence that males from outside this small population sired any of the sampled individuals. In contrast to previous results in a different population, many offspring were the result of matings within the same "pod" (long-term social group). Despite this pattern of breeding within social groups, we found no evidence of offspring produced by matings between close relatives, and the average internal relatedness of individuals was significantly less than expected if mating were random. The population's estimated effective size was <30 or about 1/3 of the current census size. Patterns of allele frequency variation were consistent with a population bottleneck.     

Protective effects of positive lysosomal modulation in Alzheimer's disease transgenic mouse models

Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid β peptide (Aβ) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aβ(1-42). Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK) was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of Aβ pathology. Systemic PADK injections in APP(SwInd) and APPswe/PS1ΔE9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both Aβ immunostaining as well as Aβ(x-42) sandwich ELISA measures in APP(SwInd) mice of 10-11 months. More extensive Aβ deposition in 20-22-month APPswe/PS1ΔE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aβ(1-38) occurs as Aβ(1-42) levels decrease in the mouse models, indicating that PADK treatment leads to Aβ truncation. Associated with Aβ clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces Aβ(1-42) accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of Aβ pathology and provides proof-of-principle for small molecule therapeutic development for AD and possibly other protein accumulation disorders.     

Characterization of the E506Q and H537A dysfunctional mutants in the E. coli ABC transporter MsbA

MsbA is a member of the ABC transporter superfamily that is specifically found in Gram-negative bacteria and is homologous to proteins involved in both bacterial and human drug resistance. The E506Q and H537A mutations have been introduced and used for crystallization of other members of the ABC transporter protein family, including BmrA and the ATPase domains MalK, HlyB-NBD, and MJ0796, but have not been previously studied in detail or investigated in the MsbA lipid A exporter. We utilized an array of biochemical and EPR spectroscopy approaches to characterize the local and global effects of these nucleotide binding domain mutations on the E. coli MsbA homodimer. The lack of cell viability in an in vivo growth assay confirms that the presence of the E506Q or H537A mutations within MsbA creates a dysfunctional protein. To further investigate the mode of dysfunction, a fluorescent ATP binding assay was used and showed that both mutant proteins maintain their ability to bind ATP, but ATPase assays indicate hydrolysis is severely inhibited by each mutation. EPR spectroscopy data using previously identified and characterized reporter sites within the nucleotide binding domain along with ATP detection assays show that hydrolysis does occur over time in both mutants, though more readily in the H537A protein. DEER spectroscopy demonstrates that both proteins studied are purified in a closed dimer conformation, indicating that events within the cell can induce a stable, closed conformation of the MsbA homodimer that does not reopen even in the absence of nucleotide.     

Effects of the L511P and D512G mutations on the Escherichia coli ABC transporter MsbA

MsbA is a member of the ABC transporter superfamily and is homologous to ABC transporters linked to multidrug resistance. The nucleotide binding domains (NBDs) of these proteins include conserved motifs that are involved in ATP binding, including conserved SALD residues (D-loop) that are diagnostic in identifying ABC transporters but whose roles have not been identified. Within the D-loop, single point mutations L511P and D512G were discovered by random mutational analysis of MsbA to disrupt protein function in the cell [Polissi, A., and Georgopoulos, C. (1996) Mol. Microbiol. 20, 1221-1233] but have not been further studied in MsbA or in detail in any other ABC transporter. In these studies, we show that both L511P and D512G mutants of MsbA are able to bind ATP at near-wild-type levels but are unable to maintain cell viability in an in vivo growth assay, verifying the theory that they are dysfunctional at some point after ATP binding. An ATPase assay further suggests that the L511P mutation prevents effective ATP hydrolysis, and an ATP detection assay reveals that only small amounts of ATP are hydrolyzed; D512G is able to hydrolyze ATP at a rate 3-fold faster than that of the wild type. EPR spectroscopy studies using reporter sites within the NBDs also indicate that at least some hydrolysis occurs in L511P or D512G MsbA but show fewer spectral changes than observed for the same reporters in the wild-type background. These studies indicate that L511 is necessary for efficient ATP hydrolysis and D512 is essential for conformational rearrangements required for flipping lipid A.     

Syndecan contributes to heart cell specification and lumen formation during Drosophila cardiogenesis

The transmembrane proteoglycan Syndecan contributes to cell surface signaling of diverse ligands in mammals, yet in Drosophila, genetic evidence links Syndecan only to the Slit receptor Roundabout and to the receptor tyrosine phosphatase LAR. Here we characterize the requirement for syndecan in the determination and morphogenesis of the Drosophila heart, and reveal two phases of activity, indicating that Syndecan is a co-factor in at least two signaling events in this tissue. There is a stochastic failure to determine heart cell progenitors in a subset of abdominal hemisegments in embryos mutant for syndecan, and subsequent to Syndecan depletion by RNA interference. This phenotype is sensitive to gene dosage in the FGF receptor (Heartless), its ligand, Pyramus, as well as BMP-ligand Decapentaplegic (Dpp) and co-factor Sara. Syndecan is also required for lumen formation during assembly of the heart vessel, a phenotype shared with mutations in the Slit and Integrin signaling pathways. Phenotypic interactions of syndecan with slit and Integrin mutants suggest intersecting function, consistent with Syndecan acting as a co-receptor for Slit in the Drosophila heart.     

Parental efficacy and child behavior in a community sample of children with and without attention-deficit hyperactivity disorder (ADHD)

Most studies of attention-deficit hyperactivity disorder (ADHD) youth have obtained data from the perspective of either children or parents, but not both simultaneously. The purpose of this study was to examine child and parent perspectives on parenting in a large community-based sample of children with and without ADHD. We identified children in grades 4?C6 and their parents through surveys administered to a random sample of public schools. We used multivariable logistic regression to determine independent associations between child and parent characteristics and the presence of ADHD while controlling for covariates and clustering by school. Sufficient data were achieved for 2,509 child/parent dyads. Ten percent of youths (n?=?240) had been diagnosed with ADHD. Compared with those without ADHD, those with ADHD were more commonly male (67.9 vs. 48.0?%, p?<?.001) and age 12 or over (16.3 vs. 10.3?%). After adjusting for covariates and clustering, compared to children without ADHD, children with ADHD were significantly more likely to report lower self-regulation (OR?=?0.68, 95?% CI?=?0.53, 0.88) and higher levels of rebelliousness (OR?=?2.00, 95?% CI?=?1.52, 2.69). Compared with parents whose children did not have ADHD, parents of children with ADHD rated their overall parental efficacy substantially lower (OR?=?0.23, 95?% CI?=?0.15, 0.33). However, child assessment of parenting style was similar by ADHD. Despite the internal challenges community-based youth with ADHD face, many parents of ADHD youth exhibit valuable parental skills from the perspective of their children. Feedback of this information to parents may improve parental self-efficacy, which is known to be positively associated with improved ADHD outcomes.