Pathological cell-cell interactions elicited by a neuropathogenic form of mutant Huntingtin contribute to cortical pathogenesis in HD mice

Expanded polyglutamine (polyQ) proteins in Huntington's disease (HD) as well as other polyQ disorders are known to elicit a variety of intracellular toxicities, but it remains unclear whether polyQ proteins can elicit pathological cell-cell interactions which are critical to disease pathogenesis. To test this possibility, we have created conditional HD mice expressing a neuropathogenic form of mutant huntingtin (mhtt-exon1) in discrete neuronal populations. We show that mhtt aggregation is a cell-autonomous process. However, progressive motor deficits and cortical neuropathology are only observed when mhtt expression is in multiple neuronal types, including cortical interneurons, but not when mhtt expression is restricted to cortical pyramidal neurons. We further demonstrate an early deficit in cortical inhibition, suggesting that pathological interactions between interneurons and pyramidal neurons may contribute to the cortical manifestation of HD. Our study provides genetic evidence that pathological cell-cell interactions elicited by neuropathogenic forms of mhtt can critically contribute to cortical pathogenesis in a HD mouse model.     

An EP overexpression screen for genetic modifiers of Notch pathway function in Drosophila melanogaster

The Notch pathway comprises a signal transduction cascade required for the proper formation of multiple tissues during metazoan development. Originally described in Drosophila for its role in nervous system formation, the pathway has attracted much wider interest owing to its fundamental roles in a range of developmental and disease-related processes. Despite extensive analysis, Notch signaling is not completely understood and it appears that additional components of the pathway remain to be identified and characterized. Here, we describe a novel genetic strategy to screen for additional Notch pathway genes. The strategy combines partial loss of function for pathway activity with Enhancer-promoter (EP)-induced overexpression of random loci across the dorsoventral wing margin. Mastermind (Mam) is a nuclear component of the Notch signaling cascade. Using a GAL4-UAS-driven dominant-negative form of Mam, we created a genotype that exhibits a completely penetrant dominant wing-nicking phenotype. This phenotype was assayed for enhancement or suppression after outcrossing to several thousand EP lines. The screen identified known components or modifiers of Notch pathway function, as well as several potential new components. Our results suggest that a genetic screen that combines partial loss of function with random gene overexpression might be a useful strategy in the analysis of developmental pathways.     

Structural characterization of lacticin 3147, a two-peptide lantibiotic with synergistic activity

Lantibiotics are antibacterial peptides isolated from bacterial sources that exhibit activity toward Gram-positive organisms and are usually several orders of magnitude more potent than traditional antibiotics such as penicillin. They contain a number of unique structural features including dehydro amino acid and lanthionine (thioether) residues. Introduced following ribosomal translation of the parent peptide, these moieties render conventional methods of peptide analysis ineffective. We report herein a new method using nickel boride (Ni(2)B), in the presence of deuterium gas, to reduce dehydro side chains and reductively desulfurize lanthionine bridges found in lantibiotics. Using this approach, it is possible to identify and distinguish the original locations of dehydro side chains and lanthionine bridges by traditional peptide sequencing (Edman degradation) followed by mass spectrometry. The strategy was initially verified using nisin A, a structurally well characterized lantibiotic, and subsequently extended to the novel two-component lantibiotic, lacticin 3147, produced by Lactococcus lactis subspecies lactis DPC3147. The primary structures of both lacticin 3147 peptides were then fully assigned by use of multidimensional NMR spectroscopy, showing that lacticin 3147 A1 has a specific lanthionine bridging pattern which resembles the globular type-B lantibiotic mersacidin, whereas the A2 peptide is a member of the elongated type-A lantibiotic class. Also obtained by NMR were solution conformations of both lacticin 3147 peptides, indicating that A1 may adopt a conformation similar to that of mersacidin and that the A2 peptide adopts alpha-helical structure. These results are the first of their kind for a synergistic lantibiotic pair (only four such pairs have been reported to date).     

ATAD2B is a phylogenetically conserved nuclear protein expressed during neuronal differentiation and tumorigenesis

ATAD2 is an E2F target gene that is highly expressed in gastrointestinal and breast carcinomas. Here we characterize a related gene product, ATAD2B. Both genes are evolutionarily conserved, with orthologues present in all eukaryotic genomes examined. Human ATAD2B shows a high degree of similarity to ATAD2. Both contain an AAA domain and a bromodomain with amino acid sequences sharing 97% and 74% identity, respectively. The expression of ATAD2B was studied in the chicken embryo using a polyclonal antibody raised against a recombinant fragment of human ATAD2B. Immunohistochemistry revealed transient nuclear expression in subpopulations of developing neurons. The transient nature of the expression was confirmed by immunoblotting homogenates of the developing telencephalon. Cell fractionation was used to confirm the nuclear localization of ATAD2B in the developing nervous system: anti-ATAD2B recognizes a smaller band (approximately 160 kDa) in the nuclear fraction and a larger band (approximately 300 kDa) in the membrane fraction, suggesting that posttranslational processing of ATAD2B may regulate its transport to the nucleus. The expression of ATAD2B was also studied in human tumors. Oncomine and immunohistochemistry reveal ATAD2B expression in glioblastoma and oligodendroglioma; ATAD2B immunostaining was also elevated in human breast carcinoma. In tumors ATAD2B appears to be cytoplasmic or membrane bound, and not nuclear. Our observations suggest that ATAD2B may play a role in neuronal differentiation and tumor progression.     

Cannabinoid WIN 55,212-2 regulates TRPV1 phosphorylation in sensory neurons

Cannabinoids are known to have multiple sites of action in the nociceptive system, leading to reduced pain sensation. However, the peripheral mechanism(s) by which this phenomenon occurs remains an issue that has yet to be resolved. Because phosphorylation of TRPV1 (transient receptor potential subtype V1) plays a key role in the induction of thermal hyperalgesia in inflammatory pain models, we evaluated whether the cannabinoid agonist WIN 55,212-2 (WIN) regulates the phosphorylation state of TRPV1. Here, we show that treatment of primary rat trigeminal ganglion cultures with WIN led to dephosphorylation of TRPV1, specifically at threonine residues. Utilizing Chinese hamster ovary cell lines, we demonstrate that Thr(144) and Thr(370) were dephosphorylated, leading to desensitization of the TRPV1 receptor. This post-translational modification occurred through activation of the phosphatase calcineurin (protein phosphatase 2B) following WIN treatment. Furthermore, knockdown of TRPA1 (transient receptor potential subtype A1) expression in sensory neurons by specific small interfering RNA abolished the WIN effect on TRPV1 dephosphorylation, suggesting that WIN acts through TRPA1. We also confirm the importance of TRPA1 in WIN-induced dephosphorylation of TRPV1 in Chinese hamster ovary cells through targeted expression of one or both receptor channels. These results imply that the cannabinoid WIN modulates the sensitivity of sensory neurons to TRPV1 activation by altering receptor phosphorylation. In addition, our data could serve as a useful strategy in determining the potential use of certain cannabinoids as peripheral analgesics.     

Chronic hyperglycemia predisposes to exaggerated inflammatory response and leukocyte dysfunction in Akita mice

The role of polymorphonuclear neutrophils (PMN) in mediating diabetic tissue damage to the periodontium was investigated in a novel model of chronic hyperglycemia, the Akita mouse. Induction of acute peritoneal inflammation in wild-type (WT) and Akita mice resulted in exaggerated IL-6 response in Akita mice (2.9-fold increase over WT values) and a markedly increased chemokine response (KC, 2.6-fold; MCP-1, 2.6-fold; and MIP-1alpha, 4.4-fold increase over WT values). Chemotaxis to both fMLP and WKYMVm was significantly reduced in isolated Akita PMN compared with WT PMN as measured in a Boyden chamber. Superoxide release in contrast was significantly increased in Akita PMN as measured with cytochrome c reduction. Bone marrow-derived Akita PMN showed partial translocation of p47phox to the cell membrane without external stimulation, suggesting premature assembly of the superoxide-producing NADPH oxidase in hyperglycemia. In vivo studies revealed that ligature-induced periodontal bone loss is significantly greater in Akita mice compared with WT. Moreover, intravital microscopy of gingival vessels showed that leukocyte rolling and attachment to the vascular endothelium is enhanced in periodontal vessels of Akita mice. These results indicate that chronic hyperglycemia predisposes to exaggerated inflammatory response and primes leukocytes for marginalization and superoxide production but not for transmigration. Thus, leukocyte defects in hyperglycemia may contribute to periodontal tissue damage by impairing the innate immune response to periodontal pathogens as well as by increasing free radical load in the gingival microvasculature.