Genetic diversity analysis using lowly polymorphic dominant markers: the example of AFLP in pigs

DNA markers are commonly used for large-scale evaluation of genetic diversity in farm animals, as a component of the management of animal genetic resources. AFLP markers are useful for such studies as they can be generated relatively simply; however, challenges in analysis arise from their dominant scoring and the low level of polymorphism of some markers. This paper describes the results obtained with a set of AFLP markers in a study of 59 pig breeds. AFLP fingerprints were generated using four primer combinations (PC), yielding a total of 148 marker loci, and average harmonic mean of breed sample size was 37.3. The average proportion of monomorphic populations was 63% (range across loci: 3%-98%). The moment-based method of Hill and Weir (2004, Mol Ecol 13:895-908) was applied to estimate gene frequencies, gene diversity (F(ST)), and Reynolds genetic distances. A highly significant average F(ST) of 0.11 was estimated, together with highly significant PC effects on gene diversity. The variance of F(ST) across loci also significantly exceeded the variance expected under the hypothesis of AFLP neutrality, strongly suggesting the sensitivity of AFLP to selection or other forces. Moment estimates were compared to estimates derived from the square root estimation of gene frequency, as currently applied for dominant markers, and the biases incurred in the latter method were evaluated. The paper discusses the hypotheses underlying the moment estimations and various issues relating to the biallelic, dominant, and lowly polymorphic nature of this set of AFLP markers and to their use as compared to microsatellites for measuring genetic diversity.     

Interferon-stimulated gene ISG12b2 is localized to the inner mitochondrial membrane and mediates virus-induced cell death

Interferons (IFNs) are crucial for host defence against viruses. Many IFN-stimulated genes (ISGs) induced by viral infection exert antiviral effects. Microarray analysis of gene expression induced in liver tissues of mice on dengue virus (DENV) infection has led to identification of the ISG gene ISG12b2. ISG12b2 is also dramatically induced on DENV infection of Hepa 1-6 cells (mouse hepatoma cell line). Here, we performed biochemical and functional analyses of ISG12b2. We demonstrate that ISG12b2 is an inner mitochondrial membrane (IMM) protein containing a cleavable mitochondrial targeting sequence and multiple transmembrane segments. Overexpression of ISG12b2 in Hepa 1-6 induced release of cytochrome c from mitochondria, disruption of the mitochondrial membrane potential, and activation of caspase-9, caspase-3, and caspase-8. Treatment of ISG12b2-overexpressing Hepa 1-6 with inhibitors of pan-caspase, caspase-9, or caspase-3, but not caspase-8, reduced apoptotic cell death, suggesting that ISG12b2 activates the intrinsic apoptotic pathway. Of particular interest, we further demonstrated that ISG12b2 formed oligomers, and that ISG12b2 was able to mediate apoptosis through both Bax/Bak-dependent and Bax/Bak-independent pathways. Our study demonstrates that the ISG12b2 is a novel IMM protein induced by IFNs and regulates mitochondria-mediated apoptosis during viral infection.     

Aminothienopyridazine inhibitors of tau aggregation: evaluation of structure-activity relationship leads to selection of candidates with desirable in vivo properties

Previous studies demonstrated that members of the aminothienopyridazine (ATPZ) class of tau aggregation inhibitors exhibit a promising combination of in vitro activity as well as favorable pharmacokinetic properties (i.e., brain-penetration and oral bioavailability). Here we report the synthesis and evaluation of several new analogues. These studies indicate that the thienopyridazine core is essential for inhibition of tau fibrillization in vitro, while the choice of the appropriate scaffold decoration is critical to impart desirable ADME-PK properties. Among the active, brain-penetrant ATPZ inhibitors evaluated, 5-amino-N-cyclopropyl-3-(4-fluorophenyl)-4-oxo-3,4-dihydrothieno[3,4-d]pyridazine-1-carboxamide (43) was selected to undergo maximum tolerated dose and one-month tolerability testing in mice. The latter studies revealed that this compound is well-tolerated with no notable side-effects at an oral dose of 50mg/kg/day.     

Protection of tea melanin on hydrazine-induced liver injury

The protective activity of melanin derived from tea (MDFT) was studied using hydrazine as a DNA-reactive chemical agent. Intra-peritoneal administration of MDFT at the doses of 5 or 20 mg/kg dose-dependently prevented liver toxicity induced by hydrazine in rats. It normalized rises in serum alanine transferase activity and a decrease in the glutathione level in the liver. It also reduced the hepatic malondialdehyde concentration. Monitoring the intensity of chemiluminescence showed that MDFT could prevent the production of free radicals that are generated owing to metabolic transformation of hydrazine. It also prevented the formation 8-hydroxy-deoxyguanosine (8-OH-dG) DNA adducts. The results obtained in vivo and in vitro suggest that MDFT confers marked protection of the liver against hydrazine-induced oxidative toxicity.     

Honeycomb structure in the lamina lucida of epidermal basement membrane during metamorphosis of Rana temporaria ornativentris

In this study we examine the structure of the lamina lucida during metamorphosis of Rana temporaria ornativentris. During the metamorphosis of anuran larvae, both the epidermal cells and the dermal connective tissues in the tail regenerate. The basal surface of the epidermis becomes irregular and the epidermal basement membrane detaches from the epidermal cells, showing a widened lamina lucida. In this widened lamina we observed a geometrical honeycomb structure and a ladder structure. Each side of the honeycomb structure was approximately 40 nm and the intervals of the ladder structure were approximately 50 nm. From our observations we believe that the honeycomb and ladder appearances are different aspects of the same structure. At the beginning of metamorphosis anchoring filaments were prominent in the lamina lucida and, when the lamina lucida was tangentially cut, the lamina lucida showed the honeycomb structure. These results suggest that both the honeycomb and the ladder structures observed in the widened lamina lucida originate from constituents of the lamina lucida and become morphologically evident during the epidermal-dermal separation.     

Membrane topology and nicastrin-enhanced endoproteolysis of APH-1, a component of the gamma-secretase complex

APH-1, presenilin, nicastrin, and Pen-2 are proteins with varying membrane topologies that compose the gamma-secretase complex, which is responsible for the intramembrane proteolysis of several substrates including the amyloid precursor protein. APH-1 is known to be necessary for gamma-secretase activity, but its precise function in the complex is not fully understood, and its membrane topology has not been described, although it is predicted to traverse the membrane seven times. To investigate this, we used selective permeabilization of the plasma membrane and immunofluorescence microscopy to show that the C terminus of the APH-1 resides in the cytosolic space. Insertion of N-linked glycosylation sites into each of the hydrophilic loop domains and the N terminus of APH-1 showed that the N-terminal domain as well as loops 2, 4, and 6 could be glycosylated, whereas loops 1, 3, and 5 were not. Thus, APH-1 topologically resembles a seven-transmembrane domain receptor with the N terminus and even-numbered loops facing the endoplasmic reticulum lumen, and the C terminus and odd-numbered loops reside in the cytosolic space. By using these glycosylation mutants, we provide evidence that the association between nicastrin and APH-1 may occur very soon after APH-1 synthesis and that the interaction between these two proteins may rely more heavily on the transmembrane domains of APH-1 than on the loop domains. Furthermore, we found that APH-1 can be processed by several endoproteolytic events. One of these cleavages is strongly up-regulated by co-expression of nicastrin and generates a stable C-terminal fragment that associates with nicastrin.     

Endoproteolysis of beta-secretase (beta-site amyloid precursor protein-cleaving enzyme) within its catalytic domain. A potential mechanism for regulation

Sequential proteolysis of the amyloid precursor protein (APP) by beta- and gamma-secretase activities yields the amyloid beta peptide that is widely deposited in the brains of individuals with Alzheimer's disease. The membrane-anchored aspartyl protease beta-site APP-cleaving enzyme (BACE) exhibits all of the characteristics of a beta-secretase and has been shown to cleave APP at its beta-site in vitro and in vivo. We found that BACE undergoes cleavage on a surface-exposed alpha-helix between amino acid residues Leu-228 and Ala-229, generating stable N- and C-terminal fragments that remain covalently associated via a disulfide bond. The efficiency of BACE endoproteolysis was observed to depend heavily on cell and tissue type. In contrast to brain where holoprotein was predominant, BACE was found primarily as endoproteolyzed fragments in pancreas, liver, and muscle. In addition, we observed a marked up-regulation of BACE endoproteolysis in C2 myoblasts upon differentiation into multinucleated myotubes, a well established model system of muscle tissue specification. As in liver, BACE exists as endoproteolyzed fragments in the hepatic cell line, HepG2. We found that HepG2 cells are capable of generating amyloid beta peptide, suggesting that endoproteolyzed BACE retains measurable beta-secretase activity. We also found that BACE endoproteolysis occurs only after export from the endoplasmic reticulum, is enhanced in the trans-Golgi network, and is sensitive to inhibitors of vesicular acidification. The membrane-bound proteases tumor necrosis factor alpha-converting enzyme and furin were not found to be responsible for this cleavage nor was BACE observed to mediate its own endoproteolysis by an autocatalytic mechanism. Thus, we characterize a specific processing event that may serve to regulate the enzymatic activity of BACE on a post-translational level.     

Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein

alpha-Synucleinopathies are neurodegenerative disorders that range pathologically from the demise of select groups of nuclei to pervasive degeneration throughout the neuraxis. Although mounting evidence suggests that alpha-synuclein lesions lead to neurodegeneration, this remains controversial. To explore this issue, we generated transgenic mice expressing wild-type and A53T human alpha-synuclein in CNS neurons. Mice expressing mutant, but not wild-type, alpha-synuclein developed a severe and complex motor impairment leading to paralysis and death. These animals developed age-dependent intracytoplasmic neuronal alpha-synuclein inclusions paralleling disease onset, and the alpha-synuclein inclusions recapitulated features of human counterparts. Moreover, immunoelectron microscopy revealed that the alpha-synuclein inclusions contained 10-16 nm wide fibrils similar to human pathological inclusions. These mice demonstrate that A53T alpha-synuclein leads to the formation of toxic filamentous alpha-synuclein neuronal inclusions that cause neurodegeneration.     

Exogenous α-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death

Inclusions composed of α-synuclein (α-syn), i.e., Lewy bodies (LBs) and Lewy neurites (LNs), define synucleinopathies including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Here, we demonstrate that preformed fibrils generated from full-length and truncated recombinant α-syn enter primary neurons, probably by adsorptive-mediated endocytosis, and promote recruitment of soluble endogenous α-syn into insoluble PD-like LBs and LNs. Remarkably, endogenous α-syn was sufficient for formation of these aggregates, and overexpression of wild-type or mutant α-syn was not required. LN-like pathology first developed in axons and propagated to form LB-like inclusions in perikarya. Accumulation of pathologic α-syn led to selective decreases in synaptic proteins, progressive impairments in neuronal excitability and connectivity, and, eventually, neuron death. Thus, our data contribute important insights into the etiology and pathogenesis of PD-like α-syn inclusions and their impact on neuronal functions, and they provide a model for discovering therapeutics targeting pathologic α-syn-mediated neurodegeneration.     

P301S mutant human tau transgenic mice manifest early symptoms of human tauopathies with dementia and altered sensorimotor gating

Tauopathies are neurodegenerative disorders characterized by the accumulation of abnormal tau protein leading to cognitive and/or motor dysfunction. To understand the relationship between tau pathology and behavioral impairments, we comprehensively assessed behavioral abnormalities in a mouse tauopathy model expressing the human P301S mutant tau protein in the early stage of disease to detect its initial neurological manifestations. Behavioral abnormalities, shown by open field test, elevated plus-maze test, hot plate test, Y-maze test, Barnes maze test, Morris water maze test, and/or contextual fear conditioning test, recapitulated the neurological deficits of human tauopathies with dementia. Furthermore, we discovered that prepulse inhibition (PPI), a marker of sensorimotor gating, was enhanced in these animals concomitantly with initial neuropathological changes in associated brain regions. This finding provides evidence that our tauopathy mouse model displays neurofunctional abnormalities in prodromal stages of disease, since enhancement of PPI is characteristic of amnestic mild cognitive impairment, a transitional stage between normal aging and dementia such as Alzheimer's disease (AD), in contrast with attenuated PPI in AD patients. Therefore, assessment of sensorimotor gating could be used to detect the earliest manifestations of tauopathies exemplified by prodromal AD, in which abnormal tau protein may play critical roles in the onset of neuronal dysfunctions.