A Hereditary Spastic Paraplegia Mouse Model Supports a Role of ZFYVE26/SPASTIZIN for the Endolysosomal System

Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells.     

Thymic epithelial cells use macroautophagy to turn their inside out for CD4 T cell tolerance

During development in the thymus, each T lymphocyte is equipped with one, essentially unique, T cell receptor (TCR)-specificity. Due to its random nature, this process inevitably also leads to the emergence of potentially dangerous T lymphocytes that may recognize ‘self.’ Nevertheless, autoimmune tissue destruction, the cause of diseases such as multiple sclerosis and diabetes, is the exception rather than the rule. This state of immunological self-tolerance is to a large degree based upon a process called ‘negative selection’: prior to joining the circulating lymphocyte pool, immature T cells test their receptor on self-antigens within the thymic microenvironment, and TCR engagement at this immature stage elicits an apoptotic suicide program. We now find evidence that macroautophagy supports the tolerogenic presentation of self-antigens in the thymus.     

Acetylenic 2-phenylethylamides and new isobutylamides from Acmella oleracea (L.) R. K. Jansen,a Brazilian spice with larvicidal activity on Aedes aegypti

Ethanol extract obtained from dried leaves of Acmella oleracea afforded after a liquid/liquid partition procedure a larvicidal hexane fraction (LC₅₀ = 145.6 ppm) and a non larvicidal dichloromethane one. From the inactive fraction, three amides were identified, two new structures, named deca-6,9-dihydroxy-(2E,7E)-dienoic acid isobutylamide (1), deca-8,9-dihydroxy-(2E,6Z)-dienoic acid isobutylamide (2) and the known nona-2,3-dihydroxy-6,8-diynoic acid 2-phenylethylamide (3). Bioassay-guided chromatographic fractionation of the hexane partition led to the identification of an amide mixture, nona-(2Z)-en-6,8-diynoic acid 2-phenylethylamide (4) and deca-(2Z)-en-6,8-diynoic acid 2-phenylethlylamide (5). This mixture was active against Aedes aegypti larvae at LC₅₀ = 7.6 ppm. Low toxicity of crude extracts and derived fractions on Artemia salina nauplies showed the possibility of using them to control the A. aegypti mosquito larvae. This is the first report on larvicidal activity of acetylenic 2-phenylethylamides and their identification in A. oleracea leaves.     

Camphor Pathway Redux: Functional Recombinant Expression of 2,5- and 3,6-Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453 with Their Cognate Flavin Reductase Catalyzing Baeyer-Villiger Reactions

Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (−) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (K_m = 32 μM), and it catalyzes the reduction of flavin mononucleotide (FMN) (K_m = 3.6 μM; k_cat = 283 s⁻¹) in preference to flavin adenine dinucleotide (FAD) (K_m = 19 μM; k_cat = 128 s⁻¹). Sequence determination of ∼40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE_25–1 for 2,5-DKCMO-1 and camE_25–2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE₃₆). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and ∼533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates.     

Hippocampal Neuroligin-2 Overexpression Leads to Reduced Aggression and Inhibited Novelty Reactivity in Rats

Disturbances of the excitation/inhibition (E/I) balance in the brain were recently suggested as potential factors underlying disorders like autism and schizophrenia resulting in associated behavioral alterations including changes in social and emotional behavior as well as abnormal aggression. Neuronal cell adhesion molecules (nCAMs) and mutations in these genes were found to be strongly implicated in the pathophysiology of these disorders. Neuroligin2 (nlgn2) is a postsynaptic cell adhesion molecule, which is predominantly expressed at inhibitory synapses and required for synapse specification and stabilization. Changes in the expression of nlgn2 were shown to result in alterations of social behavior as well as altered inhibitory synaptic transmission, hence modifying the E/I balance. In our study, we focused on the role of nlgn2 in the dorsal hippocampus in the regulation of emotional and social behaviors. To this purpose, we injected an AAV construct overexpressing nlgn2 in the hippocampus of rats and investigated the effects on behavior and on markers for the E/I ratio. We could show an increase in GAD65, a GABA-synthesizing protein in neuronal terminals, and furthermore, reduced exploration of novel stimuli and less offensive behavior. Our data suggest nlgn2 in the hippocampus to be strongly implicated in maintaining the E/I balance in the brain and thereby modulating social and emotional behavior.     

Rhipsalis (Cactaceae): loss and gain of floral rewards is mirrored in range sizes and distribution patterns of species

Autogamous species are usually distinguishable from xenogamous relatives by smaller flowers, fewer or even no floral rewards and lower pollen–ovule (P/O) ratios. Many Rhipsalis spp. are small flowered, selfing and include the most widespread species in Cactaceae. However, Rhipsalis also includes a large number of narrowly endemic species and is most diverse in the Atlantic rainforests of Brazil. To investigate the evolution of floral function and the correlation between floral function and range size, we analysed display size, floral reward and P/O ratios of Rhipsalis and its closest relatives, reconstructed ancestral traits and related these patterns to the distributions and range sizes of the species. Display size and sugar amount are reduced in subgenera Goniorhipsalis and Rhipsalis and secondarily increased in Phyllarthrorhipsalis, whereas the P/O ratio is decreased in subgenera Rhipsalis and Phyllarthrorhipsalis. We interpret this pattern as a switch from a predominantly xenogamous to an autogamous reproductive system, followed by a return to a predominantly xenogamous system. None of the floral parameters shows significant correlations with range size, except for display size. Nevertheless, those species with the smallest flowers, lowest sugar amounts per flower and lowest P/O ratios occur either outside southeastern Brazil and/or have comparatively large distribution ranges. Almost all Rhipsalis spp. occurring outside the Atlantic rainforests are restricted to the clade formed by subgenera Rhipsalis and Phyllarthrorhipsalis. Thus, we believe that the evolution of an autogamous reproduction system enabled this lineage of Rhipsalis to diversify and spread in the Atlantic rainforests, in the rest of the Neotropics and even spread to the Old World, where it is the only member of the family.     

Metabolite targeting: development of a comprehensive targeted metabolomics platform for the assessment of diabetes and its complications

Biomarker studies for metabolic disorders like diabetes mellitus (DM) are an important approach towards a better understanding of the underlying pathophysiological mechanisms of diseases (Roberts and Gerszten in Cell Metab 18:43–50, 2013; Wilson et al. in Proteome Res 4:591–598, 2005). Furthermore, screening of potential metabolic biomarkers opens the opportunity of early diagnosis as well as therapy and drug monitoring of metabolic disorders (Rhee et al. in J Clin Invest 10:1–10, 2011; Wang et al. in Nat Med 17:448–458, 2011; Wenk in Nat Rev Drug Discov 4:594–610, 2005). The aim of the present study was to develop methods for the quantitative determination of 74 potential metabolite biomarkers for DM and diabetic nephropathy (DN) in serum. Several studies have shown that the concentrations of many polar metabolites like amino or organic acids are changed in subjects suffering from diabetes (Wang et al. in Nat Med 17:448–458, 2011; Yuan et al. in J Chromatogr B 813:53–58, 2007). Analyzing polar analytes presents a challenge in liquid chromatography (LC) coupled with ESI–MS/MS (Gika et al. in J Sep Sci 31:1598–1608, 2008; Spagou et al. in J Sep Sci 33:716–727, 2010). Considering those reasons we decided to develop a specific HILIC–ESI–QqQ–MS/MS-method for quantitative determination of these polar metabolites. A subsequent method validation was carried out for both HILIC and RP chromatography with respect to the guidelines of the Food and Drug Administration (FDA in Food and Drug Administration: Guidance for industry, bioanalytical method validation, 2001). The HILIC and RP LC–MS methods were successfully validated. Furthermore, the HILIC method presented here was applied to serum samples of GIPR^dn transgenic mice, a diabetic strain developing DN, and non transgenic littermate controls. Significant, diabetes-associated changes were observed for the concentrations of 21 out of 62 metabolites. The new methods described here accurately quantify 74 metabolites known to be regulated in diabetes, allowing for direct comparison between studies and laboratories. Thus, these methods may be highly adoptable in clinical research, providing a starting point for early diagnosis and metabolic screening.