ALBERT

Beschreibung: Mutations in the leucine-rich repeat kinase 2 ( LRRK2 ) gene cause late-onset Parkinson's disease (PD). Emerging evidence suggests a role for LRRK2 in the endocytic pathway. Here, we show that LRRK2 is released in extracellular microvesicles (i.e. exosomes) from cells that natively express LRRK2. LRRK2 localizes to collecting duct epithelial cells in the kidney that actively secrete exosomes into urine. Purified urinary exosomes contain LRRK2 protein that is both dimerized and phosphorylated. We provide a quantitative proteomic profile of 1673 proteins in urinary exosomes and find that known LRRK2 interactors including 14-3-3 are some of the most abundant exosome proteins. Disruption of the 14-3-3 LRRK2 interaction with a 14-3-3 inhibitor or through acute LRRK2 kinase inhibition potently blocks LRRK2 release in exosomes, but familial mutations in LRRK2 had no effect on secretion. LRRK2 levels were overall comparable but highly variable in urinary exosomes derived from PD cases and age-matched controls, although very high LRRK2 levels were detected in some PD affected cases. We further characterized LRRK2 exosome release in neurons and macrophages in culture, and found that LRRK2-positive exosomes circulate in cerebral spinal fluid (CSF). Together, these results define a pathway for LRRK2 extracellular release, clarify one function of the LRRK2 14-3-3 interaction and provide a foundation for utilization of LRRK2 as a biomarker in clinical trials.

Beschreibung: We examine cues used in assortative mating in a sympatric species flock of pupfishes ( Cyprinodon ) from San Salvador Island, The Bahamas. The species are morphologically, genetically, and ecologically distinct. We focus on 2 species, a generalist detritivore and a predator/scale-eater. Experiments show that premating isolation is based primarily on visual cues and is asymmetrical. It is well developed in the less abundant scale-eater but is much less well developed in the more abundant detritivore. Responses of detritivore females from 3 lakes to conspecific and scale-eater males suggest that abundance of the predator also affects development of premating isolating mechanisms in the prey species. These results highlight the importance of frequency-dependent selection and predators in the evolution of premating isolating mechanisms.

Beschreibung: MUS81 plays important cellular roles in the restart of stalled replication forks, the resolution of recombination intermediates and in telomere length maintenance. Although the actions of MUS81-EME1 have been extensively investigated, MUS81 is the catalytic subunit of two human structure-selective endonucleases, MUS81-EME1 and MUS81-EME2. Little is presently known about the activities of MUS81-EME2. Here, we have purified MUS81-EME2 and compared its activities with MUS81-EME1. We find that MUS81-EME2 is a more active endonuclease than MUS81-EME1 and exhibits broader substrate specificity. Like MUS81-EME1, MUS81-EME2 cleaves 3'-flaps, replication forks and nicked Holliday junctions, and exhibits limited endonuclease activity with intact Holliday junctions. In contrast to MUS81-EME1, however, MUS81-EME2 cuts D-loop recombination intermediates and in so doing disengages the D-loop structure by cleaving the 3'-invading strand. Additionally, MUS81-EME2 acts on 5'-flap structures to cleave off a duplex arm, in reactions that cannot be promoted by MUS81-EME1. These studies suggest that MUS81-EME1 and MUS81-EME2 exhibit similar and yet distinct DNA structure selectivity, indicating that the two MUS81 complexes may promote different nucleolytic cleavage reactions in vivo .

Beschreibung: The Leucine rich repeat kinase 2 ( LRRK2 ) gene is genetically and biochemically linked to several diseases that involve innate immunity. LRRK2 protein is highly expressed in phagocytic cells of the innate immune system, most notably in myeloid cells capable of mounting potent pro-inflammatory responses. Knockdown of LRRK2 protein in these cells reduces pro-inflammatory responses. However, the effect of LRRK2 pathogenic mutations that cause Parkinson's disease on myeloid cell function is not clear but could provide insight into LRRK2-linked disease. Here, we find that rats expressing G2019S LRRK2 have exaggerated pro-inflammatory responses and subsequent neurodegeneration after lipopolysaccharide injections in the substantia nigra, with a marked increase in the recruitment of CD68 myeloid cells to the site of injection. While G2019S LRRK2 expression did not affect immunological homeostasis, myeloid cells expressing G2019S LRRK2 show enhanced chemotaxis both in vitro in two-chamber assays and in vivo in response to thioglycollate injections in the peritoneum. The G2019S mutation enhanced the association between LRRK2 and actin-regulatory proteins that control chemotaxis. The interaction between G2019S LRRK2 and actin-regulatory proteins can be blocked by LRRK2 kinase inhibitors, although we did not find evidence that LRRK2 phosphorylated these interacting proteins. These results suggest that the primary mechanism of G2019S LRRK2 with respect to myeloid cell function in disease may be related to exaggerated chemotactic responses.

Beschreibung: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common known cause of inherited Parkinson's disease (PD), and LRRK2 is a risk factor for idiopathic PD. How LRRK2 function is regulated is not well understood. Recently, the highly conserved 14-3-3 proteins, which play a key role in many cellular functions including cell death, have been shown to interact with LRRK2. In this study, we investigated whether 14-3-3s can regulate mutant LRRK2-induced neurite shortening and kinase activity. In the presence of 14-3-3 overexpression, neurite length of primary neurons from BAC transgenic G2019S-LRRK2 mice returned back to wild-type levels. Similarly, 14-3-3 overexpression reversed neurite shortening in neuronal cultures from BAC transgenic R1441G-LRRK2 mice. Conversely, inhibition of 14-3-3s by the pan-14-3-3 inhibitor difopein or dominant-negative 14-3-3 further reduced neurite length in G2019S-LRRK2 cultures. Since G2019S-LRRK2 toxicity is likely mediated through increased kinase activity, we examined 14-3-3's effects on LRRK2 kinase activity. 14-3-3 overexpression reduced the kinase activity of G2019S-LRRK2, while difopein promoted the kinase activity of G2019S-LRRK2. The ability of 14-3-3 to reduce LRRK2 kinase activity required direct binding of 14-3-3 with LRRK2. The potentiation of neurite shortening by difopein in G2019S-LRRK2 neurons was reversed by LRRK2 kinase inhibitors. Taken together, we conclude that 14-3-3 can regulate LRRK2 and reduce the toxicity of mutant LRRK2 through a reduction of kinase activity.

Beschreibung: Surface winds have declined in many regions of the world over the past few decades. These trends are referred to as global stilling and have recently been observed in the Western Cape Province of South Africa. The potential consequences of such changes on ecosystem function and productivity are a particular concern for the highly diverse and endemic local flora, largely associated with the fynbos biome. Yet, few studies have directly examined the impact of wind in the region. In this study, we explored the importance of wind and other drivers of plant transpiration ( E ) in a stand of Leucospermum conocarpodendron (L.) Buek trees on the Cape Peninsula. Wind speeds can be high in the Cape and could play an important role in influencing the rate of E . Overall, the influence of wind appeared to be significantly greater at night than during the day. While daytime E responded most strongly to changes in solar radiation ( R 2  = 0.79) and vapour pressure deficit ( R 2  = 0.57–0.67), night-time E ( E n ) was primarily driven by wind speed ( R 2  = 0.30–0.59). These findings have important implications for stilling and other aspects of climate change. Since E n was found to be a regular and significant ( P  〈 0.00) component of total daily E (10–27%), plants may conserve water should stilling continue. Still, the extent of this could be offset by strong daytime drivers. As such, plant water consumption will most likely increase in response to a warmer and drier climate. Changes in other biophysical variables are, however, clearly important to consider in the current debate on the impact of climate change.

Beschreibung: It is widely accepted that stabilization of the continental crust requires the presence of sub-continental lithospheric mantle. However, the degree of melt depletion required to stabilize the lithosphere and whether widespread refertilization is a significant process remain unresolved. Here, major and trace element, including platinum group elements (PGE), characterization of 40 mantle xenoliths from 13 localities is used to constrain the melt depletion, refertilization and metasomatic history of lithospheric mantle underneath the micro-continent Zealandia. Our previously published Re–Os isotopic data for a subset of these xenoliths indicate Phanerozoic to Paleoproterozoic ages and, reinterpreted with the new major and trace element data presented here, demonstrate that a large volume (〉2 million km 3 ) of lithospheric mantle with an age of 1·99 ± 0·21 Ga is present below the much younger crust of Zealandia. A peritectic melting model using moderately incompatible trace elements (e.g. Yb) in bulk-rocks demonstrates that these peridotites experienced a significant range of degrees of partial melting, between 3 and 28%. During subsolidus equilibration clinopyroxene gains significant rare earth elements (REE), which then leads to the underestimation of the degree of partial melting by ≤12% in fertile xenoliths. A new approach taking into account the effects of subsolidus re-equilibration on clinopyroxene composition effectively removes discrepancies in the calculated degree of melting and provides consistent estimates of between 4 and 29%. The estimated amount of melting is independent of the Re–Os model ages of the samples. The PGE patterns record simple melt depletion histories and the retention of primary base metal sulfides in the majority of the xenoliths. A rapid decrease in Pt/Ir N observed at c . 1·0 wt % Al 2 O 3 is a direct result of the exhaustion of sulfide in the mantle residue at c . 20–25% partial melting and the inability of Pt to form a stable alloy phase. Major elements preserve evidence for refertilization by a basaltic component that resulted in the formation of secondary clinopyroxene and low-forsterite olivine. The majority of xenoliths show the effects of cryptic metasomatic overprinting, ranging from minor to strong light REE enrichments in bulk-rocks (La/Yb N = 0·16–15·9). Metasomatism is heterogeneous, with samples varying from those with weak REE enrichment and notable positive Sr and U–Th anomalies and negative Nb–Ta anomalies in clinopyroxene to those that have extremely high concentrations of REE, Th–U and Nb. Chemical compositions are consistent with a carbonatitic component contributing to the metasomatism of the lithosphere under Zealandia. Notably, the intense metasomatism of the samples did not affect the PGE budget of the peridotites as this was controlled by residual sulfides.

Beschreibung: Mutations in the leucine-rich repeat kinase 2 ( LRRK2 ) gene are the most common known genetic cause of Parkinson's disease, and LRRK2 is also linked to Crohn's and Hansen's disease. LRRK2 is expressed in many organs in mammals but is particularly abundant in the kidney. We find that LRRK2 protein is predominantly localized to collecting duct cells in the rat kidney, with much lower expression in other kidney cells. While genetic knockout (KO) of LRRK2 expression is well-tolerated in mice and rats, a unique age-dependent pathology develops in the kidney. The cortex and medulla of LRRK2 KO rat kidneys become darkly pigmented in early adulthood, yet aged animals display no overt signs of kidney failure. Accompanying the dark pigment we find substantial macrophage infiltration in LRRK2 KO kidneys, suggesting the presence of chronic inflammation that may predispose to kidney disease. Unexpectedly, the dark kidneys of the LRRK2 KO rats are highly resistant to rhabdomyolysis-induced acute kidney injury compared with wild-type rats. Biochemical profiling of the LRRK2 KO kidneys using immunohistochemistry, proteomic and lipidomic analyses show a massive accumulation of hemoglobin and lipofuscin in renal tubules that account for the pigmentation. The proximal tubules demonstrate a corresponding up-regulation of the cytoprotective protein heme oxygenase-1 (HO-1) which is capable of mitigating acute kidney injury. The unusual kidney pathology of LRRK2 KO rats highlights several novel physiological roles for LRRK2 and provides indirect evidence for HO-1 expression as a protective mechanism in acute kidney injury in LRRK2 deficiency.

Beschreibung: CATNAP (Compile, Analyze and Tally NAb Panels) is a new web server at Los Alamos HIV Database, created to respond to the newest advances in HIV neutralizing antibody research. It is a comprehensive platform focusing on neutralizing antibody potencies in conjunction with viral sequences. CATNAP integrates neutralization and sequence data from published studies, and allows users to analyze that data for each HIV Envelope protein sequence position and each antibody. The tool has multiple data retrieval and analysis options. As input, the user can pick specific antibodies and viruses, choose a panel from a published study, or supply their own data. The output superimposes neutralization panel data, virus epidemiological data, and viral protein sequence alignments on one page, and provides further information and analyses. The user can highlight alignment positions, or select antibody contact residues and view position-specific information from the HIV databases. The tool calculates tallies of amino acids and N-linked glycosylation motifs, counts of antibody-sensitive and -resistant viruses in conjunction with each amino acid or N-glycosylation motif, and performs Fisher's exact test to detect potential positive or negative amino acid associations for the selected antibody. Website name: CATNAP (Compile, Analyze and Tally NAb Panels). Website address: http://hiv.lanl.gov/catnap .