CEP162 is an axoneme-recognition protein promoting ciliary transition zone assembly at the cilia base

The transition zone (TZ) is a specialized compartment found at the base of cilia, adjacent to the centriole distal end, where axonemal microtubules (MTs) are heavily cross-linked to the surrounding membrane to form a barrier that gates the ciliary compartment. A number of ciliopathy molecules have been found to associate with the TZ, but factors that directly recognize axonemal MTs to specify TZ assembly at the cilia base remain unclear. Here, through quantitative centrosome proteomics, we identified an axoneme-associated protein, CEP162, tethered specifically at centriole distal ends to promote TZ assembly. CEP162 interacts with core TZ components, and mediates their association with MTs. Loss of CEP162 arrests ciliogenesis at the stage of TZ assembly. Abolishing its centriolar tethering, however, allows CEP162 to stay on the growing end of the axoneme, and ectopically assemble TZ components at cilia tips. This generates extra-long cilia with strikingly swollen tips that actively release ciliary contents into the extracellular environment. CEP162 is thus an axoneme-recognition protein “pre-tethered” at centriole distal ends prior to ciliogenesis to promote and restrict TZ formation specifically at the cilia base

Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I

Mammalian MutL homologues function in DNA mismatch repair (MMR) after replication errors and in meiotic recombination. Both functions are initiated by a heterodimer of MutS homologues specific to either MMR (MSH2–MSH3 or MSH2–MSH6) or crossing over (MSH4–MSH5). Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects. We show herein that two distinct complexes involving MLH3 are formed during murine meiosis. The first is a stable association between MLH3 and MLH1 and is involved in promoting crossing over in conjunction with MSH4–MSH5. The second complex involves MLH3 together with MSH2–MSH3 and localizes to repetitive sequences at centromeres and the Y chromosome. This complex is up-regulated in Pms2−/− males, but not females, providing an explanation for the sexual dimorphism seen in Pms2−/− mice. The association of MLH3 with repetitive DNA sequences is coincident with MSH2–MSH3 and is decreased in Msh2−/− and Msh3−/− mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis

Increased localization of APP-C99 in mitochondria-associated ER membranes causes mitochondrial dysfunction in Alzheimer disease

In the amyloidogenic pathway associated with Alzheimer disease (AD), the amyloid precursor protein (APP) is cleaved by beta-secretase to generate a 99-aa C-terminal fragment (C99) that is then cleaved by c-secretase to generate the beta-amyloid (Ab) found in senile plaques. In previous reports, we and others have shown that c-secretase activity is enriched in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) and that ER-mitochondrial connectivity and MAM function are upregulated in AD. We now show that C99, in addition to its localization in endosomes, can also be found in MAM, where it is normally processed rapidly by c-secretase. In cell models of AD, however, the concentration of unprocessed C99 increases in MAM regions, resulting in elevated sphingolipid turnover and an altered lipid composition of both MAM and mitochondrial membranes. In turn, this change in mitochondrial membrane composition interferes with the proper assembly and activity of mitochondrial respiratory supercomplexes, thereby likely contributing to the bioenergetic defects characteristic of AD.We thank Drs. Orian Shirihai and Marc Liesa (UCLA) for assistance with the Seahorse measurements, Dr. Huaxi Xu (Sanford Burnham Institute) for the APP-DKO MEFs and Dr. Mark Mattson (NIH) for the PS1 knock-in mice, Drs. Arancio and Teich for the APP-KO mice tissues used in these studies, Dr. Hua Yang (Columbia University) for mouse husbandry, and Drs. Marc Tambini, Ira Tabas, and Serge Przedborski for helpful comments. This work was supported by the Fundacion Alfonso Martin Escudero (to M.P.); the Alzheimer's Drug Discovery Foundation, the Ellison Medical Foundation, the Muscular Dystrophy Association, the U.S. Department of Defense W911NF-12-1-9159 and W911F-15-1-0169), and the J. Willard and Alice S. Marriott Foundation (to E.A.S.); the U.S. National Institutes of Health (P01-HD080642 and P01-HD032062 to E.A.S.; NS071571 and HD071593 to M.F.M.; R01-NS056049 and P50-AG008702 to G.D.P.; 1S10OD016214-01A1 to G.S.P. and F.P.M, and K01-AG045335 to E.A.-G.), the Lucien Cote Early Investigator Award in Clinical Genetics from the Parkinson's Disease Foundation (PDF-CEI-1364 and PDF-CEI-1240) to C.G.-L., and National Defense Science and Engineering Graduate Fellowship (FA9550-11-C-0028) to R.R.A.S

Alzheimer’s-associated upregulation of mitochondria-associated ER membranes after traumatic brain injury

23 p.-5 fig.-3 tab.-1 graph. abst.Traumatic brain injury (TBI) can lead to neurodegenerative diseases such as Alzheimer’s disease (AD) through mechanisms that remain incompletely characterized. Similar to AD, TBI models present with cellular metabolic alterations and modulated cleavage of amyloid precursor protein (APP). Specifically, AD and TBI tissues display increases in amyloid-β as well as its precursor, the APP C-terminal fragment of 99 a.a. (C99). Our recent data in cell models of AD indicate that C99, due to its affinity for cholesterol, induces the formation of transient lipid raft domains in the ER known as mitochondria-associated endoplasmic reticulum (ER) membranes (“MAM” domains). The formation of these domains recruits and activates specific lipid metabolic enzymes that regulate cellular cholesterol trafficking and sphingolipid turnover. Increased C99 levels in AD cell models promote MAM formation and significantly modulate cellular lipid homeostasis. Here, these phenotypes were recapitulated in the controlled cortical impact (CCI) model of TBI in adult mice. Specifically, the injured cortex and hippocampus displayed significant increases in C99 and MAM activity, as measured by phospholipid synthesis, sphingomyelinase activity and cholesterol turnover. In addition, our cell type-specific lipidomics analyses revealed significant changes in microglial lipid composition that are consistent with the observed alterations in MAM-resident enzymes. Altogether, we propose that alterations in the regulation of MAM and relevant lipid metabolic pathways could contribute to the epidemiological connection between TBI and AD.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by the U.S. National Institutes of Health (T32-DK007647 to RRA; R21NS125395 to LS; S10-OD016214 and P30-CA013330 to FPM; R01-EB029523 to WM; R01-NS095803 to SGK; R01-NS088197 to RJD; R01-AG056387 to EA-G) and the U.S. Department of Defense (National Defense Science and Engineering Graduate Fellowship, FA9550-11-C-0028, to RRA).Peer reviewe

Isolation of a Rickettsial Pathogen from a Non-Hematophagous Arthropod

Rickettsial diversity is intriguing in that some species are transmissible to vertebrates, while others appear exclusive to invertebrate hosts. Of particular interest is Rickettsia felis, identifiable in both stored product insect pests and hematophagous disease vectors. To understand rickettsial survival tactics in, and probable movement between, both insect systems will explicate the determinants of rickettsial pathogenicity. Towards this objective, a population of Liposcelis bostrychophila, common booklice, was successfully used for rickettsial isolation in ISE6 (tick-derived cells). Rickettsiae were also observed in L. bostrychophila by electron microscopy and in paraffin sections of booklice by immunofluorescence assay using anti-R. felis polyclonal antibody. The isolate, designated R. felis strain LSU-Lb, resembles typical rickettsiae when examined by microscopy. Sequence analysis of portions of the Rickettsia specific 17-kDa antigen gene, citrate synthase (gltA) gene, rickettsial outer membrane protein A (ompA) gene, and the presence of the R. felis plasmid in the cell culture isolate confirmed the isolate as R. felis. Variable nucleotide sequences from the isolate were obtained for R. felis-specific pRF-associated putative tldD/pmbA. Expression of rickettsial outer membrane protein B (OmpB) was verified in R. felis (LSU-Lb) using a monoclonal antibody. Additionally, a quantitative real-time PCR assay was used to identify a significantly greater median rickettsial load in the booklice, compared to cat flea hosts. With the potential to manipulate arthropod host biology and infect vertebrate hosts, the dual nature of R. felis provides an excellent model for the study of rickettsial pathogenesis and transmission. In addition, this study is the first isolation of a rickettsial pathogen from a non-hematophagous arthropod

Assessing Implicit Odor Localization in Humans Using a Cross-Modal Spatial Cueing Paradigm

Navigation based on chemosensory information is one of the most important skills in the animal kingdom. Studies on odor localization suggest that humans have lost this ability. However, the experimental approaches used so far were limited to explicit judgements, which might ignore a residual ability for directional smelling on an implicit level without conscious appraisal.A novel cueing paradigm was developed in order to determine whether an implicit ability for directional smelling exists. Participants performed a visual two-alternative forced choice task in which the target was preceded either by a side-congruent or a side-incongruent olfactory spatial cue. An explicit odor localization task was implemented in a second experiment.No effect of cue congruency on mean reaction times could be found. However, a time by condition interaction emerged, with significantly slower responses to congruently compared to incongruently cued targets at the beginning of the experiment. This cueing effect gradually disappeared throughout the course of the experiment. In addition, participants performed at chance level in the explicit odor localization task, thus confirming the results of previous research.The implicit cueing task suggests the existence of spatial information processing in the olfactory system. Response slowing after a side-congruent olfactory cue is interpreted as a cross-modal attentional interference effect. In addition, habituation might have led to a gradual disappearance of the cueing effect. It is concluded that under immobile conditions with passive monorhinal stimulation, humans are unable to explicitly determine the location of a pure odorant. Implicitly, however, odor localization seems to exert an influence on human behaviour. To our knowledge, these data are the first to show implicit effects of odor localization on overt human behaviour and thus support the hypothesis of residual directional smelling in humans

HIV Nef and Antiretroviral Therapy Have an Inhibitory Effect on Autophagy in Human Astrocytes that May Contribute to HIV-Associated Neurocognitive Disorders

A significant number of people living with HIV (PLWH) develop HIV-associated neurocognitive disorders (HAND) despite highly effective antiretroviral therapy (ART). Dysregulated macroautophagy (autophagy) is implicated in HAND pathogenesis. The viral protein Nef, expressed even with suppressive ART, and certain antiretrovirals affect autophagy in non-CNS cells. Astrocytes, vital for CNS microenvironment homeostasis and neuronal health, require autophagy for their own homeostasis. We hypothesized that extracellular Nef and/or ART impact astrocyte autophagy, thus contributing to HAND. We studied in-bulk and selective autophagic flux in primary human astrocytes treated with extracellular Nef and/or a combination of tenofovir+emtricitabine+raltegravir (ART) using Western blotting, a tandem fluorescent LC3 reporter, and transmission electron microscopy/morphometry. We show that after 24 h treatment, Nef and ART decrease autophagosomes through different mechanisms. While Nef accelerates autophagosome degradation without inducing autophagosome formation, ART inhibits autophagosome formation. Combination Nef+ART further depletes autophagosomes by inducing both abnormalities. Additionally, extracellular Nef and/or ART inhibit lysosomal degradation of p62, indicating Nef and/or ART affect in-bulk and selective autophagy differently. Dysregulation of both autophagic processes is maintained after 7 days of Nef and/or ART treatment. Persistent autophagy dysregulation due to chronic Nef and/or ART exposure may ultimately result in astrocyte and neuronal dysfunction, contributing to HAND