Early mitochondrial abnormalities in hippocampal neurons cultured from Fmr1 pre‐mutation mouse model

Pre‐mutation CGG repeat expansions (55–200 CGG repeats; pre‐CGG) within the fragile‐X mental retardation 1 (FMR1) gene cause fragile‐X‐associated tremor/ataxia syndrome in humans. Defects in neuronal morphology, early migration, and electrophysiological activity have been described despite appreciable expression of fragile‐X mental retardation protein (FMRP) in a pre‐CGG knock‐in (KI) mouse model. The triggers that initiate and promote pre‐CGG neuronal dysfunction are not understood. The absence of FMRP in a Drosophila model of fragile‐X syndrome was shown to increase axonal transport of mitochondria. In this study, we show that dissociated hippocampal neuronal culture from pre‐CGG KI mice (average 170 CGG repeats) express 42.6% of the FMRP levels and 3.8‐fold higher Fmr1 mRNA than that measured in wild‐type neurons at 4 days in vitro. Pre‐CGG hippocampal neurons show abnormalities in the number, mobility, and metabolic function of mitochondria at this early stage of differentiation. Pre‐CGG hippocampal neurites contained significantly fewer mitochondria and greatly reduced mitochondria mobility. In addition, pre‐CGG neurons had higher rates of basal oxygen consumption and proton leak. We conclude that deficits in mitochondrial trafficking and metabolic function occur despite the presence of appreciable FMRP expression and may contribute to the early pathophysiology in pre‐CGG carriers and to the risk of developing clinical fragile‐X‐associated tremor/ataxia syndrome.     

KymoAnalyzer: a software tool for the quantitative analysis of intracellular transport in neurons

In axons, proper localization of proteins, vesicles, organelles, and other cargoes is accomplished by the highly regulated coordination of kinesins and dyneins, molecular motors that bind to cargoes and translocate them along microtubule (MT) tracks. Impairment of axonal transport is implicated in the pathogenesis of multiple neurodegenerative disorders including Alzheimer's and Huntington's diseases. To understand how MT‐based cargo motility is regulated and to delineate its role in neurodegeneration, it is critical to analyze the detailed dynamics of moving cargoes inside axons. Here, we present KymoAnalyzer, a software tool that facilitates the robust analysis of axonal transport from time‐lapse live‐imaging sequences. KymoAnalyzer is an open‐source software that automatically classifies particle trajectories and systematically calculates velocities, run lengths, pauses, and a wealth of other parameters that are characteristic of motor‐based transport. We anticipate that laboratories will easily use this package to unveil previously uncovered intracellular transport details of individually‐moving cargoes inside neurons.      

Carapace epithelia are rich in large filamentous actin bundles in Daphnia magna,Daphnia pulex,and Sida crystallina (Crustacea: Cladocera)

Cladocerans (water fleas) are planktonic crustaceans that typically have a bivalved carapace. Each valve of the carapace consists of two cuticle‐secreting epithelial layers that are separated by a hemolymphatic chamber and joined by pillar structures. Ultrastructural analyses in several species of Cladocera have shown that the carapace epithelia and pillars contain filamentous structures of unknown composition. In the present study we used a fluorescent phalloidin conjugate to show that the carapaces of three cladocerans, Daphnia magna, D. pulex, and Sida crystallina, are rich in large bundles of filamentous actin (F‐actin). In D. magna we employed confocal microscopy and orthogonal views of three‐dimensional reconstructions to show that these bundles extend radially from foci in the pillars towards the integument surfaces, and their structure is consistent with that of contractile stress fibers. Using a fluorescent lipophilic stain, DiOC₆(3), we show that the F‐actin bundles are distributed in membrane‐rich regions within the carapace epithelia, and that, in the superficial epithelium, these may be large membrane‐bound organelles. In D. magna, the F‐actin bundles are present in embryonic, juvenile instar, and adult, developmental stages, and through development the bundles become larger, contain more F‐actin, and become more widely spaced. We present an alignment of the deduced amino acid sequences of six putative D. pulex actin genes, and discuss the implications that their respective sequences have on the likelihood of their inclusion into the F‐actin bundles of the carapace. Our identification of these large F‐actin bundles within the pillars of three cladocerans provides new insight into the role these structures play in influencing carapace dynamics within this order.     

An evolutionary balance: conservation vs innovation in ciliate membrane trafficking

As most of eukaryotic diversity lies in single‐celled protists, they represent unique opportunities to ask questions about the balance of conservation and innovation in cell biological features. Among free‐living protists the ciliates offer ease of culturing, a rich array of experimental approaches, and versatile molecular tools, particularly in Tetrahymena thermophila and Paramecium tetraurelia. These attributes have been exploited by researchers to analyze a wealth of cellular structures in these large and complex cells. This mini‐review focuses on 3 aspects of ciliate membrane dynamics, all linked with endolysosomal trafficking. First is nutrition based on phagocytosis and maturation of food vacuoles. Secondly, we discuss regulated exocytosis from vesicles that have features of both dense core secretory granules but also lysosome‐related organelles. The third topic is the targeting, breakdown and resorption of parental nuclei in mating partners. For all 3 phenomena, it is clear that elements of the canonical membrane‐trafficking system have been retained and in some cases repurposed. In addition, there is evidence that recently evolved, lineage‐specific proteins provide determinants in these pathways.      

Motor neuropathy‐associated mutation impairs Seipin functions in neurotransmission

Gain‐of‐toxic‐function mutations in Seipin (Asparagine 88 to Serine (N88S) and Serine 90 to Leucine (S90L) mutations, both of which disrupt the N‐glycosylation) cause autosomal dominant motor neuron diseases. However, the mechanism of how these missense mutations lead to motor neuropathy is unclear. Here, we analyze the impact of disruption of N‐glycosylation of Seipin on synaptic transmission by over‐expressing mutant Seipin in cultured cortical neurons via lentiviral infection. Immunostaining shows that over‐expressed Seipin is partly colocalized with synaptic vesicle marker synaptophysin. Electrophysiological recordings reveal that the Seipin mutation significantly decreases the frequency, but not the amplitudes of miniature excitatory post‐synaptic currents and miniature inhibitory post‐synaptic currents. The amplitude of both evoked excitatory post‐synaptic currents and inhibitory post‐synaptic current is also compromised by mutant Seipin over‐expression. The readily releasable pool and vesicular release probability of synaptic vesicles are both altered in neurons over‐expressing Seipin‐N88S, whereas neither γ‐amino butyric acid (GABA) nor α‐Amino‐3‐hydroxy‐5‐methyl‐4‐ isoxazolepropionic acid (AMPA) induced whole cell currents are affected. Moreover, electron microscopy analysis reveals decreased number of morphologically docked synaptic vesicles in Seipin‐N88S‐expressing neurons. These data demonstrate that Seipin‐N88S mutation impairs synaptic neurotransmission, possibly by regulating the priming and docking of synaptic vesicles at the synapse.

     

Age-related changes in synaptic vesicle pools of axo-dendritic synapses on hippocampal CA1 pyramidal neurons in mice

The ultrastructure of symmetric (putatively inhibitory) axo-dendritic synapses on the membrane of hippocampal CA1 pyramidal neurons was investigated in young (20-day-old) and adult (1-year-old) mice. It was shown that synapses of adult animals contain, on average, fewer synaptic vesicles (SVs), and resting SVs of the reserve pool are mostly responsible for this difference. At the same time, in the synapses of adult mice SVs are localized closer to active zones, and the readily releasable pool of SVs is larger in these animals than in young mice. The observed changes in the spatial structure of SV pools presumably demonstrate the age-associated adaptation of inhibitory synapses providing the maintenance of adequate functional properties of hippocampal neuronal networks. Neirofiziologiya/Neurophysiology, Vol. 38, Nos. 5/6, pp. 407–411, September–December, 2006.     

Neuronal control of lipid metabolism by STR‐2 G protein‐coupled receptor promotes longevity in Caenorhabditis elegans

The G protein‐coupled receptor (GPCR) encoding family of genes constitutes more than 6% of genes in Caenorhabditis elegans genome. GPCRs control behavior, innate immunity, chemotaxis, and food search behavior. Here, we show that C. elegans longevity is regulated by a chemosensory GPCR STR‐2, expressed in AWC and ASI amphid sensory neurons. STR‐2 function is required at temperatures of 20°C and higher on standard Escherichia coli OP50 diet. Under these conditions, this neuronal receptor also controls health span parameters and lipid droplet (LD) homeostasis in the intestine. We show that STR‐2 regulates expression of delta‐9 desaturases, fat‐5, fat‐6 and fat‐7, and of diacylglycerol acyltransferase dgat‐2. Rescue of the STR‐2 function in either AWC and ASI, or ASI sensory neurons alone, restores expression of fat‐5, dgat‐2 and restores LD stores and longevity. Rescue of stored fat levels of GPCR mutant animals to wild‐type levels, with low concentration of glucose, rescues its lifespan phenotype. In all, we show that neuronal STR‐2 GPCR facilitates control of neutral lipid levels and longevity in C. elegans.     

Identification of precursor microRNAs within distal axons of sensory neuron

A set of specific precursor microRNAs (pre‐miRNAs) are reported to localize into neuronal dendrites, where they could be processed locally to control synaptic protein synthesis and plasticity. However, it is not clear whether specific pre‐miRNAs are also transported into distal axons to autonomously regulate intra‐axonal protein synthesis. Here, we show that a subset of pre‐miRNAs, whose mature miRNAs are enriched in axonal compartment of sympathetic neurons, are present in axons of neurons both in vivo and in vitro by quantitative PCR and by in situ hybridization. Some pre‐miRNAs (let 7c‐a and pre‐miRs‐16, 23a, 25, 125b‐1, 433, and 541) showed elevated axonal levels, while others (pre‐miRs‐138‐2, 185, and 221) were decreased in axonal levels following injury. Dicer and KSRP proteins are also present in distal axons, but Drosha is found restricted to the cell body. These findings suggest that specific pre‐miRNAs are selected for localization into distal axons of sensory neurons and are presumably processed to mature miRNAs in response to extracellular stimuli. This study supports the notion that local miRNA biogenesis effectively provides another level of temporal control for local protein synthesis in axons.

     

Single cocaine exposure does not alter striatal pre‐synaptic dopamine function in mice: an [18F]‐FDOPA PET study

Cocaine is a recreational drug of abuse that binds to the dopamine transporter, preventing reuptake of dopamine into pre‐synaptic terminals. The increased presence of synaptic dopamine results in stimulation of both pre‐ and post‐synaptic dopamine receptors, considered an important mechanism by which cocaine elicits its reinforcing properties. However, the effects of acute cocaine administration on pre‐synaptic dopamine function remain unclear. Non‐invasive imaging techniques such as positron emission tomography have revealed impaired pre‐synaptic dopamine function in chronic cocaine users. Similar impairments have been seen in animal studies, with microdialysis experiments indicating decreased basal dopamine release. Here we use micro positron emission tomography imaging techniques in mice to measure dopamine synthesis capacity and determine the effect of acute cocaine administration of pre‐synaptic dopamine function. We show that a dose of 20 mg/kg cocaine is sufficient to elicit hyperlocomotor activity, peaking 15–20 min post treatment (p < 0.001). However, dopamine synthesis capacity in the striatum was not significantly altered by acute cocaine treatment (: 0.0097 per min vs. 0.0112 per min in vehicle controls, p > 0.05). Furthermore, expression levels of two key enzymes related to dopamine synthesis, tyrosine hydroxylase and aromatic l ‐amino acid decarboxylase, within the striatum of scanned mice were not significantly affected by acute cocaine pre‐treatment (p > 0.05). Our findings suggest that while the regulation of dopamine synthesis and release in the striatum have been shown to change with chronic cocaine use, leading to a reduced basal tone, these adaptations to pre‐synaptic dopaminergic neurons are not initiated following a single exposure to the drug.

     

Age‐dependent changes in response property and morphology of a thermosensory neuron and thermotaxis behavior in Caenorhabditis elegans

Age‐dependent cognitive and behavioral deterioration may arise from defects in different components of the nervous system, including those of neurons, synapses, glial cells, or a combination of them. We find that AFD, the primary thermosensory neuron of Caenorhabditis elegans, in aged animals is characterized by loss of sensory ending integrity, including reduced actin‐based microvilli abundance and aggregation of thermosensory guanylyl cyclases. At the functional level, AFD neurons in aged animals are hypersensitive to high temperatures and show sustained sensory‐evoked calcium dynamics, resulting in a prolonged operating range. At the behavioral level, senescent animals display cryophilic behaviors that remain plastic to acute temperature changes. Excessive cyclase activity of the AFD‐specific guanylyl cyclase, GCY‐8, is associated with developmental defects in AFD sensory ending and cryophilic behavior. Surprisingly, loss of the GCY‐8 cyclase domain reduces these age‐dependent morphological and behavioral changes, while a prolonged AFD operating range still exists in gcy‐8 animals. The lack of apparent correlation between age‐dependent changes in the morphology or stimuli‐evoked response properties of primary sensory neurons and those in related behaviors highlights the importance of quantitative analyses of aging features when interpreting age‐related changes at structural and functional levels. Our work identifies aging hallmarks in AFD receptive ending, temperature‐evoked AFD responses, and experience‐based thermotaxis behavior, which serve as a foundation to further elucidate the neural basis of cognitive aging.     

A HuD‐ZBP1 ribonucleoprotein complex localizes GAP‐43 mRNA into axons through its 3′ untranslated region AU‐rich regulatory element

Localized translation of axonal mRNAs contributes to developmental and regenerative axon growth. Although untranslated regions (UTRs) of many different axonal mRNAs appear to drive their localization, there has been no consensus RNA structure responsible for this localization. We recently showed that limited expression of ZBP1 protein restricts axonal localization of both β‐actin and GAP‐43 mRNAs. β‐actin 3′UTR has a defined element for interaction with ZBP1, but GAP‐43 mRNA shows no homology to this RNA sequence. Here, we show that an AU‐rich regulatory element (ARE) in GAP‐43′s 3′UTR is necessary and sufficient for its axonal localization. Axonal GAP‐43 mRNA levels increase after in vivo injury, and GAP‐43 mRNA shows an increased half‐life in regenerating axons. GAP‐43 mRNA interacts with both HuD and ZBP1, and HuD and ZBP1 co‐immunoprecipitate in an RNA‐dependent fashion. Reporter mRNA with the GAP‐43 ARE competes with endogenous β‐actin mRNA for axonal localization and decreases axon length and branching similar to the β‐actin 3′UTR competing with endogenous GAP‐43 mRNA. Conversely, over‐expressing GAP‐43 coding sequence with its 3′UTR ARE increases axonal elongation and this effect is lost when just the ARE is deleted from GAP‐43′s 3′UTR.

     

Apical myosin XI anticipates F‐actin during polarized growth of Physcomitrella patens cells

Tip growth is essential for land colonization by bryophytes, plant sexual reproduction and water and nutrient uptake. Because this specialized form of polarized cell growth requires both a dynamic actin cytoskeleton and active secretion, it has been proposed that the F‐actin‐associated motor myosin XI is essential for this process. Nevertheless, a spatial and temporal relationship between myosin XI and F‐actin during tip growth is not known in any plant cell. Here, we use the highly polarized cells of the moss Physcomitrella patens to show that myosin XI and F‐actin localize, in vivo, at the same apical domain and that both signals fluctuate. Surprisingly, phase analysis shows that increase in myosin XI anticipates that of F‐actin; in contrast, myosin XI levels at the tip fluctuate in identical phase with a vesicle marker. Pharmacological analysis using a low concentration of the actin polymerization inhibitor latrunculin B showed that the F‐actin at the tip can be significantly diminished while myosin XI remains elevated in this region, suggesting that a mechanism exists to cluster myosin XI‐associated structures at the cell's apex. In addition, this approach uncovered a mechanism for actin polymerization‐dependent motility in the moss cytoplasm, where myosin XI‐associated structures seem to anticipate and organize the actin polymerization machinery. From our results, we inferred a model where the interaction between myosin XI‐associated vesicular structures and F‐actin polymerization‐driven motility function at the cell's apex to maintain polarized cell growth. We hypothesize this is a general mechanism for the participation of myosin XI and F‐actin in tip growing cells.     

ER arrival sites for COPI vesicles localize to hotspots of membrane trafficking

COPI‐coated vesicles mediate retrograde membrane traffic from the cis‐Golgi to the endoplasmic reticulum (ER) in all eukaryotic cells. However, it is still unknown whether COPI vesicles fuse everywhere or at specific sites with the ER membrane. Taking advantage of the circumstance that the vesicles still carry their coat when they arrive at the ER, we have visualized active ER arrival sites (ERAS) by monitoring contact between COPI coat components and the ER‐resident Dsl tethering complex using bimolecular fluorescence complementation (BiFC). ERAS form punctate structures near Golgi compartments, clearly distinct from ER exit sites. Furthermore, ERAS are highly polarized in an actin and myosin V‐dependent manner and are localized near hotspots of plasma membrane expansion. Genetic experiments suggest that the COPI?Dsl BiFC complexes recapitulate the physiological interaction between COPI and the Dsl complex and that COPI vesicles are mistargeted in dsl1 mutants. We conclude that the Dsl complex functions in confining COPI vesicle fusion sites.     

The RtcB RNA ligase is an essential component of the metazoan unfolded protein response

RNA ligation can regulate RNA function by altering RNA sequence, structure and coding potential. For example, the function of XBP1 in mediating the unfolded protein response requires RNA ligation, as does the maturation of some tRNAs. Here, we describe a novel in vivo model in Caenorhabditis elegans for the conserved RNA ligase RtcB and show that RtcB ligates the xbp‐1 mRNA during the IRE‐1 branch of the unfolded protein response. Without RtcB, protein stress results in the accumulation of unligated xbp‐1 mRNA fragments, defects in the unfolded protein response, and decreased lifespan. RtcB also ligates endogenous pre‐tRNA halves, and RtcB mutants have defects in growth and lifespan that can be bypassed by expression of pre‐spliced tRNAs. In addition, animals that lack RtcB have defects that are independent of tRNA maturation and the unfolded protein response. Thus, RNA ligation by RtcB is required for the function of multiple endogenous target RNAs including both xbp‐1 and tRNAs. RtcB is uniquely capable of performing these ligation functions, and RNA ligation by RtcB mediates multiple essential processes in vivo.     

Caenorhabditis elegans orthologs of human genes differentially expressed with age are enriched for determinants of longevity

We report a systematic RNAi longevity screen of 82 Caenorhabditis elegans genes selected based on orthology to human genes differentially expressed with age. We find substantial enrichment in genes for which knockdown increased lifespan. This enrichment is markedly higher than published genomewide longevity screens in C. elegans and similar to screens that preselected candidates based on longevity‐correlated metrics (e.g., stress resistance). Of the 50 genes that affected lifespan, 46 were previously unreported. The five genes with the greatest impact on lifespan (>20% extension) encode the enzyme kynureninase (kynu‐1), a neuronal leucine‐rich repeat protein (iglr‐1), a tetraspanin (tsp‐3), a regulator of calcineurin (rcan‐1), and a voltage‐gated calcium channel subunit (unc‐36). Knockdown of each gene extended healthspan without impairing reproduction. kynu‐1(RNAi) alone delayed pathology in C. elegans models of Alzheimer's disease and Huntington's disease. Each gene displayed a distinct pattern of interaction with known aging pathways. In the context of published work, kynu‐1, tsp‐3, and rcan‐1 are of particular interest for immediate follow‐up. kynu‐1 is an understudied member of the kynurenine metabolic pathway with a mechanistically distinct impact on lifespan. Our data suggest that tsp‐3 is a novel modulator of hypoxic signaling and rcan‐1 is a context‐specific calcineurin regulator. Our results validate C. elegans as a comparative tool for prioritizing human candidate aging genes, confirm age‐associated gene expression data as valuable source of novel longevity determinants, and prioritize select genes for mechanistic follow‐up.     

Lipid compositions in diatom Conticribra weissflogii under static and aerated culture conditions

The diatom Conticribra weissflogii is a microalga with high nutrition value, rich in docosahexaenoic acids (DHA) and eicosapentaenoic acids (EPA). In order to study the effect of culture conditions on the changes of lipid compositions, the intact lipid structural profiles and fatty acids in C. weissflogii were monitored under static and aerated culture conditions. The results showed that, lipids identified in C. weissflogii were neutral lipid triacylglycerols (TAG), betaine lipid diacylglycerylcarboxy‐N‐hydroxymethyl‐choline (DGCC), phosphatidylcholine (PC) and four classes of photosynthetic glycerolipids. The profiles of lipid metabolites of C. weissflogii were different between two culture modes, with the following characteristics under aerated conditions: TAGs increased significantly, whereas the levels of sulfoquinovosyl diacylglycerol (SQDG), monogalactosyldiacylglycerol (MGDG), and DGCC decreased. Furthermore, higher contents of EPA‐rich TAG and EPA/DHA‐rich DGCC were detected at the end of stationary phase, while EPA/DHA‐rich PC, EPA‐rich MGDG and EPA‐rich digalactosyldiacylglycerol (DGDG) were obtained in the exponential phase under static conditions. Meanwhile, the contents of almost all classes of the essential fatty acids (EFAs)‐enriched lipids increased at onset of stationary phase under aerated conditions. Taken together, given that the high levels of EFAs are required for artificial rearing of marine organisms, aeration is critically important for increasing the production rate and the contents of EFA molecules and therefore increasing the nutritional value of the microalgae.     

Intracellular traffic of the lysine and glutamic acid rich protein KERP1 reveals features of endomembrane organization in Entamoeba histolytica

The development of amoebiasis is influenced by the expression of the lysine and glutamic acid rich protein 1 (KERP1), a virulence factor involved in Entamoeba histolytica adherence to human cells. Up to date, it is unknown how the protein transits the parasite cytoplasm towards the plasma membrane, specially because this organism lacks a well‐defined endoplasmic reticulum (ER) and Golgi apparatus. In this work we demonstrate that KERP1 is present at the cell surface and in intracellular vesicles which traffic in a pathway that is independent of the ER–Golgi anterograde transport. The intracellular displacement of vesicles enriched in KERP1 relies on the actin‐rich cytoskeleton activities. KERP1 is also present in externalized vesicles deposited on the surface of human cells. We further report the interactome of KERP1 with its association to endomembrane components and lipids. The model for KERP1 traffic here proposed hints for the first time elements of the endocytic and exocytic paths of E. histolytica.     

Morphological Analysis of the Interaction of Charged Surfactant Vesicles (SVs) with Human Cultured Cells

We analyzed the binding and fusogenic properties of surfactant vesicles (SVs), composed of ionic and nonionic surfactants and cholesterol, with the surface of different human lymphoid cells. The influence of charge on SVs-cell interaction was evaluated by monitoring the presence of fluorescent sodium calcein artificially entrapped in the vesicles using optical fluorescence microscopy and laser scanning confocal microscopy. Our results clearly indicate that only negatively charged vesicles bind and fuse with the plasma membrane of human lymphoid cells, and the number of SVs bound to the cell surface was variable among the positive cells. Thin section electron microscopy illustrated that the fusogenic events of SVs with the cell plasma membrane mostly occurred at smooth and nonvillous regions of the cell surface. Taken together, our results suggest that binding and fusion of SVs with the cell plasma membrane might be dependent on interactions with specific membrane components that preferentially recognize negatively charged SVs.     

Specific KIF1A–adaptor interactions control selective cargo recognition

Intracellular transport in neurons is driven by molecular motors that carry many different cargos along cytoskeletal tracks in axons and dendrites. Identifying how motors interact with specific types of transport vesicles has been challenging. Here, we use engineered motors and cargo adaptors to systematically investigate the selectivity and regulation of kinesin-3 family member KIF1A–driven transport of dense core vesicles (DCVs), lysosomes, and synaptic vesicles (SVs). We dissect the role of KIF1A domains in motor activity and show that CC1 regulates autoinhibition, CC2 regulates motor dimerization, and CC3 and PH mediate cargo binding. Furthermore, we identify that phosphorylation of KIF1A is critical for binding to vesicles. Cargo specificity is achieved by specific KIF1A adaptors; MADD/Rab3GEP links KIF1A to SVs, and Arf-like GTPase Arl8A mediates interactions with DCVs and lysosomes. We propose a model where motor dimerization, posttranslational modifications, and specific adaptors regulate selective KIF1A cargo trafficking.