Visualizing and analyzing intracellular transport of organelles and other cargos in astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the “tri-partite” synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons, where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo-and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes’ normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins

Dark Viscous Fluid coupled with Dark Matter and future singularity

We study effects of viscous fluid coupled with dark matter in our universe. We consider bulk viscosity in the cosmic fluid and we suppose the existence of a coupling between fluid and dark matter, in order to reproduce a stable de Sitter universe protected against future-time singularities. More general inhomogeneous fluids are studied related to future singularities.Comment: 11 page

Mars and frame-dragging: study for a dedicated mission

In this paper we preliminarily explore the possibility of designing a dedicated satellite-based mission to measure the general relativistic gravitomagnetic Lense-Thirring effect in the gravitational field of Mars. The focus is on the systematic error induced by the multipolar expansion of the areopotential and on possible strategies to reduce it. It turns out that the major sources of bias are the Mars'equatorial radius R and the even zonal harmonics J_L, L = 2,4,6... of the areopotential. An optimal solution, in principle, consists of using two probes at high-altitudes (a\approx 9500-9600 km) and different inclinations, and suitably combining their nodes in order to entirely cancel out the bias due to \delta R. The remaining uncancelled mismodelled terms due to \delta J_L, L = 2,4,6,... would induce a bias \lesssim 1%, according to the present-day MGS95J gravity model, over a wide range of admissible values of the inclinations. The Lense-Thirring out-of-plane shifts of the two probes would amount to about 10 cm yr^-1.Comment: LaTex2e, 16 pages, 5 figures, no tables. To appear in General Relativity and Gravitatio

Viscous Fluids and Gauss-Bonnet Modified Gravity

We study effects of cosmic fluids on finite-time future singularities in modified $f(R,G)$-gravity, where $R$ and $G$ are the Ricci scalar and the Gauss-Bonnet invariant, respectively. We consider the fluid equation of state in the general form, $\omega=\omega(\rho)$, and we suppose the existence of a bulk viscosity. We investigate quintessence region ($\omega>-1$) and phantom region ($\omega<-1$) and the possibility to change or avoid the singularities in $f(R,G)$-gravity. Finally, we study the inclusion of quantum effects in large curvatures regime.Comment: 14 page

βiI-spectrin promotes mouse brain connectivity through stabilizing axonal plasma membranes and enabling axonal organelle transport

βII-spectrin is the generally expressed member of the β-spectrin family of elongated polypeptides that form micrometer-scale networks associated with plasma membranes. We addressed in vivo functions of βII-spectrin in neurons by knockout of βII-spectrin in mouse neural progenitors. βII-spectrin deficiency caused severe defects in long-range axonal connectivity and axonal degeneration. βII-spectrin- null neurons exhibited reduced axon growth, loss of actin-spectrinbased periodic membrane skeleton, and impaired bidirectional axonal transport of synaptic cargo. We found that βII-spectrin associates with KIF3A, KIF5B, KIF1A, and dynactin, implicating spectrin in the coupling of motors and synaptic cargo. βII-spectrin required phosphoinositide lipid binding to promote axonal transport and restore axon growth. Knockout of ankyrin-B (AnkB), a βII-spectrin partner, primarily impaired retrograde organelle transport, while double knockout of βII-spectrin and AnkB nearly eliminated transport. Thus, βII-spectrin promotes both axon growth and axon stability through establishing the actin- spectrin-based membrane-associated periodic skeleton as well as enabling axonal transport of synaptic cargo

Spectrin mutations that cause spinocerebellar ataxia type 5 impair axonal transport and induce neurodegeneration in Dmsophila

Spinocerebellar ataxia type 5 (SCA5) is an autosomal dominant neurodegenerative disorder caused by mutations in the SPBTN2 gene encoding β-IIIspectrin. To investigate the molecular basis of SCA5, we established a series of transgenic Drosophila models that express human β-III-spectrin or fly β-spectrin proteins containing SCA5 mutations. Expression of the SCA5 mutant spectrin in the eye causes a progressive neurodegenerative phenotype, and expression in larval neurons results in posterior paralysis, reduced synaptic terminal growth, and axonal transport deficits. These phenotypes are genetically enhanced by both dynein and dynactin loss-of-function mutations. In summary, we demonstrate that SCA5 mutant spectrin causes adult-onset neurodegeneration in the fly eye and disrupts fundamental intracellular transport processes that are likely to contribute to this progressive neurodegenerative disease

A PIK3C3-Ankyrin-B-Dynactin pathway promotes axonal growth and multiorganelle transport

Axon growth requires long-range transport of organelles, but how these cargoes recruit their motors and how their traffic is regulated are not fully resolved. In this paper, we identify a new pathway based on the class III PI3-kinase (PIK3C3), ankyrin-B (AnkB), and dynactin, which promotes fast axonal transport of synaptic vesicles, mitochondria, endosomes, and lysosomes. We show that dynactin associates with cargo through AnkB interactions with both the dynactin subunit p62 and phosphatidylinositol 3-phosphate (PtdIns(3)P) lipids generated by PIK3C3. AnkB knockout resulted in shortened axon tracts and marked reduction in membrane association of dynactin and dynein, whereas it did not affect the organization of spectrin-actin axonal rings imaged by 3D-STORM. Loss of AnkB or of its linkages to either p62 or PtdIns(3)P or loss of PIK3C3 all impaired organelle transport and particularly retrograde transport in hippocampal neurons. Our results establish new functional relationships between PIK3C3, dynactin, and AnkB that together promote axonal transport of organelles and are required for normal axon length

Linking the trans-Planckian and the information loss problems in black hole physics

The trans-Planckian and information loss problems are usually discussed in the literature as separate issues concerning the nature of Hawking radiation. Here we instead argue that they are intimately linked, and can be understood as "two sides of the same coin" once it is accepted that general relativity is an effective field theory.Comment: 10 pages, 2 figures. Replaced with the version to be published in General Relativity and Gravitatio

ANK2 autism mutation targeting giant ankyrin-B promotes axon branching and ectopic connectivity

Giant ankyrin-B (ankB) is a neurospecific alternatively spliced variant of ANK2, a high-confidence autism spectrum disorder (ASD) gene. We report that a mouse model for human ASD mutation of giant ankB exhibits increased axonal branching in cultured neurons with ectopic CNS axon connectivity, as well as with a transient increase in excitatory synapses during postnatal development. We elucidate a mechanism normally limiting axon branching, whereby giant ankB localizes to periodic axonal plasma membrane domains through L1 cell-adhesion molecule protein, where it couples microtubules to the plasma membrane and prevents microtubule entry into nascent axon branches. Giant ankB mutation or deficiency results in a dominantly inherited impairment in selected communicative and social behaviors combined with superior executive function. Thus, gain of axon branching due to giant ankB-deficiency/mutation is a candidate cellular mechanism to explain aberrant structural connectivity and penetrant behavioral consequences in mice as well as humans bearing ASD-related ANK2 mutations

A Mutation in the Borcs7 Subunit of the Lysosome Regulatory BORC Complex Results in Motor Deficits and Dystrophic Axonopathy in Mice

Lysosomes play a critical role in maintenance of the integrity of neuronal function, and mutations in genes that contribute to lysosome formation, transport, and activity are associated with neurodegenerative disorders. Recently, the multisubunit complex, BLOC-one-related complex (BORC), has been shown to be involved in positioning lysosomes within the cytoplasm, although the consequences of altered BORC function in adult animals have not been established. We show that a spontaneous truncation mutation in the mouse Borcs7 gene, identified through whole-genome sequencing followed by genetic complementation, results in progressive axonal dystrophy with dramatic impairment of motor function. Furthermore, mice homozygous for deletion of the entire Borcs7 coding sequence die shortly after birth, and neurons cultured from these animals show impaired centrifugal transport of lysosomes. This identifies BORCS7 as a central factor in axonal transport of lysosomes and a possible target for improving disease-related disturbances in this important function. BORC is a multisubunit complex that regulates lysosomal positioning. Snouwaert et al. report that a truncation mutation in Borcs7, a subunit of this complex, results in reduced lysosomal transport, progressive axonal dystrophy, and impaired motor function in mice. Loss of Borcs7 causes juxtanuclear clustering of neuronal lysosomes and perinatal mortality