In vitro assessment of protamine toxicity with neural cells, its therapeutic potential to counter chondroitin sulfate mediated neuron inhibition, and its effects on reactive astrocytes.

Multiple therapies have been studied to ameliorate the neuroinhibitory cues present after traumatic injury to the central nervous system. Two previous in vitro studies have demonstrated the efficacy of the FDA-approved cardiovascular therapeutic, protamine (PRM), to overcome neuroinhibitory cues presented by chondroitin sulfates; however, the effect of a wide range of PRM concentrations on neuronal and glial cells has not been evaluated. In this study, we investigate the therapeutic efficacy of PRM with primary cortical neurons, hippocampal neurons, mixed glial cultures, and astrocyte cultures. We show the threshold for PRM toxicity to be at or above 10 µg/ml depending on the cell population, that 10 µg/ml PRM enables neurons to overcome the inhibitory cues presented by chondroitin sulfate type A, and that soluble PRM allows neurons to more effectively overcome inhibition compared to a PRM coating. We also assessed changes in gene expression of reactive astrocytes with soluble PRM and determined that PRM does not increase their neurotoxic phenotype and that PRM may reduce brevican production and serpin transcription in cortical and spinal cord astrocytes. This is the first study to thoroughly assess the toxicity threshold of PRM with neural cells and study astrocyte response after acute exposure to PRM in vitro.

Kinesin Regulation in the Proximal Axon is Essential for Dendrite-selective Transport.

Neurons are polarized and typically extend multiple dendrites and one axon. To maintain polarity, vesicles carrying dendritic proteins are arrested upon entering the axon. To determine whether kinesin regulation is required for terminating anterograde axonal transport, we overexpressed the dendrite-selective kinesin KIF13A. This caused mistargeting of dendrite-selective vesicles to the axon and a loss of dendritic polarity. Polarity was not disrupted if the kinase MARK2/Par1b was coexpressed. MARK2/Par1b is concentrated in the proximal axon, where it maintains dendritic polarity-likely by phosphorylating S1371 of KIF13A, which lies in a canonical 14-3-3 binding motif. We probed for interactions of KIF13A with 14-3-3 isoforms and found that 14-3-3ß and 14-3-3ζ bound KIF13A. Disruption of MARK2 or 14-3-3 activity by small molecule inhibitors caused a loss of dendritic polarity. These data show that kinesin regulation is integral for dendrite-selective transport. We propose a new model in which KIF13A that moves dendrite-selective vesicles in the proximal axon is phosphorylated by MARK2. Phosphorylated KIF13A is then recognized by 14-3-3, which causes dissociation of KIF13A from the vesicle and termination of transport. These findings define a new paradigm for the regulation of vesicle transport by localized kinesin tail phosphorylation, to restrict dendrite-selective vesicles from entering the axon.

Polarized transport requires AP-1-mediated recruitment of KIF13A and KIF13B at the trans-Golgi.

Neurons are polarized cells that require accurate membrane trafficking to maintain distinct protein complements at dendritic and axonal membranes. The Kinesin-3 family members KIF13A and KIF13B are thought to mediate dendrite-selective transport, but the mechanism by which they are recruited to polarized vesicles and the differences in the specific trafficking role of each KIF13 have not been defined. We performed live-cell imaging in cultured hippocampal neurons and found that KIF13A is a dedicated dendrite-selective kinesin. KIF13B confers two different transport modes, dendrite- and axon-selective transport. Both KIF13s are maintained at the trans-Golgi network by interactions with the heterotetrameric adaptor protein complex AP-1. Interference with KIF13 binding to AP-1 resulted in disruptions to both dendrite- and axon-selective trafficking. We propose that AP-1 is the molecular link between the sorting of polarized cargoes into vesicles and the recruitment of kinesins that confer polarized transport.

Poly(pro-curcumin) Materials Exhibit Dual Release Rates and Prolonged Antioxidant Activity as Thin Films and Self-Assembled Particles.

Curcumin is a natural polyphenol that exhibits remarkable antioxidant and anti-inflammatory activities; however, its clinical application is limited in part by its physiological instability. Here, we report the synthesis of curcumin-derived polyesters that release curcumin upon hydrolytic degradation to improve curcumin stability and solubility in physiological conditions. Curcumin was incorporated in the polymer backbone by a one-pot condensation polymerization in the presence of sebacoyl chloride and polyethylene glycol (PEG, Mn = 1 kDa). The thermal and mechanical properties, surface wettability, self-assembly behavior, and drug-release kinetics all depend sensitively on the mole percentage of curcumin incorporated in these statistical copolymers. Curcumin release was triggered by the hydrolysis of phenolic esters on the polymer backbone, which was confirmed using a PEGylated curcumin model compound, which represented a putative repeating unit within the polymer. The release rate of curcumin was controlled by the hydrophilicity of the polymers. Burst release (2 days) and extended release (>8 weeks) can be achieved from the same polymer depending on curcumin content in the copolymer. The materials can quench free radicals for at least 8 weeks and protect primary neurons from oxidative stress in vitro. Further, these copolymer materials could be processed into both thin films and self-assembled particles, depending on the solvent-based casting conditions. Finally, we envision that these materials may have potential for neural tissue engineering application, where antioxidant release can mitigate oxidative stress and the inflammatory response following neural injury.

A novel labeling strategy reveals that myosin Va and myosin Vb bind the same dendritically polarized vesicle population.

Neurons are specialized cells with a polarized geometry and several distinct subdomains that require specific complements of proteins. Delivery of transmembrane proteins requires vesicle transport, which is mediated by molecular motor proteins. The myosin V family of motor proteins mediates transport to the barbed end of actin filaments, and little is known about the vesicles bound by myosin V in neurons. We developed a novel strategy to visualize myosin V-labeled vesicles in cultured hippocampal neurons and systematically characterized the vesicle populations labeled by myosin Va and Vb. We find that both myosins bind vesicles that are polarized to the somatodendritic domain where they undergo bidirectional long-range transport. A series of two-color imaging experiments showed that myosin V specifically colocalized with two different vesicle populations: vesicles labeled with the transferrin receptor and vesicles labeled by low-density lipoprotein receptor. Finally, coexpression with Kinesin-3 family members found that myosin V binds vesicles concurrently with KIF13A or KIF13B, supporting the hypothesis that coregulation of kinesins and myosin V on vesicles is likely to play an important role in neuronal vesicle transport. We anticipate that this new assay will be applicable in a broad range of cell types to determine the function of myosin V motor proteins.

The posttranslational modification of tubulin undergoes a switch from detyrosination to acetylation as epithelial cells become polarized.

Tubulin posttranslational modifications (PTMs) have been suggested to provide navigational cues for molecular motors to deliver cargo to spatially segregated subcellular domains, but the molecular details of this process remain unclear. Here we show that in Madin-Darby Canine Kidney (MDCK) epithelial cells, microtubules express several tubulin PTMs. These modifications, however, are not coordinated, and cells have multiple subpopulations of microtubules that are marked by different combinations of PTMs. Furthermore these subpopulations show differential sensitivity to both drug- and cold-induced depolymerization, suggesting that they are functionally different as well. The composition and distribution of modified microtubules change as cells undergo the morphogenesis associated with polarization. Two-dimensionally polarized spreading cells have more detyrosinated microtubules that are oriented toward the leading edge, but three-dimensionally polarized cells have more acetylated microtubules that are oriented toward the apical domain. These data suggest that the transition from 2D polarity to 3D polarity involves both a reorganization of the microtubule cytoskeleton and a change in tubulin PTMs. However, in both 2D polarized and 3D polarized cells, the modified microtubules are oriented to support vectorial cargo transport to areas of high need.