We have analyzed the polarity orientation of microtubules in the axons and dendrites of cultured rat hippocampal neurons. As previously reported of axons from other neurons, microtubules in these axons are uniform with respect to polarity; (+)-ends are directed away from the cell body toward the growth cone. In sharp contrast, microtubules in the mid-region of the dendrite, -75 Jtm from the cell body, are not of uniform polarity orientation. Roughly equal proportions of these microtubules are oriented with (+)-ends directed toward the growth cone and ( + )-ends directed .toward the cell body. At distances within 15 ,um of the growth cone, however, microtubule polarity orientation in dendrites is similar to that in axons; (+)-ends are uniformly directed toward the growth cone. These findings indicate a clear difference between axons and dendrites with respect to microtubule organization, a difference that may underlie the differential distribution of organelles within the neuron.Vertebrate neurons generate and maintain two morphologically and functionally distinct types of neurites, axons and dendrites (1-6). It has long been recognized that axons and dendrites differ in their complements of cytoplasmic organelles (1, 6). Most notable in this regard, ribosomes and Golgi elements are present in dendrites but are absent from axons. What is the basis for the nonuniform distribution of organelles in neurons? Several lines of evidence indicate that the distribution of organelles in a cell reflects active transport processes that selectively convey organelles from their sites of synthesis and assembly to other locations in the cell (7, 8).These observations raise the possibility that many of the differences between the organelle composition of axons and dendrites are produced by differences in the organization of the transport systems that convey materials from the cell body into the axon or dendrite.The transport of organelles is a microtubule-based process; microtubules provide the substrate for organelle translocation and, by virtue of their intrinsic polarity, influence the directionality of transport (7-9). The intrinsic polarity of microtubules is based on the asymmetry of the tubulin subunit and its self-assembly characteristics; the (+)-end is preferred for subunit addition over the (-)-end (10, 11). Microtubule-based translocators convey organelles specifically toward either the ( + )-or the ( -)-end ofthe microtubule (7-9). In the axon, microtubules are uniform with respect to polarity, with the (+ )-ends directed away from the cell body (12-15). Thus, only those organelles that translocate toward (+ )-ends of microtubules will be conveyed from the cell body into the axon.Do microtubules in dendrites have the same polarity orientation as those in axons? To date, information concerning the polarity orientation of dendritic microtubules derives from a few atypical cell types. In the dendrite-like processes of teleost retinal cone cells (16) and frog primary olfactory neurons (17), microtubules are unifo...

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The time course and specificity of the changes in dendritic morphology following deafferentation were examined in nucleus laminaris of young chickens. The dendrites of nucleus laminaris neurons are segregated into dorsal and ventral domains, which are innervated separately from the ipsilateral and contralateral nucleus magnocellularis, respectively. Transection of the crossed dorsal cochlear tract deafferents the ventral dendrites of nucleus laminaris bilaterally without interrupting the matching input to the dorsal dendrites. In 10-day-old chicks, atrophy of the ventral dendrites began immediately after transecting the tract; the ventral dendrites were 10% shorter by 1 hour and 16% shorter by 2 hours after deafferentation. The length of the ventral dendrites progressively decreased over the next 2 weeks, resulting in at least a 60% loss of ventral dendrite 16 days after surgery. The dorsal dendrites of the same cells, whose afferents remained intact, did not change in length during the time course of this study. However, 16 days after the lesion, spines appeared on the normally smooth dorsal and ventral dendrites. The time course of dendritic atrophy and its restriction to the deafferented postsynaptic surface are related to possible mechanisms by which afferents regulate and maintain their target neurons.

Recent data demonstrate that the introduction into skeletal muscle of an adenoassociated viral (AAV) vector expressing blood coagulation factor IX (F.IX) can result in long-term expression of the transgene product and amelioration of the bleeding diathesis in animals with hemophilia B. These data suggest that biologically active F.IX can be synthesized in skeletal muscle. Factor IX undergoes extensive posttranslational modifications in the liver, the normal site of synthesis. In addition to affecting specific activity, these posttranslational modifications can also affect recovery, half-life in the circulation, and the immunogenicity of the protein. Before initiating a human trial of an AAV-mediated, muscle-directed approach for treating hemophilia B, a detailed biochemical analysis of F.IX synthesized in skeletal muscle was carried out. As a model system, human myotubes transduced with an AAV vector expressing F.IX was used. F.IX was purified from conditioned medium using a novel strategy designed to purify material representative of all species of rF.IX in the medium. Purified F.IX was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, chemical ␥-carboxyglutamyl analysis, carbohydrate analysis, assays for tyrosine sulfation, and serine phosphorylation, and for specific activity.Results show that myotube-synthesized F.IX has specific activity similar to that of liver-synthesized F.IX. Posttranslational modifications critical for specific activity, including removal of the signal sequence and propeptide, and ␥-carboxylation of the N-terminal glutamic acid residues, are also similar, but carbohydrate analysis and assessment of tyrosine sulfation and serine phosphorylation disclose differences. In vivo experiments in mice showed that these differences affect recovery but not half-life of muscle-synthesized F.IX. (Blood. 2001;97:130-138)