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First published online 3 February 2004
doi: 10.1242/jcs.00785

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Cdk5 regulates axonal transport and phosphorylation of neurofilaments in cultured neurons

¹ Center for Cellular Neurobiology and Neurodegeneration Research, Departments of Biological Sciences and Biochemistry, University of Massachusetts, Lowell, One University Avenue, Lowell, MA 01854, USA
² Department of Pharmacology, Vanderbilt University, Nashville, TN 37232-8548, USA
³ Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611, USA
⁴ Laboratory of Neurochemistry, NIH, NINDS, Bethesda, MD 20892, USA

View larger version (84K): [in a new window]   Fig. 1. Characterization of exogenous NF subunits and their distribution in DRG neurons. (A) Coomassie Blue staining (CBB) following SDS-gel electrophoresis of NFs isolated from bovine spinal cords, and immunoblot analysis of this preparation (NFs) following biotinylation and of chromatographically separated NF-L as indicated. (B) Representative DRG neuron 2 hours after microinjection with a mixture of biotin-L and fluorescein-conjugated tracer, followed by extraction with Triton X-100 and immunostaining with an anti-biotin antibody followed by a Texas-Red-conjugated secondary antibody. Notice the retention of biotin-L along axons (arrows), indicating its incorporation into Triton-X-100-insoluble structures. (C) A second microinjected neuron processed for anti-biotin immunoreactivity following extraction under conditions that induce splaying of NFs, more clearly revealing individual filamentous profiles. (D) Region of a DRG axon extracted with Triton X-100 and processed for immuno-EM (directed against biotin) 2 hours after injection. Notice the association of colloidal gold particles with filamentous profiles (arrows). (E) Representative DRG neurons (arrows), interspersed with non-neuronal cells, fixed and processed for SMI-31 immunoreactivity 3 days after plating. One neuron (large arrowhead) was microinjected 2 hours before fixation with biotin-L. Neurons in culture have expressed and transported endogenous NFs by this time, as shown by perikaryal and axonal SMI-31 immunoreactivity. The single microinjected neuron also displays prominent perikaryal and axonal biotin immunoreactivity. (F) Immunoblot probed with an antibody (SMI-32) against non-phosphorylated epitopes common to NF-H and NF-M, of material immunoprecipitated from a homogenate of a NB2a/d1 culture that had been transfected the previous day with GFP-M. Notice the appearance of an SMI-32-immunoreactive band of 172 kDa (arrow), which corresponds to the expected migratory position of NF-M (145 kDa) conjugated to GFP (30 kDa) (Yabe et al., 2001a,b). (G) Perikaryon and proximal axonal neurite of a cell transfected with GFP-M the previous day. Notice the incorporation of GFP-M into filamentous profiles.

View larger version (74K): [in a new window]   Fig. 2. Roscovitine increases the translocation of biotin-L into axons. DRG neurons were injected with a mixture of biotin-L and fluorescein-conjugated tracer. Following a 2 hour incubation, cells were fixed and processed for biotin immunoreactivity. Injected cells were located by fluorescein tracer and the distribution of biotin-L (arrows) was then monitored. Micrographs present images of representative neurons, arrows denote axons. The accompanying graph presents a densitometric analyses of the proportion of biotin-L (mean densitometric value ± standard error of the mean) in perikarya from multiple neurons. Notice the marked decrease in levels of biotin-L in in perikarya of neurons treated with roscovitine, indicative of increased translocation into axons.

View larger version (55K): [in a new window]   Fig. 3. Modulation of Cdk5-p35 activity regulates phospho-NF levels in neuronal perikarya. Micrographs present representative DRG neurons treated for 2 hours with 100 µM roscovotine or microinjected with Cdk5-p35 (together with fluorescein-conjugated tracer) as indicated. Following a 2 hour incubation, cultures were fixed and processed for immunofluorescence with RT97 without extraction. Axons are indicated by arrows. The corresponding phase-contrast image is also presented for the microinjected neuron to facilitate visualization. The accompanying graph presents the mean (± standard deviation) relative perikaryal fluorescence for 10 to 50 cells. Notice the reduction in perikaryal RT97 immunoreactivity by roscovitine and the increase in RT97 immunoreactivity in cells injected with Cdk5-p35.

View larger version (40K): [in a new window]   Fig. 4. Purified Cdk5-p35 phosphorylates NF subunits in cell-free analyses. (A) Autoradiographic analyses following SDS-gel electrophoresis of Triton-X-100-insoluble cytoskeletons from day 17 embryonic chicken brains incubated for 2 hours with [32P]orthophosphate with or without Cdk5 and p35. Migratory position of NF-H, NF-M and NF-L are indicated. The accompanying graph presents the relative density of 200 kDa NF-H incubated for 2 hours with [32P]orthophosphate with or without Cdk5 and p35. Notice the marked increase in radiolabel associated with NF subunits and, in particular, that associated with NF-H following incubation with Cdk5-p35. (B) Immunoblot analyses of 100 µg Triton-X-100-insoluble cytoskeletons incubated for 2 hours at 30°C ±25 µg Cdk5 and 25 µg p35 probed with RT97 and R39 that reacts with all NF subunits regardless of phosphorylation state (Jung et al., 1998) as an index of total NF-H; only the 200 kDa region of immunoblots is presented. The accompanying graph shows the percentage increase in immunoreactivity for each antibody following incubation with Cdk5-p35. Notice the specific increase in phospho-NF (RT97) immunoreactivity.

View larger version (25K): [in a new window]   Fig. 5. Microinjection of Cdk5-p35 decreases translocation of biotin-L into axons. Micrographs present representative DRG neurons injected with a mixture of biotin-L and fluorescein-conjugated tracer with and without Cdk5-p35, then processed for biotin immunoreactivity 2 hours after injection without extraction. The accompanying graph shows the densitometric analyses of the percentage of biotin-L in axons from multiple neurons. Notice the marked reduction in axonal transport of biotin-L in neurons that have been microinjected with Cdk5-p35.

View larger version (41K): [in a new window]   Fig. 6. Overexpression of Cdk5-p35 increases NF phospho-epitopes in perikarya. Panels present DRG neurons 24 hours after transfection with a construct expressing GFP-M in the absence (–) or presence (+) of constructs that express Cdk5 and p35, followed by immunofluoresce analyses with RT97 followed by Texas-Red-conjugated secondary antibody. Transfected cells were localized by GFP fluorescence (under fluorescein optics) and RT97 immunoreactivity was then imaged under rhodamine optics. The graph shows the mean percentage of RT97 immunoreactivity (± standard deviation, expressed in arbitrary densitometric units) that had translocated into axons. Notice that perikaryal RT97 immunoreactivity (the result of increased levels of phosphorylated NF-H) was significantly (P<0.05) increased in response to the overexpression of Cdk5-p35.

View larger version (43K): [in a new window]   Fig. 7. Modulation of Cdk5-p35 activity regulates transport of GFP-M into axons. DRG neurons 24 hours after the transfection with a construct expressing GFP-M, in the absence (–) or presence (+) of constructs expressing Cdk5 and p35 are shown. The graph shows the mean percentage of GFP fluorescence (± standard error of the mean, arbitrary densitometric units) that had translocated into axons in cultures transfected with GFP-M alone, GFP-M plus Cdk5-p35 or GFP-M followed by 100 µM roscovitine for the final 2 hours of incubation, before visualization. Notice that transport of GFP-NF-M into axons was significantly (P<0.05) reduced in cultures transfected with Cdk5-p35 and significantly enhanced (P<0.05) in cultures treated with roscovitine.

View larger version (80K): [in a new window]   Fig. 8. Overexpression of Cdk5-p35 increases NF bundling in perikarya. DRG neurons before (–) and 24 hours after (+) transfection with constructs expressing Cdk5 and p35 are shown in all panels (first two panels have no +/– label). Neurons in panels `NF-H' and `NF-L' were immunostained with antibodies directed against these subunits to reveal the distribution of endogenous NFs with (right) and without (left) overexpressing Cdk5-p35. Panels labeled `Biotin-L' and `GFP-M' show representative perikarya of neurons 2 hours after microinjection of biotin-L with (+) or without (–) Cdk5-p35 (followed by immunostaining with anti-biotin) or 24 hours following transfection with a construct expressing GFP-M with (+) or without (–) Cdk5-p35. Notice that perikarya of neurons overexpressing Cdk5-p35 displayed thick filamentous profiles containing endogenous subunits, microinjected subunits and subunits expressed following transfection. By contrast, endogenous and exogenous subunits in perikarya of neurons not overexpressing Cdk5-p35 were diffuse, punctate or present as relatively fine filaments that exhibited a much smaller caliber than those of cells overexpressing Cdk5-p35. Panels labeled `RT97' show representative neurons transfected with GFP-M with (+) or without (–) Cdk5-p35 then immunostained with RT97 to reveal the distribution of NF phospho-epitopes in perikarya. Notice the increase in perikaryal RT97 immunoreactivity in perikarya of neurons transfected with Cdk5-p35 and the association of RT97 with the resultant thick filamentous profiles, indicating that perikaryal NF bundles contain phospho-NFs.

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