The non-Federal forest lands, which comprise three-fourths of the Nation's forest lands, are the key to meeting projected future needs for all forest products and uses. At the same time, the Federal role in State and Private forestry cooperative programs is being critically questioned. Public attitudes toward Federal expenditures, and the possibility of sunset legislation, may change the nature of Forest Service cooperative programs. One possible approach would be for each State to develop a comprehensive forestry plan suited to both State and national needs, perhaps related to a national program expressed through the Resources Planning Act process, as a basis for granting Federal funds for cooperative forestry activities.
Background and Purpose The scale of stroke impairment characteristics by Brott and associates, the National Institutes of Health (NIH) Stroke Scale, has been used widely in various studies of stroke outcome; however, the measurement properties of the items applied to patients during medical rehabilitation have not been evaluated thoroughly. This study evaluated the extent to which scale items cohere to define a unidimensional construct and have a useful range for application to patients during medical rehabilitation. Methods Rating scale (or Rasch) analysis of the 15 NIH Stroke Scale items was conducted using the BIGSTEPS computer program to evaluate (1) the range of impairment assessed by the items, (2) the items’ coherence with an underlying construct of impairment, and (3) range of impairment measured in rehabilitation patients. We sought to maximize the range of impairment measured by conducting analyses recursively; at each subsequent step, the worst fitting item was deleted or rescored. The sample comprised 1291 admission and discharge records from 693 rehabilitation inpatients with stroke. Results Thirteen items arrayed the sample across a sufficient range of impairment. The limb ataxia item fit poorly and was deleted; lower ratings for this item were associated with higher scores on the total scale. Pupillary response was also deleted because ratings reflected poor congruence with the total score. Best language was rescored because intermediate ratings were inconsistently related to the total score. Patients with hemorrhagic strokes had poorer fitting measures than did patients with ischemic strokes. Conclusions The items in a revised NIH Stroke Scale worked well together to define the severity of impairment resulting from stroke that is observed during medical rehabilitation. Directions regarding limb ataxia should be modified to indicate untestability due to hemiplegia.
The goal of KidSat was to provide young students with the opportunity to participate directly in the NASA space program and to enhance learning in the process. The KidSat pilot project was focused on using a color digital camera, mounted on the space shuttle, to take pictures of the Earth. These could be used to enhance middle school curricula. The project not only benefited middle school students, who were essentially the Science Team, responsible for deciding where to take pictures, but it also benefited high school students and undergraduates, who were essentially the Project Team, responsible for the development and implementation of the project. KidSat flew on three missions as part of the pilot project: STS-76, STS-81, and STS-86. This document describes the goals, project elements, results, and data for the three KidSat missions that made up the pilot program. It serves as a record for this pilot project and may be used as a reference for similar projects. It can also be a too] in using the data to its fullest extent. The KidSat Web page remains on-line at http://kidsat.jpl.nasa.gov/kidsat, and the images may be downloaded in their full resolution.
Huntingtin-associated protein-1 (HAP1) was initially identified as an interacting partner of huntingtin, the Huntington disease protein. Unlike huntingtin that is ubiquitously expressed throughout the brain and body, HAP1 is enriched in neurons, suggesting that its dysfunction could contribute to Huntington disease neuropathology. Growing evidence has demonstrated that HAP1 and huntingtin are anterogradely transported in axons and that the abnormal interaction between mutant huntingtin and HAP1 may impair axonal transport. However, the exact role of HAP1 in anterograde transport remains unclear. Here we report that HAP1 interacts with kinesin light chain, a subunit of the kinesin motor complex that drives anterograde transport along microtubules in neuronal processes. The interaction of HAP1 with kinesin light chain is demonstrated via a yeast two-hybrid assay, glutathione S-transferase pull down, and coimmunoprecipitation. Furthermore, HAP1 is colocalized with kinesin in growth cones of neuronal cells. We also demonstrated that knocking down HAP1 via small interfering RNA suppresses neurite outgrowth of PC12 cells. Analysis of live neuronal cells with fluorescence microscopy and fluorescence recovery after photobleaching demonstrates that suppressing the expression of HAP1 or deleting the HAP1 gene inhibits the kinesin-dependent transport of amyloid precursor protein vesicles. These studies provide a molecular basis for the participation of HAP1 in anterograde transport in neuronal cells. Huntingtin-associated protein-1 (HAP1) was initially identified as an interacting partner of huntingtin, the Huntington disease protein. Unlike huntingtin that is ubiquitously expressed throughout the brain and body, HAP1 is enriched in neurons, suggesting that its dysfunction could contribute to Huntington disease neuropathology. Growing evidence has demonstrated that HAP1 and huntingtin are anterogradely transported in axons and that the abnormal interaction between mutant huntingtin and HAP1 may impair axonal transport. However, the exact role of HAP1 in anterograde transport remains unclear. Here we report that HAP1 interacts with kinesin light chain, a subunit of the kinesin motor complex that drives anterograde transport along microtubules in neuronal processes. The interaction of HAP1 with kinesin light chain is demonstrated via a yeast two-hybrid assay, glutathione S-transferase pull down, and coimmunoprecipitation. Furthermore, HAP1 is colocalized with kinesin in growth cones of neuronal cells. We also demonstrated that knocking down HAP1 via small interfering RNA suppresses neurite outgrowth of PC12 cells. Analysis of live neuronal cells with fluorescence microscopy and fluorescence recovery after photobleaching demonstrates that suppressing the expression of HAP1 or deleting the HAP1 gene inhibits the kinesin-dependent transport of amyloid precursor protein vesicles. These studies provide a molecular basis for the participation of HAP1 in anterograde transport in neuronal cells. Huntingtin-associated protein-1 (HAP1) 2The abbreviations used are: HAP1, huntingtin-associated protein 1; KLC, kinesin light chain; KHC, kinesin heavy chain; WT-wild type; GFP, green fluorescent protein; YFP, yellow fluorescent protein; RFP, red fluorescent protein; siRNA-small interfering RNA; APP, amyloid precursor protein; FRAP-fluorescence recovery after photobleaching; GST, glutathione S-transferase; NGF, nerve growth factor. was the first protein identified to interact with huntingtin (htt), the Huntington disease (HD) protein (1Li X.J. Li S.H. Sharp A.H. Nucifora Jr., F.C. Schilling G. Lanahan A. Worley P. Snyder S.H. Ross C.A. Nature. 1995; 378: 398-402Crossref PubMed Scopus (541) Google Scholar, 2Li X.J. Li S.H. Trends Pharmacol. Sci. 2005; 26: 1-3Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Htt contains a polyglutamine (polyQ) stretch in its N terminus, and expansion of this glutamine repeat (>37 glutamines) causes selective neurodegeneration. However, the underlying mechanisms of the specific neuropathology of HD remain unclear, especially in light of the widespread expression of htt. It is believed that the expanded polyQ confers an abnormal protein conformation and affects the function of other neuronal proteins (3Perutz M.F. Trends Biochem. Sci. 1999; 24: 58-63Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). This idea, or the theory of gain of function, is strongly supported by the fact that polyQ expansion causes htt to abnormally interact with other proteins (4Harjes P. Wanker E.E. Trends Biochem. Sci. 2003; 28: 425-433Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, 5Li S. Li X.J. Trends Genet. 2004; 20: 146-154Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar). HAP1 is a good candidate for htt-mediated pathology, because its binding to the N-terminal region of htt is enhanced by expanded polyQ tracts, and its expression is enriched in the brain (1Li X.J. Li S.H. Sharp A.H. Nucifora Jr., F.C. Schilling G. Lanahan A. Worley P. Snyder S.H. Ross C.A. Nature. 1995; 378: 398-402Crossref PubMed Scopus (541) Google Scholar). The critical role of HAP1 in neuronal function has been demonstrated in HAP1 knock-out mice. Deletion of the mouse HAP1 gene leads to retarded growth, depressed feeding behavior, and postnatal death of these mice (6Chan E.Y. Nasir J. Gutekunst C.A. Coleman S. Maclean A. Maas A. Metzler M. Gertsenstein M. Ross C.A. Nagy A. Hayden M.R. Hum. Mol. Genet. 2002; 11: 945-959Crossref PubMed Scopus (74) Google Scholar, 7Li S.H. Yu Z.X. Li C.L. Nguyen H.P. Zhou Y.X. Deng C. Li X.J. J. Neurosci. 2003; 23: 6956-6964Crossref PubMed Google Scholar, 8Dragatsis I. Zeitlin S. Dietrich P. Hum. Mol. Genet. 2004; 20: 20Google Scholar). This phenotype may be caused by the degeneration of hypothalamic neurons that control feeding behavior (7Li S.H. Yu Z.X. Li C.L. Nguyen H.P. Zhou Y.X. Deng C. Li X.J. J. Neurosci. 2003; 23: 6956-6964Crossref PubMed Google Scholar). Several studies have suggested that HAP1 is involved in neuronal transport of organelles or molecules. HAP1 is required for vesicular transport of brain-derived neurotrophic factor along microtubules, and mutant htt impairs this transport concomitant with its increased interactions with HAP1 and dynactin p150 (9Gauthier L.R. Charrin B.C. Borrell-Pages M. Dompierre J.P. Rangone H. Cordelieres F.P. De Mey J. MacDonald M.E. Lessmann V. Humbert S. Saudou F. Cell. 2004; 118: 127-138Abstract Full Text Full Text PDF PubMed Scopus (923) Google Scholar). HAP1 might also be involved in endocytotic trafficking of membrane receptors. Evidence to support this idea includes the interaction of HAP1 with Hrs, which plays a critical role in the endocytosis of epidermal growth factor receptor (10Li Y. Chin L.S. Levey A.I. Li L. J. Biol. Chem. 2002; 277: 28212-28221Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). A recent study by Kittler et al. (11Kittler J.T. Thomas P. Tretter V. Bogdanov Y.D. Haucke V. Smart T.G. Moss S.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12736-12741Crossref PubMed Scopus (198) Google Scholar) provides further evidence that HAP1 is involved in the internalization and recycling of the GABAA receptor and that HAP1 overexpression leads to an increase in the level of GABAA (11Kittler J.T. Thomas P. Tretter V. Bogdanov Y.D. Haucke V. Smart T.G. Moss S.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12736-12741Crossref PubMed Scopus (198) Google Scholar). In addition, HAP1 interacts with the type 1 inositol (1Li X.J. Li S.H. Sharp A.H. Nucifora Jr., F.C. Schilling G. Lanahan A. Worley P. Snyder S.H. Ross C.A. Nature. 1995; 378: 398-402Crossref PubMed Scopus (541) Google Scholar, 4Harjes P. Wanker E.E. Trends Biochem. Sci. 2003; 28: 425-433Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, 5Li S. Li X.J. Trends Genet. 2004; 20: 146-154Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar)-triphosphate receptor (InsP3R1), forming an InsP3R1-HAP1-A-htt ternary complex in which mutant htt enhances the sensitivity of InsPR1 to IP3 (12Tang T.S. Tu H. Chan E.Y. Maximov A. Wang Z. Wellington C.L. Hayden M.R. Bezprozvanny I. Neuron. 2003; 39: 227-239Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 13Tang T.S. Tu H. Orban P.C. Chan E.Y. Hayden M.R. Bezprozvanny I. Eur. J. Neurosci. 2004; 20: 1779-1787Crossref PubMed Scopus (53) Google Scholar). The biochemical evidence to support the role of HAP1 in intracellular trafficking includes the interaction of HAP1 with dynactin p150 Glued (dynactin p150), a dynein-associated protein (14Engelender S. Sharp A.H. Colomer V. Tokito M.K. Lanahan A. Worley P. Holzbaur E.L. Ross C.A. Hum. Mol. Genet. 1997; 6: 2205-2212Crossref PubMed Scopus (280) Google Scholar, 15Li S.H. Gutekunst C.A. Hersch S.M. Li X.J. J. Neurosci. 1998; 18: 1261-1269Crossref PubMed Google Scholar). p150 Glued and dynein drive retrograde transport in neuronal cells, whereas kinesin directs plus-end (anterograde) movement (16Waterman-Storer C.M. Karki S.B. Kuznetsov S.A. Tabb J.S. Weiss D.G. Langford G.M. Holzbaur E.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12180-12185Crossref PubMed Scopus (214) Google Scholar, 17Vale R.D. Cell. 2003; 112: 467-480Abstract Full Text Full Text PDF PubMed Scopus (1526) Google Scholar). The cotransport of HAP1 and htt in both anterograde and retrograde directions (18Block-Galarza J. Chase K.O. Sapp E. Vaughn K.T. Vallee R.B. DiFiglia M. Aronin N. Neuroreport. 1997; 8: 2247-2251Crossref PubMed Scopus (123) Google Scholar) and the sequestration by mutant htt of soluble dynein and kinesin (19Gunawardena S. Her L.S. Brusch R.G. Laymon R.A. Niesman I.R. Gordesky-Gold B. Sintasath L. Bonini N.M. Goldstein L.S. Neuron. 2003; 40: 25-40Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar, 20Trushina E. Dyer R.B. Badger 2nd, J.D. Ure D. Eide L. Tran D.D. Vrieze B.T. Legendre-Guillemin V. McPherson P.S. Mandavilli B.S. Van Houten B. Zeitlin S. McNiven M. Aebersold R. Hayden M. Parisi J.E. Seeberg E. Dragatsis I. Doyle K. Bender A. Chacko C. McMurray C.T. Mol. Cell. Biol. 2004; 24: 8195-8209Crossref PubMed Scopus (421) Google Scholar) further raise the possibility that HAP1 participates in kinesin-mediated transport in neurons. The native conventional kinesin complex (379-386 kDa) consists of two heavy chains (KHC) (110-120 kDa) and two light chains (KLC-1 and KLC-2) (60-70 kDa) (21Bloom G.S. Wagner M.C. Pfister K.K. Brady S.T. Biochemistry. 1988; 27: 3409-3416Crossref PubMed Scopus (218) Google Scholar, 22Kuznetsov S.A. Vaisberg E.A. Shanina N.A. Magretova N.N. Chernyak V.Y. Gelfand V.I. EMBO J. 1988; 7: 353-356Crossref PubMed Scopus (134) Google Scholar). The N-terminal heads of KHC move along microtubules. The C-terminal tails are linked with light chains (KLC) that are thought to interact with membrane-bound organelles and regulate KHC activity (23Hirokawa N. Sato-Yoshitake R. Kobayashi N. Pfister K.K. Bloom G.S. Brady S.T. J. Cell Biol. 1991; 114: 295-302Crossref PubMed Scopus (198) Google Scholar, 24Pfister K.K. Wagner M.C. Stenoien D.L. Brady S.T. Bloom G.S. J. Cell Biol. 1989; 108: 1453-1463Crossref PubMed Scopus (193) Google Scholar, 25Yang J.T. Saxton W.M. Stewart R.J. Raff E.C. Goldstein L.S. Science. 1990; 249: 42-47Crossref PubMed Scopus (192) Google Scholar, 26Scholey J.M. Heuser J. Yang J.T. Goldstein L.S. Nature. 1989; 338: 355-357Crossref PubMed Scopus (171) Google Scholar). A number of associated proteins participate in linking motor proteins to various cargos (17Vale R.D. Cell. 2003; 112: 467-480Abstract Full Text Full Text PDF PubMed Scopus (1526) Google Scholar). Here we demonstrate that HAP1 interacts with the kinesin motor complex and that suppressing the expression of HAP1 inhibits kinesin-associated transport in neurons, providing the biochemical basis for the involvement of HAP1 in anterograde transport in neuronal cells. Reagents—The following antibodies were used: rabbit polyclonal antibodies against HAP1-A, HAP1-B, and HAP1 (27Li S.H. Gutekunst C.A. Hersch S.M. Li X.J. J. Neurochem. 1998; 71: 2178-2185Crossref PubMed Scopus (41) Google Scholar); a mouse antibody against kinesin heavy chain (Chemicon); and a rabbit antibody against kinesin light chain-2 was generated using GST fusion proteins containing a rat KLC-2 fragment (amino acids 124-411) by Covance Inc. Other antibodies used in the study were those against tubulin (Sigma) and the hemagglutinin epitope 12CA5 (Cell Signaling). HAP1 constructs were generated in our previous studies (15Li S.H. Gutekunst C.A. Hersch S.M. Li X.J. J. Neurosci. 1998; 18: 1261-1269Crossref PubMed Google Scholar). We also fused GFP or RFP to the N terminus of HAP1-A or HAP1-B in the PRK vector for examining its dynamic movement in cultured cells. The C-terminal region of HAP1-A at the BglII site of HAP1 cDNA was removed, resulting in a truncated HAP1 encoding 1-473 amino acids of HAP1 (27Li S.H. Gutekunst C.A. Hersch S.M. Li X.J. J. Neurochem. 1998; 71: 2178-2185Crossref PubMed Scopus (41) Google Scholar). The full-length KLC-2 cDNA was isolated by screening a rat brain cDNA library (Stratagene) and inserted into the PRK expression vector. Amyloid precursor protein (APP)-YFP cDNA was a gift from Dr. Lawrence S. B. Goldstein at University of California in San Diego (28Kamal A. Stokin G.B. Yang Z. Xia C.H. Goldstein L.S. Neuron. 2000; 28: 449-459Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). PC12 cell lines stably expressing antisense HAP1 RNA were generated in our previous studies (7Li S.H. Yu Z.X. Li C.L. Nguyen H.P. Zhou Y.X. Deng C. Li X.J. J. Neurosci. 2003; 23: 6956-6964Crossref PubMed Google Scholar). Yeast Two-hybrid Assays—HAP1 (1-599 amino acids) fused to the GAL-4 DNA binding domain was used as a bait to screen a rat brain cDNA library (1Li X.J. Li S.H. Sharp A.H. Nucifora Jr., F.C. Schilling G. Lanahan A. Worley P. Snyder S.H. Ross C.A. Nature. 1995; 378: 398-402Crossref PubMed Scopus (541) Google Scholar). Two cDNAs were identified to contain a partial KLC-2 (amino acids 80-396) fragment that was able to interact with HAP1 in yeast. Filter and liquid β-galactosidase assays with different HAP1 fragments were performed using the same method as described previously (1Li X.J. Li S.H. Sharp A.H. Nucifora Jr., F.C. Schilling G. Lanahan A. Worley P. Snyder S.H. Ross C.A. Nature. 1995; 378: 398-402Crossref PubMed Scopus (541) Google Scholar). Protein Interaction Assays and Western Blotting—In vitro binding assays were performed as described (1Li X.J. Li S.H. Sharp A.H. Nucifora Jr., F.C. Schilling G. Lanahan A. Worley P. Snyder S.H. Ross C.A. Nature. 1995; 378: 398-402Crossref PubMed Scopus (541) Google Scholar, 15Li S.H. Gutekunst C.A. Hersch S.M. Li X.J. J. Neurosci. 1998; 18: 1261-1269Crossref PubMed Google Scholar). Protein A-Sepharose (Sigma) was added to the mixture for an additional 1 h incubation. The beads were precipitated by gravity and washed twice in lysis buffer, which were then resolved by SDS-PAGE and detected by Western blotting. For immunoprecipitation of HAP1 from mouse brain, hypothalamus was dissected from postnatal day one pups. Three to four hypothalami from wild type or HAP1 knock-out pups were combined and homogenized with a handheld Kontes pellet pestle motorized homogenizer in 0.2% Triton X-100 in a phosphate-buffered saline solution. Samples were then sonicated for 10 s at low power and were gently rocked for 15 min at 4 °C, followed by centrifugation at 4 °C at 16,000 × g for 10 min. Samples were adjusted to 500 μl of protein at 1.5 μg/μl and preabsorbed by protein A-agarose beads (25 μl, Sigma 1406-5G) for 1 h at 4 °C with gentle rocking. Supernatants were collected, and 20 μl of rabbit anti-HAP1-A (EM31) were added for incubation at 4 °C for 4 h. Next, 12.5 μl of protein A beads were added and incubated with gentle rocking at 4 °C for 1 h. Samples were spun in a tabletop microcentrifuge for 10-15 s. Beads were washed three times with lysis buffer and finally resuspended in 80 μl of lysis buffer for Western blotting. For immunoprecipitation of PC12 cells, PC12 cells were lysed in a Nonidet P-40-containing buffer. Anti-HAP1-A (10 μl) was then added to lysates (500 μl of 1 μg/μl solution) to incubate overnight at 4 °C with gentle rocking. Protein A beads (15 μl) were then added to the tube for immunoprecipitation. For Western blots, cultured cells or brain tissues were solubilized in 1% SDS, resuspended in SDS sample buffer, and sonicated for 10 s. The total lysate was used for Western blotting with the ECL kit (Amersham Biosciences). Neuronal Culture and Imaging Analysis—Primary neurons were cultured using the method similar to that described previously (29Li H. Li S.H. Yu Z.X. Shelbourne P. Li X.J. J. Neurosci. 2001; 21: 8473-8481Crossref PubMed Google Scholar). Olfactory bulb neurons isolated from wild type and HAP1 knock-out mice at postnatal day one were plated on lysine-coated coverglass in two-well chamber slides from Nalge Nunc International Lab-Tek (catalog number 155379). Cells were cultured in B27-supplemented neurobasal medium (Invitrogen). PC12 cells were cultured in Dulbecco's modified Eagle's medium with 5% fetal bovine serum and 10% horse serum containing 50-100 ng/ml nerve growth factor (NGF) for 3 days. Control cells were cultured without NGF. For immunofluorescence light microscopy, light micrographs were taken using a 63× objective lens (LD-Achroplan 63×/0.75) on a Zeiss microscope (Axiovert 200 MOT) attached to a digital camera (Hamamatsu Orca-100). Micrographs were taken using Openlab software (Improvision Inc). Confocal image analysis was performed with a Ziess LSM 510 NLO confocal microscope system. For transfection of PC12 cells and imaging analysis of live transfected cells, cells were plated on two-chambered coverglass (Lab-Tek 155379) and transfected with GFP-HAP1-A or GFP-HAP1-B by Lipofectamine 2000 (Invitrogen 18324-020) for 5 h. Cells were then loaded for 10-15 min with red MitoTracker (Molecular Probes) at a concentration of 1:1000 in CO2-independent medium from Invitrogen (18045-088) at 37 °C. After two washes with CO2-independent medium, CO2-independent medium containing 100 ng/ml NGF (Sigma) was added to stimulate differentiation. Cells were then immediately placed in a heated stage and imaged using the Zeiss LSM 510 confocal microscope with a 40x objective lens. Images were scanned at 512 × 512 resolution, scan speed of 7, and a mean of 4 scans for an overall scan time of 3.93 s per image. The mitotracker dye (Molecular Probe) was imaged at a laser wavelength of 514 nm; the GFP fusion proteins were imaged at 488 nm. Scans were taken every 3 min over a course of 3.5 h. Movies were created using Metamorph software showing 6 frames/s. For vesicular movement assays, cultured olfactory bulb neurons or PC12 cells were plated in 35-mm culture dishes. After culturing PC12 cells for 24 h and primary neurons for 96 h, cells were transfected with APP-YFP DNA using Lipofectamine 2000. Wild type cells served as a control, and the identity of the cells was blinded. Cells were examined 24-36 h later in CO2-independent medium using the 63× objective lens of the fluorescence light microscope. Cells were observed for 1 min to score those having visible vesicle movement in neurites. For each culture dish, 20 cells were randomly selected. The number of cells showing vesicular movement was divided by the total number of transfected cells examined to obtain a percentage of transfected cells having vesicle movement. For each cell type, three plates were assayed, and the data are expressed as mean ± S.E. Adenoviral HAP1-siRNA Preparation—The testing of small interfering RNA (siRNA) sequences revealed that one siRNA (GAAGTATGTCCTCCAGCAA of 1695-1713 nucleotides of the rat HAP1 gene) was able to effectively inhibit the expression of HAP1. This siRNA was inserted into an adenoviral vector that independently expresses GFP under the control of the CMV promoter. A vector that expresses GFP alone or a scrambled HAP1 siRNA (GCGCGCTTTGTAGGATTCG) served as the control. Recombinant adenoviruses were generated and purified by Welgen Inc. (Worcester, MA). The viral titer was determined by measuring the number of infected HEK293 cells expressing GFP. All viral stocks were adjusted to 1 × 108 plaque-forming units/μl before their use. Fluorescence Recovery after Photobleaching (FRAP) Assay—Cultured olfactory neurons were transfected with APP-YFP DNA construct. After 18-36 h, cells were incubated with CO2-independent medium. Chamber slides were then placed into a heated microscope stage and maintained at 37 °C. Cells were imaged using a Zeiss LSM 510 confocal microscope microimaging system (Zeiss LSM 510 Meta from Carl Zeiss Microimaging) and software (LSM 510 version 302 SP2). All imaging was done with a 63× oil immersion objective lens. For imaging, the Zeiss Argon laser (maximum power of 30 milliwatt) was adjusted to a wavelength of 488 nm. Laser power was set to 75%, and transmittance was set at 4%. Transfected cells were then selected by the presence of YFP fluorescent puncta. A time series of images was collected. Total scanning time was set at 3.93 s with a wait time of 3 s between scans. Three scans were taken before photobleaching using the conditions listed above. The distal one-third to one-quarter of the longest process (in an effort to select the tips of axons over dendrites) was outlined as a region of interest to be photobleached. To photobleach cells, the laser transmittance was adjusted to 100% for both the 477 nm and 488 nm line of the argon laser. Seventy bleaching iterations were performed, lasting a total of ∼10-15s. Immediately after photobleaching, scans were taken as described above for up to 5 min. Regions of interest were also established for an unbleached cell region and for a region outside of cells at each time point. These values were used to correct background and for overall fluorescence loss because of scanning. For each wild type or HAP1 knock-out animal, 3 cells were examined and their fluorescence data averaged at each time point. A total of 13 cells from four mice were examined and the data averaged for each genotype. Statistical Analysis—Error bars in the vesicle movement assays reflect S.E., as computed for three trials of 20 cells/trial. Differences were found to be statistically significant using the Student's t test for both primary neurons (p = 0.02) and antisense RNA expressing PC12 cells (p = 0.02). Error bars in the FRAP assays reflect the S.E. for 13 cells from four wild type and four knock-out mice. Interaction of HAP1 with Kinesin—Rodent HAP1 consists of two isoforms, HAP1-A and HAP1-B, which have different C-terminal sequences (1Li X.J. Li S.H. Sharp A.H. Nucifora Jr., F.C. Schilling G. Lanahan A. Worley P. Snyder S.H. Ross C.A. Nature. 1995; 378: 398-402Crossref PubMed Scopus (541) Google Scholar). Yeast two-hybrid analysis revealed that a C-terminal fragment of HAP1 present in both rodent isoforms binds kinesin light chain-2 (KLC-2, Fig. 1), a subtype of KLC that is highly homologous to kinesin light chain-1 (KLC-1) with 71.1% amino acid identity (30Rahman A. Friedman D.S. Goldstein L.S. J. Biol. Chem. 1998; 273: 15395-15403Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The coiled-coil domains of HAP1 do not appear to mediate the binding of HAP1 to KLC, as HAP1 fragments that contained coiled-coil domains but lacked C-terminal amino acids failed to yield a positive interaction (Fig. 1B). To confirm the interaction between HAP1 and KLC, we performed a GST binding assay in which GST or GST-HAP1 fusion proteins were incubated with HEK293 cell lysates containing transfected KLC-2. A GST fusion protein consisting of a HAP1 fragment (160-599 amino acids), but not 160-445 amino acids, precipitated KLC-2, confirming the yeast two-hybrid results (Fig. 1C). To examine the in vivo interaction of HAP1 with KLC, we performed HAP1 immunoprecipitation of mouse brains. We used hypothalamic tissue for HAP1 immunoprecipitation, as it is enriched in HAP1. The hypothalamic tissue of HAP1 knock-out mice we previously created (7Li S.H. Yu Z.X. Li C.L. Nguyen H.P. Zhou Y.X. Deng C. Li X.J. J. Neurosci. 2003; 23: 6956-6964Crossref PubMed Google Scholar) was used as a control to confirm that precipitation of kinesin is dependent on the presence of HAP1. We generated a rabbit anti-KLC using the conserved region (124-411 amino acids) of KLC-2 as an antigen. This antibody reacts with both KLC-1 and KLC-2 in the brain. Western blotting revealed that KLC-1 is the major form of KLC in the hypothalamus and was coprecipitated by the antibody to HAP1. Only when HAP1 was present did HAP1 immunoprecipitation coprecipitate kinesin (Fig. 1D), indicating that KLC associates with HAP1 in vivo. Colocalization and Association of HAP1-A with Kinesin—To investigate whether HAP1 colocalizes with kinesin in cells, we examined PC12 cells that show elongated neurites in response to NGF. Without NGF treatment, KHC and KLC were mainly distributed in the cell body or soma (data not shown). After NGF treatment, PC12 cells developed long neurites. Consistent with a previous report (31Morfini G. Quiroga S. Rosa A. Kosik K. Caceres A. J. Cell Biol. 1997; 138: 657-669Crossref PubMed Scopus (66) Google Scholar), KHC and KLC were distributed in the growing tips of cellular processes (Fig. 2A). We have shown previously that HAP1-A is enriched in neurite tips, and HAP1-B is largely confined to the cell body (32Li S.H. Li H. Torre E.R. Li X.J. Mol. Cell Neurosci. 2000; 16: 168-183Crossref PubMed Scopus (55) Google Scholar). Therefore, we wanted to know whether kinesin and HAP1-A are colocalized in neurite tips. We used mouse anti-KHC and rabbit antibodies to HAP1 in immunofluorescence double labeling. KHC and HAP1-A were clearly colocalized in the same neurite tips (Fig. 2B). In contrast, most HAP1-B remained in the body of PC12 cells (Fig. 2B). Using immunoprecipitation, we then asked whether HAP1-A preferentially binds to kinesin. It is known that HAP1-A interacts with HAP1-B to form a dimer in cells (27Li S.H. Gutekunst C.A. Hersch S.M. Li X.J. J. Neurochem. 1998; 71: 2178-2185Crossref PubMed Scopus (41) Google Scholar). As expected, immunoprecipitation of either HAP1-A or HAP1-B from PC12 cells pulled down both HAP1-A and HAP1-B. Because PC12 cells express more HAP1-B than HAP1-A, the HAP1-B immunoprecipitation yielded a much greater amount of HAP1-B than HAP1-A. Importantly, more KLC was precipitated by anti-HAP1-A than by anti-HAP1-B. Because KLC binds KHC to form an active motor complex, we probed the same blot with anti-KHC. The increased level of KHC in the HAP1-A immunoprecipitation as compared with the HAP1-B immunoprecipitation also supports the idea that HAP1-A is more likely than HAP1-B to associate with the kinesin complex (Fig. 2C). Trafficking of HAP1 and Neurite Outgrowth—Microtubule-dependent transport is required for neurite outgrowth. We have previously reported that overexpression of HAP1-A significantly increases the number of PC12 cells containing extended neurites (29.4%) as compared with HAP-1B overexpression (9.7%) and vector transfection (4.7%) (32Li S.H. Li H. Torre E.R. Li X.J. Mol. Cell Neurosci. 2000; 16: 168-183Crossref PubMed Scopus (55) Google Scholar). However, it remains unclear whether this neurite promotion is related to the trafficking function of HAP1. To test this hypothesis, we first examined the effect of HAP1 on live NGF-stimulated PC12 cells. We expressed GFP- or RFP-linked HAP1-A or HAP1-B in these cells and then examined the dynamic subcellular localization of the different HAP1 isoforms. We observed that RFP-HAP1-A was able to move anterogradely from the cell body to neurite terminal (supplemental movie 1). Time-lapse imaging clearly shows that cells expressing GFP-HAP1-A are able to extend neurites. More importantly, GFP-HAP1-A is localized in the extended neurites and growth cone tips (Fig. 3A). However, GFP-HAP1-B remained in the cell body of PC12 cells and seemed to inhibit neurite outgrowth when compared with nontransfected cells labeled with a red mitochondrial staining (Fig. 3A). We also expressed a truncated HAP1 (amino acids 1-473), which lacks the C-terminal kinesin light chain binding domain. Overexpression of this mutated HAP1 was unable to promote neurite outgrowth of PC12 cells (Fig. 3B). As HAP1-B and HAP1-A are known to form heterodimers, it is possible that overexpression of HAP1-B sequesters all of the free endogenous HAP1-A of the cell, preventing cells from extending neurites. These results are consistent with the finding that HAP1-A preferentially binds kinesin and support the idea that the interaction of HAP1-A with kinesin may be involved in neurite outgrowth in some neurons. Altering the Expression of HAP1 in Neurons via siRNA—We next wanted to test the idea that HAP1-associated trafficking is indeed involved in the neurite outgrowth in PC12 cells. To do so, we used siRNA to reduce the expression of endogenous HAP1 in PC12 cells. We identified one siRNA (see sequences under "Materials and Methods") that was able to effectively inhibit the expression of HAP1. This siRNA was inserted into an adenoviral vector that also independently expresses GFP. Thus, GFP-positive cells are likely to express HAP1 siRNA. Infection of PC12 cells with this adenoviral siRNA effectively reduced endogenous levels of HAP1 in PC12 cells (Fig. 4A). Consequently, HAP1 siRNA reduced the number PC12 cells showing neurites longer than two cell bodies in the presence of NGF (Fig. 4B). This reduction by adenoviral HAP1 siRNA (20.95% ± 0.18, n = 3) is statistically significant (p < 0.01) as compared with adenoviral GFP infection (47.4% ± 1.94, n = 3), confirming the role of HAP1 in neurite growth of PC12 cells. We also tes
This paper presents experimental data evaluating the merits of using a fun and engaging therapy protocol over a less engaging one in the context of a low-cost robot/computer motivating rehabilitation system for stroke rehabilitation called TheraDrive. The preliminary results suggest that there is a small advantage of the engaging therapy over the rote therapy in reducing motor impairment, improving ADL function, and improving stability. The more engaging protocol has an advantage in maintaining engagement and interest in therapy.
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