Axonal Transport, Amyloid Precursor Protein, Kinesin-1, and the Processing Apparatus: Revisited

Nonspecific binding of AID and GST-KLC1 fusion proteins containing TRs. A, Schematic representation of GST-KLC1 deletion mutants used in GSTpull-down assays. For details, see Materials and Methods. B, Pull-down assays show that all GST-KLC constructs containing two or more TRs bind to in vitro-translated GFP-AID constructs and with lower affinity to the GFP-only control. Neither GST alone nor GST-KLC-CC (both lacking TRs) pulled down GFP-AID or GFP. The fact that TR-containing constructs pulled down both GFP-AID and to a lower extent the highly soluble protein GFP suggests that these are nonspecific protein-protein interactions. C, In addition to GFP-AID, both GFP-ALID1 and GFP-ALID2 were retained to a very similar extent on GST-KLCΔCT. Identical results were obtained with GST-KLC-TR or GST-KLC-CC/TR beads (data not shown). D, Schematic representation of GFP-APP intracellular domain deletion mutants used in GST pull downs (see Materials and Methods). E, GST-KLC-CC/TR binds with similar affinities to GFP-AID and GFP-AID constructs with deletions of the BaSS (G-AIDΔB), PEER (G-AIDΔP), or NPTY (G-AIDΔN) domains of APP. These represent domains that have been shown previously to affect trafficking of APP and map to the domain proposed by Kamal et al. (2000) to bind TR domain in KLC1. F, Beads containing GST-KLC with TR domains also pull down recombinant Fe65 with high affinity, again suggesting nonspecific binding of TR domains to proteins in this assay system. G, GST pull downs with GST fused to the intracellular domain of APP demonstrated binding to the known APP interacting proteins Fe65 and Numb, whereas no binding of KLC1 was observed under identical conditions.

Kinesin-1 and APP do not cofractionate or coimmunoprecipitate from brain lysate. A, Subcellular fractionation of mouse brain was performed as described previously (Morfini et al., 2002) to generate three vesicle fractions (V0, V1, and V2) and a cytosolic fraction (Cyt). Under the conditions of fractionation, KHCs and KLCs are found in all fractions, but KLCs exhibit differences in stoichiometry and isoform composition. In contrast, APP immunoreactivity is associated primarily with V0 and V1 fractions, with only trace amounts in V2 and low levels in the cytosol. V2 contains high levels of KHC, KLC1, KLC2, synaptophysin (P38), and synapsin I. This suggests that kinesin-1 and APP do not cofractionate in mouse brain. B, Immunoprecipitates of kinesin-1 from mouse brain show that Coomassie blue-stained levels of kinesin-1 heavy chain are obtained (CB), but no band corresponding to APP was seen. Immunoblots of whole-brain lysate show that kinesin-1 heavy chain (H2) is readily resolved from APP (22C11) in this gel system. These are distinguishable even when a mixture of both antibodies (H2+22C11) is used for immunoblotting. C, Immunoprecipitation of kinesin-1 from mouse brain using antibodies against either kinesin-1 heavy chains (SUK4 or H2) or light chains (63-90, L1, L2, or KLC-All) precipitated both KHC and KLC subunits (Pfister et al., 1989; Stenoien and Brady, 1997). Nevertheless, APP could not be detected in immunoprecipitates using kinesin-1 antibodies. Brain lysate (L) was a positive control. Negative controls, i.e., protein G beads without primary antibodies (B) or with normal mouse IgG show immunoprecipitation specificity. Similarly, kinesin-1 subunits were not detectable in immunoprecipitates with APP antibodies [Ct-APP and JH; see KHC (H2) for kinesin-1 heavy chain, KLC (63-90) for kinesin-1 light chain]. Antigens in immunoprecipitates were visualized using specific antibodies against kinesin-1 subunits (H2 or 63-90) or APP (22C11 or Ct-APP). D, Immunoblot analysis showed detectable amounts of the scaffolding protein JIP3/SYD in kinesin-1 immunoprecipitates (H2 IPP). Conversely, anti-JIP3 antibodies (SYd IPP) coprecipitate small but detectable amounts of KHC. Immunoprecipitations with normal mouse (NM IPP) or normal rabbit (NR IPP) IgGs were used as negative controls. Note the relative levels of JIP3/SYD compared with original protein input. E, Immunodepletion of kinesin-1 and APP from mouse brain lysate exhibits different depletion patterns. Immunodepletion of KHC from lysate with H2 comparing IPs and corresponding supernatant samples shows a gradual reduction in the level of both KHC (H2) and KLC (63-90) in the pellet (lanes 1-4) and the supernatant (lanes 13-16) after depletion cycles. In contrast, no change in APP level could be detected (Ct-APP, lanes 1-4 and 13-16, respectively). Similarly, a reduction in APP level was observed after depletion using APP-specific antibodies AB5352 (Ct-APP, pellet, lanes 9-12; supernatant, lanes 21-24), but no change in the level of kinesin-1 could be detected (KHC, lanes 9-12, 21-24; KLC, lanes 9-12, 21-24).

PS1 is not cotransported with APP in the same vesicular compartment by fast axonal transport. A, Top, Schematic diagram of sciatic nerve double ligation and nerve segments examined for protein expression. Bottom, Expression level of APP and PS1 in nerve segments at varying distances from the site of ligation in sciatic nerve of nontransgenic mice. APP expression level increases toward the site of ligation, whereas it decreases away from the distal ligature (CT15 panel, lanes 1-5). In contrast, PS1 expression level is comparable in intact sciatic nerve and nerve segments at varying distances from the site of ligation (PS1-NTF panel, lanes 1-7). B, APP and PS1 immunoreactivity in cryostat sections of the proximal and distal ends of ligated sciatic nerve. A, B, APP is detected using CT15 antibodies. Note the accumulation of APP proximal to the ligature (A), whereas weak immunoreactivity is observed at the distal stump (B). C, D, PS1 immunoreactivity as detected by affinity-purified αPS1-NTF antibodies; C, proximal stump; D, distal stump. Note that neither accumulation at the proximal stump nor reduced immunoreactivity at the distal stump can be detected. E, Specificity of immunostaining for PS1 was confirmed using αPS1-NTF antibodies preincubated with GST-PS1-NTF (see Materials and Methods). F, Immunostaining of mouse sciatic nerve cryostat sections using secondary antibodies only (fluorescein-conjugated goat anti-rabbit). Scale bar, 200 μm.