Disruption of fast axonal transport is a pathogenic mechanism for intraneuronal amyloid beta

oAβ Is a Potent Inhibitor of FAT.

Our previous studies found reduced anterograde FAT of specific synaptic cargoes in different AD murine models known to accumulate intracellular Aβ in the axonal compartment progressively (23). To evaluate the intraxonal effects of Aβ on FAT directly, we perfused heterogeneous preparations of synthetic Aβ42 into isolated extruded axoplasms dissected from the squid Loligo pealeii. Perfusion of synthetic Aβ42 at 2 μM severely reduced both kinesin-1-based anterograde and cytoplasmic dynein (cDyn)-based retrograde FAT (Fig. 1A). Reconstitution of synthetic Aβ in aqueous solutions produces a mixture of different Aβ assemblies caused by preexisting molecular structures in lyophilized stocks (27). To determine the contribution of specific Aβ structural assemblies to inhibition of FAT, we prepared Aβ42 under defined conditions that yield uniform solutions of oligomers or fibrils (27). These unaggregated, oligomeric, and fibrillar Aβ42 preparations (100 nM) were perfused into isolated axoplasms to evaluate the effects on FAT. Neither uAβ (Fig. 1B) nor fAβ (Fig. 1C) altered FAT in either direction. In contrast, perfusion of 100 nM oAβ inhibited both directions of FAT significantly (Fig. 1D), and inhibition of FAT was seen as low as 10 nM oAβ (data not shown). Collectively, these results indicate that oAβ has a toxic activity that inhibits FAT when present intracellularly. This toxic effect is independent of transcription and translation, as extruded axoplasms are separated from cell bodies. The oAβ inhibition of FAT is also independent of energy metabolism changes, because 5 mM ATP is added to assays of vesicle motility with oAβ, thereby ruling out the possibility that oAβ affects FAT by altering mitochondrial ATP production.

oAβ-Induced FAT Inhibition Results from Endogenous CK2 Activation.

To determine the specific molecular mechanism by which oAβ inhibits FAT, the characteristic profile of FAT inhibition was evaluated. The ability of nanomolar levels of oAβ to inhibit FAT suggested that the molecular mechanism of inhibition is likely to be enzymatic (24) rather than a physical blockade. Abnormal phosphorylation of different neuronal cytoskeletal proteins is a characteristic hallmark in AD and our previous studies in isolated axoplasm have identified multiple kinase and phosphatase activities that can inhibit FAT (22, 24, 26, 28). A number of different phosphotransferase activities are altered in brains of AD patients, AD animal models, and cell lines exposed to Aβ. The list of abnormal kinase activities in AD is extensive, among them several unrelated serine/threonine protein kinases including GSK3 (23, 29, 30), p38 (31), JNK (31), cdk5 (32, 33), and CK2 (34). Interestingly, many of these enzymes are involved in regulation of FAT (28, 29).

Similarities in the profile of FAT inhibition produced by activation of several members of the stress-activated protein kinases (SAPK) and oAβ (24) led us to test the role of axonal SAPK in oAβ-induced FAT inhibition. Co-perfusion of homogeneous oAβ42 (Fig. 2A) with 5 μM SB203580, a well-characterized inhibitor for multiple SAPKs (24), did not prevent inhibition FAT by oAβ, making a role for endogenous SAPKs unlikely.

CK2 is another kinase that can inhibit both anterograde and retrograde FAT (28). Based on results showing that heterogeneous Aβ could directly stimulate CK2 kinase activity in vitro (35), the role of endogenous CK2 activation in oAβ-induced FAT inhibition was evaluated. 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) is a potent and highly specific ATP-competitive inhibitor of CK2 (36) derived from 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB). Co-perfusion of oAβ42with DMAT (Fig. 2B) blocked the oAβ-inhibitory effects on both directions of FAT entirely. A newly developed CK2 inhibitor also derived from TBB, tetrabromocinnamic acid (TBCA), has proved to be even more specific for CK2 (37), and TBCA also blocked inhibition of FAT by oAβ (Fig. 2C). Finally, a peptide substrate specific for CK2 was used as a competitive inhibitor of CK2 activity, and it also prevented inhibition of FAT by oAβ (Fig. 2D). Thus, selective inhibition of CK2 can prevent oAβ-induced inhibition of FAT.

Perfusion of oAβ Increased Phosphorylation of Squid Kinesin-1 Light Chains by Endogenous CK2 Activity.

To assess the ability of CK2 to phosphorylate a molecular motor for FAT, we evaluated the phosphorylation status of squid kinesin-1 after oAβ perfusion using the 63–90 monoclonal antibody against KLCs (38). The 63–90 antibody recognizes dephosphorylated KLCs (22, 23). KLCs in immunoprecipitates (IP) from murine brain were recognized by 63–90 (Fig. 3A, lane 1) and immunoreactivity increased after treatment with Antarctic phosphatase (Fig. 3A, lane 4). When kinesin-1 IP were treated with CK2, KLCs immunoreactivity is dramatically reduced (Fig. 3A, lanes 5 and 6), consistent with a CK2 phosphorylation site within the 63–90 epitope. When the extent of endogenous squid KLCs phosphorylation was evaluated by immnunoblot with 63–90 (Fig. 3B), KLC immunoreactivity was significantly reduced in axoplasms perfused with oAβ relative to uAβ-perfused axoplasms. These data suggest that oAβ activates endogenous CK2 activity, leading to a concomitant increase in squid KLC phosphorylation.

CK2 Mimics the Inhibitory Effect of oAβ on FAT by Directly Phosphorylating Kinesin-1.

If oAβ effects on FAT are caused by activation of CK2, then recombinant CK2 should have the same effect on FAT when perfused into axoplasm. Indeed, both directions of FAT were inhibited by CK2, and the inhibition seen with CK2 was qualitatively indistinguishable from the oAβ effects on FAT (Fig. 4A). In vitro kinase assays showed that both kinesin-1 heavy chain (KHC) and KLCs from murine brain are phosphorylated by recombinant CK2 (Fig. 4B), consistent with previous results (39). Together these experiments suggest that inhibition of FAT by oAβ is mediated by activation of endogenous CK2 and phosphorylation of kinesin-1.

oAβ-Induced FAT Inhibition Results from Kinesin-1 Release of Its Vesicular Cargoes.

Phosphorylation of kinesin-1 by various kinases can regulate its activities and affect anterograde FAT (22, 24, 28). Previous studies on phosphorylation of KLC at the 63–90 epitope suggest that this site regulates binding of kinesin-1 to vesicle cargoes (23, 40). To determine whether oAβ leads to release of kinesin-1 from cargoes, we isolated vesicle fractions by flotation assays from axoplasms treated for 50 minutes with either uAβ or oAβ, and evaluated the association of kinesin-1 with membrane fractions by immunoblot with the kinesin-1 KHC-specific antibody H2. A dramatic reduction of kinesin-1 immunoreactivity was seen on axonal vesicles from oAβ-perfused relative to uAβ-perfused axoplasms (Fig. 5, lanes 1 and 2). There was a comparable reduction of kinesin-1 immunoreactivity on axonal vesicles perfused with active CK2 relative to those perfused with CK2 buffer control (Fig. 5, lanes 3 and 4). An antibody to the integral membrane protein Trk (41) was used to verify the presence of vesicles and to serve as a loading control. Collectively, these results suggest that one mechanism by which oAβ mediates FAT inhibition is by increasing CK2 activity and phosphorylating KLCs, leading to release of kinesin-1 from vesicles.

Antibodies and Reagents.

The following antibodies were used: H2, a monoclonal antibody (mAb) against kinesin-1 heavy chains (51); 63–90, a monoclonal antibody (mAb) that preferentially recognizes dephosphorylated kinesin-1 light chains (22); and TrkB, a rabbit polyclonal antibody from Santa Cruz Biotechnology. Protein kinase inhibitors were obtained from Calbiochem, including SB 203580 and CK2 inhibitors (DMAT and TBCA). Inhibitors were diluted in DMSO or ethanol as appropriate and kept at −80 °C until use. CK2 substrate peptide was obtained from Sigma. Active CK2 and Antarctic Phosphatase were from New England Biolabs.

Preparation of Aβ42 Solutions.

For heterogeneous Aβ solutions, synthetic Aβ42 peptide (Bachem) was prepared as previously described (52). These heterogeneous Aβ solutions were perfused into extruded axoplasms isolated from the squid Loligo pealeii at 2-μM concentration. Vesicle motility was evaluated for 50 minutes after perfusion. For defined solutions of unaggregated, oligomeric, and fibrillar Aβ42 conformations, synthetic Aβ42 from California Peptide, Inc. were meticulously prepared as previously described (27). Solutions of defined Aβ42 conformations were perfused as above at 100 nM.

Atomic Force Microscopy.

Peptide solutions were characterized using a NanoScope IIIa scanning probe work station equipped with a MultiMode head using a vertical engage E-series piezoceramic scanner (Veeco, Santa Barbara, CA). AFM probes were single-crystal silicon microcantilevers with 300-kHz resonant frequency and 42 Newton/meter spring constant model OMCL-AC160TS-W2 (Olympus). A 20-μl quanity of sample solution (diluted from 100 μM to 10 μM (or 30 μM for fibrils) in deionized MilliQ water) was spotted on freshly cleaved mica, incubated at room temperature for 3 minutes, rinsed with 0.02 μm of filtered (Whatman Anotop 10) MilliQ water (Millipore), and blown dry with tetrafluoroethane (CleanTex MicroDuster III). For Aβ solutions in F12 media, the cleaved mica was pretreated with 3 μl of 1M HCl and rinsed before addition of sample. Some samples were imaged under dry helium. Image data were acquired at scan rates between 1 and 2 Hz with drive amplitude and contact force kept to a minimum. Data were processed to remove vertical offset between scan lines by applying zero order flattening polynomials using Nanoscope software (Version 5.31r1, Veeco).

Motility Studies in Isolated Axoplasm.

Axoplasms were extruded from giant axons of the squid Loligo pealeii provided by the Marine Biological Laboratory as previously described in detail (53). Vesicle motility was analyzed using a Zeiss Axiomat microscope equipped with DIC optics. Vesicle velocities were assessed as previously described (54).

Immunochemical Methods.

Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophorisis (SDS-PAGE) on 4–12% Bis-Tris gels (NuPage minigels, Invitrogen), using Mops Running Buffer (Invitrogen) and transferred to polyvinylidene fluoride (PVDF) membranes as previously described (52). Immunoblots were blocked with 5% nonfat dried milk, in phosphate-buffered saline, pH 7.4, and probed with different polyclonal and monoclonal antibodies. Primary antibody binding was detected with horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibody (Jackson Immunoresearch) and visualized by chemiluminescence (ECL, Amersham). Kinesin immunoprecipitation was performed from murine brain by our published procedure (55). Isolation of membrane vesicle fractions from axoplasms was as previously described (40). Two axoplasms from the same animal were prepared and incubated with appropriate effectors (CK2 buffer, active CK2, uAβ, or oAβ) and vesicle fractions evaluated by immunoblot using H2 and Trk antibodies. Trk served as protein loading control and vesicle fraction marker.

In Vitro Kinesin-1 Phosphorylation.

Immunoprecipitated kinesin-1 was either phosphorylated by CK2 (250 U) or was previously dephosphorylated by Antartic Phosphatase (15 U) following NEB protocols and then phosphorylated by CK2. Kinase reactions were started by addition of 100 μM of radiolabeled ATP. After 1 hour at 30 °C, reactions were stopped by addition of 60 μl sample buffer. Proteins were separated by SDS-PAGE and the gels dried and exposed to a phosphoroimager screen for analysis (55).

Statistical Analysis.

All experiments were repeated at least three times. Unless otherwise stated, the data were analyzed by two-sample t test for pairwise comparisons or analysis of variance followed by a post hoc Student-Newman-Keul's test to make all possible comparisons. Data were expressed as mean ± SEM; significance was assessed at P < 0.05 or 0.01 as noted.