Alzheimer's presenilin 1 mutations impair kinesin-based axonal transport - PubMed (original) (raw)

Alzheimer's presenilin 1 mutations impair kinesin-based axonal transport

Gustavo Pigino et al. J Neurosci. 2003.

Abstract

Several lines of evidence indicate that alterations in axonal transport play a critical role in Alzheimer's disease (AD) neuropathology, but the molecular mechanisms that control this process are not understood fully. Recent work indicates that presenilin 1 (PS1) interacts with glycogen synthase kinase 3beta (GSK3beta). In vivo, GSK3beta phosphorylates kinesin light chains (KLC) and causes the release of kinesin-I from membrane-bound organelles (MBOs), leading to a reduction in kinesin-I driven motility (Morfini et al., 2002b). To characterize a potential role for PS1 in the regulation of kinesin-based axonal transport, we used PS1-/- and PS1 knock-inM146V (KIM146V) mice and cultured cells. We show that relative levels of GSK3beta activity were increased in cells either in the presence of mutant PS1 or in the absence of PS1 (PS1-/-). Concomitant with increased GSK3beta activity, relative levels of KLC phosphorylation were increased, and the amount of kinesin-I bound to MBOs was reduced. Consistent with a deficit in kinesin-I-mediated fast axonal transport, densities of synaptophysin- and syntaxin-I-containing vesicles and mitochondria were reduced in neuritic processes of KIM146V hippocampal neurons. Similarly, we found reduced levels of PS1, amyloid precursor protein, and synaptophysin in sciatic nerves of KIM146V mice. Thus PS1 appears to modulate GSK3beta activity and the release of kinesin-I from MBOs at sites of vesicle delivery and membrane insertion. These findings suggest that mutations in PS1 may compromise neuronal function by affecting GSK-3 activity and kinesin-I-based motility.

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Figures

Figure 1.

Figure 1.

Endogenous PS1 and GSK3β exhibit similar expression profiles during neuronal development, partially colocalize in the same vesicular compartment, and coimmunoprecipitate from brain homogenates. A, Western blot analysis of MBO and soluble fractions during development of hippocampal neurons in culture at 1, 3, and 10 d in vitro (DIV). The figure shows samples of three different homogenates obtained at each time point. Note that the expression pattern is similar for PS1 and GSK3β. Both proteins are highly enriched in the MBO fraction, particularly at 10 DIV. PS1 FL, Full-length PS1; PS1 CTF, C-terminus fragment of PS1. The antibodies include anti-GSK3β (334–348), 1:1000, and anti-PS1 (AD3L), 1:2000. B, PS1 and GSK3β colocalize on a subpopulation of MBOs and are enriched in a growth cone fraction. The Western blots show the localization of PS1, GSK3β, and kinesin-I heavy (KHC) and light (KLC) chains in three vesicle fractions (V0, V1, and V2) and supernatant (Cyt). Significant amounts of PS1 and GSK3β colocalize in V1 where kinesin-I is enriched. PS1 and GSK3β are highly enriched in growth cone fractions (GCs). PS1 NTF, N-terminus fragment of PS1. The antibodies include anti-PS1 (PSN2), 1:1000; anti-KHC (H2), 1:2000; anti-KLC (63–90), 1:2000; and anti-KLC ALL, 1:2000. C, Coimmunoprecipitation of PS1 and GSK3β from Cos cell lysates cotransfected with PS1 and GSK3β expression vectors (Cos Lys) and mouse brain homogenates (Brain Lys). A sample of transfected Cos cell lysates was included as a positive control for GSK3β staining (Lys). The Cos cell lysate that was immunoprecipitated with anti-PS1 antibody (Anti-PS1) shows immunoreactivity with anti-GSK3β in the Western blot. Similarly, anti-PS1 immunoprecipitate from mouse brain homogenate shows immunoreactivity for GSK3β. No GSK3β immunoreactivity is observed in immunoprecipitates for nonimmune mouse IgG (Ctrl IgG) or protein A-Sepharose beads alone (Beads). Immunoprecipitations were performed with anti-PS1 antibody PSN2. Western blot was revealed with anti-GSK3β (Calbiochem).

Figure 2.

Figure 2.

Endogenous membrane-associated PS1 and GSK3β colocalize in cultured neurons. A, B, Double-immunofluorescence staining of a 2 DIV hippocampal neuron stained with anti-GSK3β (A) and anti-PS1 (B) antibodies. Cultures were extracted before fixation to retain selectively the membrane-associated proteins (see Materials and Methods). PS1 and GSK3β are enriched in growth cones of developing neuritic processes (arrows). The antibodies include anti-PS1 (PSN2), 1:100, and anti-total GSK3β (334–348), 1:400. Scale bar, 20 μm. C–E, Confocal images showing colocalization of PS1 and GSK3β in the growth cone central region and filopodia (yellow in merged image E; arrows). F, The degree of colocalization between membrane-associated GSK3β and PS1 in the confocal image pair from C, D was assessed by ratio image analysis and was pseudocolored according to the degree of colocalization of both fluorescent signals. White denotes the areas of highest immunoreactivity overlap.

Figure 3.

Figure 3.

Increased activation of GSK3β in PS1 -/- and PS1 KI M146V fibroblasts and neuronal cells expressing PS1 mutations M146V, I143T, and D9. A, Protein expression analysis of PS1 wild-type (WT), PS1 -/- (KO), and PS1 KI M146V (KI) fibroblasts. The figure shows samples of two different WT, KO, and KI cultures. Reduced levels of GSK3β-Pser9 (inactive form) were detected in PS1 KO and KI as compared with WT fibroblasts. No changes in the expression levels of total GSK3β, kinesin-I heavy chain (KHC), and tubulin were observed. Note the absence of PS1 in KO fibroblast and similar levels of PS1 expression in WT and KI fibroblasts. B, NT2 neuronal cells were transfected with PS1 WT and PS1 mutant M143T, M146V, and D9 constructs. No significant changes were detected in total GSK3β (GSK3β total) and tubulin levels (data not shown). The histogram represents the amount of GSK3β-Pser9 expressed as a percentage of the level in control cells (transfected with GFP). Values are the mean ± SE; n = 3 independent experiments. *p < 0.02 relative to GFP by Student's t test. Transfection and quantitative Western blot analysis were performed as described in Materials and Methods. C, Expression of PS1 mutations does not affect cell viability. Sister cultures to the ones used for the experiment shown in B were used to assess cell viability. The results showed no changes in cell survival associated with the expression of PS1 mutations. Viability was assessed by using a propidium iodide exclusion assay as described in Materials and Methods. At least 200 cells were scored per culture in triplicate cultures. The histogram represents the mean ± SE.

Figure 4.

Figure 4.

Enhanced KLC phosphorylation in PS1 KI M146V hippocampal neurons and mouse brain. A, The 63–90 epitope on kinesin-I light chains (KLC) is sensitive to phosphorylation. V1 vesicle fractions were incubated without (Control) and with ATP. The samples were separated in an 8% gel and transferred; immunoblots were prepared with the H2 (kinesin-I heavy chain) and 63–90 antibodies. Notice the increase in relative molecular weight of KLCs after incubation with ATP. This shift is caused by the phosphorylation of KLCs by vesicle-associated kinase(s) and correlates with the detachment of kinesin-I from membranes (Morfini et al., 2002b). Immunoreactivity with 63–90, but not H2, antibody is diminished after the incubation of vesicles with ATP. When transfer membranes are dephosphorylated with alkaline phosphatase (+AP), 63–90 immnunoreactivity is recovered, indicating that the epitope for the Ab 63–90 is sensitive to phosphorylation. The same fractions were separated in a 4–20% gel, and Western blot was performed with the anti-KLC ALL antibody, which recognizes KLC irrespective of its phosphorylation state. Equal amounts of KLC were observed in all samples. B, C, Western blot analysis of WT and PS1 KI M146V total brain homogenates and cortical neuronal cultures. Samples from two WT and two KI brains and cortical cultures are shown. Note the reduction in KLCs staining with Ab 63–90 in PS1 KI M146V brain and cortical culture samples, indicating increased phosphorylation of KLCs in PS1 KI brain and cortical culture homogenates. Similar protein levels of KHC that have been detected with H2 antibody, which recognizes KHC, rule out changes in kinesin-I expression in PS1 KI mouse brain tissue. Similar levels of tubulin and GSK3β rule out general changes in protein expression. Similar levels of KLC immunoreactivity with KLC (ALL) antibody also are observed in cortical cultures. D, Western blot analysis of WT, PS1 KI M146V, and PS1 KO fibroblasts. Note the reduction in Ab 63–90 immunoreactivity. E, Kinesin-I release assay. V1 vesicles were incubated with 1 m

m

ATP for 30 min at 37°C and centrifuged at 120,000 × g; pellet (P) and supernatant (S) fractions were analyzed by Western blot. After incubation with ATP (+) the KLC and KHC are released from the vesicle fraction (P) and found in the supernatant while the vesicle markers synaptophysin (Syn) syntaxin-I (Synt) and APP remain in the vesicle fraction (P). Omission of ATP (-) results in the complete recovery of kinesin-I with the pellet fraction. Note the reduction in Ab 63–90 immunoreactivity after kinesin-I light chain phosphorylation.

Figure 5.

Figure 5.

PS1 mutations enhance the release of kinesin-I from MBO-enriched fractions. A, Western blot analysis of KHC and KLC in MBO-enriched fractions from PS1 WT, PS1 KO, and PS1 KI M146V fibroblasts. KHC and KLC were detected with antibodies H2 and 63–90, respectively. Samples of two different cultures were assayed. Note the reduction in KHC and KLC in PS1 KI and KO as compared with WT fibroblasts. Similar tubulin levels were observed in all samples. These results suggest a reduced amount of kinesin-I associated with MBOs in KI and KO fibroblasts. B, Western blot shows reduced levels of both kinesin-I heavy and light chains (KHC and KLC) in MBO fractions prepared from NT2 neuronal cell cultures expressing the PS1 KI M146V mutation. Similar tubulin levels were detected in PS1 WT and M146V. KHC and KLC were detected with antibodies H2 and 63–90, respectively. The histogram shows the result of the quantification of KHC levels in MBO-enriched fractions prepared from transfected cultures expressing PS1 WT and PS1 mutants M143T, M146V, and D9. The amount of KHC in MBO-enriched fractions was expressed as a percentage of the level in control cells (transfected with GFP). Values are the mean ± SE; n = 3 independent experiments. *p < 0.01 relative to GFP by Student's t test. KHCs were detected with antibody H2; similar results were obtained for KLCs (data not shown). Tubulin levels did not change significantly among samples. MBO fractions, transfection, and quantitative Western blot analysis were performed as described in Materials and Methods.

Figure 6.

Figure 6.

Reduced density of synaptic vesicles in hippocampal neurons expressing PS1 mutations. A, Immunofluorescence with anti-synaptophysin (Syn) and anti syntaxin-I (Synt) antibodies of PS1 WT and KI hippocampal neurons at 4 d in culture. Note the reduced number of vesicle clusters (arrows) in KI processes. The gray scale in the images was inverted for clarity. Scale bar, 20 μm. B, Reduced density of synaptophysin-containing vesicles in PS1 KI M146V hippocampal neurons. WT and PS1 KI M146V hippocampal cultures were fixed and double-immunostained with anti-tubulin class III and anti-synaptophysin antibodies. The density of synaptophysin-immunoreactive vesicles was assessed as described (Grace et al., 2002). The numbers represent the mean ± SE; n = 4 independent experiments. *p < 0.05 relative to WT by Student's t test. C, WT and PS1 KI M146V hippocampal neurons exhibit similar viability. Neuronal viability was assessed at 4 DIV by a propidium iodide exclusion assay. No significant differences in the number of viable neurons were detected between WT and PS1 KI M146V cultures. At least 200 neurons were scored per culture in triplicate cultures. The numbers represent the mean ± SE. D, WT and PS1 KI M146V hippocampal neurons exhibit similar caspase activities. No significant differences in caspase activity were observed between WT and PS1 KI M146V cultures. The activity of caspases 2, 3, 6, 8, and 9 was measured in triplicate cultures. Caspase activity in PS1 KI M146V cultures was expressed as a percentage after the activity of each caspase in WT cultures had been normalized as 100. E, A significant reduction in synaptophysin-immunoreactive vesicles was observed in hippocampal neurons expressing PS1 mutations. Transfected neurons were identified by cotransfection with a GFP expression vector (Pigino et al., 2001). Images of transfected neurons were captured at a final magnification of 400×, and the number of synaptophysin-immunoreactive dots was scored. At least 30 cells were analyzed per experimental condition. The numbers represent the mean ± SE; n = 3 independent experiments. *p < 0.02 relative to control (GFP) by Student's t test.

Figure 7.

Figure 7.

Reduced levels of synaptic proteins in sciatic nerves of PS1 KI M146V mice. Sciatic nerves and spinal cord segments from two WT and two KI animals were dissected and homogenized. Protein (15 mg) was loaded in each lane. Western blots were developed with the indicated antibodies. Note the significant reduction in PS1, APP, and synaptophysin (Syn) in sciatic nerves, but not in the spinal cord, of PS1 KI mice. A reduction in GSK3β-Pser9 and 63–90 immunoreactivity is evident in both sciatic nerve and spinal cord samples from KI mice. No significant changes between PS1 WT and KI samples were detected in the level of total GSK3β, KHC, SNAP25, or tubulin.

Figure 8.

Figure 8.

Reduced mitochondrial density in neuritic processes of PS1 KI M146V hippocampal neurons. A–D, Immunofluorescence of PS1 WT and KI M146V (KI) hippocampal neurons stained with an antibody against cytochrome c. Merged images of phase contrast and fluorescence are shown. The gray scale in the images was inverted for clarity. A, B, Representative WT and KI hippocampal neurons fixed at 2 DIV. Note the presence of mitochondria in cell bodies and neuritic processes. C, D, Higher magnification images of neuritic processes. Note the presence of mitochondria along the processes. Scale bars: (in B) A, B, 10 μm; (in D) C, D, 5 μm. E, Assessment of mitochondrial density in neuritic processes and cell bodies of PS1 WT and KI hippocampal neurons. A significant decrease in mitochondrial density was observed in neuronal processes, but not in cell bodies, of KI neurons. PS1 WT and KI hippocampal neurons were fixed at day 2 in culture. To visualize mitochondria, we immunostained the cultures with a monoclonal antibody specific for cytochrome c (1:500), an electron-transporting mitochondrial resident protein. Mitochondrial density in neurites and cell bodies was assessed by image analysis. The images were captured at a final magnification of 400 ×, and the numbers of mitochondria were scored in the cell body and neuritic processes of randomly selected neurons as described in Materials and Methods. At least 20 cells were analyzed per experimental condition. The numbers represent the mean ± SE; n = 3 independent experiments. *p < 0.01 relative to WT by Student's t test.

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