Conceived and designed the experiments: KIA KI. Performed the experiments: KIA SAH CS AG LZ KI. Analyzed the data: KIA SAH KI. Contributed reagents/materials/analysis tools: KIA KI. Wrote the paper: KIA KI.
The authors have declared that no competing interests exist.
The amyloid-β 42 (Aβ42) is thought to play a central role in the pathogenesis of Alzheimer's disease (AD). However, the molecular mechanisms by which Aβ42 induces neuronal dysfunction and degeneration remain elusive. Mitochondrial dysfunctions are implicated in AD brains. Whether mitochondrial dysfunctions are merely a consequence of AD pathology, or are early seminal events in AD pathogenesis remains to be determined. Here, we show that Aβ42 induces mitochondrial mislocalization, which contributes to Aβ42-induced neuronal dysfunction in a transgenic
Alzheimer's disease (AD) is a progressive neurodegenerative disease without effective therapies. Pathologically, AD is defined by an extensive loss of neurons and by formation of two characteristic protein deposits, extracellular amyloid plaques (APs) and intracellular neurofibrillary tangles (NFTs). The major components of APs and NFTs are the 40 or 42 amino acid amyloid-β peptides (Aβ40 or Aβ42) and the hyperphosphorylated microtubule associated protein tau, respectively
Molecular genetic studies of early-onset familial AD patients have identified causative mutations in genes encoding APP and presenilins (PS1 and PS2), and these mutations increase Aβ42 production and/or Aβ aggregation
Several lines of evidence indicate that mitochondrial function is impaired in the brains of AD patients
In order to identify genes and pathways that are involved in Aβ42-induced toxicity
Using this
Using mito-GFP transgene, a reporter construct in which GFP is fused to a mitochondrial targeting signal
(A) A schematic view of the mushroom body. (B) Signal intensities of mito-GFP in axon bundle tips, dendrites, and cell bodies of the mushroom body structure in control and Aβ42 flies at 5 days after eclosion (dae) and 21 dae. Ratios relative to control are shown (mean±SD, n = 6–10; *, p<0.05, Student's t-test). Representative images at 21 dae are shown at the top. (C) Signal intensities of mito-GFP in the central complex, focusing on the ellipsoid body, a circular neuropil region, in control and Aβ42 fly brains at 21 dae. mito-GFP signals are shown as ratios relative to control (mean±SD, n = 3–5; *, p<0.05, Student's t-test). (D) Signal intensities of CD8-GFP in axon bundle tips and dendrites in control and Aβ42 fly lines at 21 dae are shown as ratios relative to control (mean±SD, n = 6–10). (E) Signal intensities of tubulin-GFP (tub-GFP) in axon bundle tips and dendrites in controls and Aβ42 fly lines at 26 dae are shown as ratios relative to control (mean±SD, n = 6–10). (F) Signal intensities of synaptotagmin-GFP (syt-GFP) in axon bundle tips and cell bodies in controls and Aβ42 fly lines at 24 dae are shown as ratios relative to control (mean±SD, n = 6–10). (C–E) No significant difference was detected between control and Aβ42 fly brains (p>0.05, Student's t-test). Male flies with the pan-neuronal elav-GAL4 driver were used for all experiments shown in
The mito-GFP signal in the axons and dendrites of the mushroom body structure was significantly decreased in the Aβ42 fly brains (
Mitochondrial mislocalization observed in the Aβ42 fly brains is not due to overexpression of exogeneous protein, since neuronal expression human α-synuclein
The reduction in mitochondria in the axons and dendrites is unlikely to be due to degeneration of the mushroom body structure, since neurodegeneration in the Aβ42 fly brain is not prominent at 5 dae
Mitochondria are transported along microtubules by the motor proteins. To test whether Aβ42-induced mitochondrial mislocalization is due to an overall disruption of microtubule-based transport in neurons, we analyzed distributions of tubulin fused to GFP (tub-GFP) and the presynaptic protein synaptotagmin fused to GFP (syt-GFP) in axons and dendrites. Aβ42 expression did not result in any significant difference in the distributions of tub-GFP in axons and dendrites (
Mitochondrial damage and dysfunction have been shown to alter mitochondrial localization. We examined whether Aβ42 caused severe mitochondrial damage at the ages at which we observed mitochondrial mislocalization. We compared the amount of mitochondrial genomes and the levels of ATP in the brains dissected from control and Aβ42 flies, and found that they were not significantly different (
(A) Quantitation of mitochondrial DNA in the brains dissected from Aβ42 and control flies at 5 dae using quantitative Real Time-PCR. DNA levels are shown as ratios relative to control (mean±SD, n = 6). No significant difference was detected (p>0.05, Student's t-test). Co I, cytochrome c oxidase subunit I; Co III, cytochrome c oxidase subunit III, Cyt B, cytochrome
Apoptosis can cause mitochondrial fragmentation and fission/fusion defects, which can result in mitochondrial mislocalization
To test whether mitochondrial mislocalization contributes to Aβ42 toxicity, we examined the effect of a genetic reduction in mitochondrial transport on Aβ42-induced locomotor defects. Aβ42 flies show age-dependent, progressive locomotor dysfunction starting from 14 dae, which can be detected by climbing assay
(A) Neuronal expression of the RNAi transgene reduces milton mRNA levels. The milton mRNA levels in heads were quantified by qRT-PCR (n = 6; *p<0.01, Student's t-test). (B) Neuronal expression of the RNAi transgene reduces milton protein levels. The milton protein levels in brans were quantified by Western blotting with anti-
A heterozygous
cAMP is generated from ATP, and depletion of mitochondria in axons has been shown to disrupt cAMP/PKA signaling, which limits mobilization of the synaptic vesicle reserve pool in presynaptic terminals, and reduces synaptic strength
(A) Early onset of Aβ42-induced locomotor defects in the
Next, we tested whether an increase in the cAMP level by a genetic reduction of the
Since PKA activity is regulated by cAMP levels, we examined whether PKA activity is involved in Aβ42-induced toxicity. Knockdown of the catalytic subunit of PKA (PKA-C1) in neurons using UAS-PKA-C1-RNAi driven by the pan-neuronal elav-GAL4 driver enhanced Aβ42-induced locomotor defects, while neuronal knockdown of PKA-C1 by itself did not cause locomotor defects at this stage (
PKA activity is suppressed by binding of the regulatory subunits (PKA-R) to the catalytic subunit, and overexpression of PKA-R decreases, while knockdown of PKA-R increases, PKA activity. The transgenic fly lines EP2162 and EY11550 overexpress PKA-R2 in neurons when combined with the pan-neuronal elav-GAL4 driver. We found that neuronal overexpression of PKA-R2 significantly enhanced Aβ42-induced locomotor defects, while overexpression of PKA-R2 by itself did not affect locomotor function (
We further examined the effects of a reduction in neuronal PKA-R2 expression on Aβ42-induced locomotor dysfunctions. Knockdown of PKA-R2 in neurons using an RNAi transgene with the pan-neuronal elav-GAL4 driver suppressed the locomotor defects in Aβ42 flies, while PKA-R2 knockdown by itself did not affect locomotor function (
Because rut, dnc, and the PKA complex is enriched in the axons and dendrites in fly neurons
Neuronal knock-down of PKA-C1 or PKA-R2 did not affect the accumulation of Aβ42 (
To test whether PKA activity is reduced in the Aβ42 fly brain, we compared PKA-C1 and PKA-R2 protein levels and total PKA activity in extracts from dissected brains from Aβ42 and control flies. These parameters were not significantly different (
(A) Total protein levels of PKA-C1 (Left) or PKA-R2 (Right) in Aβ42 fly brains. Western blot using anti-PKA-C1 or anti-PKA-R2. Signal intensities were quantified and are shown as ratios relative to control (mean±SD, n = 5; p>0.05, Student's t-test). (B) PKA activity in brain extracts from control or Aβ42 flies at 21 dae. The PKA activity was normalized to the protein level and is shown as a ratio relative to control. No significant differences were observed (n = 4; p>0.05, Student's t-test). (C) Whole-mount immunostaining of brains of control and Aβ42 flies at 14dae using anti-PKA-C1 (green). (D) cAMP in head extracts from control flies and Aβ42 fly lines at 25 dae. The amount of cAMP was normalized to total protein. No significant differences were observed (n = 6; p>0.05, Student's t-test). A representative standard curve and control experiment are shown in
To further investigate whether the levels of putative PKA substrate phosphoproteins are reduced in the Aβ42 fly brain, we performed Western blot analysis using a phospho-PKA substrate antibody (anti-RRxpS/T). We first identified the signals whose reductions were correlated with the decreased PKA activity in the dissected fly brains. Neuronal knockdown of PKA-C1 markedly reduced the signal intensities of phosphoproteins migrating at 24 kDa and 38 kDa (
The signals of the 24 kDa and 38 kDa phosphoproteins were significantly reduced in the brains dissected from three independent Aβ42 fly lines (
In mammals, PKA activates cAMP-response element binding protein (CREB) via direct phosphorylation at Ser133, and
(A) Anti-RRxpS/T detects phosphorylation of
We have shown that mitochondria are reduced in the axons and dendrites in the Aβ42 fly brain (
(A) Age-dependent locomotor defects in the flies with a neuronal knockdown of milton using UAS-milton RNAi transgenic fly lines. Asterisks indicate the significant difference in the percentage of the flies stayed at the bottom (mean±SD, n = 5, p<0.05, Student's t-test). (B) The signal intensities of the 24 kDa and 38 kDa (arrows) proteins, but not the 30 kDa (arrowhead) protein, were reduced in brain extracts of flies with a neuronal knockdown of milton at 8 dae. The signal intensities were normalized to the tubulin level and are shown as ratios relative to controls. Asterisks indicate significant differences from control (n = 4; *, p<0.05, Student's t-test). The pan-neuronal elav-GAL4 driver was used. Since both the elav-GAL4 and UAS-milton-RNAi are on X chromosome, female flies were used in the left panel in A and B. Male flies with the elav-GAL4 driver were used in the right panel in A.
Elucidation of mechanisms underlying Aβ42-induced toxicities is crucial to understanding the complex pathogenesis of AD. An altered distribution of mitochondria has been reported in the brains of AD patients and in cellular and animal models of Aβ toxicity
In Aβ42 fly brains, Aβ42 is accumulated intraneuronally and extracellulary
Some reports have shown that Aβ is present within mitochondria and induces mitochondrial damage
In the Aβ42 fly brain, mitochondrial mislocalization occurred without severe mitochondrial damage (
In neurons, mitochondria undergo fission perinuclearly in the cell body and are transported along microtubule or actin bundles
Mitochondrial transport is regulated by several intracellular signals. Elevation of intracellular Ca2+, which occurs in regions of high metabolic demand such as nerve terminals and postsynaptic specializations, arrests microtubule-based mitochondrial movement. Mitochondria are linked to motors by the mitochondrial membrane GTPase Miro
The motility of mitochondria is thought to be interrelated with the fission-fusion machinery and an imbalance between mitochondrial fission and fusion induced by Aβ42 may result in reduced mitochondria in the axons and dendrites
Impaired regulation of the cAMP/PKA pathway has been reported in the brains of AD patients. Decreases in levels of specific adenylyl cyclase (AC) isoforms and disruption of AC/cAMP signal transduction have been detected in AD brains
Our results suggest that Aβ42-induced mitochondrial mislocalization causes local, but not global, alterations in cAMP/PKA activity, such as in the axons and dendrites.
In addition to cAMP/PKA signaling, the loss of ATP caused by the reduction of mitochondria in neurites could disrupt many biological processes and lead to neuronal dysfunction. Other major functions of mitochondria in neurons includes the regulation of Ca2+, which is important for synaptic plasticity, and cell survival
Our study demonstrates that mislocalization of mitochondria underlies Aβ42-induced toxicity
Transgenic fly lines carrying the human Aβ42 was established in the background of the Canton-S
Fly brains were dissected in cold PBS, fixed in PBS containing 4% paraformaldehyde (Electron Microscopy Sciences), and then placed under vacuum in PBS containing 4% paraformaldehyde and 0.25% Triton X-100. The fluorescence intensity in the mushroom body regions was analyzed using a confocal microscope (Carl Zeiss LSM 510) and quantified using NIH image.
Fly brains were dissected in cold PBS and frozen on dry ice, and genomic DNA was extracted. 20 brains were homogenated in 100 mM Tris-HCl pH 7.5, 100 mM EDTA, 100 mM NaCl, and 0.5% SDS, and incubate at 65°C for 30 min. Samples were treated with 1.5 M potassium acetate and 4 M LiCl, and incubated for 65°C for 30 min, and centrifuged. Supernatant was treated was phenol/chloroform, added isoprophanol, and centrifuged. Precipitated gemonic DNA was rinsed with 70% ethanol and subjected to quantitative real time-PCR (Applied Biosystems). The average threshold cycle value (Ct) was calculated from five replicates per sample. Levels of Co I, Co III and CytB DNA were standardized relative to that of rp49. Relative expression values were determined by the deltaCt method according to quantitative PCR Analysis User Bulletin (Applied Biosystems). Primers were designed using NIH primer blast as follows: Co I,
ATP contents in dissected brains without eye pigments were analyzed using ATP Bioluminescence Assay Kit CLSII (Roche, Mannheim). PKA activity in dissected brains without eye pigments was measured with MESACUP Protein Kinase Assay Kit (MBL, Woburn, MA) in the presence or absence of 2 µM cAMP. cAMP levels was measured with cAMP-screen system (Applied Biosystems, Foster City, CA). ATP, PKA and cAMP levels were calculated by standard curves and normalized by protein levels.
Probosces were removed from decapitated heads, which were then immersion-fixed overnight in 4% glutaraldehyde and 2% paraformaldehyde in 0.1 M PBS. Samples were post-fixed 1 hr in ferrocyanide-reduced osmium tetroxide (1% osmium tetroxide and 1.5% potassium ferrocyanide in distilled water). Fixation was followed by dehydration in a graded ethanol series and infiltration with Epon-Araldite resin (2 hr in 50% resin in acetone and 24 hr in 100% resin) using constant rotation. After transferring the samples to flat-bottom BEEM capsules with fresh resin, the samples were polymerized overnight at 60°C. Cured blocks containing fly heads were examined with a dissection microscope and heads with a suitable orientation (posterior oriented flat to the block surface) were selected for thin sectioning. Semi thin sections stained with toluidine blue were examined by light microscopy to localize the mushroom body region. Thin sections (120 nm) of entire heads were collected on nickel grids (100 mesh, Veco-EMS). Thin sections were stained for 5 minutes in lead citrate stain. Sections were examined and micrographs collected using a Hitachi H700T TEM.
Fly brains were fixed in PBS containing 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), treated with 25 µg/ml proteinase K for 30 min, and incubated with In Situ Cell Death Detection Kit, Fluorescein (Roche, Mannheim) for 1 hr at 37°C. The brains were analyzed using a confocal microscope (Carl Zeiss LSM 510).
For each sample, 30–40 flies were collected and frozen. Heads were mechanically isolated, and total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's protocol with an additional centrifugation step (11,000×g for 10 min) to remove cuticle membranes prior to the addition of chloroform. Total RNA was reverse-transcribed using Superscript II reverse transcriptase (Invitrogen), and the resulting cDNA was used as a template for PCR on a 7500 fast real time PCR system (Applied Biosystems). The average threshold cycle value (Ct) was calculated from five replicates per sample. Expression of milton was standardized relative to actin. Relative expression values were determined by the deltaCt method according to quantitative PCR Analysis User Bulletin (Applied Biosystems). Primers were designed using NIH primer blast as follows: milton,
Dissected brains were homogenized in Tris-glycine sample buffer (Invitrogen) and centrifuged at 13,000 rpm for 10 min, and the supernatants were separated on 6% or 10% Tris-glycine gels (Invitrogen) and transferred to nitrocellulose membranes (Invitrogen). The membranes were blocked with 5% nonfat dry milk (Nestlé) and blotted with the primary antibody (anti-
Climbing assay was performed as previously described
Fly brains were dissected in cold PBS, fixed in PBS containing 4% paraformaldehyde (Electron Microscopy Sciences), and then placed under vacuum in PBS containing 4% paraformaldehyde and 0.25% Triton X-100. After permeabilization with PBS containing 2% Triton X-100, the brains were stained with rabbit polyclonal anti-PKA-C1 antibody (a gift from Dr. D. Kalderon) followed by detection with biotin-XX goat anti-mouse IgG and streptavidin-Texas Red conjugate (Molecular Probes). The brains were analyzed using a confocal microscope (Carl Zeiss LSM 510).
For sequential extractions of Aβ42, fly heads were homogenized in RIPA buffer (50 mM Tris-HCl, pH 8.0, 0.5% sodium deoxycholate, 1% Triton X-100, 150 mM NaCl) containing 1% SDS. Lysates were centrifuged at 100,000×g for 1 h, and supernatants were collected (SDS-soluble fraction). SDS-insoluble pellets were further homogenized in 70% formic acid (Sigma) followed by centrifugation at 13,000 rpm for 20 min, and the supernatants were collected (formic acid fraction). Formic acid was evaporated by SpeedVac (Savant, SC100), and protein was resuspended in dimethyl sulfoxide (Sigma). Protein extracts were separated on 10–20% Tris-Tricine gels (Invitrogen) and transferred to nitrocellulose membranes. The membranes were boiled in phosphate-buffered saline (PBS) for 3 min, blocked with 5% nonfat dry milk, blotted with the 6E10 antibody (Signet), incubated with appropriate secondary antibody and developed using ECL plus Western Blotting Detection Reagents (GE Healthcare).
For thioflavin S (TS) staining, the dissected brains were permeabilized and incubated in 50% EtOH containing 0.1% TS (Sigma) overnight. After washing in 50% EtOH and PBS, the brains were analyzed using a confocal microscope. The numbers of TS-positive deposits were quantified from four hemispheres from three flies per genotype. The fluorescence intensity in Kenyon cell regions was analyzed using a confocal microscope (Carl Zeiss LSM 510) and quantified using NIH image.
For the analysis of neurodegeneration in Kenyon cell region, heads were fixed in 4% paraformaldehyde, processed to embed in paraffin blocks, and sectioned at a thickness of 6 µm. Sections were placed on slides, stained with hematoxylin and eosin (Vector Laboratories), and examined by bright field microscopy. To quantify neurodegeneration, images of the sections were captured, and the areas of the vacuoles were measured using NIH Image.
Mitochondria are mislocalized in cholinergic neurons in the Aβ42 fly brain. Mito-GFP in axon bundle tips, dendrites, and cell bodies of cholinergic neurons in the mushroom body in control and Aβ42 fly brains. The Cha-GAL4 driver was used to express transgene in cholinergic neurons. Signal intensities in control and Aβ42 flies at 35 dae were quantified and are shown as ratios relative to control (mean ± SD, n = 6–10; *, p<0.05, Student's t-test). Representative images are shown at the top. Male flies were used.
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α-synuclein did not cause significant alteration of mitochondria localization in the fly brain. Mito-GFP in axon bundle tips, dendrites, and cell bodies in the mushroom body in control and α-synuclein fly brains. Transgene expression was driven by the pan-neuronal elav-GAL4 driver. Signal intensities in control and α-synuclein flies at 20 dae were quantified and are shown as ratios relative to control (mean ± SD, n = 6–10; *, p<0.05, Student's t-test). Representative images are shown at the top. Male flies were used.
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Modification of Aβ42-induced locomotor defects by PKA activity was confirmed in an independent Aβ42 transgenic line. (A) Enhancement of Aβ42-induced locomotor defects by neuronal knockdown of PKA-C1 (Aβ42+PKA-C1 RNAi). (B) Enhancement of Aβ42-induced locomotor defects by overexpression of PKA-R2. (C) Suppression of Aβ42-induced locomotor defects by neuronal knockdown of PKA-R2 (PKA-R2 RNAi). Transgene expression was driven by the pan-neuronal elav-GAL4 driver. The average percentage of flies at the top (white), middle (light gray), or bottom (dark gray) of the assay vials is shown (mean ± SD, n = 5). Asterisks indicate the significant difference in the percentage of the flies stayed at the bottom (p<0.05, Student's t-test). Male flies were used.
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Accumulation of Aβ42 was not affected by neuronal knockdown of PKA-C1 or PKA-R2 in fly brains. The effect of neuronal knockdown of PKA-C1 (A) or PKA-R2 (B) on Aβ42 accumulation in fly brains. Transgene expression was driven by the pan-neuronal elav-GAL4 driver. Aβ42 in brains from flies at 25 dae in the detergent soluble (RIPA/1%SDS) or insoluble (70%FA) fraction was detected by Western blotting. Aβ42 levels were normalized to tubulin levels and are shown as ratios relative to controls. Representative blots are shown on the left, and means ± SD are plotted on the right. No significant differences were detected (n = 5; p>0.05, Student's t-test). Male flies were used.
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The number of Thioflavin S-positive Aβ42-deposits was not affected by neuronal knockdown of PKA-C1. The effect of neuronal knockdown of PKA-C1 on the formation of Aβ42-deposits. Thioflavin S (TS) staining of Kenyon cell body regions of the brain of flies expressing Aβ42 in the presence or absence of a PKA-C1 knockdown at 25 dae. Aβ42 expression was driven by the pan-neuronal elav-GAL4 driver. The numbers of TS-positive deposits in the Kenyon cell body regions are presented as the mean ± SD. No significant difference was detected (n = 4; p>0.05, Student's t-test). Male flies were used.
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Aβ42-induced neurodegeneration is not affected by neuronal knockdown of PKA-C1 or PKA-R2. The effect of neuronal knockdown of PKA-C1 or PKA-R2 on Aβ42-induced neurodegeneration in fly brains. Transgene expression was driven by the pan-neuronal elav-GAL4 driver. Representative images of Kenyon cell bodies in flies expressing Aβ42 alone (Top), Aβ42 and PKA-C1 RNAi (Middle), or Aβ42 and PKA-R2 RNAi (Bottom) at 28 dae are shown on the left. Neurodegeneration, as reflected by the presence of vacuoles, is indicated by the arrows. Percentages of the area lost in the cell body regions are shown as means ± SD (n = 7–9 hemispheres). No significant differences from controls were detected (p>0.05, Student's t-test). Male flies were used.
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An example of standard curves and control experiments for cAMP assay. The cAMP levels were measured using the cAMP-Screen assay kit (Applied Biosystems) according to the manufacturer's instruction. This assay is a competitive ELISA. Low levels of cAMP result in a high signal, while high levels result in a low signal. (Top) An example of standard curves. (Bottom) An example of readings with fly head lysates. Notice that the well containing fly head lysates without anti-cAMP antibody produced very low signal.
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We thank Drs. Mel. B. Feany, Dan Kalderon, William M. Saxton, Thomas L. Schwarz, Tim Tully, Jerry C. -P. Yin, Yi Zhong, Konrad E. Zinsmaier, and the Bloomington stock center and the VDRC for fly stocks and antibodies. We also thank Drs. Mark Fortini, Miki Fujioka, Jim Jaynes, and Diane Merry for their insightful comments on the manuscript and Ms. Linda Granger and Ms. Christine Hostetter for technical helps. KIA and KI would like to dedicate this manuscript to the memory of their friend, Goemon Ando.