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Tau Accumulation Causes Mitochondrial Distribution Deficits in Neurons in a Mouse Model of Tauopathy and in Human Alzheimer's Disease Brain

2011; Elsevier BV; Volume: 179; Issue: 4 Linguagem: Inglês

10.1016/j.ajpath.2011.07.004

ISSN

1525-2191

Autores

Katherine J. Kopeikina, George A. Carlson, Rose Pitstick, Adam E. Ludvigson, Alan Peters, Jennifer I. Luebke, Robert M. Koffie, Matthew P. Frosch, Bradley T. Hyman, Tara L. Spires‐Jones,

Tópico(s)

Neuroscience and Neuropharmacology Research

Resumo

Neurofibrillary tangles (NFT), intracellular inclusions of abnormal fibrillar forms of microtubule associated protein tau, accumulate in Alzheimer's disease (AD) and other tauopathies and are believed to cause neuronal dysfunction, but the mechanism of tau-mediated toxicity are uncertain. Tau overexpression in cell culture impairs localization and trafficking of organelles. Here we tested the hypothesis that, in the intact brain, changes in mitochondrial distribution occur secondary to pathological changes in tau. Array tomography, a high-resolution imaging technique, was used to examine mitochondria in the reversible transgenic (rTg)4510, a regulatable transgenic, mouse model and AD brain tissue. Mitochondrial distribution is progressively disrupted with age in rTg4510 brain, particularly in somata and neurites containing Alz50-positive tau aggregates. Suppression of soluble tau expression with doxycycline resulted in complete recovery of mitochondrial distribution, despite the continued presence of aggregated tau. The effect on mitochondrial distribution occurs without concomitant alterations in neuropil mitochondrial size, as assessed by both array tomography and electron microscopy. Similar mitochondrial localization alterations were also observed in human AD tissue in Alz50+ neurons, confirming the relevance of tau to mitochondrial trafficking observed in this animal model. Because abnormalities reverted to normal if soluble tau was suppressed in rTg4510 mice, even in the continued presence of fibrillar tau inclusions, we suggest that soluble tau plays an important role in mitochondrial abnormalities, which likely contribute to neuronal dysfunction in AD. Neurofibrillary tangles (NFT), intracellular inclusions of abnormal fibrillar forms of microtubule associated protein tau, accumulate in Alzheimer's disease (AD) and other tauopathies and are believed to cause neuronal dysfunction, but the mechanism of tau-mediated toxicity are uncertain. Tau overexpression in cell culture impairs localization and trafficking of organelles. Here we tested the hypothesis that, in the intact brain, changes in mitochondrial distribution occur secondary to pathological changes in tau. Array tomography, a high-resolution imaging technique, was used to examine mitochondria in the reversible transgenic (rTg)4510, a regulatable transgenic, mouse model and AD brain tissue. Mitochondrial distribution is progressively disrupted with age in rTg4510 brain, particularly in somata and neurites containing Alz50-positive tau aggregates. Suppression of soluble tau expression with doxycycline resulted in complete recovery of mitochondrial distribution, despite the continued presence of aggregated tau. The effect on mitochondrial distribution occurs without concomitant alterations in neuropil mitochondrial size, as assessed by both array tomography and electron microscopy. Similar mitochondrial localization alterations were also observed in human AD tissue in Alz50+ neurons, confirming the relevance of tau to mitochondrial trafficking observed in this animal model. Because abnormalities reverted to normal if soluble tau was suppressed in rTg4510 mice, even in the continued presence of fibrillar tau inclusions, we suggest that soluble tau plays an important role in mitochondrial abnormalities, which likely contribute to neuronal dysfunction in AD. Tau, a microtubule associated protein, is the major constituent of neurofibrillary tangles (NFT). These deposits are the most common neuronal inclusions in Alzheimer's disease (AD).1Spires-Jones T.L. Stoothoff W.H. de Calignon A. Jones P.B. Hyman B.T. Tau pathophysiology in neurodegeneration: a tangled issue.Trends Neurosci. 2009; 32: 150-159Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 2Gendron T.F. Petrucelli L. The role of tau in neurodegeneration.Mol Neurodegener. 2009; 4: 13Crossref PubMed Scopus (296) Google Scholar As a result of their correlation with synapse and neuronal loss and severity and duration of dementia in AD,3Arriagada P.V. Growdon J.H. Hedley-Whyte E.T. Hyman B.T. Neurofibrillary tangles but not senile plaques parallel duration and severity of Alzheimer's disease.Neurology. 1992; 42: 631-639Crossref PubMed Google Scholar, 4Gomez-Isla T. Hollister R. West H. Mui S. Growdon J.H. Petersen R.C. Parisi J.E. Hyman B.T. 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Differential effect of three-repeat and four-repeat tau on mitochondrial axonal transport.J Neurochem. 2009; 111: 417-427Crossref PubMed Scopus (113) Google Scholar Disruptions in mitochondrial distribution, morphology and function have been linked to several diseases and implicated as early pathogenic steps in neurodegenerative processes.22Lin M.T. Beal M.F. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases.Nature. 2006; 443: 787-795Crossref PubMed Scopus (4599) Google Scholar, 23Querfurth H.W. LaFerla F.M. Alzheimer's disease.N Engl J Med. 2010; 362: 329-344Crossref PubMed Scopus (3704) Google Scholar, 24Swerdlow R.H. Burns J.M. Khan S.M. The Alzheimer's disease mitochondrial cascade hypothesis.J Alzheimers Dis. 2010; 20: S265-S279PubMed Google Scholar, 25Wang X. Schwarz T.L. 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In the current study, we tested the hypothesis that mitochondrial trafficking defects would occur in vivo as a consequence of tau over-expression and/or its mislocalization and aggregation, and also asked whether defects in mitochondrial trafficking could be detected in human AD. Evidence of an imbalance of mitochondrial fission and fusion processes has been observed in Alzheimer's brain,26Wang X. Su B. Lee H.G. Li X. Perry G. Smith M.A. Zhu X. Impaired balance of mitochondrial fission and fusion in Alzheimer's disease.J Neurosci. 2009; 29: 9090-9103Crossref PubMed Scopus (857) Google Scholar, 27Wang X. Su B. Zheng L. Perry G. Smith M.A. Zhu X. The role of abnormal mitochondrial dynamics in the pathogenesis of Alzheimer's disease.J Neurochem. 2009; 109: 153-159Crossref PubMed Scopus (215) Google Scholar, 28Westermann B. Mitochondrial fusion and fission in cell life and death.Nat Rev Mol Cell Biol. 2010; 11: 872-884Crossref PubMed Scopus (1349) Google Scholar, 29Du H. Guo L. Yan S. 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We investigated somatic and neuritic mitochondrial distribution and mitochondrial volumes in neurons and neuropil of rTg4510 mice, which over-express a human mutant form of tau (P301L) known to lead to dementia. The rTg4510 mice demonstrate age-related cognitive impairment, accumulation of NFT and neuronal loss but also harbor a doxycycline-regulatory domain that can be used as an “off-switch” for this mutant tau over-expression. Doxycycline treatment results in stabilization of neuronal number and recovery of cognitive function even in the face of continued accumulation of NFT.9Santacruz K. Lewis J. Spires T. Paulson J. Kotilinek L. Ingelsson M. Guimaraes A. DeTure M. Ramsden M. McGowan E. Forster C. Yue M. Orne J. Janus C. Mariash A. Kuskowski M. Hyman B. Hutton M. Ashe K.H. Tau suppression in a neurodegenerative mouse model improves memory function.Science. 2005; 309: 476-481Crossref PubMed Scopus (1548) Google Scholar, 10Spires T.L. Orne J.D. SantaCruz K. Pitstick R. Carlson G.A. Ashe K.H. Hyman B.T. Region-specific dissociation of neuronal loss and neurofibrillary pathology in a mouse model of tauopathy.Am J Pathol. 2006; 168: 1598-1607Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar Since mitochondria are quite small, on the order of the limits of light microscopy, we used a recently developed high-resolution microscopy technique, array tomography, for precise localization of mitochondria as well as accompanying mitochondrial volume quantification.32Koffie R.M. Meyer-Luehmann M. Hashimoto T. Adams K.W. Mielke M.L. Garcia-Alloza M. Micheva K.D. Smith S.J. Kim M.L. Lee V.M. Hyman B.T. Spires-Jones T.L. Oligomeric amyloid beta associates with postsynaptic densities and correlates with excitatory synapse loss near senile plaques.Proc Natl Acad Sci USA. 2009; 106: 4012-4017Crossref PubMed Scopus (624) Google Scholar, 33Micheva K.D. Smith S.J. Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits.Neuron. 2007; 55: 25-36Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar Array tomography overcomes the approximate 1 μm z-resolution limitation of conventional confocal, and multiphoton microscopy, which is larger than the width of a single mitochondrion, by ultrathin sectioning of samples into ribbons of 70 nm sections followed by immunofluorescence imaging and three-dimensional reconstructions of structures of interest. This allows precise quantification of the number, volume, and protein labeling of small structures including mitochondria in a high-throughput automated fashion with thousands of mitochondria imaged per case. Electron micrographs were used to confirm mitochondrial volume data derived from the array tomography method, which validates the use of this automated technique for volume measurements. We also applied array tomography to human brain tissue to observe mitochondrial distribution changes in AD, highlighting the efficacy of this technique for human pathological analyses. Our data demonstrate that early in the course of disease, mitochondrial distribution is altered, particularly in those cells or neurites bearing aggregates of tau. These patterns persist and become more severe with age. Doxycycline treatment of a subset of rTg4510 (regulatable transgenic) mice remarkably restored mitochondrial distribution to near normal, even in the continued presence of aggregated misfolded tau. Interestingly, these distribution changes were not accompanied by alterations in mitochondrial volume in the neuropil. Mitochondrial distribution in human AD brain demonstrated patterns that mirrored those seen in rTg4510 mice. Taken together, our findings indicate that mitochondrial distribution changes occur in vivo as a consequence of tau over-expression, and may be predominantly due to soluble tau species. For this study we used a well-characterized, regulatable mouse model of tauopathy that over-expresses human mutant (P301L) tau that can be suppressed with doxycycline (dox) treatment. Mice were generated as previously described.9Santacruz K. Lewis J. Spires T. Paulson J. Kotilinek L. Ingelsson M. Guimaraes A. DeTure M. Ramsden M. McGowan E. Forster C. Yue M. Orne J. Janus C. Mariash A. Kuskowski M. Hyman B. Hutton M. Ashe K.H. Tau suppression in a neurodegenerative mouse model improves memory function.Science. 2005; 309: 476-481Crossref PubMed Scopus (1548) Google Scholar, 10Spires T.L. Orne J.D. SantaCruz K. Pitstick R. Carlson G.A. Ashe K.H. Hyman B.T. Region-specific dissociation of neuronal loss and neurofibrillary pathology in a mouse model of tauopathy.Am J Pathol. 2006; 168: 1598-1607Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar The responder transgene contains cDNA of human four-repeat tau with the P301L mutation downstream of a tetracycline-operon-responsive element (TRE). The activator transgene consists of a tet-off open reading frame downstream of Ca2+ calmodulin kinase II promoter elements. This bigenic system results in over-expression of human mutant tau in forebrain structures when both promoter and activator are present. Littermate animals with only the activator transgene, which don't over-express tau were used as controls. Two age groups, 5.5 and 8.5 month old, of tau over-expressing (rTg4510) and control animals (nonTg) were used (n = 4 per group). Doxycycline was administered at 200ppm in the food for 6 weeks between 7 and 8.5 months of age in a subset of rTg4510 and nonTg animals (n = 4 per group), leading to suppression of tau as previously described.9Santacruz K. Lewis J. Spires T. Paulson J. Kotilinek L. Ingelsson M. Guimaraes A. DeTure M. Ramsden M. McGowan E. Forster C. Yue M. Orne J. Janus C. Mariash A. Kuskowski M. Hyman B. Hutton M. Ashe K.H. Tau suppression in a neurodegenerative mouse model improves memory function.Science. 2005; 309: 476-481Crossref PubMed Scopus (1548) Google Scholar Animals were housed and treated in accordance with institutional guidelines and those of the National Institutes of Health. Tissue from the superior temporal gyrus of subjects with either an AD diagnosis or no cognitive impairment was obtained from the Alzheimer Disease Research Center at Massachusetts General Hospital. All human tissue was handled in agreement with local and national IRB guidelines. Ten AD cases (61–90 years of age) and four cognitively normal controls (62–75 years of age) were included in this study (Table 1).Table 1AD Diagnoses in 10 PatientsCaseAgeSexDiagnosis175MNo cognitive impairment (prior stroke in contralateral hemisphere)266MNo cognitive impairment369MNo cognitive impairment462MNo cognitive impairment589MAD; Braak stage VI/VI661MAD; Braak stage V/VI777MAD; Braak stage V/VI874FAD; Braak stage VI/VI990MAD; Braak stage IV/VI1080FAD; Braak stage VI/VI1183FAD; Braak stage VI/VI1284MAD; Braak stage V/VI1381MAD; Braak stage VI/VI1484FAD; Braak stage VI/VIBraak staging defines pathological progression of AD in six stages (I-VI), with VI being the most severe.AD, Alzheimer's disease; F, female; M, male. Open table in a new tab Braak staging defines pathological progression of AD in six stages (I-VI), with VI being the most severe. AD, Alzheimer's disease; F, female; M, male. Tissue was prepared for array tomography as previously described.32Koffie R.M. Meyer-Luehmann M. Hashimoto T. Adams K.W. Mielke M.L. Garcia-Alloza M. Micheva K.D. Smith S.J. Kim M.L. Lee V.M. Hyman B.T. Spires-Jones T.L. Oligomeric amyloid beta associates with postsynaptic densities and correlates with excitatory synapse loss near senile plaques.Proc Natl Acad Sci USA. 2009; 106: 4012-4017Crossref PubMed Scopus (624) Google Scholar, 33Micheva K.D. Smith S.J. Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits.Neuron. 2007; 55: 25-36Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar, 34Micheva K.D. Busse B. Weiler N.C. O'Rourke N. Smith S.J. Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers.Neuron. 2010; 68: 639-653Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar Brains were removed from mice immediately after euthanasia by CO2 inhalation. Human tissue samples were collected within 24 hours of autopsy. Small blocks (∼1 mm3) of primary somatosensory cortex from mice or temporal cortex from human cases were fixed by immersion in 4% paraformaldehyde and 2.5% sucrose in phosphate-buffered saline solution (PBS) for 3 hours at room temperature. Tissue was then dehydrated through a graded series of ethanols, and into LRWhite resin (Electron Microscopy Sciences, Hatfield PA), embedded in gelatin capsules with LRWhite and polymerized at 53C for 24 hours. Blocks were then removed from gelatin capsules and cut into ribbons of 7 to 150 ultra-thin 70 nm sections with a Jumbo Histo Diamond Knife (Diatome, Hatfield PA) and mounted on gel-subbed coverslips (Fisher Scientific, Pittsburgh PA; 12-544-E; No. 1.5; 0.16- to 0.19-μm thick). Immunostaining for analysis of mitochondrial localization was performed as follows for both mouse and human cases. Ribbons were washed in 50 mmol/L glycine in Tris-buffered saline (TBS) and blocked in 0.05% Tween and 0.1% BSA in TBS. Primary antibodies mouse IgM Alz50 (a generous gift of Peter Davies, Albert Einstein College of Medicine), mouse IgG anti-tubulin (Sigma, St. Louis MO), and rabbit anti-VDAC/porin (Abcam, Cambridge MA) were diluted 1:50 in block buffer and applied to ribbons for 2 hours then rinsed off with TBS. Fluorescent secondary antibodies, donkey anti-mouse IgM Cy3, donkey anti-mouse IgG Alexa-Fluor 488, and donkey anti-rabbit Cy5 (Jackson ImmunoResearch, Westgrove PA) were diluted 1:100 in block buffer and incubated on ribbons for 30 minutes. 1024 × 1024 pixel images of regions of interest (cell bodies or neurites) within a single 70 nm section were acquired with a Leica DMRE confocal microscope (Wetzlar, Germany) and a 63 × 1.4 numerical aperture Plan Apochromatic oil objective. For analysis of mitochondrial volume, ribbons of at least 10 ultrathin sections were stained as described above with rabbit anti-VDAC/porin (Abcam) at 1:200 followed by fluorescent secondary donkey anti-rabbit Cy3 at 1:100. Areas of interest, containing cell bodies (used as fiduciary markers) were imaged on 10 to 12 serial sections. Images of 1024 × 1024 pixels were collected with a Zeiss Axioplan LSM510 confocal/multiphoton microscope (Ziess, Thornwood, NY) with a 63 × 1.2 numerical aperture Plan Apochromatic water objective. To confirm that astrocytic processes were not included in analyses, we performed two array immunostains of rTg4510 mouse tissue as described above but using the following antibodies. The first included mouse IgM Alz50 (1:100), mouse IgG anti-tubulin (1:300), rabbit anti-glial fibrillary acidic protein (1:300; Sigma), and secondaries goat anti-rabbit Cy3, goat anti-mouse IgG Alexa-Fluor 488, goat anti-mouse IgM Cy5 (1:100) and DAPI (Invitrogen, Eugene OR). The second included mouse IgM Alz50 (1:100), rat anti-tubulin (1:100; Abcam), mouse anti-glutamine synthetase (1:50; Millipore, Billerica MA) and secondaries goat anti-mouse IgG Alexa-Fluor 488 (1:50), goat anti-rat Cy3 and goat anti-mouse IgM Cy5 (1:100) and DAPI. Single section images of 1024 × 1024 pixels were collected with a Zeiss Axioplan LSM510 confocal/multiphoton microscope with a 63 × 1.4 numerical aperture Plan Apochromatic oil DIC objective. For whole cell reconstruction by array tomography a ribbon of 150 80 nm sections was stained as described previously with rabbit anti-VDAC/porin (1:300), mouse IgG anti-tubulin (1:300), and mouse IgM Alz50 (1:100) followed by goat anti-rabbit Alexa Fluor 488 (1:100), chicken anti-mouse IgG Alexa-Fluor 647 (1:300), goat anti-mouse IgM Cy3 (1:100) and DAPI. A region of interest (1024 × 1024 pixels, zoom 3) containing the entirety of an Alz50+ and neighboring Alz50− cell was serially collected from each of 109 sections with a Zeiss Axioplan LSM510 confocal/multiphoton microscope and a 63 × 1.4 numerical aperture Plan Apochromatic oil DIC objective. Electron micrographs of two 8.5-month-old rTg4510 and one age-matched nonTg were acquired as previously described.35Ludvigson A.E. Luebke J.I. Lewis J. Peters A. Structural abnormalities in the cortex of the rTg4510 mouse model of tauopathy: a light and electron microscopy study.Brain Struct Funct. 2010; 216: 31-42Crossref PubMed Scopus (17) Google Scholar In short, mice were anesthetized with an intraperitoneal injection of sodium pentobarbital and transcardially perfused with fixative solution containing 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1M cacodylate buffer (pH 7.2–7.4) at 37°C. Following perfusion, the heads of the mice, with calvaria removed, were submerged in a solution of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1M cacodylate buffer and kept at 4°C for 12 hours. One-millimeter thick coronal slices of the dorsal premotor cortex were obtained and divided into smaller pieces for embedding. Tissue was rinsed in 0.1M sodium cacodylate buffer, osmicated with 1% osmium tetroxide in cacodylate buffer, dehydrated through ascending ethyl alcohol concentrations, rinsed with propylene oxide and immersed in 1:1 propylene oxide and Araldite 502 plastic (Ernest F. Fullam, Inc, Redding CA) overnight. Cortical pieces were placed in pure Araldite, rotated for 6 hours, transferred to Beem capsules and hardened at 60°C. An RMC MT6000-XL ultramicrotome (Boeckeler, Tuscon AZ) was used to cut thin sections, which were mounted on copper grids then stained with uranyl acetate and lead citrate and photographed using a JEOL 100S electron microscope (JEOL, USA, Peabody MA). Negatives were scanned at 800 dpi with an Epson Perfection V700 photo scanner. Images were viewed and analyzed with Image J (National Institutes of Health open software; http://rsbweb.nih.gov/ij). Analysis of mitochondrial localization was performed as previously described.18Stoothoff W. Jones P.B. Spires-Jones T.L. Joyner D. Chhabra E. Bercury K. Fan Z. Xie H. Bacskai B. Edd J. Irimia D. Hyman B.T. Differential effect of three-repeat and four-repeat tau on mitochondrial axonal transport.J Neurochem. 2009; 111: 417-427Crossref PubMed Scopus (113) Google Scholar Approximately 50 cells and 40 neurites were imaged for each animal or human case. The mitochondrial channel of each image was opened and re-named for blinded thresholding. Once thresholds were determined, the tubulin channel was used to identify and circle the somatic or neuritic cytoplasm (excluding the nucleus). This region of interest (ROI) was then applied to the thresholded mitochondrial image and the ‘Analyze Particles’ feature of Image J applied to determine percentage of the ROI occupied by mitochondria. For each cell or neurite analyzed, presence or absence of Alz50 staining was also determined. This percent area occupied by mitochondria in the soma and neurites is referred to as mitochondrial distribution and is used as a readout for the ability of mitochondria to be trafficked to all parts of the cell body and neurites as in our previous study, which showed changes in mitochondrial distribution in the soma and axon were associated with reduced anterograde trafficking of mitochondria and fewer mitochondria reaching the periphery of the cell body (resulting in perinuclear clumping) and fewer reaching the axon.18Stoothoff W. Jones P.B. Spires-Jones T.L. Joyner D. Chhabra E. Bercury K. Fan Z. Xie H. Bacskai B. Edd J. Irimia D. Hyman B.T. Differential effect of three-repeat and four-repeat tau on mitochondrial axonal transport.J Neurochem. 2009; 111: 417-427Crossref PubMed Scopus (113) Google Scholar In the whole cell reconstruction, the tubulin images were opened sequentially and converted to a stack. An ROI for an Alz50+ and an Alz50− cell was defined in each image. These ROIs were then applied to the corresponding, individually thresholded mitochondrial channel image and the ‘Analyze Particles’ feature of Image J applied to determine percentage of the ROI occupied by mitochondria. The area of each ROI was also measured. These outputs were used to calculate a percent volume fraction occupied by mitochondria in an Alz50+ and an Alz50− cell (sum of total area occupied by mitochondria in ea

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