Macroautophagy deficiency mediates age-dependent neurodegeneration through a phospho-tau pathway
© Inoue et al.; licensee BioMed Central Ltd. 2012
Received: 22 May 2012
Accepted: 5 September 2012
Published: 21 September 2012
Macroautophagy is an evolutionarily conserved mechanism for bulk intracellular degradation of proteins and organelles. Pathological studies have implicated macroautophagy defects in human neurodegenerative disorders of aging including Alzheimer’s disease and tauopathies. Neuronal deficiency of macroautophagy throughout mouse embryonic development results in neurodevelopmental defects and early postnatal mortality. However, the role of macroautophagy in mature CNS neurons, and the relationship with human disease neuropathology, remains unclear. Here we describe mice deficient in an essential macroautophagy component, Atg7, specifically within postnatal CNS neurons.
Postnatal forebrain-specific Atg7 conditional knockout (cKO) mice displayed age-dependent neurodegeneration and ubiquitin- and p62-positive inclusions. Phosphorylated tau was significantly accumulated in Atg7 cKO brains, but neurofibrillary tangles that typify end-stage human tauopathy were not apparent. A major tau kinase, glycogen synthase kinase 3β (GSK3β), was also accumulated in Atg7 cKO brains. Chronic pharmacological inhibition of tau phosphorylation, or genetic deletion of tau, significantly rescued Atg7-deficiency-mediated neurodegeneration, but did not suppress inclusion formation.
These data elucidate a role for macroautophagy in the long-term survival and physiological function of adult CNS neurons. Neurodegeneration in the context of macroautophagy deficiency is mediated through a phospho-tau pathway.
The primary etiologies of neurodegenerative disorders, including Alzheimer’s disease (AD), frontotemporal dementia (FTD) and Parkinson’s disease (PD), remain largely unknown, but common pathological features suggest a role for altered protein degradation. For instance, proteinaceous intracellular inclusions composed in part of aggregated α-synuclein protein, termed Lewy bodies, typify PD brain pathology, whereas neurofibrillary tangles (NFT) and Pick bodies containing phosphorylated tau protein are commonly found in the context of taupathies such as AD and FTD. Rare, inherited familial forms of neurodegenerative diseases  are caused by mutations in genes encoding these accumulated proteins, such as α-synuclein [2, 3] in PD and tau in FTD, but the vast majority of patients do not harbor known mutations. Thus, it has been hypothesized that in these ‘sporadic’ cases, pathological inclusions may reflect broadly defective protein degradation through mechanisms such as the ubiquitin-proteasome system (UPS)  and macroautophagy [5, 6]. The latter is of particular interest because of its apparent role in the degradation of protein aggregates and inclusions .
Macroautophagy is a pathway of bulk cytoplasmic protein and organelle degradation characterized by double-membrane vesicles that engulf cargo and target it to lysosomes for degradation . The pathway is typically induced in the context of starvation or other stressors. Defects in the macroautophagy process may theoretically occur at a variety of steps, from the initial formation of a pre-autophagosome limiting membrane, to the ultimate fusion of mature autophagosomes with the lysosomal compartment . Macroautophagy defects have been well described on pathological analyses of brain sections from patients with a variety of neurodegenerative disorders, including AD, PD and FTD [5, 10]. Furthermore, inherited genetic forms of neurodegeneration are associated with mutations in the macroautophagy-lysosomal pathway [11, 12]. Finally, as macroautophagy dysfunction is a well-documented feature of aging, it has been implicated in the age-dependent nature of the major neurodegenerative disorders [5, 9, 10].
Genetically altered mice that are deficient in essential macroautophagy pathway components, Atg5 or Atg7, throughout neural development, display reduced neuronal survival and harbor ubiquitin-positive inclusions in the cell soma [13–16]. But surprisingly, prevention of inclusion formation in the context of Atg7-deficiency by a second genetic ablation of p62, which encodes an ubiquitin-binding protein associated with autophagosomes, does not suppress neurodegeneration, arguing against a toxic role for inclusions . Thus, the mechanism of neuronal loss with macroautophagy deficiency, and how this relates to neurodegeneration, remains unclear.
Here we generated conditional Atg7-deficient mice specifically within mature CNS neurons. Atg7-deficient neurons were defective in the initiation of macroautophagy, and displayed a progressive degeneration with prominent inclusions that harbor ubiquitin, p62, phosphorylated tau and GSK3β. The mutant mice exhibited behavioral deficits consistent with the pathological changes. Furthermore, pharmacological or genetic suppression of tau phosphorylation effectively inhibited neurodegeneration in the context of Atg7-deficiency in vivo.
Slowly progressive degeneration of postnatal Atg7-deficient hippocampal CA1 neurons
Furthermore, numerous ubiquitin-positive inclusions were apparent in essentially all Atg7-deficient CA1 cell bodies from 2-month of age, whereas these were never seen in the control CamK-Atg7 cWT mice (Figure 1e). These inclusions were stained positive for p62 [17, 21], which is a component of the macroautophagy machinery pathway (Additional file 1), and further confirmed the macroautophagy defect in forebrain neurons. In contrast, such inclusions were absent from the CA3 neurons (data not shown). Further analysis by electron microscopy revealed that these inclusions were composed of both filamentous and vesicular elements (Figure 1f).
We further compared CamK-Atg7 cKO neurodegeneration with the effect of Atg7 deficiency in a second population of mature CNS neurons, midbrain dopamine (DA) neurons. To this end, we generated animals that express CRE under the control of the dopamine transporter (Dat) gene regulatory elements, and are homozygous for the floxed Atg7 allele (Dat Cre/+ Atg7 flox/flox ; Dat-Atg7 cKO mice rather than CamK-Atg7 cKO mice). Dat-Atg7 cKO mice displayed a very similar pathological progression to CamK-Atg7 cKO mice with cytoplasmic ubiquitin- and p62-positive inclusions, albeit the process is selective for midbrain DA neurons as expected (Additional file 2c,d). Neurodegeneration progresses appeared more rapid in the Dat-Atg7 cKO mouse model than the CamK-Atg7 cKO mouse model (25% midbrain DA neuron lost at 2-months of age and 38% lost at 4-month; Additional file 2a,b).
Atg7deficiency in mouse postnatal forebrain neurons results in physiological and behavioral deficits
Next, we assessed forebrain-dependent fear conditioning in CamK-Atg7 cKO mice and CamK-Atg7 cWT mice. CamK-Atg7 cKO mice did not show any increase in the ratio of freezing at their basal level. However, CamK-Atg7 cKO mice showed a significant impairment in contextual fear conditioning relative to control CamK-Atg7 cWT animals (Figure 2d). Furthermore, the cKO mice showed significant reduced freezing ratio in cued fear conditioning, whereas the basal freezing (‘Pre-Test’) was not changed (Figure 2e). Taken together, these data demonstrate forebrain physiological dysfunction, consistent with the selective forebrain pathology of CamK-Atg7 cKO mice.
Phospho-tau-positive inclusions in Atg7-deficient neurons
GSK3β staining at phospho-tau inclusions in Atg7-deficient neurons
Given the accumulation of phosphorylated -- but not total -- tau in Atg7-deficient neurons (Figure 4e), we hypothesized that a kinase that is known to phosphorylate tau, such as GSK3β, may be altered. Immunostaining of cortical neurons revealed dramatic re-localization of GSK3β, including both active (epitope at Tyr216) and inactive (epitope at Ser9) phosphorylated forms, to phospho-tau-positive and ubiquitin/p62-positive inclusions in Atg7-deficient neurons (Figure 4a-c). Western blot analysis confirmed that total and phosphorylated forms of GSK3α/β were increased in forebrain tissue extracts from CamK-Atg7 cKO mice, compared to CamK-Atg7 cWT mice (Figure 4d). Another kinase implicated in phosphorylation of tau, CDK5, did not appear to be re-localized to the inclusions in Atg7-deficient neurons  (Additional file 4d). Inclusions in Atg7-deficient neurons stained positively for a second microtubule-associated GSK3β substrate, phospho-CRMP2  (Additional file 4a,b). In contrast, β-Catenin, a well-described GSK3β substrate in the context of Wnt signaling pathway, did not appear altered in staining in Atg7-deficient neurons (Additional file 4c). Thus, accumulated GSK3β in the context of Atg7-deficiency appears to display substrate specificity, perhaps related to subcellular re-localization at inclusions.
Pharmacological or genetic inhibition of phospho-tau accumulation can rescue neuronal cell death in vivo
To examine the causality between phospho-tau and neurodegeneration in the context of Atg7-deficiency, we sought to determine whether neurons deficient in Atg7 could be effectively protected in vivo through the modulation of phospho-tau production. We focused these ‘rescue’ studies on Dat-Atg7 cKO mice (rather than CamK-Atg7 cKO mice) because the neurodegeneration progresses more rapidly in Dat-Atg7 cKO mouse model than CamK-Atg7 cKO mouse model, as noted above, and the degenerative and pathological processes are restricted to a single cell type in the Dat-Atg7 cKO mice (midbrain DA neurons; Additional file 2a,b). Dat-Atg7 cKO mice also displayed a very similar pathological progression to CamK-Atg7 cKO mice with cytoplasmic ubiquitin- and p62-positive inclusions (Additional file 2c,d) that further stain for phospho-tau and GSK3β (Additional file 2e,f). Thus, analysis of pathology in Dat-Atg7 cKO mice affords a more facile and accurate quantification of the cell autonomous impact of macroautophagy on the loss of mature CNS neurons.
Next, we examined the effect of tau-deficiency  in Dat-Atg7 cKO mice. We generated Dat-Atg7/tau double cKO (Dat Cre/+ Atg7 flox/flox tau -/- ) mice, and compared the loss of midbrain DA neuron in Dat-Atg7 single cKO (Dat Cre/+ Atg7 flox/flox tau +/+ or Dat Cre/+ Atg7 flox/flox tau +/- ) and Dat-Atg7/tau double cKO mice. The loss of midbrain DA neurons in Dat-Atg7 cKO mice was significantly rescued in Dat-Atg7/tau double cKO mice at the age of 3-month (Figure 5c,d). Again, the formation of ubiquitin-positive inclusion was not changed in Dat-Atg7/tau double cKO mice (Additional file 5d,e). Consistent with the previous report that tau-deficiency alone led to no abnormality in the brain [37, 38], neither neurodegeneration nor ubiquitin/p62-positive inclusions was seen in the midbrain DA neurons of tau KO mice (Figure 5c,d and Additional file 5d,e). Taken together, these approaches support a model whereby accumulation of phospho-tau contributes to neurodegeneration in the context of macroautophagy-deficiency, whereas the formation of ubiquitin/p62-positive inclusions is independent of phospho-tau signaling.
Here we investigated mechanisms of neurodegeneration downstream of Atg7-deficiency, and describe the pathological accumulation of GSKβ and phospho-tau proteins. A striking feature of neuropathology in the context of Atg7-deficiency is the redistribution of GSK3β to inclusions. We note that both GSK3β and phospho-tau are reported to be found in inclusions in tauopathy patient brain [39–43]. However, it is important to emphasize that Atg7-deficiency does not appear to induce a full tauopathy pathology, as not all phospho-tau epitopes are observed (e.g., PHF1 antibody is negative, Figure 4e), and amyloid staining with Thioflavin S, as well as electron microscopic analysis, do not support the presence of mature NFTs. A similar phospho-tau pattern has previously been suggested to represent an early ‘pre-tangle’ pathological state , thought to reflect non-fibrillar tau aggregation prior to assembly into PHFs. Such non-fibrillar hyperphosphorylated tau, rather than mature NFTs, may be the relevant toxic form in vivo in the context of neurodegeneration and behavioral impairment . Hoozemans et al. reported phospho-tau-positive pre-tangles with accumulation of GSK3β, ubiquitin and p62 in postmortem specimens of AD patients , reminiscent of pathology in Atg7-deficient neurons in vivo. Phospho-tau pathology as seen in Atg7-deficient animals may broadly relate to neuronal dysfunction in neurodegeneration, as macroautophagy deficiency and phospho-tau are commonly observed in a broad array of neurodegenerative disorders including AD, PD, tauopathy, huntington disease, amyotrophic lateral sclerosis, and Gaucher disease [6, 46–49]. Although genetic mutations in ATG7 have not been described in human disease, mutations within other components of the macroautophagy-lysosomal pathway underlie tauopathies , consistent with our observations in the mouse model.
The in vivo pharmacological and genetic ‘rescue’ studies herein suggest a role for phospho-tau accumulation in neurodegeneration downstream of Atg7-deficiency. In contrast, prior attempts to rescue macroautophagy-deficiency associated neurodegeneration by preventing the formation of aggregates, by generation of double-knockout mice deficient in Atg7 as well as p62, were unsuccessful , suggesting that inclusion formation per se is insufficient for degeneration. It is interesting to note that nonetheless, p62 deletion does rescue the Atg7 deficiency-associated cell loss in hepatocytes , and thus degenerative pathways downstream of macroautophagy loss appear cell type-specific. Furthermore, within the CNS, various neuronal subtypes appear to be differentially affected by macroautophagy deficiency. Purkinje neurons deficient in Atg7 display axonal swellings and are rapidly lost . TH-positive midbrain DA neurons display axonal dystrophy and degeneration, ubiquitin/p62-positive inclusions, and delayed cell loss and locomotor dysfunction . Although tau pathology was not investigated in these other models, staining for the Parkinson’s disease associated proteins α-synuclein and leucine rich repeat kinase-2 (LRRK2) was reported in Atg7-deficient DA neurons . We failed to detect evidence of α-synuclein accumulation in our analysis of either midbrain DA neuron-selective or forebrain neuron-selective Atg7-deficient mice detailed above (data not shown). Such discrepancies may reflect differences in the selectivity or timing of the CRE-mediated deletion strains used in the different studies, or selective sensitivity to macroautophagy loss across distinct neuron types. We note that phospho-tau pathology was apparent in the context of either midbrain DA neuron-selective or forebrain neuron-selective Atg7-deficiency.
The molecular basis of GSK3β and phospho-tau accumulation in Atg7-deficient neurons remains to be elucidated. We cannot exclude the possibility that GSK3β accumulation is a secondary effect of phospho-tau accumulation. A recent study described intracellular redistribution of GSK3β to multivesicular bodies, albeit in the context of Wnt pathway modulation . As multivesicular bodies directly associate with the macroautophagy machinery, it is possible that GSK3β degradation is selectively modified with macroautophagy loss . Although GSK3β is a strong candidate for the relevant upstream kinase, we hypothesize the involvement of other kinase pathways, particularly given the multiple targets of the pharmacological kinase inhibitor used, Alsterpaullone. Furthermore, Alsterpaullone-mediated protection may be mediated through targets in addition to tau, which would be of further interest.
We propose a role for basal macroautophagy in regulating the metabolism of phospho-tau proteins at physiological or pre-pathological state (Figure 5e). In the context of macroautophagy loss, GSK3β and phospho-tau are accumulated, reminiscent of early pathology that precedes human tauopathy. It is interesting to note that both GSK3β and tau are believed to be potent upstream regulators of macroautophagy [55–58]. We hypothesize that this may reflect a feedback loop, where defective macroautophagy leads progressively to more accumulation of phospho-tau and GSK3β, and in turn the accumulated phospho-tau and GSK3β both induce macroautophagy activity. Initially such feedback may be effective, although the accumulated proteins form inclusions. But once macroautophagy deficiency is complete, as in late-stage disease or in knockout mice, this feedback would be ineffective. Thus, such a feedback circuit may be an important pathway to rejuvenate the macroautophagy pathway, which is known to wane with aging .
Atg7 cKO in mouse forebrain neurons led to an age-dependent neurodegeneration with ubiquitin/p62-positive and phospho-tau/GSK3β inclusions, but not the full pathological features of NFTs in tauopathy. Pharmacological or genetic inhibition of tau phosphorylation in vivo successfully rescued neurodegeneration in the context of macroautophagy-deficiency. As GSK3β and tau are also upstream inducers of macroautophagy, this implicates a negative feedback loop in human pathology.
CamK-Cre transgenic mice, Dat Cre/+ mice, Atg7 flox/flox mice, hAPP-Tg and tau KO mice, used in this study were generated previously [19, 20, 37, 60–62]. CamK-Cre Tg and tau KO mice were purchased from Jackson Laboratories. All animals were maintained in the animal facility of the Columbia University Medical Center. Experimental protocols were approved by the Institutional Animal Care and Use Committee. Genomic DNA extracted from mouse tails was amplified by PCR for genotyping using standard methods. The PCR primers are the followings: 5’-AGA TGT TCG CGA TTA TC-3’, 5’-AGC TAC ACC AGA GAC GG-3’ for Cre transgene; 5’-TGC TCT GTG AAC TGC CCT GTT T-3’, 5’-TGT TCC TGT GCA CTG CCT CAT T-3’ for Atg7 wild-type allele; 5’-CTT GGG TGG AGA GGC TAT TC-3’, 5’-AGG TGA GAT GAC AGG AGA TC-3’ for Atg7 floxed allele.
Mice were perfused and fixed in 4% paraformaldehyde and post-fixed at 4°C overnight, 50 μm coronal brain sections were generated using a vibratome. The sections were blocked with PBS containing 5% normal donkey serum [NDS], 0.2% Triton X-100 [Tx] for 1 h, and incubated with the solution (PBS, 1% NDS, 0.2% Tx) containing primary antibody at 4°C overnight. The following antibodies were used; anti-TH (P60101, Pel-Freez), anti-TuJ1 (MMS-435P, COVANCE), anti-MAP2 (AB5622, Millipore), anti-cleaved caspase-3 [Asp175] (#9661, Millipore), anti-active caspase-3 (AB3623, Cell Signaling Technology), anti-ubiquitin (Sigma-Aldrich), anti-p62 (03-GP62-C, American Research Products), anti-Aβ [4G8] (SIG39200, COVANCE), anti-Aβ [6E10] (SIG39300, COVANCE), anti-αSynuclein (610786, BD Bioscience) (AB5038, Millipore) (ab1903, ab24715, Abcam), anti-phosph-tau TG3 and PHF1 (gifts from Dr. Peter Davies, Alberts Einstein College of Medicine), anti-phospho-tau AT8, AT100, AT180, and AT270 (Pierce), anti-total GSK3β (#9315, Cell Signaling Technology), anti-phospho-GSK3α/β [Y279/Y216] (ab52188, Abcam), anti-phospho-GSK3β [S9] (ab30619, Abcam), anti-total CRMP2 (#9393, Cell Signaling Technology), anti-phospho-CRMP2 [T514] (#9397, Cell Signaling Technology), anti-Cdk5 (MAB5410, Millipore), anti-p35/25 (#2680, Cell Signaling Technology), anti-β-catenin (#9581, 9587, Cell Signaling Technology), and anti-β-catenin (#610154, BD Biosciecnes). For secondary detection, Cy3- or FITC-conjugated antibodies were incubated for 1 h (Jackson ImmunoResearch). Photographs were taken using a Zeiss LSM 510 Meta confocal microscope.
To obtain neuronal cell count, 50 μm coronal brain sections were made using a vibratome. In order to count CA1 neurons, the first 30 sections from the rostral hippocampus were stained with rabbit anti-MAP2 antibody (AB5622, Millipore) at a dilution of 1:500, as well as NeuroTraceTM Fluorescent Nissl stain (N21480, Invitrogen). MAP2-positive neurons were visualized using a Cy3-conjugated secondary antibody (Jackson ImmunoResearch). MAP2 and Nissl double-positive neurons in the CA1 regions were counted manually. In order to count TH-positive neurons, sections covering the entire substantia nigra (25-30 sections / mouse) were stained with sheep anti-TH antibody (P60101, Pel-Freez) at a dilution of 1:250. TH-positive neurons were visualized using the ABC Kit (PK6106, Vector Laboratories) and DAB (SK4100, Vector Laboratories). TH-positive neurons in the substantia nigral regions were counted manually under the light microscope.
Electron microscopic analysis was as described . Anesthetized mice were perfused and fixed in PBS containing 4% paraformaldehyde and 0.5% gultaralaldehyde. The brains were post-fixed at 4°C for 2 h, and the 80 μm vibratome sections were made. The sections were treated in 1% osmium tetroxide, then dehydrated in pure ethanol and infiltrated overnight with Epon 812. Epon was polymerized at 60°C for 24 h, cooled and embedded in a larger Epon capsule. Ultrathin sections were cut with an MT5000 ultramicrotome, stained with uranyl acetate and lead citrate. Images were taken with a JOEL 100S Electron Microscope (JOEL USA).
Preparation of soluble and insoluble fractions was performed as described with some modifications . Cortical and hippocampal tissues from mouse brains were homogenized in 5× volume of ice-cold 0.25M sucrose buffer (50mM Tris-HCl [pH7.6]) containing protease inhibitors (P8340, Sigma) and phosphatase inhibitors (#78420, Thermo Scientific). The homogenized tissues were centrifuged at 500× g for 10 min at 4°C. The supernatants were lysed with an equal volume of cold sucrose buffer containing 1% Triton X-100. The lysates were centrifuged at 13,000× g for 15 min at 4°C. The supernatants contained the soluble fraction. The pellets were resuspended in 1% SDS in PBS (insoluble fraction). Both fractions were subjected to standard Western Blotting analysis. The antibodies used here are: anti-phospho-tau AT8, AT100, AT180, AT270, TG3 and PHF1, anti-Tau1 and anti-Actin (ab3280, Abcam). Horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch) and SuperSignal West Pico or Dura (#34077, 34075, Pierce) were used for detection.
Brains from CamK-Atg7 cWT and cKO mice littermates (~12 weeks of age) were quickly removed and transverse hippocampal slices (400 μm) were isolated with a Leica VT1200 Vibratome (Leica, Bannockburn, IL), and placed in ice-cold cutting solution (CS: 110 mM Sucrose, 60 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 28 mM NaHCO3, 0.5 mM CaCl2, 7 mM MgCl2, 5 mM Glucose, 0.6 mM Ascorbate. Slices were placed in an interface chamber (Scientific Systems Design, Mississauga, Ontario, Canada) and maintained at 32°C in ACSF (2 ml/min) containing 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 25 mM D-glucose, 2 mM CaCl2, and 1 mM MgCl2. All solutions were constantly caboxygenated with 95% O2 + 5% CO2. Slices were allowed to recover for 120 min on the electrophysiology rig prior to experimentation. Bipolar stimulating electrodes (92:8 Pt:Y) were placed at the border of area CA3 and area CA1 along the Schaffer-Collateral pathway. ACSF-filled glass recording electrodes (1–3 MΩ) were placed in stratum radiatum of area CA1. Basal synaptic transmission was assessed for each slice by applying gradually increasing stimuli (0.5–15V), using a stimulus isolator (A-M Systems, Carlsborg, WA) and determining the input:output relationship. All subsequent stimuli applied to slices was equivalent to the level necessary to evoke a fEPSP that was ~40% of the maximal initial slope that could be evoked. Synaptic efficacy was continuously monitored (0.05 Hz). Sweeps were averaged together every 2 min. fEPSPs were amplified (A-M Systems Model 1800) and digitized (Digidata 1440, Molecular Devices, Sunnyvale, CA) prior to analysis (pClamp, Molecular Devices, Sunnyvale, CA). Stable baseline synaptic transmission was established for 30 min. Slices were given high-frequency stimulation (HFS) to induce long-term potentiation (LTP) using one train of 100 Hz for one second. Stimulus intensity of the HFS was matched to the intensity used in the baseline recordings. fEPSP initial slopes from averaged traces were normalized to those recorded during baseline. Two-way RM-ANOVA were used for electrophysiological data analysis with p < 0.05 as significance criteria.
10-13-mon-old male CamK-Atg7 cWT or CamK-Atg7 cKO mice were used (n = 8 - 10). The mice were placed in a conditioning chamber (Med Associates) for 2 min before the onset of a tone (conditioned stimulus) (30 s, 85 dB sound at 2800 Hz) and conditioned by a single electrical foot shock (0.45 mA) in the last 2 s. The mice were left in the chamber for another 30 s and placed back into their home cage. Contextual fear learning was measured in the same chamber 24 h after the training by monitoring the freezing for 5 min without electrical shock. Cued fear learning was measured 24 h after the contextual testing. The mice were placed in a novel chamber for 2 min (pre-conditioning). After that, the mice were exposed to the conditioned stimulus for 3 min, and the freezing was monitored. Freezing behavior was scored using FreezeView software (Med Associates Inc.).
Five-week-old Dat-Atg7 cWT and Dat-Atg7 cKO mice were treated with Alsterpaullone (A1136, A.G. Scientific) . The drug was dissolved in saline containing 20% DMSO/ 25% Tween80, sonicated, and injected intraperitoneally at a dose of 5 mg/kg every day for 3 weeks. After the final injection, the mice were perfused and processed for histological analyses. We used Dat-Atg7 cWT mice as controls for Dat-Atg7 cKO mice, to address potential phenotypes due to Cre transgene inserted at the DAT locus .
All comparisons between groups were made using the Mann-Whitney U-test (for two samples) or non-repeated measures ANOVA (for multiple samples). The values are expressed as the means ± S.E. A p value less than 0.05 is considered significant.
We would like to thank G. Di Paulo, and O. Hobert for suggestions and comments on the manuscript, R. Hen for generously providing Dat Cre/+ mice, P. Davies for generously providing phospho-tau antibodies, E. Kominami, T. Chiba, and K. Tanaka for generously providing Atg7 flox/flox mice, J.Q. Trojanowski and D. Dickson for electron microscopic analysis, and T. Iwasato, J. Dunning, C. Doege, H. Rhinn, D. MacLeod, W. Vanti, S. Vasishta for technical help. This work was supported by grants from Kanae Foundation for the Promotion of Medical Science, and Research Foundation ITSUU Laboratory to K.I. K.I. was a postdoctoral fellow of New York Stem Cell Foundation. This work was supported by grants from the Michael J. Fox Foundation, NINDS, and NIA to A.A.
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