Selective Molecular Alterations in the Autophagy Pathway in Patients with Lewy Body Disease and in Models of α-Synucleinopathy methods
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Cases and Neuropathological Evaluation
For routine neuropathological diagnosis, paraffin sections from neocortical, limbic and subcortical regions were stained with haematoxylin and eosin (H&E) or thioflavine-S,, and Braak stage was assessed. Based on previously published clinical and pathological findings, cases were subdivided into: 1) non-demented age-matched controls, 2) AD cases, and 3) DLB cases. All cases met the Consortium to Establish a Registry for AD (CERAD) and National Institute of Aging (NIA) criteria for diagnosis and displayed neuritic plaques and tangle formation in the neocortex and limbic system,. The diagnosis of DLB was based on the clinical presentation of dementia and the pathological findings of LBs in the locus coeruleus, substantia nigra (SN), or nucleus basalis of Meynert, as well as in cortical regions. LBs were detected using H&E stain or anti-ubiquitin and anti-α-syn antibodies as r...
a-Synuclein Transgenic Mice and Treatments
Additional experiments with the α-syn tg mice included treatments with the autophagy activator rapamycin (Sigma-Aldrich, St. Louis, MO). Because rapamycin poorly crosses into the CNS, it was infused intra-cerebrally into the lateral ventricle of 9-month old mice at a concentration of 20mg/kg. Briefly as previously described, mice were anesthetized and under sterile conditions a 26 gauge stainless steel cannula was implanted stereotaxically into the lateral ventricle using the bregma as a reference (Franklin and Paxinos, bregma 0.5mm; 1.1mm lateral; depth 3mm) and secured to the cranium using superglue. The cannula was connected via a 5 mm coil of V3 Biolab vinyl to a model 1007D osmotic minipump (Alzet, Cupertino, CA) surgically placed subcutaneously beneath the shoulder. The solutions were delivered at a flow rate of 0.5ul per hour for 2 weeks. The pump was left for an addition...
Construction of Lentiviral Vectors and Injection of LV-Atg7 into α-Synuclein Transgenic Mice
A total of 12 α-syn tg mice from line D (9 months old) were injected with 3 µl of the LV preparations (2.5 × 10 7 TU) into the temporal cortex and hippocampus (using a 5 µl Hamilton syringe). Briefly, as previously described, mice were placed under anesthesia on a Koft stereotaxic apparatus and coordinates (hippocampus: AP -2.0 mm, lateral 1.5 mm, depth 1.3 mm and cortex: AP -.5 mm, lateral 1.5 mm, depth 1.0 mm) were determined as per the Franklin and Paxinos Atlas. The LVs were delivered using a Hamilton syringe connected to a hydraulic system to inject the solution at a rate of 1 µl every 2 min. To allow diffusion of the solution into the brain tissue, the needle was left for an additional 5 min after the completion of the injection. Mice received unilateral injections (right side) to allow comparisons against the contralateral side, with either L...
Construction of Lentiviral Vectors and Injection of LV-Atg7 into α-Synuclein Transgenic Mice
Following NIH guidelines for the humane treatment of animals, mice were anesthetized with chloral hydrate and flush-perfused transcardially with 0.9% saline. Brains and peripheral tissues were removed and divided sagittally. The right hemibrain was post-fixed in phosphate-buffered 4% PFA (pH 7.4) at 4°C for 48 hours for neuropathological analysis, while the left hemibrain was snap-frozen and stored at -70°C for subsequent RNA and protein analysis.
Cell Culture and Treatments
To investigate whether LC3 levels are modulated by α-syn or Atg7 over-expression or knockdown, LC3 levels were analyzed in coverslips with LC3-GFP. B103 cells were grown as described above and were then plated onto poly L-lysine coated glass coverslips at a density of 5 × 10 4 cells. Five hours after plating, cells were infected with the LV-αsyn and/or LV-Atg7 or LV-shAtg7 (or controls) and incubated for 48 hours. All coverslips were also co-infected with an LV expressing LC3-GFP at an MOI of 40. Cultures were then washed 2 × with serum-free DMEM and then fed either complete media or serum-free media for 12 hours before fixation with 4% PFA. Briefly as previously described, coverslips were treated with Prolong Gold antifading reagent with DAPI (Invitrogen) and imaged with the LSCM to determine the number of GFP-positive granular structures consistent with autophagoly...
Immunohistochemistry, Image Analysis and Laser Scanning Confocal Microscopy
Briefly, as previously described, free-floating 40 µm thick vibratome sections were washed with Tris buffered saline (TBS, pH 7.4), pre-treated in 3% H 2 O 2, and blocked with 10% serum (Vector Laboratories, Burlingame, CA), 3% bovine serum albumin (Sigma), and 0.2% gelatin in TBS-Tween (TBS-T). For human brains, sections from the temporal cortex were used; for the mice sagittal sections from the complete brain were studied. Sections were incubated at 4°C overnight with the primary antibodies. Sections were then incubated in secondary antibody (1∶75, Vector), followed by Avidin D-horseradish peroxidase (HRP, ABC Elite, Vector) and reacted with diaminobenzidine (DAB, 0.2 mg/ml) in 50 mM Tris (pH 7.4) with 0.001% H 2 O 2. Control experiments consisted of incubation with pre-immune rabbit serum. To investigate the effects of postmortem delay and fixation on the levels...
Western Blot Analysis
Immunoblot analysis was performed as previously described. Briefly, brain homogenates (temporal cortex for human tissues and cortex for mice) or cultured cells were solubilized in lysis buffer (1% Triton X-100, 10% glycerol, 50 mM HEPES, pH 7.4, 140 mM NaCl, 1mM EDTA, 1mM Na 3 VO 4, 20 mM β-glycerophosphate, and proteinase inhibitor cocktails). Brain homogenates were separated into cytosolic and membrane fractions by centrifugation at 100,000 × g for 1 hr at 4C. Isolation of lysosomal fractions from brain tissue was performed by differential centrifugation in a sucrose-based buffer essentially as previously described. Briefly, tissues were minced with a razor blade and homogenized in ice-cold 50 mM Tris/HCl buffer, pH = 7.4, containing 0.25 M sucrose, 10 mM EDTA, 3 mM MgCl 2, with protease and phosphatase inhibitor cocktails (Calbiochem) added fresh (sucrose/Tr...
Lentivirus Delivery of Atg7 Rescues α-Synuclein Accumulation and Neuronal Deficits in Transgenic Mice
For panels A-M, vibratome sections from non tg and α-syn tg mice that received LV injections into the cortex and hippocampus were immunolabeled with antibodies against Atg7 or α-syn and imaged with a digital microscope. Panels A′-M′ represent higher-power images from the hippocampus of the corresponding low-power panels in panels A-M. For panels O-T, effects of rapamycin treatment on α-syn accumulation, autophagy and neuronal integrity in the brains of α-syn tg mice. For panels A-M, vibratome sections from the hippocampus of non tg and α-syn tg mice were immunolabeled with an antibody against MAP2 and imaged with a laser scanning confocal microscope, and images were obtained from the temporal cortex. (A-F) Representative sections from the brains of non tg (A, B) and α-syn tg mice (C-F) that received in...
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Quantify the primary readouts for this experiment: Protein expression by western blot.
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Report representative outputs alongside summary comparisons for Protein expression by western blot.
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Evidence quotes (8)
For routine neuropathological diagnosis, paraffin sections from neocortical, limbic and subcortical regions were stained with haematoxylin and eosin (H&E) or thioflavine-S,, and Braak stage was assessed. Based on previously published clinical and pathological findings, cases were subdivided into: 1) non-demented age-matched controls, 2) AD cases, and 3) DLB cases. All cases met the Consortium to Establish a Registry for AD (CERAD) and National Institute of Aging (NIA) criteria for diagnosis and displayed neuritic plaques and tangle formation in the neocortex and limbic system,. The diagnosis of DLB was based on the clinical presentation of dementia and the pathological findings of LBs in the locus coeruleus, substantia nigra (SN), or nucleus basalis of Meynert, as well as in cortical regions. LBs were detected using H&E stain or anti-ubiquitin and anti-α-syn antibodies as recommended by the Consortium on DLB criteria for a pathologic diagnosis of DLB. In addition to the presence of LBs, the great majority of these cases displayed sufficient plaques and tangles to be classified as Braak stages III-IV. Specifically, DLB cases had abundant plaques in the neocortex...
Additional experiments with the α-syn tg mice included treatments with the autophagy activator rapamycin (Sigma-Aldrich, St. Louis, MO). Because rapamycin poorly crosses into the CNS, it was infused intra-cerebrally into the lateral ventricle of 9-month old mice at a concentration of 20mg/kg. Briefly as previously described, mice were anesthetized and under sterile conditions a 26 gauge stainless steel cannula was implanted stereotaxically into the lateral ventricle using the bregma as a reference (Franklin and Paxinos, bregma 0.5mm; 1.1mm lateral; depth 3mm) and secured to the cranium using superglue. The cannula was connected via a 5 mm coil of V3 Biolab vinyl to a model 1007D osmotic minipump (Alzet, Cupertino, CA) surgically placed subcutaneously beneath the shoulder. The solutions were delivered at a flow rate of 0.5ul per hour for 2 weeks. The pump was left for an additional 2 weeks and mice were euthanized one month after the initiation of the infusions. Brains were removed and divided sagittally. One hemibrain was postfixed in phosphate-buffered 4% PFA, pH 7.4, at 4°C for 48 h and sectioned at 40 µm with a Vibratome 2000 (Leica, Nussloch, Germany) and pla...
A total of 12 α-syn tg mice from line D (9 months old) were injected with 3 µl of the LV preparations (2.5 × 10 7 TU) into the temporal cortex and hippocampus (using a 5 µl Hamilton syringe). Briefly, as previously described, mice were placed under anesthesia on a Koft stereotaxic apparatus and coordinates (hippocampus: AP -2.0 mm, lateral 1.5 mm, depth 1.3 mm and cortex: AP -.5 mm, lateral 1.5 mm, depth 1.0 mm) were determined as per the Franklin and Paxinos Atlas. The LVs were delivered using a Hamilton syringe connected to a hydraulic system to inject the solution at a rate of 1 µl every 2 min. To allow diffusion of the solution into the brain tissue, the needle was left for an additional 5 min after the completion of the injection. Mice received unilateral injections (right side) to allow comparisons against the contralateral side, with either LV-Atg7 (n = 6), or LV-control (n = 6). Additional controls were performed by injecting non tg littermates with either LV-Atg7 (n = 6), or LV-control (n = 6). Mice survived for 1 month after the lentiviral injection. As an additional control for LV inje...
Following NIH guidelines for the humane treatment of animals, mice were anesthetized with chloral hydrate and flush-perfused transcardially with 0.9% saline. Brains and peripheral tissues were removed and divided sagittally. The right hemibrain was post-fixed in phosphate-buffered 4% PFA (pH 7.4) at 4°C for 48 hours for neuropathological analysis, while the left hemibrain was snap-frozen and stored at -70°C for subsequent RNA and protein analysis.
To investigate whether LC3 levels are modulated by α-syn or Atg7 over-expression or knockdown, LC3 levels were analyzed in coverslips with LC3-GFP. B103 cells were grown as described above and were then plated onto poly L-lysine coated glass coverslips at a density of 5 × 10 4 cells. Five hours after plating, cells were infected with the LV-αsyn and/or LV-Atg7 or LV-shAtg7 (or controls) and incubated for 48 hours. All coverslips were also co-infected with an LV expressing LC3-GFP at an MOI of 40. Cultures were then washed 2 × with serum-free DMEM and then fed either complete media or serum-free media for 12 hours before fixation with 4% PFA. Briefly as previously described, coverslips were treated with Prolong Gold antifading reagent with DAPI (Invitrogen) and imaged with the LSCM to determine the number of GFP-positive granular structures consistent with autophagolysosomes using semiautomatic image analysis system and the ImageQuant software. For each condition an average of 50 cells were analyzed.
Briefly, as previously described, free-floating 40 µm thick vibratome sections were washed with Tris buffered saline (TBS, pH 7.4), pre-treated in 3% H 2 O 2, and blocked with 10% serum (Vector Laboratories, Burlingame, CA), 3% bovine serum albumin (Sigma), and 0.2% gelatin in TBS-Tween (TBS-T). For human brains, sections from the temporal cortex were used; for the mice sagittal sections from the complete brain were studied. Sections were incubated at 4°C overnight with the primary antibodies. Sections were then incubated in secondary antibody (1∶75, Vector), followed by Avidin D-horseradish peroxidase (HRP, ABC Elite, Vector) and reacted with diaminobenzidine (DAB, 0.2 mg/ml) in 50 mM Tris (pH 7.4) with 0.001% H 2 O 2. Control experiments consisted of incubation with pre-immune rabbit serum. To investigate the effects of postmortem delay and fixation on the levels of mTor immunoreactivity, preliminary studies were performed in a subset of cases (n = 5) with postmortem delay ranging from 4-48 h.
Immunoblot analysis was performed as previously described. Briefly, brain homogenates (temporal cortex for human tissues and cortex for mice) or cultured cells were solubilized in lysis buffer (1% Triton X-100, 10% glycerol, 50 mM HEPES, pH 7.4, 140 mM NaCl, 1mM EDTA, 1mM Na 3 VO 4, 20 mM β-glycerophosphate, and proteinase inhibitor cocktails). Brain homogenates were separated into cytosolic and membrane fractions by centrifugation at 100,000 × g for 1 hr at 4C. Isolation of lysosomal fractions from brain tissue was performed by differential centrifugation in a sucrose-based buffer essentially as previously described. Briefly, tissues were minced with a razor blade and homogenized in ice-cold 50 mM Tris/HCl buffer, pH = 7.4, containing 0.25 M sucrose, 10 mM EDTA, 3 mM MgCl 2, with protease and phosphatase inhibitor cocktails (Calbiochem) added fresh (sucrose/Tris buffer). After filtration through a mesh to remove cell debris and capsular fragments, the homogenate was centrifuged at 1000 g for 5 min to obtain the nuclear fraction (pellet 1, P1). The supernatant was centrifuged again at 10,000 g for 10 min to collect the mitochondrial fraction (pellet 2, P2...
For panels A-M, vibratome sections from non tg and α-syn tg mice that received LV injections into the cortex and hippocampus were immunolabeled with antibodies against Atg7 or α-syn and imaged with a digital microscope. Panels A′-M′ represent higher-power images from the hippocampus of the corresponding low-power panels in panels A-M. For panels O-T, effects of rapamycin treatment on α-syn accumulation, autophagy and neuronal integrity in the brains of α-syn tg mice. For panels A-M, vibratome sections from the hippocampus of non tg and α-syn tg mice were immunolabeled with an antibody against MAP2 and imaged with a laser scanning confocal microscope, and images were obtained from the temporal cortex. (A-F) Representative sections from the brains of non tg (A, B) and α-syn tg mice (C-F) that received injections with LV-control (A-D) or LV-Atg7 (E, F) and were immunolabeled with an antibody against Atg7. Images show sections from the hemisphere ipsilateral (ipsi) or contralateral (contra) to the sites of injection. (G) Semi-quantitative image analysis of Atg7 immunoreactivity in non tg and &#...
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