Quantitative propagation of assembled human Tau from Alzheimer's disease brain in microfluidic neuronal cultures
2020; Elsevier BV; Volume: 295; Issue: 37 Linguagem: Inglês
10.1074/jbc.ra120.013325
ISSN1083-351X
AutoresAntigoni Katsikoudi, Elena Ficulle, Annalisa Cavallini, Gary J. Sharman, Amelie Guyot, Michele Zagnoni, Brian J. Eastwood, Michael Hutton, Suchira Bose,
Tópico(s)Cholinesterase and Neurodegenerative Diseases
ResumoTau aggregation and hyperphosphorylation is a key neuropathological hallmark of Alzheimer's disease (AD), and the temporospatial spread of Tau observed during clinical manifestation suggests that Tau pathology may spread along the axonal network and propagate between synaptically connected neurons. Here, we have developed a cellular model that allows the study of human AD-derived Tau propagation from neuron to neuron using microfluidic devices. We show by using high-content imaging techniques and an in-house developed interactive computer program that human AD-derived Tau seeds rodent Tau that propagates trans-neuronally in a quantifiable manner in a microfluidic culture model. Moreover, we were able to convert this model to a medium-throughput format allowing the user to handle 16 two-chamber devices simultaneously in the footprint of a standard 96-well plate. Furthermore, we show that a small molecule inhibitor of aggregation can block the trans-neuronal transfer of Tau aggregates, suggesting that the system can be used to evaluate mechanisms of Tau transfer and find therapeutic interventions. Tau aggregation and hyperphosphorylation is a key neuropathological hallmark of Alzheimer's disease (AD), and the temporospatial spread of Tau observed during clinical manifestation suggests that Tau pathology may spread along the axonal network and propagate between synaptically connected neurons. Here, we have developed a cellular model that allows the study of human AD-derived Tau propagation from neuron to neuron using microfluidic devices. We show by using high-content imaging techniques and an in-house developed interactive computer program that human AD-derived Tau seeds rodent Tau that propagates trans-neuronally in a quantifiable manner in a microfluidic culture model. Moreover, we were able to convert this model to a medium-throughput format allowing the user to handle 16 two-chamber devices simultaneously in the footprint of a standard 96-well plate. Furthermore, we show that a small molecule inhibitor of aggregation can block the trans-neuronal transfer of Tau aggregates, suggesting that the system can be used to evaluate mechanisms of Tau transfer and find therapeutic interventions. Alzheimer's disease (AD) is the most common cause of dementia, a neurological disorder that is currently believed to affect 35.6 million people worldwide and estimated to triple by 2050. A key neuropathological hallmark of AD and other tauopathies is the abnormal folding and hyperphosphorylation of Tau protein, which leads to generation of Tau filaments and neurofibrillary tangles. During the clinical manifestation of AD, a temporospatial spreading of Tau-positive neurofibrillary lesions is observed, suggesting that once Tau pathology is initiated it may spread along the axonal network and propagate between connected neuronal cells; moreover, the extent of Tau pathology strongly correlates with symptom severity and neuronal cell loss (1Braak H. Braak E. Neuropathological stageing of Alzheimer-related changes.Acta Neuropathol. 1991; 82 (1759558): 239-25910.1007/BF00308809Crossref PubMed Scopus (11172) Google Scholar). To classify Tau pathology in AD, Braak and Braak (1Braak H. Braak E. Neuropathological stageing of Alzheimer-related changes.Acta Neuropathol. 1991; 82 (1759558): 239-25910.1007/BF00308809Crossref PubMed Scopus (11172) Google Scholar) developed a six-tiered system of disease staging based on silver-stained, hyperphosphorylated Tau aggregates (1Braak H. Braak E. Neuropathological stageing of Alzheimer-related changes.Acta Neuropathol. 1991; 82 (1759558): 239-25910.1007/BF00308809Crossref PubMed Scopus (11172) Google Scholar, 2Braak H. Alafuzoff I. Arzberger T. Kretzschmar H. Del Tredici K. Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry.Acta Neuropathol. 2006; 112 (16906426): 389-40410.1007/s00401-006-0127-zCrossref PubMed Scopus (1733) Google Scholar, 3Braak H. Tredici K.D. Alzheimer's pathogenesis: is there neuron-to-neuron propagation?.Acta Neuropathol. 2011; 121 (21516512): 589-59510.1007/s00401-011-0825-zCrossref PubMed Scopus (245) Google Scholar). According to this staging system, hyperphosphorylated Tau accumulates first in the entorhinal cortex and locus coeruleus before the disease becomes symptomatic; the propagation of Tau pathology beyond entorhinal cortex and locus coeruleus by neuron-to-neuron transmission is initiated after accumulation of a high β-amyloid load in isocortical regions and is associated with symptoms of AD, according to pathological, clinical, and biomarker data (4Vasconcelos B. Stancu I.-C. Buist A. Bird M. Wang P. Vanoosthuyse A. Van Kolen K. Verheyen A. Kienlen-Campard P. Octave J.-N. Baatsen P. Moechars D. Dewachter I. Heterotypic seeding of Tau fibrillization by pre-aggregated Aβ provides potent seeds for prion-like seeding and propagation of Tau-pathology in vivo.Acta Neuropathol. 2016; 131 (26739002): 549-56910.1007/s00401-015-1525-xCrossref PubMed Scopus (95) Google Scholar). This temporal and spatial pattern of spreading observed in tauopathies supports the theory of trans-synaptic spreading of Tau, which has also been demonstrated in vivo and in cell culture models (5Ahmed Z. Cooper J. Murray T.K. Garn K. McNaughton E. Clarke H. Parhizkar S. Ward M.A. Cavallini A. Jackson S. Bose S. Clavaguera F. Tolnay M. Lavenir I. Goedert M. et al.A novel in vivo model of Tau propagation with rapid and progressive neurofibrillary tangle pathology: the pattern of spread is determined by connectivity, not proximity.Acta Neuropathol. 2014; 127 (24531916): 667-68310.1007/s00401-014-1254-6Crossref PubMed Scopus (296) Google Scholar, 6Clavaguera F. Hench J. Lavenir I. Schweighauser G. Frank S. Goedert M. Tolnay M. Peripheral administration of Tau aggregates triggers intracerebral tauopathy in transgenic mice.Acta Neuropathol. 2014; 127 (24362441): 299-30110.1007/s00401-013-1231-5Crossref PubMed Scopus (101) Google Scholar, 7Calafate S. Buist A. Miskiewicz K. Vijayan V. Daneels G. de Strooper B. de Wit J. Verstreken P. Moechars D. Synaptic contacts enhance cell-to-cell Tau pathology propagation.Cell Rep. 2015; 11 (25981034): 1176-118310.1016/j.celrep.2015.04.043Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 8Kaufman S.K. Del Tredici K. Thomas T.L. Braak H. Diamond M.I. Tau seeding activity begins in the transentorhinal/entorhinal regions and anticipates phospho-Tau pathology in Alzheimer's disease and PART.Acta Neuropathol. 2018; 136 (29752551): 57-6710.1007/s00401-018-1855-6Crossref PubMed Scopus (96) Google Scholar). However, alternative, nonsynaptic transfer of Tau between synaptically connected neurons has not been excluded (9Walsh D.M. Selkoe D.J. A critical appraisal of the pathogenic protein spread hypothesis of neurodegeneration.Nat. Rev. Neurosci. 2016; 17 (26988744): 251-26010.1038/nrn.2016.13Crossref PubMed Scopus (189) Google Scholar, 10Braak H. Braak E. Development of Alzheimer-related neurofibrillary changes in the neocortex inversely recapitulates cortical myelogenesis.Acta Neuropathol. 1996; 92 (8841666): 197-20110.1007/s004010050508Crossref PubMed Scopus (296) Google Scholar, 11Yan M.H. Wang X. Zhu X. Mitochondrial defects and oxidative stress in Alzheimer disease and Parkinson disease.Free Radic. Biol. Med. 2013; 62 (23200807): 90-10110.1016/j.freeradbiomed.2012.11.014Crossref PubMed Scopus (443) Google Scholar, 12Nave K.A. Werner H.B. Myelination of the nervous system: mechanisms and functions.Annu. Rev. Cell Dev. Biol. 2014; 30 (25288117): 503-53310.1146/annurev-cellbio-100913-013101Crossref PubMed Scopus (483) Google Scholar, 13Maphis N. Xu G. Kokiko-Cochran O.N. Jiang S. Cardona A. Ransohoff R.M. Lamb B.T. Bhaskar K. Reactive microglia drive Tau pathology and contribute to the spreading of pathological Tau in the brain.Brain. 2015; 138 (25833819): 1738-175510.1093/brain/awv081Crossref PubMed Scopus (269) Google Scholar, 14Asai H. Ikezu S. Tsunoda S. Medalla M. Luebke J. Haydar T. Wolozin B. Butovsky O. Kügler S. Ikezu T. Depletion of microglia and inhibition of exosome synthesis halt Tau propagation.Nat. Neurosci. 2015; 18 (26436904): 1584-159310.1038/nn.4132Crossref PubMed Scopus (787) Google Scholar, 15Katsinelos T. Zeitler M. Dimou E. Karakatsani A. Müller H.-M. Nachman E. Steringer J.P. Ruiz de Almodovar C. Nickel W. Jahn T.R. Unconventional secretion mediates the trans-cellular spreading of Tau.Cell Rep. 2018; 23 (29768203): 2039-205510.1016/j.celrep.2018.04.056Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Cellular and animal models recapitulating features of tauopathies provide a useful tool to investigate the causes and consequences of Tau aggregation. Cellular models are useful for understanding disease mechanisms, screening, and profiling compounds that interfere with Tau aggregation. Microfluidic devices represent a miniaturized alternative to recapitulate disease conditions (16Park J.W. Kim H.J. Kang M.W. Jeon N.L. Advances in microfluidics-based experimental methods for neuroscience research.Lab. Chip. 2013; 13 (23306275): 509-52110.1039/c2lc41081hCrossref PubMed Scopus (65) Google Scholar). These devices were first created to study axonal damage and repair (17Taylor A.M. Blurton-Jones M. Rhee S.W. Cribbs D.H. Cotman C.W. Jeon N.L. A microfluidic culture platform for CNS axonal injury, regeneration and transport.Nat. Methods. 2005; 2 (16094385): 599-60510.1038/nmeth777Crossref PubMed Scopus (833) Google Scholar), but their rapid development has now received attention from multiple scientific fields. Typically, to facilitate neuronal culture, microfluidic devices are fabricated using gas-permeable silicone elastomer polydimethylsiloxane (PDMS). The most common device layout used to create synaptically connected but environmentally isolated neuronal populations (18Robertson G. Bushell T.J. Zagnoni M. Chemically induced synaptic activity between mixed primary hippocampal co-cultures in a microfluidic system.Integr. Biol. (Camb.). 2014; 6 (24796407): 636-64410.1039/c3ib40221eCrossref PubMed Scopus (30) Google Scholar, 19MacKerron C. Robertson G. Zagnoni M. Bushell T.J. A microfluidic platform for the characterisation of CNS active compounds.Sci. Rep. 2017; 7 (29146949): 1569210.1038/s41598-017-15950-0Crossref PubMed Scopus (13) Google Scholar) comprises two microfluidic culture compartments where neuronal cells grow processes and subsequently become connected by an array of microchannels through which axonal growth is guided; whether axons or dendrites can reach the opposite culture compartment depends on the length and shape of the microchannels (17Taylor A.M. Blurton-Jones M. Rhee S.W. Cribbs D.H. Cotman C.W. Jeon N.L. A microfluidic culture platform for CNS axonal injury, regeneration and transport.Nat. Methods. 2005; 2 (16094385): 599-60510.1038/nmeth777Crossref PubMed Scopus (833) Google Scholar, 20Holloway P.M. Hallinan G.I. Hegde M. Lane S.I.R. Deinhardt K. West J. Asymmetric confinement for defining outgrowth directionality.Lab. Chip. 2019; 19 (30899932): 1484-148910.1039/C9LC00078JCrossref PubMed Google Scholar, 21Park J. Koito H. Li J. Han A. Microfluidic compartmentalized co-culture platform for CNS axon myelination research.Biomed. Microdevices. 2009; 11 (19554452): 1145-115310.1007/s10544-009-9331-7Crossref PubMed Scopus (134) Google Scholar). This structure enables axons from one culture compartment to form synaptic connections with dendrites from the other compartment. The main positive features of the microfluidic platform are small reaction volumes, leading to minimal reagent usage, and the control over spatial and temporal separation of neuronal populations, which allows simulation of a neuronal network (22Zilberzwige-Tal S. Gazit E. Go with the flow: microfluidics approaches for amyloid research.Chem. Asian J. 2018; 13 (30117682): 3437-344710.1002/asia.201801007Crossref PubMed Scopus (9) Google Scholar). Because one of the main pathophysiological characteristics of AD is neuronal cell death with loss of synapses and neuronal network within the brain, microfluidic devices provide an ideal platform to study neuronal connectivity and spread of Tau pathology. Over the past two decades, microfluidics technology has significantly advanced, and microfluidic devices have now been employed in multiple publications to model neuronal networks and mimic Tau spreading in human tauopathies (7Calafate S. Buist A. Miskiewicz K. Vijayan V. Daneels G. de Strooper B. de Wit J. Verstreken P. Moechars D. Synaptic contacts enhance cell-to-cell Tau pathology propagation.Cell Rep. 2015; 11 (25981034): 1176-118310.1016/j.celrep.2015.04.043Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 23Wu J.W. Herman M. Liu L. Simoes S. Acker C.M. Figueroa H. Steinberg J.I. Margittai M. Kayed R. Zurzolo C. Di Paolo G. Duff K.E. Small misfolded Tau species are internalized via bulk endocytosis and anterogradely and retrogradely transported in neurons.J. Biol. Chem. 2013; 288 (23188818): 1856-187010.1074/jbc.M112.394528Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 24Dujardin S. Lécolle K. Caillierez R. Bégard S. Zommer N. Lachaud C. Carrier S. Dufour N. Aurégan G. Winderickx J. Hantraye P. Déglon N. Colin M. Buée L. Neuron-to-neuron wild-type Tau protein transfer through a trans-synaptic mechanism: relevance to sporadic tauopathies.Acta Neuropathol. Commun. 2014; 2 (24479894): 1410.1186/2051-5960-2-14Crossref PubMed Scopus (156) Google Scholar, 25Usenovic M. Niroomand S. Drolet R.E. Yao L. Gaspar R.C. Hatcher N.G. Schachter J. Renger J.J. Parmentier-Batteur S. Internalized Tau oligomers cause neurodegeneration by inducing accumulation of pathogenic Tau in human neurons derived from induced pluripotent stem cells.J. Neurosci. 2015; 35 (26490863): 14234-1425010.1523/JNEUROSCI.1523-15.2015Crossref PubMed Scopus (126) Google Scholar, 26Takeda S. Wegmann S. Cho H. DeVos S.L. Commins C. Roe A.D. Nicholls S.B. Carlson G.A. Pitstick R. Nobuhara C.K. Costantino I. Frosch M.P. Müller D.J. Irimia D. Hyman B.T. et al.Neuronal uptake and propagation of a rare phosphorylated high-molecular-weight Tau derived from Alzheimer's disease brain.Nat. Commun. 2015; 6 (26458742): 849010.1038/ncomms9490Crossref PubMed Scopus (183) Google Scholar, 27Wu J.W. Hussaini S.A. Bastille I.M. Rodriguez G.A. Mrejeru A. Rilett K. Sanders D.W. Cook C. Fu H. Boonen R.A.C.M. Herman M. Nahmani E. Emrani S. Figueroa Y.H. Diamond M.I. et al.Neuronal activity enhances Tau propagation and Tau pathology in vivo.Nat. Neurosci. 2016; 19 (27322420): 1085-109210.1038/nn.4328Crossref PubMed Scopus (355) Google Scholar, 28Wang Y. Balaji V. Kaniyappan S. Krüger L. Irsen S. Tepper K. Chandupatla R. Maetzler W. Schneider A. Mandelkow E. Mandelkow E.-M. The release and trans-synaptic transmission of Tau via exosomes.Mol. Neurodegener. 2017; 12 (28086931): 510.1186/s13024-016-0143-yCrossref PubMed Scopus (311) Google Scholar). However, quantification of Tau pathology and spreading in these models was challenging, the robustness of the signal window could not be determined, and the throughput of the assays was minimal. Here, we designed microfluidic plates so that the user could handle 16 two-chamber devices simultaneously in the footprint of a standard 96-well plate, thus increasing throughput of information and facilitating interfacing with high-content imaging (HCI) equipment. We used primary nontransgenic rat cortical neurons (RCNs) seeded with human (h) AD Tau as a model for human sporadic tauopathies and were able set up a quantifiable and reproducible imaging assay in a medium-throughput microfluidic format for detecting formation and propagation of Tau aggregates. This model system could be ideal for testing the effect of potential Tau therapeutics that modulate Tau trans-neuronal propagation. Furthermore, we believe this to be the first time that a robust, reproducible methodology has been established for quantifying aggregates in microfluidic chambers, because this technology has previously struggled with delivering quantitative outcomes. This is despite the broad and increasing use of this technology. Protein aggregate propagation is central to many neurodegenerative disease progressions; therefore, the method can be further adapted to measure propagation of any prion-like disease. To extract seeding-competent material from h AD brains, we used a Sarkosyl extraction protocol adapted from Greenberg and Davies (29Greenberg S.G. Davies P. A preparation of Alzheimer paired helical filaments that displays distinct Tau proteins by polyacrylamide gel electrophoresis.Proc. Natl. Acad. Sci. U.S.A. 1990; 87 (2116006): 5827-583110.1073/pnas.87.15.5827Crossref PubMed Scopus (649) Google Scholar). This purified hAD seed was analyzed via AlphaScreen to determine Tau concentration, and 18 nm (optimal concentration as shown in Fig. S1) was added to RCNs at DIV 7; their cell lysate was biochemically characterized for Sarkosyl-insoluble Tau levels at DIV 21. Western blotting analysis of their Sarkosyl-insoluble pellet demonstrates that hAD seed when added to RCNs seeds rodent Tau as shown using the rodent specific antibody, T49 (Fig. 1A) that resulted in the formation of AT8-positive, Sarkosyl-insoluble Tau (Fig. 1B). We have analyzed 3R and 4R Tau expression in a time course in RCN (Fig. S2). The expression of 3R and 4R Tau increased over time, peaking at DIV 15 and then started to decline. We hypothesize that the endogenous aggregates that would template from hAD seed (starting from DIV 7) will contain both 3R and 4R Tau. By HCI we show that when hAD seed was added to RCNs at DIV 7 and the cells were fixed at DIV 21 and stained with the same rodent specific Tau antibody T49, the hAD seed could induce neuritic thread-like inclusions in a 96-well plate assay (Fig. 1C). In summary, hAD seed can seed rodent Tau to form neuritic thread-like inclusions that are comprised of Sarkosyl-insoluble 3R and 4R Tau, consistent with findings in mouse cortical neurons (30Guo J.L. Narasimhan S. Changolkar L. He Z. Stieber A. Zhang B. Gathagan R.J. Iba M. McBride J.D. Trojanowski J.Q. Lee V.M.Y. Unique pathological Tau conformers from Alzheimer's brains transmit Tau pathology in nontransgenic mice.J. Exp. Med. 2016; 213 (27810929): 2635-265410.1084/jem.20160833Crossref PubMed Scopus (197) Google Scholar). Dendrite-specific MAP2 staining (Fig. 2, A and C) revealed no co-localization with T49 staining, whereas microtubule-specific MAP1B staining (Fig. 2, B and D) showed co-localization. Because the T49 inclusions did not co-localize with the dendrite-specific marker MAP2, we conclude that the T49-positive Tau inclusions are axonal and somatic because they co-localized with MAP1B, a neuronal marker that is present in axonal and somatic microtubules. In developing neurons, MAP1B and Tau are the main MAPs found in axons, whereas MAP2 is found in dendrites (31Avila J. Dominguez J. Diaz-Nido J. Regulation of microtubule dynamics by microtubule-associated protein expression and phosphorylation during neuronal development.Int. J. Dev. Biol. 1994; 38 (8074993): 13-25PubMed Google Scholar).Figure 2T49-positive inclusions are localized in the axons and soma of RCNs. A–D, sparse co-localization between the T49 neuritic thread-like inclusions in RCNs and MAP2, a specific dendritic marker (A and zoomed image in C), but good co-localization with MAP1B, a neuronal cytoskeleton marker (B and zoomed image in D), indicating that the inclusions observed after seeding with hAD seed are not dendritic but somatic and axonal. Arrowheads in the D indicate co-localization between MAP1B and T49. Images were acquired with Opera Phenix and 20× objective. Figure bar, 50 μm; zoomed image bar, 25 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We moved on to test whether our hAD seed preparation could seed RCNs cultured in two-compartment microfluidic devices. For our initial experimental setup, RCNs were cultured over the course of 3 weeks in two-compartment PDMS microfluidic devices that were irreversibly plasma-bonded on the glass surface of 6-well imaging plates (Fig. 3A). The hAD seed was added on the seeded side, and the cells were fixed in devices; this plate format was used for HCI to detect aggregated pathological Tau at the propagation side (Fig. 3B). What is characteristic about the microfluidic devices used is that only the axons (Fig. 3C, Microtubule Associated Protein Tau (MAPT) staining) can grow through the 450-μm-long microgroove barriers that separate the two compartments but not the dendrites (Fig. 3C, MAP2 staining). This supports the idea that microfluidic devices can be used as an in vitro cellular model to study trans-neuronal and axonal propagation of aggregated Tau and is consistent with previous literature reports with recombinant and transgenic fibrils in transgenic cellular models (27Wu J.W. Hussaini S.A. Bastille I.M. Rodriguez G.A. Mrejeru A. Rilett K. Sanders D.W. Cook C. Fu H. Boonen R.A.C.M. Herman M. Nahmani E. Emrani S. Figueroa Y.H. Diamond M.I. et al.Neuronal activity enhances Tau propagation and Tau pathology in vivo.Nat. Neurosci. 2016; 19 (27322420): 1085-109210.1038/nn.4328Crossref PubMed Scopus (355) Google Scholar, 32Takeda S. Commins C. DeVos S.L. Nobuhara C.K. Wegmann S. Roe A.D. Costantino I. Fan Z. Nicholls S.B. Sherman A.E. Trisini Lipsanopoulos A.T. Scherzer C.R. Carlson G.A. Pitstick R. Peskind E.R. et al.Seed-competent high-molecular-weight Tau species accumulates in the cerebrospinal fluid of Alzheimer's disease mouse model and human patients.Ann. Neurol. 2016; 80 (27351289): 355-36710.1002/ana.24716Crossref PubMed Scopus (61) Google Scholar). Here, we have demonstrated that seeding RCNs with hAD seed results in propagation of endogenously generated rodent Tau in a microfluidic two-compartment model as revealed by a higher objective magnification (Fig. 3, D and E). Characteristically, the morphology of the aggregated neuronal Tau formed after seeding RCNs with hAD seed resembled the neuritic thread-like pathology observed previously in mouse cortical neurons (30Guo J.L. Narasimhan S. Changolkar L. He Z. Stieber A. Zhang B. Gathagan R.J. Iba M. McBride J.D. Trojanowski J.Q. Lee V.M.Y. Unique pathological Tau conformers from Alzheimer's brains transmit Tau pathology in nontransgenic mice.J. Exp. Med. 2016; 213 (27810929): 2635-265410.1084/jem.20160833Crossref PubMed Scopus (197) Google Scholar). The inclusions observed in the propagation side of the microfluidic devices exhibited the same neuritic thread-like pathology as observed in the seeded side (Fig. 3, D and E) and in the 96-well assay (Fig. 1C). Optimization of the seeding concentration for the hAD seed in 96-well RCN cultures revealed that 18 nm (0.75 μg/ml) is a concentration that provides a statistically significant signal window (Fig. S1). The parameters considered were the count of T49 neuritic thread-like inclusions and the hAD seed dose response. Concentrations beyond 18 nm of hAD seed demonstrated a saturation in the signal window and occasional cell toxicity (Fig. S1). During our immunocytochemistry protocol, we used methanol as previously described (30Guo J.L. Narasimhan S. Changolkar L. He Z. Stieber A. Zhang B. Gathagan R.J. Iba M. McBride J.D. Trojanowski J.Q. Lee V.M.Y. Unique pathological Tau conformers from Alzheimer's brains transmit Tau pathology in nontransgenic mice.J. Exp. Med. 2016; 213 (27810929): 2635-265410.1084/jem.20160833Crossref PubMed Scopus (197) Google Scholar) to fix the cells and remove soluble proteins. We observed diffuse background staining, mostly evident in the unseeded cells in both compartments, which could represent residual soluble protein (Fig. 3, D and E). Furthermore, to determine whether the propagation observed is a real effect and not an artifact of the two-compartment model caused by the axonal growth being longer than the microgrooves distance, three-compartment microfluidic devices were used, keeping the experimental parameters consistent with the two-compartment model described above. When RCNs were plated in the first (C1), middle (C2), and third (C3) compartments, hAD seed was added to C1 on DIV 7, and cells were fixed on DIV 21, T49 staining revealed inclusions in C3 (Fig. 4B). When RCNs were plated only in C1 and C3, leaving the middle compartment C2 empty, T49 staining revealed no inclusions in C3 (Fig. 4C). Plating cells only in the outer chambers decreased the probability of forming synapses, suggesting that a lack of synapse formation is a barrier to propagation, consistent with findings by Calafate et al. (7Calafate S. Buist A. Miskiewicz K. Vijayan V. Daneels G. de Strooper B. de Wit J. Verstreken P. Moechars D. Synaptic contacts enhance cell-to-cell Tau pathology propagation.Cell Rep. 2015; 11 (25981034): 1176-118310.1016/j.celrep.2015.04.043Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). However, neuritic inclusions are observed in C2. This could be because axons that grow through the microgrooves (Fig. 3C) make the detection of neuritic inclusions obvious in C2. These control experiments showed that hAD seeding results in true propagation because there is no axonal overlap between neurons in C1 and C3.Figure 4Demonstration that propagation of endogenously aggregated rodent Tau is a real effect and not a device artifact in three-chamber microfluidic devices. A, RCNs were plated on DIV 0 in compartments C1, C2, and C3 of a three-compartment microfluidic device with the volume gradient increasing from C1 to C3 and the neurons of C1 were seeded with 18 nm of hAD seed on DIV 7. B, the cells were fixed 2 weeks later following the timelines of the two-compartment microfluidic devices and stained for rodent specific Tau, T49. T49-positive inclusions were detected in C3. C, when no RCNs were plated in C2, there were no T49-positive inclusions detected in compartment C3, suggesting that there was no axonal growth overlap between the two neuronal populations. Image acquisition with Opera Phenix 20× magnification, image tiled montages with zooms for each compartment in the yellow squares. Figure bar, 50 μm; zoomed image bar, 12.5 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although the propagation of Tau fibrils has been demonstrated before, the evidence has been qualitative rather than quantitative. To demonstrate the robustness of our microfluidic assay, we developed a novel high-content imaging pipeline to quantify the propagation of hAD-seeded rodent Tau inclusions in our microfluidic devices. Fig. 5 summarizes the main steps of the Harmony software analysis pipeline used to quantify the neuritic thread-like inclusions when RCNs in two-compartment microfluidic devices were seeded with the hAD seed. By processing the raw images for their fluorescent background and using machine learning to identify the T49-positive neuritic thread-like regions (Tau-positive zones), we quantified the number of T49-positive neuritic thread-like inclusions (Fig. 5). To quantify and separate our results between the two microfluidic compartments, (i.e. seeded and propagation compartments), we developed a Java computer program named the Cell Counter. Fig. 6 summarizes the main steps followed when using the Cell Counter. This computer program enabled us to automate and quantify Tau pathology in a neuronal microfluidic model.Figure 6A computer program for the visualization and processing of the T49 neuritic-like thread inclusions in the seeded (top channel) and propagation (bottom channel) side of two-compartment microfluidic devices. Typical example of RCNs in a two-compartment microfluidic device. The cells were processed with HCI, and the results were loaded into the Cell Counter computer program. A and B illustrate the pixel visualization of the nuclei (Hoechst staining), and C shows the T49 neuritic-like threads in seeded (top) and propagation (bottom) compartments. The regions of interest in the top and bottom are auto-selected and processed for the number of nuclei and T49 neuritic-like threads by the program, but it also allows for human review and override by exception.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To optimize the experimental and analysis conditions, we ran a factorial experiment to assess the impact of seeding day, fixing day, and plate on the seeding and propagation side. RCNs plated in microfluidic devices in 6-well plates (one 2-compartment device per well) were seeded on DIV 3, 7, or 10 (seeding day, or SD) and fixed on DIV 18, 21, or 24 (final day, or FD). The standard conditions were SD = 7, FD = 21, and this along with SD/FD combinations 3/18, 3/24, 10/18, and 10/24 were tested on five 6-well plates containing microfluidic devices. Each plate had unseeded wells to assess the assay background conditions. The experiment was analyzed by mixed models analysis of variance. The results showed background levels did vary substantially across plates (>60% of total variation) and between seeding and propagation sides, indicating on-plate controls for background levels are required for this technology, and estimates of background levels should be subtracted from all seeded wells. The results showed that total cell counts decreased as SD is postponed from DIV 3 to DIV 10 and as FD is postponed from DIV 18 to DIV
Referência(s)