Summary

Using Live-Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons

Published: December 22, 2023
doi:

Summary

Here, we demonstrate how to combine transfection of primary hippocampal rodent neurons with live-cell confocal imaging to analyze pathological protein-induced effects on axonal transport and identify mechanistic pathways mediating these effects.

Abstract

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal transport is disrupted in multiple neurodegenerative diseases, and projection neurons are particularly vulnerable because of the need to transport cellular materials over long distances and sustain substantial axonal mass. Pathological modifications of several disease-related proteins negatively affect transport, including tau, amyloid-β, α-synuclein, superoxide dismutase, and huntingtin, providing a potential common mechanism by which pathological proteins exert toxicity in disease. Methods to study these toxic mechanisms are necessary to understand neurodegenerative disorders and identify potential therapeutic interventions.

Here, cultured primary rodent hippocampal neurons are co-transfected with multiple plasmids to study the effects of pathological proteins on fast axonal transport using live-cell confocal imaging of fluorescently-tagged cargo proteins. We begin with the harvest, dissociation, and culturing of primary hippocampal neurons from rodents. Then, we co-transfect the neurons with plasmid DNA constructs to express fluorescent-tagged cargo protein and wild-type or mutant tau (used as an exemplar of pathological proteins). Axons are identified in live cells using an antibody that binds an extracellular domain of neurofascin, an axon initial segment protein, and an axonal region of interest is imaged to measure fluorescent cargo transport.

Using KymoAnalyzer, a freely available ImageJ macro, we extensively characterize the velocity, pause frequency, and directional cargo density of axonal transport, all of which may be affected by the presence of pathological proteins. Through this method, we identify a phenotype of increased cargo pause frequency associated with the expression of pathological tau protein. Additionally, gene-silencing shRNA constructs can be added to the transfection mix to test the role of other proteins in mediating transport disruption. This protocol is easily adaptable for use with other neurodegenerative disease-related proteins and is a reproducible method to study the mechanisms of how those proteins disrupt axonal transport.

Introduction

Neurons depend on the bidirectional transport of cargo along the axon to maintain functional synapses and neural connectivity. Axonal transport deficits are thought to be critical contributors to the pathogenesis of several neurodegenerative diseases, including Alzheimer's disease (AD) and other tauopathies, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease1,2,3. Indeed, pathological modifications to several disease-related proteins negatively affect transport (reviewed in 4). Developing methods to investigate the mechanisms by which pathological proteins exert toxicity in disease is necessary to understand neurodegenerative disorders and identify potential targets for therapeutic intervention.

Several important insights into axon transport, including the discovery of conventional kinesin, the kinase- and phosphatase-dependent pathways regulating motor proteins, and mechanisms by which pathological proteins disrupt the regulation of axon transport, were made using the isolated squid axoplasm model4,5. Perfusion of squid axoplasm with pathological forms of tau protein inhibits anterograde fast axonal transport (FAT), an effect dependent on the exposure of the phosphatase-activating domain of tau, which activates protein phosphatase 1 (PP1)6,7,8. PP1 activates glycogen synthase kinase 3 (GSK3), which in turn phosphorylates kinesin light chains causing the release of cargo. Another pathological protein in AD is amyloid-β. Oligomeric forms of amyloid-β inhibit bidirectional FAT through casein kinase 2, which phosphorylates kinesin light chains9. Furthermore, pathological huntingtin protein harboring a polyglutamine expansion and a familial ALS-linked SOD1 mutant each disrupt axonal transport in the squid axoplasm through c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activity, respectively10,11.

While the squid axoplasm model continues to be a valuable tool in understanding the effects of pathological proteins on axonal transport, limited access to the equipment and specimens prevents it from being more widely used. We developed a transport assay using live-cell confocal microscopy imaging of rodent (mouse and rat) primary neurons. This model represents an easily adaptable and manipulatable mammalian neuron-based approach using widely available cell sources and microscopy systems. For example, a variety of pathological proteins (e.g., harboring disease-related modifications) are expressed to identify how specific modifications of these proteins affect transport in axons. Similarly, a variety of fluorescently-tagged cargo proteins can be used to examine cargo-specific changes. Moreover, the underlying molecular mechanisms are studied relatively easily by targeting expression (i.e., knockdown or overexpression) of selected proteins that may mediate these effects. This method also is easily adapted for primary neurons derived from a wide variety of animal models.

We present a detailed protocol describing the live-cell axon transport assay that was previously used in primary hippocampal neurons to show that mutant tau proteins associated with frontotemporal lobar dementias (FTLD; P301L or R5L tau) increase the pausing frequency of fluorescently-labeled cargo proteins bidirectionally12. Furthermore, the knockdown of the PP1γ isoform rescued the pausing effects12. This provides support for the model of pathological tau-induced disruption that is mediated through aberrant activation of a signaling pathway initiated by PP1 as described above6,7,12. In a separate study, we showed that pseudophosphorylation of tau at S199/S202/T205 (the pathogenic AT8 phosphoepitope relevant to tauopathies) increased cargo pause frequency and anterograde segment velocity. These effects were dependent on the N-terminal phosphatase activating domain of tau13. These examples highlight the utility of this model for identifying the mechanisms of how pathological proteins disrupt axon transport in mammalian neurons.

This paper provides a detailed description of the method beginning with the harvest, dissociation, and culturing of primary mouse hippocampal neurons, followed by the transfection of the neurons with cargo proteins fused with a fluorescent protein, and finally, the live-cell imaging and image analysis approach. We demonstrate how this method is used to study the effects of modified tau on the bidirectional transport of the vesicle-associated protein, synaptophysin, as an example. However, there is flexibility in the pathogenic protein and transport cargo protein of interest, which makes this a versatile approach to study axonal transport.

Protocol

These protocols were approved by the Michigan State University Institutional Animal Care and Use Committee. This protocol has been successfully applied to Tau Knockout mice in the C57BL/6J background and wild-type Sprague Dawley rats. Other strains should be acceptable as well. 1. Primary hippocampal neuron harvest Coat a 4-well glass bottom chamber slide with filtered 0.5 mg/mL poly-d-lysine (PDL) in borate buffer (12.5 mM sodium borate decahydrate and 50 mM boric…

Representative Results

Using these methods, we characterized axonal transport in the presence of wild-type or disease-related forms of tau protein to examine potential mechanisms of pathological tau-induced neurotoxicity in disease12,13. The KymoAnalyzer software calculates and pools a variety of different parameters from all kymographs within a given folder. Transport rates are calculated only when cargo is in motion (segmental velocity) as well as the overall rate, including pauses (…

Discussion

There is growing evidence that multiple pathological proteins associated with a variety of neurodegenerative disorders disrupt fast axonal transport in neurons. This represents a potential common mechanism of neurotoxicity across these diseases. To better understand the process by which these proteins disrupt transport, we need tools and models that allow us to address specific questions. The method described here allows the examination of mechanisms engaged by pathological proteins to negatively affect cargo transport i…

Declarações

The authors have nothing to disclose.

Acknowledgements

We thank Chelsea Tiernan and Kyle Christensen for their efforts in developing and optimizing aspects of these protocols. This work was supported by National Institutes of Health (NIH) Grants R01 NS082730 (N.M.K.), R01 AG044372 (N.M.K.), R01 AG067762 (N.M.K.), and F31 AG074521 (R.L.M.); NIH/National Institute on Aging, Michigan Alzheimer's Disease Research Center Grant 5P30AG053760 (N.M.K. and B.C.); Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Alzheimer's Research Program Award W81XWH-20-1-0174 (B.C.); Alzheimer's Association Research Grants 20-682085 (B.C.); and the Secchia Family Foundation (N.M.K.).

Materials

0.4% Trypan blue Gibco 15250-061
1.7 mL microcentrifuge tubes DOT RN1700-GMT
2.5% trypsin Gibco 15090-046
3 mL syringe with 21 G needle Fisher 14-826-84
10 mL plastic syringe Fisher 14-823-2A
14 G needle Fisher 14-817-203
15 G needle Medline SWD200029Z
16 G needle Fisher 14-817-104
18 G needle Fisher 14-840-97
22 G needle Fisher 14-840-90
32% paraformaldehyde Fisher 50-980-495
AlexaFluor 647 goat anti-rabbit IgG (H+L) Invitrogen A21244 RRID:AB_2535813
Amphotericin B Gibco 15290-026
Arruga Micro Embryonic Capsule Forceps, Curved; 4"  Roboz RS-5163 autoclave
B-27 Supplement (50x), serum free Gibco A3582801
BioCoat 24-well Poly D lysine plates  Fisher 08-774-124
boric acid Sigma B6768-1KG
Calcium chloride Sigma C7902
Castroviejo 3 1/2" Long 8 x 0.15 mm Angle Sharp Scissors Roboz RS-5658 autoclave
Cell counting device automatic or manual
Confocal microscope with live cell chamber attachment
Confocal imaging software
D-(+)-glucose Sigma G7528
DNase I (Worthington) Fisher NC9185812
Dulbecco's Phosphate Buffered Saline Gibco 14200-075
EGTA Fisher O2783-100
Fatal-Plus Solution Vortech Pharmaceuticals, LTD NDC 0298-9373-68 sodium pentobarbital; other approved methods of euthanasia may be used 
Fetal bovine serum Invitrogen 16000044
Gentamicin Reagent Solution Gibco 15710-072
GlutaMAX Gibco 35050-061 glutamine substitute
Hanks' Balanced Salt Solution Gibco 24020-117
ImageJ version 1.51n ImageJ Life-Line version 2017 May 30: https://imagej.net/software/fiji/downloads
KymoAnalyzer (version 1.01) Encalada Lab Package includes all 6 macros: https://www.encalada.scripps.edu/kymoanalyzer
Lipofectamine 3000 Invitrogen 100022050 Use with P3000 transfection enhancer reagent
Magnesium chloride Fisher AC223211000
MES hydrate Sigma M8250
Micro Dissecting Scissors 3.5" Straight Sharp/Sharp Roboz RS-5910 autoclave
Neurobasal Plus medium Gibco A3582901
Neurofascin (A12/18) Mouse IgG2a UC Davis/NIH NeuroMab 75-172 RRID:AB_2282826; 250 ng/mL; Works in rat neurons, NOT in mouse neurons
Neurofascin 186 (D6G60) Rabbit IgG Cell Signaling 15034 RRID:AB_2773024; 500 ng/mL; Works in mouse neurons, we have not tested in rat neurons
newborn calf serum Gibco 16010-167
Opti-MEM Gibco 31985-062
P3000 Invitrogen 100022057
Petri dish, 100 x 10 mm glass Fisher 08-748B For dissection; autoclave
Petri dish, 100 x 20 mm glass Fisher 08-748D To place uterine horns in; autoclave
Poly-D-lysine Sigma P7886-100MG
Polypropylene conical centrifuge tubes (15 mL) Fisher 14-955-238
Polypropylene conical centrifuge tubes (50 mL) Fisher 14-955-238
Potassium chloride Fisher P217-500
Sodium acetate Sigma S5636
sodium borate decahydrate VWR MK745706
Straight-Blade Operating Scissors Blunt/Sharp Fisher 13-810-2 autoclave
Syringe Filters, 0.22 µm VWR 514-1263
Thumb dressing forceps, serrated, 4.5" Roboz RS-8100 autoclave
µ-Slide 4 Well Glass Bottom Ibidi 80427

Referências

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Mueller, R. L., Kanaan, N. M., Combs, B. Using Live-Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons. J. Vis. Exp. (202), e66156, doi:10.3791/66156 (2023).

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