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

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 disease^1,2,3. 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 model^4,5. 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, respectively^10,11.

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 above^6,7,12. 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.