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Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses
Published on: June 14, 2020
Basic Science and Pathogenesis
Colleen R Zaccard1, Euan Parnell1, Marc Dos Santos1
1Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
Background:
Microglia, the brain's innate immune cells, regulate synaptic function by remodeling postsynaptic spines or pruning complement-tagged presynaptic terminals via complement receptors (CRs). In Alzheimer's disease (AD), microglia dysfunction drives chronic inflammation and synapse loss through aberrant pruning and unknown mechanisms. Tunneling nanotubes (TNTs) are long, ultrafine membranous connections that facilitate rapid cell-cell communication. TNTs have been implicated in neuro-immune crosstalk but remain understudied due to their nanoscale and lack of specific markers. Plasma membrane-organizing tetraspanins CD9 and CD81 are enriched in the TNT proteome of cell lines and regulate TNT stability and vesicle trafficking, suggesting their utility for TNT-specific labeling.
Method:
Human primary microglia, isolated postmortem from cognitively normal elderly, were transduced with a tdTomato (tdT) AAV to visualize TNTs. Microglia were polarized for 24 hours with vehicle (control), oligomeric amyloid-beta (oAβ) + IFN-γ (pro-inflammatory), or IL-4 + IL-13 (anti-inflammatory) cocktails. Immuno-labeled CR3/CR4 localization to TNTs versus cell bodies was assessed using enhanced resolution (ER) confocal microscopy. Similarly, microglia were treated with 5FAM-oAβ + vehicle or IFN-γ to assess oAβ localization to TNTs during inflammation. Microglial TNTs were analyzed in live microglia-excitatory neuron (iEN) co-cultures under control, pro-inflammatory, and anti-inflammatory conditions. Finally, CD9 and CD81 were evaluated as candidate microglial TNT markers using ER confocal and super-resolution microscopy.
Result:
Pro-inflammatory conditions significantly increased TNT number per microglia and enhanced CR3/CR4 localization on TNTs compared to cell bodies, suggesting complement-dependent functions. Pro-inflammatory cues elevated oAβ localization to TNTs, suggesting roles in pathological protein trafficking. In live microglia-iEN co-cultures, microglial TNT frequency, length, and dynamic interactions with axons, dendrites, and presumptive spines were enhanced in response to inflammation. CD81 particularly localized to microglial TNTs under varied conditions, suggesting its utility as a TNT-specific marker in vivo.
Conclusion:
Microglia respond to AD-relevant pro-inflammatory cues with increased TNT expression, CR and oAβ localization to TNTs, and enhanced TNT-mediated interactions with synaptic elements. Thus, TNT composition and functions are environmental context dependent. Inflammation-inducible TNTs may participate in complement-dependent synaptic remodeling and/or pathological protein trafficking. CD81 represents a promising marker for in vivo investigations of the microglial TNT-synapse interface during chronic inflammation, aging, and AD.
Insights
Microglia use tunneling nanotubes (TNTs) to interact with synapses, increasing their numbers and connections during inflammation. CD81 shows promise as a marker for studying these microglial TNTs in Alzheimer's disease.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia regulate synaptic function via complement receptors (CRs) and spine remodeling.
- Microglia dysfunction in Alzheimer's disease (AD) contributes to inflammation and synapse loss.
- Tunneling nanotubes (TNTs) facilitate cell-cell communication but are understudied in microglia due to lack of specific markers.
Purpose of the Study:
- Investigate microglial TNT formation and function under varying inflammatory conditions.
- Determine the role of CRs and amyloid-beta (oAβ) in microglial TNTs.
- Evaluate CD9 and CD81 as potential markers for microglial TNTs.
Main Methods:
- Human primary microglia were cultured and treated with pro-inflammatory (oAβ + IFN-γ) or anti-inflammatory (IL-4 + IL-13) stimuli.
- TNTs were visualized using tdTomato AAV transduction.
- Localization of CR3/CR4 and 5FAM-oAβ to TNTs was assessed via confocal microscopy.
- Microglial-neuron co-cultures were used to study TNT interactions with synaptic elements.
- CD9 and CD81 expression on TNTs was analyzed using super-resolution microscopy.
Main Results:
- Pro-inflammatory conditions increased microglial TNT number and CR3/CR4 localization on TNTs.
- Oligomeric amyloid-beta (oAβ) localized to TNTs under pro-inflammatory conditions.
- Microglial TNTs showed increased frequency, length, and dynamic interactions with neuronal components in co-cultures.
- CD81 was consistently localized to microglial TNTs.
Conclusions:
- Microglia increase TNT expression and interactions with synapses in response to inflammation.
- Inflammation-dependent microglial TNTs may mediate complement-dependent synaptic remodeling and pathological protein transport.
- CD81 is a promising marker for in vivo studies of microglial TNTs in neuroinflammation and AD.
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