Effects of TrkB-ICD overexpression on the morphology, activity and transcriptome of cultured primary cortical neurons
In Alzheimer's disease (AD), amyloid ß (Aß)-triggered cleavage of TrkB-FL impairs brain-derived neurotrophic factor (BDNF) signaling, thereby compromising neuronal survival, differentiation, as well as synaptic transmission and plasticity. In addition to compromising canonical BDNF signalling pathways, TrkB-FL cleavage produces an intracellular fragment (TrkB-ICD), which was shown to accumulate in the nucleus and to display tyrosine kinase activity. To dissect the role of TrkB-ICD overexpression from the loss of endogenous signaling throught TrkB-FL, we used lentiviruses to overexpress the TrkB-ICD sequence in cultured primary cortical neurons and performed morphological, electrophysiological and transcriptomics studies. While TrkB-ICD overexpression did not affect cell survival, it caused a significant decrease in the number of dendritic spines, both compared to untransduced and GFP-transduced neurons. Furthermore, TrkB-ICD overexpressing neurons presented a hyperpolarized resting membrane potential and increased frequency of miniature excitatory postsynaptic currents (mEPSCs). Finally, TrkB-ICD overexpression was associated with the upregulation of genes involved in (i) neuronal survival, growth and differentiation; (ii) neuronal cytoarchitecture and spine morphology; (iii) neurodegenerative processes, including AD; and (iv) synaptic transmission and plasticity. Overall, these results show that TrkB-ICD overexpression causes dendritic spine loss, alters excitatory synaptic transmission and causes transcriptome-wide changes, namely in genes coding for proteins involved in synaptic processes. Overall design: To investigate the effects of TrkB-ICD overexpression, we transduced cultured primary cortical neurons at DIV3 with lentiviruses (LVs) expressing TrkB-ICD or enhanced green fluorescent protein [EGFP] (as a control), under the regulation of the neuron-specific CaMKIIa promoter. We also sequenced untransduced neurons from the same cell culture batches, as an additional control. For each condition, 3 biological replicates (i.e., from 3 independent cultures) were prepared. We performed gene expression profiling analysis using data obtained from RNA-seq of the 3 experimental conditions mentioned above, using 3 biological replicates for each. Using the gene expression profiles, we performed comparative analyses, namely differential gene expression and pathway enrichment using Gene Ontology (GO) terms.