Pyk2 overexpression in postsynaptic neurons blocks amyloid β1–42-induced synaptotoxicity in microfluidic co-cultures

Abstract

Recent meta-analyses of genome-wide association studies identified a number of genetic risk factors of Alzheimer’s disease; however, little is known about the mechanisms by which they contribute to the pathological process. As synapse loss is observed at the earliest stage of Alzheimer’s disease, deciphering the impact of Alzheimer’s risk genes on synapse formation and maintenance is of great interest. In this paper, we report a microfluidic co-culture device that physically isolates synapses from pre- and postsynaptic neurons and chronically exposes them to toxic amyloid β peptides secreted by model cell lines overexpressing wild-type or mutated (V717I) amyloid precursor protein. Co-culture with cells overexpressing mutated amyloid precursor protein exposed the synapses of primary hippocampal neurons to amyloid β1-42 molecules at nanomolar concentrations and induced a significant decrease in synaptic connectivity, as evidenced by distance-based assignment of postsynaptic puncta to presynaptic puncta. Treating the cells with antibodies that target different forms of amyloid β suggested that low molecular weight oligomers are the likely culprit. As proof of concept, we demonstrate that overexpression of protein tyrosine kinase 2 beta (Pyk2) –an Alzheimer’s disease genetic risk factor involved in synaptic plasticity and shown to decrease in Alzheimer’s disease brains at gene expression and protein levels– selectively in postsynaptic neurons is protective against amyloid β1-42-induced synaptotoxicity. In summary, our lab-on-a-chip device provides a physiologically-relevant model of Alzheimer’s disease-related synaptotoxicity, optimal for assessing the impact of risk genes in pre- and postsynaptic compartments.

Alzheimer’s genetic risk factor FERMT2 (Kindlin-2) controls axonal growth and synaptic plasticity in an APP-dependent manner

“…This program assigns each postsynaptic spot to the nearest presynaptic spot (within a distance threshold of 1 μm) and calculates the number of such assignments for all presynaptic puncta. The percentage of presynaptic spots not assigned by any postsynaptic spot was consistently used as a readout of synaptic connectivity [26].…”

“…6). The effects of shRNA expression (DIV1) on synaptic connectivity were assessed by confocal microscopy of synaptic markers (DIV14) followed by three-dimensional image segmentation and quantification [26]. Underexpression of FERMT2 in the presynaptic chamber led to a decrease in synaptic connectivity, whereas no such effect was observed when underexpressing FERMT2 in the postsynaptic compartment (Fig.…”

The Non-receptor Tyrosine Kinase Pyk2 in Brain Function and Neurological and Psychiatric Diseases

“…The presence of a potentially toxic proteolytic fragment of Src was observed in 5XFAD and Pyk2 KO mice, which was corrected by Pyk2 viral expression (Giralt et al, 2018). In further support of the positive role of Pyk2, its overexpression in hippocampal neurons in a microfluidic culture system also protected these neurons from Aβ 1−42 toxicity (Kilinc et al, 2020). These results suggest that in some conditions a functional deficit of Pyk2 could play a role in AD model.…”

Alzheimer’s Disease: Current Perspectives and Advances in Physiological Modeling

“…In addition to co-culturing abilities, as described in Park et al, multi-chambered microfluidic devices offer the ability to isolate structures of interest while maintaining a physiologically relevant cell culture. Kilinic et al [ 126 ] took advantage of this ability by isolating the synapses of rat primary neurons and selectively exposing them to physiological concentrations of amyloid beta, thus examining the effects of Aβ on synaptotoxicity. Such multi-chambered devices also provide a platform by which to separate pre- and postsynaptic neurons, a feature that researchers have used to quantitatively study the axonal transfer of tau in rodent cortical neurons [ 127 ].…”