DISTINCT PRESYNAPTIC HOMEOSTATIC ADAPTATIONS IN HUMAN SYNAPSES REVEAL A LINK TO TDP-43

Presynaptic forms of homeostatic plasticity stabilize synaptic transmission across diverse synapses and species. However, evidence for presynaptic homeostatic adaptations in human synapses is lacking.
Here, we investigated homeostatic plasticity in human iPSC-derived, NGN2-induced excitatory cortical neurons co-cultured with human astrocytes. Chronic action potential blockade with tetrodotoxin (TTX, 24 h) increased both, miniature EPSC (mEPSC) amplitude and frequency, suggesting synaptic upscaling and presynaptic compensation. Furthermore, Synapsin-1 immunofluorescence was enhanced after TTX treatment, supporting presynaptic adaptation.
We next perturbed synaptic transmission by partially blocking AMPA receptors. Acute GYKI-53655 treatment reduced both mEPSC and AP-evoked EPSC (eEPSC) amplitudes. After 24 h, mEPSC amplitudes remained reduced, whereas eEPSC amplitudes recovered toward baseline, indicating presynaptic compensation. In addition, paired-pulse ratio was decreased, and short-term depression enhanced, implying increased release probability. In contrast to TTX, Synapsin-1 levels were unchanged, suggesting mechanistically distinct presynaptic adaptations. Multi-electrode array recordings revealed stable firing rates during chronic GYKI exposure, followed by a 4-fold increase in mean firing rate upon washout after 24 h, consistent with network-level homeostatic adaptations.
Loss of TDP-43 was previously shown to reduce UNC13A levels, which in turn disrupts presynaptic homeostatic plasticity in Drosophila. We therefore next investigated whether TDP-43 similarly regulates presynaptic homeostatic mechanisms in human neurons. TDP-43 knockdown reduced Unc13A protein levels, mEPSC frequency, and eEPSC amplitude, suggesting impaired baseline synaptic transmission. Intriguingly, TDP-43 knockdown disrupted presynaptic homeostatic compensation in response to chronic GYKI treatment.
Together, our results demonstrate distinct presynaptic adaptations in human synapses and implicate TDP-43 in presynaptic homeostatic plasticity.