Regulation of synapse density by Pumilio RNA-binding proteins

The ability of neurons to send and receive signals underlies the most essential functions of the brain. Thus, the formation and function of new synapses must be tightly regulated. Local protein synthesis is essential for presynaptic terminal formation and persists at mature synapses. We have recently discovered a role for Pumilio 2 as a negative regulator of axonal localization and translation of its target mRNAs. Pumilio RNA-binding proteins regulate a large number of synaptic mRNAs encoding proteins essential for neurotransmission and neuron projection development and are developmentally downregulated in the brain, corresponding with the increased translation of their target mRNAs in axons. Here, I tested the hypothesis that Pumilio proteins constrain the formation and/or maturation of synapses at early stages of neuronal maturation by regulating synaptic mRNAs. I found that simultaneous downregulation of Pumilio 1 and 2 together induces an increase in synapses in primary hippocampal neurons, while downregulation of Pumilio 1 or Pumilio 2 individually results in a reduction in synapse density. The increase in synapses seen with dual Pumilio knockdown corresponds with an increase in both excitatory and inhibitory presynaptic markers as well as an increase in Snap25 translation. Notably, this increase in synapses persists even when Pumilios are knocked down at later stages of maturation after developmental downregulation has already occurred. This suggests that remaining low levels of Pumilio proteins continue to play a significant role in plasticity and regulation of the synapse at later stages of neuronal maturation, potentially throughout the lifespan of an organism.