Excitatory Neuron-Derived Cytokine Interleukin-34 Influences Microglia Function in Development and Disease

Abstract

Neuron-microglia interactions dictate the development and function of neuronal circuits in the brain. However, the factors that support and broadly regulate these processes across developmental stages are largely unknown. My thesis work is based on the hypothesis that interleukin-34 (IL34), a cytokine released by neurons, is regulated by development, increases the maturation of microglia, and dictates microglia-neuron interactions. Here, I show that IL34 mRNA and protein is upregulated in neurons in the second week of postnatal life and that this increase coincides with increases in microglia number and expression of mature, homeostatic markers, e.g., TMEM119. I also find that IL34 mRNA is higher in excitatory (compared to inhibitory) neurons. Global genetic KO of IL34 reduced microglia numbers and prevented the functional maturation of microglia, and excitatory-neuron specific KO of IL34 similarly impacted microglia and increased aberrant microglial phagocytosis of excitatory thalamocortical synapses in the ACC at postnatal day (P)15. Acute, low dose blocking of IL34 at P15 in mice decreased microglial TMEM119 protein and increased microglial phagocytosis of excitatory thalamocortical synapses during an inappropriate time in development. In contrast, viral overexpression of IL34 early in life (P1-P8) caused early maturation of microglia and prevented microglial phagocytosis of thalamocortical synapses during the appropriate neurodevelopmental refinement window. Taken together, these findings establish IL34 as a key regulator of neuron-microglia crosstalk in postnatal brain development, controlling both microglial maturation and synapse engulfment.There is ample evidence that developmental microglia processes are reactivated in diseases such as Alzheimer’s, however the signals that regulate these processes remain elusive. Recent Alzheimer’s genome-wide association studies (GWAS) have identified a stop-gain mutation in the human IL34 gene, implicating dysfunctional IL34 as conferring risk for developing AD. In the second part of my thesis work, I set out to test whether decreased IL34 signaling in AD induces a maladaptive phagocytic microglial phenotype that contributes to neurodegeneration and memory loss. To test this hypothesis, we generated double mutant mice by crossing IL34LacZ/+ heterozygous mice with 5XFAD mutant mice. We found that this additional mutation exacerbates an anxiolytic phenotype in the elevated plus maze at 6 months of age, reduces amyloid plaque load, and increases microglial synaptic pruning in 6-month-old females. We next used a viral approach to overexpress IL34 in hippocampal neurons of 8-month-old WT and 5XFAD mice and found that increased IL34 levels are not sufficient to rescue an anxiolytic phenotype in the elevated plus maze but are able to partially rescue hyperactivity in the elevated plus maze. IL34 overexpression dramatically increased microglia numbers in the hippocampus, without impacting plaque load. Taken together, our results suggest that a heterozygous mutation in IL34 reduces plaque load in male and female 5XFAD mice and increases synaptic pruning in 6-month-old female 5XFAD mice. Further, IL34 overexpression in the hippocampus does not impact anxiolytic deficits in the elevated plus maze but does partially rescue hyperactivity in 5XFAD mice, increases hippocampal microglia, and does not reduce plaque load. These data support our hypothesis that lower levels of IL34 early in disease progression causes microglia to be more phagocytic, negatively influencing behavior and synapses, and provides the first rationale supporting the therapeutic potential behind targeting IL34 in Alzheimer’s disease.