Suppression of spastin Mutant Phenotypes by Pak3 Loss Implicates a Role for Reactive Glia in AD-HSP.

Emily F Ozdowski,Nina T Sherwood,Helena Abbott,Jill S Wentzell,Stefanie M Engert

doi:10.3389/fnins.2020.00912

Abstract

Neurodegenerative mechanisms due to mutations in spastin currently center on neuronal defects, primarily in microtubule and endomembrane regulation. Spastin loss in Drosophila larvae compromises neuronal microtubule distribution, alters synaptic bouton morphology, and weakens synaptic transmission at glutamatergic neuromuscular junction (NMJ) synapses. Pak3, a p21-activated kinase that promotes actin polymerization and filopodial projections, is required for these spastin mutant defects; animals lacking both genes have normal NMJs. Here we show that Pak3 is expressed in central and peripheral glial populations, and reduction of Pak3 specifically in subperineurial glial cells is sufficient to suppress the phenotypes associated with spastin loss. Subperineurial glia in the periphery ensheathe motor neuron axons and have been shown to extend actin-based projections that regulate synaptic terminals during normal NMJ development. We find that these subperineurial glial projections are Pak3-dependent and nearly twice as frequent in spastin mutants, while in Pak3, spastin double mutants, neither glial projections nor synaptic defects are observed. Spastin deficiency thus increases Pak3-dependent subperineurial glia activity, which is in turn required for neuronal defects. Our results demonstrate a central role for Pak3-mediated, altered glial behavior in the neuronal defects due to spastin loss, and suggest that a similar reactive glia-mediated mechanism may underlie human AD-HSP pathogenesis.

Highlights

The identification of human SPAST as the most common gene mutated in Autosomal-Dominant Hereditary Spastic Paraplegia two decades ago was a major advance, enabling multiple model systems to be leveraged toward understanding the requirement for Spastin in nervous system function (Hazan et al, 1999)
Forward genetic screen for modifiers of Spastin activity, we previously showed that the actin regulator Pak3 is required for the manifestation of morphological and functional synaptic defects due to spastin loss (Ozdowski et al, 2011)
We have determined that Pak3 functions in larval subperineurial glia cells, where it promotes projections that extend into the neuromuscular junction (NMJ) synaptic arbor

Summary

Introduction

The identification of human SPAST as the most common gene mutated in Autosomal-Dominant Hereditary Spastic Paraplegia two decades ago was a major advance, enabling multiple model systems to be leveraged toward understanding the requirement for Spastin in nervous system function (Hazan et al, 1999). Just as SPAST mutations impair the longest axons of the central nervous system in humans, diminishing patient mobility, homozygous deletion of Drosophila spastin (the spastin5.75 allele) impairs mobility in adult flies, with the distal-most limbs appearing the weakest These adult flies are rarely viable, and homozygous null larvae appear healthy, electrophysiological analysis of the larval neuromuscular junction (NMJ), a well-established model for vertebrate glutamatergic central synapses (Collins and DiAntonio, 2007), reveals reduced synaptic transmission due to defects at the presynaptic terminal (Sherwood et al, 2004; Ozdowski et al, 2011). These subcellular, morphological, and functional phenotypes in spastin mutants are all significantly rescued by low-level, neuronspecific expression of fly – or human – wild type Spastin, demonstrating that Spastin function is well-conserved between flies and humans, and is required within neurons (Sherwood et al, 2004; Du et al, 2010)

Methods

Results

Conclusion