Cerebellar axonopathy in Shivers horses identified by spatial transcriptomic and proteomic analyses

Abstract

Background: Shivers in horses is characterized by abnormal hindlimb movement when walking backward and is proposed to be caused by a Purkinje cell (PC) axonopathy based on histopathology. Objectives: Define region-specific differences in gene expression within the lateral cerebellar hemisphere and compare cerebellar protein expression between Shivers horses and controls. Animals: Case-control study of 5 Shivers and 4 control geldings ≥16.2 hands in height. Methods: Using spatial transcriptomics, gene expression was compared between Shivers and control horses in PC soma and lateral cerebellar hemisphere white matter, consisting primarily of axons. Tandem-mass-tag (TMT-11) proteomic analysis was performed on lateral cerebellar hemisphere homogenates. Results: Differences in gene expression between Shivers and control horses were evident in principal component analysis of axon-containing white matter but not PC soma. In white matter, there were 455/1846 differentially expressed genes (DEG; 350 ↓DEG, 105 ↑DEG) between Shivers and controls, with significant gene set enrichment of the Toll-Like Receptor 4 (TLR4) cascade, highlighting neuroinflammation. There were 50/936 differentially expressed proteins (DEP). The 27 ↓DEP highlighted loss of axonal proteins including intermediate filaments (5), myelin (3), cytoskeleton (2), neurite outgrowth (2), and Na/K ATPase (1). The 23 ↑DEP were involved in the extracellular matrix (7), cytoskeleton (7), redox balance (2), neurite outgrowth (1), signal transduction (1), and others. Conclusion and Clinical Importance: Our findings support axonal degeneration as a characteristic feature of Shivers. Combined with histopathology, these findings are consistent with the known distinctive response of PC to injury where axonal changes occur without a substantial impact on PC soma.