Microtubules, Membranes, and Movement: New Roles for Stathmin-2 in Axon Integrity

Abstract

Neurons establish functional connections responsible for how we perceive and react to the world around us. Communication from a neuron to its target cell occurs through a long projection called an axon. Axon distances can exceed 1 m in length in humans and require a dynamic microtubule cytoskeleton for growth during development and maintenance in adulthood. Stathmins are microtubule-associated proteins that function as relays between kinase signaling and microtubule polymerization. In this review, we describe the prolific role of Stathmins in microtubule homeostasis with an emphasis on emerging roles for Stathmin-2 (Stmn2) in axon integrity and neurodegeneration. Stmn2 levels are altered in Amyotrophic Lateral Sclerosis and loss of Stmn2 provokes motor and sensory neuropathies. There is growing potential for employing Stmn2 as a disease biomarker or even a therapeutic target. Meeting this potential requires a mechanistic understanding of emerging complexity in Stmn2 function. In particular, Stmn2 palmitoylation has a surprising contribution to axon maintenance through undefined mechanisms linking membrane association, tubulin interaction, and axon transport. Exploring these connections will reveal new insight on neuronal cell biology and novel opportunities for disease intervention.

Keywords: ALS; Stmn2; axon degeneration; cytoskeleton; neurodegeneration; palmitoylation.

Figure 1.. Domain structure of the Stathmin family.

All Stathmins possess tandem tubulin binding regions (TBR). These regions display highest sequence homology between family members. A divergent proline-rich domain (PrD) contains multiple serine residues that undergo phosphorylation. Stmn2, Stmn3, and Stmn4 have an N-terminal membrane-targeting domain (MTD) with two cysteines modified by palmitoylation. Crosshatches in the Stmn4 MTD represent multiple splice isoforms in this domain. The relative position of phosphorylation events within the four Stathmin proteins are noted. Phosphorylation sites in Stmn4 and the TBR1 of Stmn2 and Stmn3 are predicted from homologous serines in Stmn1 yet still require experimental validation. Created with BioRender.com.

Figure 2.. Stmn2 proteostasis in axon segments.

Stmn2 is synthesized, folded, and palmitoylated in the soma where it is attached to vesicles and delivered into the axon via kinesin-mediated anterograde transport. Palmitoylated Stmn2 proteins are targeted for degradation via localized DLK-JNK signaling. DLK is activated by microtubule (MT) damage and would facilitate local repair by accelerating Stmn2 degradation thereby releasing more tubulin heterodimer for polymerization. Acyl-protein thioesterases (APTs) could release Stmn2 from vesicles at specific locations within axon segments, protecting Stmn2 from DLK signaling. Moreover, Stmn2 protein generated from local axon translation would bypass palmitoylation reactions at the trans-Golgi. A key unanswered question is whether palmitoylated Stmn2 binds and sequesters tubulin heterodimer in the same way as non-palmitoylated Stmn2. Further, Stmn2 might interact with kinesin motors and adaptors to alter vesicle trafficking of other cargo proteins. Created with BioRender.com.

Figure 3.. Reduced nuclear TDP-43 causes mis-splicing of Stmn2 pre-mRNA.

Nuclear-localized TDP-43 binds to GU-motifs in a cryptic exon within the Stmn2 pre-mRNA and suppresses recognition of a 3’ splice site at this exon. Proper splicing generates a mature Stmn2 mRNA transcript that is exported from the nucleus and translated into a full-length protein. ALS-linked mutations cause TDP-43 misfolding, aggregation, and accumulation in the cytosol. Depletion of nuclear-localized TDP-43 results in aberrant Stmn2 splicing and inclusion of this cryptic exon. A premature polyadenylation site within this exon triggers transcription termination and accumulation of a truncated mRNA transcript. An in-frame stop codon would generate a 16 amino acid polypeptide lacking any functional motifs from full length Stmn2 and likely disposal through protein degradation. Created with BioRender.com.

Figure 4.. Axonal depletion of Stmn2 in diseased neurons disrupts microtubule homeostasis.

In healthy neurons Stmn2 is efficiently delivered into axon segments and regulated by kinase signaling to tune microtubule polymerization by controlling the pool of available tubulin heterodimer. Phosphorylation inhibits Stmn2 binding to tubulin heterodimers and boosts the pool of available substrate for microtubule polymerization. A normal balance between tubulin polymerization and depolymerization is critical for motor-dependent vesicular transport which sustains neuromuscular junctions (NMJs). Stmn2 is depleted in motor neurons possessing ALS-linked mutations in TDP-43 due to mis-splicing and premature polyadenylation. Additionally, neuronal stress upregulates of DLK-JNK signaling which accelerates Stmn2 degradation. Axons from diseased neurons will have chronically low Stmn2 levels, an imbalance in microtubule polymerization versus depolymerization, and corresponding impairments in protein trafficking that destabilize NMJs. Loss of Stmn2 and microtubule dysfunction reduces regenerative capacity which will manifest in progressive motor dysfunction. Created with BioRender.com.