Date of Award

12-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Neuroscience

Research Advisor

Mondira Kundu, MD, PhD

Committee

Kristin Marie Hamre, PhD Joseph T. Opferman, PhD David J. Solecki, PhD Paul J. Taylor, MD, PhD

Keywords

Axon guidance, ER stress, ER-to-Golgi trafficking, FIP200, SEC16A, ULK/ATG1

Abstract

Mammalian UNC-51–like kinases 1 and 2 (ULK1 and ULK2), Caenorhabditis elegans UNC-51 and Drosophila melanogaster Atg1 are redundant serine/threonine kinases that regulate flux through the autophagy pathway in response to various types of cellular stress. C. elegans UNC-51 and D. melanogaster Atg1 also promote axonal growth and defasciculation, and disruption of these genes results in defects in axon guidance in invertebrates. Germline Ulk1/2-deficient mice die perinatally. Therefore, we used a conditional-knockout approach to investigate the roles of ULK1/2 in the brain. Mice lacking Ulk1 and Ulk2 in their central nervous systems (CNS) showed defects in axonal pathfinding and defasciculation affecting the corpus callosum (CC), anterior commissure (AC), corticothalamic axons (CTAs) and thalamocortical axons (TCAs) and mossy fibers. These defects led to impaired midline crossing of callosal axons, anterior commissure hypoplasia and disorganization of the somatosensory cortex. The axon guidance defects observed in Ulk1/2 double knockout (dko) and in CNS-specific (Nestin-Cre) Ulk1/2 conditional double knockout (cdko) mice were not recapitulated in mice lacking other autophagy genes (i.e. Atg7 or Fip200), and was associated with abnormal localization of the axon guidance molecule, transient axonal glycoprotein-1 (TAG-1) in the distal CTAs. Approximately 40% of the Ulk1/2 cdko animals died shortly after birth; the remaining animals survived up to 4 months. Although the mice showed neuronal degeneration, specifically in the hippocampal CA1 region, the neurons showed no accumulation of P62+/ubiquitin+ inclusions or abnormal membranous structures, which are observed in mice lacking other autophagy genes, such as Atg7, and Fip200. Rather, neuronal death was associated with activation of the unfolded protein response (UPR) pathway. An unbiased proteomics approach identified SEC16A as a novel ULK1/2-interacting partner. ULK-mediated phosphorylation of SEC16A regulated the assembly of endoplasmic reticulum (ER) exit sites and ER-to-Golgi trafficking of specific cargo such as, the serotonin transporter SERT, and did not require other autophagy proteins (e.g. ATG13). The defect in ER-to-Golgi trafficking activated the UPR pathway in ULK-deficient cells; both processes were reversed upon expression of SEC16A with a phosphomimetic substitution. Thus, the regulation of ER-to-Golgi trafficking by ULK1/2 is essential for cellular homeostasis. Moreover, the defect in SERT trafficking may also contribute to the disrupted formation of the barrel cortex in the Ulk1/2 cdko mice. Together, these data highlight the autophagy-independent role of ULK1 and ULK2 in maintaining cellular homeostasis and regulating axon guidance in the mammalian brain.

ORCID

http://orcid.org/0000-0001-6898-9757

DOI

10.21007/etd.cghs.2016.0419

Comments

One year embargo expires November 2017.

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