The autophagy-inducing kinases, ULK1 and ULK2, regulate axon guidance in the developing mouse forebrain via a noncanonical pathway

Abstract

Mammalian ULK1 (unc-51 like kinase 1) and ULK2, Caenorhabditis elegans UNC-51, and Drosophila melanogaster Atg1 are 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; disruption of these genes results in defective axon guidance in invertebrates. Although disrupting ULK1/2 function impairs normal neurite outgrowth in vitro, the role of ULK1 and ULK2 in the developing brain remains poorly characterized. Here, we show that ULK1 and ULK2 are required for proper projection of axons in the forebrain. Mice lacking Ulk1 and Ulk2 in their central nervous systems showed defects in axonal pathfinding and defasciculation affecting the corpus callosum, anterior commissure, corticothalamic axons and thalamocortical axons. These defects impaired the midline crossing of callosal axons and caused hypoplasia of the anterior commissure and disorganization of the somatosensory cortex. The axon guidance defects observed in ulk1/2 double-knockout mice and central nervous system-specific (Nes-Cre) Ulk1/2-conditional double-knockout mice were not recapitulated in mice lacking other autophagy genes (i.e., Atg7 or Rb1cc1 [RB1-inducible coiled-coil 1]). The brains of Ulk1/2-deficient mice did not show stem cell defects previously attributed to defective autophagy in ambra1 (autophagy/Beclin 1 regulator 1)- and Rb1cc1-deficient mice or accumulation of SQSTM1 (sequestosome 1) or ubiquitin deposits. Together, these data demonstrate that ULK1 and ULK2 regulate axon guidance during mammalian brain development via a noncanonical (i.e., autophagy-independent) pathway.

Axonal autophagosome maturation defect through failure of ATG9A sorting underpins pathology in AP-4 deficiency syndrome

“…70% of complex HSP patients presenting with progressive spasticity, intellectual impairment and thin corpus callosum are accounted for by mutations in SPG11/spatacsin and ZFYVE26/spastizin [6], both having roles in autophagosome maturation and endolysosomal function [57]. Thin corpus callosum has also been identified in autophagy-related CNS specific KO mouse models including ulk1 ulk2 double knockout and atg9a [25,61], and axonal extension is concomitantly reduced in neurons cultured from these lines [25,56,62,63]. Given this striking similarity to ap4e1 KO mice, we provide evidence towards thinning of the corpus callosum in AP-4 deficiency arising developmentally through defective axonal extension as a result of reduced axonal autophagosome biogenesis.…”

AP-4 mediated ATG9A sorting underlies axonal and autophagosome biogenesis defects in a mouse model of AP-4 deficiency syndrome

“…Notably, both spatacsin and spastizin have roles in autophagosome maturation and endolysosomal function (Vantaggiato et al, 2013). More recently, thin corpus callosum has been identified in autophagy-related CNS specific KO mouse models including ULK1/ULK2 dKO (Wang et al, 2017;Yamaguchi et al, 2017). Importantly, axonal extension is reduced in cultured primary neurons prepared from all of these lines (Khundadze et al, 2013;Pérez-Brangulí et al, 2014;Yamaguchi et al, 2017;Zhou et al, 2007).…”

Autophagy-Independent Functions of the Autophagy Machinery

“…Mice with conditional Ulk1/Ulk2 co-deletion in the brain surviving the first 24 h after birth resemble their Atg5-and Atg7-deficient counterparts as they display abnormal limb-clasping reflexes, but they do not develop cerebellar ataxia, suggesting that the neuronal phenotype caused by the lack of Ulk1 and Ulk2 may not result from an autophagic defect . Indeed, Ulk1 À/À Ulk2 À/À neurons do not exhibit accumulation of autophagic substrates such as sequestosome 1 (SQSTM1, best known as p62) but manifest signs of the unfolded protein response Wang et al, 2018), an intracellular pathway of adaptation to accumulation of unfolded polypeptides in the ER lumen (Galluzzi et al, 2018b). Consistently, both ULK1 and ULK2 can phosphorylate SEC16 homolog A, endoplasmic reticulum export factor (SEC16A) to drive the anterograde transport of ER-derived vesicles to the GA, and this pathway is insensitive to depletion of ATG13 (which is required for the pro-autophagic functions of ULKs), ATG14, and ATG7 .…”