Altered distribution of ATG9A and accumulation of axonal aggregates in neurons from a mouse model of AP-4 deficiency syndrome

Abstract

The hereditary spastic paraplegias (HSP) are a clinically and genetically heterogeneous group of disorders characterized by progressive lower limb spasticity. Mutations in subunits of the heterotetrameric (ε-β4-μ4-σ4) adaptor protein 4 (AP-4) complex cause an autosomal recessive form of complicated HSP referred to as “AP-4 deficiency syndrome”. In addition to lower limb spasticity, this syndrome features intellectual disability, microcephaly, seizures, thin corpus callosum and upper limb spasticity. The pathogenetic mechanism, however, remains poorly understood. Here we report the characterization of a knockout (KO) mouse for the AP4E1 gene encoding the ε subunit of AP-4. We find that AP-4 ε KO mice exhibit a range of neurological phenotypes, including hindlimb clasping, decreased motor coordination and weak grip strength. In addition, AP-4 ε KO mice display a thin corpus callosum and axonal swellings in various areas of the brain and spinal cord. Immunohistochemical analyses show that the transmembrane autophagy-related protein 9A (ATG9A) is more concentrated in the trans-Golgi network (TGN) and depleted from the peripheral cytoplasm both in skin fibroblasts from patients with mutations in the μ4 subunit of AP-4 and in various neuronal types in AP-4 ε KO mice. ATG9A mislocalization is associated with increased tendency to accumulate mutant huntingtin (HTT) aggregates in the axons of AP-4 ε KO neurons. These findings indicate that the AP-4 ε KO mouse is a suitable animal model for AP-4 deficiency syndrome, and that defective mobilization of ATG9A from the TGN and impaired autophagic degradation of protein aggregates might contribute to neuroaxonal dystrophy in this disorder.

Loss of ap4s1 in zebrafish leads to neurodevelopmental defects resembling spastic paraplegia 52

“…We next examined spinal motor axons and found that morphants exhibited shorter axonal length, indicating that SPG52 may be important for the outgrowth of motor axons. This is consistent with impaired neuron outgrowth found in cultured neurons from Ap4e1 knockout mice 16,17 and similar to results seen in several zebrafish models of HSP including spastin, 18 atl1 , 19 and spatacsin 20 . Seizures are found in about two‐thirds of AP‐4‐HSP patients and febrile seizures are common early in life 3 .…”

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

“…Further, we found slowed SQSTM1/p62 accumulation in KO neurons during these treatments ( Figure 4(j, l); Table S1). Interestingly, steady state levels of LC3 were unaffected in cultured KO neurons (Figure 4(j,k)), but also in hippocampal lysates prepared from animals at 1 month (Figure 4(m,n); relative LC3-II:I ratio: WT 1 ± 0.012, KO 0.82 ± 0.13, p = 0.24; relative LC3-II: WT 1 ± 0.077, KO 1 ± 0.22, p = 0.99; relative LC3-I: WT 1 ± 0.089, KO 1.21 ± 0.14, p = 0.29; t-test), in agreement with data recently reported [30]. Further, we found a robust decrease in SQSTM1/p62 in KO animals at 1 month ( Figure 4(o,p); relative SQSTM1/p62 protein: WT 1 ± 0.11, KO 0.59 ± 0.05, p = 0.025; t-test), similar to that reported in ap4b1 null mice [13].…”

“…Similar to our in vivo findings, endogenous ATG9A was increased in KO neurons (Figure 3(a,b); relative neuronal ATG9A signal: WT 1 ± 0.06, KO 2.8 ± 0.2, p < 0.0001; t-test), and other cell types including astrocytes ( Figure S3 (a); relative astrocytic ATG9A signal: WT 1 ± 0.11 KO 2.42 ± 0.27, p < 0.0001; t-test). Given the known localization of AP-4 to the TGN in cell lines, we hypothesized that the sorting of ATG9A may be dependent on AP-4, and thus ATG9A may be retained within the TGN in neurons [26,29,30]. We firstly confirmed neuronal AP-4 localization at the TGN, through staining of endogenous AP4E1 and the TGN resident marker, GOLGA1/ Golgin-97/GOLG97 ( Figure S3(b)).…”

Novel insights into the clinical and molecular spectrum of congenital disorders of autophagy

“…AP‐4 is composed of four subunits (β4, ε, μ4, σ4) forming an obligate complex and has been shown to be involved in protein trafficking from the trans‐Golgi network to early and late endosomes . Recent studies in cultured cells have demonstrated that the autophagy protein ATG9A is a cargo of AP‐4 and that loss of AP‐4 leads to a mislocalization of ATG9A, potentially impacting the transport and function of ATG9A . Dysregulation of autophagy has been reported in AP‐4 knockout cells and Ap4e1 knockout mice …”