Combined generalized and focal epilepsy with reflex features in Adaptor protein complex 4-associated hereditary spastic paraplegias: A cohort observational study.

BACKGROUND: Patients with genetic deficiency of the adaptor protein complex 4 (AP-4) exhibit earlyonset developmental delay, spastic diplegia, intellectual disability, speech impairment. The phenotype overlaps with other hereditary spastic paraplegias and cerebral palsies. Febrile seizures are common at onset. Epilepsy has been described in more than half of cases, arising in early infancy often with status epilepticus, but no typical seizure semiology or electroencephalographic features have been identified thus far. PURPOSE: We aimed to specifically investigate the epileptological characteristics of the syndrome to unveil possible biomarkers of seizure development and prognosis in AP-4 deficiency. METHODS: Observational cohort study on patients with bi-allelic pathogenic variants in AP-4 subunits and epilepsy. We focused on the seizure semiology, electroencephalographic characteristics and response to antiseizure medications. RESULTS: Patients harboured pathogenic variants in AP4S1 (n = 5) or AP4M1 (n = 1). The phenotype included spastic paraparesis, intellectual disability, speech/language impairment, microcephaly, and MRI evidence of hypoplasia of the corpus callosum. In 66 % of the patients, febrile seizures preceded the onset of epilepsy, which spanned from infancy to adolescence (range=14 months-13 years). Absences (66 %) and focal motor seizures (50 %) were common. No patient met the criteria for drug-resistance. Peculiar electroencephalographic features arose after the epilepsy onset and persisted at long-term follow-up: bilateral and asynchronous focal discharges combined with independent diffuse spike-wave-discharges (100 %) and reflex abnormalities (66 %). CONCLUSION: In AP-4 complex disease, epilepsy could arise beyond early infancy, until adolescence, with variable combination of generalized and focal seizures. The prognosis was favourable. We observed a common electroencephalographic signature - combined focal/generalized and reflex abnormalities - which may constitute a biomarker of AP-4 deficiency with epilepsy, applicable to inform genetic testing and disentangle the differential diagnosis.

Comprehensive genotype-phenotype correlation in AP-4 deficiency syndrome; Adding data from a large cohort of Iranian patients.

Mutations in adaptor protein complex-4 (AP-4) genes have first been identified in 2009, causing a phenotype termed as AP-4 deficiency syndrome. Since then several patients with overlapping phenotypes, comprised of intellectual disability (ID) and spastic tetraplegia have been reported. To delineate the genotype-phenotype correlation of the AP-4 deficiency syndrome, we add the data from 30 affected individuals from 12 out of 640 Iranian families with ID in whom we detected disease-causing variants in AP-4 complex subunits, using next-generation sequencing. Furthermore, by comparing genotype-phenotype findings of those affected individuals with previously reported patients, we further refine the genotype-phenotype correlation in this syndrome. The most frequent reported clinical findings in the 101 cases consist of ID and/or global developmental delay (97%), speech disorders (92.1%), inability to walk (90.1%), spasticity (77.2%), and microcephaly (75.2%). Spastic tetraplegia has been reported in 72.3% of the investigated patients. The major brain imaging findings are abnormal corpus callosum morphology (63.4%) followed by ventriculomegaly (44.5%). Our result might suggest the AP-4 deficiency syndrome as a major differential diagnostic for unknown hereditary neurodegenerative disorders.

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

Autosomal recessive spastic paraplegia 52 is caused by biallelic mutations in AP4S1 which encodes a subunit of the adaptor protein complex 4 (AP-4). Using next-generation sequencing, we identified three novel unrelated SPG52 patients from a cohort of patients with cerebral palsy. The discovered variants in AP4S1 lead to reduced AP-4 complex formation in patient-derived fibroblasts. To further understand the role of AP4S1 in neuronal development and homeostasis, we engineered the first zebrafish model of AP-4 deficiency using morpholino-mediated knockdown of ap4s1. In this model, we discovered several phenotypes mimicking SPG52, including altered CNS development, locomotor deficits, and abnormal neuronal excitability.

Adaptor protein complex 4 deficiency: a paradigm of childhood-onset hereditary spastic paraplegia caused by defective protein trafficking.

Deficiency of the adaptor protein complex 4 (AP-4) leads to childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). This study aims to evaluate the impact of loss-of-function variants in AP-4 subunits on intracellular protein trafficking using patient-derived cells. We investigated 15 patient-derived fibroblast lines and generated six lines of induced pluripotent stem cell (iPSC)-derived neurons covering a wide range of AP-4 variants. All patient-derived fibroblasts showed reduced levels of the AP4E1 subunit, a surrogate for levels of the AP-4 complex. The autophagy protein ATG9A accumulated in the trans-Golgi network and was depleted from peripheral compartments. Western blot analysis demonstrated a 3-5-fold increase in ATG9A expression in patient lines. ATG9A was redistributed upon re-expression of AP4B1 arguing that mistrafficking of ATG9A is AP-4-dependent. Examining the downstream effects of ATG9A mislocalization, we found that autophagic flux was intact in patient-derived fibroblasts both under nutrient-rich conditions and when autophagy is stimulated. Mitochondrial metabolism and intracellular iron content remained unchanged. In iPSC-derived cortical neurons from patients with AP4B1-associated SPG47, AP-4 subunit levels were reduced while ATG9A accumulated in the trans-Golgi network. Levels of the autophagy marker LC3-II were reduced, suggesting a neuron-specific alteration in autophagosome turnover. Neurite outgrowth and branching were reduced in AP-4-HSP neurons pointing to a role of AP-4-mediated protein trafficking in neuronal development. Collectively, our results establish ATG9A mislocalization as a key marker of AP-4 deficiency in patient-derived cells, including the first human neuron model of AP-4-HSP, which will aid diagnostic and therapeutic studies.

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

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-ß4-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.

Adaptor protein complex-4 (AP-4) deficiency causes a novel autosomal recessive cerebral palsy syndrome with microcephaly and intellectual disability.

BACKGROUND: Cerebral palsy is a heterogeneous group of neurodevelopmental brain disorders resulting in motor and posture impairments often associated with cognitive, sensorial, and behavioural disturbances. Hypoxic-ischaemic injury, long considered the most frequent causative factor, accounts for fewer than 10% of cases, whereas a growing body of evidence suggests that diverse genetic abnormalities likely play a major role. METHODS AND RESULTS: This report describes an autosomal recessive form of spastic tetraplegic cerebral palsy with profound intellectual disability, microcephaly, epilepsy and white matter loss in a consanguineous family resulting from a homozygous deletion involving AP4E1, one of the four subunits of the adaptor protein complex-4 (AP-4), identified by chromosomal microarray analysis. CONCLUSION: These findings, along with previous reports of human and mouse mutations in other members of the complex, indicate that disruption of any one of the four subunits of AP-4 causes dysfunction of the entire complex, leading to a distinct 'AP-4 deficiency syndrome'.

Accumulation of AMPA receptors in autophagosomes in neuronal axons lacking adaptor protein AP-4.

AP-4 is a member of the adaptor protein complexes, which control vesicular trafficking of membrane proteins. Although AP-4 has been suggested to contribute to basolateral sorting in epithelial cells, its function in neurons is unknown. Here, we show that disruption of the gene encoding the beta subunit of AP-4 resulted in increased accumulation of axonal autophagosomes, which contained AMPA receptors and transmembrane AMPA receptor regulatory proteins (TARPs), in axons of hippocampal neurons and cerebellar Purkinje cells both in vitro and in vivo. AP-4 indirectly associated with the AMPA receptor via TARPs, and the specific disruption of the interaction between AP-4 and TARPs caused the mislocalization of endogenous AMPA receptors in axons of wild-type neurons. These results indicate that AP-4 may regulate proper somatodendritic-specific distribution of its cargo proteins, including AMPA receptor-TARP complexes and the autophagic pathway in neurons.

A novel human primary immunodeficiency syndrome caused by deficiency of the endosomal adaptor protein p14.

Lysosome-related organelles have versatile functions, including protein and lipid degradation, signal transduction and protein secretion. The molecular elucidation of rare congenital diseases affecting endosomal-lysosomal biogenesis has given insights into physiological functions of the innate and adaptive immune system. Here, we describe a previously unknown human primary immunodeficiency disorder and provide evidence that the endosomal adaptor protein p14, previously characterized as confining mitogen-activated protein kinase (MAPK) signaling to late endosomes, is crucial for the function of neutrophils, B cells, cytotoxic T cells and melanocytes. Combining genetic linkage studies and transcriptional profiling analysis, we identified a homozygous point mutation in the 3' untranslated region (UTR) of p14 (also known as MAPBPIP), resulting in decreased protein expression. In p14-deficient cells, the distribution of late endosomes was severely perturbed, suggesting a previously unknown role for p14 in endosomal biogenesis. These findings have implications for understanding endosomal membrane dynamics, compartmentalization of cell signal cascades, and their role in immunity.