scholarly journals High-throughput imaging of ATG9A distribution as a diagnostic functional assay for adaptor protein complex 4-associated hereditary spastic paraplegia

2021 ◽  
Author(s):  
Darius Ebrahimi-Fakhari ◽  
Julian E Alecu ◽  
Barbara Brechmann ◽  
Marvin Ziegler ◽  
Kathrin Eberhardt ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Complex ◽  
Hereditary Spastic Paraplegia ◽  
Transmembrane Protein ◽  
Adaptor Protein ◽  
Molecular Testing ◽  
Functional Assay ◽  
Spastic Paraplegia ◽  
Characteristic Analysis ◽  
Missense Variants

Abstract Adaptor protein complex 4 (AP-4)-associated hereditary spastic paraplegia is caused by biallelic loss-of-function variants in AP4B1, AP4M1, AP4E1 or AP4S1, which constitute the four subunits of this obligate complex. While the diagnosis of AP-4-associated hereditary spastic paraplegia relies on molecular testing, the interpretation of novel missense variants remains challenging. Here we address this diagnostic gap by using patient-derived fibroblasts to establish a functional assay that measures the subcellular localization of ATG9A, a transmembrane protein that is sorted by AP-4. Using automated high-throughput microscopy, we determine the ratio of the ATG9A fluorescence in the trans-Golgi-network versus cytoplasm and ascertain that this metric meets standards for screening assays (Z’-factor robust > 0.3, strictly standardized mean difference > 3). The ‘ATG9A ratio’ is increased in fibroblasts of 18 well-characterized AP-4-associated hereditary spastic paraplegia patients (mean: 1.54 ± 0.13 vs. 1.21 ± 0.05 (standard deviation) in controls) and receiver-operating-characteristic analysis demonstrates robust diagnostic power (area under the curve: 0.85, 95% confidence interval: 0.849–0.852). Using fibroblasts from two individuals with atypical clinical features and novel biallelic missense variants of unknown significance in AP4B1, we show that our assay can reliably detect AP-4 function. Our findings establish the ‘ATG9A ratio’ as a diagnostic marker of AP-4-associated hereditary spastic paraplegia.

scholarly journals Defining the clinical, molecular and imaging spectrum of adaptor protein complex 4-associated hereditary spastic paraplegia

Brain ◽  
2020 ◽  
Author(s):  
Darius Ebrahimi-Fakhari ◽  
Julian Teinert ◽  
Robert Behne ◽  
Miriam Wimmer ◽  
Angelica D'Amore ◽  
...  
Keyword(s):  
Disease Severity ◽  
Protein Complex ◽  
Hereditary Spastic Paraplegia ◽  
Adaptor Protein ◽  
Allelic Loss ◽  
Foot Deformities ◽  
Periventricular White Matter ◽  
Spastic Paraplegia ◽  
Clinical Imaging ◽  
The Mean

Abstract Bi-allelic loss-of-function variants in genes that encode subunits of the adaptor protein complex 4 (AP-4) lead to prototypical yet poorly understood forms of childhood-onset and complex hereditary spastic paraplegia: SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). Here, we report a detailed cross-sectional analysis of clinical, imaging and molecular data of 156 patients from 101 families. Enrolled patients were of diverse ethnic backgrounds and covered a wide age range (1.0–49.3 years). While the mean age at symptom onset was 0.8 ± 0.6 years [standard deviation (SD), range 0.2–5.0], the mean age at diagnosis was 10.2 ± 8.5 years (SD, range 0.1–46.3). We define a set of core features: early-onset developmental delay with delayed motor milestones and significant speech delay (50% non-verbal); intellectual disability in the moderate to severe range; mild hypotonia in infancy followed by spastic diplegia (mean age: 8.4 ± 5.1 years, SD) and later tetraplegia (mean age: 16.1 ± 9.8 years, SD); postnatal microcephaly (83%); foot deformities (69%); and epilepsy (66%) that is intractable in a subset. At last follow-up, 36% ambulated with assistance (mean age: 8.9 ± 6.4 years, SD) and 54% were wheelchair-dependent (mean age: 13.4 ± 9.8 years, SD). Episodes of stereotypic laughing, possibly consistent with a pseudobulbar affect, were found in 56% of patients. Key features on neuroimaging include a thin corpus callosum (90%), ventriculomegaly (65%) often with colpocephaly, and periventricular white-matter signal abnormalities (68%). Iron deposition and polymicrogyria were found in a subset of patients. AP4B1-associated SPG47 and AP4M1-associated SPG50 accounted for the majority of cases. About two-thirds of patients were born to consanguineous parents, and 82% carried homozygous variants. Over 70 unique variants were present, the majority of which are frameshift or nonsense mutations. To track disease progression across the age spectrum, we defined the relationship between disease severity as measured by several rating scales and disease duration. We found that the presence of epilepsy, which manifested before the age of 3 years in the majority of patients, was associated with worse motor outcomes. Exploring genotype-phenotype correlations, we found that disease severity and major phenotypes were equally distributed among the four subtypes, establishing that SPG47, SPG50, SPG51 and SPG52 share a common phenotype, an ‘AP-4 deficiency syndrome’. By delineating the core clinical, imaging, and molecular features of AP-4-associated hereditary spastic paraplegia across the age spectrum our results will facilitate early diagnosis, enable counselling and anticipatory guidance of affected families and help define endpoints for future interventional trials.

scholarly journals Clinical and pathogenic themes in hereditary spastic paraplegia

Brain ◽  
2020 ◽  
Vol 143 (10) ◽  
pp. 2864-2866
Author(s):  
Thomas T Warner
Keyword(s):  
Protein Complex ◽  
Hereditary Spastic Paraplegia ◽  
Adaptor Protein ◽  
Spastic Paraplegia

This scientific commentary refers to ‘Defining the clinical, molecular and imaging spectrum of adaptor protein complex 4-associated hereditary spastic paraplegia’, by Ebrahimi-Fakhari etal. (doi:10.1093/brain/awz307).

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

2020 ◽  
Vol 29 (2) ◽  
pp. 320-334 ◽  
Author(s):  
Robert Behne ◽  
Julian Teinert ◽  
Miriam Wimmer ◽  
Angelica D’Amore ◽  
Alexandra K Davies ◽  
...  
Keyword(s):  
Protein Complex ◽  
Protein Trafficking ◽  
Hereditary Spastic Paraplegia ◽  
Adaptor Protein ◽  
Spastic Paraplegia ◽  
Childhood Onset ◽  
Trans Golgi Network ◽  
Wide Range ◽  
Golgi Network ◽  
The Impact

Abstract 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.

scholarly journals UNC93B1 mediates differential trafficking of endosomal TLRs

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Bettina L Lee ◽  
Joanne E Moon ◽  
Jeffrey H Shu ◽  
Lin Yuan ◽  
Zachary R Newman ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Autoimmune Diseases ◽  
Protein Complex ◽  
Secretory Pathway ◽  
Transmembrane Protein ◽  
Adaptor Protein ◽  
Regulatory Effects ◽  
Copii Vesicles

UNC93B1, a multipass transmembrane protein required for TLR3, TLR7, TLR9, TLR11, TLR12, and TLR13 function, controls trafficking of TLRs from the endoplasmic reticulum (ER) to endolysosomes. The mechanisms by which UNC93B1 mediates these regulatory effects remain unclear. Here, we demonstrate that UNC93B1 enters the secretory pathway and directly controls the packaging of TLRs into COPII vesicles that bud from the ER. Unlike other COPII loading factors, UNC93B1 remains associated with the TLRs through post-Golgi sorting steps. Unexpectedly, these steps are different among endosomal TLRs. TLR9 requires UNC93B1-mediated recruitment of adaptor protein complex 2 (AP-2) for delivery to endolysosomes while TLR7, TLR11, TLR12, and TLR13 utilize alternative trafficking pathways. Thus, our study describes a mechanism for differential sorting of endosomal TLRs by UNC93B1, which may explain the distinct roles played by these receptors in certain autoimmune diseases.

The role of AP-4 in cargo export from the trans-Golgi network and hereditary spastic paraplegia

2020 ◽  
Vol 48 (5) ◽  
pp. 1877-1888
Author(s):  
Rafael Mattera ◽  
Raffaella De Pace ◽  
Juan S. Bonifacino
Keyword(s):  
Hereditary Spastic Paraplegia ◽  
Adaptor Protein ◽  
Deficiency Syndrome ◽  
Small Gtpases ◽  
Neurological Dysfunction ◽  
Dependent Manner ◽  
Spastic Paraplegia ◽  
Trans Golgi Network ◽  
Cytosolic Domains ◽  
Golgi Network

Heterotetrameric adaptor protein (AP) complexes play key roles in protein sorting and transport vesicle formation in the endomembrane system of eukaryotic cells. One of these complexes, AP-4, was identified over 20 years ago but, up until recently, its function remained unclear. AP-4 associates with the trans-Golgi network (TGN) through interaction with small GTPases of the ARF family and recognizes transmembrane proteins (i.e. cargos) having specific sorting signals in their cytosolic domains. Recent studies identified accessory proteins (tepsin, RUSC2 and the FHF complex) that co-operate with AP-4, and cargos (amyloid precursor protein, ATG9A and SERINC3/5) that are exported from the TGN in an AP-4-dependent manner. Defective export of ATG9A from the TGN in AP-4-deficient cells was shown to reduce ATG9A delivery to pre-autophagosomal structures, impairing autophagosome formation and/or maturation. In addition, mutations in AP-4-subunit genes were found to cause neurological dysfunction in mice and a form of complicated hereditary spastic paraplegia referred to as ‘AP-4-deficiency syndrome’ in humans. These findings demonstrated that mammalian AP-4 is required for the development and function of the central nervous system, possibly through its role in the sorting of ATG9A for the maintenance of autophagic homeostasis. In this article, we review the properties and functions of AP-4, and discuss how they might explain the clinical features of AP-4 deficiency.

scholarly journals Genetic testing of hereditary spastic paraplegia

2015 ◽  
Vol 156 (3) ◽  
pp. 113-117 ◽  
Author(s):  
Kinga Hadzsiev ◽  
László Balikó ◽  
Katalin Komlósi ◽  
Anett Lőcsei-Fekete ◽  
Györgyi Csábi ◽  
...  
Keyword(s):  
Hereditary Spastic Paraplegia ◽  
Molecular Testing ◽  
Spastic Paraplegia ◽  
Chain Reaction ◽  
Hereditary Spastic Paraplegias ◽  
Gene Testing ◽  
New Information ◽  
Common Causes ◽  
Polymerase Chain ◽  
Dependent Probe

Introduction: Hereditary spastic paraplegia is the overall term for clinically and genetically diverse disorders characterized with progressive and variable severe lower extremity spasticity. The most common causes of autosomal dominantly inherited hereditary spastic paraplegias are different mutations of the spastin gene with variable incidence in different ethnic groups, ranging between 15–40%. Mutations in the spastin gene lead to loss of spastins function, causing progressive neuronal failure, which results in axon degeneration finally. Aim: The molecular testing of spastin gene is available in the institution of the authors since January, 2014. The experience gained with the examination of the first eleven patients is described in this article. Method: After polymerase chain reaction, Sanger sequencing was performed to examine the 17 exons of the spastin gene. Multiplex ligation-dependent probe amplification was performed to detect greater rearrangements in the spastin gene. Eight of the patients were examined in the genetic counseling clinic of the authors and after detailed phenotype assessment spastin gene testing was obtained. The other three patients were referred to the laboratory from different outpatient clinics. Results: Out of the 11 examined patients, four different pathogenic mutations were found in 5 patients. Conclusions: The first Hungarian data, gained with the examination of spastin gene are presented in this article. The five patients, in whom mutations were detected, represent 45.5% of all tested patients with hereditary spastic paraplegia, which is similar to those published in the international literature. Molecular testing and subsequent detailed genotype-phenotype correlations of the Hungarian patients may serve valuable new information about the disease, which later on may influence our therapeutic possibilities and decisions. Orv. Hetil., 2015, 156(3), 113–117.

Systematic Analysis of Brain MRI Findings in Adaptor Protein Complex 4–Associated Hereditary Spastic Paraplegia

Neurology ◽  
2021 ◽  
pp. 10.1212/WNL.0000000000012836
Author(s):  
Darius Ebrahimi-Fakhari ◽  
Julian E Alecu ◽  
Marvin Ziegler ◽  
Gregory Geisel ◽  
Catherine Jordan ◽  
...  
Keyword(s):  
White Matter ◽  
Corpus Callosum ◽  
Brain Development ◽  
Hereditary Spastic Paraplegia ◽  
Anterior Commissure ◽  
Brain Mri ◽  
Adaptor Protein ◽  
Spastic Paraplegia ◽  
Mri Findings

Background and Objectives:AP-4-associated hereditary spastic paraplegia (AP-4-HSP: SPG47, SPG50, SPG51, SPG52) is an emerging cause of childhood-onset hereditary spastic paraplegia and mimic of cerebral palsy. This study aims to define the spectrum of brain MRI findings in AP-4-HSP and to investigate radio-clinical correlations.Methods:A systematic qualitative and quantitative analysis of 107 brain MRI studies from 76 individuals with genetically-confirmed AP-4-HSP and correlation with clinical findings including surrogates of disease severity.Results:We define AP-4-HSP as a disorder of gray and white matter and demonstrate that abnormal myelination is common and that metrics of reduced white matter volume correlate with severity of motor symptoms. We identify a common diagnostic imaging signature consisting of (1) a thin splenium of the corpus callosum, (2) an absent or thin anterior commissure, (3) characteristic signal abnormalities of the forceps minor (“ears of the grizzly sign”), and (4) periventricular white matter abnormalities. The presence of two or more of these findings has a sensitivity of ∼99% for detecting AP-4-HSP, while the combination of all four is found in ∼45% of cases. Compared to other HSP with a thin corpus callosum, the absent anterior commissure appears to be specific to AP-4-HSP. Our analysis further identified a subset of AP-4-HSP patients with polymicrogyria, underscoring the role of AP-4 in early brain development. Of clinical importance, these patients displayed a higher prevalence of seizures and status epilepticus, many at a young age.Discussion:Our findings define the MRI spectrum of AP-4-HSP providing opportunities for early diagnosis, identification of individuals at risk for complications, and a window into the role of the AP-4 complex in brain development and neurodegeneration.

scholarly journals Adaptor Protein Complex 4 Deficiency Causes Severe Autosomal-Recessive Intellectual Disability, Progressive Spastic Paraplegia, Shy Character, and Short Stature

2011 ◽  
Vol 88 (6) ◽  
pp. 788-795 ◽  
Author(s):  
Rami Abou Jamra ◽  
Orianne Philippe ◽  
Annick Raas-Rothschild ◽  
Sebastian H. Eck ◽  
Elisabeth Graf ◽  
...  
Keyword(s):  
Intellectual Disability ◽  
Short Stature ◽  
Protein Complex ◽  
Autosomal Recessive ◽  
Adaptor Protein ◽  
Spastic Paraplegia

scholarly journals The Mouse Homolog of the Bovine Leukemia Virus Receptor Is Closely Related to the δ Subunit of Adaptor-Related Protein Complex AP-3, Not Associated with the Cell Surface

1998 ◽  
Vol 72 (1) ◽  
pp. 593-599 ◽  
Author(s):  
Takako Suzuki ◽  
Hidetoshi Ikeda
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Protein Complex ◽  
Bovine Leukemia Virus ◽  
Transmembrane Protein ◽  
Leukemia Virus ◽  
Adaptor Protein ◽  
Type I ◽  
Related Protein ◽  
Virus Receptor

ABSTRACT A mouse cDNA (mBLVR1) which was highly homologous to the bovine cDNA of the bovine leukemia virus receptor (BLVR) gene was cloned. The mBLVR1 cDNA, of 4,730 bp, covered nearly the full length of the mRNA (about 5 kb) and included an open reading frame (ORF) encoding a protein of 1,199 amino acids. While the bovine BLVR protein was thought to be a type I transmembrane protein, the deduced protein coded by mBLVR1 did not appear to be a typical transmembrane protein. The ORF of mBLVR1 ended at a site 280 amino acids upstream of the termination codon of the bovine BLVR ORF, so the deduced mouse BLVR protein lacked the corresponding transmembrane and cytoplasmic regions of the predicted bovine BLVR protein. No significant hydrophobic region was found in the mouse protein. Recently, a human cDNA which was highly homologous (69.6% homology) to the mouse BLVR gene was reported. The cDNA encodes the δ subunit of the human adaptor-related protein complex AP-3, which aligned almost collinearly with the mouse BLVR protein. AP-3 and all other related adaptor protein complexes have been shown to be associated with intracellular vesicles but not with the cell surface. Thus, the mouseBLVR homolog appeared to be the mouse AP-3 δ subunit itself or closely related to it, but the bovine BLVR gene seemed slightly different from the adaptor subunit gene family.

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