Mutations In WDR45 Research Articles

Lipid droplet accumulation in Wdr45-deficient cells caused by impairment of chaperone-mediated autophagic degradation of Fasn

Backgroundβ-Propeller protein-associated neurodegeneration (BPAN) is a genetic neurodegenerative disease caused by mutations in WDR45. The impairment of autophagy caused by WDR45 deficiency contributes to the pathogenesis of BPAN; however, the pathomechanism of this disease is largely unknown. Lipid dyshomeostasis is involved in neurogenerative diseases, but whether lipid metabolism is affected by Wdr45 deficiency and whether lipid dyshomeostasis contributes to the progression of BPAN are unclear.MethodsWe generated Wdr45 knockout SN4741 cell lines using CRISPR‒Cas9-mediated genome editing, then lipid droplets (LDs) were stained using BODIPY 493/503. Chaperone-mediated autophagy was determined by RT-qPCR and western blotting. The expression of fatty acid synthase (Fasn) was detected by western blot in the presence or absence of the lysosomal inhibitor NH4Cl and the CMA activator AR7. The interaction between Fasn and HSC70 was analyzed using coimmunoprecipitation (Co-IP) assay. Cell viability was measured by a CCK-8 kit after treatment with the Fasn inhibitor C75 or the CMA activator AR7.ResultsDeletion of Wdr45 impaired chaperone-mediated autophagy (CMA), thus leading to lipid droplet (LD) accumulation. Moreover, Fasn can be degraded via CMA, and that defective CMA leads to elevated Fasn, which promotes LD formation. LD accumulation is toxic to cells; however, cell viability was not rescued by Fasn inhibition or CMA activation. Inhibition of Fasn with a low concentration of C75 did not affect cell viability but decreases LD density.ConclusionsThese results suggested that Fasn is essential for cell survival but that excessive Fasn leads to LD accumulation in Wdr45 knockout cells.

Human WIPI β-propeller function in autophagy and neurodegeneration.

The four human WIPI β-propellers, WIPI1 through WIPI4, belong to the ancient PROPPIN family and fulfill scaffold functions in the control of autophagy. In this context, WIPI β-propellers function as PI3P effectors during autophagosome formation and loss of WIPI function negatively impacts autophagy and contributes to neurodegeneration. Of particular interest are mutations in WDR45, the human gene that encodes WIPI4. Sporadic WDR45 mutations are the cause of a rare human neurodegenerative disease called BPAN, hallmarked by high brain iron accumulation. Here, we discuss the current understanding of the functions of human WIPI β-propellers and address unanswered questions with a particular focus on the role of WIPI4 in autophagy and BPAN.

Antioxidants Prevent Iron Accumulation and Lipid Peroxidation, but Do Not Correct Autophagy Dysfunction or Mitochondrial Bioenergetics in Cellular Models of BPAN.

Neurodegeneration with brain iron accumulation (NBIA) is a group of rare neurogenetic disorders frequently associated with iron accumulation in the basal nuclei of the brain. Among NBIA subtypes, β-propeller protein-associated neurodegeneration (BPAN) is associated with mutations in the autophagy gene WDR45. The aim of this study was to demonstrate the autophagic defects and secondary pathological consequences in cellular models derived from two patients harboring WDR45 mutations. Both protein and mRNA expression levels of WDR45 were decreased in patient-derived fibroblasts. In addition, the increase of LC3B upon treatments with autophagy inducers or inhibitors was lower in mutant cells compared to control cells, suggesting decreased autophagosome formation and impaired autophagic flux. A transmission electron microscopy (TEM) analysis showed mitochondrial vacuolization associated with the accumulation of lipofuscin-like aggregates containing undegraded material. Autophagy dysregulation was also associated with iron accumulation and lipid peroxidation. In addition, mutant fibroblasts showed altered mitochondrial bioenergetics. Antioxidants such as pantothenate, vitamin E and α-lipoic prevented lipid peroxidation and iron accumulation. However, antioxidants were not able to correct the expression levels of WDR45, neither the autophagy defect nor cell bioenergetics. Our study demonstrated that WDR45 mutations in BPAN cellular models impaired autophagy, iron metabolism and cell bioenergetics. Antioxidants partially improved cell physiopathology; however, autophagy and cell bioenergetics remained affected.

Comprehensive analysis of autophagic functions of WIPI family proteins and their implications for the pathogenesis of β-propeller associated neurodegeneration.

β-propellers that bind polyphosphoinositides (PROPPINs) are an autophagy-related protein family conserved throughout eukaryotes. The PROPPIN family includes Atg18, Atg21, and Hsv2 in yeast and WD-repeat protein interacting with phosphoinositides (WIPI)1-4 in mammals. Mutations in the WIPI genes are associated with human neuronal diseases, including β-propeller associated neurodegeneration (BPAN) caused by mutations in WDR45 (encoding WIPI4). In contrast to yeast PROPPINs, the functions of mammalian WIPI1-WIPI4 have not been systematically investigated. Although the involvement of WIPI2 in autophagy has been clearly shown, the functions of WIPI1, WIPI3, and WIPI4 in autophagy remain poorly understood. In this study, we comprehensively analyzed the roles of WIPI proteins by using WIPI-knockout (single, double, and quadruple knockout) HEK293T cells and recently developed HaloTag-based reporters, which enable us to monitor autophagic flux sensitively and quantitatively. We found that WIPI2 was nearly essential for autophagy. Autophagic flux was unaffected or only slightly reduced by single deletion of WIPI3 (encoded by WDR45B) or WIPI4 but was profoundly reduced by double deletion of WIPI3 and WIPI4. Furthermore, we revealed variable effects of BPAN-related missense mutations on the autophagic activity of WIPI4. BPAN is characterized by neurodevelopmental and neurodegenerative abnormalities, and we found a possible association between the magnitude of the defect of the autophagic activity of WIPI4 mutants and the severity of neurodevelopmental symptoms. However, some of the BPAN-related missense mutations, which produce neurodegenerative signs, showed almost normal autophagic activity, suggesting that non-autophagic functions of WIPI4 may be related to neurodegeneration in BPAN.

Clinical Exome Sequencing in Patients with Undifferentiated General Developmental Delay and Intellectual Disabilities

Introduction. Each fifth neurodevelopmental disorder is diagnosed in massively parallel sequencing only.
 Objective: to present the experience of exome sequencing in children with undifferentiated general developmental delay and intellectual disabilities.
 Materials and methods. We assessed 33 patients (19 males and 14 females) at the age of 4.5 2.4 years with general developmental delay and intellectual disabilities. We studied patients' medical and family histories and their neurological statuses as well as the findings of neuropsychological testing and clinical exome sequencing.
 Results.The effectiveness of clinical exome sequencing in the sample was 39.4% (MECP2, WDR45, SYNJ1, ADAR, PMM2, SHANK3, KMT5B, UBE3A, PTPN11, CTNNB1, and MTOR mutations). The pathogenic variants were significantly more prevalent in the patients with motor development delay, 53.8% of patients (P .05). Most incident conditions included insomnias (46.2%), autism spectrum disorders (38.5%), developmental regression (38.5%), and epilepsy (38.5%). Genetic disorders were more common in the female patients .
 Conclusion. With the study results, we can suppose that ca. 40% of patients with undifferentiated general developmental delay and intellectual disabilities had genetic disorders and, therefore, needed further evaluation with molecular genetic testing.

Beta-propeller protein-associated neurodegeneration: A clinical update with a case report.

Unlabelled Image.

Expanding the Spectrum of Early Neuroradiologic Findings in β Propeller Protein-Associated Neurodegeneration.

β propeller protein-associated neurodegeneration (BPAN) is the most common neurodegeneration with brain iron accumulation disorder. Typical radiologic findings are T2 hypointensity in the substantia nigra and globus pallidus, as well as a T1 halolike substantia nigra hyperintense signal surrounding a hypointense central area. However, these findings are often subtle or absent on initial scans, risking diagnostic delay. In this study, we sought to investigate radiologic findings that could aid in the early diagnosis of BPAN. A retrospective cohort study was performed in a national referral center, including all pediatric patients with confirmed pathogenic WDR45 mutations and consistent clinical semiology. MR imaging findings were independently reported by 2 pediatric neuroradiologists. Fifteen patients were included in the study, and 27 scans were available for review. The initial neuroimaging study was undertaken at a mean age of 3.2 years. Iron deposition was uncommon in patients younger than 4 years of age. Neuroradiologic features from very early on included dentate, globus pallidus, and substantia nigra swelling, as well as a thin corpus callosum and small pontine volume. Optic nerve thinning was also present in all patients. Our study highlights the key early MR imaging features of BPAN. Iron deposition in the globus pallidus and substantia nigra is not common in children younger than 4 years of age; clinicians should not be deterred from suspecting BPAN in the presence of the findings described in this study and the appropriate clinical context.

Physiological significance of WDR45, a responsible gene for \u03b2-propeller protein associated neurodegeneration (BPAN), in brain development

WDR45 plays an essential role in the early stage of autophagy. De novo heterozygous mutations in WDR45 have been known to cause β-propeller protein-associated neurodegeneration (BPAN), a subtype of neurodegeneration with brain iron accumulation (NBIA). Although BPAN patients display global developmental delay with intellectual disability, the neurodevelopmental pathophysiology of BPAN remains largely unknown. In the present study, we analyzed the physiological role of Wdr45 and pathophysiological significance of the gene abnormality during mouse brain development. Morphological and biochemical analyses revealed that Wdr45 is expressed in a developmental stage-dependent manner in mouse brain. Wdr45 was also found to be located in excitatory synapses by biochemical fractionation. Since WDR45 mutations are thought to cause protein degradation, we conducted acute knockdown experiments by in utero electroporation in mice to recapitulate the pathophysiological conditions of BPAN. Knockdown of Wdr45 caused abnormal dendritic development and synaptogenesis during corticogenesis, both of which were significantly rescued by co-expression with RNAi-resistant version of Wdr45. In addition, terminal arbors of callosal axons were less developed in Wdr45-deficient cortical neurons of adult mouse when compared to control cells. These results strongly suggest a pathophysiological significance of WDR45 gene abnormalities in neurodevelopmental aspects of BPAN.

A novel WDR45 mutation in a 6-year-old boy with autism, Lennox-Gastaut syndrome, and ADHD

Beta-propeller protein-associated neurodegeneration, i.e., neurodegeneration with brain iron accumulation type 5, is due to mutations in the WDR45 gene. We report a case where a novel pathogenic mutation was discovered.

The BPAN and intellectual disability disease proteins WDR45 and WDR45B modulate autophagosome-lysosome fusion

ABSTRACT WDR45 and WDR45B are β-propeller proteins belonging to the WIPI (WD repeat domain, phosphoinositide interacting) family. Mutations in WDR45 and WDR45B are genetically linked with beta-propeller protein-associated neurodegeneration (BPAN) and intellectual disability (ID), respectively. WDR45 and WDR45B are homologs of yeast Atg18. Atg18 forms a complex with Atg2 for autophagosome biogenesis, probably by transferring lipids from the ER to phagophores. We revealed that WDR45 and WDR45B are critical for autophagosome-lysosome fusion in neural cells. WDR45 and WDR45B, but not their disease-related mutants, bind to the tether protein EPG5 and facilitate its targeting to late endosomes/lysosomes. In Wdr45 Wdr45b-deficient cells, the formation of tether-SNARE fusion machinery is compromised. The macroautophagy/autophagy deficiency in wdr45 wdr45b DKO cells is ameliorated by suppression of O-GlcNAcylation, which promotes autophagosome maturation. Thus, our results provide insights into the pathogenesis of WDR45- and WDR45B-related neurological diseases.

WDR45 Mutation Impairs the Autophagic Degradation of Transferrin Receptor and Promotes Ferroptosis.

WDR45 is an autophagy-related protein that involves in the formation of autophagosome. Mutations in WDR45 lead to the impairment of autophagy which is associated with the human β-propeller protein-associated neurodegeneration (BPAN). However, the relationship between autophagy and brain iron accumulation in patients with BPAN remains unclear. Here, we demonstrated that transferrin receptor (TfRC) which is critical for the iron import of cells was degraded via autophagy. TfRC was accumulated after the inhibition of autophagy by treatment with autophagic inhibitor chloroquine or knockdown of ATG2A. The intracellular iron content was increased in cells overexpressing TfRC or mutant WDR45, however, ferritin H (FTH) chain was decreased. Increased TfRC and simultaneously decreased FTH consequently resulted in an elevated level of ferrous iron (Fe2+) which further promoted cell ferroptosis, demonstrated by the increased lipid peroxidation and reactive oxygen species (ROS) and the decreased glutathione peroxidase 4 (GPX4) and cell viability. Taken together, these findings provide a piece of important evidence that WDR45 deficiency impairs autophagic degradation of TfRC, therefore leading to iron accumulation, and the elevated iron promotes ferroptosis which may contribute to the progression of BPAN.

WDR45 Gene and Its Role in Pediatric Epilepsies

AbstractWD repeat domain 45 (WDR45) gene has been increasingly found in patients with developmental delay (DD) and epilepsy. Previously, WDR45 de novo mutations were reported in sporadic adult and pediatric patients presenting iron accumulation, while heterozygous mutations were associated with β-propeller protein-associated neurodegeneration (BPAN), a subtype of neurodegeneration with brain iron accumulation disorders, characterized by extrapyramidal movement disorders and abnormal accumulation of iron in the basal ganglia. Overall, people harboring WDR45 mutations have moderate to severe DD and different types of seizures. The phenotype of adult patients is characterized by extrapyramidal movement, dystonia, parkinsonism, language impairment, and involvement of the substantia nigra and in the globus pallidus at brain magnetic resonance imaging. Importantly, there are no findings of brain iron accumulation in brain in BPAN patients in the first decade of life, thus suggesting a progressive course of the disease. Comparatively, the main phenotype of pediatric patients is epilepsy with early onset, most of which present infantile spasms and arrest or regression of psychomotor development. The phenotype of patients with WDR45 mutations is variable, being different if caused by somatic mosaicism or germline mutations, and presenting with a different spectrum of manifestations in males and females. The treatment of affected individuals is symptomatic. Regarding the seizures, specific, gene-based approaches with specific antiepileptic drugs are not currently available. The early diagnosis of BPAN could be useful in some aspects, such as providing families a supportive treatment to their affected children.

β-propeller proteins WDR45 and WDR45B regulate autophagosome maturation into autolysosomes in neural cells

Mutations in WDR45 and WDR45B cause the human neurological diseases β-propeller protein-associated neurodegeneration (BPAN) and intellectual disability (ID), respectively. WDR45 and WDR45B, along with WIPI1 and WIPI2, belong to a WD40 repeat-containing phosphatidylinositol-3-phosphate (PI(3)P)-binding protein family. Their yeast homolog Atg18 forms a complex with Atg2 and is required for autophagosome formation in part by tethering isolation membranes (IMs) (autophagosome precursor) to the endoplasmic reticulum (ER) to supply lipid for IM expansion in the autophagy pathway. The exact functions of WDR45/45B are unclear. We show here that WDR45/45B are specifically required for neural autophagy. In Wdr45/45b-depleted cells, the size of autophagosomes is decreased, and this is rescued by overexpression of ATG2A, providing invivo evidence for the lipid transfer activity of ATG2-WIPI complexes. WDR45/45B are dispensable for the closure of autophagosomes but essential for the progression of autophagosomes into autolysosomes. WDR45/45B interact with the tether protein EPG5 and target it to late endosomes/lysosomes to promote autophagosome maturation. In the absence of Wdr45/45b, formation of the fusion machinery, consisting of SNARE proteins and EPG5, is dampened. BPAN- and ID-related mutations of WDR45/45B fail to rescue the autophagy defects in Wdr45/45b-deficient cells, possibly due to their impaired binding to EPG5. Promoting autophagosome maturation by inhibiting O-GlcNAcylation increases SNARE complex formation and facilitates the fusion of autophagosomes with late endosomes/lysosomes in Wdr45/45b double knockout (DKO) cells. Thus, our results uncover a novel function of WDR45/45B in autophagosome-lysosome fusion and provide molecular insights into the development of WDR45/WDR45B mutation-associated diseases.

Phenotypic and Imaging Spectrum Associated With WDR45

BackgroundMutations in the X-linked gene WDR45 cause neurodegeneration with brain iron accumulation type 5. Global developmental delay occurs at an early age with slow progression to dystonia, parkinsonism, and dementia due to progressive iron accumulation in the brain. MethodsWe present 17 new cases and reviewed 106 reported cases of neurodegeneration with brain iron accumulation type 5. Detailed information related to developmental history and key time to event measures was collected. ResultsWithin this cohort, there were 19 males. Most individuals were molecularly diagnosed by whole-exome testing. Overall 10 novel variants were identified across 11 subjects. All individuals were affected by developmental delay, most prominently in verbal skills. Most individuals experienced a decline in motor and cognitive skills. Although most individuals were affected by seizures, the spectrum ranged from provoked seizures to intractable epilepsy. The imaging findings varied as well, often evolving over time. The classic iron accumulation in the globus pallidus and substantia nigra was noted in half of our cohort and was associated with older age of image acquisition, whereas myelination abnormalities were associated with younger age. ConclusionsWDR45 mutations lead to a progressive and evolving disorder whose diagnosis is often delayed. Developmental delay and seizures predominate in early childhood, followed by a progressive decline of neurological function. There is variable expressivity in the clinical phenotypes of individuals with WDR45 mutations, suggesting that this gene should be considered in the diagnostic evaluation of children with myelination abnormalities, iron deposition, developmental delay, and epilepsy depending on the age at evaluation.

Epileptic encephalopathy and brain iron accumulation due to WDR45 mutation

Epileptic encephalopathy and brain iron accumulation due to WDR45 mutation

Functional evidence for a de novo mutation in WDR45 leading to BPAN in a Chinese girl.

BackgroundBeta‐propeller protein‐associated neurodegeneration (BPAN, OMIM 300894) is an X‐linked neurodegenerative disorder caused by mutations in WDR45. WDR45 is required for autophagy, defect in WDR45 impaired autophagy which contributes for the pathogenesis of BPAN. Previously, we reported a novel de novo mutation (c.1040_1041del, p.Glu347GlyfsTer7) in WDR45 (NM_007075) in a 3‐year‐old Chinese girl with BPAN.MethodsThe protein structure was constructed using SWISS‐MODEL and the isoelectric point (pI) was predicted by the online pI/Mw tool at ExPASy. The functional effects of this mutation were predicted by two online software programs: PROVEN and MutationTaster. Stable overexpression of Flag‐tagged wild‐type or mutant WDR45 in HeLa cells was constructed. Protein levels of LC3 and p62 were analyzed by western blot upon treatment with/without autophagy inhibitor Bafilomycin A1, the formation of LC3 puncta were analyzed in HeLa cells transfected with mCherry‐LC3 by confocal microscopy.ResultsThe mutation resulted in a shift of pI from 6.74 to 8.84 and was predicted to be pathogenic. The protein levels of LC3‐II and p62 were increased in cells overexpression of wild‐type and mutant WDR45 while the protein levels were not increased in cells overexpression of mutant WDR45 upon treatment with autophagy inhibitor Bafilomycin A1. Results from confocal microscopy revealed that LC3‐positive puncta were increased in cells expressing both wild‐type and mutant WDR45 while the number of LC3‐positive puncta was not increased in cells expressing mutant WDR45 upon treatment with Bafilomycin A1.ConclusionOur study evidenced that this novel mutation in WDR45 impaired autophagy in cells thus this mutation is the cause for BPAN in this patient.

Autistic Parkinsonism: NBIA with Mosaic WDR45 Mutation and DaTscan positive dopa-Responsiveness (P3.8-022)

Autistic Parkinsonism: NBIA with Mosaic WDR45 Mutation and DaTscan positive dopa-Responsiveness (P3.8-022)

Substantia Nigra Swelling and Dentate Nucleus T2 Hyperintensity May Be Early Magnetic Resonance Imaging Signs of β-Propeller Protein-Associated Neurodegeneration.

Mutations in WDR45 cause β-propeller protein-associated neurodegeneration (BPAN), a type of neurodegeneration with brain iron accumulation (NBIA). We reviewed clinical and MRI findings in 4 patients with de novo WDR45 mutations. Psychomotor delay and movement disorders were present in all cases; early-onset epileptic encephalopathy was present in 3. In all cases, first MRI showed: prominent bilateral SN enlargement, bilateral dentate nuclei T2-hyperintensity, and corpus callosum thinning. Iron deposition in the SN and globus pallidus (GP) only became evident later. Diffuse cerebral atrophy was present in 3 cases. In this series, SN swelling and dentate nucleus T2 hyperintensity were early signs of BPAN, later followed by progressive iron deposition in the SN and GP. When clinical suspicion is raised, MRI is crucial for identifying early features suggesting this type of NBIA.

Beta-propeller protein-associated neurodegeneration (BPAN) as a genetically simple model of multifaceted neuropathology resulting from defects in autophagy

Autophagy is an essential and conserved cellular homeostatic process. Defects in the core and accessory components of the autophagic machinery would most severely impact terminally differentiated cells, such as neurons. The neurodevelopmental/neurodegenerative disorder β-propeller protein-associated neurodegeneration (BPAN) resulted from heterozygous or hemizygous germline mutations/pathogenic variant of the X chromosome gene WDR45, encoding WD40 repeat protein interacting with phosphoinositides 4 (WIPI4). This most recently identified subtype of the spectrum of neurodegeneration with brain iron accumulation diseases is characterized by a biphasic mode of disease manifestation and progression. The first phase involves early-onset of epileptic seizures, global developmental delay, intellectual disability and autistic syndrome. Subsequently, Parkinsonism and dystonia, as well as dementia, emerge in a subacute manner in adolescence or early adulthood. BPAN disease phenotypes are thus complex and linked to a wide range of other neuropathological disorders. WIPI4/WDR45 has an essential role in autophagy, acting as a phosphatidylinositol 3-phosphate binding effector that participates in autophagosome biogenesis and size control. Here, we discuss recent updates on WIPI4's mechanistic role in autophagy and link the neuropathological manifestations of BPAN's biphasic infantile onset (epilepsy, autism) and adolescent onset (dystonic, Parkinsonism, dementia) phenotypes to neurological consequences of autophagy impairment that are now known or emerging in many other neurodevelopmental and neurodegenerative disorders. As monogenic WDR45 mutations in BPAN result in a large spectrum of disease phenotypes that stem from autophagic dysfunctions, it could potentially serve as a simple and unique genetic model to investigate disease pathology and therapeutics for a wider range of neuropathological conditions with autophagy defects.

Chapter 19 - Neurodegeneration with brain iron accumulation

Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of disorders affecting children and adults. These rare disorders are often first suspected when increased basal ganglia iron is observed on brain magnetic resonance imaging. For the majority of NBIA disorders the genetic basis has been delineated, and clinical testing is available. The four most common NBIA disorders include pantothenate kinase-associated neurodegeneration (PKAN) due to mutations in PANK2, phospholipase A2-associated neurodegeneration caused by mutation in PLA2G6, mitochondrial membrane protein-associated neurodegeneration from mutations in C19orf12, and beta-propeller protein-associated neurodegeneration due to mutations in WDR45. The ultrarare NBIA disorders are caused by mutations in CoASY, ATP13A2, and FA2H (causing CoA synthase protein-associated neurodegeneration, Kufor-Rakeb disease, and fatty acid hydroxylase-associated neurodegeneration, respectively). Together, these genes account for disease in approximately 85% of patients diagnosed with an NBIA disorder. New NBIA genes are being recognized with increasing frequency as a result of whole-exome sequencing, which is also facilitating early ascertainment of patients whose phenotype is often nonspecific.