Loss of the endoplasmic reticulum protein Tmem208 affects cell polarity, development, and viability.

Nascent proteins destined for the cell membrane and the secretory pathway are targeted to the endoplasmic reticulum (ER) either posttranslationally or cotranslationally. The signal-independent pathway, containing the protein TMEM208, is one of three pathways that facilitates the translocation of nascent proteins into the ER. The in vivo function of this protein is ill characterized in multicellular organisms. Here, we generated a CRISPR-induced null allele of the fruit fly ortholog CG8320/Tmem208 by replacing the gene with the Kozak-GAL4 sequence. We show that Tmem208 is broadly expressed in flies and that its loss causes lethality, although a few short-lived flies eclose. These animals exhibit wing and eye developmental defects consistent with impaired cell polarity and display mild ER stress. Tmem208 physically interacts with Frizzled (Fz), a planar cell polarity (PCP) receptor, and is required to maintain proper levels of Fz. Moreover, we identified a child with compound heterozygous variants in TMEM208 who presents with developmental delay, skeletal abnormalities, multiple hair whorls, cardiac, and neurological issues, symptoms that are associated with PCP defects in mice and humans. Additionally, fibroblasts of the proband display mild ER stress. Expression of the reference human TMEM208 in flies fully rescues the loss of Tmem208, and the two proband-specific variants fail to rescue, suggesting that they are loss-of-function alleles. In summary, our study uncovers a role of TMEM208 in development, shedding light on its significance in ER homeostasis and cell polarity.

Loss-of-function variants in TIAM1 are associated with developmental delay, intellectual disability, and seizures.

TIAM Rac1-associated GEF 1 (TIAM1) regulates RAC1 signaling pathways that affect the control of neuronal morphogenesis and neurite outgrowth by modulating the actin cytoskeletal network. To date, TIAM1 has not been associated with a Mendelian disorder. Here, we describe five individuals with bi-allelic TIAM1 missense variants who have developmental delay, intellectual disability, speech delay, and seizures. Bioinformatic analyses demonstrate that these variants are rare and likely pathogenic. We found that the Drosophila ortholog of TIAM1, still life (sif), is expressed in larval and adult central nervous system (CNS) and is mainly expressed in a subset of neurons, but not in glia. Loss of sif reduces the survival rate, and the surviving adults exhibit climbing defects, are prone to severe seizures, and have a short lifespan. The TIAM1 reference (Ref) cDNA partially rescues the sif loss-of-function (LoF) phenotypes. We also assessed the function associated with three TIAM1 variants carried by two of the probands and compared them to the TIAM1 Ref cDNA function in vivo. TIAM1 p.Arg23Cys has reduced rescue ability when compared to TIAM1 Ref, suggesting that it is a partial LoF variant. In ectopic expression studies, both wild-type sif and TIAM1 Ref are toxic, whereas the three variants (p.Leu862Phe, p.Arg23Cys, and p.Gly328Val) show reduced toxicity, suggesting that they are partial LoF variants. In summary, we provide evidence that sif is important for appropriate neural function and that TIAM1 variants observed in the probands are disruptive, thus implicating loss of TIAM1 in neurological phenotypes in humans.

De novo variants in EMC1 lead to neurodevelopmental delay and cerebellar degeneration and affect glial function in Drosophila.

BACKGROUND: The endoplasmic reticulum (ER)-membrane protein complex (EMC) is a multi-protein transmembrane complex composed of 10 subunits that functions as a membrane-protein chaperone. Variants in EMC1 lead to neurodevelopmental delay and cerebellar degeneration. Multiple families with biallelic variants have been published, yet to date, only a single report of a monoallelic variant has been described, and functional evidence is sparse. METHODS: Exome sequencing was used to investigate the genetic cause underlying severe developmental delay in three unrelated children. EMC1 variants were modeled in Drosophila, using loss-of-function (LoF) and overexpression studies. Glial-specific and neuronal-specific assays were used to determine whether the dysfunction was specific to one cell type. RESULTS: Exome sequencing identified de novo variants in EMC1 in three individuals affected by global developmental delay, hypotonia, seizures, visual impairment and cerebellar atrophy. All variants were located at Pro582 or Pro584. Drosophila studies indicated that imbalance of EMC1-either overexpression or knockdown-results in pupal lethality and suggest that the tested homologous variants are LoF alleles. In addition, glia-specific gene dosage, overexpression or knockdown, of EMC1 led to lethality, whereas neuron-specific alterations were tolerated. DISCUSSION: We establish de novo monoallelic EMC1 variants as causative of a neurological disease trait by providing functional evidence in a Drosophila model. The identified variants failed to rescue the lethality of a null allele. Variations in dosage of the wild-type EMC1, specifically in glia, lead to pupal lethality, which we hypothesize results from the altered stoichiometry of the multi-subunit protein complex EMC.

Variant functional assessment in Drosophila by overexpression: what can we learn?

The last decade has been highlighted by the increased use of next-generation DNA sequencing technology to identify novel human disease genes. A critical downstream part of this process is assigning function to a candidate gene variant. Functional studies in Drosophila melanogaster, the common fruit fly, have made a prominent contribution in annotating variant impact in an in vivo system. The use of patient-derived knock-in flies or rescue-based, "humanization", approaches are novel and valuable strategies in variant testing but have been recently widely reviewed. An often-overlooked strategy for determining variant impact has been GAL4/upstream activation sequence-mediated tissue-defined overexpression in Drosophila. This mini-review will summarize the recent contribution of ectopic overexpression of human reference and variant cDNA in Drosophila to assess variant function, interpret the consequence of the variant, and in some cases infer biological mechanisms.

TM2D genes regulate Notch signaling and neuronal function in Drosophila.

TM2 domain containing (TM2D) proteins are conserved in metazoans and encoded by three separate genes in each model organism species that has been sequenced. Rare variants in TM2D3 are associated with Alzheimer's disease (AD) and its fly ortholog almondex is required for embryonic Notch signaling. However, the functions of this gene family remain elusive. We knocked-out all three TM2D genes (almondex, CG11103/amaretto, CG10795/biscotti) in Drosophila and found that they share the same maternal-effect neurogenic defect. Triple null animals are not phenotypically worse than single nulls, suggesting these genes function together. Overexpression of the most conserved region of the TM2D proteins acts as a potent inhibitor of Notch signaling at the γ-secretase cleavage step. Lastly, Almondex is detected in the brain and its loss causes shortened lifespan accompanied by progressive motor and electrophysiological defects. The functional links between all three TM2D genes are likely to be evolutionarily conserved, suggesting that this entire gene family may be involved in AD.

Neuronal ROS-induced glial lipid droplet formation is altered by loss of Alzheimer's disease-associated genes.

A growing list of Alzheimer's disease (AD) genetic risk factors is being identified, but the contribution of each variant to disease mechanism remains largely unknown. We have previously shown that elevated levels of reactive oxygen species (ROS) induces lipid synthesis in neurons leading to the sequestration of peroxidated lipids in glial lipid droplets (LD), delaying neurotoxicity. This neuron-to-glia lipid transport is APOD/E-dependent. To identify proteins that modulate these neuroprotective effects, we tested the role of AD risk genes in ROS-induced LD formation and demonstrate that several genes impact neuroprotective LD formation, including homologs of human ABCA1, ABCA7, VLDLR, VPS26, VPS35, AP2A, PICALM, and CD2AP Our data also show that ROS enhances Aß42 phenotypes in flies and mice. Finally, a peptide agonist of ABCA1 restores glial LD formation in a humanized APOE4 fly model, highlighting a potentially therapeutic avenue to prevent ROS-induced neurotoxicity. This study places many AD genetic risk factors in a ROS-induced neuron-to-glia lipid transfer pathway with a critical role in protecting against neurotoxicity.

De Novo Variants in CDK19 Are Associated with a Syndrome Involving Intellectual Disability and Epileptic Encephalopathy.

We identified three unrelated individuals with de novo missense variants in CDK19, encoding a cyclin-dependent kinase protein family member that predominantly regulates gene transcription. These individuals presented with hypotonia, global developmental delay, epileptic encephalopathy, and dysmorphic features. CDK19 is conserved between vertebrate and invertebrate model organisms, but currently abnormalities in CDK19 are not known to be associated with a human disorder. Loss of Cdk8, the fly homolog of CDK19, causes larval lethality, which is suppressed by expression of human CDK19 reference cDNA. In contrast, the CDK19 p.Tyr32His and p.Thr196Ala variants identified in the affected individuals fail to rescue the loss of Cdk8 and behave as null alleles. Additionally, neuronal RNAi-mediated knockdown of Cdk8 in flies results in semi-lethality. The few eclosing flies exhibit severe seizures and a reduced lifespan. Both phenotypes are fully suppressed by moderate expression of the CDK19 reference cDNA but not by expression of the two variants. Finally, loss of Cdk8 causes an obvious loss of boutons and synapses at larval neuromuscular junctions (NMJs). Together, our findings demonstrate that human CDK19 fully replaces the function of Cdk8 in the fly, the human disease-associated CDK19 variants behave as strong loss-of-function variants, and deleterious CDK19 variants underlie a syndromic neurodevelopmental disorder.

Loss of Neuron Navigator 2 Impairs Brain and Cerebellar Development.

Cerebellar hypoplasia and dysplasia encompass a group of clinically and genetically heterogeneous disorders frequently associated with neurodevelopmental impairment. The Neuron Navigator 2 (NAV2) gene (MIM: 607,026) encodes a member of the Neuron Navigator protein family, widely expressed within the central nervous system (CNS), and particularly abundant in the developing cerebellum. Evidence across different species supports a pivotal function of NAV2 in cytoskeletal dynamics and neurite outgrowth. Specifically, deficiency of Nav2 in mice leads to cerebellar hypoplasia with abnormal foliation due to impaired axonal outgrowth. However, little is known about the involvement of the NAV2 gene in human disease phenotypes. In this study, we identified a female affected with neurodevelopmental impairment and a complex brain and cardiac malformations in which clinical exome sequencing led to the identification of NAV2 biallelic truncating variants. Through protein expression analysis and cell migration assay in patient-derived fibroblasts, we provide evidence linking NAV2 deficiency to cellular migration deficits. In model organisms, the overall CNS histopathology of the Nav2 hypomorphic mouse revealed developmental anomalies including cerebellar hypoplasia and dysplasia, corpus callosum hypo-dysgenesis, and agenesis of the olfactory bulbs. Lastly, we show that the NAV2 ortholog in Drosophila, sickie (sick) is widely expressed in the fly brain, and sick mutants are mostly lethal with surviving escapers showing neurobehavioral phenotypes. In summary, our results unveil a novel human neurodevelopmental disorder due to genetic loss of NAV2, highlighting a critical conserved role of the NAV2 gene in brain and cerebellar development across species.

De novo mutations in TOMM70, a receptor of the mitochondrial import translocase, cause neurological impairment.

The translocase of outer mitochondrial membrane (TOMM) complex is the entry gate for virtually all mitochondrial proteins and is essential to build the mitochondrial proteome. TOMM70 is a receptor that assists mainly in mitochondrial protein import. Here, we report two individuals with de novo variants in the C-terminal region of TOMM70. While both individuals exhibited shared symptoms including hypotonia, hyper-reflexia, ataxia, dystonia and significant white matter abnormalities, there were differences between the two individuals, most prominently the age of symptom onset. Both individuals were undiagnosed despite extensive genetics workups. Individual 1 was found to have a p.Thr607Ile variant while Individual 2 was found to have a p.Ile554Phe variant in TOMM70. To functionally assess both TOMM70 variants, we replaced the Drosophila Tom70 coding region with a Kozak-mini-GAL4 transgene using CRISPR-Cas9. Homozygous mutant animals die as pupae, but lethality is rescued by the mini-GAL4-driven expression of human UAS-TOMM70 cDNA. Both modeled variants lead to significantly less rescue indicating that they are loss-of-function alleles. Similarly, RNAi-mediated knockdown of Tom70 in the developing eye causes roughening and synaptic transmission defect, common findings in neurodegenerative and mitochondrial disorders. These phenotypes were rescued by the reference, but not the variants, of TOMM70. Altogether, our data indicate that de novo loss-of-function variants in TOMM70 result in variable white matter disease and neurological phenotypes in affected individuals.

Bi-allelic Variants in IQSEC1 Cause Intellectual Disability, Developmental Delay, and Short Stature.

We report two consanguineous families with probands that exhibit intellectual disability, developmental delay, short stature, aphasia, and hypotonia in which homozygous non-synonymous variants were identified in IQSEC1 (GenBank: NM_001134382.3). In a Pakistani family, the IQSEC1 segregating variant is c.1028C>T (p.Thr343Met), while in a Saudi Arabian family the variant is c.962G>A (p.Arg321Gln). IQSEC1-3 encode guanine nucleotide exchange factors for the small GTPase ARF6 and their loss affects a variety of actin-dependent cellular processes, including AMPA receptor trafficking at synapses. The ortholog of IQSECs in the fly is schizo and its loss affects growth cone guidance at the midline in the CNS, also an actin-dependent process. Overexpression of the reference IQSEC1 cDNA in wild-type flies is lethal, but overexpression of the two variant IQSEC1 cDNAs did not affect viability. Loss of schizo caused embryonic lethality that could be rescued to 2nd instar larvae by moderate expression of the human reference cDNA. However, the p.Arg321Gln and p.Thr343Met variants failed to rescue embryonic lethality. These data indicate that the variants behave as loss-of-function mutations. We also show that schizo in photoreceptors is required for phototransduction. Finally, mice with a conditional Iqsec1 deletion in cortical neurons exhibited an increased density of dendritic spines with an immature morphology. The phenotypic similarity of the affecteds and the functional experiments in flies and mice indicate that IQSEC1 variants are the cause of a recessive disease with intellectual disability, developmental delay, and short stature, and that axonal guidance and dendritic projection defects as well as dendritic spine dysgenesis may underlie disease pathogenesis.

Drosophila Voltage-Gated Sodium Channels Are Only Expressed in Active Neurons and Are Localized to Distal Axonal Initial Segment-like Domains.

In multipolar vertebrate neurons, action potentials (APs) initiate close to the soma, at the axonal initial segment. Invertebrate neurons are typically unipolar with dendrites integrating directly into the axon. Where APs are initiated in the axons of invertebrate neurons is unclear. Voltage-gated sodium (NaV) channels are a functional hallmark of the axonal initial segment in vertebrates. We used an intronic Minos-Mediated Integration Cassette to determine the endogenous gene expression and subcellular localization of the sole NaV channel in both male and female Drosophila, para Despite being the only NaV channel in the fly, we show that only 23 ± 1% of neurons in the embryonic and larval CNS express para, while in the adult CNS para is broadly expressed. We generated a single-cell transcriptomic atlas of the whole third instar larval brain to identify para expressing neurons and show that it positively correlates with markers of differentiated, actively firing neurons. Therefore, only 23 ± 1% of larval neurons may be capable of firing NaV-dependent APs. We then show that Para is enriched in an axonal segment, distal to the site of dendritic integration into the axon, which we named the distal axonal segment (DAS). The DAS is present in multiple neuron classes in both the third instar larval and adult CNS. Whole cell patch clamp electrophysiological recordings of adult CNS fly neurons are consistent with the interpretation that Nav-dependent APs originate in the DAS. Identification of the distal NaV localization in fly neurons will enable more accurate interpretation of electrophysiological recordings in invertebrates.SIGNIFICANCE STATEMENT The site of action potential (AP) initiation in invertebrates is unknown. We tagged the sole voltage-gated sodium (NaV) channel in the fly, para, and identified that Para is enriched at a distal axonal segment. The distal axonal segment is located distal to where dendrites impinge on axons and is the likely site of AP initiation. Understanding where APs are initiated improves our ability to model neuronal activity and our interpretation of electrophysiological data. Additionally, para is only expressed in 23 ± 1% of third instar larval neurons but is broadly expressed in adults. Single-cell RNA sequencing of the third instar larval brain shows that para expression correlates with the expression of active, differentiated neuronal markers. Therefore, only 23 ± 1% of third instar larval neurons may be able to actively fire NaV-dependent APs.

IRF2BPL Is Associated with Neurological Phenotypes.

Interferon regulatory factor 2 binding protein-like (IRF2BPL) encodes a member of the IRF2BP family of transcriptional regulators. Currently the biological function of this gene is obscure, and the gene has not been associated with a Mendelian disease. Here we describe seven individuals who carry damaging heterozygous variants in IRF2BPL and are affected with neurological symptoms. Five individuals who carry IRF2BPL nonsense variants resulting in a premature stop codon display severe neurodevelopmental regression, hypotonia, progressive ataxia, seizures, and a lack of coordination. Two additional individuals, both with missense variants, display global developmental delay and seizures and a relatively milder phenotype than those with nonsense alleles. The IRF2BPL bioinformatics signature based on population genomics is consistent with a gene that is intolerant to variation. We show that the fruit-fly IRF2BPL ortholog, called pits (protein interacting with Ttk69 and Sin3A), is broadly detected, including in the nervous system. Complete loss of pits is lethal early in development, whereas partial knockdown with RNA interference in neurons leads to neurodegeneration, revealing a requirement for this gene in proper neuronal function and maintenance. The identified IRF2BPL nonsense variants behave as severe loss-of-function alleles in this model organism, and ectopic expression of the missense variants leads to a range of phenotypes. Taken together, our results show that IRF2BPL and pits are required in the nervous system in humans and flies, and their loss leads to a range of neurological phenotypes in both species.

Regulation of myeloid cell phagocytosis by LRRK2 via WAVE2 complex stabilization is altered in Parkinson's disease.

Leucine-rich repeat kinase 2 (LRRK2) has been implicated in both familial and sporadic Parkinson's disease (PD), yet its pathogenic role remains unclear. A previous screen in Drosophila identified Scar/WAVE (Wiskott-Aldrich syndrome protein-family verproline) proteins as potential genetic interactors of LRRK2 Here, we provide evidence that LRRK2 modulates the phagocytic response of myeloid cells via specific modulation of the actin-cytoskeletal regulator, WAVE2. We demonstrate that macrophages and microglia from LRRK2-G2019S PD patients and mice display a WAVE2-mediated increase in phagocytic response, respectively. Lrrk2 loss results in the opposite effect. LRRK2 binds and phosphorylates Wave2 at Thr470, stabilizing and preventing its proteasomal degradation. Finally, we show that Wave2 also mediates Lrrk2-G2019S-induced dopaminergic neuronal death in both macrophage-midbrain cocultures and in vivo. Taken together, a LRRK2-WAVE2 pathway, which modulates the phagocytic response in mice and human leukocytes, may define an important role for altered immune function in PD.

LRRK2(I2020T) functional genetic interactors that modify eye degeneration and dopaminergic cell loss in Drosophila.

Progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta is the primary cause for motor symptoms observed in Parkinson's disease (PD). Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most commonly linked contributor to familial PD. LRRK2 is suggested to be involved in a wide variety of cellular processes, but deciphering its role in the pathogenesis of PD has been difficult. Modelling PD in rodents has been a persistent challenge for the field. However, the fruit fly has been exploited to recapitulate PD gene related dopaminergic cell loss. Using the GAL4-UAS system and established models of hLRRK2 induced eye degeneration in Drosophila, we conducted an unbiased suppressor/enhancer screen to uncover genetic modifiers of LRRK2. We have identified 36 candidate interactors that modify LRRK2 induced toxicity in the Drosophila eye. Importantly, we determined that a subset of these interactors also modified hLRRK2(I2020T) induced dopaminergic neuronal loss in the fly brain and uncovered 16 candidates that modify dopaminergic cell loss. Our results suggest LRRK2 may be involved in a wide variety of cellular processes and the results from this screen provide an important genetic resource for further evaluation of LRRK2 function.

BAG2 Gene-mediated Regulation of PINK1 Protein Is Critical for Mitochondrial Translocation of PARKIN and Neuronal Survival.

Emerging evidence has demonstrated a growing genetic component in Parkinson disease (PD). For instance, loss-of-function mutations in PINK1 or PARKIN can cause autosomal recessive PD. Recently, PINK1 and PARKIN have been implicated in the same signaling pathway to regulate mitochondrial clearance through recruitment of PARKIN by stabilization of PINK1 on the outer membrane of depolarized mitochondria. The precise mechanisms that govern this process remain enigmatic. In this study, we identify Bcl2-associated athanogene 2 (BAG2) as a factor that promotes mitophagy. BAG2 inhibits PINK1 degradation by blocking the ubiquitination pathway. Stabilization of PINK1 by BAG2 triggers PARKIN-mediated mitophagy and protects neurons against 1-methyl-4-phenylpyridinium-induced oxidative stress in an in vitro cell model of PD. Collectively, our findings support the notion that BAG2 is an upstream regulator of the PINK1/PARKIN signaling pathway.

Progressive dopaminergic cell loss with unilateral-to-bilateral progression in a genetic model of Parkinson disease.

DJ-1 mutations cause autosomal recessive early-onset Parkinson disease (PD). We report a model of PD pathology: the DJ1-C57 mouse. A subset of DJ-1-nullizygous mice, when fully backcrossed to a C57BL/6 [corrected] background, display dramatic early-onset unilateral loss of dopaminergic (DA) neurons in their substantia nigra pars compacta, progressing to bilateral degeneration of the nigrostriatal axis with aging. In addition, these mice exhibit age-dependent bilateral degeneration at the locus ceruleus nucleus and display mild motor behavior deficits at aged time points. These findings effectively recapitulate the early stages of PD. Therefore, the DJ1-C57 mouse provides a tool to study the preclinical aspects of neurodegeneration. Importantly, by exome sequencing, we identify candidate modifying genes that segregate with the phenotype, providing potentially critical clues into how certain genes may influence the penetrance of DJ-1-related degeneration in mice.

Cdk8/CDK19 promotes mitochondrial fission through Drp1 phosphorylation and can phenotypically suppress pink1 deficiency in Drosophila.

Cdk8 in Drosophila is the orthologue of vertebrate CDK8 and CDK19. These proteins have been shown to modulate transcriptional control by RNA polymerase II. We found that neuronal loss of Cdk8 severely reduces fly lifespan and causes bang sensitivity. Remarkably, these defects can be rescued by expression of human CDK19, found in the cytoplasm of neurons, suggesting a non-nuclear function of CDK19/Cdk8. Here we show that Cdk8 plays a critical role in the cytoplasm, with its loss causing elongated mitochondria in both muscles and neurons. We find that endogenous GFP-tagged Cdk8 can be found in both the cytoplasm and nucleus. We show that Cdk8 promotes the phosphorylation of Drp1 at S616, a protein required for mitochondrial fission. Interestingly, Pink1, a mitochondrial kinase implicated in Parkinson's disease, also phosphorylates Drp1 at the same residue. Indeed, overexpression of Cdk8 significantly suppresses the phenotypes observed in flies with low levels of Pink1, including elevated levels of ROS, mitochondrial dysmorphology, and behavioral defects. In summary, we propose that Pink1 and Cdk8 perform similar functions to promote Drp1-mediated fission.

DJ-1 protects the nigrostriatal axis from the neurotoxin MPTP by modulation of the AKT pathway.

Loss-of-function DJ-1 (PARK7) mutations have been linked with a familial form of early onset Parkinson disease. Numerous studies have supported the role of DJ-1 in neuronal survival and function. Our initial studies using DJ-1-deficient neurons indicated that DJ-1 specifically protects the neurons against the damage induced by oxidative injury in multiple neuronal types and degenerative experimental paradigms, both in vitro and in vivo. However, the manner by which oxidative stress-induced death is ameliorated by DJ-1 is not completely clear. We now present data that show the involvement of DJ-1 in modulation of AKT, a major neuronal prosurvival pathway induced upon oxidative stress. We provide evidence that DJ-1 promotes AKT phosphorylation in response to oxidative stress induced by H(2)O(2) in vitro and in vivo following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment. Moreover, we show that DJ-1 is necessary for normal AKT-mediated protective effects, which can be bypassed by expression of a constitutively active form of AKT. Taken together, these data suggest that DJ-1 is crucial for full activation of AKT upon oxidative injury, which serves as one explanation for the protective effects of DJ-1.

A defect in mitochondrial fatty acid synthesis impairs iron metabolism and causes elevated ceramide levels.

In most eukaryotic cells, fatty acid synthesis (FAS) occurs in the cytoplasm and in mitochondria. However, the relative contribution of mitochondrial FAS (mtFAS) to the cellular lipidome is not well defined. Here we show that loss of function of Drosophila mitochondrial enoyl coenzyme A reductase (Mecr), which is the enzyme required for the last step of mtFAS, causes lethality, while neuronal loss of Mecr leads to progressive neurodegeneration. We observe a defect in Fe-S cluster biogenesis and increased iron levels in flies lacking mecr, leading to elevated ceramide levels. Reducing the levels of either iron or ceramide suppresses the neurodegenerative phenotypes, indicating an interplay between ceramide and iron metabolism. Mutations in human MECR cause pediatric-onset neurodegeneration, and we show that human-derived fibroblasts display similar elevated ceramide levels and impaired iron homeostasis. In summary, this study identifies a role of mecr/MECR in ceramide and iron metabolism, providing a mechanistic link between mtFAS and neurodegeneration.