Functional interpretation of ATAD3A variants in neuro-mitochondrial phenotypes.

BACKGROUND: ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane-anchored protein involved in diverse processes including mitochondrial dynamics, mitochondrial DNA organization, and cholesterol metabolism. Biallelic deletions (null), recessive missense variants (hypomorph), and heterozygous missense variants or duplications (antimorph) in ATAD3A lead to neurological syndromes in humans. METHODS: To expand the mutational spectrum of ATAD3A variants and to provide functional interpretation of missense alleles in trans to deletion alleles, we performed exome sequencing for identification of single nucleotide variants (SNVs) and copy number variants (CNVs) in ATAD3A in individuals with neurological and mitochondrial phenotypes. A Drosophila Atad3a Gal4 knockin-null allele was generated using CRISPR-Cas9 genome editing technology to aid the interpretation of variants. RESULTS: We report 13 individuals from 8 unrelated families with biallelic ATAD3A variants. The variants included four missense variants inherited in trans to loss-of-function alleles (p.(Leu77Val), p.(Phe50Leu), p.(Arg170Trp), p.(Gly236Val)), a homozygous missense variant p.(Arg327Pro), and a heterozygous non-frameshift indel p.(Lys568del). Affected individuals exhibited findings previously associated with ATAD3A pathogenic variation, including developmental delay, hypotonia, congenital cataracts, hypertrophic cardiomyopathy, and cerebellar atrophy. Drosophila studies indicated that Phe50Leu, Gly236Val, Arg327Pro, and Lys568del are severe loss-of-function alleles leading to early developmental lethality. Further, we showed that Phe50Leu, Gly236Val, and Arg327Pro cause neurogenesis defects. On the contrary, Leu77Val and Arg170Trp are partial loss-of-function alleles that cause progressive locomotion defects and whose expression leads to an increase in autophagy and mitophagy in adult muscles. CONCLUSION: Our findings expand the allelic spectrum of ATAD3A variants and exemplify the use of a functional assay in Drosophila to aid variant interpretation.

A conserved role for AMP-activated protein kinase in NGLY1 deficiency.

Mutations in human N-glycanase 1 (NGLY1) cause the first known congenital disorder of deglycosylation (CDDG). Patients with this rare disease, which is also known as NGLY1 deficiency, exhibit global developmental delay and other phenotypes including neuropathy, movement disorder, and constipation. NGLY1 is known to regulate proteasomal and mitophagy gene expression through activation of a transcription factor called "nuclear factor erythroid 2-like 1" (NFE2L1). Loss of NGLY1 has also been shown to impair energy metabolism, but the molecular basis for this phenotype and its in vivo consequences are not well understood. Using a combination of genetic studies, imaging, and biochemical assays, here we report that loss of NGLY1 in the visceral muscle of the Drosophila larval intestine results in a severe reduction in the level of AMP-activated protein kinase α (AMPKα), leading to energy metabolism defects, impaired gut peristalsis, failure to empty the gut, and animal lethality. Ngly1-/- mouse embryonic fibroblasts and NGLY1 deficiency patient fibroblasts also show reduced AMPKα levels. Moreover, pharmacological activation of AMPK signaling significantly suppressed the energy metabolism defects in these cells. Importantly, the reduced AMPKα level and impaired energy metabolism observed in NGLY1 deficiency models are not caused by the loss of NFE2L1 activity. Taken together, these observations identify reduced AMPK signaling as a conserved mediator of energy metabolism defects in NGLY1 deficiency and suggest AMPK signaling as a therapeutic target in this disease.

Elevated COUP-TFII expression in dopaminergic neurons accelerates the progression of Parkinson's disease through mitochondrial dysfunction.

Parkinson's disease (PD) is a neurodegenerative disorder featuring progressive loss of midbrain dopaminergic (DA) neurons that leads to motor symptoms. The etiology and pathogenesis of PD are not clear. We found that expression of COUP-TFII, an orphan nuclear receptor, in DA neurons is upregulated in PD patients through the analysis of public datasets. We show here that through epigenetic regulation, COUP-TFII contributes to oxidative stress, suggesting that COUP-TFII may play a role in PD pathogenesis. Elevated COUP-TFII expression specifically in DA neurons evokes DA neuronal loss in mice and accelerates the progression of phenotypes in a PD mouse model, MitoPark. Compared to control mice, those with elevated COUP-TFII expression displayed reduced cristae in mitochondria and enhanced cellular electron-dense vacuoles in the substantia nigra pars compacta. Mechanistically, we found that overexpression of COUP-TFII disturbs mitochondrial pathways, resulting in mitochondrial dysfunction. In particular, there is repressed expression of genes encoding cytosolic aldehyde dehydrogenases, which could enhance oxidative stress and interfere with mitochondrial function via 3,4-dihydroxyphenylacetaldehyde (DOPAL) buildup in DA neurons. Importantly, under-expression of COUP-TFII in DA neurons slowed the deterioration in motor functions of MitoPark mice. Taken together, our results suggest that COUP-TFII may be an important contributor to PD development and a potential therapeutic target.

Loss- or Gain-of-Function Mutations in ACOX1 Cause Axonal Loss via Different Mechanisms.

ACOX1 (acyl-CoA oxidase 1) encodes the first and rate-limiting enzyme of the very-long-chain fatty acid (VLCFA) ß-oxidation pathway in peroxisomes and leads to H2O2 production. Unexpectedly, Drosophila (d) ACOX1 is mostly expressed and required in glia, and loss of ACOX1 leads to developmental delay, pupal death, reduced lifespan, impaired synaptic transmission, and glial and axonal loss. Patients who carry a previously unidentified, de novo, dominant variant in ACOX1 (p.N237S) also exhibit glial loss. However, this mutation causes increased levels of ACOX1 protein and function resulting in elevated levels of reactive oxygen species in glia in flies and murine Schwann cells. ACOX1 (p.N237S) patients exhibit a severe loss of Schwann cells and neurons. However, treatment of flies and primary Schwann cells with an antioxidant suppressed the p.N237S-induced neurodegeneration. In summary, both loss and gain of ACOX1 lead to glial and neuronal loss, but different mechanisms are at play and require different treatments.

Loss of Oxidation Resistance 1, OXR1, Is Associated with an Autosomal-Recessive Neurological Disease with Cerebellar Atrophy and Lysosomal Dysfunction.

We report an early-onset autosomal-recessive neurological disease with cerebellar atrophy and lysosomal dysfunction. We identified bi-allelic loss-of-function (LoF) variants in Oxidative Resistance 1 (OXR1) in five individuals from three families; these individuals presented with a history of severe global developmental delay, current intellectual disability, language delay, cerebellar atrophy, and seizures. While OXR1 is known to play a role in oxidative stress resistance, its molecular functions are not well established. OXR1 contains three conserved domains: LysM, GRAM, and TLDc. The gene encodes at least six transcripts, including some that only consist of the C-terminal TLDc domain. We utilized Drosophila to assess the phenotypes associated with loss of mustard (mtd), the fly homolog of OXR1. Strong LoF mutants exhibit late pupal lethality or pupal eclosion defects. Interestingly, although mtd encodes 26 transcripts, severe LoF and null mutations can be rescued by a single short human OXR1 cDNA that only contains the TLDc domain. Similar rescue is observed with the TLDc domain of NCOA7, another human homolog of mtd. Loss of mtd in neurons leads to massive cell loss, early death, and an accumulation of aberrant lysosomal structures, similar to what we observe in fibroblasts of affected individuals. Our data indicate that mtd and OXR1 are required for proper lysosomal function; this is consistent with observations that NCOA7 is required for lysosomal acidification.

cindr, the Drosophila Homolog of the CD2AP Alzheimer's Disease Risk Gene, Is Required for Synaptic Transmission and Proteostasis.

The Alzheimer's disease (AD) susceptibility gene, CD2-associated protein (CD2AP), encodes an actin binding adaptor protein, but its function in the nervous system is largely unknown. Loss of the Drosophila ortholog cindr enhances neurotoxicity of human Tau, which forms neurofibrillary tangle pathology in AD. We show that Cindr is expressed in neurons and present at synaptic terminals. cindr mutants show impairments in synapse maturation and both synaptic vesicle recycling and release. Cindr associates and genetically interacts with 14-3-3ζ, regulates the ubiquitin-proteasome system, and affects turnover of Synapsin and the plasma membrane calcium ATPase (PMCA). Loss of cindr elevates PMCA levels and reduces cytosolic calcium. Studies of Cd2ap null mice support a conserved role in synaptic proteostasis, and CD2AP protein levels are inversely related to Synapsin abundance in human postmortem brains. Our results reveal CD2AP neuronal requirements with relevance to AD susceptibility, including for proteostasis, calcium handling, and synaptic structure and function.

Pancreatic Cell Fate Determination Relies on Notch Ligand Trafficking by NFIA.

Notch is activated globally in pancreatic progenitors; however, for progenitors to differentiate into endocrine cells, they must escape Notch activation to express Neurogenin-3. Here, we find that the transcription factor nuclear factor I/A (NFIA) promotes endocrine development by regulating Notch ligand Dll1 trafficking. Pancreatic deletion of NFIA leads to cell fate defects, with increased duct and decreased endocrine formation, while ectopic expression promotes endocrine formation in mice and human pancreatic progenitors. NFIA-deficient mice exhibit dysregulation of trafficking-related genes including increased expression of Mib1, which acts to target Dll1 for endocytosis. We find that NFIA binds to the Mib1 promoter, with loss of NFIA leading to an increase in Dll1 internalization and enhanced Notch activation with rescue of the cell fate defects after Mib1 knockdown. This study reveals NFIA as a pro-endocrine factor in the pancreas, acting to repress Mib1, inhibit Dll1 endocytosis and thus promote escape from Notch activation.

Ari-1 Regulates Myonuclear Organization Together with Parkin and Is Associated with Aortic Aneurysms.

Nuclei are actively positioned and anchored to the cytoskeleton via the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex. We identified mutations in the Parkin-like E3 ubiquitin ligase Ariadne-1 (Ari-1) that affect the localization and distribution of LINC complex members in Drosophila. ari-1 mutants exhibit nuclear clustering and morphology defects in larval muscles. We show that Ari-1 mono-ubiquitinates the core LINC complex member Koi. Surprisingly, we discovered functional redundancy between Parkin and Ari-1: increasing Parkin expression rescues ari-1 mutant phenotypes and vice versa. We further show that rare variants in the human homolog of ari-1 (ARIH1) are associated with thoracic aortic aneurysms and dissections, conditions resulting from smooth muscle cell (SMC) dysfunction. Human ARIH1 rescues fly ari-1 mutant phenotypes, whereas human variants found in patients fail to do so. In addition, SMCs obtained from patients display aberrant nuclear morphology. Hence, ARIH1 is critical in anchoring myonuclei to the cytoskeleton.

The Krebs Cycle Enzyme Isocitrate Dehydrogenase 3A Couples Mitochondrial Metabolism to Synaptic Transmission.

Neurotransmission is a tightly regulated Ca2+-dependent process. Upon Ca2+ influx, Synaptotagmin1 (Syt1) promotes fusion of synaptic vesicles (SVs) with the plasma membrane. This requires regulation at multiple levels, but the role of metabolites in SV release is unclear. Here, we uncover a role for isocitrate dehydrogenase 3a (idh3a), a Krebs cycle enzyme, in neurotransmission. Loss of idh3a leads to a reduction of the metabolite, alpha-ketoglutarate (αKG), causing defects in synaptic transmission similar to the loss of syt1. Supplementing idh3a flies with αKG suppresses these defects through an ATP or neurotransmitter-independent mechanism. Indeed, αKG, but not glutamate, enhances Syt1-dependent fusion in a reconstitution assay. αKG promotes interaction between the C2-domains of Syt1 and phospholipids. The data reveal conserved metabolic regulation of synaptic transmission via αKG. Our studies provide a synaptic role for αKG, a metabolite that has been proposed as a treatment for aging and neurodegenerative disorders.

Recurrent De Novo and Biallelic Variation of ATAD3A, Encoding a Mitochondrial Membrane Protein, Results in Distinct Neurological Syndromes.

ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane protein implicated in mitochondrial dynamics, nucleoid organization, protein translation, cell growth, and cholesterol metabolism. We identified a recurrent de novo ATAD3A c.1582C>T (p.Arg528Trp) variant by whole-exome sequencing (WES) in five unrelated individuals with a core phenotype of global developmental delay, hypotonia, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. We also describe two families with biallelic variants in ATAD3A, including a homozygous variant in two siblings, and biallelic ATAD3A deletions mediated by nonallelic homologous recombination (NAHR) between ATAD3A and gene family members ATAD3B and ATAD3C. Tissue-specific overexpression of borR534W, the Drosophila mutation homologous to the human c.1582C>T (p.Arg528Trp) variant, resulted in a dramatic decrease in mitochondrial content, aberrant mitochondrial morphology, and increased autophagy. Homozygous null bor larvae showed a significant decrease of mitochondria, while overexpression of borWT resulted in larger, elongated mitochondria. Finally, fibroblasts of an affected individual exhibited increased mitophagy. We conclude that the p.Arg528Trp variant functions through a dominant-negative mechanism that results in small mitochondria that trigger mitophagy, resulting in a reduction in mitochondrial content. ATAD3A variation represents an additional link between mitochondrial dynamics and recognizable neurological syndromes, as seen with MFN2, OPA1, DNM1L, and STAT2 mutations.

Loss of Frataxin induces iron toxicity, sphingolipid synthesis, and Pdk1/Mef2 activation, leading to neurodegeneration.

Mutations in Frataxin (FXN) cause Friedreich's ataxia (FRDA), a recessive neurodegenerative disorder. Previous studies have proposed that loss of FXN causes mitochondrial dysfunction, which triggers elevated reactive oxygen species (ROS) and leads to the demise of neurons. Here we describe a ROS independent mechanism that contributes to neurodegeneration in fly FXN mutants. We show that loss of frataxin homolog (fh) in Drosophila leads to iron toxicity, which in turn induces sphingolipid synthesis and ectopically activates 3-phosphoinositide dependent protein kinase-1 (Pdk1) and myocyte enhancer factor-2 (Mef2). Dampening iron toxicity, inhibiting sphingolipid synthesis by Myriocin, or reducing Pdk1 or Mef2 levels, all effectively suppress neurodegeneration in fh mutants. Moreover, increasing dihydrosphingosine activates Mef2 activity through PDK1 in mammalian neuronal cell line suggesting that the mechanisms are evolutionarily conserved. Our results indicate that an iron/sphingolipid/Pdk1/Mef2 pathway may play a role in FRDA.

WAC Regulates mTOR Activity by Acting as an Adaptor for the TTT and Pontin/Reptin Complexes.

The ability to sense energy status is crucial in the regulation of metabolism via the mechanistic Target of Rapamycin Complex 1 (mTORC1). The assembly of the TTT-Pontin/Reptin complex is responsive to changes in energy status. Under energy-sufficient conditions, the TTT-Pontin/Reptin complex promotes mTORC1 dimerization and mTORC1-Rag interaction, which are critical for mTORC1 activation. We show that WAC is a regulator of energy-mediated mTORC1 activity. In a Drosophila screen designed to isolate mutations that cause neuronal dysfunction, we identified wacky, the homolog of WAC. Loss of Wacky leads to neurodegeneration, defective mTOR activity, and increased autophagy. Wacky and WAC have conserved physical interactions with mTOR and its regulators, including Pontin and Reptin, which bind to the TTT complex to regulate energy-dependent activation of mTORC1. WAC promotes the interaction between TTT and Pontin/Reptin in an energy-dependent manner, thereby promoting mTORC1 activity by facilitating mTORC1 dimerization and mTORC1-Rag interaction.

Correction: the retromer complex is required for rhodopsin recycling and its loss leads to photoreceptor degeneration.

[This corrects the article DOI: 10.1371/journal.pbio.1001847.].

A voltage-gated calcium channel regulates lysosomal fusion with endosomes and autophagosomes and is required for neuronal homeostasis.

Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis.

A TRPV channel in Drosophila motor neurons regulates presynaptic resting Ca2+ levels, synapse growth, and synaptic transmission.

Presynaptic resting Ca(2+) influences synaptic vesicle (SV) release probability. Here, we report that a TRPV channel, Inactive (Iav), maintains presynaptic resting [Ca(2+)] by promoting Ca(2+) release from the endoplasmic reticulum in Drosophila motor neurons, and is required for both synapse development and neurotransmission. We find that Iav activates the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin, which is essential for presynaptic microtubule stabilization at the neuromuscular junction. Thus, loss of Iav induces destabilization of presynaptic microtubules, resulting in diminished synaptic growth. Interestingly, expression of human TRPV1 in Iav-deficient motor neurons rescues these defects. We also show that the absence of Iav causes lower SV release probability and diminished synaptic transmission, whereas Iav overexpression elevates these synaptic parameters. Together, our findings indicate that Iav acts as a key regulator of synaptic development and function by influencing presynaptic resting [Ca(2+)].

Mitochondrial fusion but not fission regulates larval growth and synaptic development through steroid hormone production.

Mitochondrial fusion and fission affect the distribution and quality control of mitochondria. We show that Marf (Mitochondrial associated regulatory factor), is required for mitochondrial fusion and transport in long axons. Moreover, loss of Marf leads to a severe depletion of mitochondria in neuromuscular junctions (NMJs). Marf mutants also fail to maintain proper synaptic transmission at NMJs upon repetitive stimulation, similar to Drp1 fission mutants. However, unlike Drp1, loss of Marf leads to NMJ morphology defects and extended larval lifespan. Marf is required to form contacts between the endoplasmic reticulum and/or lipid droplets (LDs) and for proper storage of cholesterol and ecdysone synthesis in ring glands. Interestingly, human Mitofusin-2 rescues the loss of LD but both Mitofusin-1 and Mitofusin-2 are required for steroid-hormone synthesis. Our data show that Marf and Mitofusins share an evolutionarily conserved role in mitochondrial transport, cholesterol ester storage and steroid-hormone synthesis.

The retromer complex is required for rhodopsin recycling and its loss leads to photoreceptor degeneration.

Rhodopsin mistrafficking can cause photoreceptor (PR) degeneration. Upon light exposure, activated rhodopsin 1 (Rh1) in Drosophila PRs is internalized via endocytosis and degraded in lysosomes. Whether internalized Rh1 can be recycled is unknown. Here, we show that the retromer complex is expressed in PRs where it is required for recycling endocytosed Rh1 upon light stimulation. In the absence of subunits of the retromer, Rh1 is processed in the endolysosomal pathway, leading to a dramatic increase in late endosomes, lysosomes, and light-dependent PR degeneration. Reducing Rh1 endocytosis or Rh1 levels in retromer mutants alleviates PR degeneration. In addition, increasing retromer abundance suppresses degenerative phenotypes of mutations that affect the endolysosomal system. Finally, expressing human Vps26 suppresses PR degeneration in Vps26 mutant PRs. We propose that the retromer plays a conserved role in recycling rhodopsins to maintain PR function and integrity.

Crag is a GEF for Rab11 required for rhodopsin trafficking and maintenance of adult photoreceptor cells.

Rhodopsins (Rhs) are light sensors, and Rh1 is the major Rh in the Drosophila photoreceptor rhabdomere membrane. Upon photoactivation, a fraction of Rh1 is internalized and degraded, but it remains unclear how the rhabdomeric Rh1 pool is replenished and what molecular players are involved. Here, we show that Crag, a DENN protein, is a guanine nucleotide exchange factor for Rab11 that is required for the homeostasis of Rh1 upon light exposure. The absence of Crag causes a light-induced accumulation of cytoplasmic Rh1, and loss of Crag or Rab11 leads to a similar photoreceptor degeneration in adult flies. Furthermore, the defects associated with loss of Crag can be partially rescued with a constitutive active form of Rab11. We propose that upon light stimulation, Crag is required for trafficking of Rh from the trans-Golgi network to rhabdomere membranes via a Rab11-dependent vesicular transport.

Drosophila neuroligin 2 is required presynaptically and postsynaptically for proper synaptic differentiation and synaptic transmission.

Trans-synaptic adhesion between Neurexins (Nrxs) and Neuroligins (Nlgs) is thought to be required for proper synapse organization and modulation, and mutations in several human Nlgs have shown association with autism spectrum disorders. Here we report the generation and phenotypic characterization of Drosophila neuroligin 2 (dnlg2) mutants. Loss of dnlg2 results in reduced bouton numbers, aberrant presynaptic and postsynaptic development at neuromuscular junctions (NMJs), and impaired synaptic transmission. In dnlg2 mutants, the evoked responses are decreased in amplitude, whereas the total active zone (AZ) numbers at the NMJ are comparable to wild type, suggesting a decrease in the release probability. Ultrastructurally, the presynaptic AZ number per bouton area and the postsynaptic density area are both increased in dnlg2 mutants, whereas the subsynaptic reticulum is reduced in volume. We show that both presynaptic and postsynaptic expression of Dnlg2 is required to restore synaptic growth and function in dnlg2 mutants. Postsynaptic expression of Dnlg2 in dnlg2 mutants and wild type leads to reduced bouton growth whereas presynaptic and postsynaptic overexpression in wild-type animals results in synaptic overgrowth. Since Nlgs have been shown to bind to Nrxs, we created double mutants. These mutants are viable and display phenotypes that closely resemble those of dnlg2 and dnrx single mutants. Our results provide compelling evidence that Dnlg2 functions both presynaptically and postsynaptically together with Neurexin to determine the proper number of boutons as well as the number of AZs and size of synaptic densities during the development of NMJs.