Dominantly acting variants in ARF3 have disruptive consequences on Golgi integrity and cause microcephaly recapitulated in zebrafish


 Vesicle biogenesis, trafficking and signaling via the ER-Golgi network support essential processes during development and their disruption can lead to neurodevelopmental disorders and neurodegeneration. We report that de novo missense variants in ARF3, encoding a small GTPase regulating Golgi structure and function, cause a neurodevelopmental disease showing microcephaly and progressive cortical atrophy, with microsomia and rib anomalies in severely affected subjects, suggesting a pleiotropic effect. All microcephaly-associated variants clustered in the guanine nucleotide binding pocket and perturbed the biochemical behavior of the protein by stabilizing it in a GTP-bound state. Functional analysis proved the disruptive consequences of the variants on Golgi integrity, and brain and body plan formation. In-depth analysis in zebrafish embryos expressing ARF3 mutants traced back the developmental alterations to defective gastrulation cell movements as the earliest detectable effect. Our findings document a role of ARF3 in Golgi homeostasis and demonstrate an obligate dependence for early development.


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The Golgi apparatus (GA) is a polarized, membrane network-built organelle 103 responsible for transporting, modifying, and packaging proteins and lipids into vesicles 104 for their delivery to targeted destinations 1,2 . It is organized as a series of flattened, 105 stacked pouches (known as cisternae) that are held together by matrix proteins and 106 microtubules, and are structured into two major networks, the cis and trans-Golgi  [6][7][8] . In these disorders, which have collectively been termed as 122 "Golgipathies", recurrent features include microcephaly, central nervous system (CNS) 123 defects (e.g., delayed myelination, cortical atrophy, abnormal corpus callosum, and 124 pontocerebellar hypoplasia) and developmental delay (DD)/intellectual disability 125 Dominantly acting variants in ARF3 have disruptive consequences on Golgi integrity and cause microcephaly recapitulated in zebrafish Fasano, Muto, Radio, et al. 6 The five members of the ADP-ribosylation factors (ARF) family of small GTPases 127 (ARF1, ARF3-6) regulate key events in vesicular biogenesis, transport and various GA 128 functions, and participate in the control of bidirectional membrane trafficking required 129 for secretion, endocytosis and recycling [10][11][12][13] . These proteins bind to guanine 130 nucleotides with high affinity and specificity, and have a slow intrinsic competence to 131 hydrolyze GTP to GDP 12,14 . ARF proteins are characterized by a unique myristoylated interact with effectors in its GTP-bound state [15][16][17] . Similarly to other members of the 136 RAS superfamily, release of GDP is stimulated by specific guanine nucleotide 137 exchange factors (ARFGEFs), which indirectly favor binding to GTP 12,18,19 . As a 138 consequence of the conformational change promoted by GTP, the N-terminal 139 myristoylated region is exposed, allowing anchoring of the GTPase to the cytoplasmic 140 leaflet of membranes of different organelles, including cis and trans-Golgi, plasma 141 membrane (PM) and endosomes, where these proteins exert their function 12,14,17,20,21 .

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The intrinsic slow GTPase activity of ARFs is substantially accelerated by specific 143 GTPase-activating proteins (ARFGAPs), which result in protein inactivation and 144 release from membranes 12,[21][22][23] . 145 To exert their function, ARF proteins interact with a number of effectors, most of which 146 are coat proteins and adaptors 1,12 ; they can also recruit non-coat GA-specific proteins 147 to membranes (e.g., golgin-160 and GCC88) 24 , which are important for GA structure 148 homeostasis 25,26 . Evidence shows that ARF proteins actually contribute to the control 149 of GA and organelles structural organization and function 13,27,28 , and GA dynamics Dominantly acting variants in ARF3 have disruptive consequences on Golgi integrity and cause microcephaly recapitulated in zebrafish Fasano, Muto,Radio,et al. 7 during cell division and cytokinesis in precursor cells [29][30][31] . By controlling GA structure, 151 function, cargo sorting and ER-GA targeted trafficking, ARFs actively participate to the 152 fine regulation of key events during embryogenesis (i.e., cell polarity establishment and 153 migration in early gastrulation, neuronal maturation and tissue morphogenesis) 32 .

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ARF3 mutations cause a developmental disorder characterized by microcephaly 173 and cortical atrophy 174 In the frame of a research program dedicated to subjects affected by unclassified 175 diseases, trio-based exome sequencing allowed us to identify a previously unreported  Table 7). The same Lys-to-Glu substitution at codon 127 in Subject 1 201 was recently reported to affect the corresponding residue in ARF1 in a patient with DD, 202 microcephaly, periventricular heterotopia, progressive cerebral atrophy and epilepsy 34 .  Table 7).

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Affected subjects showed variable degree of DD/ID (   Disease-associated ARF3 variants variably affect protein stability and function 230 The identified disease-associated variants affected residues spotted throughout the 231 coding sequence with exception of the C-terminus region ( Figure 1a). First, we 232 investigated the functional consequences of each amino acid substitution by using a 233 three-dimensional structure of the GTPase recently solved by X-ray diffraction 43 . We 234 noted that all residues cluster within or close to the GTP/GDP binding pocket ( Figure   235 1b). Specifically, Lys 127 is one of the four residues of the NKXD motif directly mediating 236 binding to GTP/GDP by binding to the ribose ring 16 , and substitution of the positively 237 charged residue with a negatively charge glutamate was predicted to affect proper 238 nucleotide binding (Figure 1c). Similarly, Thr 32 contributes to stabilize the GTP/GDP 239 binding via direct hydrogen bonding with one oxygen atom of the a phosphate ( Figure   240 1c). While conservative, the Thr to Asn substitution was predicted to result in a steric 241 hindrance. Asp 93 does not directly contact GTP, even though it participates to the 242 overall general structure of the nucleotide binding pocket by a direct hydrogen bond Dominantly acting variants in ARF3 have disruptive consequences on Golgi integrity and cause microcephaly recapitulated in zebrafish Fasano, Muto, Radio, et al.

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with the lateral chain of Lys 127 (Figure 1d). Since the high GTP:GDP ratio within cells, 244 these three changes were anticipated to favor an active, GTP-bound state of the 245 GTPase, bypassing the requirement for a GEF, as previously reported for pathogenic 246 mutations affecting RAS proteins 44,45 . On the other hand, Pro 47 and Asp 67 were 247 predicted to affect ARF3 GTPase activity. Specifically, Pro 47 is located within the switch   Table 9). We assessed the structural  In its active GTP-bound state, ARF3 is able to bind to the Golgi-associated gamma-      581 The structural impact of the disease-associated missense changes was assessed 582 using the available three-dimensional structures of human ARF3 complexed with GTP 583 and V. vulnificus multifunctional-autoprocessing repeats-in-toxin (MARTX) (PDB 584 6ii6) 43 .The structure was visualized using the VMD visualization software 81 .       Kolmogorov-Smirnov tests) were run to assess normal distribution of the data.

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Parametric data with more experimental groups were analyzed with Anova test, non- The authors declare no competing interests.

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Data availability 848 The data generated in this work are available upon request from the corresponding 849 authors.