# GOLGI DYNAMICS IN PHYSIOLOGICAL AND PATHOLOGICAL CONDITIONS

EDITED BY : Jaakko Saraste, Vladimir Lupashin, Yanzhuang Wang and Kristian Prydz PUBLISHED IN : Frontiers in Cell and Developmental Biology

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ISSN 1664-8714 ISBN 978-2-88963-539-9 DOI 10.3389/978-2-88963-539-9

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# GOLGI DYNAMICS IN PHYSIOLOGICAL AND PATHOLOGICAL CONDITIONS

Topic Editors: Jaakko Saraste, University of Bergen, Norway Vladimir Lupashin, University of Arkansas for Medical Sciences, United States Yanzhuang Wang, University of Michigan, United States Kristian Prydz, University of Oslo, Norway

Citation: Saraste, J., Lupashin, V., Wang, Y., Prydz, K., eds. (2020). Golgi Dynamics in Physiological and Pathological Conditions. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88963-539-9

# Table of Contents

*05 Editorial: Golgi Dynamics in Physiological and Pathological Conditions* Kristian Prydz, Vladimir Lupashin, Yanzhuang Wang and Jaakko Saraste

*09 Identification of CDC42 Effectors Operating in FGD1-Dependent Trafficking at the Golgi*

Mikhail Egorov and Roman Polishchuk

*20 Golgi Complex Dynamics and its Implication in Prevalent Neurological Disorders*

Mario O. Caracci, Luz M. Fuentealba and María-Paz Marzolo

Christian Makhoul, Prajakta Gosavi and Paul A. Gleeson

*90 Defects in COG-Mediated Golgi Trafficking Alter Endo-Lysosomal System in Human Cells*

Zinia D'Souza, Jessica Bailey Blackburn, Tetyana Kudlyk, Irina D. Pokrovskaya and Vladimir V. Lupashin

*110 CREB3 Transcription Factors: ER-Golgi Stress Transducers as Hubs for Cellular Homeostasis*

Luciana Sampieri, Pablo Di Giusto and Cecilia Alvarez

Shaheena Shaik, Himani Pandey, Satish Kumar Thirumalasetti and Nobuhiro Nakamura

*166 The Structure and Function of Acylglycerophosphate Acyltransferase 4/ Lysophosphatidic Acid Acyltransferase Delta (AGPAT4/LPAAT*d*)* Mikhail A. Zhukovsky, Angela Filograna, Alberto Luini, Daniela Corda and Carmen Valente

	- Peter M. Luo and Michael Boyce

Sarah Oddoux, Davide Randazzo, Aster Kenea, Bruno Alonso, Kristien J. M. Zaal and Evelyn Ralston

*316 BML-265 and Tyrphostin AG1478 Disperse the Golgi Apparatus and Abolish Protein Transport in Human Cells*

Gaelle Boncompain, Nelly Gareil, Sarah Tessier, Aurianne Lescure, Thouis R. Jones, Oliver Kepp, Guido Kroemer, Elaine Del Nery and Franck Perez

*329 Protein Amphipathic Helix Insertion: A Mechanism to Induce Membrane Fission*

Mikhail A. Zhukovsky, Angela Filograna, Alberto Luini, Daniela Corda and Carmen Valente

# Editorial: Golgi Dynamics in Physiological and Pathological Conditions

#### Kristian Prydz 1 \*, Vladimir Lupashin2 \*, Yanzhuang Wang3 \* and Jaakko Saraste4 \*

1 Department of Biosciences, University of Oslo, Oslo, Norway, 2 Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, United States, 3 Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, United States, 4 Department of Biomedicine and Molecular Imaging Center, University of Bergen, Bergen, Norway

Keywords: Golgi apparatus, Golgi dynamics, Golgi structure, Golgi function, membrane trafficking

#### **Editorial on the Research Topic**

#### Edited and reviewed by:

Georg Haase, INSERM U1106 Institut de Neurosciences des Systèmes, France

#### \*Correspondence:

Kristian Prydz<EMAIL_ADDRESS>Vladimir Lupashin<EMAIL_ADDRESS>Yanzhuang Wang<EMAIL_ADDRESS>Jaakko Saraste<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Membrane Traffic, a section of the journal Frontiers in Cell and Developmental Biology

> Received: 08 December 2019 Accepted: 10 January 2020 Published: 29 January 2020

#### Citation:

Prydz K, Lupashin V, Wang Y and Saraste J (2020) Editorial: Golgi Dynamics in Physiological and Pathological Conditions. Front. Cell Dev. Biol. 8:7. doi: 10.3389/fcell.2020.00007 **Golgi Dynamics in Physiological and Pathological Conditions**

The internal reticular apparatus, first reported in 1898, and later called the Golgi apparatus, was initially observed by Camillo Golgi after his refinement of the silver nitrate technique for staining cells in the nervous system, for which he received the Nobel Prize in Physiology or Medicine in 1906. The true existence of the Golgi apparatus was, however, disputed for decades, and many scientists regarded it as a staining artifact (Palade and Claude, 1949). Only after the electron microscope became a more generally available tool for cell biologists was the Golgi apparatus accepted as an authentic cellular structure, and its existence demonstrated in practically all eukaryotic cells, from yeast to man. However, for reasons that are not completely understood, the Golgi assumes various forms in different cell types, ranging from tubular networks or individual cisternae (budding yeast S. cerevisiae) to separate cisternal stacks (e.g., invertebrates and plants) and a ribbon-like structure of interconnected stacks (vertebrates). As reflected in this collection, much of the recent efforts to define the mechanism of Golgi structure formation have been put on Golgi structural proteins including Golgins and GRASPs, actin and microtubule cytoskeletons, Rabs, and other GTPases, as well as certain other proteins (Ahat et al.; Egorov and Polishchuk; Lowe; Phuyal and Farhan; Satoh et al.; Shaik et al.). Although these proteins have their own characteristic functions, they coordinate with each other to maintain the structural and functional integrity of the Golgi.

The fascinating structure of the Golgi stacks is partly determined by GRASP proteins which also participate in a number of additional cellular processes, such as unconventional secretion and autophagy (Ahat et al.). In vertebrate cells, the multiple stacks are further organized into the Golgi ribbon, a continuous structure where the cisternal stacks are joined together by lateral connections (Saraste and Prydz). Several proteins have been shown to be involved in the regulation of ribbon structure, among these GRASP55 and GRASP65 (Ahat et al.) and the tethering protein giantin (Satoh et al.). Although the ribbon-like organization of the Golgi remains enigmatic, it is highly interesting with respect to certain pathological situations. Indeed, fragmentation of the Golgi ribbon is encountered in a number of neurological diseases (Caracci et al.; Makhoul et al.). The consequences of Golgi structural abnormalities on its function and cellular activities may vary in different experimental or disease conditions. For example, knocking down the GRASP proteins accelerates intra-Golgi trafficking (Xiang et al., 2013; Lee et al., 2014), while inhibition of GM130 and p115 results in the accumulation of COPI vesicles and reduced membrane trafficking (Seemann et al., 2000). Similarly, Aβ (amyloid β) induced Golgi reorganization in Alzheimer's disease has been proposed to increase APP (amyloid precursor protein) trafficking and Aβ production (Joshi et al., 2014), while GM130 knockout slows down ER-Golgi trafficking, resulting in Purkinje neuron loss and ataxia in mice (Liu et al., 2017). In addition, both the morphology (Makhoul et al.) and the internal environment (such as lumenal pH) of the Golgi apparatus are altered in cancer cells, leading to changes in glycosylation (Kellokumpu). Future investigations of the cause and effect of Golgi defects in disease will undoubtedly yield exciting findings essential for the understanding of both Golgi function and disease development.

Structural reorganization of the Golgi is also an integral part of naturally occurring processes, as best demonstrated in the case of cell division. During the late G2 phase, as cells prepare for mitosis, the Golgi ribbon is unlinked into individual stacks, which subsequently undergo further disassembly and vesiculation. In the course of these events, repositioning of Golgi elements is typically observed in the perinuclear region of many vertebrate cells, controlled by the duplicated centrosome and centrosome-nucleated microtubules of the forming mitotic spindle (Mascanzoni et al.). Notably, the pulling apart of the centrosomes during the late G2/early mitosis is accompanied by the evidently equal partitioning of the intermediate compartment (IC), a permanent membrane network that–unlike the Golgi stacks—keeps its properties during mitosis (Saraste and Marie, 2018).

The role of the Golgi apparatus as an important way station in anterograde trafficking along the secretory pathway was established during the 1960s and 1970s (see Farquhar and Palade, 1998, for a review), while retrograde transport via this organelle was described much later (Sandvig et al., 1992). In addition, the role of the Golgi apparatus as an "educational" site for glycoproteins, proteoglycans, and glycolipids is welldescribed and generally accepted. Accordingly, the majority of newly synthesized proteins that enter the Golgi apparatus at its cis-side carry N-linked glycans of identical structure, but leave the trans-Golgi region equipped with highly diverse glycans, specific for the actual species, the cell type, as well as the cell's developmental stage or degree of differentiation (Fisher et al.; Akintayo and Stanley). The ability of various transiting cargo molecules to obtain a healthy output of these and other Golgi modifications, however, requires mechanisms of membrane homeostasis and transport that are still subject to active investigation and dispute (Mironov and Beznoussenko; Saraste and Prydz; Makhoul et al.; Pantazopoulou and Glick). Not only the evaluation of competing Golgi models (Mironov and Beznoussenko), but also performing mathematical modeling (Fisher et al.) can advance our understanding of how cargo molecules that enter the cis-face of the Golgi apparatus are modified during Golgi transit, and how their final structures will turn out. The ongoing attempts to correlate the structural and functional dynamics of the Golgi apparatus are still absolutely required to achieve this goal. While the mechanisms of how different cargo molecules traffic through the Golgi stacks are still under debate, it remains even less clear how precise localization of Golgi resident proteins is achieved within the polarized stacks.

The swarms of COPI vesicles observed at the outskirts of the Golgi membranes are essential for normal Golgi function, although their engagement is still not fully understood due to partial knowledge of their exact cargo selection and composition, places of origin and destination, delivery mechanisms, interaction partners and regulatory modes (Pantazopoulou and Glick; Luo and Boyce; D'Souza et al.). The Rho GTPase Cdc42 is involved in the regulation of actin filament- and microtubuledependent Golgi positioning, in addition to interacting with COPI vesicles or tubules, thus potentially promoting anterograde transport toward the leading edge of migrating cells (Phuyal and Farhan). The trafficking capacity of these transport intermediates can be regulated in a number of ways (Luo and Boyce). For example, defects in the octameric COG complex that functions as a tether in COPI vesicle-mediated retrograde transport, not only affect traditional Golgi functions like glycosylation and sorting, but also exert effects elsewhere in the cell, in particular within the endo-lysosomal system (D'Souza et al.). Knockdown of the COG3 subunit of COG–or the ZW10 subunit of NRZ/Dsl1, another member of the CATCHR family of multisubunit tethering complexes–leads to the dispersal of the Golgi apparatus throughout the cytoplasm of metazoan cells (Zolov and Lupashin, 2005; Sun et al., 2007) in a process that requires both Rab GTPases and kinesin motor proteins (Liu et al.). The dynamic nature of the Golgi apparatus is underlined by the fact that while a large number of proteins are required to maintain its normal organization, treatments affecting a single structural or machinery component are often sufficient to destabilize its structure, as exemplified by the EGFR tyrosine kinase inhibitors BML-265 and AG1478 (Boncompain et al.).

In addition to the COPI transport machineries, the dynamic nature and maintenance of the Golgi apparatus crucially depend on efficient mechanisms of membrane fusion and fission. Both processes are influenced by lipid modifying enzymes, such as acylglycerophosphate acyltransferases (Zhukovsky et al.), membrane curvature-sensing proteins, and fission inducing-proteins (Zhukovsky et al.). To understand Golgi function completely it will be important to reconstitute Golgi fusion and fission in vitro using purified components and endogenous cargo.

A question that has been touched upon, but is far from being solved, is whether all the stacks in a Golgi ribbon handle the same cargo and have identical enzymatic contents to carry out the same protein and lipid modifications. While the nonlinked, wide-spread Golgi stacks in Drosophila cells differ in their enzymatic repertoire (Yano et al., 2005), the apical and basolateral routes in mammalian epithelial MDCK cells have also been shown to treat the same cargo molecule differently (Prydz et al., 2008). A related question is whether there may be lateral segregation of cargo within cisternae of the same Golgi stack. Interestingly, this was recently shown to be the case for HeLa cells, where two proteins heading for the endolysosomal system gradually underwent lateral segregation while passing through the Golgi (Chen et al., 2017). Here, the paper by Ernst et al. discusses recent findings showing that acylation can influence the lateral positioning of proteins in Golgi cisternae, and as a consequence, their anterograde transport efficiency (Ernst et al.). While anterograde transport of cargo also takes place after the stacks of the Golgi ribbon have been dispersed, for instance, due to breakdown of microtubules, an intact ribbon does seem to be important for the transport of large cargo molecules (Lavieu et al., 2014). In a new view of the Golgi ribbon, the non-compact zones linking the cisternal stacks are jointly occupied by the permanent IC elements and recycling endosomes (RE). As mentioned above, as a prelude for cell division, the stacks that undergo reversible break-down disconnect from these centrosome-linked compartments, which as a consequence would be allowed move to the spindle poles for partitioning into the forming daughter cells (Saraste and Marie, 2018; Saraste and Prydz).

Indeed, the Golgi apparatus is in intimate communication with both pre- and post-Golgi compartments. A number of important proteins recycle between the endoplasmic reticulum (ER) and the Golgi apparatus. YIPFα1A, a member of the YIPF protein family, functions at ER exit sites and interacts with COPII components, but can also localize to the IC and cis-Golgi, interact with YIPβ1A, and recycle back to the ER (Shaik et al.). Members of the CREB3 family of transcription factors move from the ER to the Golgi apparatus when the cell receives a proper signal. The activation of these proteins in the Golgi apparatus is based on two sequential proteolytic cleavage events. Upon cleavage, the N-terminal portions of the proteins, which are localized to the cytoplasmic side of the Golgi membrane, are released and become free to move into the nucleus (Sampieri et al.).

At the trans-side of the Golgi stacks, the trans-Golgi network (TGN) is an important site for Golgi exit of cargo molecules destined for various organelles of the endomembrane system and different plasma membrane domains. With the continuous improvement of the resolution of fluorescence-based light microscopes, it is now possible to observe the segregation of

#### REFERENCES

various cargo molecules and the membrane carriers that exit the Golgi at the level of the TGN (Huang et al.).

To fully (or at least better) understand the Golgi apparatus in various physiological and pathological conditions, one has to examine this organelle in a variety of tissues at different stages of differentiation, development or degeneration, for instance in neuronal (Caracci et al.; Rabouille and Haase, 2016) and muscle cells (Oddoux et al.). This is important to understand the adaptability of the Golgi apparatus to the cellular requirements, ensuring a healthy glycan output, as exemplified in this collection by the analysis of glycans of oocytes and sperm cells (Akintayo and Stanley). Equally important is to study a growing number of genetic diseases—such as the Aarskog-Scott syndrome (Egorov and Polishchuk), a faciogenital dysplasia caused by mutations in a GEF protein (FGD1) regulating the Rho GTPase Cdc42–that are found to affect Golgi structure and function.

Altogether, topical reviews, hypothesis and theory articles, and original studies in this collection illustrate our recent progress in understanding Golgi biology, and also outline a specific set of yet unanswered Golgi-related questions. For example, exactly how do cargo and resident proteins travel to, through and out of the Golgi? What are the exact modes, carriers and molecular machineries of bi-directional Golgi trafficking? How are Golgi structure and its various functions modified during normal (differentiation, development, etc.) and abnormal (diseases, drugs, pathogens, etc.) circumstances?

#### AUTHOR CONTRIBUTIONS

KP, VL, YW, and JS prepared and wrote the manuscript.

#### FUNDING

This work was supported by the NIH Grant GM083144 to VL and by the National Institute of Health (Grants GM112786, GM105920, and GM130331), MCubed and the Fastforward Protein Folding Disease Initiative of the University of Michigan to YW.

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cells. Proc. Natl. Acad. Sci. U.S.A. 102, 13467–13472. doi: 10.1073/pnas.050 6681102

Zolov, S. N., and Lupashin, V. V. (2005). Cog3p depletion blocks vesiclemediated Golgi retrograde trafficking in HeLa cells. J. Cell Biol. 168, 747–759. doi: 10.1083/jcb.200412003

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Prydz, Lupashin, Wang and Saraste. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Identification of CDC42 Effectors Operating in FGD1-Dependent Trafficking at the Golgi

#### Mikhail Egorov\* and Roman Polishchuk\*

Telethon Institute of Genetics and Medicine, Naples, Italy

Loss of function mutations in the FGD1 gene cause a rare X-linked disease, faciogenital dysplasia (FGDY, also known as Aarskog-Skott syndrome), which is associated with bone and urogenital abnormalities. The FGD1 gene encodes à CDC42-specific guanine nucleotide exchange factor. The mutations are frequently located in the DH module of FGD1 preventing its transformation to the active form. We previously reported that Golgi-associated FGD1 regulates post-Golgi transport of some conventional and bonespecific proteins in a CDC42-dependent manner. However, the downstream targets of FGD1/CDC42 signaling that operate to support transport from the Golgi remain elusive. Here, we demonstrate that Golgi-localized CDC42 effectors might be involved in FGD1 mediated post-Golgi transport, probably through coordination of Golgi membrane and cytoskeleton dynamics. Overexpression of effector-specific CDC42 mutants (exhibiting preferential affinities for PAK1, IQGAP1, N-WASP, or PAR6) only partially rescue membrane trafficking in FGD1-deficient cells, indicating that the orchestrated activities of several downstream targets of CDC42 are required to support FGD1-mediated export from the Golgi. Our findings provide new insights into understanding the molecular mechanisms behind FGD1/CDC42-dependent transport events and uncover new targets whose potential might be explored for correction of membrane trafficking in FGDY.

 FGDY is caused by loss-of-function mutations in the FGD1 gene that encodes a 961 amino acid FGD1 protein that acts as a specific GEF for the small Rho GTPase CDC42 (Pasteris et al., 1994; Zheng et al., 1996). The predicted frequency of clinical manifestation of FGDY is about 1:25,000 (Orrico et al., 2015). The FGD1 protein is expressed in regions of active osteogenesis in developing long bones (Gorski et al., 2000). In humans, the highest levels of FGD1 expression have been observed in bone tissue, kidney, liver, lung, heart and brain (Pasteris et al., 1994; Olson, 1996). Thus, the pattern of postnatal FGD1 expression strongly correlates with clinical manifestations of FGDY. Moreover, the FGD1/CDC42 signaling machinery has an important role in osteogenetic differentiation in hMSC and may persist throughout adult life (Gao et al., 2011). Similarly to other

#### Edited by:

Roberto Weigert, National Institute of Dental and Craniofacial Research (NIDCR), United States

#### Reviewed by:

Vladimir Lupashin, University of Arkansas for Medical Sciences, United States Catherine Jackson, UMR7592 Institut Jacques Monod (IJM), France

#### \*Correspondence:

Mikhail Egorov<EMAIL_ADDRESS>Roman Polishchuk<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Membrane Traffic, a section of the journal Frontiers in Cell and Developmental Biology

> Received: 12 July 2018 Accepted: 16 January 2019 Published: 04 February 2019

#### Citation:

Egorov M and Polishchuk R (2019) Identification of CDC42 Effectors Operating in FGD1-Dependent Trafficking at the Golgi. Front. Cell Dev. Biol. 7:7. doi: 10.3389/fcell.2019.00007

genetic diseases, there is no specific treatment for patients with FGDY, surgical intervention being the only option to correct some abnormalities and increase the quality of life.

Although FGD1 was detected at the Golgi (Estrada et al., 2001; Egorov et al., 2009), the mechanism by which the FGD1/CDC42 machinery regulates the transport of cargo at the Golgi apparatus remains unclear. We reported previously that FGD1 silencing strongly suppressed CDC42 activity at the membranes of trans-Golgi network (TGN). As a result, formation of post-Golgi transport intermediates was compromised, apparently due to an impairment of TGN membrane extension along microtubules (Egorov et al., 2009). In particular, this interaction is important for docking of nascent carriers to the microtubules (MTs) that trigger trafficking events. The failure of FGD1 to coordinate membrane and cytoskeletal dynamics at the Golgi could explain the membrane transport delay induced by loss-of-function FGD1 mutations and might be considered as one of the key mechanisms of FGDY pathogenesis.

Given the importance of the FGD1/CDC42 machinery in regulating post-Golgi membrane transport, we investigated whether and to what extent the downstream targets of Golgilocalized CDC42 mediate FGD1-dependent signal transduction at the Golgi (McCallum et al., 1998; Matas et al., 2004; Cau and Hall, 2005; Valente et al., 2012; Matsui et al., 2015). Our findings indicate that suppression of either PAK1 or IQGAP1 genes resembles the inhibitory effects of FGD1 silencing on post-Golgi transport, while their activation only partially overrides the TGN transport block induced by FGD1 deficiency. Expression of effector-specific CDC42 mutants revealed that other downstream components of the FGD1/CDC42 signaling pathway including N-WASP and PAR6 might be involved in FGD1-mediated trafficking events. Further characterization of the proposed signaling pathways may help to uncover the key druggable molecules with therapeutic potential for FGDY.

#### RESULTS

To determine whether Golgi-associated CDC42 targets such as IQGAP1, PAK1 and N-WASP operate in FGD1/CDC42 dependent trafficking events, we assessed the ability of these proteins to influence post-Golgi transport by monitoring constitutive export. We reasoned that CDC42 effectors whose suppression would cause the aberrant FGDY-like "secretory" phenotype (Egorov et al., 2009) are likely to be involved in FGD1/CDC42-medited regulation of trafficking events at the Golgi. To address this issue, we silenced genes of interest in HeLa cells with specific siRNAs and analyzed the impact of gene suppression on post-Golgi transport of VSVG, as previously described (Polishchuk et al., 2003). VSVG was synchronized within the TGN using a 20◦C block. We found that VSVG strongly co-localized with the trans-Golgi marker TGN46 in both control and IQGAP1-, PAK1-, or N-WASP-depleted cells at the end of the 20◦C temperature block (**Figure 1A**, upper row; see **Figure 1C** for silencing efficiency), indicating that suppression of these CDC42 effectors did not impact on the delivery of cargo proteins to the TGN. Release from the 20◦C block triggered the formation of numerous VSVG-containing carriers from the Golgi apparatus in control cells. To block the fusion of carriers with the plasma membrane (which facilitates quantification), 0.5% tannic acid treatment was employed (Egorov et al., 2009). Unlike control cells, where numerous post-Golgi carriers formed within 1 h after the release of the 20◦C block, IQGAP1- or PAK1-depleted cells exhibited a significant reduction in the number of TGNderived VSVG carriers (**Figure 1A**, bottom row). Morphometric analysis confirmed that suppression of either IQGAP1 or PAK1 resulted in a significant decrease of VSVG-containing post-Golgi carriers (**Figure 1B**). In contrast, inhibition of N-WASP did not significantly affect the post-Golgi transport of VSVG (**Figures 1A,B**) indicating that N-WASP may operate mainly in a different transport route from the Golgi (Matas et al., 2004; Egea et al., 2013).

The inhibitory impact of IQGAP1 and PAK1 depletion on export from the TGN was confirmed by electron microscopy analysis. The general morphology of the TGN in HeLa cells lacking either PAK1 or IQGAP1 proteins were assessed using TGN38-HRP transfection (Egorov et al., 2009). The inspection of thin sections revealed a TGN38-HRP-associated DAB precipitate, which decorated the lumen of both tubular and round membrane profiles in the TGN area of control HeLa cells (**Figure 1D**). The silenced cells exhibited much longer and larger TGN profiles with 2–3 labeled cisternae in some Golgi stacks (**Figures 1D,E**). This phenotype indicates significantly reduced consumption of TGN membranes in both IQGAP1- and PAK1-deficient cells due to a delay of protein export at the level of the TGN. In summary, our observations suggest that suppression of the CDC42 downstream targets IQGAP1 and PAK1 inhibits export from the Golgi in a manner similar to what is observed in FGD1-deficient cells (Egorov et al., 2009).

To further test the impact of IQGAP1/PAK1 proteins on the FGD1-dependent mechanism of post-Golgi transport, we investigated their ability to rescue VSVG trafficking in FGD1 deficient cells. To this end, FGD1-silenced HeLa cells were co-transfected with VSVG-EGFP and plasmids encoding either PAK1-wt or PAK1-Thr423 (a constitutively active mutant) (Jaffer and Chernoff, 2002), and VSVG transport was analyzed upon a release from the 20◦C block as described above. The loss of FGD1 function led to a delay of VSVG-GFP transport, while expression of PAK1-wt and PAK1-Thr423 resulted in a moderate increase in the number of post-Golgi VSVG-positive transport carriers in FGD1-deficient cells (**Figures 2A,B**). It appears that overexpression of PAK1 rescues post-Golgi trafficking to some extent but cannot completely overcome the transport block at the TGN induced by FGD1 knockdown.

We also evaluated the ability of IQGAP1 to facilitate protein export from the TGN in FGD1-silenced cells using the same experimental strategy. Expression of IQGAP1-WT induced a modest but significant recovery of VSVG export from the Golgi in FGD1-deficient cells (**Figures 2A,B**). To show that the IQGAP1-mediated rescue of trafficking in FGD1-deficient cells indeed requires CDC42, we used the IQGAP1 mutant (IQGAP1- T1050AX2), which fails to interact with its upstream activator CDC42/RAC1 (Fukata et al., 2002). We observed that expression of this particular IQGAP1 mutant was unable to rescue VSVG

FIGURE 1 | Down-stream targets of CDC42 regulate post-Golgi transport in HeLa cells. Cells were treated with either control non-targeting duplex or specific siRNAs of IQGAP1, PAK1, and N-WASP for 72 h. Cells were then processed for evaluation of post-Golgi trafficking (A,B), efficiency of silencing (C), or TGN morphology (D,E). (A) After the 20◦C block, VSVG accumulated at the Golgi level where it overlapped with the TGN46 marker (top row). Release of the secretory material from the Golgi (32◦C 60 min; see bottom row) in the presence of 0.5% tannic acid resulted in the accumulation of VSVG carriers (arrows) in control and N-WASP-depleted cells, while most VSVG still remained in the Golgi (empty arrows) in IQGAP1- and PAK1-silenced cells. Scale bar, 15 µm. (B) Quantification of VSVG-containing carriers after 60 min of release from the 20◦C block (n = 30 cells; ∗∗∗p < 0,001, t-test). (C) Western blotting showed the efficient reduction in the expression of corresponding proteins after treatment with siRNAs for N-WASP, IQGAP1, and PAK1. The level of actin was used as a loading marker. (D) Control, IQGAP1 or PAK1-silenced cells were transfected with TGN38-HRP, fixed and subsequently processed for electron microscopy analysis (see methods). Control HeLa cells showed the TGN-38-HRP-associated DAB precipitate in several tubular and rounded profiles at the trans side of the Golgi stack. Unlike control cells, the silencing of either the IQGAP1 gene or the PAK1 gene resulted in a much larger TGN, comprised of numerous additional labeled cisternae and tubular profiles. Scale bar: control and siRNA PAK1 200 nm, siRNA IQGAP1 500 nm. (E) Quantification of the increase in the area of the TGN compartment in IQGAP1- and PAK1-silenced cells (n = 20 stacks; ∗∗∗p < 0.001, t-test).

FIGURE 2 | CDC42 effectors partially rescue post-Golgi transport of VSVG in FGD1-deficient HeLa cells. HeLa cells were treated with targeted FGD1 siRNA and co-transfected with VSVG-GFP and a plasmid encoding CDC42 effector (indicated at the corresponding panel). Cells were subsequently exposed to the 20◦C block for 2 h and then shifted to 32◦C for 60 min in the presence of tannic acid. (A) Expression of either the PAK1 or the IQGAP1 mutant partially rescued the VSVG-GFP transport in FGDY cells; (B) Quantification of post-Golgi VSVG-GFP- containing carriers per cell in the experiments shown in panel A (n = 30 cells; ∗p < 0.005, t-test). (C) Co-expression of VSVG-GFP and different constitutively active CDC42 mutants (see Table 1) partially rescued transport of VSVG-GFP in FGD1-silenced cells. (D) Quantification of post-Golgi VSVG-GFP-containing carriers per cell in the experiments shown in panel C (n = 30 cells; ∗∗∗p < 0.001, ∗p < 0.005, t-test). Scale bars, 20 µm.

transport in FGD1-silenced cells even after 60 min of release from the TGN (**Figures 2A,B**). These findings demonstrate that IQGAP1 might potentially operate as a FGD1/CDC42 signal conductor to regulate transport from the Golgi. However, none of these downstream CDC42 effectors were sufficient alone to completely restore trafficking in FGD1-deficient cells.

To further characterize the FGD1 signaling pathway that is mediated by CDC42 effectors, we used constitutively active CDC42 mutants, which are defective in binding to different downstream targets of CDC42 (Li et al., 1999; Guo et al., 2010; See **Table 1**). We first tested whether expression of the CDC42/A37 mutant, which is defective in interacting with PAK1, could overcome the transport block induced by FGD1 lossof-function using the traffic pulse protocol (**Figures 2C,D**). We noted a significant (but not full) recovery of VSVG-EGFP post-Golgi transport in FGD1-deficient cells expressing this CDC42 mutant. Thus, PAK1 does not appear to operate as an indispensible effector in post-Golgi trafficking events that are regulated through the FGD1/CDC42 axis. This observation is in line with a previous report showing a limited ability of FGD1 to regulate the activity of PAK1 (Nagata et al., 1998).

Another mutant, CDC42/H63, which is defective in binding to IQGAP1, had a similar impact. Overexpression of this mutant in FGD1-deficient cells showed a moderate improvement in VSVG-EGFP transport (**Figures 2C,D**), indicating that CDC42 can sustain post-Golgi trafficking through other effectors. Indeed, this mutant binds PAR6, N-WASP and to some extent PAK1 (**Table 1**).

Next we tested whether expression of the CDC42 mutants, which do not interact with N-WASP/PAR6 (K40) or with PAR6 (AA173-174), could restore transport in FGD1-deficient cells (**Figures 2C,D**). We observed no statistically significant recovery of VSVG-GFP post-Golgi transport in FGD1-silenced cells after K40 expression and only a slight increase (10.3%) in trafficking in the case of CDC42-AA173-174 expression. This suggests that FGD1/CDC42-dependent post-Golgi trafficking might require N-WASP and PAR6, whose roles in this process have to be further investigated.

Finally, we tested CDC42 constitutively active mutants that have no binding limitations toward any of the effectors (L-28 and QQ186-187). Overexpression of these mutants resulted in significant recovery of VSVG trafficking in FGD1-depleted cells.

+, binding to the indicated effectors; −, no binding activity; Li et al. (1999) and Guo et al. (2010).

Interestingly, expression of the QQ186-187 mutant, which has a lower binding affinity for PAR6, was less efficient in recovering VSVG transport compared to CDC42/L28. This implies that only a CDC42 form that is fully active toward all downstream effectors is able to completely rescue the Golgi export block caused by FGD1 loss-of-function (**Figures 2C,D**). Thus, the recovery post-Golgi trafficking in FGD1-deficient cells appears to require the synergistic action of several CDC42 effectors at a certain ratio. Alternatively, the limited effect on the transport by some of the mutants might be due to their weak association with the Golgi apparatus. Additional experiments will be necessary to fully characterize the consequences of these interactions.

To further explore possible mechanisms downstream of FGD1/CDC42 signaling at the Golgi, we assessed the levels of CDC42 effectors (PAK1/IQGAP1) under conditions of FGD1 silencing. Surprisingly, Western blot revealed that both PAK1 and IQGAP signals increased in FGD1-deficient cells by 78 and 19.5%, respectively (**Figures 3A–C**). Using an antibody that specifically recognizes the phosphorylated forms of PAK1- 3, we found that Golgi-associated levels of phospho-PAK were elevated as judged by immunofluorescence inspection (**Figures 4A–F**). Taking into account that only PAK1 (but not PAK2/3) resides at the Golgi, we assume that the increase in phospho-PAK in the TGN46 compartment reflects activation of PAK1 at the Golgi in FGD1-deficient cells (**Figures 4G,H**). It appears that PAK1 becomes more active at the Golgi in the absence of functional FGD1. Thus, the up-regulation/activation of CDC42 might explain a weak compensatory response supporting cell homeostasis in conditions of a mutated FGD1 protein. This response, however, does not appear to be sufficient for recovery of normal post-Golgi trafficking rates.

Driven by the hypothesis that FGD1 could function in the signaling machinery that regulates the interaction between nascent TGN membranes and MTs (Egorov et al., 2009), we investigated the involvement of FGD1 and CDC42 effectors in both the nucleation and growth of the Golgi-associated subset of MTs in HeLa cells. To this end, we employed a well-known approach based on the regrowth of MTs in interphase cells after their nocodazole-induced depolymerization (Efimov et al., 2007). Immunofluorescence revealed that control cells quickly recovered the Golgi-associated pool of MTs 2–10 min after nocodazole washout (**Figures 5A,E**). By contrast, there was a significant delay of MT recovery in the case of FGD1 suppression since only a few MT profiles emerged from the Golgi elements in FGD1-silenced cells (**Figures 5B,E**). This effect of FGD1 depletion on Golgi-associated MT growth might contribute to inhibiting the formation of post-Golgi carriers, which exit from the TGN along MT tracks (Polishchuk et al., 2003; Egorov et al., 2009). Similarly to FGD1 knockdown, the silencing of either PAK1 or IQGAP1 by specific siRNAs affected the efficiency of MT regrowth from the Golgi units (**Figures 5C–E**) suggesting that both CDC42 effectors regulate the dynamics of MTs emerging from the Golgi. Importantly, in these experiments we found that the rate of MT growth from the MTOC was not affected, implying the involvement of FGD1, PAK1 and IQGAP in MT dynamics operates only at Golgi stacks.

The function, stabilization and dynamics of MTs are tightly regulated by numerous MT-associated proteins. One of these is the well-characterized MT non-motor plus-end-binding protein CLASP (cytoplasmic linker associated protein (1) that has been shown to drive nucleation of non-centrosomal MTs originating from the TGN (Efimov et al., 2007). We reasoned that the dynamics of CLASP1 at the Golgi might be affected by FGD1 loss-of-function. Confocal microscopy showed a very clear but faint CLASP1 signal at the Golgi membranes in control cells (**Figures 6A–C**). By contrast, FGD1-silenced cells exhibited a strong increase in CLASP1 levels at the Golgi (**Figures 6D–F**) indicating that molecular coordination of Golgi membranes and MT dynamics are likely to be impaired by the loss of FGD1.

# DISCUSSION

Our findings indicate that the impact of FGD1 on post-Golgi trafficking is indeed mediated by CDC42 effectors. However, none of the CDC42 effectors appears to be absolutely indispensible: rather, it is the orchestrated activity of several such effectors that is required to support export of cargo proteins from the TGN. We have shown that direct Golgi-associated downstream targets of CDC42, such IQGAP1 and PAK1, are involved in regulating constitutive post-Golgi membrane transport. PAK1 has been already reported to operate in the fission of post-Golgi carriers from the TGN (Valente et al., 2012), while the involvement of IQGAP1 in post-Golgi trafficking has not yet been observed despite a well-documented association of IQGAP with the Golgi (McCallum et al., 1998). Our observations suggest that FGD1, IQGAP1 and PAK1 are required (i) to support formation of post-Golgi carriers and (ii) to allow nucleation of Golgi-associated MTs, which are used by post-Golgi carriers as highways to reach their target destinations (Polishchuk et al., 2003; Egorov et al., 2009). However, both IQGAP and PAK1 exhibited a limited ability to restore the impaired TGN export in FGD1-deficient cells when expressed alone. Indeed, the impact of their overexpression on post-Golgi trafficking in the absence of FGD1 appears to be incomplete, probably due to the lack of activity of other effectors coordinated by the FGD1/CDC42 machinery or by their weaker association with TGN membranes when CDC42 is missing. On the other hand, we found that the levels of both IQGAP1 and PAK1 were increased in cells lacking FGD1.

This indicates that cells might try to compensate for the loss of FGD1 through the up-regulation of the CDC42 effectors PAK1 and IQGAP1. These compensatory mechanisms may target PAK1 and IQGAP1 through FGD1 independent signaling pathways in an attempt to circumvent the FGD1 deficiency. In this regard, FGD1 has been shown to play a limited role in PAK1 activation (Nagata et al., 1998) indicating that additional mechanisms might be involved to stimulate PAK1 activity at the Golgi. However, such activation does not appear to be sufficient to recover post-Golgi transport, probably because other components of the FGD1/CDC42-controlled molecular network remain inactive or cannot be recruited to the Golgi.

The complexity of FGD1-regulated post-Golgi transport might imply the coexistence of several CDC42-dependent signaling pathways, some of which may include N-WASP as an effector. This hypothesis is supported by our findings showing that CDC42 mutants with impaired N-WASP binding have a limited capacity to support VSVG export from the Golgi in FGD1-deficient cells. N-WASP regulates actin dynamics and the formation of transport carriers at the Golgi (Egea et al., 2013). We cannot rule out that actin-based mechanisms of post-Golgi carrier formation are also controlled by the FGD1/CDC42 machinery. WASP, as a CDC42 target, might be a part of this mechanism as well, but our RNAi results suggest that WASP is probably indispensable for post-Golgi trafficking.

Our findings also suggest that one of the plausible mechanisms by which FGD1 regulates post-Golgi transport requires the stabilization of a Golgi-emanating subset of MTs, which serve as tracks for the formation of post-Golgi carriers and for their further translocation to the target membrane (Polishchuk et al., 2003; Egorov et al., 2009). Indeed, nucleation of MTs at the Golgi membranes was strongly inhibited by silencing

of FGD1 or by suppression of the CDC42 effectors IQGAP and PAK1. MT assembly at Golgi membranes is driven by MT-associated proteins such as CLASPs (Efimov et al., 2007; Miller et al., 2009). It has been shown recently that the dynamics of CLASP2 association with the Golgi is regulated by a novel protein complex, PAR6/PAR3/aPKC, that has CDC42 and PAR6

as targets (Matsui et al., 2015). This protein complex controls CLASP2 phosphorylation at sites responsible for its interaction with GCC185 at the TGN (Matsui et al., 2015). Disruption of this PAR6 complex results in aberrant CLASP accumulation at the Golgi (Matsui et al., 2015). Notably, FGD1 knockdown causes a similar increase in CLASP association with Golgi membranes (see **Figure 6**). Thus, it is tempting to speculate that the missing link between the FGD1/CDC42 machinery and MTs could be constituted by the PAR/PKC-CLASP complex (**Figure 7**). In this scenario, the lack of FGD1 activity might impact on the

bar, 28 µm.

fcell-07-00007 January 31, 2019 Time: 18:45 # 9

PAR6 complex and, hence, further affect the dynamics of CLASP. This, in turn, would cause either MT instability or a low rate of MT regrowth, thus affecting the formation and translocation of Golgi-derived transport carriers and post-Golgi trafficking in general. Indeed, abnormal CLASP accumulation and impaired nucleation of MTs at the Golgi in FGD1-deficient cells supports this hypothesis. We also found that loss of interaction with PAR6 reduces the ability of constitutively active CDC42 mutants to rescue transport in FGD1-deficient cells. This suggests that PAR6 is involved in the FGD1/CDC42 pathway that regulates MT dynamics at TGN membranes.

Further studies of the molecular players involved in FGD1/CDC42-dependent regulation of post-Golgi trafficking represents a challenging task in finding a cure for FGDY. Although we found that activation of some CDC42 effectors may partially compensate for FGD1 deficiency, none of these effectors alone circumvent the consequences of FGD1 loss. Therefore, drugs or treatments that stimulate multiple FGD1/CDC42 targets have to be considered for eventual therapeutic approaches. Furthermore, considering the abundance of FGD1 in bones and its importance for the transport of bone proteins (Egorov et al., 2009), pharmacological activation of FGD1/CDC42 effectors deserves serious evaluation as a potential strategy to facilitate bone regeneration after damage or surgery. In this context, significant efforts will be required to identify key druggable nodes in the FGD1/CDC42 signaling pathway(s) operating in the bone-specific post-Golgi secretory pathway(s).

# MATERIALS AND METHODS

#### Antibodies and Reagents

The following antibodies were used: anti-TGN46 1:1000 (IF) (AbD Serotec, United Kingdom); anti-Giantin 1:500 (IF) (Abcam, United Kingdom); anti-myc 1:250 (IF); anti-flag

1:250 (IF); anti-HA 1:500 (IF) and P5D4 Cy3-conjugated anti-VSVG 1:500 (IF) from Sigma-Aldrich (Italy); anti-N-WASP 1:1000(WB); anti-IQGAP1 1:1000(WB); phosphoPAK1- 3 1:500 (IF), 1:1000 (WB); and PAK1 (N-20) 1:500 (IF), 1:1000 (WB) were purchased from Santa Cruz Biotechnology (United States); anti-FGD1 1:1000 (WB) made by G. Di Tullio (CMNS, Italy); GAPDH 1:1000 (WB), CLASP1 1:300 (IF) provided by I. Kaverina (Vanderbilt University, United States).

The following plasmids were used: IQGAP1-T1050AX2 flag (provided by K. Kaibuchi, Japan); IQGAP1-WT-myc (Columbia University); PAK1-WT-myc, PAK1-Thr423-myc (provided by M. Gimona); CDC42 mutants were tagged with HA (a gift form Y. Zheng, United States); VSVG-EGFP (provided by J. Lippincott-Schwarz, NIH/NICHD, United States).

# Cell Culture, Transfections, and Infection With VSV

HeLa cells (ATCC, United States) were cultured in DMEM (Invitrogen, Italy) supplemented with 10% FBS. Both non-targeted (control) and target-specific siRNAs were obtained from Sigma Aldrich (Italy) and transfected by Oligofectamine (Invitrogen, Italy). The efficiency of silencing was assessed for each experiment by Western blotting. Plasmid transfection was performed using Lipofectamine 2000 (Invitrogen, Italy). The infection of HeLa with VSV was performed as previously described (Polishchuk et al., 2003). The nododazole-washout assay was performed according to a published protocol (Efimov et al., 2007).

### Western Blotting

RNAi-treated HeLa cells were lysed in 50 mM Tris–HCl, pH 8.8, containing 0.2% SDS and protease inhibitor cocktail (Roche, France). Sixty micrograms of cell lysates were run on 7% SDS– PAGE and subsequently transferred to nitrocellulose. Blots were probed with specific antibodies of interest using the ECL-based detection method (Amersham Pharmacia Biotech, Piscataway, NJ, United States).

#### Immunofluorescence and Confocal Microscopy

For immunofluorescence analysis, HeLa cells were fixed with 4% paraformaldehyde in phosphate-buffered saline at room temperature for 30 min, permeabilized with 0.2% saponin for 30 min, and then blocked with 2% bovine serum albumin (BSA) for 30 min. The cells were labeled with the primary antibodies and secondary antibodies of interest conjugated to Alexa Fluor 488 and 568. Cells were mounted with mowiol and analyzed on LSM 510 META or LSM710 confocal microscopes (Carl Zeiss, Germany) with the 63 × Apo NA 1.4 objective. Numbers of VSVG carriers and numbers of MTs emerging from the Golgi were quantified in confocal images using ImageJ software (NIH, United States) as described (Egorov et al., 2009). Colocalization of p-PAK or CLASP1 with TGN46 was evaluated using either ImageJ (NIH, United States) or Zen Black (Zeiss, Germany) software.

#### Electron Microscopy

For electron microscopy analysis, cells of interest expressing TGN38-HRP were directly fixed with 2% glutaraldehyde

(pH 7.4) and incubated with buffered 3,30 -diaminodenzidine DAB, according to Egorov et al. (2009). After dehydration the cells were embedded in Epon812. Embedded cells were sectioned with a 45◦ degree diamond knife (Diatome, Switzerland) using a Leica ultramicrotome (Leica, Austria). The thin 70 nm sections were imaged with a Tecnai-12 electron microscope (FEI, The Netherlands) equipped with an Ultra View CCD digital camera (Soft Imaging System, Munich, Germany).

#### Statistical Analysis

All statistical data are presented as a mean value ± standard deviation. The unpaired Student t-test was used to calculate differences between two sets of data. Statistical significance between different sets of data are indicated in the text and figures as follows: ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001. All results are representative of three independent experiments.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

#### FUNDING

This work was supported by Telethon, Italy (Grant #TGM16YCBDM09 to RP) and by AIRC Italy (Grant #IG17118).

#### ACKNOWLEDGMENTS

The authors thank Cathal Wilson for critical reading of the manuscript.

phosphorilation to generate cell polarity. Mol. Biol. Cell. 26, 751–761. doi: 10.1091/mbc.E14-09-1382

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Egorov and Polishchuk. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Golgi Complex Dynamics and Its Implication in Prevalent Neurological Disorders

#### Mario O. Caracci, Luz M. Fuentealba and María-Paz Marzolo\*

Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile

Coupling of protein synthesis with protein delivery to distinct subcellular domains is essential for maintaining cellular homeostasis, and defects thereof have consistently been shown to be associated with several diseases. This function is particularly challenging for neurons given their polarized nature and differential protein requirements in synaptic boutons, dendrites, axons, and soma. Long-range trafficking is greatly enhanced in neurons by discrete mini-organelles resembling the Golgi complex (GC) referred to as Golgi outposts (GOPs) which play an essential role in the development of dendritic arborization. In this context, the morphology of the GC is highly plastic, and the polarized distribution of this organelle is necessary for neuronal migration and polarized growth. Furthermore, synaptic components are readily trafficked and modified at GOP suggesting a function for this organelle in synaptic plasticity. However, little is known about GOPs properties and biogenesis and the role of GOP dysregulation in pathology. In this review, we discuss current literature supporting a role for GC dynamics in prevalent neurological disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and epilepsy, and examine the association of these disorders with the wide-ranging effects of GC function on common cellular pathways regulating neuronal excitability, polarity, migration, and organellar stress. First, we discuss the role of Golgins and Golgi-associated proteins in the regulation of GC morphology and dynamics. Then, we consider abnormal GC arrangements observed in neurological disorders and associations with common neuronal defects therein. Finally, we consider the cell signaling pathways involved in the modulation of GC dynamics and argue for a master regulatory role for Reelin signaling, a well-known regulator of neuronal polarity and migration. Determining the cellular pathways involved in shaping the Golgi network will have a direct and profound impact on our current understanding of neurodevelopment and neuropathology and aid the development of novel therapeutic strategies for improved patient care and prognosis.

Keywords: Golgins, GOPs, CLASP2, neurodegeneration, epilepsy, LRRK2, Reelin, synaptic activity

# INTRODUCTION

The Golgi complex (GC) is composed of distinct compartments or cisternae: the cis-, medialand trans-Golgi and the trans-Golgi network (TGN) (Chen et al., 2017). The cis-Golgi, located immediately after the endoplasmic reticulum (ER), is where proteins are sorted based on carboxyl terminal signal peptides to either continue moving through the Golgi cisternae or return to the

#### Edited by:

Vladimir Lupashin, University of Arkansas for Medical Sciences, United States

#### Reviewed by:

Leslie K. Climer, Baylor University, United States Juan S. Bonifacino, National Institutes of Health (NIH), United States

> \*Correspondence: María-Paz Marzolo<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Membrane Traffic, a section of the journal Frontiers in Cell and Developmental Biology

> Received: 18 March 2019 Accepted: 18 April 2019 Published: 07 May 2019

#### Citation:

Caracci MO, Fuentealba LM and Marzolo M-P (2019) Golgi Complex Dynamics and Its Implication in Prevalent Neurological Disorders. Front. Cell Dev. Biol. 7:75. doi: 10.3389/fcell.2019.00075

ER, or, in the case of lysosomal enzymes, be tagged with mannose-6-phosphate to be directed to the lysosomes. The medial-Golgi is enriched in glycosyl-transferase enzymes and conducts several post-translational modifications of recently translated proteins. The trans-most Golgi cisterna branches toward a tubular and reticulating compartment called the TGN, considered the central cargo sorting station of the cell (Guo et al., 2014). Both clathrinand non-clathrin coated vesicles, as well as tubules, are formed at the TGN to deliver lipid and proteins to their final destination, mainly plasma membrane and endosomal compartments (Lowe, 2011; Guo et al., 2014). Golgi cisternae are laterally linked to form the Golgi ribbon structure. The stability of this complex structure and the maintenance of cisternae-specific protein makeup largely depends on resident structural proteins and their interaction with both microtubules and the actin cytoskeleton (Lowe, 2011).

Excellent reviews have addressed the biology of the GC in an array of cell types (Hanus and Ehlers, 2008; Nakano and Luini, 2010; Lowe, 2011; Ramírez and Couve, 2011; Muschalik and Munro, 2018). However, little attention has been given to the role of GC in pathological conditions associated with the central nervous system (CNS). Increased GC fragmentation has been reported in several neurological disorders, including Alzheimer's disease (AD) (Joshi et al., 2015), amyotrophic lateral sclerosis (ALS) (Sundaramoorthy et al., 2015), Creutzfeldt-Jakob disease (Sakurai et al., 2000), and Parkinson's disease (PD) (Gosavi et al., 2002), among others. Compellingly, this fragmentation is often observed at early stages of the disease thus making it unlikely that GC fragmentation is a result of apoptotic cell death (Gosavi et al., 2002; Liazoghli et al., 2005; van Dis et al., 2014).

In the present review, we summarize relevant information regarding the regulation of GC dynamics in neurons and associate it with common cellular and molecular defects identified in neurodevelopmental and neurodegenerative disorders. First, we describe the distinct role and organization of the GC in neurons, where it participates of relevant developmental processes such as neuronal polarity and migration. Afterward, we discuss the role of Golgins in CNS development and disease. We then address the convergence of well-known neurological disorders with abnormal GC architecture observed therein. Finally, we link all cellular pathways discussed in the previous sections with known cellular outcomes of Reelin signaling activation, a cell signaling pathway consistently associated with CNS development that includes neuronal polarity and Golgi/cytoskeletal dynamics (Santana and Marzolo, 2017).

#### ARCHITECTURE AND FUNCTION OF THE GC IN NEURONS

The role of the GC in polarized cells such as neurons is particularly challenging given the differential protein composition of each neuronal compartment and the long distances that newly synthesized and modified proteins need to traverse to get to their subcellular destinations (Bentley and Banker, 2016). The polarized distribution of proteins in the dendritic and axonal compartments is essential to maintaining neuronal homeostasis, which is highly relevant for neuronal chemical and electrical communication throughout the CNS (Bentley and Banker, 2016). Furthermore, during CNS development, neuronal polarization is also essential, as neuronal migration and positioning within the developing brain ultimately impact neuronal connectivity (Bentley and Banker, 2016). One of the first relevant processes in neuronal polarization is evidenced by the change in localization and polarity of the GC, in a highly regulated process that is essential for axonal specification and dendritic growth. The Serine/Threonine Kinase 25 (STK25) is a crucial mediator of GC polarity; STK25 downregulation inhibits axonal specification in hippocampal neurons, and conversely, overexpression of STK25 leads to the development of multiple axons (Santana and Marzolo, 2017). Golgi morphology is also significantly disrupted in STK25 silenced neurons, consistent with loss of dendritic polarity of the GC (Matsuki et al., 2010). STK25 expression is also a determinant for establishing neuronal polarity and appropriate migration in developing mouse neocortex and hippocampus (Matsuki et al., 2013; Rao et al., 2018). Additionally, the knockdown of STK25-interacting partners, the pseudokinase STE20-Related Kinase Adaptor (STRAD), Liver kinase B1 (LKB1) and Golgi matrix protein 130 kDa (GM130) reproduces such polarity and migration deficits (Matsuki et al., 2010, 2013; Orlova et al., 2010), suggesting a pivotal role for the STK25-STRAD-GM130-LKB1 complex, in GC polarity and neuronal development.

As Golgi positioning determines neuronal polarity, dendritic, and axonal development show differential reliance on secretory trafficking (Ye et al., 2007). For instance, limiting ER-to-Golgi trafficking decreases membrane supply toward the dendrites without affecting the axon (Ye et al., 2007). More importantly, secretory membrane trafficking and dendritic development are highly dependent on discrete units of ER and GC located within the dendritic compartment which fulfill essential functions in protein synthesis, maturation and sorting to target membranes (Hanus and Ehlers, 2008).

Endoplasmic reticulum-related structures referred to as ER exit sites (ERES) and ER-Golgi intermediate compartments (ERGICs) are widely distributed throughout the soma and dendritic compartment, in stark contrast to discrete Golgi units referred to as Golgi outposts (GOPs) which are mostly found in proximal dendrites of cultured hippocampal neurons and in apical dendrites of pyramidal neurons in vivo (Ramírez and Couve, 2011). Several protein markers forcis-, medial-, and trans-Golgi have been identified in GOPs. However, their abundance and colocalization with one another are highly dynamic, suggesting that GOPs form by discrete Golgi compartments which only transiently interact with one another (Zhou et al., 2014), distinct from the classical GC ribbon morphology observed in non-polarized cells or neuronal soma.

Dendrites containing GOPs exhibit higher complexity compared to the ones lacking these structures. Furthermore, dendritic polarization of the somatic Golgi precedes the increase of dendritic complexity (Horton et al., 2005). The frequent location of GOPs at dendritic branch points, in conjunction with time-lapse analysis, showed that dendritic branches dynamically extend as GOPs move toward a developing

branch (Ye et al., 2007). Finally, disruption of Golgi polarity by overexpression of Golgi reassembly-stacking protein of 65 kDa (GRASP65), a binding partner of GM130, leads to loss of dendritic arbor polarity and uniform growth of dendritic branches (Horton et al., 2005).

Golgi outposts are not only important for dendritic development; in fact, proteins associated with synaptic function also traffic through these distal GC structures. For instance, the kainate receptor GluK2 readily colocalizes to GOPs 30 min after synchronous release from the ER, and activation of protein kinase C (PKC) reduces GluK2 ER exit (Evans et al., 2017). In contrast, PKC-dependent phosphorylation of synaptic scaffold synapse-associated protein 97 (SAP97) protein is necessary for interacting with ADAM10 and facilitating its exit from GOPs into the synaptic compartment (Saraceno et al., 2014). Trafficking of N-methyl-D-aspartate (NMDA) receptors also bypasses somatic GC and localizes to GOPs where it colocalizes with SAP97 (Jeyifous et al., 2009). Finally, specialized GOPs enriched in glycosylation machinery and positive for the TGN resident protein calneuron-2 (Mikhaylova et al., 2009) referred to as Golgi satellites (GS), have been described to be widely distributed in the dendritic arbor and only occasionally associated with GC cisternae markers (Mikhaylova et al., 2016). Interestingly, the motility of GS is substantially reduced upon treatment with the GABA receptor antagonist bicuculine and with BDNF treatment, suggesting a regulatory role for these GS in activity-dependent neuronal plasticity (Mikhaylova et al., 2016). Indeed, GS colocalized with synaptic transmembrane proteins including synaptic cell adhesion proteins Neuroligin-1 and Neuroplastin-55, synaptic scaffold protein Homer1 and ionotropic receptor subunit GluA1 (Mikhaylova et al., 2016). The role of GOPs in synaptic plasticity has been controversial as they are mostly found in the largest dendrite of pyramidal neurons; however, the description of specialized and widely distributed GS which readily colocalize with synaptic components provides a fresh impulse to this idea and warrants further investigation.

Despite the pivotal role of GOPs in neuronal function and development, we know little about their biogenesis. Livecell imaging experiments have shown that GOP biogenesis at apical dendrites requires GC deployment from the soma and subsequent tubule fission which involves the activation of GClocalized RhoA GTPase and downstream effectors, LIM motifcontaining protein kinase 1 (LIMK1) and cofilin (Quassollo et al., 2015). These data suggest that GOPs generate from an existing GC cisterna rather than synthesized de novo at the dendritic compartment and depends on the regulation of the actin cytoskeleton. More recently GOPs biogenesis was determined to be highly dependent on the secretory pathway, as silencing of Sec23 and Sec31, components of the COPII complex mediating anterograde transport from the ER to the cis-Golgi (Fromme et al., 2008), decreases the number of GOPs in Drosophila Dendritic arbor (Da) neurons (Chung et al., 2017). Moreover, expression of these subunits is enhanced by CrebA expression (CREB3L in mammals), a master transcriptional regulator of secretory trafficking-associated genes (Fox et al., 2010). Accordingly, CrebA overexpression in Drosophila Da neurons increased GOP abundance in dendrites (Chung et al., 2017).

To sum up, the highly polarized nature of neurons is highly dependent on GC positioning. Furthermore, long-range protein synthesis and transport are mediated by discrete units of GC such as GOPs and GS. Over the next sections, we will discuss how the disruption of physiological GC function directly impacts in neurological disorders.

### STRUCTURAL PROTEINS OF THE GC AND THEIR ROLE IN NEURODEVELOPMENT AND DISEASE

Golgins are a set of proteins characterized by the presence of coiled-coil domains that play a substantial role in maintaining GC morphology (Muschalik and Munro, 2018). Golgins associate with several proteins including small GTPases of the Rab and Arl families, that control their tethering function and membrane recruitment (Cheung and Pfeffer, 2016). Loss-offunction approaches targeting several Golgins have proven their role in maintaining GC architecture in cell culture systems, but little is known of their role in the development and function of the nervous system or neuropathology.

Animal knockout (KO) models for Golgins such as Giantin or GMAP 210 have shown them to be essential for healthy development, as these animals exhibit defects in craniofacial and skeletal development (Smits et al., 2010; Stevenson et al., 2017). On the other hand, KO of Golgin-84, a protein closely related to Giantin, does not show any significant developmental abnormalities, and compound mutants for both of these Golgins do not show any additional defects (McGee et al., 2017). While these models highlight the importance of Golgins in embryonic development, some Golgins might be dispensable; indeed, several Golgins are associated with human diseases (Toh and Gleeson, 2016), but only a few have been linked to obvious neurological defects.

In this regard, neuronal GM130 KO mice showed severe motor defects similar to ataxia (Liu et al., 2017). GM130 is one of the most studied Golgins and is known to maintain Golgi ribbon morphology (Lowe et al., 1998). Furthermore, GM130 is necessary for maintaining the interaction of multi-cisternae structures within distal dendrites in Drosophila (Zhou et al., 2014). GM130 KO in mice leads to Purkinje cell degeneration in the cerebellum and impaired secretory trafficking. The latter ultimately affects dendritic growth, as previously described in Drosophila (Zhou et al., 2014; Liu et al., 2017), and these defects were both associated with Golgi fragmentation and abnormal positioning (Liu et al., 2017). On the other hand, GM130 overexpression is observed in in vitro models for mucopolysaccharidosis type IIIB (MPSIIIB) (Roy et al., 2011), a lysosomal storage disorder featuring strong neurological symptoms such as intellectual disability and progressive dementia (Kan et al., 2014). Most notably, overexpression of GM130 alone mimicked MPSIIIB cellular defects observed in HeLa cells (Roy et al., 2011). In conclusion, it appears both gain- and loss-of-function of GM130 drastically impact the morphology and function of the GC and could be a promising target for pharmacological intervention.

Mutations in the Golgin bicaudal D homolog 2 (BICD2) has been described in spinal muscular atrophy related disorders (Neveling et al., 2013). BICD2 participates in vesicular trafficking through direct interaction with dynactin and dynein motor proteins (Splinter et al., 2012) in association with active Rab6 (Cheung and Pfeffer, 2016). Overexpression of BIDC2 missense mutants affecting the coiled-coil domain, in HeLa cells, leads to severe GC fragmentation (Neveling et al., 2013). Most notably, the expression of these mutants in neurons leads to increased motility of BICD2/dynein–dynactin complexes and reduced neurite outgrowth (Huynh and Vale, 2017).

In conclusion, GC structural proteins play important roles in development and disease; however, there remains much to elucidate regarding the direct role of Golgins in inheritable neurological disorders and neurodevelopment (**Figure 1**).

#### DISRUPTION OF GC ARCHITECTURE IN NEUROLOGICAL DISORDERS

#### Alzheimer's Disease

Alzheimer's disease is a neurodegenerative disorder featuring progressive neuronal deterioration and cognitive impairment

trafficking through microtubules; mutations are related to Spinal muscular atrophy disorders. Giantin tethers COPI vesicles to the GC; the knockout animals manifest craniofacial defects. GM130 localizes in cis-Golgi and GOPs, participates in Golgi positioning and is involved in the maintenance of Golgi ribbon morphology and multi-cisternae structures; knockout animals display motor defects, cerebellar Purkinje cell degeneration, and aberrant dendritic growth, while overexpression is linked with a model of the lysosomal storage disorder Mucopolysaccharidosis type IIIB (MPSIIIB).

(Verheijen and Sleegers, 2018). It is widely accepted that AD arises from abnormal proteolytic processing of the amyloid precursor protein (APP), which leads to the generation of Aβ peptides, whose aggregation and deposition enhances neuronal cytotoxicity (Ittner and Götz, 2011). Aside from the amyloid cascade hypothesis, the appearance of neurofibrillary tangles of hyperphosphorylated Tau, a protein commonly associated with the microtubule network, is also a common histopathological observation in AD (Ittner and Götz, 2011).

Interestingly, disruption of GC architecture is observed in neuropathological processes associated with AD. There is growing interest in studying these phenomena in neurodegenerative disorders, as GC fragmentation is thought not to be the result of apoptosis or cell death associated pathways but rather precede them (Nakagomi et al., 2008).

The integrity of GC morphology regulates APP processing and has been extensively reviewed elsewhere (Joshi et al., 2015). Aβ triggers GC fragmentation through activation of cyclindependent kinase-5 (CDK5), which phosphorylates GRASP65. In turn, loss of Golgi integrity increases Aβ production by enhancing amyloidogenic protein cleavage (Joshi et al., 2014). Interestingly, activation of CDK5 by itself increases GC fragmentation through direct phosphorylation of GM130 (Sun et al., 2008). Moreover, overactivation of this kinase is common in AD and other neurodegenerative disorders including PD and ALS (Qu et al., 2007; Shukla et al., 2012; Klinman and Holzbaur, 2015). Overexpression of GRASP65 rescues GC morphology and increases accumulation of APP in the GC compartment, which, strikingly, is accompanied by an increase in non-amyloidogenic processing (Joshi et al., 2014). Indeed, more recently, the TGN has been identified as a major site for α-secretase cleavage and inhibition of APP trafficking from the TGN has been found to increase sAPPα levels (Andersen et al., 2005; Tan and Gleeson, 2019). Thus, GC integrity regulates proteolytic cleavage of APP in physiological and pathological settings (**Figure 2A**).

Golgi Fragmentation is induced in cell culture by overexpression of wild type (WT) and AD-associated mutant Tau (Liazoghli et al., 2005). Besides, the presence of neurofibrillary tangles in AD patients' brains correlates with abnormal GC morphology and decreased surface area in the neocortex and hippocampus (Antón-Fernández et al., 2017). The increased neuronal activity triggered by KCl-mediated neuronal depolarization induces GC fragmentation by a CDK5 dependent mechanism (Mohamed et al., 2017). Concomitantly, KCl-induced depolarization increases secretion of Tau protein into the extracellular medium in WT neurons, which is inhibited by pharmacological blockade of CDK5, indicating that GC maintenance is a crucial regulator of Tau secretion (Mohamed et al., 2017). In this context, knockdown of the small GTPase Rab1A, which mediates ER to GC trafficking and whose deficiency is known to induce Golgi fragmentation (Haas et al., 2007), also increases Tau secretion independently of hyperexcitability (Mohamed et al., 2017). This correlation between neuronal hyperexcitability and Tau deposition is of particular interest since seizures are often comorbid with AD. Furthermore, hyperphosphorylated Tau can be detected in brains of patients with refractory epilepsy and AD patients with

seizures show an accelerated cognitive decline (Tai et al., 2016; Vossel et al., 2016).

Altogether these data highlight the importance of Golgi morphology maintenance and its overall impact on the deposition of both Aβ peptide and Tau in the extracellular medium which, in turn, may enhance the spreading of cytotoxicity throughout the brain (Pignataro and Middei, 2017; Yamada, 2017). Thus, understanding the cellular basis of GC fragmentation could inform the development of drugs to slow down AD progression.

#### Parkinson's Disease

Parkinson's Disease (PD) is the second most common neurodegenerative disorder after AD and is diagnosed based on motor abnormalities such as rigidity, bradykinesia, abnormal posture, and tremors (Błaszczyk, 2016). Determining the genetic basis for PD has been an elusive task. Nevertheless, a few genes have been linked to familial type PD including the encoding of the extensively studied α-synuclein (SCNA) and leucine-rich repeat kinase 2 (LRRK2) (Wood-Kaczmar et al., 2006).

Overexpression of α-synuclein in rat substantia nigra leads to GC fragmentation, a phenotype that is reversed by co-expression of Rab1A, which enhances ER to GC trafficking (Haas et al., 2007) (**Figure 2B**). While Rab1A expression is not able to rescue the neurodegeneration completely, it improves motor function in affected animals (Coune et al., 2011). Most notably, LRRK2 interacts and phosphorylates Rab1A, a phosphorylation event that is significantly induced by LRRK2 G2019A, a PD-associated mutant with increased kinase activity (Jeong et al., 2018). Additionally, overexpression of phospho-dead Rab1A induces neurodegeneration in cultured neurons (Jeong et al., 2018). However, despite these observations, the regulatory role of Rab1A phosphorylation remains unclear. LRRK2 also phosphorylates and directly interacts with Rab7L1, a TGN resident Rab GTPase that has also been identified as a candidate gene for PD (Fujimoto et al., 2018). LRRK2-mediated phosphorylation of Rab7L1 increases TGN fragmentation and PD-associated LRRK2 mutants demonstrate highly increased Rab7L1 phosphorylation rates (Fujimoto et al., 2018).

The retromer complex (VPS35-VPS26-VPS29), which participates in retrograde cargo retrieval from endosomes back to the TGN and recycling cargo back to the plasma membrane, has also been associated with PD (Zhang et al., 2018). Knockdown of VPS26 induces GC fragmentation (Seaman, 2004) and nonsynonymous mutations in this subunit have been reported in PD patients (Gustavsson et al., 2015). This fragmentation is reduced by downregulation of the Golgin GCC88, which finely regulates GC morphology through interaction with the actin cytoskeleton (Makhoul et al., 2019). The retromer complex also appears to have a direct correlation with LRRK2 function, for instance, the brain tissue of patients featuring LRRK2 PD-associated mutations show reduced expression of retromer complex component VPS35 (Zhao et al., 2017). More interestingly, expression of PD-associated VPS35 D620N mutant elevates phosphorylation of Rab GTPases (Namely Rab8A, Rab10, and Rab12) and increases overall kinase activity of LRRK2 to a greater degree than PD pathogenic mutations (Mir et al., 2018).

Overall, these data point to an extensive regulatory network where proper functioning of LRRK2 kinase activity can modulate traffic between the GC and the ER through regulation of Rab GTPases. Furthermore, retrograde trafficking toward the GC in association with the Retromer complex is also affected by LRRK2 activity, thus regulating membrane protein availability and affecting the overall organellar organization.

Overexpression of LRRK2 G2019A is known to reduce dendritic arborization (Lin et al., 2010), which suggests that this protein might play a role in GOP biogenesis or function. Lrrk (Drosophila homolog of LRRK2) colocalizes with medial Golgi marker α-mannosidase II-GFP compartments in both the soma and dendrites of Drosophila Da neurons (Lin et al., 2015). Notably, Lrrk-positive GOPs are mostly stationary while other GOPs exhibit dynamic anterograde and retrograde movement within dendrites (Lin et al., 2015). The direct interaction of Lrrk with Lva, a golgin in Drosophila with no known orthologs (Sisson et al., 2000), prevents the association of GOPs to dynein–dynactin complexes, thus reducing motility. Consistently, decreased expression of Lrrk reduced GOPs anterograde movement and increased dendritic development (Lin et al., 2015). Remarkably, expression of LRRK2 G2019A is associated with an enhancement of retrograde GOPs movement, which is consistent with a suppressed dendritic arborization seen in neurons expressing this variant (Lin et al., 2015). In conclusion, LRRK2 function not only modulates GC structure but also plays an essential role in regulating GOPs positioning in maturing dendrites, which could underlie dendrite degeneration seen in animal models expressing mutant LRRK2.

The dysregulation of LRRK2 has been extensively studied and directly linked to mitochondrial dysfunction, oxidative stress, and synaptic dysfunction among others (Li et al., 2014). On the other hand, over this section, we have enlisted a wide array of protein interactors and phosphorylation targets which point out to an LRRK2 signaling hub residing at the GC that can directly impact on the organelle dynamics.

#### Huntington's Disease

Huntington's disease (HD) is a neurodegenerative disorder characterized by progressive motor and cognitive impairment (Munoz-Sanjuan and Bates, 2011). It is genetically linked to an abnormal CAG repeat expansion in the first exon of HTT, a gene encoding the protein huntingtin (HTT), which participates in several cellular pathways including secretory and endosomal trafficking, transcriptional regulation, autophagy, and ciliogenesis (Saudou and Humbert, 2016). CAG repeats result in a poly-glutamine extension in the translated protein product, which is prone to aggregation and generates cellular toxicity (Labbadia and Morimoto, 2013). HD is one of several so-called poly-Q diseases, each one associated with a different gene locus but sharing similar pathological outcomes (Nath and Lieberman, 2017).

The Golgi adaptor acyl-coenzyme A binding domain protein 3 (ACBD3) is a GC resident protein which interacts directly with giantin and regulates GC morphology (Sohda et al., 2001). ACBD3 is overexpressed in the striatum of HD patients and in

HD model mice where it interacts directly with mutant HTT (**Figure 2C**). The treatment of Q7 and Q111 striatal cell lines with monensin, an ionophore that disrupts acidic organelles including the GC and lysosomes used to model the Golgi stress response, increases ACBD3 levels in both cell lines with a greater induction in Q111, the cell line with higher polyglutamine repeats in HTT (Sbodio et al., 2013).

ACBD3 has also been implicated in glycosphingolipid synthesis in the GC through direct interaction with the transporter of glucosylceramide precursors, human four phosphate adaptor protein 2 (FAPP2) (Liao et al., 2018). Knockdown of ACBD3 leads to mislocalization of FAPP2 from the TGN to the cytoplasm, fragmenting the GC and severely affecting cellular lipidic profile (Liao et al., 2018). Glycosphingolipid metabolism has received considerable attention regarding neurological diseases such as HD, ALS, hereditary spastic paraplegia, lysosomal storage diseases, among others (Desplats et al., 2007; Robert et al., 2009; Boukhris et al., 2013; Dodge et al., 2015).

Metabolic stress related to amino acid deprivation is associated with HD. Low levels of cysteine γ-lyase (CSE), the enzyme directing cysteine biosynthesis has been documented in HD. Cysteine depletion triggers a stress response that enhances ATF4 expression, which is also reduced in HD and ultimately leads to increased oxidative stress and cytotoxicity (Sbodio et al., 2016). ATF4 is a stress-induced transcription factor that directs the expression of adaptative genes necessary to withstand stressors such as hypoxia, amino acid deprivation and organelle stress (Wortel et al., 2017). Q111 cells are highly sensitive to low cysteine medium and show reduced expression of both ATF4 and CSE. However, pre-treatment with low dose monensin rescues these expression levels and increases cell survival rates relative to control Q7 cells, without affecting GC morphology (Sbodio et al., 2018). Neurodegeneration in HD is a result of multiple stress pathways which can be successfully modulated with monensin and are also related to the expression of GC structural proteins, thus suggesting an important role for GC integrity in HD.

More recently, expression of poly-Q-containing Machado-Joseph's protein associated with spinocerebellar ataxia type 3 (Matos et al., 2018) in Da neurons led to reduced dendritic branching and severe reduction of Mannosidase II-EGP positive GOPs in dendrites (Chung et al., 2017). This effect correlates with diminished expression of genes linked to the secretory pathway, including a master transcriptional regulator of secretory trafficking genes, CrebA (Fox et al., 2010; Chung et al., 2017). Indeed, upregulation of CrebA rescues GOP deficiency in poly-Q expressing Da Neurons (Chung et al., 2017). Nuclear localization of poly-Q containing proteins is known to disrupt normal transcriptional programming and perturb nuclear protein turnover (Mohan et al., 2014). However, its potential role in regulating secretory trafficking in neurons through transcriptional repression opens a new door for understanding the cellular pathways involved in poly-Q diseases.

As described above, HD and poly-Q diseases feature a wide array of pathological pathways ranging from metabolic regulation toward basic transcriptional activity issues. Nevertheless, the convergence of these pathways toward GC dynamics regulation offers a novel approach for understanding and ultimately treat these neurodegenerative disorders.

#### Epilepsy

Epilepsy is a condition associated with recurrent, unprovoked seizures which result from a hypersynchronous discharge of neurons in the brain (Stafstrom and Carmant, 2015). The delicate balance between inhibitory and excitatory transmission is often disrupted, and most of the genes associated with epilepsy are known to regulate neuronal connectivity, synaptic discharge, and signaling, as well as ion channel function and trafficking (Meisler and Kearney, 2005; Poduri et al., 2013; Maljevic et al., 2019).

Neuronal hyperexcitability has been shown to regulate the GC, as chronic exposure to slightly elevated concentrations of KCl (5 nM) inducing neuronal depolarization for 2 days leads to increased GC fragmentation (Thayer et al., 2013). Additionally, pharmacological hyperactivation of neuronal activity through treatment with bicuculine or APV, GABAA, and NMDA receptor antagonists induces Golgi fragmentation (Thayer et al., 2013) (**Figure 2D**). Remarkably these changes in GC architecture could be reversed after returning neurons to the normal culture medium (Thayer et al., 2013), showing that GC structure is highly dynamic and remodels itself upon external pharmacological signals. Bicuculine-induced Golgi fragmentation can be prevented by pre-treatment with Ca2+/calmodulin-dependent protein kinase II/IV (CAMKII/IV) inhibitors, suggesting a high dependence of calcium signaling pathways in activity-induced GC fragmentation (Thayer et al., 2013). Several voltage-gated calcium channels associate with epilepsy (Rajakulendran and Hanna, 2016) and calcium signaling mediated by CAMKII has been consistently associated with neurological disease including epilepsy, schizophrenia, and autism spectrum disorders (Robison, 2014). Calcium homeostasis also relies on GC integrity in concert with ER and mitochondrial reservoirs (Lissandron et al., 2010; Yang et al., 2015). However, little is known of the role of GC calcium storage in cellular homeostasis or disease.

Golgi positioning defects could also play a role in the onset of seizure disorders, as the proliferation, migration, and differentiation of neural progenitors are ultimately responsible for shaping neuronal connectivity in the developing brain. Mutations in STRAD, a core protein in the Golgi positioning complex, were identified through microarray analysis in patients with polyhydramnios megalencephaly and symptomatic epilepsy (PMSE) syndrome (Puffenberger et al., 2007). Expression of a PMSE-associated 180-amino acid, C-terminal truncating mutation of STRAD in adult neuronal progenitor cells leads to the disassembly of the GC, reduced expression of GM130 and severe defects in dendritic development (Rao et al., 2018). The reported defects in dendritic growth were similar to the ones observed in STK25-deficient neurons. Furthermore, the knockdown of GM130 in adult neuronal progenitors reproduced this observation (Rao et al., 2018), suggesting

an underlying role of GC integrity in the onset of this rare neurodevelopmental disorder.

The mammalian target of rapamycin (mTOR)-related signaling pathway has acquired increasing importance in seizure disorders, as somatic mutations of upstream and downstream signaling components, underlie cortical lamination deficits and the onset of focal cortical dysplasia and hemimegalencephaly, both neurodevelopmental disorders associated with seizures (Poduri et al., 2013). Most notably, a recent study pointed out that GC fragmentation associated with slight overexpression of Golgin GCC88 decreases mTOR activity and increases autophagy in HeLa cells (Gosavi et al., 2018), suggesting that disruption of GC morphology could lead to reduced mTOR activity in neurological disorders. Conversely, human PMSE brains and brains from STRAD knockdown mice show an enhanced activity of mTORC1 and cortical heterotopias resembling mTORC1 deficient lamination defects (Orlova et al., 2010). Increased mTORC1 activity was associated with LKB1 mislocalization to the nucleus (Orlova et al., 2010), a protein kinase which inhibits mTOR activity through activation of AMPK (Shaw et al., 2004).

 Moreover, the availability of several pharmacological and genetic approaches both in vivo and in vitro can often lead to conflicting interpretations; however, we can conclude that both approaches ultimately lead to GC fragmentation associated to increased synaptic activity. Additionally, the direct role of the GC in regulating mTOR activation, a protein kinase consistently associated with epilepsy, gives us new insight for treating so-called "mTORopathies" (Crino, 2015). In this context, mTOR inhibitor, rapamycin has been successfully used to attenuate seizures and cortical dysplasia in animal models featuring enhanced mTOR activity (Hsieh et al., 2016).

#### REELIN SIGNALING REGULATES GC DYNAMICS AND MORPHOLOGY

In the next section, we will analyze the role of Reelin signaling in the regulation of these processes commonly affected in neurological disorders, and how this pathway plays an important role in the structuring and dynamics of the CG. Indeed, Reelin signaling dysfunction has been documented in several neurodevelopmental and neurodegenerative disorders including AD, PD, HD, and epilepsy (Haas et al., 2002; McCullough et al., 2012; Bodea et al., 2014; Baek et al., 2015; Cuchillo-Ibanez et al., 2016). Furthermore, Reelin signaling plays a central role in regulating neuronal migration and polarity during development and also regulates neuronal plasticity in the adult brain (Santana and Marzolo, 2017; Wasser and Herz, 2017).

In this context, Reelin was first described as a protein absent in the mouse mutant reeler which features defective neuronal migration in the neocortex and hippocampus, resulting in the inversion of cortical layering (Frotscher, 2010). Most notably, reeler recapitulates phenotypes observed in GM130 KO mice (Liu et al., 2017) including ataxia and cerebellar atrophy (Yuasa et al., 1993; Magdaleno et al., 2002) suggesting that Reelin participates in pathways associated with Golgi structural regulation during early brain development.

Reelin is a large, secreted glycoprotein that binds to the ApoE receptor 2 (ApoER2) and/or very low density lipoprotein receptor (VLDLR) leading to Src-family kinases Fyn- and Src-dependent tyrosine phosphorylation of the adaptor protein Dab1, which in turn regulates several downstream pathways, including PI3K-Rac1/Cdc42, PI3K-AKT-GSK3β, and Crk/CrkL-Rap1 among others (**Figure 3**) (Santana and Marzolo, 2017).

Through the PI3K-Cdc42/Rac1 pathway, Reelin is an upstream activator of LIMK1 and cofilin, which, as previously discussed, participate in GC tubule fragmentation (Quassollo et al., 2015). However, it is not yet known if Reelin induces the formation of GOPs. What has been established is that Reelin signaling plays a role in GC deployment and positioning (**Figure 3**). The GC appears condensed near the nucleus in Dab1 and Reelin KO neocortical pyramidal neurons as opposed to extended toward the apical layer in control neurons (Matsuki et al., 2010). Strikingly, acute treatment with Reelinconditioned medium in cultured hippocampal neurons leads to rapid deployment of the GC into the largest neurite. Furthermore, GC deployment is significantly reduced by expression of STK25, suggesting opposing roles for Reelin- and STK25 mediated GC modification (Matsuki et al., 2010). In nocodazoletreated hippocampal neurons, Reelin specifically promotes the reconstitution of disrupted dendritic GC, but not somatic GC. Reelin also induces the formation of plus-end dynamic microtubules labeled with the microtubule plus end binding protein 3 (EB3) (Meseke et al., 2013a). Other studies have shown that the GTPases Rac1 and Cdc42 (via its GEF aPIX) are involved in GC translocation and deployment (Jossin and Cooper, 2011; Meseke et al., 2013b). Cdc42 is found in the plasma membrane and the GC where it is known to participate in determining cell polarity and regulation of the actin cytoskeleton. Activation of Cdc42 also leads to reducing retrograde transport from the GC and increased intra-GC anterograde trafficking via COPI vesicles (Farhan and Hsu, 2016). It remains unclear whether Reelin activation of Cdc42 impacts the processes of cisternal maturation, and retrograde or anterograde trafficking.

More recently, Reelin signaling modulation of the microtubule nucleating protein CLASP2 has further strengthened the role of this pathway in the regulation of neuronal cell polarity and migration through regulation of GC morphology (Dillon et al., 2017). CLASP proteins are found in the GC and act as acentrosomal nucleation sites for the microtubule network (Efimov et al., 2007). CLASP2 associates with the TGN resident Golgin GCC185, a protein known to be necessary for maintaining ribbon formation of the Golgi stack (Brown et al., 2011). The overexpression of CLASP2 enhances dendritic branching and Golgi condensation (Beffert et al., 2012), thus supporting an important role for CLASP2 in GC dynamics. In line with this evidence, CLASP2 function has been consistently associated with dendritic and axonal development, synaptic function, neuronal progenitor proliferation, and migration (Beffert et al., 2012; Dillon et al., 2017).

releasing it from GCC185. Unphosphorylated CLASP2 can bind pDab1 and GCC185, not bound to the microtubules, leads to dismantling of Golgi ribbon morphology. pDab1 via PI3K and ITSN activates Cdc42. The Cdc42/Rac1 GEF αPix/Cool2 has been involved in Reelin-induced Golgi translocation after axon specification (not shown in the figure) and deployment to the apical dendrite. (2) Active Cdc42 and Rac1 induce the positioning of microtubule-associated Golgi vesicles possibly via regulation that involves dynein and microtubule-associated Lis1, necessary for Golgi translocation and deployment. Besides, Reelin signaling promotes the formation of dynamic plus end microtubules labeled with EB3. (3) Also, Cdc42/Rac1 activate LIMK1 that phosphorylates and inactivates cofilin, leading to actin fiber formation, required for Golgi deployment and possibly GOPs formation. (4) Furthermore, active Cdc42 could regulate Golgi transport by reducing COPI retrograde trafficking and promoting tubular Golgi formation at the TGN. (5) Src also phosphorylates NMDAR, increasing Ca2+ influx and activating CAMKII, which phosphorylates CREB and, together with CBP regulates gene expression, thus, conceivably inducing genes to promote enhanced secretion and possibly GOP biogenesis.

CLASP2 directly interacts with Dab1 and triggers phosphorylation by GSK3β (Dillon et al., 2017). CLASP2 features multiple GSK3β phosphorylation sites, allowing for a delicate tuning of microtubule binding activity which differentially affects dendritic and axonal growth (Hur et al., 2011). Furthermore, Reelin signaling increases inhibitory phosphorylation of GSK3β mediated by AKT (Beffert et al., 2002), adding to a complex regulation of microtubules through regulation of this kinase. GSK3β can be found in the TGN of HeLa cells, and its depletion causes Golgi fragmentation and deregulation of anterograde trafficking, diverting the cation-independent mannose-6-phosphate receptor, CI-M6PR, from transport to the pre-lysosomal compartment into the exocytic pathway (Adachi et al., 2010). GSK3β localization at the GC is dependent on TGN Golgin p230 and also determines CLASP2 phosphorylation status and localization, as GSK3β

depletion leads to increased peripheral localization of CLASP2 (Adachi et al., 2010).

An exciting and recently described Reelin signaling modulator is the multi-scaffold protein Intersectin (ITSN), which acts as a guanine nucleotide exchange factor for Cdc42 and also functions as an essential scaffold for endocytic and exocytic processes at the pre-synapse, making it an interesting protein to study in pathological conditions associated with neurotransmitter release such as epilepsies (Gerth et al., 2017). Pharmacological inhibition of the interaction between ITSN and Cdc42 reduces GC motility and disrupts GC organization through modification of the actin cytoskeleton (Friesland et al., 2013). ITSN1 also interacts directly with Golgin GCC88 in HeLa cells and localizes to the TGN. Most notably, GCC88-induced GC fragmentation is significantly reduced in ITSN1-silenced HeLa cells, further suggesting a role for this protein in actin regulation (Makhoul et al., 2019).

ITSN1 and ITSN2 interact directly with VLDLR and Dab1, and Reelin-mediated Dab1 phosphorylation is significantly reduced in ITSN1 KO neurons abrogating Reelin-mediated long term potentiation (LTP) enhancement (Jakob et al., 2017) thus directly affecting learning and memory processes (Nicoll, 2017). Moreover, double KO of ApoER2 and ITSN1 show laminar defects reminiscent of ApoER2/VLDLR double KO (Jakob et al., 2017). While these findings directly implicate ITSN in Reelin signal transduction associated with neuronal migration and plasticity, the recent description of ITSN residing at the TGN opens the possibility for novel functions of this protein in Reelin-associated GC regulation. In conclusion, Reelin signaling regulates both the actin and microtubule cytoskeleton and in turn these accessory proteins directly impact on Reelin pathway activation.

Throughout this article, we have also argued for an essential role of neuronal activity-dependent modulation of GC architecture. Hyperexcitability is a major driver of GC fragmentation and may account for common morphological GC features observed in neuropathological conditions (Thayer et al., 2013). It is also interesting that increased synaptic activity modulates Reelin signaling. As already mentioned, it is wellestablished that Reelin signaling enhances LTP in hippocampal slices through binding of ApoER2 and increased recruitment of AMPAR to the cell surface (Weeber et al., 2002; Beffert et al., 2005; Qiu et al., 2006) and downstream activation of cAMP response element-binding protein (CREB) transcriptional activator (Telese et al., 2015). More recently, stimulation of synaptic activity by a short incubation with 10 mM KCl was shown to induce dendritic clustering of ApoER2 in hippocampal neurons without modifying its total expression (Pfennig et al., 2017). Increased activity also enhances Dab1 phosphorylation and localization at synaptic sites (Pfennig et al., 2017) suggesting that alterations in synaptic activity modulate Reelin signaling activation through ApoER2.

On the other hand, the secretion of Reelin itself appears independent of calcium-mediated exocytosis that is modulated by synaptic activity, although it is sensitive to brefeldin A, suggesting that this process requires a fully functioning GC (Lacor et al., 2000). Interestingly, the incubation of neurons with kainic acid, as an in vitro model for epileptic seizures, leads to GC dispersion and decreased proteolytic processing of Reelin evidenced by an increase in the full length 400 kDa protein and a decrease in the 320 kDa secreted Reelin. Furthermore, the glycosylation level of the 320 and 400 kDa secreted Reelin is significantly decreased in kainic acid-treated neurons (Kaneko et al., 2016). Altogether, these data suggest, that Reelin post-translational modifications and secretion might be sensitive to glutamate and kainate receptor agonists, and further suggests a regulatory role for neuronal excitability in Reelin pathway signal transduction.

Overall, Reelin signaling appears to impact GC dynamics during development directly and might play a role in the interplay between cell signaling and activity-dependent plasticity which also regulates the GC. Nevertheless, how Reelin signaling directly impacts secretory trafficking remains to be determined. In this regard, a few clues can be found through the examination of transcriptional regulatory pathways. Several lines of evidence point out that efficient secretory pathway functioning is essential for the establishment of GOPs in dendrites. Indeed, overexpression of CrebA in Da neurons increased the number of GOPs by upregulating genes associated with ER-Golgi anterograde trafficking (Chung et al., 2017). Most notably, CREBbinding protein (CBP) has been shown to interact directly with CrebA in Drosophila (Anderson et al., 2005) and CBP modulation also regulates GOP abundance (Chung et al., 2017).

CREB-binding protein and related transcriptional partners including CREB and p300 have been associated with Rubinstein– Taybi syndrome, a neurodevelopmental disorder within the autism spectrum (Roelfsema et al., 2005). CBP-dependent transcription is highly dependent on calcium influx, induces transcription of early genes associated with synaptic plasticity and is important for LTP in the hippocampus (Wood et al., 2005). Nevertheless, CBP also controls transcription in other cell signaling pathways. For instance, the recent identification of LRP8-Reelin-regulated neuronal enhancers for genes activated by acute treatment with Reelin showed that these are modulated by a transcriptional complex, which including CREB and CBP (Telese et al., 2015). Similarly, Reelin treatment enhances the transcription of early genes associated with neuronal plasticity in cortical neurons (Telese et al., 2015). While gene ontology analysis of Reelin-induced genes did not show enrichment in genes associated with protein trafficking, likely due to the acute nature of the stimuli, the convergence of related transcriptional activators further strengthens a possible role for Reelin signaling in the modulation of Golgi dynamics. Particularly interesting genes enhanced by Reelin treatment include the stress response effectors ATF4, PERK, and ETS2 (Telese et al., 2015). These genes are associated with transcriptional regulation induced by Golgi stress (Baumann et al., 2018; Sbodio et al., 2018), suggesting that Reelin-mediated transcriptional activation might precede major changes in Golgi protein composition and directly impact the secretory pathway.

Altogether we can conclude that Reelin signaling impacts GC dynamics through 4 distinct mechanisms: (1) The activation of LIMK1, Cdc42, and GSK3β all well-known downstream effectors of Reelin signaling. (2) The direct interaction and regulation of cytoskeletal scaffolds with core receptor components ApoER2, VLDLR, Dab1 which could suggest a structural role for these

proteins at the GC, distinct from their role as Reelin receptors at the plasma membrane. (3) The regulation of synaptic plasticity which modulated GC dynamics in both physiological and pathological conditions (4) and finally, while there is a robust description of several protein kinases that become activated upon induction of the Reelin pathway, we have only scratched the surface of the transcriptional regulatory network activating downstream. In this context, future transcriptomic and proteomic approaches could unveil new components associating Reelin signaling with GC dynamics.

### CONCLUDING REMARKS

One hundred and twenty years have passed since the Golgi complex was first described, and a formidable body of information regarding its structure, protein composition, homeostatic dynamics, and trafficking has since been elucidated. Different from other cellular pathways, our basic knowledge of the dynamic regulation of the GC and its function in protein trafficking has preceded our knowledge of how defects in this organelle's homeostasis affect health and disease. In this article, we have discussed the current information linking GC dysfunction with neurodegenerative and neurodevelopmental disorders. It is clear that GC dynamics is directly linked to common neuronal defects observed in neurological disorders including abnormal proteolytic processing, dendritic arborization, neuronal migration, and synaptic plasticity. While each of these processes encompasses a broader and more complex protein network, our review should prompt a more thorough investigation of the GC in the onset, diagnosis, and treatment of neurological disorders.

Expanding our knowledge in this area will significantly aid clinical investigation as genome-wide association studies and large-scale exome sequencing initiatives have consistently unveiled variants and protein networks directly associated with both endosomal and secretory trafficking pathways which ultimately converge in the GC (Nuytemans et al., 2013; Novarino et al., 2014; Raghavan et al., 2018; Striano and Nobile, 2018). More importantly, a large body of data directly points to a central role for cell signaling cues in the regulation of Golgi dynamics in both development and disease. Indeed, Reelin

#### REFERENCES

signaling has been implicated in neuronal polarity, migration and synaptic function, and its direct interaction with downstream effectors linked with the regulation of GC architecture places this pathway as a potential master regulator of GC biology and a pharmacological target. Indeed, chemical modulators for Cdc42, LIMK1, and GSK3β are currently available (Hong et al., 2013; Petrilli et al., 2014) and several FDA-approved GSK3β inhibitors are already being used in the treatment of neurological diseases (Caracci et al., 2016).

Finally, we have extensively discussed changes in GC architecture. However, there is little and often conflicting information about how Golgi disassembly orfragmentation affects integral trafficking of transmembrane proteins and ionotropic channels towards the cell surface or synaptic compartment. Protein translation and ER stress are well-known to be associated with neurological diseases. Here, we have comprehensively described the GC alterations in neuropathology. Future work connecting the role of the GC in neuropathology with its established participation in delivering protein cargo to organelles, endosomes and the plasma membrane, will consolidate our understanding of the secretory pathway and unveil novel cellular paradigms for understanding prevalent neurological diseases.

### AUTHOR CONTRIBUTIONS

MOC wrote and designed the first draft of the manuscript and made a relevant contribution to the general idea of the review article. LMF made the figures and wrote the legend section of the manuscript. M-PM supervised all steps of the writing process, including the structure of the article and figures, the list of references, and wrote and edited the final version of the manuscript. All authors contributed to manuscript revision read and approved the submitted version.

# FUNDING

This work was supported by the CONICYT-PCHA/National Graduate Program/2016- 21160410 to MOC and 2017-21171004 to LMF and Fondo Nacional de Ciencia y Tecnología, FONDECYT of Chile, project 1150444 to M-PM.

related to tau hyperphosphorylation. Neurobiol. Dis. 97, 11–23. doi: 10.1016/j. nbd.2016.10.005

induces neurodegeneration. Mol. Neurodegener. 13:8. doi: 10.1186/s13024-018- 0240-1

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Caracci, Fuentealba and Marzolo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Roles for Golgi Glycans in Oogenesis and Spermatogenesis

#### Ayodele Akintayo and Pamela Stanley\*

Department of Cell Biology, Albert Einstein College of Medicine, New York, NY, United States

Glycosylation of proteins by N- and O-glycans or glycosaminoglycans (GAGs) mostly begins in the endoplasmic reticulum and is further orchestrated in the Golgi compartment via the action of >100 glycosyltransferases that reside in this complex organelle. The synthesis of glycolipids occurs in the Golgi, also by resident glycosyltransferases. A defect in the glycosylation machinery may impair the functions of glycoproteins and other glycosylated molecules, and lead to a congenital disorder of glycosylation (CDG). Spermatogenesis in the male and oogenesis in the female are tightly regulated differentiation events leading to the production of functional gametes. Insights into roles for glycans in gamete production have been obtained from mutant mice following deletion or inactivation of genes that encode a glycosylation activity. In this review, we will summarize the effects of altering the synthesis of N-glycans, O-glycans, proteoglycans, glycophosphatidylinositol (GPI) anchored proteins, and glycolipids during gametogenesis in the mouse. Glycosylation genes whose deletion causes embryonic lethality have been investigated following conditional deletion using various Cre recombinase transgenes with a cell-type specific promoter. The potential effects of mutations in corresponding glycosylation genes of humans will be discussed in relation to consequences to fertility and potential for use in contraception.

Keywords: glycosylation, glycans, Golgi, spermatogenesis, oogenesis, fertility

#### INTRODUCTION

The mammalian glycome is defined by the genes that encode activities required for the synthesis of glycosylated proteins and lipids. These include genes that encode glycosyltransferases, nucleotide sugar synthases, nucleotide sugar modifiers, nucleotide sugar transporters and glycosidases required to prune glycans during synthesis. Also required for optimal glycosylation are proteins that maintain the structure and environment of the secretory pathway. A summary of the classes of glycan synthesized by mammals is given in **Figure 1**. The actual complement of glycan structures expressed in the endoplasmic reticulum (ER), in Golgi compartments, at the cell surface, or secreted by a mammalian cell will depend on the glycosylation-related genes that are active in that cell, and that spectrum will likely vary at different stages of development or differentiation. With few exceptions, all glycoproteins, proteoglycans and glycolipids are synthesized in the secretory pathway, although N-glycans and glycosylphosphatidylinositol (GPI) anchors begin their synthesis on the cytoplasmic side of the ER membrane, before an immature glycan is flipped to the luminal side. Maturation continues in the ER and Golgi compartments. Thus, effects on the structure or biochemical environment of the ER and Golgi compartments may affect glycosylation and alter the nature of the glycans produced. The glycans on mature glycoproteins and glycolipids expressed

#### Edited by:

Vladimir Lupashin, University of Arkansas for Medical Sciences, United States

#### Reviewed by:

Taroh Kinoshita, Osaka University, Japan Michael Tiemeyer, University of Georgia, United States

> \*Correspondence: Pamela Stanley<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Membrane Traffic, a section of the journal Frontiers in Cell and Developmental Biology Received: 17 March 2019

Accepted: 21 May 2019 Published: 07 June 2019

#### Citation:

Akintayo A and Stanley P (2019) Roles for Golgi Glycans in Oogenesis and Spermatogenesis. Front. Cell Dev. Biol. 7:98. doi: 10.3389/fcell.2019.00098

at the cell surface or secreted from a cell are the functional glycans involved in recognition by glycan binding proteins on other cells, and in the extracellular matrices of a tissue. Glycoprotein exceptions to synthesis in the secretory pathway are proteins modified on Ser or Thr by O-GlcNAc in the cytoplasm or nucleus (Zachara et al., 2015), glycogen that is also synthesized in the cytoplasm, and hyaluronan synthesized at the plasma membrane and extruded to the extracellular matrix (Vasconcelos et al., 2013).

A commonly used strategy to determine if a particular glycan or sugar residue is necessary for the development or differentiation of a specific cell type is to inactivate or delete a glycosylation gene. However, this has the drawback that many glycosylated molecules of different functions will be altered. To address functions for glycans in individual glycoproteins, one or more known glycosylation sites can be mutated to preclude their modification. However, relatively few glycans are added at a known amino acid, making this approach arduous, although useful once glycosylation sites have been established. Usually it is important not to eliminate glycosylation of a glycoprotein altogether since this often leads to defective folding, aggregation and degradation and/or inability to exit the ER.

This review will describe the consequences for oogenesis (**Figure 2**) and spermatogenesis (**Figure 3**) of altering glycan synthesis by targeted inactivation of glycosylation genes responsible for the synthesis of Golgi glycans in the mouse. These glycans are identified in **Figures 1**, **4**. Until recently, glycans in the ovary and testis have been investigated by immunohistochemistry and other histochemical assays and by using glycan binding proteins such as plant lectins (Lee and Damjanov, 1984; Wu et al., 1984; Lohr et al., 2010). However, the application of matrix-assisted laser desorption mass spectrometry imaging (MALDI-MSI) has begun to reveal the molecular nature of glycans along with their location in complex tissues (Drake et al., 2018).

#### GOLGI GLYCANS IMPORTANT FOR OOGENESIS IN MAMMALS

Deletion or inactivation of glycosyltransferases that function in the Golgi compartment may have profound or relatively mild effects (Stanley, 2016). For example, inactivation of the Mgat1 gene which blocks synthesis of all complex and hybrid N-glycans, leads to embryonic lethality (Ioffe and Stanley, 1994; Metzler et al., 1994). By contrast, inactivation of Mgat5, which prevents the addition of a single β1,6 branch of complex N-glycans, has much milder consequences (Dennis et al., 2002; Partridge et al., 2004). Conditional deletion of Mgat1 in oocytes using the ZP3-Cre transgene (Lewandoski et al., 1997) was used to identify roles for complex and hybrid N-glycans in differentiation of oocytes from the primordial follicle stage (**Figure 2**). Loss of MGAT1 and hybrid and complex N-glycans allowed progression of mutant oocytes to ovulation and fertilization (Shi et al., 2004). Heterozygous embryos from mutant egg and wild type sperm give rise to embryos that develop normally. Mgat1 null oocytes fertilized by Mgat1 null sperm also give Mgat1[-/-] embryos that develop to ∼E9.5 as observed in a global knockout (Ioffe and Stanley, 1994; Metzler et al., 1994). However, closer examination revealed that females with Mgat1 null oocytes have ∼25% more empty deciduae than control females, and the zona pellucida of mutant oocytes is very thin. Zona pellucida (ZP) glycoproteins lacked complex N-glycans showing that deletion of Mgat1 was efficient. While all embryos from Mgat1 null oocytes implant, not all continue to develop, suggesting that a proportion of oocytes do not give rise to fully functional embryos. Defective oogenesis became more apparent when females were stimulated to ovulate by hormonal treatment. Females producing Mgat1 null oocytes gave ∼50% fewer ovulated eggs, even though they generated equivalent numbers of antral follicles to controls. In addition, embryos arising from Mgat1 null eggs were slow in developing through the early stages of embryogenesis prior to implantation. Thus, MGAT1 and complex N-glycans (**Figure 4A**) are required for the development of fully competent oocytes and ovulated eggs. In addition, these experiments clearly demonstrated that complex or hybrid N-glycans are not required for sperm binding or fertilization. Further investigations revealed that oocytes lacking MGAT1 have aberrant development of preovulatory follicles (retarded folliculogenesis), and structural alterations of the cumulus in cumulus-oocyte complexes (COC; Williams and Stanley, 2009). The reason for sub-optimal development of oocytes that lack MGAT1 may be related to a reduction in growth factor signaling since complex N-glycans regulate the retention of growth factor receptors at the cell surface, and thereby regulate signaling duration (Boscher et al., 2011).

Whole body deletion of Man2a1 which encodes an alpha-mannosidase II that acts after MGAT1 to prepare the substrate of MGAT2 (**Figure 4A**), does not result in obvious defects in female fertility (Chui et al., 1997). Global deletion of both Man2a1 and Man2a2 (previously called alpha-mannosidase IIX) causes embryonic or perinatal lethality (Akama et al., 2006) and oogenesis in conditional mutants has not been examined. Mgat2 null mice also die perinatally (Ye and Marth, 2004) in an inbred mouse model, and only 30% of the surviving females in an outbred mouse background were fertile. However, no further description of the basis for the fertility defect, and no targeted deletion in oocytes have been reported. The sugar added after GlcNAc to complex N-glycans in the trans Golgi compartment is Gal (**Figure 4A**). Deletion of B4galt1 also causes perinatal lethality in one mouse strain (Lu et al., 1997) but not in another (Asano et al., 1997). Females lacking B4GALT1 were fertile but had a slight reduction in litter size when compared to wild type (Asano et al., 1997).

The deletion of another Golgi glycosyltransferase by ZP3-Cre that affects oogenesis gives rise to a phenotype distinct from the loss of Mgat1. The glycosyltransferase C1GALT1 (also known as T synthase) transfers Gal to GalNAc attached at Ser/Thr residues, and initiates the synthesis of core 1 and 2 O-GalNAc glycans in the Golgi (**Figures 1**, **4B**). Deletion of C1galt1 is embryonic lethal at ∼E13.5 (Xia et al., 2004). Conditional deletion by ZP3-Cre in primary follicles (**Figure 2**) is efficient and strikingly gives rise to an increase in the number of eggs and pups produced by females that lack C1GALT1 in oocytes (Williams and Stanley, 2008). The increase of ∼30–50% in ovulated eggs leads to sustained increases

FIGURE 1 | Cell Surface Glycans in Mammals. The diagram depicts one or more glycans from each class of mammalian glycan. The diagram is modified from Figure in Stanley (2016) with permission. Sugar symbols are according to the Symbol Nomenclature for Glycans (Varki et al., 2015).

in litter size. While follicular development is enhanced, there is no increase in apoptosis of mutant oocytes (Williams and Stanley, 2008). In addition, oocytes lacking C1GALT1 generate more multiple oocyte follicles (MOF) at late stages of folliculogenesis. The formation of MOFs may be due to the compromised follicular basal lamina apparent in follicles containing C1galt1 null oocytes (Christensen et al., 2015; Grasa et al., 2016). In fact, robust fusion of mutant follicles is observed in in vitro cultures containing follicles with C1galt1 mutant oocytes, whereas no MOF arose in cultured control oocytes (Christensen et al., 2017). The enhanced folliculogenesis and fertility in females with oocytes lacking C1GALT1 reflects the enhanced sensitivity

of follicles containing mutant oocytes to stimulation by follicle stimulating hormone (FSH), reduced apoptosis and altered BMP15-GDF9 signaling (Grasa et al., 2015).

Interestingly, when both Mgat1 and C1galt1 were deleted by ZP3-Cre, the results were significantly worse for folliculogenesis, ovulation and fertility than deletion of either one alone (Williams and Stanley, 2011). Removing N-glycans and core 1 and 2 O-GalNAc glycans together abolishes the high fertility phenotype observed in C1galt1 mutant females and worsens the folliculogenesis defects observed in Mgat1 null mice, resulting in an overall reduction in egg production and fertility. Nevertheless, eggs lacking both complex and hybrid N-glycans as well as core 1 and 2 O-glycans (and therefore all Gal residues on N- and O-GalNAc glycans) were fertilized, showing that all these glycans are dispensable for fertilization. However, follicle production was greatly reduced, as were the number of ovulated double mutant eggs, and females had, at most, one litter (Williams et al., 2007; Grasa et al., 2012). The extracellular matrix of the cumulus in the cumulus-oocyte complexes (COCs) with double mutant oocytes was also altered (Lo et al., 2018). Ultimately, the combined inactivation of Mgat1 and C1galt1 in oocytes led to premature ovarian failure (POF), a novel model for investigation of this syndrome in human premature ovarian insufficiency (POI; Williams and Stanley, 2011; Grasa et al., 2016). In vitro culture of double mutant follicles has shown that a subset retains the potential to develop to antral follicles in vitro if obtained prior to the development of POF (Kaune et al., 2017). In summary, the generation of complex or hybrid N-glycans as well as core 1 and 2 O-GalNAc glycans in the Golgi is essential for fully functional oogenesis and the production of a complete complement of developmentally-competent eggs and embryos.

Another class of O-glycans is found only on Ser/Thr of epidermal growth factor-like (EGF) repeats at specific consensus sequences (**Figure 1**; Haltiwanger et al., 2017). Notch receptors and canonical Notch ligands are highly modified by these O-glycans because of the large number of EGF repeats in their extracellular domains. O-fucose, O-glucose and O-GlcNAc residues are transferred to proteins with EGF repeats in the ER by POFUT1, POGLUT1 and EOGT respectively, and further elongated in Golgi compartments (Takeuchi and Haltiwanger, 2014). Deletion of the initiating glycosyltransferase genes is embryonic lethal for O-fucose and O-glucose glycans but not for O-GlcNAc glycans (Varshney and Stanley, 2018). Female mice lacking secretory pathway O-GlcNAc glycans are fertile. Surprisingly, females with ZP3-Cre-mediated deletion of Pofut1 leading to the loss of all O-fucose glycans in oocytes, also have normal fertility, and thus apparently normal oogenesis (Shi et al., 2005), despite proposed roles for Notch signaling in oogenesis (Xu and Gridley, 2013; Feng et al., 2014). By contrast, the global deletion of Lfng, a Golgi GlcNAc-transferase which adds GlcNAc to O-fucose-modified EGF repeats, leads to infertility in female mice due to defective follicular development and meiotic maturation of oocytes (Hahn et al., 2005). Because the loss of O-fucose on EGF repeats has no effect on female fertility, the Lfng null phenotype must not be due to defect(s) in oocytes but in some other cell(s) required for normal oogenesis (**Figure 4E**). No effects on female fertility were reported in mice lacking the related GlcNAc-transferases Mfng or Rfng (Moran et al., 2009).

Only in a few cases have specific roles in oogenesis and/or female fertility been reported for mice with disrupted synthesis of glycosaminoglycans (GAG), glycosylphosphatidylinositol (GPI) anchors or glycolipids. While female mice lacking dermatan-4-O-sulfotransferase 1 (CHST14) are fertile when crossed with wild type males and Chst14 null males are also fertile when crossed to wild type females, homozygous null females crossed with homozygous null males give no progeny (Akyuz et al., 2013; **Figure 4F**). GAGs as well as glycolipids and other glycans of glycoproteins are main components of the extracellular matrix and, it is known that the composition and structure of the extracellular matrix is critical for the development of follicles and eggs in the ovary (Nagyova, 2018). The extracellular matrix and COC are significantly compromised in mice with oocytes lacking MGAT1 (Williams and Stanley, 2009) or both MGAT1 and C1GALT1 (Lo et al., 2018). ZP3 promoter-directed conditional deletion of Piga, the enzyme that initiates GPI-anchor synthesis in the endoplasmic reticulum (ER), gives rise to females in which folliculogenesis and egg production appear normal but mutant eggs lacking PIGA and thus GPI-anchored proteins at the cell surface are unable to fuse with sperm (Alfieri et al., 2003). One reason for this may be the absence of the GPI-anchored protein JUNO at the egg membrane. JUNO is essential for sperm binding to the egg via its ligand Izumo1 (Bianchi et al., 2014).

Despite the indications from mouse mutants that fertility would be expected to be affected in women with altered

glycosylation, a literature search did not reveal known genetic bases associated with glycosylation genes that correlate with defective oogenesis, fertility or POI in humans. Nor do the results in mice to date suggest that disruption of glycosylation would be an effective female contraceptive.

#### GOLGI GLYCANS IMPORTANT FOR SPERMATOGENESIS IN MAMMALS