Association of poly(rC)-binding protein-2 with sideroflexin-3 through TOM20 as an iron entry pathway to mitochondria.

Iron is essential for all the lives and mitochondria integrate iron into heme and Fe-S clusters for diverse use as cofactors. Here, we screened mitochondrial proteins in KU812 human chronic myelogenous leukemia cells by glutathione S-transferase pulldown assay with PCBP2 to identify mitochondrial receptors for PCBP2, a major cytosolic Fe(II) chaperone. LC-MS analyses identified TOM20, sideroflexin-3 (SFXN3), SFXN1 and TOM70 in the affinity-score sequence. Stimulated emission depletion microscopy and proteinase-K digestion of mitochondria in HeLa cells revealed that TOM20 is located in the outer membrane of mitochondria whereas SFXN3 is located in the inner membrane. Although direct association was not observed between PCBP2 and SFXN3 with co-immunoprecipitation, proximity ligation assay demonstrated proximal localization of PCBP2 with TOM20 and there was a direct binding between TOM20 and SFXN3. Single knockdown either of PCBP2 and SFXN3 in K562 leukemia cells significantly decreased mitochondrial catalytic Fe(II) and mitochondrial maximal respiration. SFXN3 but not MFRN1 knockout (KO) in mouse embryonic fibroblasts decreased FBXL5 and heme oxygenase-1 (HO-1) but increased transferrin uptake and induced ferritin, indicating that mitochondrial iron entry through SFXN3 is distinct. MFRN1 KO revealed more intense mitochondrial Fe(II) deficiency than SFXN3 KO. Insufficient mitochondrial heme synthesis was evident under iron overload both with SFXN3 and MFRN KO, which was partially reversed by HO-1 inhibitor. Conversely, SFXN3 overexpression caused cytosolic iron deficiency with mitochondrial excess Fe(II), which further sensitized HeLa cells to RSL3-induced ferroptosis. In conclusion, we discovered a novel pathway of iron entry into mitochondria from cytosol through PCBP2-TOM20-SFXN3 axis.

Protocol for the isolation of GFP-expressing ferroptosis-dependent extracellular vesicles in in vitro cell culture models.

Extracellular vesicles (EVs) are complex structures that transport various DNA, RNA, and protein. Recently, new EV secretion mechanisms have been identified through the iron regulatory system in mammalian cells. We revealed that ferroptosis increases EV secretion, which is named ferroptosis-dependent EVs (FedEVs). Here, we describe a step-by-step procedure to isolate GFP-expressing FedEVs for in vitro analysis. The FedEVs are further analyzed by imaging and flow cytometry analysis. For complete details on the use and execution of this protocol, please refer to Ito et al.1.

[An overview of iron metabolism and new insights into the role of ferritin secretion].

For approximately 4 billion years after the birth of life, iron, the fourth most abundant metal on earth, has played a critical role in redox reactions. Iron is a trace metal that is required for life activity, and no organism can survive without it. However, it has recently been discovered that iron causes life-threatening toxicity as organisms live longer. Ten years have passed since the discovery of iron-dependent cell death (ferroptosis), and the interest in iron is growing rapidly in various fields. Many diseases, such as neurodegenerative diseases and cancers, have been linked to abnormalities in iron metabolism, and completely new treatment methods based on iron regulation have been developed. In this article, we will assess the secretory mechanism ferritin, an iron storage protein that we recently discovered, as well as the role of secreted ferritin in diseases.

New iron export pathways acting via holo-ferritin secretion.

Ferritin is a spherical nanocage protein for iron storage, composed of 24 light- or heavy-polypeptide chain subunits. A single ferritin molecule can carry up to 4500 iron atoms in its core, which plays an important role in suppressing intracellular iron toxicity. Serum ferritin levels are used as a marker for the total amount of iron stored in the body. Most serum ferritin is iron-free (apo-ferritin) and it is unclear how ferritin is released from cells. Ferritin is secreted into serum via extracellular vesicles (EVs) or the secretory autophagy pathway but not via the classical endoplasmic reticulum (ER)-to-Golgi secretion pathway. We recently discovered that the level of tetraspanin CD63, a common EV marker, is post-transcriptionally regulated by the intracellular iron level and both CD63 and ferritin expression is induced by iron loading. Ferritin is incorporated into CD63(+)-EVs through the ferritin-specific autophagy adapter molecule, NCOA4, and then secreted from cells. EV production differs drastically depending on cell type and physiological conditions. Extracellular matrix detached cells express pentaspanin prominin 2 and prominin 2(+)-EVs secrete ferritin independently of NCOA4 trafficking. Ferritin is tightly bound to iron in EVs and functions as an iron-carrier protein in the extracellular environment. Cells can suppress ferroptosis by secreting holo-ferritin, which reduces intracellular iron concentration. However, this exposes the neighboring cells receiving the secreted holo-ferritin to a large excess of iron. This results in cellular toxicity through increased generation of reactive oxygen species (ROS). Here we review the machinery by which ferritin is incorporated into EVs and its role as an intercellular communication molecule.

Iron links endogenous and exogenous nanoparticles.

Current progress in biology and medical science is based on the observation at the level of nanometers via electron microscopy and computation. Of note, the size of most cells in higher species exists in a limited range from 5 to 50 µm. Recently, it was demonstrated that endogenous extracellular nanoparticles play a role in communication among various cellular types in a variety of contexts. Among them, exosomes in serum have been established as biomarkers for human diseases by analyzing the cargo molecules. No life on the earth can survive without iron. However, excess iron can be a risk for carcinogenesis in rodents and humans. Nano-sized molecules may cause unexpected bioeffects, including carcinogenesis, which is a process to establish cellular iron addiction with ferroptosis-resistance. Asbestos and carbon nanotubes are the typical examples, leading to carcinogenesis by the alteration of iron metabolism. Recently, we found that CD63, one of the representative markers of exosomes, is under the regulation of iron-responsive element/iron-regulatory protein system. This is a safe strategy to share excess iron in the form of holo-ferritin between iron-sufficient and -deficient cells. On the other hand, damaged cells may secrete holo-ferritin-loaded exosomes as in the case of macrophages in ferroptosis after asbestos exposure. These holo-ferritin-loaded exosomes can cause mutagenic DNA damage in the recipient mesothelial cells. Thus, there is an iron link between exogenous and endogenous nanoparticles, which requires further investigation for better understanding and the future applications.

Newly uncovered biochemical and functional aspects of ferritin.

Iron homeostasis is strictly regulated at both the systemic and cellular levels by complex mechanisms because of its indispensability and toxicity. Among the various iron-regulatory proteins, ferritin is the earliest discovered regulator of iron metabolism and is a molecule that safely retains excess intracellular iron in the cores of its shells. Two types of ferritin, cytosolic ferritin and mitochondrial ferritin (FTMT), have been identified in a range of organisms from plants to humans. FTMT was identified approximately 60 years after the discovery of cytosolic ferritin. Cytosolic ferritin expression is regulated in an iron-responsive manner. Recently, the molecular mechanisms of iron-dependent degradation of cytosolic ferritin or its secretion into serum have been clarified. FTMT, which shares a high degree of sequence homology with cytosolic ferritin, has distinct functions and is regulated in different ways from cytosolic ferritin. Although knowledge of the physiological role of FTMT is still incomplete, recent studies have shed light on the function and regulation of FTMT. The accumulating biological evidence of both ferritins has made it possible to deepen our knowledge about iron metabolism and its significance in diseases. In this review, we discuss the biological properties of both ferritins, focusing on their newly uncovered behaviors.

Matrigel-based organoid culture of malignant mesothelioma reproduces cisplatin sensitivity through CTR1.

Organoids are a three-dimensional (3D) culture system that simulate actual organs. Therefore, tumor organoids are expected to predict precise response to chemotherapy in patients. However, to date, few studies have studied the drug responses in organoids of malignant mesothelioma (MM). The poor prognosis of MM emphasizes the importance of establishing a protocol for generating MM-organoid for research and clinical use. Here, we established murine MM organoids from p53+/- or wild-type C57BL/6 strain by intraperitoneal injection either with crocidolite or carbon nanotube. Established MM-organoids proliferated in Matrigel as spheroids. Subcutaneous injection assays revealed that the MM-organoids mimicked actual tissue architecture and maintained the original histological features of the primary MM. RNA sequencing and pathway analyses revealed that the significant expressional differences between the 2D- and 3D-culture systems were observed in receptor tyrosine kinases, including IGF1R and EGFR, glycosylation and cholesterol/steroid metabolism. MM-organoids exhibited a more sensitive response to cisplatin through stable plasma membrane localization of a major cisplatin transporter, copper transporter 1/Slc31A1 (Ctr1) in comparison to 2D-cultures, presumably through glycosylation and lipidation. The Matrigel culture system facilitated the localization of CTR1 on the plasma membrane, which simulated the original MMs and the subcutaneous xenografts. These results suggest that the newly developed protocol for MM-organoids is useful to study strategies to overcome chemotherapy resistance to cisplatin.

Iron and liver cancer: an inseparable connection.

Iron is an essential element for all organisms. Iron-containing proteins play critical roles in cellular functions. The biological importance of iron is largely attributable to its chemical properties as a transitional metal. However, an excess of 'free' reactive iron damages the macromolecular components of cells and cellular DNA through the production of harmful free radicals. On the contrary, most of the body's excess iron is stored in the liver. Not only hereditary haemochromatosis but also some liver diseases with mild-to-moderate hepatic iron accumulation, such as chronic hepatitis C, alcoholic liver disease and nonalcoholic steatohepatitis, are associated with a high risk for liver cancer development. These findings have attracted attention to the causative and promotive roles of iron in the development of liver cancer. In the last decade, accumulating evidence regarding molecules regulating iron metabolism or iron-related cell death programmes such as ferroptosis has shed light on the relationship between hepatic iron accumulation and hepatocarcinogenesis. In this review, we briefly present the current molecular understanding of iron regulation in the liver. Next, we describe the mechanisms underlying dysregulated iron metabolism depending on the aetiology of liver diseases. Finally, we discuss the causative and promotive roles of iron in cancer development.

Ferroptosis-dependent extracellular vesicles from macrophage contribute to asbestos-induced mesothelial carcinogenesis through loading ferritin.

Asbestos-associated diseases remain a social burden worldwide. Our previous studies identified asbestos-induced iron-rich milieu for mesothelial cells with ceaseless macrophage ferroptosis. However, molecular mechanisms how this mutagenic milieu influences mesothelial cells have not been elucidated yet. Here, we propose a novel mechanism that extracellular vesicles (EVs) mediate asbestos-associated mutagenic factors to mesothelial cells. In a mice model of intraperitoneal crocidolite injection, mutagenic milieu highly expressed CD63, an exosomal marker. We then used a GFP-CD63 labeled THP-1 macrophage model exposed to crocidolite/iron, which generated EVs under ferroptotic process. We observed that MeT-5A mesothelial cells can receive and internalize these EVs. Furthermore, we comprehensively analyzed the ferroptosis-dependent EVs (FedEVs) for transported proteins and identified ferritin heavy/light chains as major components. Therefore, we inferred that FedEVs transport iron from ferroptotic macrophages to mesothelial cells. RNA sequencing revealed that the mesothelial cells receiving higher amounts of the FedEVs were mitotic, especially at the S and G2/M phases, by the use of Fucci mesothelial cells. Nuclear 8-hydroxy-2'-deoxyguanosine and γ-H2AX were significantly increased in the recipient mesothelial cells after exposure to FedEVs. Collectively, we here demonstrate a novel mechanism that FedEVs act as a key mutagenic mediator by transporting iron, which contribute to asbestos-induced mesothelial carcinogenesis.

CD63 is regulated by iron via the IRE-IRP system and is important for ferritin secretion by extracellular vesicles.

Extracellular vesicles (EVs) transfer functional molecules between cells. CD63 is a widely recognized EV marker that contributes to EV secretion from cells. However, the regulation of its expression remains largely unknown. Ferritin is a cellular iron storage protein that can also be secreted by the exosome pathway, and serum ferritin levels classically reflect body iron stores. Iron metabolism-associated proteins such as ferritin are intricately regulated by cellular iron levels via the iron responsive element-iron regulatory protein (IRE-IRP) system. Herein, we present a novel mechanism demonstrating that the expression of the EV-associated protein CD63 is under the regulation of the IRE-IRP system. We discovered a canonical IRE in the 5' untranslated region of CD63 messenger RNA that is responsible for regulating its expression in response to increased iron. Cellular iron loading caused a marked increase in CD63 expression and the secretion of CD63+ EVs from cells, which were shown to contain ferritin-H and ferritin-L. Our results demonstrate that under iron loading, intracellular ferritin is transferred via nuclear receptor coactivator 4 (NCOA4) to CD63+ EVs that are then secreted. Such iron-regulated secretion of the major iron storage protein ferritin via CD63+ EVs, is significant for understanding the local cell-to-cell exchange of ferritin and iron.

LRRK2-phosphorylated Rab10 sequesters Myosin Va with RILPL2 during ciliogenesis blockade.

Activating mutations in LRRK2 kinase causes Parkinson's disease. Pathogenic LRRK2 phosphorylates a subset of Rab GTPases and blocks ciliogenesis. Thus, defining novel phospho-Rab interacting partners is critical to our understanding of the molecular basis of LRRK2 pathogenesis. RILPL2 binds with strong preference to LRRK2-phosphorylated Rab8A and Rab10. RILPL2 is a binding partner of the motor protein and Rab effector, Myosin Va. We show here that the globular tail domain of Myosin Va also contains a high affinity binding site for LRRK2-phosphorylated Rab10. In the presence of pathogenic LRRK2, RILPL2 and MyoVa relocalize to the peri-centriolar region in a phosphoRab10-dependent manner. PhosphoRab10 retains Myosin Va over pericentriolar membranes as determined by fluorescence loss in photobleaching microscopy. Without pathogenic LRRK2, RILPL2 is not essential for ciliogenesis but RILPL2 over-expression blocks ciliogenesis in RPE cells independent of tau tubulin kinase recruitment to the mother centriole. These experiments show that LRRK2 generated-phosphoRab10 dramatically redistributes a significant fraction of Myosin Va and RILPL2 to the mother centriole in a manner that likely interferes with Myosin Va's role in ciliogenesis.

Application of a C. trachomatis expression system to identify C. pneumoniae proteins translocated into host cells.

Chlamydia pneumoniae is a Gram-negative, obligate intracellular pathogen that causes community-acquired respiratory infections. C. pneumoniae uses a cell contact-dependent type-III secretion (T3S) system to translocate pathogen effector proteins that manipulate host cellular functions. While several C. pneumoniae T3S effectors have been proposed, few have been experimentally confirmed in Chlamydia In this study, we expressed 382 C. pneumoniae genes in C. trachomatis as fusion proteins to an epitope tag derived from glycogen synthase kinase 3ß (GSK3ß) which is the target of phosphorylation by mammalian kinases. Based on the detection of the tagged C. pneumoniae protein with anti-phospho GSK3ß antibodies, we identified 49 novel C. pneumoniae-specific proteins that are translocated by C. trachomatis into the host cytoplasm and thus likely play a role as modifiers of host cellular functions. In this manner, we identified and characterized a new C. pneumoniae effector CPj0678 that recruits the host cell protein PACSIN2 to the plasma membrane. These findings indicate that C. trachomatis provides a powerful screening system to detect candidate effector proteins encoded by other pathogenic and endosymbiotic Chlamydia species.Importance Chlamydia injects numerous effector proteins into host cells to manipulate cellular functions important for bacterial survival. Based on findings in C. trachomatis, it has been proposed that between 5-10% of the C. pneumoniae genome, a related respiratory pathogen, encodes secreted effectors. However only a few C. pneumoniae effectors have been identified and experimentally validated. With the development of methods for the stable genetic transformation of C. trachomatis, it is now possible to use C. trachomatis shuttle plasmids to express and explore the function of proteins from other Chlamydia in a more relevant bacterial system. In this study, we experimentally determined that 49 C. pneumoniae-specific proteins are translocated into the host cytoplasm by Chlamydia secretion systems, and identify a novel effector required to recruit the host factor PACSIN2 to the plasma membrane during infection.

Carcinogenesis as Side Effects of Iron and Oxygen Utilization: From the Unveiled Truth toward Ultimate Bioengineering.

Evolution from the first life on earth to humans took ~3.8 billion years. During the time there have been countless struggles among the species. Mycobacterium tuberculosis was the last major uncontrollable species against the human public health worldwide. After the victory with antibiotics, cancer has become the leading cause of death since 1981 in Japan. Considering that life inevitably depends on ceaseless electron transfers through iron and oxygen, we believe that carcinogenesis is intrinsically unavoidable side effects of using iron and oxygen. Many animal models unequivocally revealed that excess iron is a risk for carcinogenesis. This is supported by a variety of human epidemiological data on cancer risk and prognosis. Cancer is basically a disease of the genome with persistently activated oncogenes and inactivated tumor suppressor genes through which iron addiction with ferroptosis-resistance is maintained. Engineering has made a great advance in the past 50 years. In particular, nanotechnology is distinct in that the size of the engineered molecules is similar to that of our biomolecules. While some nano-molecules are found carcinogenic, there are principles to avoid such carcinogenicity with a smart possibility to use nano-molecules to specifically kill cancer cells. Non-thermal plasma is another modality to fight against cancer.

Asbestos conceives Fe(II)-dependent mutagenic stromal milieu through ceaseless macrophage ferroptosis and ß-catenin induction in mesothelium.

Asbestos is still a social burden worldwide as a carcinogen causing malignant mesothelioma. Whereas recent studies suggest that local iron reduction is a preventive strategy against carcinogenesis, little is known regarding the cellular and molecular mechanisms surrounding excess iron. Here by differentially using high-risk and low-risk asbestos fibers (crocidolite and anthophyllite, respectively), we identified asbestos-induced mutagenic milieu for mesothelial cells. Rat and cell experiments revealed that phagocytosis of asbestos by macrophages results in their distinctive necrotic death; initially lysosome-depenent cell death and later ferroptosis, which increase intra- and extra-cellular catalytic Fe(II). DNA damage in mesothelial cells, as assessed by 8-hydroxy-2'-deoxyguanosine and γ-H2AX, increased after crocidolite exposure during regeneration accompanied by ß-catenin activation. Conversely, ß-catenin overexpression in mesothelial cells induced higher intracellular catalytic Fe(II) with increased G2/M cell-cycle fraction, when p16INK4A genomic loci localized more peripherally in the nucleus. Mesothelial cells after challenge of H2O2 under ß-catenin overexpression presented low p16INK4A expression with a high incidence of deletion in p16INK4A locus. Thus, crocidolite generated catalytic Fe(II)-rich mutagenic environment for mesothelial cells by necrotizing macrophages with lysosomal cell death and ferroptosis. These results suggest novel molecular strategies to prevent mesothelial carcinogenesis after asbestos exposure.

Iron loss triggers mitophagy through induction of mitochondrial ferritin.

Mitochondrial quality is controlled by the selective removal of damaged mitochondria through mitophagy. Mitophagy impairment is associated with aging and many pathological conditions. An iron loss induced by iron chelator triggers mitophagy by a yet unknown mechanism. This type of mitophagy may have therapeutic potential, since iron chelators are clinically used. Here, we aimed to clarify the mechanisms by which iron loss induces mitophagy. Deferiprone, an iron chelator, treatment resulted in the increased expression of mitochondrial ferritin (FTMT) and the localization of FTMT precursor on the mitochondrial outer membrane. Specific protein 1 and its regulator hypoxia-inducible factor 1α were necessary for deferiprone-induced increase in FTMT. FTMT specifically interacted with nuclear receptor coactivator 4, an autophagic cargo receptor. Deferiprone-induced mitophagy occurred selectively for depolarized mitochondria. Additionally, deferiprone suppressed the development of hepatocellular carcinoma (HCC) in mice by inducing mitophagy. Silencing FTMT abrogated deferiprone-induced mitophagy and suppression of HCC. These results demonstrate the mechanisms by which iron loss induces mitophagy and provide a rationale for targeting mitophagic activation as a therapeutic strategy.

The new role of poly (rC)-binding proteins as iron transport chaperones: Proteins that could couple with inter-organelle interactions to safely traffic iron.

BACKGROUND: Intracellular iron transport is mediated by iron chaperone proteins known as the poly(rC)-binding proteins (PCBPs), which were originally identified as RNA/DNA-binding molecules. SCOPE OF REVIEW: PCBPs assume a role as not only as cytosolic iron carriers, but also as regulators of iron transport and recycling. PCBP1 is involved in the iron storage pathway that involves ferritin, while PCBP2 is involved in processes that include: iron transfer from the iron importer, divalent metal ion transporter 1; iron export mediated by ferroportin-1; and heme degradation via heme oxygenase 1. MAJOR CONCLUSIONS: Both PCBP1 and PCBP2 possess iron-binding activity and form hetero/homo dimer complexes. These iron chaperones have a subset of non-redundant functions and regulate iron metabolism independently. GENERAL SIGNIFICANCE: This intracellular iron chaperone system mediated by PCBPs provide a transport "gateway" of ferrous iron that may potentially link with dynamic, inter-organelle interactions to safely traffic intracellular iron.

Ferroptosis at the crossroads of infection, aging and cancer.

Despite significant developments and persistent efforts by scientists, cancer is one of the primary causes of human death worldwide. No form of life on Earth can survive without iron, although some species can live without oxygen. Iron presents a double-edged sword. Excess iron is a risk for carcinogenesis, while its deficiency causes anemia, leading to oxygen shortage. Every cell is eventually destined to death, either through apoptosis or necrosis. Regulated necrosis is recognized in distinct forms. Ferroptosis is defined as catalytic Fe(II)-dependent regulated necrosis accompanied by lipid peroxidation. The main observation was necrosis of fibrosarcoma cells through inhibition of cystine/glutamate antiporter with erastin, which reduced intracellular cysteine and, thus, glutathione levels. Our current understanding of ferroptosis is relative abundance of iron (catalytic Fe[II]) in comparison with sulfur (sulfhydryls). Thus, either excess iron or sulfur deficiency causes ferroptosis. Cell proliferation inevitably requires iron for DNA synthesis and energy production. Carcinogenesis is a process toward iron addiction with ferroptosis resistance. Conversely, ferroptosis is associated with aging and neurodegeneration. Ferroptosis of immune cells during infection is advantageous for infectious agents, whereas ferroptosis resistance incubates carcinogenic soil as excess iron. Cancer cells are rich in catalytic Fe(II). Directing established cancer cells to ferroptosis is a novel strategy for discovering cancer therapies. Appropriate iron regulation could be a tactic to reduce and delay carcinogenesis.

How iron is handled in the course of heme catabolism: Integration of heme oxygenase with intracellular iron transport mechanisms mediated by poly (rC)-binding protein-2.

Heme and iron are essential to almost all forms of life. The strict maintenance of heme and iron homeostasis is essential to prevent cellular toxicity and the existence of systemic and intracellular regulation is fundamental. Cytosolic heme can be catabolized and detoxified by heme oxygenases (HOs). Interestingly, free heme detoxification through HOs results in the production of free ferrous iron, which can be potentially hazardous for cells. Recently, the intracellular iron chaperone, poly (rC)-binding protein 2 (PCBP2), has been identified, which can be involved in accepting iron after heme catabolism as well as intracellular iron transport. In fact, HO1, NADPH-cytochrome P450 reductase, and PCBP2 form a functional unit that integrates the catabolism of heme with the binding and transport of iron by PCBP2. In this review, we provide an overview of our understanding of the iron chaperones and discuss the mechanism how iron chaperones bind iron released during the process of heme degradation.

DMT1 and iron transport.

Many past and recent advances in the field of iron metabolism have relied upon the discovery of divalent metal transporter 1, DMT1 in 1997. DMT1 is the major iron transporter and contributes non-heme iron uptake in most types of cell. Each DMT1 isoform exhibits different expression patterns in cell-type specificity and distinct subcellular distribution, which enables cells to uptake both transferrin-bound and non-transferrin-bound irons efficiently. DMT1 expression is regulated by iron through the translational and degradation pathways to ensure iron homeostasis. It is considered that mammalian iron transporters including DMT1 cannot transport ferric iron but ferrous iron. Being reduced to ferrous state is likely to damage cells and tissues through the production of reactive oxygen species. Recently, iron chaperones have been identified, which can provide an answer to how ferrous iron is transported safely in cytosol. We summarize DMT1 expression depending on the types of cell or tissue and the function and mechanism of one of the iron chaperones, PCBP2.

A pathway for Parkinson's Disease LRRK2 kinase to block primary cilia and Sonic hedgehog signaling in the brain.

Parkinson's disease-associated LRRK2 kinase phosphorylates multiple Rab GTPases, including Rab8A and Rab10. We show here that LRRK2 kinase interferes with primary cilia formation in cultured cells, human LRRK2 G2019S iPS cells and in the cortex of LRRK2 R1441C mice. Rab10 phosphorylation strengthens its intrinsic ability to block ciliogenesis by enhancing binding to RILPL1. Importantly, the ability of LRRK2 to interfere with ciliogenesis requires both Rab10 and RILPL1 proteins. Pathogenic LRRK2 influences the ability of cells to respond to cilia-dependent, Hedgehog signaling as monitored by Gli1 transcriptional activation. Moreover, cholinergic neurons in the striatum of LRRK2 R1441C mice show decreased ciliation, which will decrease their ability to sense Sonic hedgehog in a neuro-protective circuit that supports dopaminergic neurons. These data reveal a molecular pathway for regulating cilia function that likely contributes to Parkinson's disease-specific pathology. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).