Aging Associates with Cilium Elongation and Dysfunction in Kidney and Pancreas.

Cilia are best known and most studied for their manifold functions enabling proper embryonic development. Loss of cilia or dysfunction thereof results in a great variety of congenital malformations and syndromes. However, there are also cilia-driven conditions, which manifest only later in life, such as polycystic kidney disease. Even degenerative diseases in the central nervous system have recently been linked to alterations in cilia biology. Surprisingly though, there is very little knowledge regarding cilia in normally aged organisms absent any disease. Here, it is provided evidence that cilia in naturally aged mice are considerably elongated in the kidney and pancreas, respectively. Moreover, such altered cilia appear to have become dysfunctional as indicated by changes in cellular signaling.

Cholesterol and Phosphoinositides in Cilia Biology.

Cilia are evolutionarily conserved organelles that can be found on virtually every cell. They appear as hair-like structures emanating from the cellular surface either as single or as bundles of cilia. There, they sense external stimuli and translate them into intracellular signals. Motile cilia beat for the generation of locomotion of unicellular organisms or fluid flow in certain body cavities of vertebrate organisms. Defects in cilia are detrimental and account for the development of ciliopathies, one of the fastest-growing family of afflictions. In the past decade, membrane lipids, such as cholesterol and phosphoinositides, have emerged as essential elements in both the signal transduction via cilia and the building of cilia itself. Here, we summarize the current knowledge on the impact of cholesterol and phosphoinositides on cilium biology.

Kinase Activity Is Not Required for G Protein-Coupled Receptor Kinase 4 Restraining mTOR Signaling during Cilia and Kidney Development.

SIGNIFICANCE STATEMENT: G protein-coupled receptor kinase 4 (GRK4) regulates renal sodium and water reabsorption. Although GRK4 variants with elevated kinase activity have been associated with salt-sensitive or essential hypertension, this association has been inconsistent among different study populations. In addition, studies elucidating how GRK4 may modulate cellular signaling are sparse. In an analysis of how GRK4 affects the developing kidney, the authors found that GRK4 modulates mammalian target of rapamycin (mTOR) signaling. Loss of GRK4 in embryonic zebrafish causes kidney dysfunction and glomerular cysts. Moreover, GRK4 depletion in zebrafish and cellular mammalian models results in elongated cilia. Rescue experiments suggest that hypertension in carriers of GRK4 variants may not be explained solely by kinase hyperactivity; instead, elevated mTOR signaling may be the underlying cause. BACKGROUND: G protein-coupled receptor kinase 4 (GRK4) is considered a central regulator of blood pressure through phosphorylation of renal dopaminergic receptors and subsequent modulation of sodium excretion. Several nonsynonymous genetic variants of GRK4 have been only partially linked to hypertension, although these variants demonstrate elevated kinase activity. However, some evidence suggests that function of GRK4 variants may involve more than regulation of dopaminergic receptors alone. Little is known about the effects of GRK4 on cellular signaling, and it is also unclear whether or how altered GRK4 function might affect kidney development. METHODS: To better understand the effect of GRK4 variants on the functionality of GRK4 and GRK4's actions in cellular signaling during kidney development, we studied zebrafish, human cells, and a murine kidney spheroid model. RESULTS: Zebrafish depleted of Grk4 develop impaired glomerular filtration, generalized edema, glomerular cysts, pronephric dilatation, and expansion of kidney cilia. In human fibroblasts and in a kidney spheroid model, GRK4 knockdown produced elongated primary cilia. Reconstitution with human wild-type GRK4 partially rescues these phenotypes. We found that kinase activity is dispensable because kinase-dead GRK4 (altered GRK4 that cannot result in phosphorylation of the targeted protein) prevented cyst formation and restored normal ciliogenesis in all tested models. Hypertension-associated genetic variants of GRK4 fail to rescue any of the observed phenotypes, suggesting a receptor-independent mechanism. Instead, we discovered unrestrained mammalian target of rapamycin signaling as an underlying cause. CONCLUSIONS: These findings identify GRK4 as novel regulator of cilia and of kidney development independent of GRK4's kinase function and provide evidence that the GRK4 variants believed to act as hyperactive kinases are dysfunctional for normal ciliogenesis.

Pathway-specific effects of ADSL deficiency on neurodevelopment.

Adenylosuccinate lyase (ADSL) functions in de novo purine synthesis (DNPS) and the purine nucleotide cycle. ADSL deficiency (ADSLD) causes numerous neurodevelopmental pathologies, including microcephaly and autism spectrum disorder. ADSLD patients have normal serum purine nucleotide levels but exhibit accumulation of dephosphorylated ADSL substrates, S-Ado, and SAICAr, the latter being implicated in neurotoxic effects through unknown mechanisms. We examined the phenotypic effects of ADSL depletion in human cells and their relation to phenotypic outcomes. Using specific interventions to compensate for reduced purine levels or modulate SAICAr accumulation, we found that diminished AMP levels resulted in increased DNA damage signaling and cell cycle delays, while primary ciliogenesis was impaired specifically by loss of ADSL or administration of SAICAr. ADSL-deficient chicken and zebrafish embryos displayed impaired neurogenesis and microcephaly. Neuroprogenitor attrition in zebrafish embryos was rescued by pharmacological inhibition of DNPS, but not increased nucleotide concentration. Zebrafish also displayed phenotypes commonly linked to ciliopathies. Our results suggest that both reduced purine levels and impaired DNPS contribute to neurodevelopmental pathology in ADSLD and that defective ciliogenesis may influence the ADSLD phenotypic spectrum.

DNA Replication proteins in primary microcephaly syndromes.

Improper expansion of neural stem and progenitor cells during brain development manifests in primary microcephaly. This disease is characterized by a reduced head circumference, which correlates with a reduction in brain size. This often corresponds to a general underdevelopment of the brain and entails cognitive, behavioral and motoric retardation. In the past decade significant research efforts have been undertaken to identify genes and the molecular mechanisms underlying microcephaly. One such gene set encompasses factors required for DNA replication. Intriguingly, a growing body of evidence indicates that a substantial number of these genes mediate faithful centrosome and cilium function in addition to their canonical function in genome duplication. Here, we summarize, which DNA replication factors are associated with microcephaly syndromes and to which extent they impact on centrosomes and cilia.

Limitations and opportunities in the pharmacotherapy of ciliopathies.

Ciliopathies are a family of rather diverse conditions, which have been grouped based on the finding of altered or dysfunctional cilia, potentially motile, small cellular antennae extending from the surface of postmitotic cells. Cilia-related disorders include embryonically arising conditions such as Joubert, Usher or Kartagener syndrome, but also afflictions with a postnatal or even adult onset phenotype, i.e. autosomal dominant polycystic kidney disease. The majority of ciliopathies are syndromic rather than affecting only a single organ due to cilia being found on almost any cell in the human body. Overall ciliopathies are considered rare diseases. Despite that, pharmacological research and the strive to help these patients has led to enormous therapeutic advances in the last decade. In this review we discuss new treatment options for certain ciliopathies, give an outlook on promising future therapeutic strategies, but also highlight the limitations in the development of therapeutic approaches of ciliopathies.

miR-103/107 regulates left-right asymmetry in zebrafish by modulating Kupffer's vesicle development and ciliogenesis.

In zebrafish, cilia movement within the Kupffer's vesicle (KV) generates a fluid flow responsible for accumulating nodal signals exclusively in the left lateral plate mesoderm, thereby initiating left-right patterning (LRP). Defects in LRP cause devastating congenital disorders including congenital heart malformations due to organ mis-positioning. We identified the miR-103/107 family to be involved in regulating LRP. Depletion of miR-103/107 in zebrafish embryos resulted in malpositioned and malformed visceral organs and hearts due to disturbed LRP gene expression, indicating early defects in LRP. Additionally, loss of miR-103/107 affected KV morphogenesis and cilia formation without disturbing endoderm development. Human fibroblasts depleted of miR-103a/107 often failed to extend cilia or developed shorter cilia, indicating functional conservation between species. We identified arl6, araf and foxH1 as direct targets of miR-103/107 providing a mechanistic link to cilia development and nodal signal titration. We describe a new microRNA family controlling KV development and hence influencing establishment of internal organ asymmetry.

Elevated Hedgehog activity contributes to attenuated DNA damage responses in aged hematopoietic cells.

Accumulation of DNA damage and myeloid-skewed differentiation characterize aging of the hematopoietic system, yet underlying mechanisms remain incompletely understood. Here, we show that aging hematopoietic progenitor cells particularly of the myeloid branch exhibit enhanced resistance to bulky DNA lesions-a relevant type of DNA damage induced by toxins such as cancer drugs or endogenous aldehydes. We identified aging-associated activation of the Hedgehog (Hh) pathway to be connected to this phenotype. Inhibition of Hh signaling reverts DNA damage tolerance and DNA damage-resistant proliferation in aged hematopoietic progenitors. Vice versa, elevating Hh activity in young hematopoietic progenitors is sufficient to impair DNA damage responses. Altogether, these findings provide experimental evidence for aging-associated increases in Hh activity driving DNA damage tolerance in myeloid progenitors and myeloid-skewed differentiation. Modulation of Hh activity could thus be explored as a therapeutic strategy to prevent DNA damage tolerance, myeloid skewing, and disease development in the aging hematopoietic system.

Muscarinic receptors promote pacemaker fate at the expense of secondary conduction system tissue in zebrafish.

Deterioration or inborn malformations of the cardiac conduction system (CCS) interfere with proper impulse propagation in the heart and may lead to sudden cardiac death or heart failure. Patients afflicted with arrhythmia depend on antiarrhythmic medication or invasive therapy, such as pacemaker implantation. An ideal way to treat these patients would be CCS tissue restoration. This, however, requires precise knowledge regarding the molecular mechanisms underlying CCS development. Here, we aimed to identify regulators of CCS development. We performed a compound screen in zebrafish embryos and identified tolterodine, a muscarinic receptor antagonist, as a modifier of CCS development. Tolterodine provoked a lower heart rate, pericardiac edema, and arrhythmia. Blockade of muscarinic M3, but not M2, receptors induced transcriptional changes leading to amplification of sinoatrial cells and loss of atrioventricular identity. Transcriptome data from an engineered human heart muscle model provided additional evidence for the contribution of muscarinic M3 receptors during cardiac progenitor specification and differentiation. Taken together, we found that muscarinic M3 receptors control the CCS already before the heart becomes innervated. Our data indicate that muscarinic receptors maintain a delicate balance between the developing sinoatrial node and the atrioventricular canal, which is probably required to prevent the development of arrhythmia.

SPRTN protease and checkpoint kinase 1 cross-activation loop safeguards DNA replication.

The SPRTN metalloprotease is essential for DNA-protein crosslink (DPC) repair and DNA replication in vertebrate cells. Cells deficient in SPRTN protease exhibit DPC-induced replication stress and genome instability, manifesting as premature ageing and liver cancer. Here, we provide a body of evidence suggesting that SPRTN activates the ATR-CHK1 phosphorylation signalling cascade during physiological DNA replication by proteolysis-dependent eviction of CHK1 from replicative chromatin. During this process, SPRTN proteolyses the C-terminal/inhibitory part of CHK1, liberating N-terminal CHK1 kinase active fragments. Simultaneously, CHK1 full length and its N-terminal fragments phosphorylate SPRTN at the C-terminal regulatory domain, which stimulates SPRTN recruitment to chromatin to promote unperturbed DNA replication fork progression and DPC repair. Our data suggest that a SPRTN-CHK1 cross-activation loop plays a part in DNA replication and protection from DNA replication stress. Finally, our results with purified components of this pathway further support the proposed model of a SPRTN-CHK1 cross-activation loop.

Imbalanced mitochondrial function provokes heterotaxy via aberrant ciliogenesis.

About 1% of all newborns are affected by congenital heart disease (CHD). Recent findings identify aberrantly functioning cilia as a possible source for CHD. Faulty cilia also prevent the development of proper left-right asymmetry and cause heterotaxy, the incorrect placement of visceral organs. Intriguingly, signaling cascades such as mTor that influence mitochondrial biogenesis also affect ciliogenesis, and can cause heterotaxy-like phenotypes in zebrafish. Here, we identify levels of mitochondrial function as a determinant for ciliogenesis and a cause for heterotaxy. We detected reduced mitochondrial DNA content in biopsies of heterotaxy patients. Manipulation of mitochondrial function revealed a reciprocal influence on ciliogenesis and affected cilia-dependent processes in zebrafish, human fibroblasts and Tetrahymena thermophila. Exome analysis of heterotaxy patients revealed an increased burden of rare damaging variants in mitochondria-associated genes as compared to 1000 Genome controls. Knockdown of such candidate genes caused cilia elongation and ciliopathy-like phenotypes in zebrafish, which could not be rescued by RNA encoding damaging rare variants identified in heterotaxy patients. Our findings suggest that ciliogenesis is coupled to the abundance and function of mitochondria. Our data further reveal disturbed mitochondrial function as an underlying cause for heterotaxy-linked CHD and provide a mechanism for unexplained phenotypes of mitochondrial disease.

Pharmacological cholesterol depletion disturbs ciliogenesis and ciliary function in developing zebrafish.

Patients with an inherited inability to synthesize sufficient amounts of cholesterol develop congenital malformations of the skull, toes, kidney and heart. As development of these structures depends on functional cilia we investigated whether cholesterol regulates ciliogenesis through inhibition of hydroxymethylglutaryl-Coenzyme A reductase (HMG-CoA-R), the rate-limiting enzyme in cholesterol synthesis. HMG-CoA-R is efficiently inhibited by statins, a standard medication for hyperlipidemia. When zebrafish embryos are treated with statins cilia dysfunction phenotypes including heart defects, left-right asymmetry defects and malformation of ciliated organs develop, which are ameliorated by cholesterol replenishment. HMG-CoA-R inhibition and other means of cholesterol reduction lowered ciliation frequency and cilia length in zebrafish as well as several mammalian cell types. Cholesterol depletion further triggers an inability for ciliary signalling. Because of a reduction of the transition zone component Pi(4,5)P2 we propose that cholesterol governs crucial steps of cilium extension. Taken together, we report that cholesterol abrogation provokes cilia defects.

Resting cells rely on the DNA helicase component MCM2 to build cilia.

Minichromosome maintenance (MCM) proteins facilitate replication by licensing origins and unwinding the DNA double strand. Interestingly, the number of MCM hexamers greatly exceeds the number of firing origins suggesting additional roles of MCMs. Here we show a hitherto unanticipated function of MCM2 in cilia formation in human cells and zebrafish that is uncoupled from replication. Zebrafish depleted of MCM2 develop ciliopathy-phenotypes including microcephaly and aberrant heart looping due to malformed cilia. In non-cycling human fibroblasts, loss of MCM2 promotes transcription of a subset of genes, which cause cilia shortening and centriole overduplication. Chromatin immunoprecipitation experiments show that MCM2 binds to transcription start sites of cilia inhibiting genes. We propose that such binding may block RNA polymerase II-mediated transcription. Depletion of a second MCM (MCM7), which functions in complex with MCM2 during its canonical functions, reveals an overlapping cilia-deficiency phenotype likely unconnected to replication, although MCM7 appears to regulate a distinct subset of genes and pathways. Our data suggests that MCM2 and 7 exert a role in ciliogenesis in post-mitotic tissues.

The analysis of heterotaxy patients reveals new loss-of-function variants of GRK5.

G protein-coupled receptor kinase 5 (GRK5) is a regulator of cardiac performance and a potential therapeutic target in heart failure in the adult. Additionally, we have previously classified GRK5 as a determinant of left-right asymmetry and proper heart development using zebrafish. We thus aimed to identify GRK5 variants of functional significance by analysing 187 individuals with laterality defects (heterotaxy) that were associated with a congenital heart defect (CHD). Using Sanger sequencing we identified two moderately frequent variants in GRK5 with minor allele frequencies <10%, and seven very rare polymorphisms with minor allele frequencies <1%, two of which are novel variants. Given their evolutionarily conserved position in zebrafish, in-depth functional characterisation of four variants (p.Q41L, p.G298S, p.R304C and p.T425M) was performed. We tested the effects of these variants on normal subcellular localisation and the ability to desensitise receptor signalling as well as their ability to correct the left-right asymmetry defect upon Grk5l knockdown in zebrafish. While p.Q41L, p.R304C and p.T425M responded normally in the first two aspects, neither p.Q41L nor p.R304C were capable of rescuing the lateralisation phenotype. The fourth variant, p.G298S was identified as a complete loss-of-function variant in all assays and provides insight into the functions of GRK5.

Xpg limits the expansion of haematopoietic stem and progenitor cells after ionising radiation.

Reduced capacity of genome maintenance represents a problem for any organism, potentially causing premature death, carcinogenesis, or accelerated ageing. Strikingly though, loss of certain genome stability factors can be beneficial, especially for the maintenance of tissue stem cells of the intestine and the haematopoietic system. We therefore screened for genome stability factors negatively impacting maintenance of haematopoietic stem cells (HSC) in the context of ionising radiation (IR). We found that in vivo knock down of Xeroderma pigmentosum, complementation group G (Xpg) causes elevation of HSC numbers after IR treatment, while numbers of haematopoietic progenitors are elevated to a lesser extent. IR rapidly induces Xpg both on mRNA and on protein level. Prevention of this induction does not influence activation of the checkpoint cascade, yet attenuates late checkpoint steps such as induction of p21 and Noxa. This causes a leaky cell cycle arrest and lower levels of apoptosis, both contributing to increased colony formation and transformation rates. Xpg thus helps to adequately induce DNA damage responses after IR, thereby keeping the expansion of damaged cells under control. This represents a new function of Xpg in the response to IR, in addition to its well-characterized role in nucleotide excision repair.

Left-right asymmetry in the light of TOR: An update on what we know so far.

The internal left-right (LR) asymmetry is a characteristic that exists throughout the animal kingdom from roundworms over flies and fish to mammals. Cilia, which are antenna-like structures protruding into the extracellular space, are involved in establishing LR asymmetry during early development. Humans who suffer from dysfunctional cilia often develop conditions such as heterotaxy, where internal organs appear to be placed randomly. As a consequence to this failure in asymmetry development, serious complications such as congenital heart defects (CHD) occur. The mammalian (or mechanistic) target of rapamycin (mTOR) pathway has recently emerged as an important regulator regarding symmetry breaking. The mTOR pathway governs fundamental processes such as protein translation or metabolism. Its activity can be transduced by two complexes, which are called TORC1 and TORC2, respectively. So far, only TORC1 has been implicated with asymmetry development and appears to require very precise regulation. A number of recent papers provided evidence that dysregulated TORC1 results in alterations of motile cilia and asymmetry defects. In here, we give an update on what we know so far of mTORC1 in LR asymmetry development.

Genome instability of ageing stem cells--Induction and defence mechanisms.

The mammalian organism is comprised of tissue types with varying degrees of self-renewal and regenerative capacity. In most organs self-renewing tissue-specific stem and progenitor cells contribute to organ maintenance, and it is vital to maintain a functional stem cell pool to preserve organ homeostasis. Various conditions like tissue injury, stress responses, and regeneration challenge the stem cell pool to re-establish homeostasis (Fig. 1). However, with increasing age the functionality of adult stem cells declines and genomic mutations accumulate. These defects affect different cellular response pathways and lead to impairments in regeneration, stress tolerance, and organ function as well as to an increased risk for the development of ageing associated diseases and cancer. Maintenance of the genome appears to be of utmost importance to preserve stem cell function and to reduce the risk of ageing associated dysfunctions and pathologies. In this review, we discuss the causal link between stem cell dysfunction and DNA damage accrual, different strategies how stem cells maintain genome integrity, and how these processes are affected during ageing.

Phenotypic regulation of the sphingosine 1-phosphate receptor miles apart by G protein-coupled receptor kinase 2.

The evolutionarily conserved DRY motif at the end of the third helix of rhodopsin-like, class-A G protein-coupled receptors (GPCRs) is a major regulator of receptor stability, signaling activity, and ß-arrestin-mediated internalization. Substitution of the DRY arginine with histidine in the human vasopressin receptor results in a loss-of-function phenotype associated with diabetes insipidus. The analogous R150H substitution of the DRY motif in zebrafish sphingosine-1 phosphate receptor 2 (S1p2) produces a mutation, miles apart m(93) (mil(m93)), that not only disrupts signaling but also impairs heart field migration. We hypothesized that constitutive S1p2 desensitization is the underlying cause of this strong zebrafish developmental defect. We observed in cell assays that the wild-type S1p2 receptor is at the cell surface whereas in distinct contrast the S1p2 R150H receptor is found in intracellular vesicles, blocking G protein but not arrestin signaling activity. Surface S1p2 R150H expression could be restored by inhibition of G protein-coupled receptor kinase 2 (GRK2). Moreover, we observed that ß-arrestin 2 and GRK2 colocalize with S1p2 in developing zebrafish embryos and depletion of GRK2 in the S1p2 R150H miles apart zebrafish partially rescued cardia bifida. The ability of reduced GRK2 activity to reverse a developmental phenotype associated with constitutive desensitization supports efforts to genetically or pharmacologically target this kinase in diseases involving biased GPCR signaling.

Mutations in SPRTN cause early onset hepatocellular carcinoma, genomic instability and progeroid features.

Age-related degenerative and malignant diseases represent major challenges for health care systems. Elucidation of the molecular mechanisms underlying carcinogenesis and age-associated pathologies is thus of growing biomedical relevance. We identified biallelic germline mutations in SPRTN (also called C1orf124 or DVC1) in three patients from two unrelated families. All three patients are affected by a new segmental progeroid syndrome characterized by genomic instability and susceptibility toward early onset hepatocellular carcinoma. SPRTN was recently proposed to have a function in translesional DNA synthesis and the prevention of mutagenesis. Our in vivo and in vitro characterization of identified mutations has uncovered an essential role for SPRTN in the prevention of DNA replication stress during general DNA replication and in replication-related G2/M-checkpoint regulation. In addition to demonstrating the pathogenicity of identified SPRTN mutations, our findings provide a molecular explanation of how SPRTN dysfunction causes accelerated aging and susceptibility toward carcinoma.