Matrix Metalloproteinases and Minocycline: Therapeutic Avenues for Fragile X Syndrome

Fragile X syndrome (FXS) is the most common known genetic form of intellectual disability and autism spectrum disorders. FXS patients suffer a broad range of other neurological symptoms, including hyperactivity, disrupted circadian activity cycles, obsessive-compulsive behavior, and childhood seizures. The high incidence and devastating effects of this disease state make finding effective pharmacological treatments imperative. Recently, reports in both mouse and Drosophila FXS disease models have indicated that the tetracycline derivative minocycline may hold great therapeutic promise for FXS patients. Both models strongly suggest that minocycline acts on the FXS disease state via inhibition of matrix metalloproteinases (MMPs), a class of zinc-dependent extracellular proteases important in tissue remodeling and cell-cell signaling. Recent FXS clinical trials indicate that minocycline may be effective in treating human patients. In this paper, we summarize the recent studies in Drosophila and mouse FXS disease models and human FXS patients, which indicate that minocycline may be an effective FXS therapeutic treatment, and discuss the data forming the basis for the proposed minocycline mechanism of action as an MMP inhibitor

nNOS regulates ciliated cell polarity, ciliary beat frequency, and directional flow in mouse trachea.

Clearance of the airway is dependent on directional mucus flow across the mucociliary epithelium, and deficient flow is implicated in a range of human disorders. Efficient flow relies on proper polarization of the multiciliated cells and sufficient ciliary beat frequency. We show that NO, produced by nNOS in the multiciliated cells of the mouse trachea, controls both the planar polarity and the ciliary beat frequency and is thereby necessary for the generation of the robust flow. The effect of nNOS on the polarity of ciliated cells relies on its interactions with the apical networks of actin and microtubules and involves RhoA activation. The action of nNOS on the beat frequency is mediated by guanylate cyclase; both NO donors and cGMP can augment fluid flow in the trachea and rescue the deficient flow in nNOS mutants. Our results link insufficient availability of NO in ciliated cells to defects in flow and ciliary activity and may thereby explain the low levels of exhaled NO in ciliopathies

Neural circuit architecture defects in a Drosophila model of Fragile X syndrome are alleviated by minocycline treatment and genetic removal of matrix metalloproteinase

Fragile X syndrome (FXS), caused by loss of the fragile X mental retardation 1 (FMR1) product (FMRP), is the most common cause of inherited intellectual disability and autism spectrum disorders. FXS patients suffer multiple behavioral symptoms, including hyperactivity, disrupted circadian cycles, and learning and memory deficits. Recently, a study in the mouse FXS model showed that the tetracycline derivative minocycline effectively remediates the disease state via a proposed matrix metalloproteinase (MMP) inhibition mechanism. Here, we use the well-characterized Drosophila FXS model to assess the effects of minocycline treatment on multiple neural circuit morphological defects and to investigate the MMP hypothesis. We first treat Drosophila Fmr1 (dfmr1) null animals with minocycline to assay the effects on mutant synaptic architecture in three disparate locations: the neuromuscular junction (NMJ), clock neurons in the circadian activity circuit and Kenyon cells in the mushroom body learning and memory center. We find that minocycline effectively restores normal synaptic structure in all three circuits, promising therapeutic potential for FXS treatment. We next tested the MMP hypothesis by assaying the effects of overexpressing the sole Drosophila tissue inhibitor of MMP (TIMP) in dfmr1 null mutants. We find that TIMP overexpression effectively prevents defects in the NMJ synaptic architecture in dfmr1 mutants. Moreover, co-removal of dfmr1 similarly rescues TIMP overexpression phenotypes, including cellular tracheal defects and lethality. To further test the MMP hypothesis, we generated dfmr1;mmp1 double null mutants. Null mmp1 mutants are 100% lethal and display cellular tracheal defects, but co-removal of dfmr1 allows adult viability and prevents tracheal defects. Conversely, co-removal of mmp1 ameliorates the NMJ synaptic architecture defects in dfmr1 null mutants, despite the lack of detectable difference in MMP1 expression or gelatinase activity between the single dfmr1 mutants and controls. These results support minocycline as a promising potential FXS treatment and suggest that it might act via MMP inhibition. We conclude that FMRP and TIMP pathways interact in a reciprocal, bidirectional manner

Conditional knockout mice for the distal appendage protein CEP164 reveal its essential roles in airway multiciliated cell differentiation.

Multiciliated cells of the airways, brain ventricles, and female reproductive tract provide the motive force for mucociliary clearance, cerebrospinal fluid circulation, and ovum transport. Despite their clear importance to human biology and health, the molecular mechanisms underlying multiciliated cell differentiation are poorly understood. Prior studies implicate the distal appendage/transition fiber protein CEP164 as a central regulator of primary ciliogenesis; however, its role in multiciliogenesis remains unknown. In this study, we have generated a novel conditional mouse model that lacks CEP164 in multiciliated tissues and the testis. These mice show a profound loss of airway, ependymal, and oviduct multicilia and develop hydrocephalus and male infertility. Using primary cultures of tracheal multiciliated cells as a model system, we found that CEP164 is critical for multiciliogenesis, at least in part, via its regulation of small vesicle recruitment, ciliary vesicle formation, and basal body docking. In addition, CEP164 is necessary for the proper recruitment of another distal appendage/transition fiber protein Chibby1 (Cby1) and its binding partners FAM92A and FAM92B to the ciliary base in multiciliated cells. In contrast to primary ciliogenesis, CEP164 is dispensable for the recruitment of intraflagellar transport (IFT) components to multicilia. Finally, we provide evidence that CEP164 differentially controls the ciliary targeting of membrane-associated proteins, including the small GTPases Rab8, Rab11, and Arl13b, in multiciliated cells. Altogether, our studies unravel unique requirements for CEP164 in primary versus multiciliogenesis and suggest that CEP164 modulates the selective transport of membrane vesicles and their cargoes into the ciliary compartment in multiciliated cells. Furthermore, our mouse model provides a useful tool to gain physiological insight into diseases associated with defective multicilia

CEP164 is essential for early embryonic development and primary ciliogenesis.

<p>(A) Comparison of control (WT or heterozygous) embryos with CEP164-knockout (KO) littermates at E7.5, E9.5, and E10.5. At E7.5, KO embryos were indistinguishable from control littermates. In contrast, E9.5 and E10.5 KO embryos displayed holoprosencephaly (arrow), an edematous pericardial sac (white arrowhead), cardiac looping defects (blue arrowhead), and a truncated posterior trunk (blue asterisk). Confocal images of neural tube sections from E9.5 control and KO embryos are presented in lower right panels. Primary cilia were labeled with the ciliary marker Arl13b (green), and nuclei are visualized with DAPI staining (blue). Asterisks indicate the lumen of the neural tube. Scale bar, 35 μm. (B) Loss of primary cilia in CEP164-KO MEFs. Mouse embryonic fibroblasts (MEFs) were prepared from E8.5 CEP164-KO or control embryos, serum-starved for 48 hours to induce primary cilia, and immunostained for Arl13b (green) and the basal body marker γ-tubulin (G-tub) (red). Nuclei were stained with DAPI (blue). Scale bar, 10 μm. Quantification is shown on the right. >200 cells were counted for each of three independent MEF preparations per genotype. Error bars represent ±SEM. **, p<0.01. (C) Schematic depiction of the localization of Cby1, FAM92, and CEP164 at the ciliary base. Basal bodies and centrioles are barrel-shaped structures composed of nine microtubule triplets. During ciliogenesis, mother centrioles transform into basal bodies by acquiring accessary structures to assemble cilia. The axoneme is the detergent-insoluble cytoskeletal structure of the cilium including microtubules and their associated proteins. (D) Serum-starved MEFs were double-labeled for CEP164, Cby1, or FAM92A (green) and the ciliary marker acetylated α-tubulin (A-tub) or G-tub (red) as indicated. Nuclei were visualized by DAPI (blue). The boxed regions are enlarged in insets, highlighting the loss of CEP164, Cby1, and FAM92A centriolar localization in CEP164-KO MEFs. Arrowheads point to primary cilia. Scale bars, 10 μm.</p

CEP164 plays an important role in proper development of female and male reproductive systems.

<p>(A) H&E staining of oviduct sections from CEP164^fl/fl and FOXJ1-Cre;CEP164^fl/fl adult mice. The boxed regions are enlarged in insets. Scale bar, 10 μm. (B) IF staining of oviduct sections for A-tub (green) and DAPI (blue). Scale bar, 50 μm. (C) H&E staining of both testis and epididymis from 3-month-old CEP164^fl/fl and FOXJ1-Cre;CEP164^fl/fl mice. Asterisk denotes seminiferous tubules lacking germ cells. Scale bars, 100 μm for low magnification and 40 μm for high magnification. (D) X-gal staining of testis sections from a control WT mouse and a mouse heterozygous for the CEP164 KO-first allele that contains a <i>lacZ</i> reporter. Scale bar, 40 μm.</p

CEP164 regulates small vesicle recruitment to basal bodies in multiciliated cells.

<p>(A) TEM images of multiciliated cells from CEP164^fl/fl and FOXJ1-Cre;CEP164^fl/fl adult tracheas. Asterisks depict multiple cytoplasmic basal bodies in FOXJ1-Cre;CEP164^fl/fl trachea. Scale bar, 500 nm. (B) TEM images of cross sections through the apical region of multiciliated cells from ALId14 MTECs derived from CEP164^fl/fl and FOXJ1-Cre;CEP164^fl/fl adult tracheas. Asterisks depict multiple cytoplasmic, misoriented basal bodies in FOXJ1-Cre;CEP164^fl/fl multiciliated cells. Scale bars, 500 nm. (C) TEM images of CEP164^fl/fl and FOXJ1-Cre;CEP164^fl/fl P8 tracheas subjected to <i>ex vivo</i> culture in the presence of taxol to enrich vesicle-bound basal bodies. Arrowheads denote vesicles attached to the distal end of the cytoplasmic centriole in control CEP164^fl/fl samples. Scale bar, 200 nm.</p

Ablation of CEP164 in the FOXJ1-positive tissues results in loss of airway and ependymal multicilia and hydrocephalus.

<p>(A) Hematoxylin and eosin (H&E)-stained tracheal and sinus sections from control CEP164^fl/fl and FOXJ1-Cre;CEP164^fl/fl adult mice. Scale bar, 20 μm. (B) Shown are lateral views of postnatal day 18 (P18) mice (left panels), coronal sections of adult brains (middle panels), and IF staining for A-tub (green) and β-catenin (red) in whole mounts of the adult subventricular zone (SVZ) (right panels). Asterisks denote enlarged lateral ventricular spaces indicative of hydrocephalus. Scale bar, 25 μm.</p