3,3',5-Triiodothyroacetic Acid Transporters.

Introduction: Thyroid hormone transporters are essential for thyroid hormones to enter target cells. Monocarboxylate transporter (MCT) 8 is a key transporter and is expressed at the blood-brain barrier (BBB), in neural cells and many other tissues. Patients with MCT8 deficiency have severe neurodevelopmental delays because of cerebral hypothyroidism and chronic sequelae of peripheral thyrotoxicosis. The T3 analog 3,3',5-triiodothyroacetic acid (TRIAC) rescued neurodevelopmental features in animal models mimicking MCT8 deficiency and improved key metabolic features in patients with MCT8 deficiency. However, the identity of the transporter(s) that facilitate TRIAC transport are unknown. Here, we screened candidate transporters that are expressed at the human BBB and/or brain-cerebrospinal fluid barrier and known thyroid hormone transporters for TRIAC transport. Materials and Methods: Plasma membrane expression was determined by cell surface biotinylation assays. Intracellular accumulation of 1 nM TRIAC was assessed in COS-1 cells expressing candidate transporters in Dulbecco's phosphate-buffered saline (DPBS)/0.1% glucose or Dulbecco's modified Eagle's medium (DMEM) with or without 0.1% bovine serum albumin (BSA). Expression of Slc22a8 was determined by fluorescent in situ hybridization in brain sections from wild-type and Mct8/Oatp1c1 knockout mice at postnatal days 12, 21, and 120. Results: In total, 59 plasma membrane transporters were selected for screening of TRIAC accumulation (n = 40 based on expression at the human BBB and/or brain-cerebrospinal fluid barrier and having small organic molecules as substrates; n = 19 known thyroid hormone transporters). Screening of the selected transporter panel showed that 18 transporters facilitated significant intracellular accumulation of TRIAC in DPBS/0.1% glucose or DMEM in the absence of BSA. In the presence of BSA, substantial transport was noted for SLCO1B1 and SLC22A8 (in DPBS/0.1% glucose and DMEM) and SLC10A1, SLC22A6, and SLC22A24 (in DMEM). The zebrafish and mouse orthologs of these transporters similarly facilitated intracellular accumulation of TRIAC. Highest Slc22a8 mRNA expression was detected in mouse brain capillary endothelial cells and choroid plexus epithelial cells at early postnatal time points, but was reduced at P120. Conclusions: Human SLC10A1, SLCO1B1, SLC22A6, SLC22A8, and SLC22A24 as well as their mouse and zebrafish orthologs are efficient TRIAC transporters. These findings contribute to the understanding of TRIAC treatment in patients with MCT8 deficiency and animal models thereof.

Brain Gene Expression in Systemic Hypothyroidism and Mouse Models of MCT8 Deficiency: The Mct8-Oatp1c1-Dio2 Triad.

Background: The monocarboxylate transporter 8 (Mct8) protein is a primary thyroxine (T4) and triiodothyronine (T3) (thyroid hormone [TH]) transporter. Mutations of the MCT8-encoding, SLC16A2 gene alter thyroid function and TH metabolism and severely impair neurodevelopment (Allan-Herndon-Dudley syndrome [AHDS]). Mct8-deficient mice manifest thyroid alterations but lack neurological signs. It is believed that Mct8 deficiency in mice is compensated by T4 transport through the Slco1c1-encoded organic anion transporter polypeptide 1c1 (Oatp1c1). This allows local brain generation of sufficient T3 by the Dio2-encoded type 2 deiodinase, thus preventing brain hypothyroidism. The Slc16a2/Slco1c1 (MO) and Slc16a2/Dio2 (MD) double knockout (KO) mice lacking T4 and T3 transport, or T3 transport and T4 deiodination, respectively, should be appropriate models of AHDS. Our goal was to compare the cerebral hypothyroidism of systemic hypothyroidism (SH) caused by thyroid gland blockade with that present in the double KO mice. Methods: We performed RNA sequencing by using RNA from the cerebral cortex and striatum of SH mice and the double KO mice on postnatal days 21-23. Real-time polymerase chain reaction was used to confirm RNA-Seq results in replicate biological samples. Cell type involvement was assessed from cell type-enriched genes. Functional genomic differences were analyzed by functional node activity based on a probabilistic graphical model. Results: Each of the three conditions gave a different pattern of gene expression, with partial overlaps. SH gave a wider and highest variation of gene expression than MD or MO. This was partially due to secondary gene responses to hypothyroidism. The set of primary transcriptional T3 targets showed a tighter overlap, but quantitative gene responses indicated that the gene responses in SH were more severe than in MD or MO. Examination of cell type-enriched genes indicated cellular differences between the three conditions. Conclusions: The results indicate that the neurological impairment of AHDS is too severe to be fully explained by TH deprivation only.

Autophagy pathway upregulation in a human iPSC-derived neuronal model of Cohen syndrome with VPS13B missense mutations.

Significant clinical symptoms of Cohen syndrome (CS), a rare autosomal recessive disorder, include intellectual disability, facial dysmorphism, postnatal microcephaly, retinal dystrophy, and intermittent neutropenia. CS has been associated with mutations in the VPS13B (vacuolar protein sorting 13 homolog B) gene, which regulates vesicle-mediated protein sorting and transport; however, the cellular mechanism underlying CS pathogenesis in patient-derived neurons remains uncertain. This report states that autophagic vacuoles accumulate in CS fibroblasts and the axonal terminals of CS patient-specific induced pluripotent stem cells (CS iPSC)-derived neurons; additionally, autophagic flux was significantly increased in CS-derived neurons compared to control neurons. VPS13B knockout HeLa cell lines generated using the CRISPR/Cas9 genome editing system showed significant upregulation of autophagic flux, indicating that VSP13B may be associated with autophagy in CS. Transcriptomic analysis focusing on the autophagy pathway revealed that genes associated with autophagosome organization were dysregulated in CS-derived neurons. ATG4C is a mammalian ATG4 paralog and a crucial regulatory component of the autophagosome biogenesis/recycling pathway. ATG4C was significantly upregulated in CS-derived neurons, indicating that autophagy is upregulated in CS neurons. The autophagy pathway in CS neurons may be associated with the pathophysiology exhibited in the neural network of CS patients.

Central Hypothyroidism Impairs Heart Rate Stability and Prevents Thyroid Hormone-Induced Cardiac Hypertrophy and Pyrexia.

Background: Tachycardia, cardiac hypertrophy, and elevated body temperature are major signs of systemic hyperthyroidism, which are considered to reflect the excessive thyroid hormone (TH) action in the respective peripheral tissues. However, recent observations indicate that the central actions of TH also contribute substantially to cardiovascular regulation and thermogenesis. Methods: In this study, we dissect the individual contributions of peripheral TH action versus the central effects in body temperature regulation and cardiovascular functions by taking advantage of mice lacking the TH transporters monocarboxylate transporter 8 (MCT8) and organic anion transporting polypeptide 1C1 (OATP1C1) (M/O double knock-out [dko]), which exhibit elevated serum triiodothyronine (T3) levels while their brain is in a profoundly hypothyroid state. We compared these animals with wild-type (WT) mice that were treated orally with T3 to achieve similarly elevated serum T3 levels, but are centrally hyperthyroid. For the studies, we used radiotelemetry, infrared thermography, gene expression profiling, Western blot analyses, and enzyme linked immunosorbent assays (ELISA) assays. Results: Our analyses revealed mild hyperthermia and cardiac hypertrophy in T3-treated WT mice but not in M/O dko animals, suggesting that central actions of TH are required for these hyperthyroid phenotypes. Although the average heart rate was unaffected in either model, the M/O dko exhibited an altered heart rate frequency distribution with tachycardic bursts in active periods and bradycardic episodes during resting time, demonstrating that the stabilization of heart rate by the autonomic nervous system can be impaired in centrally hypothyroid animals. Conclusions: Our studies unravel distinct phenotypical traits of hyperthyroidism that depend on an intact central nervous system, and provide valuable insight into the cardiovascular pathology of the Allan-Herndon-Dudley syndrome, a condition caused by the lack of MCT8 in humans.

Thyroid Hormone Transporters.

Thyroid hormone transporters at the plasma membrane govern intracellular bioavailability of thyroid hormone. Monocarboxylate transporter (MCT) 8 and MCT10, organic anion transporting polypeptide (OATP) 1C1, and SLC17A4 are currently known as transporters displaying the highest specificity toward thyroid hormones. Structure-function studies using homology modeling and mutational screens have led to better understanding of the molecular basis of thyroid hormone transport. Mutations in MCT8 and in OATP1C1 have been associated with clinical disorders. Different animal models have provided insight into the functional role of thyroid hormone transporters, in particular MCT8. Different treatment strategies for MCT8 deficiency have been explored, of which thyroid hormone analogue therapy is currently applied in patients. Future studies may reveal the identity of as-yet-undiscovered thyroid hormone transporters. Complementary studies employing animal and human models will provide further insight into the role of transporters in health and disease. (Endocrine Reviews 41: 1 - 55, 2020).

Tissue-Specific Function of Thyroid Hormone Transporters: New Insights from Mouse Models.

Thyroid hormone (TH) transporters are required for cellular transmembrane passage of TH and are thus mandatory for proper TH metabolism and action. Consequently, inactivating mutations in TH transporters such as MCT8 or OATP1C1 can cause tissue- specific changes in TH homeostasis. As the most prominent example, patients with MCT8 mutations exhibit elevated serum T3 levels, whereas their CNS appear to be in a TH deficient state. Here, we will briefly summarize recent studies of mice lacking Mct8 alone or in combination with the TH transporters Mct10 or Oatp1c1 that shed light on many aspects and pathogenic events underlying global MCT8 deficiency and also underscore the contribution of Mct10 and Oatp1c1 in tissue-specific TH transport processes. Moreover, development of conditional knock-out mice that allow a cell-specific inactivation of TH transporters in distinct tissues, disclosed cell-specific changes in TH signaling, thereby highlighting the pathophysiological significance of local control of TH action.

Activated Thiol Sepharose-based proteomic approach to quantify reversible protein oxidation.

Reactive oxygen species (ROS) can act as second messengers in various signaling pathways, and abnormal oxidation contributes to multiple diseases, including cancer. Detecting and quantifying protein oxidation is crucial for a detailed understanding of reduction-oxidation reaction (redox) signaling. We developed an Activated Thiol Sepharose-based proteomic (ATSP) approach to quantify reversible protein oxidation. ATSP can enrich H2O2-sensitive thiol peptides, which are more likely to contain reactive cysteines involved in redox signaling. We applied our approach to analyze hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a type of kidney cancer that harbors fumarate hydratase (FH)-inactivating mutations and has elevated ROS levels. Multiple proteins were oxidized in FH-deficient cells, including many metabolic proteins such as the pyruvate kinase M2 isoform (PKM2). Treatment of HLRCC cells with dimethyl fumarate or PKM2 activators altered PKM2 oxidation levels. Finally, we found that ATSP could detect Src homology region 2 domain-containing phosphatase-2 and PKM2 oxidation in cells stimulated with platelet-derived growth factor. This newly developed redox proteomics workflow can detect reversible oxidation of reactive cysteines and can be employed to analyze multiple physiologic and pathologic conditions.-Xu, Y., Andrade, J., Ueberheide, B., Neel, B. G. Activated Thiol Sepharose-based proteomic approach to quantify reversible protein oxidation.

In Vitro Characterization of Human, Mouse, and Zebrafish MCT8 Orthologues.

Background: Mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) cause MCT8 deficiency, characterized by severe intellectual and motor disability and abnormal serum thyroid function tests. Various Mct8 knock-out mouse models as well as mct8 knock-out and knockdown zebrafish models are used as a disease model for MCT8 deficiency. Although important for model eligibility, little is known about the functional characteristics of the MCT8 orthologues in these species. Therefore, we here compared the functional characteristics of mouse (mm) MCT8 and zebrafish (dr) Mct8 to human (hs) MCT8. Methods: We performed extensive transport studies in COS-1 and JEG-3 cells transiently transfected with hsMCT8, drMct8, and mmMCT8. Protein expression levels and subcellular localization were assessed by immunoblotting, surface biotinylation, and immunocytochemistry. Sequence alignment and structural modeling were used to interpret functional differences between the orthologues. Results: hsMCT8, drMct8, and mmMCT8 all facilitated the uptake and efflux of 3,3'-diiodothyronine (3,3'-T2), rT3, triiodothyronine (T3), and thyroxine (T4), although the initial uptake rates of drMct8 were 1.5-4.0-fold higher than for hsMCT8 and mmMCT8. drMct8 exhibited 3-50-fold lower apparent IC50 values than hsMCT8 and mmMCT8 for all tested substrates, and substrate preference of drMct8 (3,3'-T2, T3 > T4 > rT3) differed from hsMCT8 and mmMCT8 (T3 > T4 > rT3, 3,3'-T2). Compared with hsMCT8 and mmMCT8, cis-inhibition studies showed that T3 uptake by drMct8 was inhibited at a lower concentration and by a broader spectrum of TH metabolites. Total and cell surface expression levels of drMct8 and hsMCT8 were equal and both significantly exceeded those of mmMCT8. Structural modeling located most non-conserved residues outside the substrate pore, except for H192 in hsMCT8, which is replaced by a glutamine in drMct8. However, a H192Q substituent of hsMCT8 did not alter its transporter characteristics. Conclusion: Our studies substantiate the eligibility of mice and zebrafish models for human MCT8 deficiency. However, differences in the intrinsic transporter properties of MCT8 orthologues may exist, which should be realized when comparing MCT8 deficiency in different in vivo models. Moreover, our findings may indicate that the protein domains outside the substrate channel may play a role in substrate selection and protein stability.

Connecting Versatile lncRNAs with Heterogeneous Nuclear Ribonucleoprotein K and Pathogenic Disorders.

Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA-binding protein that regulates multiple biological processes, including paraspeckles formation and cellular signal transduction. Recently, hnRNPK has been shown to interact with SINE-derived nuclear RNA localization (SIRLOIN)-containing RNAs, and orchestrate nuclear enrichment and cellular functions of long noncoding RNAs (lncRNAs). hnRNPK-lncRNAs interaction is potentially implicated in various pathogenic disorders including tumorigenesis, and Kabuki-like, Au-Kline, and Okamoto syndromes.

PIGQ glycosylphosphatidylinositol-anchored protein deficiency: Characterizing the phenotype.

PIGQ (OMIM *605754) encodes phosphatidylinositol glycan biosynthesis class Q (PIGQ) and is required for proper functioning of an N-acetylglucosamine transferase complex in a similar manner to the more established PIGA, PIGC, and PIGH. There are two previous patients reported with homozygous and apparently deleterious PIGQ mutations. Here, we provide the first detailed clinical report of a patient with heterozygous deleterious mutations associated with glycosylphosphatidylinositol-anchored protein (GPI-AP) biosynthesis deficiency. Our patient died at 10 months of age. The rare skeletal findings in this disorder expand the differential diagnosis of long bone radiolucent lesions and sphenoid wing dysplasia. This clinical report describes a new and rare disorder-PIGQ GPI-AP biosynthesis deficiency syndrome.

Functional analysis of monocarboxylate transporter 8 mutations in Japanese Allan-Herndon-Dudley syndrome patients.

Monocarboxylate transporter 8 (MCT8) facilitates T3 uptake into cells. Mutations in MCT8 lead to Allan-Herndon-Dudley syndrome (AHDS), which is characterized by severe psychomotor retardation and abnormal thyroid hormone profile. Nine uncharacterized MCT8 mutations in Japanese patients with severe neurocognitive impairment and elevated serum T3 levels were studied regarding the transport of T3. Human MCT8 (hMCT8) function was studied in wild-type (WT) or mutant hMCT8-transfected human placental choriocarcinoma cells (JEG3) by visualizing the locations of the proteins in the cells, detecting specific proteins, and measuring T3 uptake. We identified 6 missense (p.Arg445Ser, p.Asp498Asn, p.Gly276Arg, p.Gly196Glu, p.Gly401Arg, and p.Gly312Arg), 2 frameshift (p.Arg355Profs*64 and p.Tyr550Serfs*17), and 1 deletion (p.Pro561del) mutation(s) in the hMCT8 gene. All patients exhibited clinical characteristics of AHDS with high free T3, low-normal free T4, and normal-elevated TSH levels. All tested mutants were expressed at the protein level, except p.Arg355Profs*64 and p.Tyr550Serfs*17, which were truncated, and were inactive in T3 uptake, excluding p.Arg445Ser and p.Pro561del mutants, compared with WT-hMCT8. Immunocytochemistry revealed plasma membrane localization of p.Arg445Ser and p.Pro561del mutants similar with WT-hMCT8. The other mutants failed to localize in significant amount(s) in the plasma membrane and instead localized in the cytoplasm. These data indicate that p.Arg445Ser and p.Pro561del mutants preserve residual function, whereas p.Asp498Asn, p.Gly276Arg, p.Gly196Glu, p.Gly401Arg, p.Gly312Arg, p.Arg355Profs*64, and p.Tyr550Serfs*17 mutants lack function. These findings suggest that the mutations in MCT8 cause loss of function by reducing protein expression, impairing trafficking of protein to plasma membrane, and disrupting substrate channel.

A homozygous potentially pathogenic variant in the PAXBP1 gene in a large family with global developmental delay and myopathic hypotonia.

PAX binding protein 1 (PAXBP1) is an adaptor protein linking the transcription factor PAX3 and PAX7 to the histone methylation machinery. PAXBP1 is a nuclear protein and its high expression is known in brain cerebellar hemisphere and cerebellum. Moreover, it is also found in abundance in muscle precursor cells that are involved in myogenesis and skeletal muscles formation. Whole genome SNP genotyping and exome sequencing in a family with distinct syndrome of global developmental delay and hypotonia mapped the disease locus to the chromosome 21q22.11 and identified a homozygous missense variant (c.1612C>T) in the PAXBP1 gene, respectively. This variant is predicted to change the highly conserved strongly basic arginine at position 538 in the PAX7 binding domain of PAXBP1 to a neutral cysteine (p.Arg538Cys) residue. Arg538 is highly conserved and the variant is predicted to be deleterious by variety of in silico tools. Furthermore, protein modeling studies showed that in the mutant protein (Cys538), the shorter cysteine is incapable of forming hydrogen bond with the side chain of nearby Asp517 due to its reduced size and lower polarizability. As a consequence, a slight local perturbation of the loop conformation in the PAX7 binding domain of the PAXBP1 protein was observed. Our findings suggest that the pathogenic variant in PAX binding protein underlies distinct syndrome of global developmental delay and myopathic hypotonia. This clinical report should prompt a search for mutations in PAXBP1 in patients presenting with developmental delay and hypotonia. Moreover, these results imply that establishment of PAXBP1 targets and its spatiotemporal interaction will help in understanding of development of cerebellar and will provide basis for developing therapeutic approaches.

Zebrafish - An emerging model to explore thyroid hormone transporters and psychomotor retardation.

Thyroid hormones (THs) regulate a variety of fundamental physiological processes, including the development and maintenance of the brain. For decades, it was thought that THs enter the cells by passive diffusion. However, it is now clear that TH transport across the cell membrane requires specific transporter proteins that facilitate the uptake and efflux of THs. Several thyroid hormone transmembrane transporters (THTTs) have been identified, including monocarboxylate transporter 8 (MCT8), MCT10, and organic anion transporting polypeptide 1C1 (OATP1C1). The critical role of THTTs in regulating metabolism and brain function is demonstrated in the Allan-Herndon-Dudley syndrome (AHDS), an X-linked psychomotor retardation associated with mutations in the MCT8/SLC16A2 gene. In addition to traditional research on humans, cell-lines, and rodents, the zebrafish has recently emerged as an attractive model to study THTTs and neuroendocrinological-related disorders. In this review, we describe the unique contribution of zebrafish studies to the understanding of the functional role of THTTs in live animals, and how this transparent vertebrate model can be used for translational studies on TH-related disorders.

Thyroid hormone transporters--functions and clinical implications.

The cellular influx and efflux of thyroid hormones are facilitated by transmembrane protein transporters. Of these transporters, monocarboxylate transporter 8 (MCT8) is the only one specific for the transport of thyroid hormones and some of their derivatives. Mutations in SLC16A2, the gene that encodes MCT8, lead to an X-linked syndrome with severe neurological impairment and altered concentrations of thyroid hormones. Histopathological analysis of brain tissue from patients who have impaired MCT8 function indicates that brain lesions start prenatally, and are most probably the result of cerebral hypothyroidism. A Slc16a2 knockout mouse model has revealed that Mct8 is an important mediator of thyroid hormone transport, especially T3, through the blood-brain barrier. However, unlike humans with an MCT8 deficiency, these mice do not have neurological impairment. One explanation for this discrepancy could be differences in expression of the T4 transporter OATP1C1 in the blood-brain barrier; OATP1C1 is more abundant in rodents than in primates and permits the passage of T4 in the absence of T3 transport, thus preventing full cerebral hypothyroidism. In this Review, we discuss the relevance of thyroid hormone transporters in health and disease, with a particular focus on the pathophysiology of MCT8 mutations.

In vitro and mouse studies supporting therapeutic utility of triiodothyroacetic acid in MCT8 deficiency.

Monocarboxylate transporter 8 (MCT8) transports thyroid hormone (TH) across the plasma membrane. Mutations in MCT8 result in the Allan-Herndon-Dudley syndrome, comprising severe psychomotor retardation and elevated serum T3 levels. Because the neurological symptoms are most likely caused by a lack of TH transport into the central nervous system, the administration of a TH analog that does not require MCT8 for cellular uptake may represent a therapeutic strategy. Here, we investigated the therapeutic potential of the biologically active T3 metabolite Triac (TA3) by studying TA3 transport, metabolism, and action both in vitro and in vivo. Incubation of SH-SY5Y neuroblastoma cells and MO3.13 oligodendrocytes with labeled substrates showed a time-dependent uptake of T3 and TA3. In intact SH-SY5Y cells, both T3 and TA3 were degraded by endogenous type 3 deiodinase, and they influenced gene expression to a similar extent. Fibroblasts from MCT8 patients showed an impaired T3 uptake compared with controls, whereas TA3 uptake was similar in patient and control fibroblasts. In transfected cells, TA3 did not show significant transport by MCT8. Most importantly, treatment of athyroid Pax8-knockout mice and Mct8/Oatp1c1-double knockout mice between postnatal days 1 and 12 with TA3 restored T3-dependent neural differentiation in the cerebral and cerebellar cortex, indicating that TA3 can replace T3 in promoting brain development. In conclusion, we demonstrated uptake of TA3 in neuronal cells and in fibroblasts of MCT8 patients and similar gene responses to T3 and TA3. This indicates that TA3 bypasses MCT8 and may be used to improve the neural status of MCT8 patients.

Transporters MCT8 and OATP1C1 maintain murine brain thyroid hormone homeostasis.

Allan-Herndon-Dudley syndrome (AHDS), a severe form of psychomotor retardation with abnormal thyroid hormone (TH) parameters, is linked to mutations in the TH-specific monocarboxylate transporter MCT8. In mice, deletion of Mct8 (Mct8 KO) faithfully replicates AHDS-associated endocrine abnormalities; however, unlike patients, these animals do not exhibit neurological impairments. While transport of the active form of TH (T3) across the blood-brain barrier is strongly diminished in Mct8 KO animals, prohormone (T4) can still enter the brain, possibly due to the presence of T4-selective organic anion transporting polypeptide (OATP1C1). Here, we characterized mice deficient for both TH transporters, MCT8 and OATP1C1 (Mct8/Oatp1c1 DKO). Mct8/Oatp1c1 DKO mice exhibited alterations in peripheral TH homeostasis that were similar to those in Mct8 KO mice; however, uptake of both T3 and T4 into the brains of Mct8/Oatp1c1 DKO mice was strongly reduced. Evidence of TH deprivation in the CNS of Mct8/Oatp1c1 DKO mice included highly decreased brain TH content as well as altered deiodinase activities and TH target gene expression. Consistent with delayed cerebellar development and reduced myelination, Mct8/Oatp1c1 DKO mice displayed pronounced locomotor abnormalities. Intriguingly, differentiation of GABAergic interneurons in the cerebral cortex was highly compromised. Our findings underscore the importance of TH transporters for proper brain development and provide a basis to study the pathogenic mechanisms underlying AHDS.

Cellular and ultra structural evidence for cytoskeletal localization of prolyl endopeptidase-like protein in neurons.

The biochemical properties and subcellular localization of prolyl endopeptidase (PREP) in brain are well characterized and its implications in the realization of cognitive processes and in the pathogenesis of neurodegenerative disorders are a matter of intensive investigation. In contrast, very little is known about its homolog, the PREP-like protein (PREPL). In order to obtain initial hints about the involvement of PREPL in physiological processes, a differential proteomic screen was performed with human skin fibroblasts from controls and patients with PREPL deficiency (hypotonia-cystinuria syndrome). The majority of affected proteins represented cytoskeletal proteins, including caldesmon, tropomyosin α3 chain, lamin A, ß-actin, γ-actin, vimentin and zyxin. Therefore, the analysis of PREPL subcellular localization by confocal laser scanning and electron microscopy in mouse neurons was focused on the cytoskeleton. The co-localization of PREPL with cytoskeletal marker proteins such as ß-actin and microtubulin-associated protein-2 was observed, in addition to the presence of PREPL within Golgi apparatus and growth cones. In the mouse brain, PREPL is neuronally expressed and highly abundant in neocortex, substantia nigra and locus coeruleus. This mirrors to some extent the distribution pattern of PREP and points toward redundant functions of both proteins. In the human neocortex, PREPL immunostaining was found in the cytoplasm and in neuropil, in particular of layer V pyramidal neurons. This staining was reduced in the neocortex of Alzheimer's disease (AD) patients. Moreover, in AD brains, PREPL immunoreactivity was observed in the nucleus and in varicose neuritic processes. Our data indicate physiological functions of PREPL associated with the cytoskeleton, which may be affected under conditions of cytoskeletal degeneration.

Effects of tamsulosin and silodosin on isolated albino and pigmented rabbit iris dilators: possible mechanism of intraoperative floppy-iris syndrome.

PURPOSE: To determine the mechanism of intraoperative floppy-iris syndrome (IFIS) by examining the binding affinity of tamsulosin and silodosin to α-receptors and melanin pigment using control and α(2)-blocker chronically administered in rabbit models. SETTING: Department of Ophthalmology, Kitasato University School of Medicine, Kanagawa, Japan. DESIGN: Experimental study. METHODS: The study was performed in isolated albino and pigmented rabbit iris dilators using pharmacologic and morphologic examinations. RESULTS: For pharmacologic examinations, the mean pK(B) values (pK(B) = -log K(B), where -log K(B) is the equilibrium dissociation constant of the antagonist-receptor complex) of tamsulosin in albino and pigmented rabbits were 9.10 and 8.08 and those of silodosin, 10.3 and 8.11, respectively. The pK(B) values of tamsulosin and silodosin in albino rabbits were significantly higher than in pigmented rabbits. In the isolated rabbit iris dilator, the maximum contraction evoked by 10(-3) mol/L phenylephrine gradually decreased by repetitive application in the chronic α-blocker-administered models. For morphologic examinations, the sizes of the pigment granules of pigment epitheliums for the α-blocker-administered models were irregular. The shape of shared nucleus of dilator muscles and pigment epitheliums changed to lobular, and the dilator muscle layer was thinner than in the control. CONCLUSIONS: The high affinity of α-blockers for α(1)-adrenoreceptors is important in the analysis of the mechanism of IFIS. However, IFIS should not be attributed to long-term binding with receptors alone; the drug-melanin interaction causing dilator muscle atrophy is probably the other important factor in the mechanism of IFIS.