Alan P. Farwell

Thyroid Hormone Regulates the Expression of Laminin in the Developing Rat Cerebellum

In the rat cerebellum, migration of neurons from the external granular layer to the internal granular layer occurs postnatally and is dependent upon the presence of thyroid hormone. In hypothyroidism, many neurons fail to complete their migration and die. Key guidance signals to these migrating neurons are provided by laminin, an extracellular matrix protein that is fixed to the surface of astrocytes. Expression of laminin in the brain is developmentally timed to coincide with neuronal growth spurts. In this study, we examined the role of thyroid hormone on the expresssion and distribution of laminin in the rat cerebellum. We show that laminin content steadily increased 2- to 3-fold from birth to maximal levels on postnatal day 8–10 then steadily decreased to a plateau by postnatal day 12 in the euthyroid cerebellum. Immunoreactive laminin appeared in the molecular layer of the euthyroid cerebellum by postnatal day 4, reached maximal intensity by postnatal day 8–10, and was gone by postnatal day 14. In co...

Thyroid Hormone Regulates the Extracellular Organization of Laminin on Astrocytes

Astrocytes produce laminin, a key extracellular matrix guidance molecule in the developing brain. Laminin is bound to transmembrane receptors on the surface of astrocytes known as integrins, which are, in turn, bound to the microfilament meshwork inside the astrocyte. Previous studies have shown that T4 regulates the pattern of integrin distribution in astrocytes by modulating the organization of the microfilaments. In this study, the effect of thyroid hormone on the secretion and topology of laminin in astrocytes was examined. Linear arrays of secreted laminin were observed on the surface of the T4-treated astrocytes within 10 h after seeding the cells onto poly-d-lysine-coated coverslips and became an organized meshwork by 24 h. In contrast, little if any laminin was identified on the surface of either hormone-deficient or T3-treated cells until 36 h after seeding and then was restricted to punctate deposits. Secretion of laminin into the medium by hormone-deficient and T3-treated cells was significantl...

Tissue-engineered spinal cord

LIMITED SUCCESS has been reported in restoring function to spinal cord-injured rodents. Lower limb paralysis caused by complete spinal cord transection in neonatal rats less than 14 days of age may resolve, presumably because of the plasticity of the still-developing central nervous system (CNS). Less success has been achieved in functional repair of the injured adult spinal cord. Implants of immobilized nerve growth factor (NGF) appear to enhance the regrowth of ascending sensory axons across spinal cord gaps in adult rats. Limited but progressive improvement (over a 6-month period) of hind limb function in spinal cord-transected adult rats has been observed over a 6-month period after bridging the transected cord with multiple intercostal nerve grafts. In addition, much has been learned recently about the biology of stem cells. Stem cells isolated from the brain and the spinal cord of both neonatal and adult mice reportedly retain the potential to differentiate into neurons, astrocytes, and oligodendrocytes. Undifferentiated stem cells have been reported to propagate in culture by adding epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to the growth medium. Recent reports suggest that transplantation of immortalized neuronal stem cells into the spinal cord and cerebral cortex led to differentiation of such cells into site-specific neuronal and glial cells and also some functional recovery of the associated defects seen in spinal cord injury, Sly disease, and the myelin degenerative disorder found in the shiverer (shi) mouse. Thus, neuronal stem cells propagated in vitro appear to retain the developmental plasticity necessary to respond to local environmental cues. We postulated that resected segments of the spinal cord could be regenerated in adult rats by implanting spinal cord progenitor cells associated with an appropriate scaffolding or matrix.

Cloning, Expression, and Functional Characterization of the Substrate Binding Subunit of Rat Type II Iodothyronine 5′-Deiodinase

Type II iodothyronine 5′-deiodinase catalyzes the bioactivation of thyroid hormone in the brain. In astrocytes, this ∼200-kDa, membrane-bound enzyme is composed of at least one p29 subunit, an ∼60-kDa, cAMP-induced activation protein, and one or more uniden- tified catalytic subunit(s). Recently, an artificial type II-like selenodeiodinase was engineered by fusing two independent cDNAs together; however, no native type II selenodeiodinase polypeptide is translated in the brain or brown adipose tissue of rats. These data suggest that the native type II 5′-deiodinase in rat brain is unrelated to this artificial selenoprotein. In this report, we describe the cloning of the 29-kDa subunit (p29) of type II 5′-deiodinase from a λzapII cDNA library prepared from cAMP-induced astrocytes. The 3.3-kilobase (kb) cDNA encodes an ∼30-kDa, 277-amino acid long, hydrophobic protein lacking selenocysteine. Northern blot analysis showed that a 3.5-kb p29 mRNA was present in tissues showing type II 5′-deiodinase activity such as brain and cAMP-stimulated astrocytes. Domain-specific, anti-p29 antibodies specifically immunoprecipitated enzyme activity. Overexpression of exogenous p29 or a green fluorescence protein (GFP)-tagged p29 fusion protein led to a >100-fold increase in deiodinating activity in cAMP-stimulated astrocytes, and the increased activity was specifically immunoprecipitated by anti-GFP antibodies. Steady-state reaction kinetics of the enzyme in GFP-tagged p29-expressing astrocytes are identical to those of the native enzyme in brain. Direct injection of replication-deficient Ad5-p29GFP virus particles into the cerebral cortex of neonatal rats leads to a ∼2-fold increase in brain type II 5′-deiodinating activity. These data show 1) that the 3.3-kb p29 cDNA encodes an essential subunit of rat type II iodothyronine 5′-deiodinase and 2) identify the first non-selenocysteine containing subunit of the deiodinase family of enzymes.

Catalytic Activity of Type II Iodothyronine 5′-Deiodinase Polypeptide Is Dependent upon a Cyclic AMP Activation Factor

Type II iodothyronine 5′-deiodinase is an ∼200-kDa multimeric enzyme in the brain that catalyzes the deiodination of thyroxine (T4) to its active metabolite, 3,5,3′-triiodothyronine. In astrocytes, cAMP stimulation is required to express catalytically active type II iodothyronine 5′-deiodinase. The affinity ligand N-bromoacetyl-L-T4 specifically labels the 29-kDa substrate-binding subunit (p29) of this enzyme in cAMP-stimulated astrocytes. To determine the requirements for cAMP-induced activation of this enzyme, we optimized N-bromoacetyl-L-T4 labeling of p29 in astrocytes lacking type II iodothyronine 5′-deiodinase activity and examined the effects of cAMP on the hydrodynamic properties and subcellular location of the enzyme. We show that the p29 subunit is expressed in unstimulated astrocytes and requires 10-fold higher concentrations of N-bromoacetyl-L-T4 to achieve incorporation levels equal to those of p29 in cAMP-stimulated cells. Gel filtration showed that p29 was part of a multimeric membrane-associated complex in both cAMP-stimulated and unstimulated astrocytes and that cAMP stimulation led to an increase of ∼60 kDa in the mass of the holoenzyme. In unstimulated astrocytes, p29 resides in the perinuclear space. Cyclic AMP stimulation leads to the translocation of p29 to the plasma membrane coincident with the appearance of deiodinating activity. These data show that cAMP-dependent activation of type II iodothyronine 5′-deiodinase activity results from the synthesis of additional activating factor(s) that associates with inactive enzyme and leads to the translocation of enzyme polypeptide(s) from the perinuclear space to the plasma membrane.

Myosin V Plays an Essential Role in the Thyroid Hormone-dependent Endocytosis of Type II Iodothyronine 5*-Deiodinase*

In astrocytes, thyroxine modulates type II iodothyronine 5′-deiodinase levels by initiating the binding of the endosomes containing the enzyme to microfilaments, followed by actin-based endocytosis. Myosin V is a molecular motor thought to participate in vesicle trafficking in the brain. In this report, we developed an in vitro actin-binding assay to characterize the thyroid hormone-dependent binding of endocytotic vesicles to microfilaments. Thyroxine and reverse triiodothyronine (EC50 levels ∼1 nm) were >100-fold more potent than 3,5,3′-triiodothyronine in initiating vesicle binding to actin fibers in vitro. Thyroxine-dependent vesicle binding was calcium-, magnesium-, and ATP-dependent, suggesting the participation of one or more myosin motors, presumably myosin V. Addition of the myosin V globular tail, lacking the actin-binding head, specifically blocked thyroid hormone-dependent vesicle binding, and direct binding of the myosin V tail to enzyme-containing endosomes was thyroxine-dependent. Progressive NH2-terminal deletion of the myosin V tail and domain-specific antibody inhibition studies revealed that the thyroxine-dependent vesicle-tethering domain was localized to the last 21 amino acids of the COOH terminus. These data show that myosin V is responsible for thyroid hormone-dependent binding of primary endosomes to the microfilaments and suggest that this motor mediates the actin-based endocytosis of the type II iodothyronine deiodinase.

Degradation and Recycling of the Substrate-binding Subunit of Type II Iodothyronine 5′-Deiodinase in Astrocytes

Thyroxine dynamically regulates levels of type II iodothyronine 5′-deiodinase (5′D-II) by modulating enzyme inactivation and targeting the enzyme to different pathways of internalization. 5′D-II is an ∼200-kDa multimeric protein containing a 29-kDa substrate-binding subunit (p29) and an unknown number of other subunits. In the absence of thyroxine (T4), p29 is slowly endocytosed and transported to the lysosomes. T4 treatment rapidly activates an actin-mediated endocytotic pathway and targets the enzyme to the endosomes. In this study, we have characterized the influence of T4 on the intracellular trafficking of 5′D-II. We show that T4 accelerates the rate of 5′D-II inactivation by translocating the enzyme to the interior of the cell and by sequestering p29 in the endosomal pool without accelerating the rate of degradation of p29. This dichotomy between the rapid inactivation of catalytic activity and the much slower degradation of p29 is consistent with the reuse of p29 in the production of 5′D-II activity. Immunocytochemical analysis with a specific anti-p29 IgG shows that pulse affinity-labeled p29 reappears on the plasma membrane ∼2 h after enzyme internalization in the presence of T4, indicating that p29 is recycled. Despite the ability of p29 to be recycled in the T4-treated cell, 5′D-II catalytic activity requires ongoing protein synthesis, presumably of another enzyme component(s) or an accessory enzyme-related protein. In the absence of T4, enzyme inactivation and p29 degradation are temporally linked, and pulse affinity-labeled p29 is internalized and sequestered in discrete intracellular pools. These data suggest that T4 regulates fundamental processes involved with the turnover of integral membrane proteins and participates in regulating the inter-relationships between the degradation, recycling, and synthetic pathways.

Selenium-regulated translation control of heterologous gene expression: Normal function of selenocysteine-substituted gene products

In eukaryotes, the synthesis of selenoproteins depends on an exogenous supply of selenium, required for synthesis of the novel amino acid, selenocysteine, and on the presence of a “selenium translation element” in the 3′ untranslated region of mRNA. The selenium translation element is required to re‐interpret the stop codon, UGA, as coding for selenocysteine incorporation and chain elongation. Messenger RNA lacking the selenium translation element and/or an inadequate selenium supply lead to chain termination at the UGA codon. We exploited these properties to provide direct translational control of protein(s) encoded by transfected cDNAs. Selenium‐dependent translation of mRNA transcribed from target cDNA was conferred by mutation of an in‐frame UGU, coding for cysteine, to UGA, coding for either selenocysteine or termination, then fusing the mutated coding region to a 3′ untranslated region containing the selenium translation element of the human cellular glutathione peroxidase gene. In this study, the biological consequences of placing this novel amino acid in the polypeptide chain was examined with two proteins of known function: the rat growth hormone receptor and human thyroid hormone receptor β1. UGA (opal) mutant‐STE fusion constructs of the cDNAs encoding these two polypeptides showed selenium‐dependent expression and their selenoprotein products maintained normal ligand binding and signal transduction. Thus, integration of selenocysteine had little or no consequence on the functional activity of the opal mutants; however, opal mutants were expressed at lower levels than their wild‐type counterparts in transient expression assays. The ability to integrate this novel amino acid at predetermined positions in a polypeptide chain provides selenium‐dependent translational control to the expression of a wide variety of target genes, allows facile 75Se radioisotopic labeling of the heterologous proteins, and permits site‐specific heavy atom substitution.

Real-time Visualization of Processive Myosin 5a-mediated Vesicle Movement in Living Astrocytes

Recycling endosomes in astrocytes show hormone-regulated, actin fiber-dependent delivery to the endosomal sorting pool. Recycling vesicle trafficking was followed in real time using a fusion protein composed of green florescent protein coupled to the 29-kDa subunit of the short-lived, membrane-bound enzyme type 2 deiodinase. Primary endosomes budded from the plasma membrane and oscillated near the cell periphery for 1–4 min. The addition of thyroid hormone triggered the processive, centripetal movement of the recycling vesicle in linear bursts at velocities of up to 200 nm/s. Vesicle migration was hormone-specific and blocked by inhibitors of actin polymerization and myosin ATPase. Domain mapping confirmed that the hormone-dependent vesicle-binding domain was located at the C terminus of the motor. In addition, the interruption of normal dimerization of native myosin 5a monomers inactivated vesicle transport, indicating that single-headed myosin 5a motors do not transport cargo in situ. This is the first demonstration of processive hormone-dependent myosin 5a movement in living cells.