Scott Ribich

Cellular and Molecular Basis of Deiodinase-Regulated Thyroid Hormone Signaling

The iodothyronine deiodinases initiate or terminate thyroid hormone action and therefore are critical for the biological effects mediated by thyroid hormone. Over the years, research has focused on their role in preserving serum levels of the biologically active molecule T(3) during iodine deficiency. More recently, a fascinating new role of these enzymes has been unveiled. The activating deiodinase (D2) and the inactivating deiodinase (D3) can locally increase or decrease thyroid hormone signaling in a tissue- and temporal-specific fashion, independent of changes in thyroid hormone serum concentrations. This mechanism is particularly relevant because deiodinase expression can be modulated by a wide variety of endogenous signaling molecules such as sonic hedgehog, nuclear factor-kappaB, growth factors, bile acids, hypoxia-inducible factor-1alpha, as well as a growing number of xenobiotic substances. In light of these findings, it seems clear that deiodinases play a much broader role than once thought, with great ramifications for the control of thyroid hormone signaling during vertebrate development and metamorphosis, as well as injury response, tissue repair, hypothalamic function, and energy homeostasis in adults.

Promoter Choice Determines Splice Site Selection in Protocadherin α and γ Pre-mRNA Splicing

A family of mammalian protocadherin (Pcdh) proteins is encoded by three closely linked gene clusters (alpha, beta, and gamma). Multiple alpha and gamma Pcdh mRNAs are expressed in distinct patterns in the nervous system and are generated by alternative pre-mRNA splicing between different variable exons and three constant exons within each cluster. We show that each Pcdh variable exon is preceded by a promoter and that promoter choice determines which variable exon is included in a Pcdh mRNA. In addition, we provide evidence that alternative splicing of variable exons within a gene cluster occurs via a cis-splicing mechanism. However, virtually every variable exon can engage in trans-splicing with constant exons from another cluster, albeit at a far lower level.

Membrane Association and Protein Conformation of α-Synuclein in Intact Neurons EFFECT OF PARKINSON′S DISEASE-LINKED MUTATIONS

Two missense mutations (Ala-30 → Pro and Ala-53 → Thr) in the gene encoding α-synuclein are associated with rare autosomal dominant forms of familial Parkinsons disease. In addition, α-synuclein is an abundant component of Lewy bodies in sporadic Parkinsons disease and diffuse Lewy body disease. However, the normal conformation of α-synuclein, its cellular localization in neurons, and the effects of the mutations remain to be determined. In the present study, we examine these questions using sensitive fluorescence resonance energy transfer techniques. Transient transfection of α-synuclein expression constructs into primary cortical neurons and counterstaining with the lipophilic fluorescent marker, DiI, demonstrates a close association between α-synuclein and cellular membranes. Both the N- and C-terminal regions of α-synuclein are tightly associated with membranes. A weak interaction also occurs between the N and C termini themselves. The Parkinsons disease-associated mutations have no effect on membrane interaction; however, the Ala-30 → Pro mutation alters the three-dimensional conformation of α-synuclein, as measured by significantly increased fluorescence resonance energy transfer between the N and C termini.

Identification of long-range regulatory elements in the protocadherin-α gene cluster

The clustered protocadherins (Pcdh) are encoded by three closely linked gene clusters (Pcdh-α, -β, and -γ) that span nearly 1 million base pairs of DNA. The Pcdh-α gene cluster encodes a family of 14 distinct cadherin-like cell surface proteins that are expressed in neurons and are present at synaptic junctions. Individual Pcdh-α mRNAs are assembled from one of 14 “variable” (V) exons and three “constant” exons in a process that involves both differential promoter activation and alternative pre-mRNA splicing. In individual neurons, only one (and rarely two) of the Pcdh α1–12 promoters is independently and randomly activated on each chromosome. Thus, in most cells, this unusual form of monoallelic expression leads to the expression of two different Pcdh-α 1–12 V exons, one from each chromosome. The two remaining V exons in the cluster (Pcdh-αC1 and αC2) are expressed biallelically in every neuron. The mechanisms that underlie promoter choice and monoallelic expression in the Pcdh-α gene cluster are not understood. Here we report the identification of two long-range cis-regulatory elements in the Pcdh-α gene cluster, HS5–1 and HS7. We show that HS5–1 is required for maximal levels of expression from the Pcdh α1–12 and αC1 promoters, but not the Pcdh-αC2 promoter. The nearly cluster-wide requirement of the HS5–1 element is consistent with the possibility that the monoallelic expression of Pcdh-α V exons is a consequence of competition between individual V exon promoters for the two regulatory elements.

Absence of Thyroid Hormone Activation during Development Underlies a Permanent Defect in Adaptive Thermogenesis

Type 2 deiodinase (D2), which is highly expressed in brown adipose tissue (BAT), is an enzyme that amplifies thyroid hormone signaling in individual cells. Mice with inactivation of the D2 pathway (D2KO) exhibit dramatically impaired thermogenesis in BAT, leading to hypothermia during cold exposure and a greater susceptibility to diet-induced obesity. This was interpreted as a result of defective acute activation of BAT D2. Here we report that the adult D2KO BAT has a permanent thermogenic defect that stems from impaired embryonic BAT development. D2KO embryos have normal serum T3 but due to lack of D2-generated T3 in BAT, this tissue exhibits decreased expression of genes defining BAT identity [i.e. UCP1, PGC-1alpha and Dio2 (nonfunctional)], which results in impaired differentiation and oxidative capacity. Coinciding with a reduction of these T3-responsive genes, there is oxidative stress that in a cell model of brown adipogenesis can be linked to decreased insulin signaling and decreased adipogenesis. This discovery highlights the importance of deiodinase-controlled thyroid hormone signaling in BAT development, where it has important metabolic repercussions for energy homeostasis in adulthood.

Type 2 Deiodinase Expression Is Induced by Peroxisomal Proliferator-Activated Receptor-γ Agonists in Skeletal Myocytes

The thyroid hormone activating type 2 deiodinase (D2) is known to play a role in brown adipose tissue-mediated adaptive thermogenesis in rodents, but the finding of D2 in skeletal muscle raises the possibility of a broader metabolic role. In the current study, we examined the regulation of the D2 pathway in primary skeletal muscle myoblasts taken from both humans and mice. We found that pioglitazone treatment led to a 1.6- to 1.9-fold increase in primary human skeletal myocyte D2 activity; this effect was seen with other peroxisomal proliferator-activated receptor-gamma agonists. D2 activity in primary murine skeletal myotubes increased 2.8-fold in response to 5 microM pioglitazone and 1.6-fold in response to 5 nM insulin and increased in a dose-dependent manner in response to lithocholic acid (maximum response at 25 microM was approximately 3.8-fold). We compared Akt phosphorylation in primary myotubes derived from wild-type and D2 knockout (D2KO) mice: phospho-Akt was reduced by 50% in the D2KO muscle after 1 nM insulin exposure. Expression of T(3)-responsive muscle genes via quantitative RT-PCR suggests that D2KO cells have decreased thyroid hormone signaling, which could contribute to the abnormalities in insulin signaling. D2 activity in skeletal muscle fragments from both murine and human sources was low, on the order of about 0.01 fmol/min . mg of muscle protein. The phenotypic changes seen with D2KO cells support a metabolic role for D2 in muscle, hinting at a D2-mediated linkage between thyroid hormone and insulin signaling, but the low activity calls into question whether skeletal muscle D2 is a major source of plasma T(3).

Subcellular localization of α-synuclein in primary neuronal cultures: Effect of missense mutations

Numerous recent observations have implicated alpha-synuclein in the pathogenesis of several neurodegenerative diseases, including Parkinsons disease, Alzheimers disease, dementia with Lewy bodies and multiple-system atrophy. Two missense mutations in the gene for alpha-synuclein have been identified in some cases of familial Parkinsons disease and it is thought that these may disrupt the normal structure of the protein and thus promote aggregation into Lewy body filaments. Here, we examine the subcellular localization of alpha-synuclein in primary cortical neurons maintained in a monolayer culture. The protein has widespread expression throughout neurons, including the nucleus, and has a discete localization in the neurites of more mature neurons, reminiscent of synaptic specializations. Interestingly, in a subpopulation of cortical neurons transfected at 13 days in vitro, we find that alpha-synuclein appears to aggregate into distinct punctate inclusions in the cytoplasm and proximal neurites. Unlike Lewy bodies, these structures are not ubiquitin positive. These regions of alpha-synuclein accumulation are observed following transfections with wild-type, Ala30Pro or Ala53Thr alpha-synuclein; neither mutation alters their frequency.

The chemical chaperones tauroursodeoxycholic and 4-phenylbutyric acid accelerate thyroid hormone activation and energy expenditure

Exposure of cell lines endogenously expressing the thyroid hormone activating enzyme type 2 deiodinase (D2) to the chemical chaperones tauroursodeoxycholic acid (TUDCA) or 4‐phenylbutiric acid (4‐PBA) increases D2 expression, activity and T3 production. In brown adipocytes, TUDCA or 4‐PBA induced T3‐dependent genes and oxygen consumption (∼2‐fold), an effect partially lost in D2 knockout cells. In wild type, but not in D2 knockout mice, administration of TUDCA lowered the respiratory quotient, doubled brown adipose tissue D2 activity and normalized the glucose intolerance associated with high fat feeding. Thus, D2 plays a critical role in the metabolic effects of chemical chaperones.

Mice lacking Pctp /StarD2 exhibit increased adaptive thermogenesis and enlarged mitochondria in brown adipose tissue

Pctp−/− mice that lack phosphatidylcholine transfer protein (Pctp) exhibit a marked shift toward utilization of fatty acids for oxidative phosphorylation, suggesting that Pctp may regulate the entry of fatty acyl-CoAs into mitochondria. Here, we examined the influence of Pctp expression on the function and structure of brown adipose tissue (BAT), a mitochondrial-rich, oxidative tissue that mediates nonshivering thermogenesis. Consistent with increased thermogenesis, Pctp−/− mice exhibited higher core body temperatures than wild-type controls at room temperature. During a 24 h cold challenge, Pctp−/− mice defended core body temperature efficiently enough that acute, full activation of BAT thermogenic genes did not occur. Brown adipocytes lacking Pctp harbored enlarged and elongated mitochondria. Consistent with increased fatty acid utilization, brown adipocytes cultured from Pctp−/− mice exhibited higher oxygen consumption rates in response to norepinephrine. The absence of Pctp expression during brown adipogenesis in vitro altered the expression of key transcription factors, which could be corrected by adenovirus-mediated overexpression of Pctp early but not late during the differentiation. Collectively, these findings support a key role for Pctp in limiting mitochondrial oxidation of fatty acids and thus regulating adaptive thermogenesis in BAT.