<p>Correa, P. 2002. Vitamin D and its receptor in parathyroid tumors. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1186. 49 pp. Uppsala. ISBN 91-554-541-0 </p><p>Hyperparathyroidism (HPT) is characterized by tumor development in the parathyroid glands and excessive production of parathyroid hormone. Parathyroidectomy is the only considered therapy for the majority of patients. </p><p>LOH (loss of heterozygosity) analysis revealed putative tumor suppressor genes on chromosome regions 1p and 11q in tumors from patients with truly mild hypercalcemia.</p><p>Active vitamin D [1,25(OH)2D3] and its receptors, the vitamin D receptor (VDR), are essential regulators of the calcium homeostasis and are involved in HPT development. The VDR-FokI polymorphism, coupled to bone mineral density, was found not to be associated to development of primary HPT (pHPT). The total VDR mRNA levels is reduced in adenomas of pHPT as well as in hyperplastic glands of secondary HPT (sHPT). The VDR exon 1f transcripts were exclusively downregulated in the adenomas of pHPT, suggesting default regulation of the tissue-specially expressed VDR 1f promoter. The cytochrome P450 enzymes responsible for synthesis and degradation of 1,25(OH)2D3, namely vitamin D3 25-hydroxylase (25-hydroxylase), 25-hydroxyvitamin D3 1a-hydroxylase (1a-hydroxylase) and 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) were found to be expressed in normal and pathological parathyroid glands. Tumors of pHPT and sHPT demonstrated increased 1a-hydroxylase and reduced 24- and 25-hydroxylase expression, suggesting an augmented local production of active vitamin D. In contrast, parathyroid carcinomas displayed reduced expression of all three hydroxylases. The gained knowledge of vitamin D metabolism and catabolism in parathyroid tumors may indicate possibilities for novel treatment of sHPT and perhaps pHPT.</p>

myotubesProper muscle function depends upon the fine tuning of the different molecular components of the neuromuscular junction (NMJ). Synaptic acetylcholinesterase (AChE) is responsible for rapidly terminating neurotransmission. Neuroscientists in the field have elucidated many aspects of synaptic AChE structure, function, and localization during the last 75 years. Nevertheless, how the enzyme is regulated and targeted to the NMJ is not completely understood. In skeletal muscle the synaptic AChE form derives from two separate genes encoding the catalytic and the collagenic tail (ColQ) subunits respectively. ColQ-AChE expression is regulated by muscle activity; however, how this regulation takes place remains poorly understood. We found that over or down-regulation of ColQ is sufficient to change the levels of AChE activity by promoting assembly of higher order oligomeric forms including the collagen-tailed forms. Furthermore, when peptides containing the Proline Rich Attachment Domain (PRAD), the region of ColQ that interacts with the AChE, are fed to muscle cells or cell lines expressing AChE, they are taken up by the cells and retrogradely transported to the endoplasmic reticulum (ER)/Golgi network where they induce assembly of newly synthesize AChE into tetramers. This results in an increase, as a consequence, in total cell associated AChE activity and active tetramer secretion, making synthetic PRAD peptides potential candidates for the treatment of organophosphate pesticides and nerve gas poisoning. To study the developmental regulation of ColQ-AChE we determined the levels of ColQ and ColQ mRNA in primary quail muscle cells in culture and as a function of muscle activity. Surprisingly, we found dissociation between transcription and translation of ColQ from its assembly into ColQ-AChE indicating the importance of posttranslational controls in the regulation of AChE folding and assembly. Furthermore, we found that the vast majority of the ColQ molecules in QMCs are not assembled into ColQ-AChE, suggesting that they can have alternative function(s). Finally, we found that the levels of ER molecular chaperones calnexin, calreticulin, and particularly protein disulfide isomerase are regulated by muscle activity and they correlate with the levels of ColQ-AChE. More importantly, our results suggest that newly synthesized proteins compete for chaperone assistance during the folding process.

Correa, P. 2002. Vitamin D and its receptor in parathyroid tumors. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1186. 49 pp. Uppsala. ISBN 91-554-541-0 Hyperparathyroidism (HPT) is characterized by tumor development in the parathyroid glands and excessive production of parathyroid hormone. Parathyroidectomy is the only considered therapy for the majority of patients. LOH (loss of heterozygosity) analysis revealed putative tumor suppressor genes on chromosome regions 1p and 11q in tumors from patients with truly mild hypercalcemia. Active vitamin D [1,25(OH)2D3] and its receptors, the vitamin D receptor (VDR), are essential regulators of the calcium homeostasis and are involved in HPT development. The VDR-FokI polymorphism, coupled to bone mineral density, was found not to be associated to development of primary HPT (pHPT). The total VDR mRNA levels is reduced in adenomas of pHPT as well as in hyperplastic glands of secondary HPT (sHPT). The VDR exon 1f transcripts were exclusively downregulated in the adenomas of pHPT, suggesting default regulation of the tissue-specially expressed VDR 1f promoter. The cytochrome P450 enzymes responsible for synthesis and degradation of 1,25(OH)2D3, namely vitamin D3 25-hydroxylase (25-hydroxylase), 25-hydroxyvitamin D3 1a-hydroxylase (1a-hydroxylase) and 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) were found to be expressed in normal and pathological parathyroid glands. Tumors of pHPT and sHPT demonstrated increased 1a-hydroxylase and reduced 24- and 25-hydroxylase expression, suggesting an augmented local production of active vitamin D. In contrast, parathyroid carcinomas displayed reduced expression of all three hydroxylases. The gained knowledge of vitamin D metabolism and catabolism in parathyroid tumors may indicate possibilities for novel treatment of sHPT and perhaps pHPT.

Parkinson’s disease (PD) is a common neurodegenerative disorder that is characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra and motor deficits. Although the majority of PD cases are sporadic, several genetic defects in rare familial cases have been identified. Animal models of these genetic defects have been created and have provided unique insights into the molecular mechanisms of the pathogenesis of PD. However, the etiology of PD is still not well understood. Here, taking advantage of the unique features offered by zebrafish, I characterized the functions of PINK1 (PTEN-induced kinase 1) gene, which is associated with recessive familial PD, in the development and survival of DA neurons. In zebrafish, antisense morpholino knockdown of pink1 did not cause a large loss of DA neurons in the ventral diencephalon (vDC), but the patterning of these neurons and their projections were perturbed. The pink1 morphants also showed impaired response to touch stimuli and reduced swimming behaviour. Moreover, the pink1 knockdown caused a significant reduction in the number of mitochondria, as well as mitochondrial morphological defects such as smaller size or loss of cristae, thus affecting mitochondrial function. These results suggest that zebrafish pink1 plays conserved important roles in the development of DA neurons and in the mitochondrial morphology and function. To better follow DA neurons after injury or administration of toxins, I generated a transgenic zebrafish line, Tg(dat:EGFP), in which the green fluorescent protein (GFP) is expressed under the control of cis-regulatory elements of dopamine transporter (dat). In Tg(dat:EGFP) fish, all major groups of DA neurons are correctly labeled with GFP, especially the ones in the vDC, which are analogous to the ascending midbrain DA neurons in mammals. In addition, we observed that the DA neurons in the vDC could partially be replaced after severe laser cell ablation. This suggests that zebrafish may have the unique capacity of regenerating DA neurons after injury. Taken together, my studies suggested that zebrafish could be a useful alternative animal model for the study of the molecular mechanisms underlying PD and for the screening of potential therapeutic compounds for PD.

Neurons in the Locus coeruleus (LC) play an important role in the central CO2 chemosensitivity. However, the molecular mechanisms for neuronal CO2 chemosensitivity remain unclear. To demonstrate the expression of pH/CO2 sensitive ion channels, we screened the inward rectifier K+ channels (Kir) and transient receptor protein (TRP) channels, as parallel studies in this lab suggested that certain Kir and TRP channels are involved in neuronal responses to high levels of CO2. Our results showed that several members of the Kir and TRP channel families were robustly expressed in the LC neurons at the mRNA level. Of particular interest are TRPC5, Kir4.1 and Kir5.1 channels that are all pH-sensitive. The rich expression of various pH-sensitive Kir and TRP channels suggests that these ion channels are likely to play a role in the chemosensitivity of LC neurons.

Parkinson’s disease (PD) is a common neurodegenerative disorder that is characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra and motor deficits. Although the majority of PD cases are sporadic, several genetic defects in rare familial cases have been identified. Animal models of these genetic defects have been created and have provided unique insights into the molecular mechanisms of the pathogenesis of PD. However, the etiology of PD is still not well understood. Here, taking advantage of the unique features offered by zebrafish, I characterized the functions of PINK1 (PTEN-induced kinase 1) gene, which is associated with recessive familial PD, in the development and survival of DA neurons. In zebrafish, antisense morpholino knockdown of pink1 did not cause a large loss of DA neurons in the ventral diencephalon (vDC), but the patterning of these neurons and their projections were perturbed. The pink1 morphants also showed impaired response to touch stimuli and reduced swimming behaviour. Moreover, the pink1 knockdown caused a significant reduction in the number of mitochondria, as well as mitochondrial morphological defects such as smaller size or loss of cristae, thus affecting mitochondrial function. These results suggest that zebrafish pink1 plays conserved important roles in the development of DA neurons and in the mitochondrial morphology and function. To better follow DA neurons after injury or administration of toxins, I generated a transgenic zebrafish line, Tg(dat:EGFP), in which the green fluorescent protein (GFP) is expressed under the control of cis-regulatory elements of dopamine transporter (dat). In Tg(dat:EGFP) fish, all major groups of DA neurons are correctly labeled with GFP, especially the ones in the vDC, which are analogous to the ascending midbrain DA neurons in mammals. In addition, we observed that the DA neurons in the vDC could partially be replaced after severe laser cell ablation. This suggests that zebrafish may have the unique capacity of regenerating DA neurons after injury. Taken together, my studies suggested that zebrafish could be a useful alternative animal model for the study of the molecular mechanisms underlying PD and for the screening of potential therapeutic compounds for PD.

The phenylalanine hydroxylase from Chromobacterium violaceum (CvPheH) is a non-heme iron monooxygenase that catalyzes the hydroxylation of phenylalanine. This study presents the use of kinetic isotope effects (KIE) as mechanistic probes to compare the reactivity of CvPheH and that of the eukaryotic aromatic amino acid hydroxylases. This study also describes the use of different spectroscopic and kinetic techniques to identify the hydroxylating intermediate for this enzyme and the assignment of the NMR backbone resonances of CvPheH. Kinetic isotope effects on aromatic and benzylic hydroxylation were used to establish that bacterial and eukaryotic phenylalanine hydroxylases have similar reactivity. The observed KIE on aromatic hydroxylation of 1.4 was shown to be a combination of an inverse isotope effect on the hydroxylation of the amino acid and a normal isotope effect on a subsequent step in the reaction. An isotope effect on benzylic hydroxylation of 10 was found for CvPheH. This result establishes the similar reactivity for CvPheH and the eukaryotic aromatic amino acid hydroxylases and suggests the involvement of a common hydroxylating intermediate. Kinetic isotope effects were used to study the hydroxylation of the aliphatic substrate cyclohexylalanine. The Dkcat value with [1,2,2,3,3,4,4,5,5,6,6-2H11]- cyclohexylalanine is unity with wild-type CvPheH, suggesting that chemistry is not ratelimiting with this substrate. The intramolecular isotope effect calculated using [1,2,3,4,5,6-2H6]-cyclohexylalanine yields a value of 14. This result is evidence for the involvement of a reactive iron species capable of abstracting a hydrogen atom from the aliphatic carbon in cyclohexylalanine. Analysis of the CvPheH reaction using freeze-quench Mössbauer spectroscopy allowed the detection of an Fe(IV) species in the first turnover of the enzyme. Chemical quench and stopped-flow spectrophotometric methods were used to establish the kinetic competency of the Fe(IV) intermediate as the hydroxylating species. The NMR amide backbone resonances in the HSQC spectrum of CvPheH were assigned to the corresponding amino acid residues using a suite of TROSY-based threedimensional triple resonance experiments. We were able to assign 224 residues out of the 278 assignable residues in CvPheH, this constitutes 81 percent of the assignable protein sequence.

In my dissertation research we used use human wild type α-synuclein gene expression using an adeno-associated viral vector (AAV9) that induced a slowly progressive loss of dopamine (DA) neurons in the Substantia nigra (SN) as one of our animal model of Parkinson’s disease (PD). It is our hypothesis that neuroinflammation predisposes the brain to susceptibility to neurodegenerative diseases. Thus we examined the progression of a PD lesion and examined the manipulations of the immune system to understand further the inflammatory role when we administered exogenous soluble fractalkine. The specific etiology of neurodegeneration in PD is unknown, but the inflammatory mechanisms and free radicals have been postulated to play a central role. α-synuclein is believed to be the one of the main characteristic associated with PD. It has been found inside saclike structures, called lewy bodies. α-synuclein is believed to activate resident microglia worsening the degeneration of the nigrostriatal pathway due to its aggregation. Aggregation increases the production of reactive oxygen species (ROS) released from microglia. The constant release of these factors and prolonged activation of microglia could be the cause that leads to neurodegeneration in the SN. Spirulina, a blue - green algae, has been shown to have anti-oxidant and anti-inflammatory properties. For example, when rats received an intrastriatal injection of 6-OHDA and were fed a spirulina enriched diet for 4 weeks, there was a significant increase in regeneration of DA terminals into the Tyrosine Hydroxylase (TH) -negative zone of the striatum. This regeneration was accompanied by a decrease in microglia activation as determined by immunohistochemistry of major histo compatibility class II (MHC) (OX-6). This suggests that decreases in microglia activation modulate the beneficial effects of spirulina. Another important therapeutic tool we used was fractalkine as an anti-inflammatory treatment. It is known that fractalkine levels are reduced in the brain during aging. For this reason we administered exogenous fractalkine to 6-OHDA model of PD to test the hypothesis that it improved the microenvironment by reducing microglial activation.

The CYP27B1 enzyme that synthesizes 1alpha,25-(OH) 2D, is expressed in chondrocytes, suggesting that local production of 1alpha,25-(OH)2D could play an autocrine or paracrine role in the differentiation of these cells. To test this hypothesis, we have engineered mutant mice that do not express the Cyp27b1 gene in chondrocytes. This led to increased width of the hypertrophic zone of the growth plate at E15.5, increased bone mass in neonatal long bones, and increased expression of the chondrocytic differentiation markers Indian Hedgehog and PTH/PTHrP receptor. VEGF mRNA levels were decreased, accompanied by decreased PECAM-1 immunostaining, suggesting a delay in vascularization. We have also engineered mice overexpressing a Cyp27b1 transgene in chondrocytes. The transgenic mice showed a partial mirror image phenotype compared to the tissue-specific inactivation model. These results support an autocrine/paracrine role of 1alpha,25-(OH) 2D in endochondral ossification and chondrocyte development in vivo. / The CYP24A1 enzyme is involved in the catabolic breakdown of 1alpha,25-(OH)2D but also synthesizes the 24R,25-(OH) 2D metabolite. Studies in chicken suggest a role for 24R,25-(OH) 2D in fracture repair. We induced stabilized transverse mid-diaphysial fractures of the tibia in four-month-old wild-type and Cyp24a1-deficient mice. Examination of the callus sections showed delayed hard callus formation in the homozygous mutant animals compared to wild-type littermates. RT-qPCR showed perturbed levels of type X collagen transcripts in mutant mice at 14 and 21 days post-fracture, reflecting the delayed healing. Rescue of the impaired healing by subcutaneous injection of 24R,25-(OH)2D3 normalized the histological appearance of the callus, static histomorphometric index and type X collagen mRNA expression, while 1alpha,25-(OH)2D 3 did not. These results show that Cyp24a1 deficiency delays fracture repair and strongly suggest that vitamin D metabolites hydroxylated at position 24, such as 24R,25-(OH)2D, play an important role in the mechanisms leading to normal fracture healing.

Parkinson’s disease (PD) is a common neurodegenerative disorder that is characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra and motor deficits. Although the majority of PD cases are sporadic, several genetic defects in rare familial cases have been identified. Animal models of these genetic defects have been created and have provided unique insights into the molecular mechanisms of the pathogenesis of PD. However, the etiology of PD is still not well understood. Here, taking advantage of the unique features offered by zebrafish, I characterized the functions of PINK1 (PTEN-induced kinase 1) gene, which is associated with recessive familial PD, in the development and survival of DA neurons. In zebrafish, antisense morpholino knockdown of pink1 did not cause a large loss of DA neurons in the ventral diencephalon (vDC), but the patterning of these neurons and their projections were perturbed. The pink1 morphants also showed impaired response to touch stimuli and reduced swimming behaviour. Moreover, the pink1 knockdown caused a significant reduction in the number of mitochondria, as well as mitochondrial morphological defects such as smaller size or loss of cristae, thus affecting mitochondrial function. These results suggest that zebrafish pink1 plays conserved important roles in the development of DA neurons and in the mitochondrial morphology and function. To better follow DA neurons after injury or administration of toxins, I generated a transgenic zebrafish line, Tg(dat:EGFP), in which the green fluorescent protein (GFP) is expressed under the control of cis-regulatory elements of dopamine transporter (dat). In Tg(dat:EGFP) fish, all major groups of DA neurons are correctly labeled with GFP, especially the ones in the vDC, which are analogous to the ascending midbrain DA neurons in mammals. In addition, we observed that the DA neurons in the vDC could partially be replaced after severe laser cell ablation. This suggests that zebrafish may have the unique capacity of regenerating DA neurons after injury. Taken together, my studies suggested that zebrafish could be a useful alternative animal model for the study of the molecular mechanisms underlying PD and for the screening of potential therapeutic compounds for PD.