Quantitation of tyrosine hydroxylase protein levels: Spot immunolabeling with an affinity-purified antibody

doi:10.1016/0003-2697(89)90240-6

Analytical Biochemistry

Volume 181, Issue 2, September 1989, Pages 259-266

https://doi.org/10.1016/0003-2697(89)90240-6 Get rights and content

Abstract

Tyrosine hydroxylase was purified from bovine adrenal chromaffin cells and rat pheochromocytoma using a rapid (<2 days) procedure performed at room temperature. Rabbits were immunized with purified enzyme that was denatured with sodium dodecylsulfate, and antibodies to tyrosine hydroxylase were affinity-purified from immune sera. A Western blot procedure using the affinity-purified antibodies and ¹²⁵I-protein A demonstrated a selective labeling of a single M_r ≈ 62,000 band in samples from a number of different tissues. The relative lack of background ¹²⁵I-protein A binding permitted the development of a quantitative spot immunolabeling procedure for tyrosine hydroxylase protein. The sensitivity of the assay is 1–2 ng of enzyme. Essentially identical standard curves were obtained with tyrosine hydroxylase purified from rat pheochromocytoma, rat corpus striatum, and bovine adrenal medulla. An extrac of PC 12 cells (clonal rat pheochromocytoma cells) was calibrated against purified rat pheochromocytoma tyrosine hydroxylase and used as an external standard against which levels of tyrosine hydroxylase in PC12 cells and other tissues were quantified. With this procedure, qualitative assessment of tyrosine hydroxylase protein levels can be obtained in a few hours and quantitative assessment can be obtained in less than a day.

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The antibody was rabbit polyclonal anti-tyrosine hydroxylase (Pel-Freez Biologicals, Rogers, AR; P40101-0, Lot# 06432; working dilution 1:1000), which was generated using SDS-denatured tyrosine hydroxylase purified from rat pheochromocytoma. The antibody was specific for a 60 kDa band on Western blots of rat striatal lysates (Haycock, 1989). The immunostaining pattern observed was also consistent with the reported distribution of tyrosine hydroxylase and its mRNA in rat brain (Chan and Sawchenko, 1998).
The midbrain dopaminergic perikarya are differentially affected in Parkinson׳s disease (PD). This study compared the effects of a partial unilateral intrastriatal 6-hydroxydopamine (6-OHDA) lesion model of PD on the number, morphology, and nucleolar volume of dopaminergic cells in the substantia nigra pars compacta (SNpc), ventral tegmental area (VTA), and retrorubral field (RRF). Adult, male rats (n=10) underwent unilateral intrastriatal infusion of 6-OHDA (12.5 μg). Lesions were verified by amphetamine-stimulated rotation 7 days post-infusion. Rats were euthanized 14 days after treatment with 6-OHDA and brains were stained with a tyrosine hydroxylase-silver nucleolar (TH-AgNOR) stain. Dopaminergic cell number and morphology in the lesioned and intact hemispheres were quantified using stereological methods. The magnitude of decrease in planimetric volume, neuronal number, cell density, and neuronal volume resulting from 6-OHDA lesion differed between regions, with the SNpc exhibiting the greatest loss of neurons (46%), but the smallest decrease in neuronal volume (13%). The lesion also resulted in a decrease in nucleolar volume that was similar in all three regions (22–26%). These findings indicate that intrastriatal 6-OHDA lesion differentially affects dopaminergic neurons in the SNpc, VTA, and RRF; however, the resulting changes in nucleolar morphology suggest a similar cellular response to the toxin in all three cell populations.
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To test the specificity of antisera, control experiments for β-gal were done using the same immunostaining protocol in brainstem sections of animals not injected with THZ. The specificity of the rat TH antiserum was previously demonstrated (Haycock, 1989). The specificity of the secondary antibodies was tested by omission of the primary antibodies.
Antidepressants that inhibit the recapture of noradrenaline have variable effects in chronic pain which may be related to the complex role of noradrenaline in pain modulation. Whereas at the spinal cord noradrenaline blocks nociceptive transmission, both antinociception and pronociception were reported after noradrenaline release in the brain. To study the role of noradrenaline in pain modulatory areas of the brain, we elected the dorsal reticular nucleus (DRt), a key pain facilitatory area located at the medulla oblongata. Three studies were performed. First, we show that the infusion in the DRt of nomifensine, which increases local extracellular levels of noradrenaline as shown by in vivo microdialysis, also enhances pain behavioral responses during both phases of the formalin test, a classic inflammatory pain model. Then, we demonstrate that the formalin test triggers the release of noradrenaline in the DRt in a biphasic pattern that matches the two phases of the test. Finally, we show that reducing noradrenaline release into the DRt, using an HSV-1 vector which decreases the expression of tyrosine hydroxylase in noradrenergic DRt-projecting neurons, attenuates pain behavioral responses in both phases of the formalin test. The increased noradrenaline levels induced by the infusion of nomifensine at the DRt, along with the hyperalgesic effects of noradrenaline released at the DRt upon noxious stimulation, indicates that noradrenaline may enhance pain facilitation from the brain. It is important to evaluate if antidepressants that inhibit noradrenaline recapture enhance pain facilitation from the brain herein attenuating their analgesic effects.
Changes in serotoninergic and noradrenergic descending pain pathways during painful diabetic neuropathy: The preventive action of IGF1
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The percentages of control animals were considered as the basal levels (equal to 1) and the results for STZ group are presented as fold-increase over the control percentages. The specificity of each primary antibody was previously demonstrated (Haycock, 1989; Pinto et al., 2007; Pinna et al., 2007; Riedel et al., 2008; Wu et al., 2009; Malleret et al., 2010). Negative control experiments included the omission of primary or secondary antibodies.
Painful diabetic neuropathy (PDN) induces neuronal hyperactivity at the spinal cord and periaqueductal gray (PAG), a key area in descending nociceptive modulation. Since the PAG uses relay stations at serotoninergic and noradrenergic brainstem areas, we determined the serotonin and noradrenaline levels at the spinal cord of streptozotocin-diabetic rats and at those brainstem areas (serotoninergic rostroventromedial medulla and noradrenergic A₅ and A₇ cell groups). Since, during diabetes, the levels of insulin growth factor 1 (IGF1) decrease, reducing its neurotrophic effect in the brain, we also studied the effects of IGF1 treatment. One week after diabetes induction, subcutaneous injections of IGF1 (2.5 mg/kg) were performed during 3 weeks. Body weights, glycemia, and mechanical nociception were weekly evaluated until the end of the study, the time when the animals were subjected to a modified formalin test to study chemical allodynia. Serotonin and noradrenaline levels were quantified by ELISA at the spinal cord, whereas at the brainstem, the quantification was performed by immunohistochemistry against, respectively, tryptophan hydroxylase (TpH) or tyrosine hydroxylase (TH). STZ-diabetic rats exhibited mechanical hyperalgesia and chemical allodynia, along with higher spinal levels of serotonin and noradrenaline and higher numbers of neurons expressing TpH at the RVM and TH at the A₅ noradrenergic cell group. Treatment with IGF1 prevented the behavioral signs of PDN and reversed the neuronal hyperactivity at the spinal cord and ventrolateral PAG and the neurochemical changes at the spinal cord and at the brainstem. Based on the facilitatory role of serotoninergic and noradrenergic descending modulation during chronic pain, the increased serotonin and noradrenaline innervation of the dorsal horn in STZ-diabetic rats may probably account for enhanced pain during PDN. The benefits of IGF1 in PDN are probably due to blockade of the increased peripheral input to the somatosensory system, but direct central actions cannot be discarded. The value of IGF1 in PDN treatment deserves further evaluation.
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For β-gal, the controls were done in brainstem sections of animals not injected with DPZ and using the same immunostaining protocol. The specificity of the rat TH antiserum was previously demonstrated [15]. The specificity of the secondary antibodies was tested by omission of the primary antibodies.
Descending modulation of nociceptive transmission depends on the release of noradrenaline at the spinal cord. The role of noradrenaline in the control of nociceptive transmission at the supraspinal pain control system remains understudied. As chronic pain is associated with enhanced descending facilitation of nociceptive transmission, we sought to determine the role of noradrenaline in pain facilitation from the brain during neuropathic pain. We determined the action of the noradrenergic input to the dorsal reticular nucleus (DRt), a unique pain facilitatory area, using the spared nerve injury model. Injections of the α1-adrenoreceptor agonist phenylephrine into the DRt induced hyperalgesia and allodynia, indicating that α1-adrenoreceptors enhance the facilitatory action of the nucleus. This led us to reduce noradrenaline release at the DRt using a viral vector derived from the Herpes Simplex virus type 1 (HSV-1) which carried the tyrosine hydroxylase (TH) transgene in antisense orientation. The reduction of noradrenaline release, confirmed by microdialysis experiments, induced a long-lasting attenuation of pain responses, which was reverted by the local administration of phenylephrine. The present study indicates that the noradrenergic modulation of a pronociceptive area at the supraspinal pain control system accounts for pain facilitation, through the activation of α1-adrenoreceptors. The study also shows that sustained effects on chronic pain can be achieved by decreasing the release of noradrenaline in a pain facilitatory centre of the brain using gene transfer.
Quantification of MPTP-induced dopaminergic neurodegeneration in the mouse substantia nigra by laser capture microdissection
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The neurotoxin MPTP is widely used to cause damage to the dopaminergic system in rodents and non-human primates to model various aspects of Parkinson's disease. In mice, depletion of striatal dopamine is the commonly used endpoint to assess neuronal damage. However, it has proved technically challenging to quantify dopaminergic cell bodies as an index of neuronal integrity. To meet this challenge, we applied laser pressure catapult microdissection (LCM) of the substantia nigra in combination with quantitative Western blot to provide an index of dopamine neurodegeneration in mice treated with MPTP. Seven days following initiation of MPTP treatment, striatal dopamine depletion was maximal and there was histological evidence of neuronal degeneration in the substantia nigra. To index the integrity of dopamine cell bodies, tyrosine hydroxylase (TH) and β-actin were quantified by Western blot in LCM extracts. In untreated mice, TH was detected in LCM extracts of substantia nigra but was undetectable in equivalently sized extracts of cortex from the same animals. In MPTP-treated mice, there was a significant 70% reduction in TH relative to β-actin in LCM extracts as compared to vehicle-injected controls. This reduction corresponded to decreases in striatal dopamine and loss of immunocytochemically detected TH but not β-actin in the substantia nigra (SN). Thus, this method provides a quantitative means to measure dopamine neuron toxicity in the substantia nigra and, as such has potential application in evaluating regimens that may be neuroprotective or neurorestorative for dopaminergic neurons.
Differential expression of the B′βregulatory subunit of protein phosphatase 2A modulates tyrosine hydroxylase phosphorylation and catecholamine synthesis
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Specificity was confirmed in heterologous expression experiments. Other Reagents—Rabbit polyclonal antibodies recognizing total TH and Ser19-phosphorylated TH were provided by John Haycock (Louisiana State University (30, 31)). The pGEX-2T plasmid expressing glutathione S-transferase (GST)-TH (regulatory domain residues 31-164) was a gift from Drs. Lily Moy and Li-Huei Tsai (Harvard University, Cambridge, MA (32)).
Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is stimulated by N-terminal phosphorylation by several kinases and inhibited by protein serine/threonine phosphatase 2A (PP2A). PP2A is a family of heterotrimeric holoenzymes containing one of more than a dozen different regulatory subunits. In comparison with rat forebrain extracts, adrenal gland extracts exhibited TH hyperphosphorylation at Ser¹⁹, Ser³¹, and Ser⁴⁰, as well as reduced phosphatase activity selectively toward phosphorylated TH. Because the B′β regulatory subunit of PP2A is expressed in brain but not in adrenal glands, we tested the hypothesis that PP2A/B′β is a specific TH phosphatase. In catecholamine-secreting PC12 cells, inducible expression of B′β decreased both N-terminal Ser phosphorylation and in situ TH activity, whereas inducible silencing of endogenous B′β had the opposite effect. Furthermore, PP2A/B′β directly dephosphorylated TH in vitro. As to specificity, other PP2A regulatory subunits had negligible effects on TH activity and phosphorylation in situ and in vitro. Whereas B′β was highly expressed in dopaminergic cell bodies in the substantia nigra, the PP2A regulatory subunit was excluded from TH-positive terminal fields in the striatum and failed to colocalize with presynaptic markers in general. Consistent with a model in which B′β enrichment in neuronal cell bodies helps confine catecholamine synthesis to axon terminals, TH phosphorylation was higher in processes than in somata of dopaminergic neurons. In summary, we show that B′β recruits PP2A to modulate TH activity in a tissue- and cell compartment specific fashion.

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Quantitation of tyrosine hydroxylase protein levels: Spot immunolabeling with an affinity-purified antibody

Abstract

J. Biol. Chem

Anal. Biochem

J. Neurosci. Methods

Neurochem. Int

Anal. Biochem

J. Biol. Chem

Anal. Biochem

Anal. Biochem

Anal. Biochem

Adv. Cell. Neurobiol

J. Biol. Chem

Biochim. Biophys. Acta

Anal. Biochem

Exp. Cell Res

Brain Res. Bull