Catecholamine-synthesizing Enzymes Research Articles

Physiological and metabolic effects of short-term dopamine reduction in healthy horses using a tyrosine hydroxylase inhibitor (alpha-methyl-para-tyrosine)

Alpha-methyl-para-tyrosine (AMPT) is a reversible inhibitor of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. This study aimed to determine whether AMPT could reduce dopamine concentrations in horses. Six healthy adult Standardbred geldings were administered AMPT (40 mg/kg BW, orally) or placebo in a randomised crossover study design. Clinical examination findings were recorded, and blood samples were collected for up to 6 h after administration of AMPT or placebo, for measurement of blood glucose, plasma ACTH and cortisol concentrations, and plasma metabolomic analysis. Plasma prolactin concentration was determined as a proxy index of central dopamine reduction. No adverse clinical effects were detected after oral administration of AMPT, with heart rate, mean arterial pressure and blood glucose concentration not differing between AMPT treatment or placebo. Plasma prolactin concentration peaked 1 h after AMPT administration before returning to baseline at 2 h (for five horses) or 6 h (for one horse). Metabolomic analysis demonstrated a reduction in plasma dopamine (0.72-fold change; P=0.016) 1 h after AMPT treatment. Plasma ACTH and cortisol concentrations were not different between AMPT and placebo over time. A few metabolites associated with ketogenesis were increased, and certain amino acids decreased, at 1 h compared with baseline, for both AMPT treatment and placebo. Therefore, AMPT was effective in reducing both central and circulating dopamine concentrations in healthy horses following a single oral dose. Further pharmacokinetic and pharmacodynamic studies are warranted to optimise the dose and duration of AMPT treatment to achieve longer-term dopamine reduction. Plasma metabolomic findings suggested an interruption to energy flux at the time of sample collection, which may be relevant to nutritional studies in horses and warrants further investigation.

Endoplasmic reticulum stress in a circumventricular organ-hypothalamic neuronal circuit alters hepatic glucose and lipid metabolism during obesity

Non-alcoholic fatty liver disease (NAFLD) is a leading cause of chronic hepatic disorders and is associated with metabolic conditions such type 2 diabetes. Accumulating evidence points to endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) in the subfornical organ (SFO), a circumventricular nucleus situated outside of the blood-brain-barrier, as a major contributor to NAFLD development during obesity. We recently reported that ER stress in SFO neurons that project to the hypothalamic paraventricular nucleus (PVN, SFO→PVN neurons) drives liver triglyceride accumulation during obesity. However, the mechanism by which ER stress in this neural network mediates obesity-related hepatic steatosis remains unknown. Elevations in hepatic gluconeogenesis, lipogenesis, and free fatty acid uptake are all associated with the development of fatty liver. Based on this, we hypothesized that ER stress in SFO→PVN neurons may contribute to NAFLD through alterations in hepatic glucose/lipid metabolism during obesity. To investigate this, we used an intersectional viral strategy to selectively inhibit ER stress in SFO→PVN neurons in C57Bl/6J male mice that were fed a high-fat diet (HFD, 60% kCal fat) for 7 wks (n=4-5/group) and then underwent selective PVN targeting of a retrogradely transported Cre-recombinase virus (CAV2-Cre-GFP; CAV2-GFP served as a control). Concurrently, a Cre inducible vector to overexpress the ER chaperone glucose-regulated peptide 78 (GRP78; AAV-FLEX-GRP78-mStrawberry) was targeted to the SFO. This approach results in the overexpression of GRP78 specifically in SFO→PVN neurons, which has been established as an effective means of reducing ER stress. Four weeks following selective inhibition of SFO→PVN neuronal ER stress, body weight (46±2 vs 43±1 g, CAV2-GFP vs CAV2-Cre, P=0.26), food intake, and adipose mass were not different between groups. However, diminishing UPR activation in this circumventricular-hypothalamic circuit resulted in a reduction in hepatomegaly (1.8±0.1 vs 1.5±0.1 g, CAV2-GFP vs CAV2-Cre, P=0.08), hepatic triglyceride levels (4.3±0.6 vs 2.3±0.4 mM, CAV2-GFP vs CAV2-Cre, p<0.05), and plasma glucose (198±12 vs 168±8 mg/dL, CAV2-GFP vs CAV2-Cre, P=0.06). Interestingly, reducing ER stress in SFO→PVN neurons was also associated with a downregulation in hepatic mRNA levels of genes involved in hepatic gluconeogenesis (e.g. G6pase: 0.29±0.04; Pepck: 0.46±0.04 fold CAV2-GFP, both p<0.05), de novo lipogenesis (e.g. SREBP1: 0.65±0.1 fold CAV2-GFP, P=0.05) and free fatty acid synthesis and uptake (e.g. Fasn: 0.57±0.11 fold CAV2-GFP, P=0.09 and FATP5: 0.42±0.03, fold CAV2-GFP, p<0.05). Overactivation of the liver sympathetic nerves has recently been shown to contribute to obesity-related NAFLD, and inhibition of ER stress in SFO→PVN neurons resulted in a reduction in protein levels of hepatic tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis (0.58±0.03, fold CAV2-GFP, p<0.05). Notably, hepatic TH levels were significantly correlated with liver glucose/lipid metabolism-related gene expression and triglyceride content (e.g. Pepck: R2=0.57; Fasn: R2=0.80; FATP5: R2=0.77; triglycerides: R2=0.70, all p<0.05). Collectively, these results suggest that during obesity, ER stress in SFO→PVN neurons may drive liver sympathetic overactivity and subsequently disrupt hepatic glucose/lipid metabolism, thus contributing to NAFLD development. R01DK117007, R01HL141393, AHA932522. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Adrenergic microenvironment driven by cancer-associated Schwann cells contributes to chemoresistance in patients with lung cancer.

Doublecortin (DCX)-positive neural progenitor-like cells are purported components of the cancer microenvironment. The number of DCX-positive cells in tissues reportedly correlates with cancer progression; however, little is known about the mechanism by which these cells affect cancer progression. Here we demonstrated that DCX-positive cells, which are found in all major histological subtypes of lung cancer, are cancer-associated Schwann cells (CAS) and contribute to the chemoresistance of lung cancer cells by establishing an adrenergic microenvironment. Mechanistically, the activation of the Hippo transducer YAP/TAZ was involved in the acquisition of new traits of CAS and DCX positivity. We further revealed that CAS express catecholamine-synthesizing enzymes and synthesize adrenaline, which potentiates the chemoresistance of lung cancer cells through the activation of YAP/TAZ. Our findings shed light on CAS, which drive the formation of an adrenergic microenvironment by the reciprocal regulation of YAP/TAZ in lung cancer tissues.

Catecholamines and Parkinson's disease: tyrosine hydroxylase (TH) over tetrahydrobiopterin (BH4) and GTP cyclohydrolase I (GCH1) to cytokines, neuromelanin, and gene therapy: a historical overview.

The author identified the genes and proteins of human enzymes involved in the biosynthesis of catecholamines (dopamine, norepinephrine, epinephrine) and tetrahydrobiopterin (BH4): tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), dopamine β-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), and GTP cyclohydrolase I (GCH1). In Parkinson's disease (PD), the activities and levels of mRNA and protein of all catecholamine-synthesizing enzymes are decreased, especially in dopamine neurons in the substantia nigra. Hereditary GCH1 deficiency results in reductions in the levels of BH4 and the activities of TH, causing decreases in dopamine levels. Severe deficiencies in GCH1 or TH cause severe decreases in dopamine levels leading to severe neurological symptoms, whereas mild decreases in TH activity in mild GCH1 deficiency or in mild TH deficiency result in only modest reductions in dopamine levels and symptoms of DOPA-responsive dystonia (DRD, Segawa disease) or juvenile Parkinsonism. DRD is a treatable disease and small doses of L-DOPA can halt progression. The death of dopamine neurons in PD in the substantia nigra may be related to (i) inflammatory effect of extra neuronal neuromelanin, (ii) inflammatory cytokines which are produced by activated microglia, (iii) decreased levels of BDNF, and/or (iv) increased levels of apoptosis-related factors. This review also discusses progress in gene therapies for the treatment of PD, and of GCH1, TH and AADC deficiencies, by transfection of TH, AADC, and GCH1 via adeno-associated virus (AAV) vectors.

Mejoría de la inmunidad y la esperanza de vida en ratones maduros, genéticamente deficientes en la síntesis de catecolaminas, tras convivir con controles durante dos meses

IntroductionMice hemizygous in tyrosine hydroxylase (TH-HZ), the limiting enzyme in catecholamine synthesis, show premature immunosenescence, which in females is associated with a shorter lifespan than the corresponding controls (WT). The coexistence of TH-Hz with WT improves the immune function in both males and females in adulthood. ObjectiveTo test whether cohabitation for two months of mature male TH-HZ with WT improves the immune function of the former and whether this impacts the lifespan. Material and methodsMature male ICR-CD1 mice (13 ± 1 months) TH-HZ coexisted with WT (2:4 ratio in each cage) for two months. Peritoneal leukocytes were extracted from all animals at baseline, one month, and two months after cohabitation, and macrophage phagocytic capacity, macrophage and lymphocyte chemotaxis, natural killer (NK) antitumor activity, and lymphoproliferative capacity in response to the mitogens concanavalin A and lipopolysaccharide (LPS) were assessed. The animals were maintained under these conditions until their natural death. ResultsThe TH-HZ, which start, in general, with lower values than the WT in the immune functions studied, improved them after two months of cohabitation, becoming similar to those of the controls. This improvement was already observed in NK activity after one month of cohabitation.The TH-HZ presented lower mean longevity than WT, but when they cohabited with WT, it was similar to the latter. ConclusionThe coexistence of TH-HZ male mice with WT mice for two months at mature age improves these genetically modified animals’ immune response and longevity.

Myeloid cell-derived catecholamines influence bone turnover and regeneration in mice.

Catecholamine signaling is known to influence bone tissue as reuptake of norepinephrine released from sympathetic nerves into bone cells declines with age leading to osteoporosis. Further, β-adrenoceptor-blockers like propranolol provoke osteoprotective effects in osteoporotic patients. However, besides systemic adrenal and sympathetic catecholamine production, it is also known that myeloid cells can synthesize catecholamines, especially under inflammatory conditions. To investigate the effects of catecholamines produced by CD11b+ myeloid cells on bone turnover and regeneration, a mouse line with specific knockout of tyrosine hydroxylase, the rate-limiting enzyme of catecholamine synthesis, in CD11b+ myeloid cells (THflox/flox/CD11b-Cre+, referred to as THCD11b-Cre) was generated. For bone phenotyping, male mice were sacrificed at eight and twelve weeks of age and harvested bones were subjected to bone length measurement, micro-computed tomography, fluorescence-activated cell sorting of the bone marrow, gene expression analysis, histology and immunohistochemistry. Support for an age-dependent influence of myeloid cell-derived catecholamines on bone homeostasis is provided by the fact that twelve-week-old, but not eight-week-old THCD11b-Cre mice, developed an osteopenic phenotype and showed increased numbers of neutrophils and T lymphocytes in the bone marrow, while CCL2, IL-6, IL-4 and IL-10 mRNA expression was reduced in sorted myeloid bone marrow cells. To investigate the influence of myeloid cell-derived catecholamines on fracture healing, mice received a diaphyseal femur osteotomy. Three days post-fracture, immunohistochemistry revealed an increased number of macrophages, neutrophils and cytotoxic T lymphocytes in the fracture hematoma of THCD11b-Cre mice. Micro-computed tomography on day 21 showed a decreased tissue mineral density, a reduced bone volume and less trabeculae in the fracture callus indicating delayed fracture healing, probably due to the increased presence of inflammatory cells in THCD11b-Cre mice. This indicates a crucial role of myeloid cell-derived catecholamines in immune cell-bone cell crosstalk and during fracture healing.

Prefrontal‐medullary circuit activation attenuates stress reactivity

Organismal survival and adaptation to stress rely on brainstem catecholaminergic neurons. In particular, catecholaminergic neurons in the rostral ventrolateral medulla (RVLM) drive sympathetic activity and enable physiological adaptations, including vasoconstriction, corticosterone release, and glycemic mobilization. However, it is unclear how brain regions involved in the cognitive appraisal of stress regulate the activity of RVLM neurons. Our previous studies found that the rodent infralimbic prefrontal cortex (IL) integrates behavioral and physiological responses to stress. Thus, a potential IL‐to‐RVLM connection would represent a crucial link between stress appraisal and sympathetic reactivity. In the current study, we investigated a direct IL‐to‐RVLM circuit by targeting a genetically‐encoded anterograde tracer under the control of the CaMKIIα promoter to the IL of adult male and female rats. Analysis revealed that IL terminals apposed RVLM neurons expressing the catecholamine‐synthesizing enzyme dopamine beta hydroxylase. Further, IL input to catecholamine cells was widespread throughout the rostral to caudal extent of the VLM in both male and female rodents. Quantification of innervation density revealed that males had a larger proportion of VLM catecholamine neurons receiving IL inputs relative to female rats. Additionally, IL appositions were identified on GABAergic and glycinergic neurons in both sexes. Accordingly, we hypothesized that IL projections may activate local RVLM inhibitory cells to limit sympathetic output. To test this hypothesis, we injected a viral vector coding for channelrhodopsin‐2 (ChR2) in the IL of males and females. Next, a fiber optic cannula was implanted dorsal to the RVLM to evoke IL synaptic glutamate release. Animals then received photostimulation during restraint stress with blood sampled to determine stress reactivity. Compared to controls, male rats expressing ChR2 on IL terminals had suppressed glycemic stress responses (p < 0.05). In contrast, stimulation of the IL‐RVLM circuit in females did not affect glucose mobilization (p < 0.05). However, ChR2 decreased corticosterone responses to stress relative to control rats in both sexes (males, p < 0.01; females, p < 0.05). Thus, both male and female rats have a direct circuit from the IL portion of the mPFC to catecholamine‐synthesizing cells of the RVLM that limits glucocorticoid stress responses, likely through the activation of local inhibitory neurons. However, the density of this circuit is greater in males, potentially accounting for reduced glycemic responses. Ultimately, excitatory/inhibitory balance at IL synapses in the RVLM may be critical for the health consequences of stress.

Farnesoid X Receptor Activation in Brain Alters Brown Adipose Tissue Function via the Sympathetic System.

The nuclear bile acid (BA) receptor farnesoid X receptor (FXR) is a major regulator of metabolic/energy homeostasis in peripheral organs. Indeed, enterohepatic-expressed FXR controls metabolic processes (BA, glucose and lipid metabolism, fat mass, body weight). The central nervous system (CNS) regulates energy homeostasis in close interaction with peripheral organs. While FXR has been reported to be expressed in the brain, its function has not been studied so far. We studied the role of FXR in brain control of energy homeostasis by treating wild-type and FXR-deficient mice by intracerebroventricular (ICV) injection with the reference FXR agonist GW4064. Here we show that pharmacological activation of brain FXR modifies energy homeostasis by affecting brown adipose tissue (BAT) function. Brain FXR activation decreases the rate-limiting enzyme in catecholamine synthesis, tyrosine hydroxylase (TH), and consequently the sympathetic tone. FXR activation acts by inhibiting hypothalamic PKA-CREB induction of TH expression. These findings identify a function of brain FXR in the control of energy homeostasis and shed new light on the complex control of energy homeostasis by BA through FXR.

3177 – NEURAL CREST CONTRIBUTION TO THE MURINE HSC SPECIFICATION NICHE

Hematopoietic stem cells (HSCs) are developmentally specified from endothelium of the dorsal aorta (DA) in the aorta-gonad-mesonephros (AGM) region of the midgestation mouse embryo. Their emergence requires signals from neighboring cell populations in the microenvironment. We previously showed that neural crest cells (NCCs), a migratory population of cells that in mouse migrates to the DA around midgestation, are required for HSC specification in zebrafish. Although NCC-derived differentiated sympathetic neurons have been proposed to promote HSC emergence in the mouse through secretion of catecholamines, an earlier role for NCCs in HSC specification prior to sympathetic neuronal differentiation has not been demonstrated. In the fish model, the NCC requirement precedes sympathetic neuronal differentiation. In our recent studies, we have found that mouse NCCs reach the dorsal aorta between E9.5 and E10 but only express catecholamine-synthesizing enzymes after E10.5, when the definitive hematopoietic program initiates. We hypothesize that NCCs play a role in HSC specification in mouse prior to neuronal differentiation and secretion of catecholamines. To test this possibility, we have used an inducible diphtheria toxin mouse model to ablate NCCs before they reach the DA. Although we find an increase in immature hematopoietic stem and progenitor cells, we note a decrease in pre-HSCs type II. Our data suggest that NCCs play a distinct role in HSC specification at a specific maturational transition point during early specification. Hematopoietic stem cells (HSCs) are developmentally specified from endothelium of the dorsal aorta (DA) in the aorta-gonad-mesonephros (AGM) region of the midgestation mouse embryo. Their emergence requires signals from neighboring cell populations in the microenvironment. We previously showed that neural crest cells (NCCs), a migratory population of cells that in mouse migrates to the DA around midgestation, are required for HSC specification in zebrafish. Although NCC-derived differentiated sympathetic neurons have been proposed to promote HSC emergence in the mouse through secretion of catecholamines, an earlier role for NCCs in HSC specification prior to sympathetic neuronal differentiation has not been demonstrated. In the fish model, the NCC requirement precedes sympathetic neuronal differentiation. In our recent studies, we have found that mouse NCCs reach the dorsal aorta between E9.5 and E10 but only express catecholamine-synthesizing enzymes after E10.5, when the definitive hematopoietic program initiates. We hypothesize that NCCs play a role in HSC specification in mouse prior to neuronal differentiation and secretion of catecholamines. To test this possibility, we have used an inducible diphtheria toxin mouse model to ablate NCCs before they reach the DA. Although we find an increase in immature hematopoietic stem and progenitor cells, we note a decrease in pre-HSCs type II. Our data suggest that NCCs play a distinct role in HSC specification at a specific maturational transition point during early specification.

Immunohistochemical Expression of Choline Acetyltransferase and Catecholamine-Synthesizing Enzymes in Head-and-Neck and Thoracoabdominal Paragangliomas and Pheochromocytomas.

Paragangliomas (PGLs) are neural-crest-derived, non-epithelial neuroendocrine tumors distributed along the parasympathetic and sympathetic nerves. Head-and-neck PGLs (HNPGLs) have been recognized as nonchromaffin, nonfunctional, parasympathetic tumors. By contrast, thoracoabdominal paragangliomas and pheochromocytomas (PPGLs) are chromaffin, functional, sympathetic tumors. Although HNPGLs and PPGLs have the same histological structure, the zellballen pattern, composed of chief and sustentacular cells surrounded by abundant capillaries, the pathobiological differences between these types of PGLs remain unclarified. To determine the phenotypic features of these PGLs, we performed an immunohistochemical study using specific antibodies against choline acetyltransferase (ChAT), an enzyme involved in acetylcholine synthesis, and enzymes for the catecholamine-synthesis, tyrosine hydroxylase (TH), and dopamine beta-hydroxylase (DBH), in 34 HNPGLs from 31 patients, 12 thoracoabdominal PGLs from 12 patients, and 26 pheochromocytomas from 22 patients. The expression of ChAT, TH, and DBH was 100%, 23%, and 10% in the HNPGLs; 12%, 100%, and 100% in the pheochromocytomas; and 25%, 67%, and 100% in the thoracoabdominal PGLs, respectively. These results designate HNPGLs as acetylcholine-producing parasympathetic tumors, in contrast to PPGLs being catecholamine-producing tumors. The other most frequently used neuroendocrine markers are synaptophysin and chromogranin A expressed 100% and 80%, respectively, and synaptophysin was superior to chromogranin A in HNPGLs. This is the first report of HNPGLs being acetylcholine-producing tumors. Immunohistochemistry of ChAT could be greatly useful for pathologic diagnosis of HNPGL. Whether measurement of acetylcholine levels in the blood or urine could be a tumor marker of HNPGLs should be investigated soon.

Dopamine-Secreting Pheochromocytoma and Paraganglioma.

Predominantly or exclusively dopamine-secreting pheochromocytoma and paraganglioma are very rare. We report a 64-year-old woman with an adrenal incidentaloma. She was normotensive and had no symptoms of catecholamine excess. The 24-hour urine catecholamine level showed normal norepinephrine (122.9 μg/day), normal epinephrine (24.3 μg/day), and markedly elevated dopamine (148 212.4 μg/day). 123I-metaiodobenzylguanidine (MIBG) scintigraphy revealed tumor uptake. After α-blockade as preoperative management, she successfully underwent laparoscopic left adrenalectomy and was finally diagnosed with an exclusively dopamine-secreting pheochromocytoma. The tumor was histologically comprised of small polygonal cells with high cellularity and was immunohistochemically positive for all 3 catecholamine-synthesizing enzymes: tyrosine hydroxylase (very weak), dopamine β-hydroxylase (heterogeneous), and phenylethanolamine N-methyltransferase (very weak). Electron microscopy revealed very few catecholamine-containing small vesicles with a few organelles, which reflected immature cells. No biochemical or imaging evidence of recurrence or metastasis were evident 1 year after the surgery. We conducted a literature search in the PubMed database. A total of 33 cases were collected. Our case had the second-highest 24-hour urinary dopamine excretion and was the first in which immunostaining for catecholamine synthase and electron microscopy were performed together. Histological findings in our case give a possible hypothesis that the mechanism underlying a dopamine-secreting pheochromocytoma is associated with immature catecholamine vesicles in which dopamine β-hydroxylase is localized, thus resulting in inhibited conversion from dopamine to norepinephrine. We also discuss the reasons for the lack of catecholamine excess symptoms, whether preoperative management of α-blockade is needed, and the association between the prognosis and genetic mutation, with an extensive literature review.

Distribution of neuronal cells which contain dopamine β-hydroxylase, tyrosine hydroxylase, neuropeptide Y and vasoactive intestinal polypeptide in the human internal carotid nerve.

The human internal carotid nerve (ICN) occasionally has a swelling beneath the external opening of the carotid canal. In this study, the presence and distribution of neuronal cells were investigated in the bilateral ICNs of nine human cadavers. Among 44.4% of the cadavers, swellings were detected in the ICN. Their diameters ranged from 1.7 to 3.6 mm (average ±SD= 2.6± 0.7 mm). Thirty-eight percent of these swellings were large (diameter > 3 mm) and showed an oval shape. The large swelling contained many neuronal cells. However, the ICNs with or without a swelling <3 mm diameter were mostly free from neuronal cells (93.3%). Only in one human cadaver, the right ICN without a swelling had a small number of neuronal cells. By the present immunohistochemical method, ICN neurons contained catecholamine-synthesizing enzymes and neuropeptides. Dopamine-beta hydroxylase- and tyrosine hydroxylase-immunoreactivity were mostly expressed by ICN neurons. More than half of them also contained neuropeptide Y-immunoreactivity. However, vasoactive intestinal polypeptide-immunoreactive ICN neurons were relatively infrequent. Substance P- and calcitonin gene-related peptide-immunoreactive ICN neurons could not be detected. By the cell size analysis, neuropeptide Y-immunoreactive neurons were significantly smaller than neuropeptide Y-immunonegative neurons in the ICN. The present study suggests that ICN neurons have a sympathetic function in the human.

TNF\u03b1 increases tyrosine hydroxylase expression in human monocytes

Most, if not all, peripheral immune cells in humans and animals express tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis. Since TH is typically studied in the context of brain catecholamine signaling, little is known about changes in TH production and function in peripheral immune cells. This knowledge gap is due, in part, to the lack of an adequately sensitive assay to measure TH in immune cells expressing lower TH levels compared to other TH expressing cells. Here, we report the development of a highly sensitive and reproducible Bio-ELISA to quantify picogram levels of TH in multiple model systems. We have applied this assay to monocytes isolated from blood of persons with Parkinson’s disease (PD) and to age-matched, healthy controls. Our study unexpectedly revealed that PD patients’ monocytes express significantly higher levels of TH protein in peripheral monocytes relative to healthy controls. Tumor necrosis factor (TNFα), a pro-inflammatory cytokine, has also been shown to be increased in the brains and peripheral circulation in human PD, as well as in animal models of PD. Therefore, we investigated a possible connection between higher levels of TH protein and the known increase in circulating TNFα in PD. Monocytes isolated from healthy donors were treated with TNFα or with TNFα in the presence of an inhibitor. Tissue plasminogen activator (TPA) was used as a positive control. We observed that TNFα stimulation increased both the number of TH+ monocytes and the quantity of TH per monocyte, without increasing the total numbers of monocytes. These results revealed that TNFα could potentially modify monocytic TH production and serve a regulatory role in peripheral immune function. The development and application of a highly sensitive assay to quantify TH in both human and animal cells will provide a novel tool for further investigating possible PD immune regulatory pathways between brain and periphery.

Chronic propranolol treatment moderately attenuated development of N-methyl-N-nitrosourea-induced mammary carcinoma in female rats.

The sympathetic nervous system participates in the development and progression of several cancer types and this effect is mediated mainly via β-adrenergic signaling. However, the potential of β-adrenergic signaling blockade to prevent cancer development after exposure to carcinogens has not been investigated, yet. Therefore, in our study, we determined the effect of the β-blocker propranolol on the development and progression of mammary cancer induced in female rats by administration of the chemical carcinogen N-methyl-N-nitrosourea (MNU). The propranolol treatment (20 mg/kg body weight) started 12 days after MNU administration and lasted 10 weeks. We found that both saline and propranolol treatment significantly increased gene expression of the catecholamine-synthesizing enzyme tyrosine hydroxylase, indicating that repeated injection of saline or propranolol-induced stress in these two groups. However, compared to the vehicle-treated group, propranolol slightly delayed the development and moderately reduced the incidence of mammary carcinoma in animals. To evaluate the mechanisms mediating the effect of propranolol on the development of MNU-induced cancer, we investigated several parameters of the tumor microenvironment and found that propranolol increased gene expression of Casp3. Our data indicate that propranolol treatment that starts after exposure to carcinogens might represent a new, useful approach for preventing the development of cancer, especially in stressed individuals. However, the potential efficiency of propranolol treatment for preventing cancer development and progression in individuals exposed to carcinogens needs further investigation.

Catecholaminergic Innervation of the Lateral Nucleus of the Cerebellum Modulates Cognitive Behaviors.

The cerebellum processes neural signals related to rewarding and aversive stimuli, suggesting that the cerebellum supports nonmotor functions in cognitive and emotional domains. Catecholamines are a class of neuromodulatory neurotransmitters well known for encoding such salient stimuli. Catecholaminergic modulation of classical cerebellar functions have been demonstrated. However, a role for cerebellar catecholamines in modulating cerebellar nonmotor functions is unknown. Using biochemical methods in male mice, we comprehensively mapped TH+ fibers throughout the entire cerebellum and known precerebellar nuclei. Using electrochemical (fast scan cyclic voltammetry), and viral/genetic methods to selectively delete Th in fibers innervating the lateral cerebellar nucleus (LCN), we interrogated sources and functional roles of catecholamines innervating the LCN, which is known for its role in supporting cognition. The LCN has the most TH+ fibers in cerebellum, as well as the most change in rostrocaudal expression among the cerebellar nuclei. Norepinephrine is the major catecholamine measured in LCN. Distinct catecholaminergic projections to LCN arise only from locus coeruleus, and a subset of Purkinje cells that are positive for staining of TH. LC stimulation was sufficient to produce catecholamine release in LCN. Deletion of Th in fibers innervating LCN (LCN-Th-cKO) resulted in impaired sensorimotor integration, associative fear learning, response inhibition, and working memory in LCN-Th-cKO mice. Strikingly, selective inhibition of excitatory LCN output neurons with inhibitory designer receptor exclusively activated by designer drugs led to facilitation of learning on the same working memory task impaired in LCN-Th-cKO mice. Collectively, these data demonstrate a role for LCN catecholamines in cognitive behaviors.SIGNIFICANCE STATEMENT Here, we report on interrogating sources and functional roles of catecholamines innervating the lateral nucleus of the cerebellum (LCN). We map and quantify expression of TH, the rate-limiting enzyme in catecholamine synthesis, in the entire cerebellar system, including several precerebellar nuclei. We used cyclic voltammetry and pharmacology to demonstrate sufficiency of LC stimulation to produce catecholamine release in LCN. We used advanced viral techniques to map and selectively KO catecholaminergic neurotransmission to the LCN, and characterized significant cognitive deficits related to this manipulation. Finally, we show that inhibition of excitatory LCN neurons with designer receptor exclusively activated by designer drugs, designed to mimic Gi-coupled catecholamine GPCR signaling, results in facilitation of a working memory task impaired in LCN-specific TH KO mice.

Effect of partial hysterectomy on the neurons of the paracervical ganglion (PCG) of the pig.

Autonomic neurons innervating uterine horn is probably the only nerve cell population capable of periodical physiological degeneration and regeneration. One of the main sources of innervation of the uterus is paracervical ganglion (PCG). PCG is a unique structure of the autonomic nervous system. It contains components of both the sympathetic and parasympathetic nervous system. The present study examines the response of neurons of PCG innervating uterine horn to axotomy caused by partial hysterectomy in the domestic pig animal model. The study was performed using a neuronal retrograde tracing and double immunofluorescent staining for tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DβH), choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), neuronal nictric oxide synthase (nNOS), galanin, neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), somatostatin and substance P (SP). Our study showed that virtually all neurons of the porcine PCG innervating uterine horn are adrenergic and we did not confirm that PCG is the source of cholinergic fibers innervating uterine horn of the pig. After axotomy there was a decrease in expression of catecholamine-synthesizing enzymes (TH, DβH) and a strong increase in the galanin expression. The increase of the number of NPY-IR neurons in the ganglia after axotomy was observed. There were no changes in the expression of other studied substances in the PCG neurons innervating the uterine horn, what was often found in rodents studies. This indicates that neurons can respond to damage in a species-specific way.

Adipocytes and macrophages secretomes coregulate catecholamine-synthesizing enzymes.

Obesity associates with macrophage accumulation in adipose tissue where these infiltrating cells interact with adipocytes and contribute to the systemic chronic metabolic inflammation present in immunometabolic diseases. Tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) are two of the main enzymes of catecholamines (CA) synthesis. Adipocytes and macrophages produce, secrete and respond to CA, but the regulation of their synthesis in the interplay between immune and metabolic systems remains unknown. A model of indirect cell coculture with conditioned medium (CM) from RAW 264.7 macrophages with or without LPS-activation and 3T3-L1 adipocytes and preadipocytes was established to study the effect of cellular secretomes on the expression of the above enzymes. During the adipocyte differentiation process, we found a decrease of TH and PNMT expression. The secretome from LPS-activated macrophages downregulated TH and PNMT expression in preadipocytes, but not in mature adipocytes. Mature adipocytes CM induced a decrease of PNMT levels in RAW 264.7 macrophages. Pre and mature adipocytes showed a similar pattern of TH, PNMT and peroxisome proliferator-activated receptor gamma expression after exposure to pro and anti-inflammatory cytokines. We evidenced macrophages and adipocytes coregulate the expression of CA synthesis enzymes through secretome, with non-inflammatory signaling networks possibly being involved. Mediators released by macrophages seem to equally affect CA production by adipocytes, while adipocytes secretome preferentially affect AD production by macrophages. CA synthesis seems to be more determinant in early stages of adipogenic differentiation. Our results suggest that CA are key signaling molecules in the regulation of immune-metabolic crosstalk within the adipose tissue.

ЗГАСЛА ЯСКРАВА ЗІРКА В СУЗІР’Ї ВИДАТНИХ НЕЙРОНАУКОВЦІВ (спомин про Олега Горникевича)

Oleh Hornykiewicz, one of the most accomplished neuropharmacologists of the 20th century passed away on May 26, 2020 at the age of 93. In 1939, Oleh’s father, a Ukrainian Catholic priest, fled with his young family from Soviet-occupied Ukraine and settled in Vienna. During his medical studies, Oleh became fascinated by pharmacology and in 1956 was awarded a fellowship to Oxford University (U.K.) where he worked with Dr. Hermann Blaschko, a pioneer in the study of enzymes of catecholamine synthesis and metabolism. At that time, it was still thought that dopamine was merely an intermediate in the synthesis of norepinephrine. Dr. Hornykiewicz demonstrated that even after its breakdown had been blocked, administration of dopamine or its precursor L-DOPA could elevate blood pressure in animal models. Dopamine was bioactive per se. Upon his return to Vienna in 1958, the young scientist began studying dopamine in the brain. Familiar with Parkinson’s Disease and intrigued by Arvid Carlsson’s finding that L-DOPA could counteract the akinesia of the reserpenized rat, Dr. Hornyckiewicz began to suspect that a dopamine deficiency mediated the motor symptoms of Parkinson’s disease. He developed new techniques, applied these to the study of postmortem brains, and demonstrated that Parkinson’s disease was associated with a dopamine deficiency in the basal ganglia. Shortly afterwards, he and Walter Birkmeyer observed how the intravenous administration of L-DOPA could awaken mute and akinetic patients with Parkinson’s disease. For the first time, a discreet chemical was both implicated in a complex brain disorder and could be used as a treatment. This stands as one of the seminal discoveries in neuropharmacology. To this day, L-DOPA remains a mainstay in the treatment of Parkinson’s disease. Over the following half-century Dr. Hornykiewicz continued his own work, generously mentored others and established brain banks in Vienna as well as in Toronto, Canada where he worked for many years. He received numerous awards, and was nominated for but not awarded the Nobel Prize in Medicine, in a decision that has remained controversial. He will remain forever, a brilliant star in the history of neuropharmacology.

Histopathological Analysis of Tumor Microenvironment and Angiogenesis in Pheochromocytoma.

Pheochromocytomas (PHEOs) are relatively rare catecholamine-producing tumors derived from adrenal medulla. Tumor microenvironment (TME) including neoangiogenesis has been explored in many human neoplasms but not necessarily in PHEOs. Therefore, in this study, we examined tumor infiltrating lymphocytes (CD4 and CD8), tumor associated macrophages (CD68 and CD163), sustentacular cells (S100p), and angiogenic markers (CD31 and areas of intratumoral hemorrhage) in 39 cases of PHEOs in the quantitative fashion. We then compared the results with pheochromocytoma of the adrenal gland scaled score (PASS), grading system for pheochromocytoma and paraganglioma (GAPP) and the status of intra-tumoral catecholamine-synthesizing enzymes (TH, DDC, and PNMT) as well as their clinicopathological factors. Intratumoral CD8 (p = 0.0256), CD31 (p = 0.0400), and PNMT (p = 0.0498) status was significantly higher in PHEOs with PASS <4 than PASS ≧4. In addition, intratumoral CD8+ lymphocytes were also significantly more abundant in well-than moderately differentiated PHEO according to GAPP score (p = 0.0108) and inversely correlated with tumor size (p = 0.0257). Intratumoral CD68+ cells were significantly higher in PHEOs with regular or normal histological patterns than those not (p = 0.0370) and inversely correlated with tumor size (p = 0.0457). The status of CD163 was significantly positively correlated with that of CD8 positive cells (p = 0.0032). The proportion of intratumoral hemorrhage areas was significantly higher in PHEOs with PASS ≧4 (p = 0.0172). DDC immunoreactivity in tumor cells was significantly positively correlated with PASS score (p = 0.0356) and TH status was significantly higher in PHEOs harboring normal histological patterns (p = 0.0236) and cellular monotony (p = 0.0219) than those not. Results of our present study did demonstrate that abundant CD8+ and CD68+ cells could represent a histologically low-scored tumor. In particular, PHEOs with increased intratumoral hemorrhage should be considered rather malignant. In addition, abnormal catecholamine-producing status of tumor cells such as deficient PNMT and TH and increased DDC could also represent more aggressive PHEOs.

Transition-State Analogues of Phenylethanolamine N-Methyltransferase.

Phenylethanolamine N-methyltransferase (PNMT) is a critical enzyme in catecholamine synthesis. It transfers the methyl group of S-adenosylmethionine (SAM) to catalyze the synthesis of epinephrine from norepinephrine. Epinephrine has been associated with diverse human processes, including the regulation of blood pressure and respiration, as well as neurodegeneration found in Alzheimer's disease. Human PNMT (hPNMT) proceeds through an SN2 transition state (TS) in which the transfer of the methyl group is rate limiting. TS analogue enzyme inhibitors are specific for their target and bind orders of magnitude more tightly than their substrates. Molecules resembling the TS of hPNMT were designed, synthesized, and kinetically characterized. This new inhibitory scaffold was designed to mimic the geometry and electronic properties of the hPNMT TS. Synthetic efforts resulted in a tight-binding inhibitor with a Ki value of 12.0 nM. This is among the first of the TS analogue inhibitors of methyltransferase enzymes to show an affinity in the nanomolar range. Isothermal titration calorimetry (ITC) measurements indicated negative cooperative binding of inhibitor to the dimeric protein, driven by favorable entropic contributions. Structural analysis revealed that inhibitor 3 binds to hPNMT by filling the catalytic binding pockets for the cofactor (SAM) and the substrate (norepinephrine) binding sites.