Molecular Psychiatry (2012) 17, 354–358 & 2012 Macmillan Publishers Limited All rights reserved 1359-4184/12 www.nature.com/mp PERSPECTIVE Chromaffin cells: the peripheral brain SR Bornstein1, M Ehrhart-Bornstein1, A Androutsellis-Theotokis1,2, G Eisenhofer1, V Vukicevic1, J Licinio3, ML Wong3, P Calissano2, G Nistico` 2, P Preziosi2,* and R Levi-Montalcini2 1Department of Medicine III, University of Dresden, Dresden, Germany; 2European Brain Research Institute Rome, Rome, Italy and 3John-Curtin-Medical-School Canberra, Canberra, Australia

Chromaffin cells probably are the most intensively studied of the neural crest derivates. They are closely related to the nervous system, share with neurons some fundamental mechanisms and thus were the ideal model to study the basic mechanisms of neurobiology for many years. The lessons we have learned from chromaffin cell biology as a peripheral model for the brain and brain diseases pertain more than ever to the cutting edge research in neurobiology. Here, we highlight how studying this cell model can help unravel the basic mechanisms of cell renewal and regeneration both in the central nervous system (CNS) and neuroendocrine tissue and also can help in designing new strategies for regenerative therapies of the CNS. Molecular Psychiatry (2012) 17, 354–358; doi:10.1038/mp.2011.176; published online 17 January 2012 Keywords: adrenal medulla; neurobiology; neuronal differentiation; stem cells

Chromaffin cells form perhaps the most interesting of the brain. Chromaffin cells served as the model to cellular system in the human body. They are closely identify the basic concept of neurochemical transmis- related to sympathetic neurons and are one of the sion. The first secretory organelles were isolated from most intensively studied of the neural crest derivates. chromaffin cells, and chromaffin vesicles served as As an evolutionary hybrid of both the endocrine and the model neurotransmitter-containing vesicle. Many nervous system they have served as a model to of the major vesicle-associated proteins involved explore the basic mechanisms of neurophysiology, in the process of exocytosis were characterized in regulated secretion and pharmacology. Thus, adrenal chromaffin vesicles.1–5 chromaffin cells can be considered as the peripheral Sharing the same ectodermic (neural crest) origin, brain as they share with neurons some fundamental chromaffin cells are part of the so called Erspamer’s mechanisms: (1) Receive inputs of electrical and triangle (skin, gut and brain), which possess the chemical nature; (2) are able to decode and recognize same neurotransmitters, neuropeptides, transduction these signals; (3) possess the machinery to generate mechanisms.6 Much of today’s understanding of the and elaborate pattern of responses such as the release physiology and pathophysiology of neuropeptide and of catecholamines and other messengers. In conclu- monoamine neurotransmitter systems is owed to sion, the chromaffin cells, such as a neuron can be studies utilizing chromaffin cell model systems. The viewed as a secretory cell that releases its secretion at rat pheochromocytoma (PC12) cell line developed by large distance from cell body where macromolecules Greene and Tischler7 over 35 years ago remains one are synthesized, provide for rapid communication of the most well studied and to this day continues between widely separated parts of the body. In to provide a powerful model for the understanding of addition, the adrenal medulla with its chromaffin neuronal systems. This cell line and other chromaffin cells serves through the release of catecholamines for model systems have been particularly useful for communication with all the most important organs studies of exocytotic mechanisms, including the such as the heart, vascular apparatus, lungs, kidney operation of ion channels, vesicular dynamics and and also the brain. stimulus-secretion coupling. Associated methodolo- More importantly, chromaffin cells located in the gical advances in electrophysiology, from develop- center of an easily accessible peripheral organ have ment of the patch clamp to more sophisticated served for many years as a window into the functions techniques combining electrophysiological and elec- trochemical methods (for example, patch ampero- Correspondence: Dr SR Bornstein, Department of Medicine, metry) have been largely facilitated by the availability University of Dresden, Universita¨tsklinikum Carl Gustav of such model systems. Carus an der TU Dresden, Fetscherstrae 74, Dresden 01307, In cat’s adrenal gland perfused in situ it was clearly Germany. demonstrated by using different receptorial stimuli E-mail: [email protected] *Emeritus Professor Catholic University, Rome, Italy. (acetylcholine, nicotine, dimethylphenylpiperazi- Received 1 November 2011; accepted 2 November 2011; mium and so on) that chromaffin cells posses two published online 17 January 2012 distinct neurosecretory granules (the noradrenergic Chromaffin cells biology SR Bornstein et al 355 and the adrenergic ones), thus ruling out the idea nitric-oxide system of the brain. Thus, the basic that the noradrenergic ones were the precursor mechanisms of the nitric-oxide regulation both for cells in which phenylethanolamine-N-methyl- neurons and endocrine cells have been identified transferase functions to turn them into adrenergic in chromaffin cells.22 Similarly, the pituitary and chromaffin cells. In addition, also at the brain level the adrenal medulla contain the highest amount of D-amphetamine particularly increase the release of vitamin C in the human body, and mechanisms of the noradrenaline.8,9 of role vitamin C uptake and regulation of neurotrans- In addition to improving understanding of neural mitters were identified in chromaffin cells.23 development processes, chromaffin cell systems The broad role of nerve growth factor (NGF) in the have been extremely important for studies of neuro- living organism was first discovered in the adrenal degenerative processes, tumorigenesis and drug medulla.24 Indeed, Unsicker et al.25 at that time at the development. Johns Hopkins University found that immature Furthermore, due to the close relation of chromaffin chromaffin cells obtained from the adrenal medulla cells to catecholaminergic neurons they have even cultured in the presence of NGF acquire the biochem- been used for the treatment of neurodegenerative ical and morphological properties of sympathetic brain disorders such as Parkinson’s disease.10 neurons. Between 1988 and 2001, > 300 Parkinsonian patients Furthermore, experiments carried out in the CNR were treated by autologous adrenal transplants with (Italian Council for Research) laboratory of cell some improvement of the clinical symptoms. How- biology in Rome by Aloe and Levi-Montalcini26 ever, the survival rates of grafted adult chromaffin demonstrated in vivo that application of NGF into cells were only short-term and clinical improvements the rat fetus and continued for 3 weeks after birth disappeared 1–2 years after transplantation.11,12 A induced the differentiation of chromaffin cells into serious limitation in the application of adult adrenal sympathetic neurones within the adrenal gland. This medulla probably was the post-mitotic nature of most clearly established that NGF had a much wider role in cells transplanted. the living organisms than had been supposed so far. At the same time, endocrinologists have explored The central medulla markedly increased in volume the role of several central releasing hormones and as a result of the differentiation of adrenal cells into neuropeptides within the chromaffin cell systems.13 sympathetic neurons, which sprout a large number of Interestingly, the adrenal medulla in the periphery widely branching fibers. expresses a similar set of neuropeptides that occur in Given the outstanding role the chromaffin cell the brain involved in stress regulation, energy homeo- system has had in the past, it would be a mistake stasis, anxiety and pain.14,15 The concept of ectopic not to use the system for the current issues of brain hormone production was among others described and research. refined in these cells. This includes the expression of We have entered a new era of regenerative medicine corticotropin-releasing hormone, adrenocorticotro- also for neurodegenerative diseases of the brain. pin, pro-opiomelanocortin and other neuropeptides Chromaffin cells could again take the lead to explore in the adrenal medulla. The intense crosstalk of some of the mechanisms of regeneration that pertain endocrine cells, the paracrine and neurocrine path- in a similar way to the more complex central nervous ways of endocrine communication, were established system (CNS) disorders. Chromaffin cell lines are now particularly in the adrenal gland.16 Here, again the being used to explore the role of NGF in Alzheimer’s complex but accessible microenvironment of the disease. Indeed, proteins from chromaffin granules adrenal mimics the microenvironment of the brain promote survival of neurons,27 which may be due to with respect to the crosstalk of neuronal structures a number of known or yet unknown neurotrophin with different endocrine cell types.17 Furthermore, factors. Thus, NGF deprivation from differentiated the action of steroids and neurosteroids occurring PC12 cells caused overproduction of amyloid-b pep- in the brain has been widely studied in chromaffin tides, which are the most toxic protein fragments cell systems.18,19 The strict interaction between directly implicated in the development of Alzheimer’s the cortical and the medullary part of the adrenal disease, concomitantly with cell death by apoptosis.28 gland was demonstrated since long time. In fact, the The tight connection between NGF deprivation and inhibition of biosynthesis of adrenal glucocorticoids activation of the amyloidogenic pathway has been by the specific inhibitor aminoglutethimide deter- extended to hippocampal neurons.29 These studies mined both in cats and rats a significant decrease of have revealed a new property of TrkA, the high affinity catecholamines at the medullary level without NGF receptor. When deprived of NGF TrakA switches changes between the two types of chromaffin cells. from a prosurvival to a proapoptotic cell signaling Therefore, glucocorticoids excert a permissive role on system. It is reasonable to hypothesize that such the chromaffin system through a double mechanism, property is also operative in chromaffin neurons a direct one at the chromaffin cells and an indirect bearing NGF receptor and underly new avenues for one via the inhibition of CRH release at the hypo- cell signaling machineries in these cells.29 thalamic levels.20,21 NGF also promotes cell survival during endo- Whereas the peripheral nitric-oxide system occurs plasmic reticulum stress in PC12 cells.30 More- in the adrenal cortex, chromaffin cells express the over, PC12 cells are now widely used to study the

Molecular Psychiatry Chromaffin cells biology SR Bornstein et al 356 effect and signaling pathways of numerous other neurotrophic and neuroprotective peptides in brain regeneration. This includes pituitary adenylate cyclase-activating polypeptide,31,32 bone morpho- genetic protein 733 and cerebral dopamine neuro- trophic factor,34 which may restore dopaminergic neurons in degenerative diseases of the brain such as Parkinson’s disease. Sequence variations in the BDNF gene have been associated with major depression and antidepressant treatment success,35 and the role of BDNF in mediating the neuroprotective role of antidepressants has been recently explored in chromaffin cell lines.36 Furthermore, mounting evidence suggests the exis- tence of multipotent neural crest-derived progenitor cells in the adult adrenal medulla.37 Their recent identification and isolation38 sparks off new hopes for their potential use in regenerative treatment of neurodegenerative diseases such as Parkinson’s Figure 1 Adult chromaffin precursor cells behave simi- 39–41 larly to adult neural stem cells. Over a century ago, Kohn disease. Chromaffin progenitor cells share signi- demonstrated the similarities between chromaffin cells and ficant properties with neural stem cells. Similar to neurons.53–55 As a consequence, isolated chromaffin cells neurospheres, when prevented from adherence to and the PC12 cell line, derived from a rat adrenal medulla the culture dish, chromaffin progenitor cells grow pheochromocytoma have been established as models of in spheres with self-renewing capacity, which we neuronal differentiation. Recent advances in stem cell named chromospheres. They express the neural biology have generated culture methods that enable the progenitor markers nestin, vimentin, musashi 1 and propagation of primary precursor cells from non-cancerous NGF receptor, as well as Sox1 and Sox10,38 Mash137 tissue. These techniques have expanded the similarities and proteins of the Notch pathway (Vukicevic and between adult mature chromaffin cells and adult mature colleagues, under revision). Furthermore, they are neurons to their precursor cells of origin: it is now becoming increasingly understood that the precursors of chromaffin able to differentiate to mature catecholaminergic 38 cells behave similarly to neural stem cells from the CNS. (a) neurons (Vukicevic and colleagues, under revision). Neural stem cells from the fetal and adult brain and Similar to differentiating neural stem cells, where chromaffin precursors from the adult bovine adrenal Notch is a key regulator of neural stem cell main- medulla can be cultured without cell attachment to the tenance in the developing nervous system,42,43 the substrate, giving rise to spheroid (3-dimensional) structures. shift toward neuronal differentiation of chromo- (b) Like their CNS counterparts, adult bovine adrenal sphere cells is accompanied by a reduction of neural medulla precursor cells can also be cultured as monolayers progenitor markers including Notch-2, Hes (hairy and in the absence of serum, with mitogenic support from basic enhancer of split) 1, Hes 5 and nestin (Vukicevic and fibroblast growth factor (bFGF). Withdrawal of bFGF from colleagues, under revision). the culture medium induces the differentiation of these cells within 2 days. (Inset: monolayer cultures of neural Finally, it should not been forgotten that entero- stem cells in the presence of bFGF for morphology chromaffin cells derive also from the neural crest, comparison). and represent the site in which Vittorio Erspamer discovered in 1930s enteramine,44 which later was found identical to serotonin (5-HT)45 present in CNS.46 A large number of papers exist in the literature possibly as a means of preserving homeostasis and that show that other neurotransmitters, neuropep- memory. The adrenal gland, in stark contrast, is a tides and the same transduction mechanisms present remarkably plastic organ that reacts in marked fashion in the brain exist also in the gastrointestinal chro- to many physical and emotional stresses. For the maffin cells where they have important roles.47 scientist researching state change, the adrenal gland is Thus, chromaffin cells and brain neurons and their an outstanding tool case. Within it, the canary in the precursor cells share similar signaling pathways and coal mine is the chromaffin cell and its precursor cell. again constitute an ideal model to identify the innate Physical and emotional insults alter the function, pathways and targets for brain regeneration. properties and numbers of these cells in a way, which Recent efforts probing the endogenous regenerative is easy to assess and manipulate. The relevance to and repair potential of the adult CNS are met with neuroscience is direct: chromaffin precursor cells great difficulties, which are based on the system’s share many common features with their bona fide rigidity and limited access to pharmaceuticals. The CNS brethren; they can be cultured in much the same blood–brain barrier limits the number of factors that way (Figure 1), they express many common markers can be tested systemically and necessitates invasive including components of the cytoskeleton and tran- alternatives such as direct injections into the brain. scription factors and, critically, they respond to many Rigidity comes from the subtle plasticity exhibited, treatments similarly. As a research system to the

Molecular Psychiatry Chromaffin cells biology SR Bornstein et al 357 neuroscientist, the adrenal gland is a model of the new strategies for regenerative therapies of the brain brain, outside the blood–brain barrier, exhibiting in the future. augmented reactions. Within it, chromaffin precursor cells are an accessible and measurable window to the workings of CNS neural stem cells. At a time when Conflict of interest scientists strive to not pigeon-hole themselves within one organ, but search for clues that will lead to novel The authors declare no conflict of interest. therapeutic approaches in as many places as possible, the chromaffin cell system is an outstanding research companion to its more esoteric CNS counterpart. Acknowledgments In the CNS, a role of endogenous neural stem cells This work was supported by grants of the Deutsche (eNSCs) in generating new neurons is only recognized Forschungsgemeinschaft: KFO 252/1 (SRB, MEB, in two areas, the subventricular zone and the dentate AAT, GE), SFB 655 (MEB, SRB) and the Center for gyrus of the hippocampus. Yet, populations of eNSCs Regenerative Therapies Dresden Cluster of Excellence have been discovered throughout the brain and spinal to (SRB, MEB). cord of adult rodents and primates. A widespread population can be identified using expression of the transcription factor Hes3.48 Such observations have References raised roles for eNSCs other than cell replacement. Hes3 is regulated by a non-canonical Notch pathway 1 Helle KB, Reed RK, Ehrhart M, Aunis D, Hogue AR. 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