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Cellular diversity and stress-induced cell-state switches in the mammalian hypothalamus

Periodic Reporting for period 2 - Secret-Cells (Cellular diversity and stress-induced cell-state switches in the mammalian hypothalamus)

Reporting period: 2018-03-01 to 2019-08-31

Summary of the context:

Any organism that intends to survive is continuously required to adapt and respond to its environment. Within multicellular bodies, intercellular lines of communication have evolved to allow rapid metabolic commands be executed once sensing a change in environmental parameters. The vertebrate hypothalamus acts as an interface between peripheral tissues that use humoral signals and hierarchically-wired neural centers (cognitive, reward and motor circuits) that are synaptically connected to the hypothalamus to allow for near-infinite numbers of response modalities towards the environment, maximizing the organism’s ability for adaptation. Within the hypothalamus, a spectrum of neuroendocrine cells co-exist and use hormonal, neuropeptide and neurotransmitter codes for local as well as long-range intercellular communication.

A key feature of hypothalamic organization is that instead of the sheer numerical expansion of specific cell types (as seen for cortical evolution to increase computational power), the number of specialized neuroendocrine units has increased while leaving cell numbers per unit at the range of 500-10,000 cells. This has led to exceptional cellular heterogeneity in a limited brain volume. This arrangement represents a major paradigm shift as compared to laminated brain structures since cellular reserves and ensuing functional redundancy are at a minimum, and the loss of any cell type seems causal to neuroendocrine diseases, and requires life-long intervention (e.g. hormone replacement therapy), if available, to sustain functionality. During the past century, an avalanche of eminent neuroanatomy and endocrine studies have been carried out to matching. one-on-one, specific and metabolically-driven peripheral (hormonal) signals to the activation of neuroendocrine cells in the hypothalamus. Conversely, the ablation of parts of the hypothalamus (first by toxins and more recently by optical and chemogenetic tools) was used to define causality between neuroendocrine commands and behavioral (e.g. aggression, nursing, sleep-wake cycle) and metabolic (food and water intake, electrolyte balance) outcomes. However, the limited number of function-defining neurons and their many modalities that might exist in the hypothalamus make the establishment of a unified taxonomy of neuronal subtypes and their relationships a daunting task.

High-throughput single-cell RNA-sequencing and associated bioinformatic pipelines have brought a sea-change in our understanding of cellular diversity in the mammalian (human) body. Therrefore, we first took advantage of this technique in combination with a range of experimental validation strategies to generate an atlas of hypothalamic cell types (Romanov et al., 2017; Romanov et al., 2019). Initially, our focus was on the paraventricular nucleus of the hypothalamus, a small brain volume which classically is viewed to have many subterritories and being the host to many magno- and parvocellular neuronal subtypes, including those that operate the stress circuitry.

On the basis of establishing an overarching taxonomy classifying hypothalamic neuroendocrine output as well as interneurons, we have made an attempt to molecularly subclassify stress-sensing (on/off) neurons, with an intent to identify molecular features that enable their rapid and repetitive responses upon stimulation. By identifying rate-limiting molecular check-points in stress neurons we have asked if any of these molecular marks is expressed in other tissues and found commonalities with pancreatic beta cells.

We have particularly focused on secretagogin, an EF-hand calcium-binding protein, which we showed to control corticotropin-releasing hormone release upon acute stress in the hypothalamus, as well as insulin release from pancreatic beta cells. Nevertheless, and while adhering to the original work program of this ERC award, the study of secretagogin had taken us to entirely uncharted territories of developmental and addiction biology, with the most exciting results relating to beta cell differentiation and life-long modifications of beta-cell function upon maternal lifestyle choices during pregnancy.

Overall objectives:

- To define how neurotranmistter, neuropeptide (and neuromodulator) constellations identify neuroendocrine neurons (as well as other cell types) in the mammalian hypothalamus,

- To analyze transcriptional signatures upon stimuli (particularly acute stress) to produce a molecular definition of “cell state-changes” (that is, no change in identity but acute functional adaptation to the onset/offset of a stimulus), including changes in neuron-glia communication,

- Analyze the biophysics of neuropeptide co-release and how neuropeptides modulate fast neurotransmission,

- Dissect the role of secretagogin in brain and pancreas, and test if secretagogin can carry meaningful biological activity in blood/extracellularly.

The problem/issue being addressed:

The hypothalamus lacks developmental organizational principles that apply for laminated structures. Therefore, investigators have only looked at specific sub-nuclei (that is neuronal assembiles which define single functional outputs, such as feeding and nursing) so far. Yet a unified cellular taxonomy (atlas) of the hypothalamus, including knowledge on molecular features of potentially novel cell types, is missing. Moreover, how glia, tanycytes (ventricular interface cells) and neurons shape multimodal signaling units, probably combining synaptic and volume transmission, is effectively unknown. Lastly, molecular features in endocrine cells that are rate-limiting and function-determinant are superficially mapped, limiting therapeutic benefit in metabolic disorders. By focusing on acute stress as an experimental paradigm, we produced (and continue to do so) proof-of-principle data for molecular neuroendocrinology at the level of cellular and network analyses. We have achieved great progress in all specific aims, which we rate above and beyond that had initially been expected for this ERC award.

Social importance:

Neuroendocrine diseases are on a steep rising trajectory in our society. These include the obesity epidemic, reduced fertility in both women and men, stress (acute and chronic leading to neuropsychiatric conditions such as “burn-out syndrome”, depression, post-traumatic stress disoreder), sleep disorders (insomnia, disrupted sleep patterns) and pressures on interpetsonal (social) contacts (aggression, defence).

Therefore, understanding hypothalamic cellular diversity, function determination, local and long-range connectivity to orchestrate neuronal responses, and the encoding and representation of hormonal responses in conscious decision making/actions through hypothalamic connections with other brain areas is of paramount significance. In this ERC award, we have created a cellular atlas, singled out novel neuronal subtypes for analysis, associated those with specific disorders and environmental exposures, and characterized some of their key molecular marks as well as cellular and network features. This knowledge seems to us very important for novel generations of medical strategies to emerge.

Besides immediate professionals, the general public will benefit from our line of work since we can and will spearhead public outreach on risks to life-long well-being imposed by lifestyle and illicit drugs.
Results related to Theme 1

Molecular interrogation of hypothalamic organization
(Romanov et al., Nat Neurosci. (2017))
The hypothalamus contains the highest diversity of neurons in the brain. Many of these neurons can co-release neurotransmitters and neuropeptides in a use-dependent manner. Investigators have hitherto relied on candidate protein-based tools to correlate behavioral, endocrine and gender traits with hypothalamic neuron identity. In this study, we mapped neuronal identities in the hypothalamus by single-cell RNA sequencing. We distinguished 62 neuronal subtypes producing glutamatergic, dopaminergic or GABAergic markers for synaptic neurotransmission and harboring the ability to engage in task-dependent neurotransmitter switching. We identified dopamine neurons that uniquely coexpress the Onecut3 and Nmur2 genes, and placed these in the periventricular nucleus with many synaptic afferents arising from neuromedin S+ neurons of the suprachiasmatic nucleus. These neuroendocrine dopamine cells may contribute to the dopaminergic inhibition of prolactin secretion diurnally, as their neuromedin S+ inputs originate from neurons expressing Per2 and Per3 and their tyrosine hydroxylase phosphorylation is regulated in a circadian fashion. Overall, our catalog of neuronal subclasses provides new understanding of hypothalamic organization and function.

Molecular diversity of corticotropin-releasing factor-containing neurons
(Romanov et al. J Endocrinol (2017))
Hormonal responses to acute stress rely on the rapid induction of corticotropin-releasing hormone (CRH) production in the mammalian hypothalamus, with subsequent instructive steps culminating in corticosterone release at the periphery. Hypothalamic CRH neurons in the paraventricular nucleus of the hypothalamus are therefore considered as 'stress neurons'. However, significant morphological and functional diversity among neurons that can transiently produce CRH in other hypothalamic nuclei has been proposed, particularly as histochemical and molecular biology evidence associates CRH to both GABA and glutamate neurotransmission. We suggested, based on single-cell RNA sequencing and circuit mapping, that CRH production reflects a state switch in hypothalamic neurons and thus confers functional competence rather than being an identity mark of phenotypically segregated neurons. We showed that CRH mRNA transcripts can therefore be seen in GABAergic, glutamatergic and dopaminergic neuronal contingents in the hypothalamus. We then distinguished 'stress neurons' of the paraventricular nucleus that constitutively express secretagogin, a calcium sensor critical for the stimulus-driven assembly of the molecular machinery underpinning the fast regulated exocytosis of CRH at the median eminence. Cumulatively, we infer that CRH neurons are functionally and molecularly more diverse than previously thought.

A novel output pathway of CRH neurons uses volume transmission to orchestrate post-traumatic cortical sensitization
(Alpar et al., EMBOJ (2018))
Stress-induced cortical alertness is maintained by a heightened excitability of noradrenergic neurons innervating, notably, the prefrontal cortex. However, neither the signaling axis linking hypothalamic activation to delayed and lasting noradrenergic excitability nor the molecular cascade gating noradrenaline synthesis is defined. We discovered that hypothalamic corticotropin-releasing hormone-releasing neurons innervate ependymal cells of the 3rd ventricle to induce ciliary neurotrophic factor (CNTF) release for transport through the brain's aqueductal system. CNTF binding to its cognate receptors on norepinephrinergic neurons in the locus coeruleus then initiates sequential phosphorylation of extracellular signal-regulated kinase 1 and tyrosine hydroxylase with secretagogin ensuring activity dependence in both rodent and human brains. Both CNTF and secretagogin ablation occlude stress-induced cortical norepinephrine synthesis, ensuing neuronal excitation and behavioral stereotypes. Cumulatively, we identified a multimodal pathway that is rate-limited by CNTF volume transmission and poised to directly convert hypothalamic activation into long-lasting cortical excitability following acute stress.

Results Related to Theme 2

Secretagogin regulates neuroblast migration by regulating matrix metalloprotease release
(Hanics et al., PNAS (2017))
The rostral migratory stream (RMS) is viewed as a glia-enriched conduit of forward-migrating neuroblasts in which chemorepulsive signals control the pace of forward migration. We demonstrated the existence of a scaffold of neurons that receive synaptic inputs within the rat, mouse, and human fetal RMS equivalents. These neurons express secretagogin to execute an annexin V-dependent externalization of matrix metalloprotease-2 (MMP-2) for reconfiguring the extracellular matrix locally. Mouse genetics combined with pharmacological probing in vivo and in vitro demonstrated that MMP-2 externalization occurs on demand and that its loss slows neuroblast migration. Loss of function is particularly remarkable upon injury to the olfactory bulb. Cumulatively, we identified a signaling cascade that provokes structural remodeling of the RMS through recruitment of MMP-2 by a previously unrecognized neuronal constituent. Given the life-long presence of secretagogin-containing neurons in human, this mechanism might be exploited for therapeutic benefit in rescue strategies.

Secretagogin regulates beta-cell turnover
(Malenczyk et al., EMBOJ (2017))
Calcium-sensor proteins are generally implicated in insulin release through SNARE interactions. In this study, secretagogin, whose expression in human pancreatic islets correlates with their insulin content and the incidence of type 2 diabetes, is shown to orchestrate an unexpectedly distinct mechanism. Single-cell RNA-seq revealed retained expression of the TRP family members in β-cells from diabetic donors. Amongst these, pharmacological probing identified calcium-permeable transient receptor potential vanilloid type 1 channels (TRPV1) as potent inducers of secretagogin expression through recruitment of Sp1 transcription factors. Accordingly, agonist stimulation of TRPV1s failed to rescue insulin release from pancreatic islets of glucose intolerant secretagogin knock-out(-/-) mice. However, instead of merely impinging on the SNARE machinery, reduced insulin availability in secretagogin-/- mice was due to β-cell loss, which is underpinned by the collapse of protein folding and deregulation of secretagogin-dependent USP9X deubiquitinase activity. Therefore, and considering the desensitization of TRPV1s in diabetic pancreata, a TRPV1-to-secretagogin regulatory axis seems critical to maintain the structural integrity and signal competence of β-cells.

Secretagogin controls beta-cell specification by configuring permissive thranscirption factor landscape
(Malenczyk et al., Mol Metab (2018))
Specification of endocrine cell lineages in the developing pancreas relies on extrinsic signals from non-pancreatic tissues, which initiate a cell-autonomous sequence of transcription factor activation and repression switches. The steps in this pathway share reliance on activity-dependent calcium signals. However, the mechanisms by which phasic calcium surges become converted into a dynamic, cell-state-specific and physiologically meaningful code made up by transcription factors constellations remain essentially unknown. We used high-resolution histochemistry to explore the coincident expression of secretagogin and transcription factors driving β cell differentiation. Secretagogin promoter activity was tested in response to genetically manipulating Pax6 and Pax4 expression. Secretagogin null mice were produced with their pancreatic islets morphologically and functionally characterized during fetal development. A proteomic approach was utilized to identify the calcium-dependent interaction of secretagogin with subunits of the 26S proteasome and verified in vitro by focusing on Pdx1 retention. We showed that secretagogin, which controls α and β cell turnover in adult, is in fact expressed in endocrine pancreas from the inception of lineage segregation in a Pax4-and Pax6-dependent fashion. By genetically and pharmacologically manipulating secretagogin expression and interactome engagement in vitro, we found secretagogin to gate excitation-driven calcium signals for β cell differentiation and insulin production. Accordingly, secretagogin-/- fetuses retained a non-committed pool of endocrine progenitors that co-express both insulin and glucagon. We identified the calcium-dependent interaction of secretagogin with subunits of the 26S proteasome complex to prevent Pdx1 degradation through proteasome inactivation. This coincided with retained Nkx6.1 Pax4 and insulin transcription in prospective β cells. In sum, secretagogin scales the temporal availability of a calcium-dependent transcription factor network to define β cell identity.


Overall, through our published studies we have made significant advances in not only understanding the molecular and cellular organization of the mammalian hypothalamus but also, by developing novel technologies, linking molecular determinants of select neuroendocrine command neurons to the context-dependent release of hypothalamic hormones. By applying this knowledge to anther organ, the endocrine pancreas, we have discovered novel molecular mechanisms that control beta-cell specification, survival and functionality during adult life. Moreover, out studies identify the coincident expression of secretagogin in stress-responsive neurons and beta-cells as a first systems biology candidate for metabolic diseases whose cellular effects are defined by the availability of interacting partners and, more broadly, the cellular/functional context itself. Through our studies and successes, we are making timely progress with individual deliverbales of this ERC action, and produced substantial and novel data.
As per the description of tasks, we have achieved major advances in Theme 1/Aims 1,2 and Theme 2/Aim 1. These include:

i) generation of a cellular atlas of the hypothalamus (Romanov et al., Nat Neurosci (2017); Romanov et al., J Endocrinol (2017); Romanov et al. Ann Rev Neurosci (2019)),

ii) description of a novel multimodal signaling system that links stress neurons to norepinephrine centers of the brain for cortical activaton upon stress (Alpar et al., EMBOJ (2018)), and the analysis and recognition of ventricular ependymal heterogeneity (Alpar et al., Curr Opin Neurobiol (2018)),

iii) molecular dissection of secretagogin function in the brain (related to neuronal migration and replacement; Hanics et al., PNAS (2017)), spinal cord/pain perception (Zhang et al. J Clin Invest (2018)) and pancreas (for the control of beta cell turnover; Malenczyk et al., EMBOJ (2017) and particularly with Pdx1 transcription factors; Malenczyk et al., Mol Metab (2018)),

iv) In the process of these studies, we have actively collaborated on brain (dopamine cells; Rivetti di val Chervo et al., Nat Biotech (2017)), pain (Peng et al., Science (2017)), diabetes (Li et al., Cell (2017)) and lung development (Cohen et al., Cell (2018)),

v) Moreover, my mentoring of an up-and-coming new generation of researchers led to spin off on taste perception (Romanov et al., Sci Signal (2018)) and on the role of GPR55, one of the previously less characterized G protein-coupled receptors (GPCRs), in gating salivation (Korchynska et al., JCI Insight (2019)),

vi) At present, we are revising manuscripts on a novel cell type in the mammalian indusium griseum (Fuzik et al., PNAS (2019)) and on how maternal diets affect brain development of the fetus (Cinquina et al., Mol Psychiatry (2019))

vii) As a spin-off of our work, we are generating mouse lines that have been in part published (CCK-DsRed/GAD67-GFP: Calvigioni et al., Cereb Cortex (2017); Secretagogin null and tissue-specific knockout: Malenczyk et al., EMBOJ (2018); secretagogin-Cre: Alpar et al., EMBOJ (2018)) and made available to the scientific community free-of-charge.