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

Periodic Reporting for period 4 - Secret-Cells (Cellular diversity and stress-induced cell-state switches in the mammalian hypothalamus)

Reporting period: 2021-03-01 to 2021-08-31

The hypothalamus is an essential interface linking neuroendocrine, autonomic and somatomotor systems, allowing dynamic bodily adaptations to environmental cues via complex neuroendocrine processes. Hypothalamic nuclei are organized into a partially overlapping patchwork with the unprecedented diversity of resident neurons and glia underpinning the breadth of neuroendocrine modalities produced. A significant limitation to understanding hypothalamic operations at the level of intra- and extrahypothalamic neurocircuits and endocrine output is the lack of a comprehensive catalogue of neuronal subtypes. This challenge is complicated by the ability of hypothalamic neurons to undergo stimulus-dependent expressional switches in gene expression (termed, “cell-state switching”), which can bias their classification. To overcome these gaps of knowledge, we performed single-cell RNA-seq in the hypothalamic of both developing and adult laboratory rodents and described the developmental trajectories, reliance on gene regulatory networks, intercellular signal requirements and neuronal circuit diagrams for dozens of hypothalamic neurons. We have shown the direct terminal differentiation of glutamatergic neurons, which contrasts the intermittent stalling of GABA differentiation during antenatal development. Moreover, we demonstrated that dopaminergic neurons of the hypothalamus arise from GABA precursors. Hypothalamic differentiation programs lead to >60 (likely ~100) neuronal subtypes. Amongst these, we defined 10 dopamine clusters and analyzed these in detail. Moreover, we showed that acute stress does not lead to the repartitioning of single-cell RNA-seq data (that is, no new cell type arises on stress). Instead, gene regulatory networks are modified in corticotropin-releasing hormone (CRH)-containing neurons such that metabolic readiness to release CRH can ensue. We have discovered an excitatory projection by which CRH neurons innervate ependymocytes in the wall of the 3rd ventricle to facilitate the volume transmission of neurotrophins that activate norepinephrine (NE) neurons (an intracerebral multi-synpatic pathway of stress reinforcement). Thus, the conversion of synaptic signals into metabolic codes is used to excite NE neurons that innervate the cerebral cortex. The functional outcome of this signaling axis is heightened NE drive in the cerebrum, which reduces the threshold of cortical excitation upon recurrent stress episodes. During these studies, we have identified secretagogin, a calcium-sensor protein, as a molecular linchpin of CRH release. Accordingly, cell type-specific deletion of secretagogin leads to the cessation of CRH release. Nevertheless, an adverse consequence of sustained CRH hypofunction is the gradual development of diabetes because of the loss of pancreatic beta cells. Overall, our work program advanced knowledge on the diversity of neurons in the mammalian hypothalamus, identified new cell types and circuits, and defined molecular underpinnings of CRH release. On the backdrop of our results, the continued interrogation of hypothalamic neuronal subclasses can lead to improved understanding of metabolic diseases and offer their prevention.
Results related to Theme 1:

Molecular interrogation of hypothalamic organization: (Romanov et al., Nat Neurosci. (2017))
Molecular diversity of corticotropin-releasing factor-containing neurons: (Romanov et al., J Endocrinol (2017))
A novel output pathway of CRH neurons uses volume transmission to orchestrate post-traumatic cortical sensitization: (Alpar et al., EMBOJ (2018))
Hypothalamic long-distance signals by neuropeptides: (Alpar et al., Current Open Neurobiol (2019))
Adverse effects of maternal high-fat diets on brain development: (Cinquina et al., Mol Psychiatry (2020))
Developmental organization of the hypothalamus: (Romanov et al., Nature (2020))
Neurohormonal hypothalamic circuit for paternal behaviour: (Stagkourakis et al., Cell (2020))
Identification of ALK in thinness: (Orthofer et al., Cell (2020))
Life-long impairment of glucose homeostasis by prenatal prychostimulant exposure: (Korchynska et al., EMBOJ (2020))
functional heterogeneity of POMC neurones in the arcuate nucleus: (Saucisse et al., Cell Rep (2021))

Results Related to Theme 2:

Secretagogin regulates neuroblast migration by regulating matrix metalloprotease release: (Hanics et al., PNAS (2017))
Secretagogin regulates beta-cell turnover: (Malenczyk et al., EMBOJ (2017))
Secretagogin controls beta-cell specification by configuring permissive transcirption factor landscape: (Malenczyk et al., Mol Metab (2018))
Calcium-binding protein NECAB2 facilitates pain: (Zhang et al. J Clin Invest (2018))
Psychostimulant sensitivity of hypothalamic and limbic neurone identify by Patch-seq (Fuzik et al., PNAS (2019))
Secretagogin involvement in NMDA receptor turnover (Hevesi et al., PNAS, 2021)).


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.
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., Nature 2020, 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), hypothalamic neurons: Saucisse et al., Cell Rep (2021), Stagkourakis et al., Cell (2020), Orthofer et al., Cell (2020)), 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)), a novel neuron type in the mammalian indusium griseum (Fuzik et al., PNAS (2019)) and the effect of maternal diet choices on fetal brain development (Cinquina et al., Mol Psychiatry (202).

vi) We have generated mouse lines that have been 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.
Cover image for Nature Biotechnology on Patch-seq
Cover image for JCI Insight on GPR55 receptors
Cover image for the EMBO Journal on volume transmission and stress