Constitutive and conditional PACAP deletion reveals distinct phenotypes driven by developmental versus neurotransmitter actions of PACAP

Journal of Neuroendocrinology

2023 May 02

Bakalar, D;Gavrilova, O;Jiang, S;Zhang, H;Roy, S;Williams, S;Liu, N;Wisser, S;Usdin, T;Eiden, L;
| DOI: 10.1111/jne.13286

Neuropeptides may exert trophic effects during development, and then neurotransmitter roles in the developed nervous system. One way to associate peptide-deficiency phenotypes with either role is first to assess potential phenotypes in so-called constitutive knockout mice, and then proceed to specify, regionally and temporally, where and when neuropeptide expression is required to prevent these phenotypes. We have previously demonstrated that the well-known constellation of behavioral and metabolic phenotypes associated with constitutive PACAP knockout mice are accompanied by transcriptomic alterations of two types: those that distinguish the PACAP-null phenotype from wild-type in otherwise quiescent mice (cPRGs), and gene induction that occurs in response to acute environmental perturbation in wild-type mice that do not occur in knock-out mice (aPRGs). Comparing constitutive PACAP knock-out mice to a variety of temporally and regionally specific PACAP knock-outs, we show that the prominent hyperlocomotor phenotype is a consequence of early loss of PACAP expression, is associated with Fos overexpression in hippocampus and basal ganglia, and that a thermoregulatory effect previously shown to be mediated by PACAP-expressing neurons of medial preoptic hypothalamus is independent of PACAP expression in those neurons in adult mice. In contrast, PACAP dependence of weight loss/hypophagia triggered by restraint stress, seen in constitutive PACAP knock-out mice, is phenocopied in mice in which PACAP is deleted after neuronal differentiation. Our results imply that PACAP has a prominent role as a trophic factor early in development determining global central nervous system characteristics, and in addition a second, discrete set of functions as a neurotransmitter in the fully developed nervous system that support physiological and psychological responses to stress.

Urocortin-3 neurons in the perifornical area are critical mediators of chronic stress on female infant-directed behavior

Molecular psychiatry

2023 Jan 01

Abdelmesih, B;Anderson, R;Bambah-Mukku, D;Carta, I;Autry, AE;
PMID: 36476733 | DOI: 10.1038/s41380-022-01902-2

Infant avoidance and aggression are promoted by activation of the Urocortin-3 expressing neurons of the perifornical area of hypothalamus (PeFAUcn3) in male and female mice. PeFAUcn3 neurons have been implicated in stress, and stress is known to reduce maternal behavior. We asked how chronic restraint stress (CRS) affects infant-directed behavior in virgin and lactating females and what role PeFAUcn3 neurons play in this process. Here we show that infant-directed behavior increases activity in the PeFAUcn3 neurons in virgin and lactating females. Chemogenetic inhibition of PeFAUcn3 neurons facilitates pup retrieval in virgin females. CRS reduces pup retrieval in virgin females and increases activity of PeFAUcn3 neurons, while CRS does not affect maternal behavior in lactating females. Inhibition of PeFAUcn3 neurons blocks stress-induced deficits in pup-directed behavior in virgin females. Together, these data illustrate the critical role for PeFAUcn3 neuronal activity in mediating the impact of chronic stress on female infant-directed behavior.

Seasonal changes in day length induce multisynaptic neurotransmitter switching to regulate hypothalamic network activity and behavior

Science advances

2022 Sep 02

Porcu, A;Nilsson, A;Booreddy, S;Barnes, SA;Welsh, DK;Dulcis, D;
PMID: 36054362 | DOI: 10.1126/sciadv.abn9867

Seasonal changes in day length (photoperiod) affect numerous physiological functions. The suprachiasmatic nucleus (SCN)-paraventricular nucleus (PVN) axis plays a key role in processing photoperiod-related information. Seasonal variations in SCN and PVN neurotransmitter expression have been observed in humans and animal models. However, the molecular mechanisms by which the SCN-PVN network responds to altered photoperiod is unknown. Here, we show in mice that neuromedin S (NMS) and vasoactive intestinal polypeptide (VIP) neurons in the SCN display photoperiod-induced neurotransmitter plasticity. In vivo recording of calcium dynamics revealed that NMS neurons alter PVN network activity in response to winter-like photoperiod. Chronic manipulation of NMS neurons is sufficient to induce neurotransmitter switching in PVN neurons and affects locomotor activity. Our findings reveal previously unidentified molecular adaptations of the SCN-PVN network in response to seasonality and the role for NMS neurons in adjusting hypothalamic function to day length via a coordinated multisynaptic neurotransmitter switching affecting behavior.

Spatial and temporal heterogeneity in the lineage progression of fine oligodendrocyte subtypes

BMC biology

2022 May 25

Hilscher, MM;Langseth, CM;Kukanja, P;Yokota, C;Nilsson, M;Castelo-Branco, G;
PMID: 35610641 | DOI: 10.1186/s12915-022-01325-z

Oligodendrocytes are glial cells that support and insulate axons in the central nervous system through the production of myelin. Oligodendrocytes arise throughout embryonic and early postnatal development from oligodendrocyte precursor cells (OPCs), and recent work demonstrated that they are a transcriptional heterogeneous cell population, but the regional and functional implications of this heterogeneity are less clear. Here, we apply in situ sequencing (ISS) to simultaneously probe the expression of 124 marker genes of distinct oligodendrocyte populations, providing comprehensive maps of the corpus callosum, cingulate, motor, and somatosensory cortex in the brain, as well as gray matter (GM) and white matter (WM) regions in the spinal cord, at postnatal (P10), juvenile (P20), and young adult (P60) stages. We systematically compare the abundances of these populations and investigate the neighboring preference of distinct oligodendrocyte populations.We observed that oligodendrocyte lineage progression is more advanced in the juvenile spinal cord compared to the brain, corroborating with previous studies. We found myelination still ongoing in the adult corpus callosum while it was more advanced in the cortex. Interestingly, we also observed a lateral-to-medial gradient of oligodendrocyte lineage progression in the juvenile cortex, which could be linked to arealization, as well as a deep-to-superficial gradient with mature oligodendrocytes preferentially accumulating in the deeper layers of the cortex. The ISS experiments also exposed differences in abundances and population dynamics over time between GM and WM regions in the brain and spinal cord, indicating regional differences within GM and WM, and we found that neighboring preferences of some oligodendroglia populations are altered from the juvenile to the adult CNS.Overall, our ISS experiments reveal spatial heterogeneity of oligodendrocyte lineage progression in the brain and spinal cord and uncover differences in the timing of oligodendrocyte differentiation and myelination, which could be relevant to further investigate functional heterogeneity of oligodendroglia, especially in the context of injury or disease.

Area postrema neurons mediate interleukin-6 function in cancer-associated cachexia

bioRxiv : the preprint server for biology

2023 Jan 14

Sun, Q;van de Lisdonk, D;Ferrer, M;Gegenhuber, B;Wu, M;Tollkuhn, J;Janowitz, T;Li, B;
PMID: 36711916 | DOI: 10.1101/2023.01.12.523716

Interleukin-6 (IL-6) has been long considered a key player in cancer-associated cachexia 1-15 . It is believed that sustained elevation of IL-6 production during cancer progression causes brain dysfunctions, which ultimately result in cachexia 16-20 . However, how peripheral IL-6 influences the brain remains poorly understood. Here we show that neurons in the area postrema (AP), a circumventricular structure in the hindbrain, mediate the function of IL-6 in cancer-associated cachexia in mice. We found that circulating IL-6 can rapidly enter the AP and activate AP neurons. Peripheral tumor, known to increase circulating IL-6 1-5,15,18,21-23 , leads to elevated IL-6 and neuronal hyperactivity in the AP, and causes potentiated excitatory synaptic transmission onto AP neurons. Remarkably, neutralization of IL-6 in the brain of tumor-bearing mice with an IL-6 antibody prevents cachexia, reduces the hyperactivity in an AP network, and markedly prolongs lifespan. Furthermore, suppression of Il6ra , the gene encoding IL-6 receptor, specifically in AP neurons with CRISPR/dCas9 interference achieves similar effects. Silencing of Gfral-expressing AP neurons also ameliorates the cancer-associated cachectic phenotypes and AP network hyperactivity. Our study identifies a central mechanism underlying the function of peripheral IL-6, which may serve as a target for treating cancer-associated cachexia.

Elevated TNF-α leads to neural circuit instability in the absence of Interferon Regulatory Factor 8

The Journal of neuroscience : the official journal of the Society for Neuroscience

2022 Jul 01

Feinberg, PA;Becker, SC;Chung, L;Ferrari, L;Stellwagen, D;Anaclet, C;Durán-Laforet, V;Faust, TE;Sumbria, RK;Schafer, DP;
PMID: 35790400 | DOI: 10.1523/JNEUROSCI.0601-22.2022

Interferon regulatory factor 8 (IRF8) is a transcription factor necessary for the maturation of microglia, as well as other peripheral immune cells. It also regulates the transition of microglia and other immune cells to a pro-inflammatory phenotype. Irf8 is also a known risk gene for multiple sclerosis and lupus and it has recently been shown to be downregulated in schizophrenia. While most studies have focused on IRF8-dependent regulation of immune cell function, little is known about how it impacts neural circuits. Here, we show by RNAseq from Irf8-/- male and female mouse brains that several genes involved in regulation of neural activity are dysregulated. We then show these molecular changes are reflected in heightened neural excitability and a profound increase in susceptibility to lethal seizures in male and female Irf8-/- mice. Finally, we identify that TNF-α is elevated specifically in microglia in the CNS and genetic or acute pharmacological blockade of TNF-α in the Irf8-/- central nervous system (CNS) rescued the seizure phenotype. These results provide important insights into the consequences of IRF8 signaling and TNF-α on neural circuits. Our data further suggest that neuronal function is impacted by loss of IRF8, a factor involved in neuropsychiatric and neurodegenerative diseases.SIGNIFICANCE STATEMENTHere, we identify a previously unknown and key role for Interferon regulator factor 8 (IRF8) in regulating neural excitability and seizures. We further determine these effects on neural circuits are through elevated TNF-α in the CNS. As IRF8 has most widely been studied in the context of regulating the development and inflammatory signaling in microglia and other immune cells, we have uncovered a novel function. Further, IRF8 is a risk gene for multiple sclerosis (MS) and lupus, IRF8 is dysregulated in schizophrenia, and elevated TNF-α has been identified in a multitude of neurological conditions. Thus, elucidating these IRF8 and TNF-α-dependent effects on brain circuit function have profound implications for understanding underlying, therapeutically-relevant mechanisms of disease.

Epigenetic control of cellular crosstalk defines gastrointestinal organ fate and function

Nature communications

2023 Jan 30

Smith, RJ;Liang, M;Loe, AKH;Yung, T;Kim, JE;Hudson, M;Wilson, MD;Kim, TH;
PMID: 36717563 | DOI: 10.1038/s41467-023-36228-2

Epithelial-mesenchymal signaling in the gastrointestinal system is vital in establishing regional identity during organogenesis and maintaining adult stem cell homeostasis. Although recent work has demonstrated that Wnt ligands expressed by mesenchymal cells are required during gastrointestinal development and stem cell homeostasis, epigenetic mechanisms driving spatiotemporal control of crosstalk remain unknown. Here, we demonstrate that gastrointestinal mesenchymal cells control epithelial fate and function through Polycomb Repressive Complex 2-mediated chromatin bivalency. We find that while key lineage-determining genes possess tissue-specific chromatin accessibility, Polycomb Repressive Complex 2 controls Wnt expression in mesenchymal cells without altering accessibility. We show that reduction of mesenchymal Wnt secretion rescues gastrointestinal fate and proliferation defects caused by Polycomb Repressive Complex 2 loss. We demonstrate that mesenchymal Polycomb Repressive Complex 2 also regulates niche signals to maintain stem cell function in the adult intestine. Our results highlight a broadly permissive chromatin architecture underlying regionalization in mesenchymal cells, then demonstrate further how chromatin architecture in niches can influence the fate and function of neighboring cells.

The circadian clock gene, Bmal1, regulates intestinal stem cell signaling and represses tumor initiation

Cellular and molecular gastroenterology and hepatology

2021 Sep 14

Stokes, K;Nunes, M;Trombley, C;Flôres, DEFL;Wu, G;Taleb, Z;Alkhateeb, A;Banskota, S;Harris, C;Love, OP;Khan, WI;Rueda, L;Hogenesch, JB;Karpowicz, P;
PMID: 34534703 | DOI: 10.1016/j.jcmgh.2021.08.001

Circadian rhythms are daily physiological oscillations driven by the circadian clock: a 24-hour transcriptional timekeeper that regulates hormones, inflammation, and metabolism. Circadian rhythms are known to be important for health, but whether their loss contributes to colorectal cancer is not known.We tested the non-redundant clock gene, Bmal1, in intestinal homeostasis and tumorigenesis, using the Apcmin model of colorectal cancer.Bmal1 mutant, epithelium-conditional Bmal1 mutant, and photoperiod-disrupted mice bearing the Apcmin allele were assessed for tumorigenesis. Tumors and normal non-transformed tissue were characterized. Intestinal organoids were assessed for circadian transcription rhythms by RNA-sequencing, and in vivo and organoid assays were used to test Bmal1-dependent proliferation and self-renewal.Loss of Bmal1 or circadian photoperiod increases tumor initiation. In the intestinal epithelium the clock regulates transcripts involved in regeneration and intestinal stem cell signaling. Tumors have no self-autonomous clock function and only weak clock function in vivo. Apcmin clock-disrupted tumors exhibit high Yap (Hippo signaling) activity but exhibit low Wnt activity. Intestinal organoid assays reveal that loss of Bmal1 increases self-renewal in a Yap-dependent manner.Bmal1 regulates intestinal stem cell pathways, including Hippo signaling, and the loss of circadian rhythms potentiates tumor initiation.

A preoptic neuronal population controls fever and appetite during sickness

Nature

2022 Jun 01

Osterhout, JA;Kapoor, V;Eichhorn, SW;Vaughn, E;Moore, JD;Liu, D;Lee, D;DeNardo, LA;Luo, L;Zhuang, X;Dulac, C;
PMID: 35676482 | DOI: 10.1038/s41586-022-04793-z

During infection, animals exhibit adaptive changes in physiology and behaviour aimed at increasing survival. Although many causes of infection exist, they trigger similar stereotyped symptoms such as fever, warmth-seeking, loss of appetite and fatigue1,2. Yet exactly how the nervous system alters body temperature and triggers sickness behaviours to coordinate responses to infection remains unknown. Here we identify a previously uncharacterized population of neurons in the ventral medial preoptic area (VMPO) of the hypothalamus that are activated after sickness induced by lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid. These neurons are crucial for generating a fever response and other sickness symptoms such as warmth-seeking and loss of appetite. Single-nucleus RNA-sequencing and multiplexed error-robust fluorescence in situ hybridization uncovered the identity and distribution of LPS-activated VMPO (VMPOLPS) neurons and non-neuronal cells. Gene expression and electrophysiological measurements implicate a paracrine mechanism in which the release of immune signals by non-neuronal cells during infection activates nearby VMPOLPS neurons. Finally, we show that VMPOLPS neurons exert a broad influence on the activity of brain areas associated with behavioural and homeostatic functions and are synaptically and functionally connected to circuit nodes controlling body temperature and appetite. Together, these results uncover VMPOLPS neurons as a control hub that integrates immune signals to orchestrate multiple sickness symptoms in response to infection.

Hedgehog signaling promotes expansion of Meibomian Gland stem cells in vivo

Investigative Ophthalmology & Visual Science

2022 Jan 01

Zhu, X;Xu, M;Grachtchouk, M;

RESULTS : Short-term lineage tracing data showed that _Lrig1_, _Lgr6_ and _Axin2_ label basal cells in MG ducts and acini. Long-term lineage tracing results showed that clones of labeled cells persist through multiple rounds of ductal and acinar renewal and give rise to differentiated progeny, identifying _Lrig1_+, _Lgr6_+ and _Axin2+_ ductal and acinar basal cells as self-renewing SCs. Forced expression of GLI2ΔN enhanced basal proliferation, caused expansion of _Lrig1_+ SCs, and lead to replacement of lipid-filled meibocytes by proliferative and poorly differentiated acinar cells. Transcriptional profiling of GLI2ΔN-expressing and control MGs revealed that forced GLI2ΔN expression caused greatly increased expression of _Lrig1_ and _Lgr6_ and suppressed expression of meibocyte differentiation genes.

Neurotensin neurons in the extended amygdala control dietary choice and energy homeostasis

Nature neuroscience

2022 Nov 01

Furlan, A;Corona, A;Boyle, S;Sharma, R;Rubino, R;Habel, J;Gablenz, EC;Giovanniello, J;Beyaz, S;Janowitz, T;Shea, SD;Li, B;
PMID: 36266470 | DOI: 10.1038/s41593-022-01178-3

Obesity is a global pandemic that is causally linked to many life-threatening diseases. Apart from some rare genetic conditions, the biological drivers of overeating and reduced activity are unclear. Here, we show that neurotensin-expressing neurons in the mouse interstitial nucleus of the posterior limb of the anterior commissure (IPAC), a nucleus of the central extended amygdala, encode dietary preference for unhealthy energy-dense foods. Optogenetic activation of IPACNts neurons promotes obesogenic behaviors, such as hedonic eating, and modulates food preference. Conversely, acute inhibition of IPACNts neurons reduces feeding and decreases hedonic eating. Chronic inactivation of IPACNts neurons recapitulates these effects, reduces preference for sweet, non-caloric tastants and, furthermore, enhances locomotion and energy expenditure; as a result, mice display long-term weight loss and improved metabolic health and are protected from obesity. Thus, the activity of a single neuronal population bidirectionally regulates energy homeostasis. Our findings could lead to new therapeutic strategies to prevent and treat obesity.

Engineered Wnt ligands enable blood-brain barrier repair in neurological disorders

Science (New York, N.Y.)

2022 Feb 18

Martin, M;Vermeiren, S;Bostaille, N;Eubelen, M;Spitzer, D;Vermeersch, M;Profaci, CP;Pozuelo, E;Toussay, X;Raman-Nair, J;Tebabi, P;America, M;De Groote, A;Sanderson, LE;Cabochette, P;Germano, RFV;Torres, D;Boutry, S;de Kerchove d'Exaerde, A;Bellefroid, EJ;Phoenix, TN;Devraj, K;Lacoste, B;Daneman, R;Liebner, S;Vanhollebeke, B;
PMID: 35175798 | DOI: 10.1126/science.abm4459

The blood-brain barrier (BBB) protects the central nervous system (CNS) from harmful blood-borne factors. Although BBB dysfunction is a hallmark of several neurological disorders, therapies to restore BBB function are lacking. An attractive strategy is to repurpose developmental BBB regulators, such as Wnt7a, into BBB-protective agents. However, safe therapeutic use of Wnt ligands is complicated by their pleiotropic Frizzled signaling activities. Taking advantage of the Wnt7a/b-specific Gpr124/Reck co-receptor complex, we genetically engineered Wnt7a ligands into BBB-specific Wnt activators. In a "hit-and-run" adeno-associated virus-assisted CNS gene delivery setting, these new Gpr124/Reck-specific agonists protected BBB function, thereby mitigating glioblastoma expansion and ischemic stroke infarction. This work reveals that the signaling specificity of Wnt ligands is adjustable and defines a modality to treat CNS disorders by normalizing the BBB.

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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