The Methylation of Clock Genes in Perinatal Depression: Which Role for Oxytocin?

Background: Perinatal Depression (PD) is a widespread disabling condition that is hypothesized to be associated with abnormalities in circadian rhythms and neuropeptide release including oxytocin (OXT).Methods: Fourty-four pregnant women (28 with PD, and 16 controls) were evaluated through the Edinburgh Postnatal Depression Scale (EPDS), the State/Trait Anxiety Inventory Form Y (STAI-Y), and the Prenatal Attachment Inventory (PAI). A blood sample was collected from all participants, and OXT plasma levels, DNA methylation of clock genes, as well as of FOXp3 and HERV-W were measured. Linear regression analyses were performed to assess the effect of oxytocin on the methylation of selected genes. Continuous ordinal regression models was further applied to see if the score of rating scales was associated to gene methylation, adjusting for oxytocin-methylation interaction.Results: OXT plasma levels were positively associated with CRY1 methylation. Women with higher OXT plasma levels showed an association between higher degree of CRY2 methylation (thus, reduced expression) and lower EPDS (OR = 0.21; P = 0.043) and STAI-S scores (OR = 6.96; P = 0.019). Finally, with high OXT levels, hypermethylation of CRY1 was associated to higher scores on the PAI (OR = 2.74; P = 0.029) while higher methylation of HERV-W related to lower PAI scores (OR = 0.273; P = 0.019).Conclusion: Our results suggest a possible protective role played by oxytocin in the development of PD by promoting a favorable methylation profile characterized by reduced expression of CRY1 and CRY2. Moreover, oxytocin strengthens the association between maternal prenatal attachment with a favorable pattern of methylation of clock genes and HERV-W, which is essential for pregnancy outcomes.

Selective Expression of a SNARE-Cleaving Protease in Peripheral Sensory Neurons Attenuates Pain-Related Gene Transcription and Neuropeptide Release

Chronic pain is a leading health and socioeconomic problem and an unmet need exists for long-lasting analgesics. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are required for neuropeptide release and noxious signal transducer surface trafficking, thus, selective expression of the SNARE-cleaving light-chain protease of botulinum neurotoxin A (LCA) in peripheral sensory neurons could alleviate chronic pain. However, a safety concern to this approach is the lack of a sensory neuronal promoter to prevent the expression of LCA in the central nervous system. Towards this, we exploit the unique characteristics of Pirt (phosphoinositide-interacting regulator of TRP), which is expressed in peripheral nociceptive neurons. For the first time, we identified a Pirt promoter element and cloned it into a lentiviral vector driving transgene expression selectively in peripheral sensory neurons. Pirt promoter driven-LCA expression yielded rapid and concentration-dependent cleavage of SNAP-25 in cultured sensory neurons. Moreover, the transcripts of pain-related genes (TAC1, tachykinin precursor 1; CALCB, calcitonin gene-related peptide 2; HTR3A, 5-hydroxytryptamine receptor 3A; NPY2R, neuropeptide Y receptor Y2; GPR52, G protein-coupled receptor 52; SCN9A, sodium voltage-gated channel alpha subunit 9; TRPV1 and TRPA1, transient receptor potential cation channel subfamily V member 1 and subfamily A member 1) in pro-inflammatory cytokines stimulated sensory neurons were downregulated by viral mediated expression of LCA. Furthermore, viral expression of LCA yielded long-lasting inhibition of pain mediator release. Thus, we show that the engineered Pirt-LCA virus may provide a novel means for long lasting pain relief.

Nociceptor neurons impair cancer immunosurveillance

Solid tumors are innervated by nerve fibers that arise from the autonomic and sensory peripheral nervous systems. In prostate cancer, doublecortin-expressing neural progenitors initiate autonomic adrenergic neurogenesis1 which facilitates tumor development and dissemination2, via an angiogenic switch that fuels cancer growth3,4. Similarly, a loss of TP53 drives the reprogramming of tumor-innervating sensory nerves into adrenergic neurons in head and neck tumors, which promotes tumor growth5. However, the impact of tumor neo-innervation by pain-initiating sensory neurons remains unclear. We show that melanoma cells interact with nociceptors, increasing neurite outgrowth, responsiveness to noxious ligands, and neuropeptide release. In turn, CGRP, a nociceptor-produced neuropeptide, directly increases exhaustion of cytotoxic CD8+ T-cells (PD1+Lag3+Tim3+IFNγ-), limiting their capacity to eliminate melanoma. Genetic NaV1.8 or TRPV1 lineage ablation, local pharmacological silencing or blockade of neuropeptide release from tumor-innervating nociceptors, and the antagonism of the CGRP receptor RAMP1, all blunt tumor-infiltrating leukocyte exhaustion, and tumor growth, nearly tripling survival of B16F10-inoculated mice. Inversely, CD8+ T-cell exhaustion increased following optogenetic activation of tumor-innervating NaV1.8 neurons+ and was rescued in sensory neuron depleted mice treated with recombinant CGRP. In comparison to wild-type CD8+ T-cells, RAMP1-/- CD8+ T-cells were protected from undergoing exhaustion when co-transplanted into tumor-bearing Rag1 deficient mice. Single-cell RNA sequencing of patient tumors revealed that intratumoral RAMP1-expressing CD8+ T-cells are more exhausted than their RAMP1 negative counterparts. RAMP1 expression in intratumoral CD8+ T-cells was also associated with resistance to immune checkpoint inhibitor treatment, while RAMP1 overexpression within the tumor correlated with a worse clinical prognosis. We conclude that reducing CGRP release from tumor-innervating nociceptors, by eliminating its immunomodulatory action on cytotoxic CD8+ T-cells, constitutes a useful strategy to safeguard anti-tumor immunity.

Temporally and spatially partitioned neuropeptide release from individual clock neurons

Neuropeptides control rhythmic behaviors, but the timing and location of their release within circuits is unknown. Here, imaging in the brain shows that synaptic neuropeptide release by Drosophila clock neurons is diurnal, peaking at times of day that were not anticipated by prior electrical and Ca2+ data. Furthermore, hours before peak synaptic neuropeptide release, neuropeptide release occurs at the soma, a neuronal compartment that has not been implicated in peptidergic transmission. The timing disparity between release at the soma and terminals results from independent and compartmentalized mechanisms for daily rhythmic release: consistent with conventional electrical activity–triggered synaptic transmission, terminals require Ca2+ influx, while somatic neuropeptide release is triggered by the biochemical signal IP3. Upon disrupting the somatic mechanism, the rhythm of terminal release and locomotor activity period are unaffected, but the number of flies with rhythmic behavior and sleep–wake balance are reduced. These results support the conclusion that somatic neuropeptide release controls specific features of clock neuron–dependent behaviors. Thus, compartment-specific mechanisms within individual clock neurons produce temporally and spatially partitioned neuropeptide release to expand the peptidergic connectome underlying daily rhythmic behaviors.

Stac protein regulates release of neuropeptides

Neuropeptides are important for regulating numerous neural functions and behaviors. Release of neuropeptides requires long-lasting, high levels of cytosolic Ca2+. However, the molecular regulation of neuropeptide release remains to be clarified. Recently, Stac3 was identified as a key regulator of L-type Ca2+channels (CaChs) and excitation–contraction coupling in vertebrate skeletal muscles. There is a small family ofstacgenes in vertebrates with other members expressed by subsets of neurons in the central nervous system. The function of neural Stac proteins, however, is poorly understood.Drosophila melanogastercontain a singlestacgene,Dstac, which is expressed by muscles and a subset of neurons, including neuropeptide-expressing motor neurons. Here, genetic manipulations, coupled with immunolabeling, Ca2+imaging, electrophysiology, and behavioral analysis, revealed that Dstac regulates L-type CaChs (Dmca1D) inDrosophilamotor neurons and this, in turn, controls the release of neuropeptides.

Independently Regulated Multi-compartment Neuropeptide Release from a Clock Neuron Controls Circadian Behavior

Neuropeptides control many behaviors, including circadian rhythms. However, because monitoring neuropeptide release in the brain is challenging, analysis of peptidergic circuits often has relied on monitoring surrogates in the soma based on the paradigm that synaptic transmission is mediated exclusively by Ca2+ influx induced by propagating action potentials. Here live imaging demonstrates that neuropeptide release by Drosophila small ventrolateral (s-LNv) clock neurons does not conform to this paradigm. First, neuropeptide release from terminals peaks hours after sunrise, which was not evident from electrical and Ca2+ data. Second, inconsistent with global release by propagating action potentials, release from terminals is preceded by hours by release from the soma, a compartment not usually considered in peptidergic transmission. The timing of release from the two neuronal compartments reflects different mechanisms: terminals require Ca2+ influx, as expected with coupling to electrical activity, while somatic release is based on intracellular IP3 signaling. Upon cell specific disruption of the somatic mechanism, daily neuropeptide release from terminals remains rhythmic and the period of daily locomotor activity is unaffected, but behavioral rhythmicity is reduced. Thus, rhythmic bouts of anatomically, mechanistically and temporally distinct release from a single neuron control neuropeptide dependent features of circadian behavior.