Mou, TM;Lane, MV;Ireland, DDC;Verthelyi, D;Tonelli, LH;Clark, SM;
PMID: 35995342 | DOI: 10.1016/j.nbd.2022.105840
An early inflammatory insult is the most recognized risk factor associated with neurodevelopmental psychiatric disorders, even more so than genetic variants. Notably, complement component 4 (C4), a molecule involved in inflammatory responses, has been strongly associated with schizophrenia (SZ) and its role in other neurodevelopmental disorders, such as autism (ASD), is an area of active investigation. However, while C4 in SZ has been implicated in the context of synaptic pruning, little is known about its neuroinflammatory role. The subventricular zone (SVZ) is a region heavily involved in neurodevelopment and neuroimmune interactions through the lifespan; thus, it is a region wherein C4 may play a vital role in disease pathology. Using in situ hybridization with radioactive riboprobes and RNAscope, we identified robust astrocytic expression of C4 in the SVZ and in the septum pellucidum. C4 was also expressed in ependyma, neurons, and Ki67+ progenitor cells. Examination of mRNA levels showed elevated C4 in both ASD and SZ, with higher expression in SZ compared to controls. Targeted transcriptomic analysis of inflammatory pathways revealed a strong association of complement system genes with SZ, and to a lesser extent, ASD, as well as generalized immune dysregulation without a strong association with known infectious pathways. Analysis of differentially expressed genes (DEGs) showed that ASD DEGs were enriched in adaptive immune system functions such as Th cell differentiation, while SZ DEGs were enriched in innate immune system functions, including NF-κB and toll like receptor signaling. Moreover, the number of Ki67+ cells was significantly higher in ASD compared to SZ and controls. Taken together, these results support a role for C4 into inflammatory-neuroimmune dysregulation observed in SZ and ASD pathology.
Sci Transl Med. 2018 Oct 10;10(462).
Murthy SE, Loud MC, Daou I, Marshall KL, Schwaller F, Kühnemund J, Francisco AG, Keenan WT, Dubin AE, Lewin GR, Patapoutian A.
PMID: 30305457 | DOI: 10.1126/scitranslmed.aat9897
The brush of a feather and a pinprick are perceived as distinct sensations because they are detected by discrete cutaneous sensory neurons. Inflammation or nerve injury can disrupt this sensory coding and result in maladaptive pain states, including mechanical allodynia, the development of pain in response to innocuous touch. However, the molecular mechanisms underlying the alteration of mechanical sensitization are poorly understood. In mice and humans, loss of mechanically activated PIEZO2 channels results in the inability to sense discriminative touch. However, the role of Piezo2 in acute and sensitized mechanical pain is not well defined. Here, we showed that optogenetic activation of Piezo2-expressing sensory neurons induced nociception in mice. Mice lacking Piezo2 in caudal sensory neurons had impaired nocifensive responses to mechanical stimuli. Consistently, ex vivo recordings in skin-nerve preparations from these mice showed diminished Aδ-nociceptor and C-fiber firing in response to mechanical stimulation. Punctate and dynamic allodynia in response to capsaicin-induced inflammation and spared nerve injury was absent in Piezo2-deficient mice. These results indicate that Piezo2 mediates inflammation- and nerve injury-induced sensitized mechanical pain, and suggest that targeting PIEZO2 might be an effective strategy for treating mechanical allodynia.
Kashima DT, Grueter BA.
PMID: 28760987 | DOI: 10.1073/pnas.1705974114
Behavioral manifestations of drug-seeking behavior are causally linked to alterations of synaptic strength onto nucleus accumbens (NAc) medium spiny neurons (MSN). Although neuron-driven changes in physiology and behavior are well characterized, there is a lack of knowledge of the role of the immune system in mediating such effects. Toll-like receptor 4 (TLR4) is a pattern recognition molecule of the innate immune system, and evidence suggests that it modulates drug-related behavior. Using TLR4 knockout (TLR4.KO) mice, we show that TLR4 plays a role in NAc synaptic physiology and behavior. In addition to differences in the pharmacological profile of N-methyl-d-aspartate receptors (NMDAR) in the NAc core, TLR4.KO animals exhibit a deficit in low-frequency stimulation-induced NMDAR-dependent long-term depression (LTD). Interestingly, the synaptic difference is region specific as no differences were found in excitatory synaptic properties in the NAc shell. Consistent with altered NAc LTD, TLR4.KO animals exhibit an attenuation in drug reward learning. Finally, we show that TLR4 in the NAc core is primarily expressed on microglia. These results suggest that TLR4 influences NAc MSN synaptic physiology and drug reward learning and behavior.
Liu, L;Zhao, Y;An, W;Zhao, M;Ding, N;Liu, H;Ge, N;Wen, J;Zhang, X;Zu, S;Sun, W;
PMID: 37227654 | DOI: 10.1007/s12035-023-03386-9
Mechanical sensing Piezo2 channel in primary sensory neurons has been shown contribute to mechanical allodynia in somatic chronic pain conditions. Interstitial cystitis (IC)-associated pain is often triggered by bladder filling, a presentation that mimics the mechanical allodynia. In the present study, we aimed to examine the involvement of sensory Piezo2 channel in IC-associated mechanical allodynia using a commonly employed cyclophosphamide (CYP)-induced IC model rat. Piezo2 channels in dorsal root ganglia (DRGs) was knocked down by intrathecal injections of Piezo2 anti-sense oligodeoxynucleotides (ODNs) in CYP-induced cystitis rats, and mechanical stimulation-evoked referred bladder pain was measured in the lower abdomen overlying the bladder using von Frey filaments. Piezo2 expression at the mRNA, protein, and functional levels in DRG neurons innervating the bladder was detected by RNA-fluorescence in situ hybridization, western blotting, immunofluorescence, and Ca2+ imaging, respectively. We found that Piezo2 channels were expressed on most (> 90%) of the bladder primary afferents, including afferents that express CGRP, TRPV1 and stained with isolectin B4. CYP-induced cystitis was associated with Piezo2 upregulation in bladder afferent neurons at the mRNA, protein, and functional levels. Knockdown of Piezo2 expression in DRG neurons significantly suppressed mechanical stimulation-evoked referred bladder pain as well as bladder hyperactivity in CYP rats compared to CYP rats treated with mismatched ODNs. Our results suggest upregulation of Piezo2 channels is involved in the development of bladder mechanical allodynia and bladder hyperactivity in CYP-induced cystitis. Targeting Piezo2 might be an attractive therapeutic approach for IC-related bladder pain.
Piezo2 mechanosensitive ion channel is located to sensory neurons and nonneuronal cells in rat peripheral sensory pathway: implications in pain
Shin, SM;Moehring, F;Itson-Zoske, B;Fan, F;Stucky, CL;Hogan, QH;Yu, H;
PMID: 34285153 | DOI: 10.1097/j.pain.0000000000002356
Piezo2 mechanotransduction channel is a crucial mediator of sensory neurons for sensing and transducing touch, vibration, and proprioception. We here characterized Piezo2 expression and cell specificity in rat peripheral sensory pathway using a validated Piezo2 antibody. Immunohistochemistry using this antibody revealed Piezo2 expression in pan primary sensory neurons of dorsal root ganglia in naïve rats, which was actively transported along afferent axons to both central presynaptic terminals innervating the spinal dorsal horn (DH) and peripheral afferent terminals in the skin. Piezo2 immunoreactivity (IR) was also detected in the postsynaptic neurons of the DH and in the motor neurons of the ventral horn, but not in spinal glial fibrillary acidic protein-positive and Iba1-positive glia. Notably, Piezo2-IR was clearly identified in peripheral nonneuronal cells, including perineuronal glia, Schwann cells in the sciatic nerve and surrounding cutaneous afferent endings, as well as in skin epidermal Merkel cells and melanocytes. Immunoblots showed increased Piezo2 in dorsal root ganglia ipsilateral to plantar injection of complete Freund's adjuvant, and immunostaining revealed increased Piezo2-IR intensity in the DH ipsilateral to complete Freund's adjuvant injection. This elevation of DH Piezo2-IR was also evident in various neuropathic pain models and monosodium iodoacetate knee osteoarthritis pain model, compared with controls. We conclude that (1) the pan neuronal profile of Piezo2 expression suggests that Piezo2 may function extend beyond simply touch or proprioception mediated by large-sized low-threshold mechanosensitive primary sensory neurons; (2) Piezo2 may have functional roles involving sensory processing in the spinal cord, Schwann cells, and skin melanocytes; and (3) aberrant Piezo2 expression may contribute pain pathogenesis.
Li S, Uno Y, Rudolph U, Cobb J, Liu J, Anderson T, Levy D, Balu DT, Coyle JT.
PMID: 29305854 | DOI: 10.1016/j.bcp.2017.12.023
D-Serine is a co-agonist at forebrain N-methyl-D-aspartate receptors (NMDAR) and is synthesized by serine racemase (SR). Although D-serine and SR were originally reported to be localized to glia, recent studies have provided compelling evidence that under healthy physiologic conditions both are localized primarily in neurons. However, in pathologic conditions, reactive astrocytes can also express SR and synthesize D-serine. Since cultured astrocytes exhibit features of reactive astrocytes, we have characterized D-serine synthesis and the expression of enzymes involved in its disposition in primary glial cultures. The levels of SR were quite low early in culture and increased markedly in all astrocytes with the duration in vitro. The concentration of D-serine in the culture medium increased in parallel with SR expression in the astrocytes. Microglia, identified by robust expression of Iba1, did not express SR. While the levels of glial fibrillary acidic protein (GFAP), glycine decarboxylase (GLDC) and phosphoglycerate dehydrogenase (PHGDH), the initial enzyme in the pathway converting glycine to L-serine, remained constant in culture, the expression of lipocalin-2, a marker for pan-reactive astrocytes, increased several-fold. The cultured astrocytes also expressed Complement-3a, a marker for a subpopulation of reactive astrocytes (A1). Astrocytes grown from mice with a copy number variant associated with psychosis, which have four copies of the GLDC gene, showed a more rapid production of D-serine and a reduction of glycine in the culture medium. These results substantiate the conclusion that A1 reactive astrocytes express SR and release D-serine under pathologic conditions, which may contribute to their neurotoxic effects by activating extra-synaptic NMDARs.
Key role for hypothalamic interleukin-6 in food-motivated behavior and body weight regulation
López-Ferreras, L;Longo, F;Richard, J;Eerola, K;Shevchouk, O;Tuzinovic, M;Skibicka, K;
| DOI: 10.1016/j.psyneuen.2021.105284
The pro-inflammatory role of interleukin-6 (IL-6) is well-characterized. Blockade of IL-6, by Tocilizumab, is used in patients with rheumatoid arthritis and those diagnosed with cytokine storm. However, brain-produced IL-6 has recently emerged as a critical mediator of gut/adipose communication with the brain. Central nervous system (CNS) IL-6 is engaged by peripheral and central signals regulating energy homeostasis. IL-6 is critical for mediating hypophagia and weight loss effects of a GLP-1 analog, exendin-4, a clinically utilized drug. However, neuroanatomical substrates and behavioral mechanisms of brain IL-6 energy balance control remain poorly understood. We propose that the lateral hypothalamus (LH) is an IL-6-harboring brain region, key to food intake and food reward control. Microinjections of IL-6 into the LH reduced chow and palatable food intake in male rats. In contrast, female rats responded with reduced motivated behavior for sucrose, measured by the progressive ratio operant conditioning test, a behavioral mechanism previously not linked to IL-6. To test whether IL-6, produced in the LH, is necessary for ingestive and motivated behaviors, and body weight homeostasis, virogenetic knockdown by infusion of AAV-siRNA-IL6 into the LH was utilized. Attenuation of LH IL-6 resulted in a potent increase in sucrose-motivated behavior, without any effect on ingestive behavior or body weight in female rats. In contrast, the treatment did not affect any parameters measured (chow intake, sucrose-motivated behavior, locomotion, and body weight) in chow-fed males. However, when challenged with a high-fat/high-sugar diet, the male LH IL-6 knockdown rats displayed rapid weight gain and hyperphagia. Together, our data suggest that LH-produced IL-6 is necessary and sufficient for ingestive behavior and weight homeostasis in male rats. In females, IL-6 in the LH plays a critical role in food-motivated, but not ingestive behavior control or weight regulation. Thus, collectively these data support the idea that brain-produced IL-6 engages the hypothalamus to control feeding behavior.
Shi MM, Fan KM, Qiao YN, Xu JH, Qiu LJ, Li X, Liu Y, Qian ZQ, Wei CL, Han J, Fan J, Tian YF, Ren W, Liu ZQ.
PMID: 31142818 | DOI: 10.1038/s41380-019-0435-z
Stressful life events induce abnormalities in emotional and cognitive behaviour. The endogenous opioid system plays an essential role in stress adaptation and coping strategies. In particular, the µ-opioid receptor (μR), one of the major opioid receptors, strongly influences memory processing in that alterations in μR signalling are associated with various neuropsychiatric disorders. However, it remains unclear whether μR signalling contributes to memory impairments induced by acute stress. Here, we utilized pharmacological methods and cell-type-selective/non-cell-type-selective μR depletion approaches combined with behavioural tests, biochemical analyses, and in vitro electrophysiological recordings to investigate the role of hippocampal μR signalling in memory-retrieval impairment induced by acute elevated platform (EP) stress in mice. Biochemical and molecular analyses revealed that hippocampal μRs were significantly activated during acute stress. Blockage of hippocampal μRs, non-selective deletion of μRs or selective deletion of μRs on GABAergic neurons (μRGABA) reversed EP-stress-induced impairment of memory retrieval, with no effect on the elevation of serum corticosterone after stress. Electrophysiological results demonstrated that stress depressed hippocampal GABAergic synaptic transmission to CA1 pyramidal neurons, thereby leading to excitation/inhibition (E/I) imbalance in a μRGABA-dependent manner. Pharmaceutically enhancing hippocampal GABAAreceptor-mediated inhibitory currents in stressed mice restored their memory retrieval, whereas inhibiting those currents in the unstressed mice mimicked the stress-induced impairment of memory retrieval. Our findings reveal a novel pathway in which endogenous opioids recruited by acute stress predominantly activate μRGABA to depress GABAergic inhibitory effects on CA1 pyramidal neurons, which subsequently alters the E/I balance in the hippocampus and results in impairment of memory retrieval.
Yosten GL, Harada CM, Haddock CJ, Giancotti LA, Kolar GR, Patel R, Guo C, Chen Z, Zhang J, Doyle TM, Dickenson AH, Samson WK, Salvemini D.
PMID: 31999650 | DOI: 10.1172/JCI133270
Treating neuropathic pain is challenging and novel non-opioid based medicines are needed. Using unbiased receptomics, transcriptomic analyses, immunofluorescence and in situ hybridization, we found the expression of the orphan GPCR (oGPCR) Gpr160 and GPR160 increased in the rodent dorsal horn of the spinal cord (DH-SC) following traumatic nerve injury. Genetic and immunopharmacological approaches demonstrated that GPR160 inhibition in the spinal cord prevented and reversed neuropathic pain in male and female rodents without altering normal pain response. GPR160 inhibition in the spinal cord attenuated sensory processing in the thalamus, a key relay in the sensory discriminative pathways of pain. We also identified cocaine- and amphetamine-regulated transcript peptide (CARTp) as a GPR160 ligand. Inhibiting endogenous CARTp signaling in spinal cord attenuated neuropathic pain, whereas exogenous intrathecal (i.th.) CARTp evoked painful hypersensitivity through GPR160-dependent ERK and cAMP response element-binding protein (CREB). Our findings de-orphanize GPR160, identify it as a determinant of neuropathic pain and potential therapeutic target, and provide insights to its signaling pathways. CARTp is involved in many diseases including depression, reward and addiction, de-orphanization of GPR160 is a major step forward understanding the role of CARTp signaling in health and disease
Pook C, Ahrens JM, Clagett-Dame M
PMID: 32081718 | DOI: 10.1016/j.gep.2020.119099
Neuron navigator 2 (NAV2, RAINB1, POMFIL2, HELAD1, unc53H2) is essential for nervous system development. In the present study the spatial distribution of Nav2 transcript in mouse CNS during embryonic, postnatal and adult life is examined. Because multiple NAV2 proteins are predicted based on alternate promoter usage and RNA splicing, in situ hybridization was performed using probes designed to the 5' and 3' ends of the Nav2 transcript, and PCR products using primer sets spanning the length of the mRNA were also examined by real time PCR (qPCR). These studies support full-length Nav2 transcript as the predominant form in the wild-type mouse CNS. The developing cortex, hippocampus, thalamus, olfactory bulb, and granule cells (GC) within the cerebellum show the highest expression, with a similar staining pattern using either the 5'Nav2 or 3'Nav2 probe. Nav2 is expressed in GC precursors migrating over the cerebellar primordium as well as in the postmitotic premigratory cells of the external granule cell layer (EGL). It is expressed in the cornu ammonis (CA) and dentate gyrus (DG) throughout hippocampal development. In situ hybridization was combined with immunohistochemistry for Ki67, CTIP2 and Nissl staining to follow Nav2 transcript location during cortical development, where it is observed in neuroepithelial cells exiting the germinal compartments, as well as later in the cortical plate (CP) and developing cortical layers. The highest levels of Nav2 in all brain regions studied are observed in late gestation and early postnatal life which coincides with times when neurons are migrating and differentiating. A hypomorphic mouse that lacks the full-length transcript but expresses shorter transcript shows little staining in the CNS with either probe set except at the base of the cerebellum, where a shorter Nav2 transcript is detected. Using dual fluorescent probe in situ hybridization studies, these cells are identified as oligodendrocytes and are detected using both Olig1 and the 3'Nav2 probe. The identification of full-length Nav2 as the primary transcript in numerous brain regions suggests NAV2 could play a role in CNS development beyond that of its well-established role in the cerebellum
Nature. 2014 Dec 4;516(7529):121-5.
Ranade SS, Woo SH, Dubin AE, Moshourab RA, Wetzel C, Petrus M, Mathur J, Bégay V, Coste B, Mainquist J, Wilson AJ, Francisco AG, Reddy K, Qiu Z, Wood JN, Lewin GR, Patapoutian A.
PMID: 25471886 | DOI: 10.1038/nature13980.
The sense of touch provides critical information about our physical environment by transforming mechanical energy into electrical signals1. It is postulated that mechanically activated cation channels initiate touch sensation, but the identity of these molecules in mammals has been elusive2. Piezo2 is a rapidly adapting, mechanically activated ion channel expressed in a subset of sensory neurons of the dorsal root ganglion and in cutaneous mechanoreceptors known as Merkel-cell–neurite complexes3, 4. It has been demonstrated that Merkel cells have a role in vertebrate mechanosensation using Piezo2, particularly in shaping the type of current sent by the innervating sensory neuron4, 5, 6; however, major aspects of touch sensation remain intact without Merkel cell activity4, 7. Here we show that mice lacking Piezo2 in both adult sensory neurons and Merkel cells exhibit a profound loss of touch sensation. We precisely localize Piezo2 to the peripheral endings of a broad range of low-threshold mechanoreceptors that innervate both hairy and glabrous skin. Most rapidly adapting, mechanically activated currents in dorsal root ganglion neuronal cultures are absent in Piezo2 conditional knockout mice, and ex vivo skin nerve preparation studies show that the mechanosensitivity of low-threshold mechanoreceptors strongly depends on Piezo2. This cellular phenotype correlates with an unprecedented behavioural phenotype: an almost complete deficit in light-touch sensation in multiple behavioural assays, without affecting other somatosensory functions. Our results highlight that a single ion channel that displays rapidly adapting, mechanically activated currents in vitro is responsible for the mechanosensitivity of most low-threshold mechanoreceptor subtypes involved in innocuous touch sensation. Notably, we find that touch and pain sensation are separable, suggesting that as-yet-unknown mechanically activated ion channel(s) must account for noxious (painful) mechanosensation.
Zhang M, Wang Y, Geng J, Zhou S, Xiao B.
PMID: 30726728 | DOI: 10.1016/j.celrep.2019.01.056
Touch and mechanical pain represent distinct, but interactive, modalities of mechanosensation. However, the molecular mechanisms underlying these mechanotransduction processes remain incompletely understood. Here, we show that deletion of the mechanically activated and rapidly adapting Piezo2 channel in a portion of the low-threshold mechanoreceptors and a majority of the IB4-positive nociceptors impairs touch but sensitizes mechanical pain in mice. Ectopic expression of the Piezo2 homolog, the intermediately adapting Piezo1 channel, in sensory neurons can sensitize touch in normal mice and rescue defective touch of the Piezo2-knockout mice. Broad expression of Piezo1 in sensory neurons decreases, rather than evokes, mechanical pain responses. Together, our data suggest that Piezo channels can mediate touch and indirectly suppress acute pain. Tuning Piezo-mediated touch sensitivity allows us to recapitulate the inhibitory effect of touch on acute pain in mouse models.
