Recent Publications | Neurosciences | School of Medicine

Coordinated cadherin functions sculpt respiratory motor circuit connectivity

Abstract: Breathing, and the motor circuits that control it, is essential for life. At the core of respiratory circuits are Dbx1-derived interneurons, which generate the rhythm and pattern of breathing, and phrenic motor neurons (MNs), which provide the final motor output that drives diaphragm muscle contractions during inspiration. Despite their critical function, the principles that dictate how respiratory circuits assemble are unknown. Here, we show that coordinated activity of a type I cadherin (N-cadherin) and type II cadherins (Cadherin-6, -9, and -10) is required in both MNs and Dbx1-derived neurons to generate robust respiratory motor output. Both MN- and Dbx1-specific cadherin inactivation in mice during a critical developmental window results in perinatal lethality due to respiratory failure and a striking reduction in phrenic MN bursting activity. This combinatorial cadherin code is required to establish phrenic MN cell body and dendritic topography; surprisingly, however, cell body position appears to be dispensable for the targeting of phrenic MNs by descending respiratory inputs. Our findings demonstrate that type I and II cadherins function cooperatively throughout the respiratory circuit to generate a robust breathing output and reveal novel strategies that drive the assembly of motor circuits.

Alicia N Vagnozzi, Matthew T Moore, Minshan Lin, Elyse M Brozost, Ritesh KC, Aambar Agarwal, Lindsay A Schwarz, Xin Duan, Niccolò Zampieri, Lynn T Landmesser, Polyxeni Philippidou

eLife 11:e82116. February 9, 2023 https://doi.org/10.7554/eLife.82116

Hypothalamic supramammillary nucleus selectively excites hippocampal CA3 interneurons to suppress CA3 pyramidal neuron activity

Abstract: A key mode of neuronal communication between distant brain regions is through excitatory synaptic transmission mediated by long-range glutamatergic projections emitted from principal neurons. The long-range glutamatergic projection normally forms numerous en passant excitatory synapses onto both principal neurons and interneurons along its path. Under physiological conditions, the monosynaptic excitatory drive onto postsynaptic principal neurons outweighs disynaptic feedforward inhibition, with the net effect of depolarizing principal neurons. In contrast with this conventional doctrine, here we report that a glutamatergic projection from the hypothalamic supramammillary nucleus (SuM) largely evades postsynaptic pyramidal neurons (PNs), but preferentially target interneurons in the hippocampal CA3 region to predominantly provide feedforward inhibition. Using viral-based retrograde and anterograde tracing and ChannelRhodopsin2 (ChR2)-assisted patch-clamp recording in mice of either sex, we show that SuM projects sparsely to CA3 and provides minimal excitation onto CA3 PNs. Surprisingly, despite its sparse innervation, the SuM input inhibits all CA3 PNs along the transverse axis. Further, we find that SuM provides strong monosynaptic excitation onto CA3 parvalbumin (PV)-expressing interneurons evenly along the transverse axis, which likely mediates the SuM-driven feedforward inhibition. Taken together, our results demonstrate that a novel long-range glutamatergic pathway largely evades principal neurons, but rather preferentially innervates interneurons in a distant brain region to suppress principal neuron activity. Moreover, our findings reveal a new means by which SuM regulates hippocampal activity through SuM-to-CA3 circuit, independent of the previously focused projections from SuM to CA2 or dentate gyrus.

Minghua Li, Jessica L. Kinney, Yu-Qiu Jiang, Daniel K Lee, Qiwen Wu, Daehoon Lee, Wen-Cheng Xiong and Qian Sun

Journal of Neuroscience 28 April 2023, JN-RM-1910-22; DOI: https://doi.org/10.1523/JNEUROSCI.1910-22.2023

The Conditioning Lesion Response in Dorsal Root Ganglion Neurons Is Inhibited in Oncomodulin Knock-Out Mice

Regeneration can occur in peripheral neurons after injury, but the mechanisms involved are not fully delineated. Macrophages in dorsal root ganglia (DRGs) are involved in the enhanced regeneration that occurs after a conditioning lesion (CL), but how macrophages stimulate this response is not known. Oncomodulin (Ocm) has been proposed as a proregenerative molecule secreted by macrophages and neutrophils, is expressed in the DRG after axotomy, and stimulates neurite outgrowth by DRG neurons in culture. Wild-type (WT) and Ocm knock-out (KO) mice were used to investigate whether Ocm plays a role in the CL response in DRG neurons after sciatic nerve transection. Neurite outgrowth was measured after 24 and 48 h in explant culture 7 d after a CL. Sciatic nerve regeneration was also measured in vivo 7 d after a CL and 2 d after a subsequent sciatic nerve crush. The magnitude of the increased neurite outgrowth following a CL was significantly smaller in explants from Ocm KO mice than in explants from WT mice. In vivo after a CL, increased regeneration was found in WT animals but not in KO animals. Macrophage accumulation and levels of interleukin-6 (IL-6) mRNA were measured in axotomized DRG from WT and Ocm KO animals, and both were significantly higher than in sham-operated ganglia. At 6 h after axotomy, Il-6 mRNA was higher in WT than in Ocm KO mice. Our data support the hypothesis that Ocm plays a necessary role in producing a normal CL response and that its effects possibly result in part from stimulation of the expression of proregenerative macrophage cytokines such as IL-6.

Jon P. Niemi*, Talia DeFrancesco-Oranburg* , Andrew Cox , Jane A. Lindborg , Franklin D. Echevarria, Jemima McCluskey , Dwayne D. Simmons, Richard E. Zigmond

eNeuro 2022 Feb 24;9(1):ENEURO.0477-21.2022. doi: 10.1523/ENEURO.0477-21.2022.

The primary macrophage chemokine, CCL2, is not necessary after a peripheral nerve injury for macrophage recruitment and activation or for conditioning lesion enhanced peripheral regeneration

Background: Peripheral nerve injuries stimulate the regenerative capacity of injured neurons through a neuroimmune phenomenon termed the conditioning lesion (CL) response. This response depends on macrophage accumulation in affected dorsal root ganglia (DRGs) and peripheral nerves. The macrophage chemokine CCL2 is upregulated after injury and is allegedly required for stimulating macrophage recruitment and pro-regenerative signaling through its receptor, CCR2. In these tissues, CCL2 is putatively produced by neurons in the DRG and Schwann cells in the distal nerve.
Methods: Ccl2fl/fl mice were crossed with Advillin-Cre, P0-Cre, or both to create conditional Ccl2 knockouts (CKOs) in sensory neurons, Schwann cells, or both to hypothetically remove CCL2 and macrophages from DRGs, nerves or both. CCL2 was localized using Ccl2-RFPfl/fl mice. CCL2-CCR2 signaling was further examined using global Ccl2 KOs and Ccr2gfp knock-in/knock-outs. Unilateral sciatic nerve transection was used as the injury model, and at various timepoints, chemokine expression, macrophage accumulation and function, and in vivo regeneration were examined using qPCR, immunohistochemistry, and luxol fast blue staining.
Results: Surprisingly, in all CKOs, DRG Ccl2 gene expression was decreased, while nerve Ccl2 was not. CCL2-RFP reporter mice revealed CCL2 expression in several cell types beyond the expected neurons and Schwann cells. Furthermore, macrophage accumulation, myelin clearance, and in vivo regeneration were unaffected in all CKOs, suggesting CCL2 may not be necessary for the CL response. Indeed, Ccl2 global knockout mice showed normal macrophage accumulation, myelin clearance, and in vivo regeneration, indicating these responses do not require CCL2. CCR2 ligands, Ccl7 and Ccl12, were upregulated after nerve injury and perhaps could compensate for the absence of Ccl2. Finally, Ccr2gfp knock-in/knock-out animals were used to differentiate resident and recruited macrophages in the injured tissues. Ccr2gfp/gfp KOs showed a 50% decrease in macrophages in the distal nerve compared to controls with a relative increase in resident macrophages. In the DRG there was a small but insignificant decrease in macrophages.
Conclusions: CCL2 is not necessary for macrophage accumulation, myelin clearance, and axon regeneration in the peripheral nervous system. Without CCL2, other CCR2 chemokines, resident macrophage proliferation, and CCR2-independent monocyte recruitment can compensate and allow for normal macrophage accumulation.

Aaron D. Talsma, Jon P. Niemi, Joel S. Pachter, Richard E. Zigmond

J Neuroinflammation 2022 Jul 12;19(1):179. doi: 10.1186/s12974-022-02497-9.

Reorganization of postmitotic neuronal chromatin accessibility for maturation of serotonergic identity

Assembly of transcriptomes encoding unique neuronal identities requires selective accessibility of transcription factors to cis-regulatory sequences in nucleosome-embedded postmitotic chromatin. Yet, the mechanisms controlling postmitotic neuronal chromatin accessibility are poorly understood. This paper identifies the unique accessible distal enhancer landscape that defines the Pet1 neuron lineage from which all brain serotonin (5-HT) neurons are generated in mice. Heterogeneous single cell chromatin landscapes are established early in postmitotic Pet1 neurons and reveal the putative regulatory programs driving Pet1 neuron subtype identities. Distal enhancer accessibility is highly dynamic as Pet1 neurons mature, suggesting the existence of regulatory factors that reorganize postmitotic neuronal chromatin. Pet1 and Lmx1b were found to reorganize chromatin accessibility of select Pet1-lineage specific enhancers for 5-HT neurotransmission. Additionally, these factors are required to maintain chromatin accessibility during early maturation suggesting that postmitotic neuronal open chromatin is unstable and requires continuous regulatory input. Together this paper identifies postmitotic transcription factors that reorganize accessible chromatin for neuron specialization.

Zhang, X. L., Spencer, W. C., Tabuchi, N., Kitt, M. M., & Deneris, E. S. (2022).

Elife. 2022 Apr 26;11:e75970. doi: 10.7554/eLife.75970. PMID: 35471146

An adult-stage transcriptional program for survival of serotonergic connectivity

CNS synapse and axon degeneration occurs naturally as we age and is accelerated in many neurodegenerative diseases. The underlying mechanisms explaining the vulnerability of axons, especially long ones, in aging and disease are not understood but seem likely to relate to the extraordinary cell biologic challenge of sustaining long-distance axonal and synaptic integrity over decades of life. How the axonal, synaptic and energy-generating machinery of these remote morphological compartments are sustained over decades of life in mitotically quiescent cells against constant macromolecular turnover is not understood. This paper describes an adult stage self-sustaining mouse transcriptional program that protects 5-HT connectivity against degeneration. The apex transcription factor (TF) governing this GRN is the LIM homeodomain TF, Lmx1b, although the ETS TF, Pet1, functionally contributes. These TFs function in adult 5-HT neurons to maintain a broad and diverse connectivity transcriptome comprising hundreds of synapse, axon, and mitochondrial genes including all 5-HT neurotransmission genes. Adult stage loss of Lmx1b and Pet1 expression in 5-HT neurons leads to a slow progressive breakdown of 5-HT connectivity characterized by expansive loss of long distance 5-HT axon arbors throughout the brain and spinal cord, loss of 5-HT pericellular basket presynaptic terminals, abnormal spheroid formation, accumulation of APP and α-synuclein in abnormally swollen varicosities and spheroids, and mitochondrial fragmentation. Based on these findings, it is proposed that progressive decay of connectivity transcriptomes brought about by genetic or environmental disruption of adult-stage transcriptional connectivity survival programs may represent a previously unrecognized path to aging- and disease-related degeneration of adult brain circuitry.

Kitt, M. M., Tabuchi, N., Spencer, W. C., Robinson, H. L., Zhang, X. L., Eastman, B. A., Lobur, K. J., Silver, J., Mei, L., & Deneris, E. S. (2022).

In Cell Reports (Vol. 39, Issue 3, p. 110711). Elsevier BV.

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Age-Related Unstructured Spike Patterns Patterns and Molecular Localization in Drosophila Circadian Neurons

Aging decreases sleep quality by disrupting the molecular machinery that regulates the circadian rhythm. However, we do not fully understand the mechanism that underlies this process. In Drosophila, sleep quality is regulated by precisely timed patterns of spontaneous firing activity in posterior DN1 (DN1p) circadian clock neurons. How aging affects the physiological function of DN1p neurons is unknown. In this study, we found that aging altered functional parameters related to neural excitability and disrupted patterned spike sequences in DN1p neurons during nighttime. We also characterized age-associated changes in intrinsic membrane properties related to spike frequency adaptations and synaptic properties, which may account for the unstructured spike patterns in aged DN1p neurons. Because Slowpoke binding protein (SLOB) and the Na⁺/K⁺ ATPase β subunit (NaKβ) regulate clock-dependent spiking patterns in circadian networks, we compared the subcellular organization of these factors between young and aged DN1p neurons. Young DN1p neurons showed circadian cycling of HA-tagged SLOB and myc-tagged NaKβ targeting the plasma membrane, whereas aged DN1p neurons showed significantly disrupted subcellular localization patterns of both factors. The distribution of SLOB and NaKβ signals also showed greater variability in young vs. aged DN1p neurons, suggesting aging leads to a loss of actively formed heterogeneity for these factors. These findings showed that aging disrupts precisely structured molecular patterns that regulate structured neural activity in the circadian network, leading to age-associated declines in sleep quality. Thus, it is possible to speculate that a recovery of unstructured neural activity in aging clock neurons could help to rescue age-related poor sleep quality.

Nguyen, D. L., Hutson, A. N., Zhang, Y., Daniels, S. D., Peard, A. R., & Tabuchi, M. (2022)

In Frontiers in Physiology (Vol. 13).

Neddylation is critical to cortical development by regulating Wnt/b-catenin signaling.

The neocortex is characterized by a six-layered structure that is critical to brain function. Cortical development requires proper generation and differentiation of neurons that migrate out from the ventricular zone to populate the cortex. This is regulated by Wnt/β-catenin signaling, which reduces gradually during cortical development. However, how the decreasing of Wnt/β-catenin signaling is regulated is largely unclear. We demonstrate that neddylation, a ubiquitylation-like protein posttranslational modification, targets β-catenin itself to inhibit Wnt/β-catenin signaling in the process. We also show that ablating Nae1, an obligative subunit of the E1 for neddylation, from cortical progenitor cells leads to similar phenotypes in β-catenin gain-of-function mice. This study reveals a previously unappreciated role of neddylation in regulating cortical lamination by targeting β-catenin signaling.

L. Zhang, H. Jing, H. Li, W. Chen, B. Luo, H-S. Zhang, Z-Q. Dong, L. Li, H-B. Su, W-C. Xiong and L. Mei.

Proc. Nat. Acad. Sci. USA. 117:26448-26459, 2020 Oct

Topographic heterogeneity of intrinsic excitability in mouse hippocampal CA3 pyramidal neurons.

Area CA3 is a major hippocampal region that is classically thought to act as a homogeneous neural network vital for spatial navigation and episodic memories. Here, we report that CA3 pyramidal neurons exhibit marked heterogeneity of somatodendritic morphology and cellular electrical properties along both proximodistal and dorsoventral axes. These new results uncover a complex, yet orderly, pattern of topographic organization of CA3 neuronal features that may contribute to its in vivo functional diversity.

Sun Q, Jiang YQ, Lu MC.

J Neurophysiol. 2020 Oct 1;124(4):1270-1284. doi: 10.1152/jn.00147.2020. Epub 2020 Sep 16.

A role of lamin A/C in preventing neuromuscular junction decline in mice.

This study provides evidence that lamin A/C, a scaffolding component of the nuclear envelope, is critical to maintaining the NMJ in mice. Its muscle-specific mutation led to progressive NMJ degeneration in vivo. We showed that the mutation reduced the level of rapsyn, a protein necessary for acetylcholine receptor (AChR) clustering; and expression of rapsyn in muscles attenuated NMJ deficits of HSA-Lmna−/− mice. These results reveal a role of lamin A/C in NMJ maintenance and suggest that nuclear dysfunction or deficiency may contribute to NMJ deficits in aged muscles.

N. Gao, K. Zhao, Y. Cao, X. Ren, H. Jing, G. Xing, W.-C. Xiong, and Lin Mei.

J. Neurosci. 40:7203-7215, 2020 Sept (Cover)

A role of low-density lipoprotein receptor-related protein 4 (LRP4) in astrocytic Aβ clearance.

This study investigates how astrocytes, a type of non-nerve cells in the brain, may contribute to Alzheimer's disease (AD) development. We demonstrate that the low-density lipoprotein receptor-related protein 4 (LRP4) is reduced in the brain of AD patients. Mimicking the reduced levels in an AD mouse model exacerbates cognitive impairment and increases amyloid aggregates that are known to damage the brain. We show that LRP4 could promote the clearance of amyloid protein by astrocytes. Our results reveal a previously unappreciated role of LRP4 in AD development.

H. Zhang, W. Chen, Z. Tan, L. Zhang, Z. Dong, W. Cui, K. Zhao, H. Wang, H. Jing, R. Cao, C. Kim, J.G. Safar, W.-C. Xiong and L. Mei.

J. Neurosci., 40:5347:5361, 2020 Jul

Clinical features of LRP4/agrin-antibody – positive myasthenia gravis: A multicenter study.

M.H. Rivner, B.M. Quarles, J.-X. Pan, Z. Yu, J.F. Howard Jr, A. Corse, M.M. Dimachkie, C. Jackson, T. Vu, G. Small, R.P. Lisak, J. Belsh, I. Lee, R.J. Nowak, V. Baute, S. Scelsa, J.A. Fernandes, Z. Simmons, A. Swenson, R. Barohn, R.B. Sanka, C. Gooch, E. Ubogu, J. Caress, M. Pasnoor, H. Xu, L. Mei.

Muscle & Nerve 62:333–343, 2020 Jun.