High protein and mRNA expression levels of TUBB3 (class III ß-tubulin) are associated with aggressive tumor features in esophageal adenocarcinomas.

Oncotarget.

2017 Dec 11

Loeser H, Schallenberg S, von Winterfeld M, Tharun L, Alakus H, Hölscher A, Bollschweiler E, Buettner R, Zander T, Quaas A.
PMID: 29383151 | DOI: 10.18632/oncotarget.23112

Abstract

BACKGROUND:

Esophageal adenocarcinomas show an increasing incidence in the Western world and their overall survival remains low. Microtubules are multifunctional cytoskeletal proteins involved in crucial cellular roles, including maintenance of cell shape, intracellular transport, meiosis, and mitosis. Microtubulus-TUBB3 was found overexpressed in several carcinomas suggesting a significant role in cancer development. High levels of TUBB3 expression were also described to be associated with poor clinical outcome in various cancers. It was shown that overexpression of TUBB3 could be related to reduced efficiency of taxane-based targeting anticancer drugs in several cancer types.

RESULTS:

There is a statistically significant association between high TUBB3 protein and TUBB3 mRNA expression and shortened survival (p<0,0001). Prognostic impact of TUBB3 expression is seen in patients with and without multimodal treatment. Multivariate analysis revealed a strong TUBB3 expression to be an independent prognosis factor. Validation of protein expression by mRNA in situ hybridization underlines the credibility of the immunohistochemical results.

DISCUSSION:

Our study emphasized the significant importance of TUBB3 in esophageal adenocarcinoma. TUBB3 serves as an independent prognostic marker and may be a valuable biomarker for routine application in esophageal adenocarcinoma especially to address the need for adjuvant treatment in individuals following neoadjuvant therapy and surgery. Future prospective studies are needed which include the results of TUBB3 in preoperative biopsy material to proof the prognostic impact of TUBB3.

MATERIALS AND METHODS:

280 esophageal adenocarcinomas that underwent primary surgical resection or resection after neoadjuvant therapy were analyzed by mRNA-in-situ-hybridization (RNAscope™) and by immunohistochemistry (TUBB3 rabbit monoclonal antibody; Epitomics).

The fragile X syndrome protein FMRP participates in axon guidance mediated by the Wnt/planar cell polarity pathway

Neuroscience

2022 Sep 30

Marfull-Oromí, P;Onishi, K;Zou, Y;
PMID: 36191829 | DOI: 10.1016/j.neuroscience.2022.09.018

The Planar cell polarity (PCP) pathway is known to mediate the function of the Wnt proteins in growth cone guidance. Here, we show that the PCP pathway may directly influence local protein synthesis within the growth cones. We found that FMRP interacts with Fzd3. This interaction is negatively regulated by Wnt5a, which induces FMRP phosphorylation. Knocking down FMRP via electroporating shRNAs into the dorsal spinal cord lead to a randomization of anterior-posterior turning of commissural axons, which could be rescued by a FMRP rescue construct. Using RNAscope, we found that some of the FMRP target mRNAs encoding PCP components, PRICKLE2 and Celsr2, as well as regulators of cytoskeletal dynamics and components of cytoskeleton, APC, Cfl1, Map1b, Tubb3 and Actb, are present in the commissural neuron growth cones. Our results suggest that PCP signaling may regulate growth cone guidance, at least in part, by regulating local protein synthesis in the growth cones through via an interaction between Frizzled3 and FMRP.

Keratinocytes produce IL-17c to protect peripheral nervous systems during human HSV-2 reactivation

J Exp Med.

2017 Jun 29

Peng T, Chanthaphavong RS, Sun S, Trigilio JA, Phasouk K, Jin L, Layton ED, Li AZ, Correnti CE, De van der Schueren W, Vazquez J, O’Day DR, Glass IA, Knipe DM, Wald A, Corey L, Zhu J.
PMID: 28663436 | DOI: 10.1084/jem.20160581

Abstract

Despite frequent herpes simplex virus (HSV) reactivation, peripheral nerve destruction and sensory anesthesia are rare. We discovered that skin biopsies obtained during asymptomatic human HSV-2 reactivation exhibit a higher density of nerve fibers relative to biopsies during virological and clinical quiescence. We evaluated the effects of HSV infection on keratinocytes, the initial target of HSV replication, to better understand this observation. Keratinocytes produced IL-17c during HSV-2 reactivation, and IL-17RE, an IL-17c-specific receptor, was expressed on nerve fibers in human skin and sensory neurons in dorsal root ganglia. In ex vivo experiments, exogenous human IL-17cprovided directional guidance and promoted neurite growth and branching in microfluidic devices. Exogenous murine IL-17c pretreatment reduced apoptosis in HSV-2-infected primary neurons. These results suggest that IL-17c is a neurotrophic cytokine that protects peripheralnerve systems during HSV reactivation. This mechanism could explain the lack of nerve damage from recurrent HSV infection and may provide insight to understanding and treating sensory peripheral neuropathies.

Diversity amongst trigeminal neurons revealed by high throughput single cell sequencing

PLoS ONE

2017 Sep 28

Nguyen MQ, Wu Y, Bonilla LS, von Buchholtz LJ, Ryba NJP.
PMID: 28957441 | DOI: 10.1371/journal.pone.0185543

The trigeminal ganglion contains somatosensory neurons that detect a range of thermal, mechanical and chemical cues and innervate unique sensory compartments in the head and neck including the eyes, nose, mouth, meninges and vibrissae. We used single-cell sequencing and in situ hybridization to examine the cellular diversity of the trigeminal ganglion in mice, defining thirteen clusters of neurons. We show that clusters are well conserved in dorsal root ganglia suggesting they represent distinct functional classes of somatosensory neurons and not specialization associated with their sensory targets. Notably, functionally important genes (e.g. the mechanosensory channel Piezo2 and the capsaicin gated ion channel Trpv1) segregate into multiple clusters and often are expressed in subsets of cells within a cluster. Therefore, the 13 genetically-defined classes are likely to be physiologically heterogeneous rather than highly parallel (i.e., redundant) lines of sensory input. Our analysis harnesses the power of single-cell sequencing to provide a unique platform for in silico expression profiling that complements other approaches linking gene-expression with function and exposes unexpected diversity in the somatosensory system.

Astrocyte-Secreted Glypican 4 Regulates Release of Neuronal Pentraxin 1 from Axons to Induce Functional Synapse Formation.

Neuron.

2017 Oct 11

Farhy-Tselnicker I, van Casteren ACM, Lee A, Chang VT, Aricescu AR, Allen NJ.
PMID: 29024665 | DOI: 10.1016/j.neuron.2017.09.053

The generation of precise synaptic connections between developing neurons is critical to the formation of functional neural circuits. Astrocyte-secreted glypican 4 induces formation of active excitatory synapses by recruiting AMPA glutamate receptors to the postsynaptic cell surface. We now identify the molecular mechanism of how glypican 4 exerts its effect. Glypican 4 induces release of the AMPA receptor clustering factor neuronal pentraxin 1 from presynaptic terminals by signaling through presynaptic protein tyrosine phosphatase receptor δ. Pentraxin then accumulates AMPA receptors on the postsynaptic terminal forming functional synapses. Our findings reveal a signaling pathway that regulates synaptic activity during central nervous system development and demonstrates a role for astrocytes as organizers of active synaptic connections by coordinating both pre and post synaptic neurons. As mutations in glypicans are associated with neurological disorders, such as autism and schizophrenia, this signaling cascade offers new avenues to modulate synaptic function in disease.

Dual leucine zipper kinase is required for mechanical allodynia and microgliosis after nerve injury.

Elife.

2018 Jul 03

Wlaschin JJ, Gluski JM, Nguyen E, Silberberg H, Thompson JH, Chesler AT, Le Pichon CE.
PMID: 29968565 | DOI: 10.7554/eLife.33910

Neuropathic pain resulting from nerve injury can become persistent and difficult to treat but the molecular signaling responsible for its development remains poorly described. Here, we identify the neuronal stress sensor dual leucine zipper kinase (DLK; Map3k12) as a key molecule controlling the maladaptive pathways that lead to pain following injury. Genetic or pharmacological inhibition of DLK reduces mechanical hypersensitivity in a mouse model of neuropathic pain. Furthermore, DLK inhibition also prevents the spinal cord microgliosis that results from nerve injury and arises distant from the injury site. These striking phenotypes result from the control by DLK of a transcriptional program in somatosensory neurons regulating the expression of numerous genes implicated in pain pathogenesis, including the immune gene Csf1. Thus, activation of DLK is an early event, or even the master regulator, controlling a wide variety of pathways downstream of nerve injury that ultimately lead to chronic pain.

Astrocyte-Secreted Chordin-like 1 Drives Synapse Maturation and Limits Plasticity by Increasing Synaptic GluA2 AMPA Receptors.

Neuron. 2018 Oct 12.

2018 Oct 12

Blanco-Suarez E, Liu TF, Kopelevich A, Allen NJ.
PMID: 30344043 | DOI: 10.1016/j.neuron.2018.09.043

In the developing brain, immature synapses contain calcium-permeable AMPA glutamate receptors (AMPARs) that are subsequently replaced with GluA2-containing calcium-impermeable AMPARs as synapses stabilize and mature. Here, we show that this essential switch in AMPARs and neuronal synapse maturation is regulated by astrocytes. Using biochemical fractionation of astrocyte-secreted proteins and mass spectrometry, we identified that astrocyte-secreted chordin-like 1 (Chrdl1) is necessary and sufficient to induce mature GluA2-containing synapses to form. This function of Chrdl1 is independent of its role as an antagonist of bone morphogenetic proteins (BMPs). Chrdl1 expression is restricted to cortical astrocytes in vivo, peaking at the time of the AMPAR switch. Chrdl1 knockout (KO) mice display reduced synaptic GluA2 AMPARs, altered kinetics of synaptic events, and enhanced remodeling in an in vivo plasticity assay. Studies have shown that humans with mutations in Chrdl1 display enhanced learning. Thus astrocytes, via the release of Chrdl1, promote GluA2-dependent synapse maturation and thereby limit synaptic plasticity.

Nppb Neurons Are Sensors of Mast Cell-Induced Itch

Cell Rep

2019 Mar 26

Solinski HJ, Kriegbaum MC, Tseng PY, Earnest TW, Gu X, Barik A, Chesler AT and Hoon MA
PMID: 30917312 | DOI: 10.1016/j.celrep.2019.02.089

Itch is an unpleasant skin sensation that can be triggered by exposure to many chemicals, including those released by mast cells. The natriuretic polypeptide b (Nppb)-expressing class of sensory neurons, when activated, elicits scratching responses in mice, but it is unclear which itch-inducing agents stimulate these cells and the receptors involved. Here, we identify receptors expressed by Nppb neurons and demonstrate the functional importance of these receptors as sensors of endogenous pruritogens released by mast cells. Our search for receptors in Nppb neurons reveals that they express leukotriene, serotonin, and sphingosine-1-phosphate receptors. Targeted cell ablation, calcium imaging of primary sensory neurons, and conditional receptor knockout studies demonstrate that these receptors induce itch by the direct stimulation of Nppb neurons and neurotransmission through the canonical gastrin-releasing peptide (GRP)-dependent spinal cord itch pathway. Together, our results define a molecular and cellular pathway for mast cell-induced itch.

Transcriptional Programming of Human Mechanosensory Neuron Subtypes from Pluripotent Stem Cells.

Cell Rep

2020 Jan 21

Nickolls AR, Lee MM, Espinoza DF, Szczot M, Lam RM, Wang Q, Beers J, Zou J, Nguyen MQ, Solinski HJ, AlJanahi AA, Johnson KR, Ward ME, Chesler AT, B�nnemann CG
PMID: 31968264 | DOI: 10.1016/j.celrep.2019.12.062

Efficient and homogeneous in vitro generation of peripheral sensory neurons may provide a framework for novel drug screening platforms and disease models of touch and pain. We discover that, by overexpressing NGN2 and BRN3A, human pluripotent stem cells can be transcriptionally programmed to differentiate into a surprisingly uniform culture of cold- and mechano-sensing neurons. Although such a neuronal subtype is not found in mice, we identify molecular evidence for its existence in human sensory ganglia. Combining NGN2 and BRN3A programming with neural crest patterning, we produce two additional populations of sensory neurons, including a specialized touch receptor neuron subtype. Finally, we apply this system to model a rare inherited sensory disorder of touch and proprioception caused by inactivating mutations in PIEZO2. Together, these findings establish an approach to specify distinct sensory neuron subtypes in vitro, underscoring the utility of stem cell technology to capture human-specific features of physiology and disease.

Familial Alzheimer's disease-associated PSEN1 mutations affect neurodevelopment through increased Notch signaling

Stem cell reports

2023 Jun 08

Hurley, EM;Mozolewski, P;Dobrowolski, R;Hsieh, J;
PMID: 37352850 | DOI: 10.1016/j.stemcr.2023.05.018

Alzheimer's disease (AD) is the most common neurodegenerative disorder, but its root cause may lie in neurodevelopment. PSEN1 mutations cause the majority of familial AD, potentially by disrupting proper Notch signaling, causing early unnoticed cellular changes that affect later AD progression. While rodent models are useful for modeling later stages of AD, human induced pluripotent stem cell-derived cortical spheroids (hCSs) allow access to studying the human cortex at the cellular level over the course of development. Here, we show that the PSEN1 L435F heterozygous mutation affects hCS development, increasing size, increasing progenitors, and decreasing post-mitotic neurons as a result of increased Notch target gene expression during early hCS development. We also show altered Aβ expression and neuronal activity at later hCS stages. These results contrast previous findings, showing how individual PSEN1 mutations may differentially affect neurodevelopment and may give insight into fAD progression to provide earlier time points for more effective treatments.

Nav1.7 gain-of-function mutation I228M triggers age-dependent nociceptive insensitivity and C-LTMR dysregulation

Experimental neurology

2023 Mar 30

Wimalasena, NK;Taub, DG;Shim, J;Hakim, S;Kawaguchi, R;Chen, L;El-Rifai, M;Geschwind, D;Dib-Hajj, SD;Waxman, SG;Woolf, CJ;
PMID: 37003485 | DOI: 10.1016/j.expneurol.2023.114393

Gain-of-function mutations in Scn9a, which encodes the peripheral sensory neuron-enriched voltage-gated sodium channel Nav1.7, cause paroxysmal extreme pain disorder (PEPD), inherited erythromelalgia (IEM), and small fiber neuropathy (SFN). Conversely, loss-of-function mutations in the gene are linked to congenital insensitivity to pain (CIP). These mutations are evidence for a link between altered sodium conductance and neuronal excitability leading to somatosensory aberrations, pain, or its loss. Our previous work in young adult mice with the Nav1.7 gain-of-function mutation, I228M, showed the expected DRG neuron hyperexcitability, but unexpectedly the mice had normal mechanical and thermal behavioral sensitivity. We now show that with aging both male and female mice with this mutation unexpectedly develop a profound insensitivity to noxious heat and cold, as well skin lesions that span the body. Electrophysiology demonstrates that, in contrast to young mice, aged I228M mouse DRGs have a profound loss of sodium conductance and changes in activation and slow inactivation dynamics, representing a loss-of-function. Through RNA sequencing we explored how these age-related changes may produce the phenotypic changes and found a striking and specific decrease in C-low threshold mechanoreceptor- (cLTMR) associated gene expression, suggesting a potential contribution of this DRG neuron subtype to Nav1.7 dysfunction phenotypes. A GOF mutation in a voltage-gated channel can therefore produce over a prolonged time, highly complex and unexpected alterations in the nervous system beyond excitability changes.

Cross-species transcriptomic atlas of dorsal root ganglia reveals species-specific programs for sensory function

Nature communications

2023 Jan 23

Jung, M;Dourado, M;Maksymetz, J;Jacobson, A;Laufer, BI;Baca, M;Foreman, O;Hackos, DH;Riol-Blanco, L;Kaminker, JS;
PMID: 36690629 | DOI: 10.1038/s41467-023-36014-0

Sensory neurons of the dorsal root ganglion (DRG) are critical for maintaining tissue homeostasis by sensing and initiating responses to stimuli. While most preclinical studies of DRGs are conducted in rodents, much less is known about the mechanisms of sensory perception in primates. We generated a transcriptome atlas of mouse, guinea pig, cynomolgus monkey, and human DRGs by implementing a common laboratory workflow and multiple data-integration approaches to generate high-resolution cross-species mappings of sensory neuron subtypes. Using our atlas, we identified conserved core modules highlighting subtype-specific biological processes related to inflammatory response. We also identified divergent expression of key genes involved in DRG function, suggesting species-specific adaptations specifically in nociceptors that likely point to divergent function of nociceptors. Among these, we validated that TAFA4, a member of the druggable genome, was expressed in distinct populations of DRG neurons across species, highlighting species-specific programs that are critical for therapeutic development.

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	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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