Proteomic Analysis of Dorsal Root Ganglia in a Mouse Model of Paclitaxel-Induced Neuropathic Pain.

Paclitaxel is a chemotherapy drug widely used for the treatment of various cancers based on its ability to potently stabilize cellular microtubules and block division in cancer cells. Paclitaxel-based treatment, however, accumulates in peripheral system sensory neurons and leads to a high incidence rate (over 60%) of chemotherapy induced peripheral neuropathy. Using an established preclinical model of paclitaxel-induced peripheral neuropathy (PIPN), we examined proteomic changes in dorsal root ganglia (DRG) of adult male mice that were treated with paclitaxel (8 mg/kg, at 4 injections every other day) relative to vehicle-treated mice. High throughput proteomics based on liquid chromatography electrospray ionization mass spectrometry identified 165 significantly altered proteins in lumbar DRG. Gene ontology enrichment and bioinformatic analysis revealed an effect of paclitaxel on pathways for mitochondrial regulation, axonal function, and inflammatory purinergic signaling as well as microtubule activity. These findings provide insight into molecular mechanisms that can contribute to PIPN in patients.

Proteomic responses in the human dopaminergic LUHMES cell line to imidacloprid and its metabolites imidacloprid-olefin and desnitro-imidacloprid.

Neonicotinoids (neonics) are amongst the most commonly used class of pesticides globally. In the United States, imidacloprid (IMI) is extensively used for agriculture and in other common applications such as house-hold pest control. Regular exposure to IMI, and several of its known metabolites including IMI-olefin and desnitro-imidacloprid (DN-IMI), has been shown to be harmful to many organisms including mammals, birds, and fish. Studies show that neonics bind human nicotinicacetylcholine receptors (nAChRs) and cause cellular toxicity. In the dopaminergic Lund human mesencephalic (LUHMES) cell line, IMI and other neonics (10-100 µM) have been recently shown to activate intracellular calcium signaling through nAChRs. Thus, we examined proteomic responses of LUHMES cells to a 48-h treatment with 50 µM IMI, IMI-olefin, or DN-IMI. Our findings show differential effects of these neonics on cellular protein expression. Bioinformatic analysis of significantly altered proteins indicates an effect of IMI, IMI-olefin, and DN-IMI on protein synthesis and ribosomal function. These findings suggest a role for protein synthesis and transcriptional regulation in neonic-mediated dopaminergic neurotoxicity.

α7 nicotinic acetylcholine receptor interaction with G proteins in breast cancer cell proliferation, motility, and calcium signaling.

Chronic smoking is a primary risk factor for breast cancer due to the presence of various toxins and carcinogens within tobacco products. Nicotine is the primary addictive component of tobacco products and has been shown to promote breast cancer cell proliferation and metastases. Nicotine activates nicotinic acetylcholine receptors (nAChRs) that are expressed in cancer cell lines. Here, we examine the role of the α7 nAChR in coupling to heterotrimeric G proteins within breast cancer MCF-7 cells. Pharmacological activation of the α7 nAChR using choline or nicotine was found to increase proliferation, motility, and calcium signaling in MCF-7 cells. This effect of α7 nAChR on cell proliferation was abolished by application of Gαi/o and Gαq protein blockers. Specifically, application of the Gαi/o inhibitor pertussis toxin was found to abolish choline-mediated cell proliferation and intracellular calcium transient response. These findings were corroborated by expression of a G protein binding dominant negative nAChR subunit (α7345-348A), which resulted in significantly attenuating calcium signaling and cellular proliferation in response to choline. Our study shows a new role for G protein signaling in the mechanism of α7 nAChR-associated breast cancer growth.

The human acetylcholinesterase C-terminal T30 peptide activates neuronal growth through alpha 7 nicotinic acetylcholine receptors and the mTOR pathway.

Acetylcholinesterase (AChE) is a highly conserved enzyme responsible for the regulation of acetylcholine signaling within the brain and periphery. AChE has also been shown to participate in non-enzymatic activity and contribute to cellular development and aging. In particular, enzymatic cleavage of the synaptic AChE isoform, AChE-T, is shown to generate a bioactive T30 peptide that binds to the ⍺7 nicotinic acetylcholine receptor (nAChR) at synapses. Here, we explore intracellular mechanisms of T30 signaling within the human cholinergic neural cell line SH-SY5Y using high performance liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry (ESI-MS/MS). Proteomic analysis of cells exposed to (100 nM) T30 for 3-days reveals significant changes within proteins important for cell growth. Specifically, bioinformatic analysis identifies proteins that converge onto the mammalian target of rapamycin (mTOR) pathway signaling. Functional experiments confirm that T30 regulates neural cell growth via mTOR signaling and ⍺7 nAChR activation. T30 was found promote mTORC1 pro-growth signaling through an increase in phosphorylated elF4E and S6K1, and a decrease in the autophagy LC3B-II protein. These findings are corroborated in hippocampal neurons and show that T30 promotes dendritic arborization. Taken together, our findings define mTOR as a novel pathway activated by T30 interaction with the nAChR and suggest a role for this process in human disease.

A Novel Bioactive Peptide, T14, Selectively Activates mTORC1 Signalling: Therapeutic Implications for Neurodegeneration and Other Rapamycin-Sensitive Applications.

T14 modulates calcium influx via the α-7 nicotinic acetylcholine receptor to regulate cell growth. Inappropriate triggering of this process has been implicated in Alzheimer's disease (AD) and cancer, whereas T14 blockade has proven therapeutic potential in in vitro, ex vivo and in vivo models of these pathologies. Mammalian target of rapamycin complex 1 (mTORC1) is critical for growth, however its hyperactivation is implicated in AD and cancer. T14 is a product of the longer 30mer-T30. Recent work shows that T30 drives neurite growth in the human SH-SY5Y cell line via the mTOR pathway. Here, we demonstrate that T30 induces an increase in mTORC1 in PC12 cells, and ex vivo rat brain slices containing substantia nigra, but not mTORC2. The increase in mTORC1 by T30 in PC12 cells is attenuated by its blocker, NBP14. Moreover, in post-mortem human midbrain, T14 levels correlate significantly with mTORC1. Silencing mTORC1 reverses the effects of T30 on PC12 cells measured via AChE release in undifferentiated PC12 cells, whilst silencing mTORC2 does not. This suggests that T14 acts selectively via mTORC1. T14 blockade offers a preferable alternative to currently available blockers of mTOR as it would enable selective blockade of mTORC1, thereby reducing side effects associated with generalised mTOR blockade.

Nicotinic receptor components of amyloid beta 42 proteome regulation in human neural cells.

Alzheimer's disease (AD) is associated with chronic neurodegeneration often accompanied by elevated levels of the neurotoxic peptide amyloid-beta 1-42 (Aß42) in the brain. Studies show that extracellular Aß42 binds to various cell surface receptors including the human α7 nicotinic acetylcholine receptor (nAChR) and activates pathways of neurotoxicity leading to cell death. The α7 nAChR is thus considered a promising drug target for therapy against neurodegenerative disease such as AD. In this study, we use mass spectrometry-based label-free precursor ion quantification to identify proteins and pathways that are changed by a 72-hour treatment with Aß42 or Aß42 in the presence of the α7 nAChR blocker, α-bungarotoxin (Bgtx) in the human neuroblastoma SH-SY5Y cell line. Bioinformatic gene ontology enrichment analysis was used to identify and characterize proteins and pathways altered by Aß42 presentation. The results support evidence on the involvement of mitochondrial proteins in Aß42 responses and define potential mechanisms of α7 nAChR mediated amyloid toxicity. These findings can inform pharmacological strategies for drug design and treatment against amyloid disease.

Mitochondrial Disruption by Amyloid Beta 42 Identified by Proteomics and Pathway Mapping.

Alzheimer's disease (AD) is marked by chronic neurodegeneration associated with the occurrence of plaques containing amyloid ß (Aß) proteins in various parts of the human brain. An increase in several Aß fragments is well documented in patients with AD and anti-amyloid targeting is an emerging area of therapy. Soluble Aß can bind to various cell surface and intracellular molecules with the pathogenic Aß42 fragment leading to neurotoxicity. Here we examined the effect of Aß42 on network adaptations in the proteome of nerve growth factor (NGF) differentiated PC12 cells using liquid-chromatography electrospray ionization mass spectrometry (LC-ESI MS/MS) proteomics. Whole-cell peptide mass fingerprinting was coupled to bioinformatic gene set enrichment analysis (GSEA) in order to identify differentially represented proteins and related gene ontology (GO) pathways within Aß42 treated cells. Our results underscore a role for Aß42 in disrupting proteome responses for signaling, bioenergetics, and morphology in mitochondria. These findings highlight the specific components of the mitochondrial response during Aß42 neurotoxicity and suggest several new biomarkers for detection and surveillance of amyloid disease.

Nicotinic receptor targeting in physiological and environmental vulnerability: A whole of biosphere perspective.

We propose a biosphere model of convergent interactions between nicotine and neonicotinoids (neonics) within a related framework of nicotinic receptor targeting agents (NrTA) across the globe. We explore how rising global trends in the use nicotine as well as neonics impacts vulnerability, within and across species, and posit that evolutionary conservation at the nicotinic acetylcholine receptor (nAChR) provides an operational strategy map for pathogens and disease. Furthermore, we examine the effects of NrTA exposure on balance within extant and developing ecological niches, food chains, and human societies. We advocate for a global strategy for biomonitoring across agriculture, wildlife, and human centers. Such a strategy would relate emergent pathogenic and infectious diseases, amongst others, along a tractable biological stress pathway. This new framework aims to better prepare society in the face of emergent pandemics through 1. identifying primary chemical drivers that can impact emergent diseases; 2. outlining data-driven strategy options for health and environmental policy decision makers.

Does COVID19 Infect the Brain? If So, Smokers Might Be at a Higher Risk.

COVID19 is a devastating global pandemic with epicenters in China, Italy, Spain, and now the United States. While the majority of infected cases appear mild, in some cases, individuals present serious cardiorespiratory complications with possible long-term lung damage. Infected individuals report a range of symptoms from headaches to shortness of breath to taste and smell loss. To that end, less is known about how the virus may impact different organ systems. The SARS-CoV2 virus, which is responsible for COVID19, is highly similar to SARS-CoV. Both viruses have evolved an ability to enter host cells through direct interaction with the angiotensin converting enzyme (ACE) 2 protein at the surface of many cells. Published findings indicate that SARS-CoV can enter the human nervous system with evidence from both postmortem brains and detection in cerebrospinal fluid of infected individuals. Here, we consider the ability of SARS-CoV2 to enter and infect the human nervous system based on the strong expression of the ACE2 target throughout the brain. Moreover, we predict that nicotine exposure through various kinds of smoking (cigarettes, electronic cigarettes, or vape) can increase the risk for COVID19 neuroinfection based on known functional interactions between the nicotinic receptor and ACE2. We advocate for higher surveillance and analysis of neurocomplications in infected cases. SIGNIFICANCE STATEMENT: The COVID19 epidemic has spurred a global public health crisis. While many of the cases requiring hospitalization and intensive medical care center on cardiorespiratory treatment, a growing number of cases present neurological symptoms. Viral entry into the brain now appears a strong possibility with deleterious consequences and an urgent need for addressing.

Is nicotine exposure linked to cardiopulmonary vulnerability to COVID-19 in the general population?

The recent emergence of COVID-19 has resulted in a worldwide crisis, with large populations locked down and transportation links severed. While approximately 80% of infected individuals have minimal symptoms, around 15-20% need to be hospitalized, greatly stressing global healthcare systems. As of March 10, the death rate appears to be about 3.4%, although this number is highly stratified among different populations. Here, we focus on those individuals who have been exposed to nicotine prior to their exposure to the virus. We predict that these individuals are 'primed' to be at higher risk because nicotine can directly impact the putative receptor for the virus (ACE2) and lead to deleterious signaling in lung epithelial cells.

Association of a Functional Polymorphism in the CHRFAM7A Gene with Inflammatory Response Mediators and Neuropathic Pain after Spinal Cord Injury.

The alpha 7 nicotinic acetylcholine receptor, α7 nAChR, plays a central role in regulating inflammatory responses. Previous studies showed that pharmacological inhibitors of α7nAChR have a pro-inflammatory effect, increasing the circulating levels of cytokines such as tumor necrosis factor alpha (TNFα). This study focused on how genetic polymorphisms of the partially duplicated α7nAChR gene (CHRFAM7A), which is highly expressed in peripheral blood cells, contribute to functional outcome after spinal cord injury (SCI). In a cohort of 27 SCI patients and 25 emergency room consented controls (% F/M: 15/85, 24/76; mean ± SE age: 35 ± 1.38 and 35 ± 2.0 respectively), a panel of circulating cytokines, noradrenergic metabolite (normetanephrine [NMN]) levels, and clinical data were available within the first 7 days post-injury (DPI) up to 90 DPI, and were investigated in the acute/subacute (DPI 1-21) and intermediate (DPI 22-90) temporal periods. Cytokine and NMN plasma levels on different DPI were analyzed as a function of CHRFAM7A genotype. TNFα levels, as a representative of some elevated inflammatory mediators, were nearly threefold higher in individuals carrying the del-2bp variant of the CHRFAM7A gene compared with that in the no-deletion genotype (p = 0.001 analysis of variance [ANOVA]) 3 weeks DPI, and twofold higher than genotype-matched acute/subacute non-SCI injury controls within 7 days DPI. In contrast, NMN levels were initially unchanged, although after 3 weeks, NMN levels were significantly decreased in SCI individuals carrying the del-2bp variant compared with non-carriers (p = 0.011 ANOVA). Numerical pain scores over this same period post-injury were significantly elevated in SCI patients carrying the del-2bp variant relative to non-carriers (p = 0.001 ANOVA). Taken together, these data reveal that pro-inflammatory responses associated with CHRFAM7A gene variation may also be associated with differences in pain experience in patients following SCI, at least during the intermediate phase.

Alpha 7 nicotinic receptors attenuate neurite development through calcium activation of calpain at the growth cone.

The α7 nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that plays an important role in cellular calcium signaling contributing to synaptic development and plasticity, and is a key drug target for the treatment of neurodegenerative conditions such as Alzheimer's disease. Here we show that α7 nAChR mediated calcium signals in differentiating PC12 cells activate the proteolytic enzyme calpain leading to spectrin breakdown, microtubule retraction, and attenuation in neurite growth. Imaging in growth cones confirms that α7 activation decreases EB3 comet motility in a calcium dependent manner as demonstrated by the ability of α7 nAChR, ryanodine, or IP3 receptor antagonists to block the effect of α7 nAChR on growth. α7 nAChR mediated EB3 comet motility, spectrin breakdown, and neurite growth was also inhibited by the addition of the selective calpain blocker calpeptin and attenuated by the expression of an α7 subunit unable to bind Gαq and activate calcium store release. The findings indicate that α7 nAChRs regulate cytoskeletal dynamics through local calcium signals for calpain protease activity.

Ionotropic and Metabotropic Mechanisms of Allosteric Modulation of α7 Nicotinic Receptor Intracellular Calcium.

The pharmacological targeting of the α7 nicotinic acetylcholine receptor (α7) is a promising strategy in the development of new drugs for neurologic diseases. Because α7 receptors regulate cellular calcium, we investigated how the prototypical type II-positive allosteric modulator PNU120596 affects α7-mediated calcium signaling. Live imaging experiments show that PNU120596 augments ryanodine receptor-driven calcium-induced calcium release (CICR), inositol-induced calcium release (IICR), and phospholipase C activation by the α7 receptor. Both influx of calcium through the α7 nicotinic acetylcholine receptor (nAChR) channel as well as the binding of intracellular G proteins were involved in the effect of PNU120596 on intracellular calcium. This is evidenced by the findings that chelation of extracellular calcium, expression of α7D44A or α7345-348A mutant subunits, or blockade of calcium store release compromised the ability of PNU120596 to increase intracellular calcium transients generated by α7 ligand activation. Spatiotemporal stochastic modeling of calcium transient responses corroborates these results and indicates that α7 receptor activation enables calcium microdomains locally and to lesser extent in the distant cytosol. From the model, allosteric modulation of the receptor activates CICR locally via ryanodine receptors and augments IICR through enhanced calcium influx due to prolonged α7 nAChR opening. These findings provide a new mechanistic framework for understanding the effect of α7 receptor allosteric modulation on both local and global calcium dynamics.

Alpha 7 nicotinic receptor coupling to heterotrimeric G proteins modulates RhoA activation, cytoskeletal motility, and structural growth.

Nicotinic acetylcholine receptors (nAChRs) modulate the growth and structure of neurons throughout the nervous system. Ligand stimulation of the α7 nAChR has been shown to regulate the large heterotrimeric GTP-binding protein (G protein) signaling in various types of cells. Here, we demonstrate a role for α7 nAChR/G protein interaction in the activation of the small (monomeric) RhoA GTPase leading to cytoskeletal changes during neurite growth. Treatment of PC12 cells with the α7 nAChR agonist choline or PNU-282987 was associated with an increase in RhoA activity and an inhibition in neurite growth. Specifically, choline treatment was found to attenuate the velocity of microtubule growth at the growth cone and decrease the rate of actin polymerization throughout the cell. The effects of α7 nAChR activation were abolished by expression of a dominant negative α7 nAChR (α7345-348A ) deficient in G protein coupling. Proteomic analysis of immunoprecipitated α7 nAChR complexes from differentiating PC12 cells and synaptic fractions of the developing mouse hippocampus revealed the existence of Rho GTPase-regulating guanine nucleotide exchange factors within α7 nAChR interactomes. These findings underscore the role of α7 nAChR/G protein in cytoskeletal regulation during neurite growth. This image depicts the hypothesized interaction of the traditionally ionotropic α7 nicotinic acetylcholine receptor (α7 nAChR) and its ability to interact and signal through both large and small G proteins, leading to the regulation of cytoskeletal growth. Using differentiated PC12 cells, and the specific agonist choline, it was shown that α7 nAChR/G protein interactions mediate both short- and long-term neurite growth dynamics through increased RhoA activation. Activation of RhoA was shown to decrease actin polymerization, and lead to an overall decrease in neurite growth via regulation of the microtubule network. Cover Image for this issue: doi: 10.1111/jnc.13330.

Effects of Menthol on Nicotine Pharmacokinetic, Pharmacology and Dependence in Mice.

Although menthol, a common flavoring additive to cigarettes, has been found to impact the addictive properties of nicotine cigarettes in smokers little is known about its pharmacological and molecular actions in the brain. Studies were undertaken to examine whether the systemic administration of menthol would modulate nicotine pharmacokinetics, acute pharmacological effects (antinociception and hypothermia) and withdrawal in male ICR mice. In addition, we examined changes in the brain levels of nicotinic receptors of rodents exposed to nicotine and menthol. Administration of i.p. menthol significantly decreased nicotine's clearance (2-fold decrease) and increased its AUC compared to i.p. vehicle treatment. In addition, menthol pretreatment prolonged the duration of nicotine-induced antinociception and hypothermia (2.5 mg/kg, s.c.) for periods up to 180 min post-nicotine administration. Repeated administration of menthol with nicotine increased the intensity of mecamylamine-precipitated withdrawal signs in mice exposed chronically to nicotine. The potentiation of withdrawal intensity by menthol was accompanied by a significant increase in nicotine plasma levels in these mice. Western blot analyses of α4 and ß2 nAChR subunit expression suggests that chronic menthol impacts the levels and distribution of these nicotinic subunits in various brain regions. In particular, co-administration of menthol and nicotine appears to promote significant increase in ß2 and α4 nAChR subunit expression in the hippocampus, prefrontal cortex and striatum of mice. Surprisingly, chronic injections of menthol alone to mice caused an upregulation of ß2 and α4 nAChR subunit levels in these brain regions. Because the addition of menthol to tobacco products has been suggested to augment their addictive potential, the current findings reveal several new pharmacological molecular adaptations that may contribute to its unique addictive profile.

Identification and Characterization of a G Protein-binding Cluster in α7 Nicotinic Acetylcholine Receptors.

α7 nicotinic acetylcholine receptors (nAChRs) play an important role in synaptic transmission and inflammation. In response to ligands, this receptor channel opens to conduct cations into the cell but desensitizes rapidly. In recent studies we show that α7 nAChRs bind signaling proteins such as heterotrimeric GTP-binding proteins (G proteins). Here, we demonstrate that direct coupling of α7 nAChRs to G proteins enables a downstream calcium signaling response that can persist beyond the expected time course of channel activation. This process depends on a G protein-binding cluster (GPBC) in the M3-M4 loop of the receptor. A mutation of the GPBC in the α7 nAChR (α7345-348A) abolishes interaction with Gαq as well as Gßγ while having no effect on receptor synthesis, cell-surface trafficking, or α-bungarotoxin binding. Expression of α7345-348A, however, did significantly attenuate the α7 nAChR-induced Gαq calcium signaling response as evidenced by a decrease in PLC-ß activation and IP3R-mediated calcium store release in the presence of the α7 selective agonist choline. Taken together, the data provides new evidence for the existence of a GPBC in nAChRs serving to promote intracellular signaling.

Microtubule dynamics at the growth cone are mediated by α7 nicotinic receptor activation of a Gαq and IP3 receptor pathway.

The α7 nicotinic receptor (α7) plays an important role in neuronal growth and structural plasticity in the developing brain. We have recently characterized a G-protein-signaling pathway regulated by α7 that directs the growth of neurites in developing neural cells. Now we show that choline activation of α7 promotes a rise in intracellular calcium from local ER stores via Gαq signaling, leading to IP3 receptor (IP3R) activation at the growth cone of differentiating PC12 cells. A mutant α7 significantly attenuated in calcium conductance (D44A; P<0.001) was found to be unable to promote IP3R signaling and calcium store release. In addition, calcium elevation via α7 correlates with a significant attenuation in the rate of microtubule invasion of the growth cone (P<0.001). This process was also attenuated in the D44A mutant and blocked by an inhibitor of the IP3R, suggesting that calcium flow through the α7 channel and activation of the Gαq pathway are necessary for growth. Taken together, the findings reveal an inhibitory mechanism of α7 on cytoskeletal growth via the intracellular calcium activity of the receptor channel and the Gαq signaling pathway at the growth cone.-Nordman, J. C., Kabbani, N. Microtubule dynamics at the growth cone are mediated by α7 nicotinic receptor activation of a Gαq and IP3 receptor pathway.

The α4 nicotinic receptor promotes CD4+ T-cell proliferation and a helper T-cell immune response.

Smoking is a common addiction and a leading cause of disease. Chronic nicotine exposure is known to activate nicotinic acetylcholine receptors (nAChRs) in immune cells. We demonstrate a novel role for α4 nAChRs in the effect of nicotine on T-cell proliferation and immunity. Using cell-based sorting and proteomic analysis we define an α4 nAChR expressing helper T-cell population (α4(+)CD3(+)CD4(+)) and show that this group of cells is responsive to sustained nicotine exposure. In the circulation, spleen, bone marrow, and thymus, we find that nicotine promotes an increase in CD3(+)CD4(+) cells via its activation of the α4 nAChR and regulation of G protein subunit o, G protein regulated-inducer of neurite outgrowth, and CDC42 signaling within T cells. In particular, nicotine is found to promote a helper T cell 2 adaptive immunologic response within T cells that is absent in α4(-/-) mice. We thus present a new mechanism of α4 nAChR signaling and immune regulation in T cells, possibly accounting for the effect of smoking on the immune system.

Axon targeting of the alpha 7 nicotinic receptor in developing hippocampal neurons by Gprin1 regulates growth.

Cholinergic signaling plays an important role in regulating the growth and regeneration of axons in the nervous system. The α7 nicotinic receptor (α7) can drive synaptic development and plasticity in the hippocampus. Here, we show that activation of α7 significantly reduces axon growth in hippocampal neurons by coupling to G protein-regulated inducer of neurite outgrowth 1 (Gprin1), which targets it to the growth cone. Knockdown of Gprin1 expression using RNAi is found sufficient to abolish the localization and calcium signaling of α7 at the growth cone. In addition, an α7/Gprin1 interaction appears intimately linked to a Gαo, growth-associated protein 43, and CDC42 cytoskeletal regulatory pathway within the developing axon. These findings demonstrate that α7 regulates axon growth in hippocampal neurons, thereby likely contributing to synaptic formation in the developing brain.