Nitric Oxide (NO)-mediated Toxicity in Neurodegeneration

Programme Leader: Joern Steinert

Summary of Research Interests

 

NO signalling has been implicated in several neurodegenerative diseases such as Alzheimer’s (AD), Huntington’s (HD) and Parkinson’s disease (PD), but its exact contribution to neuronal death remains elusive due to the great complexity of downstream nitrergic activities. Elevated Nitric Oxide (NO) levels, as seen in many diseased states, can lead to the formation of cytotoxic peroxynitrite (ONOO-) which in turn can directly modulate a wide range of protein functions via nitration of tyrosine residues (3-Nitrotyrosination/Tyr-NO). Furthermore, at higher concentrations, toxic NO signalling can alter the functioning of proteins in a process known as S-nitrosylation (S-NO). Both signalling pathways have been widely reported, rendering it of similar functional importance to phosphorylation and ubiquitination processes. The key aim of this project is the identification of substrates subjected to this regulation by nitrergic signalling pathways. To date, little is known as to what extent NO-mediated post-translational modifications contribute to or exacerbate disease development and which key modifications cause dysfunctional neuronal signalling. The identification of novel nitrergic signalling pathways in neurodegeneration and correlation with early functional changes before disease onset will allow a better understanding of cytotoxic NO signalling related to disease progression.

 

Our group investigates cell signalling pathways involved in Nitric Oxide (NO)-induced neurotoxicity and neuroinflammation with the aim to identify putative targets for therapeutic intervention(s).

 

Key Objectives are

i) to investigate the contribution of cytotoxic NO/ONOO- to synaptic communication and transmitter release using a multi-systemic model approach (mouse, cell culture and Drosophila).

ii) to identify nitrergic mechanisms by which synaptic target proteins are regulated leading to cytotoxicity.

iii) to investigate post-translational control by NO associated with neurodegenerative signalling.

Cytotoxic Nitric Oxide Signalling in Degenerative Conditions

Left, Synaptic transmission is controlled by presynaptic proteins which are regulated by signalling molecules such as Syntaxin or MUNC complex. nNOS and cellular prion (PrPC) are co-localized within caveolin-enriched membrane rafts and PrPC negatively regulates NMDAR activity.

Right, Hyperactivity of NMDAR leading to enhanced levels of NO to induce a cytotoxic environment with activated nitrosylation and nitrotyrosination signalling. Enhanced extra-synaptic NMDAR signalling leads to caspase 6 activation and downstream pro-apoptotic pathways. Misfolded prion (PrPSC) disrupts nNOS regulation (via caveolin interaction), allows excitotoxicity by enhancing NMDAR currents and activates iNOS thereby providing additional cytotoxic levels of NO. Elevated NO levels interact with synaptic proteins (Syntaxin, MUNC complex) leading to compromised vesicle release and synaptic function or modulate calcium homeostasis by SERCA nitrotyrosination and RyR nitrosylation. miRNA-939 blocks iNOS translation thereby providing an anti-inflammatory signalling pathway and reducing NO levels.

Bottom, Concentration dependency of pro-survival and pro-death signalling. Lower NO concentrations lead to low levels of nitrosylation/nitrotyrosination and are neuroprotective, whereas higher levels of NO lead to neurotoxic signalling induced by abnormal post-translational modifications of proteins as seen in many neurodegenerative conditions.

Background

NO signalling pathways

NO is a highly diffusible molecule with a short half-life and is involved in many physiological and pathological processes. In the brain, it modulates neuronal excitability, neurotransmitter release, long-term potentiation and neurovascular coupling. Nanomolar concentrations of NO are enzymatically generated by endothelial (e)NOS and neuronal (n)NOS, whereas the inducible (i)NOS can produce micromolar levels in response to pro-inflammatory stimuli.

NO potentially acts via multiple downstream signalling mechanisms, depending on the concentration, with low levels being neuroprotective and mediate physiological signalling (e.g. neurotransmission or vasodilatation), whereas higher concentrations mediate immune/inflammatory actions and are neurotoxic. After its generation by one of the three isoenzymes (nNOS, eNOS, iNOS) NO is preserved in its molecular structure by a group of spontaneously decomposing endogenous NO donors. The chemistry of NO involves inter-related redox forms (NO., NO+, NO-) with different chemical reactivities towards distinct target groups, thus explaining the great variety of biological actions.

Approaches to allow Investigation of Nitrergic Effects at the Synapse

A, Model of a synapse illustrating pre- and postsynaptic components involved in synaptic transmission.

B, ICC of a glutamatergic synapse showing the active zones (Bruchpilot, Brp) of a Drosophila NMJ.

C, Electrophysiological recordings of miniature excitatory junctional currents (mEJC) and Paired Pulse Ratios at different frequencies and two calcium concentrations. This shows facilitation or depression depending on calcium levels and inter spike frequencies. Different frequencies are thought to recruit vesicles from different presynaptic pools, ie the reserve pool (RP) or readily releasable pool (RRP).

D, EM images of a synapse illustrating the active zone (T-Bar, AZ) with synaptic vesicles (SV) and sub-synaptic reticulum (SSR).

Relevant Publications

  1. Robinson SW, Bourgognon J, Breda C, Campesan S, Dinsdale D, Morone N, Mistry R, Smith TM, Guerra-Martin M, Challiss RAJ, Giorgini F & Steinert JR. Nitric oxide-mediated post-translational modifications control neurotransmitter release by modulating complexin farnesylation and enhancing its clamping ability. Plos Biol, in revision.
  2. Robinson SW, Gutierrez-Olmo M, Martin M, Smith TM, Morone N & Steinert JR (2017). Endogenous nitric oxide synthase activity regulates synaptic transmitter release. Opera Med Physiol, 3 (2): 31-38.
  3. Bradley SA, Steinert JR (2016). Nitric oxide mediated post-translational modifications: impacts at the synapse. Oxidative Medicine and Cellular Longevity 2016;2016:5681036.
  4. Bradley SA, Steinert JR (2015). Characterisation and comparison of temporal release profiles of nitric oxide generating donors. J Neurosci Methods 245:116-124
  5. Peretti D, Bastide A, Radford H, Verity N, Molloy C, Martin MG, Moreno JA, Steinert JR, Smith T, Dinsdale D, Willis AE, Mallucci GR (2015). RBM3 mediates structural plasticity and protective effects of cooling in neurodegeneration. Nature 518(7538):236-9
  6. Robinson SW, Nugent ML, Dinsdale D & Steinert JR (2014). Prion protein facilitates synaptic vesicle release by enhancing release probability. Hum Mol Genet 23(17):4581-96.
  7. Tozer AJB, Forsythe ID & Steinert JR (2012). Nitric oxide signalling augments neuronal voltage-gated L-type (CaV1) and P/Q-type (CaV2.1) in the mouse Medial Nucleus of the Trapezoid Body. PLoS One 7(2):e32256.
  8. Steinert JR, Campesan S, Richards P, Kyriacou CP, Forsythe ID & Giorgini F (2012). Rab11 modulates synaptic dysfunction and behavioural deficits in a Drosophila model of Huntington’s disease. Hum Mol Genet 1(21): 2912-22
  9. Steinert JR, Robinson SW, Tong H, Haustein MD, Kopp-Scheinpflug C & Forsythe ID (2011). Nitric oxide is an activity-dependent regulator of target neuron intrinsic excitability. Neuron 71, 291-305.
  10. Steinert JR, Chernova T & Forsythe ID (2010). Nitric Oxide in brain function, dysfunction and dementia. Neuroscientist 16, 435-452.
  11. Steinert JR, Chernova T & Forsythe ID (2009). Nitric Oxide can alter brain function. Adv Clin Neurosci Rehabil 9, 10-12.
  12. Steinert JR, Kopp-Scheinpflug C, Baker C, Challiss RAJ, Mistry R, Haustein MD, Griffin SJ, Tong H, Graham BP & Forsythe ID (2008). Nitric oxide is a volume transmitter regulating neuronal excitability in the auditory pathway. Neuron 60, 642-656 *
  13. Steinert JR, Kuromi H, Hellwig A, Knirr M, Wyatt AW, Kidokoro Y, Schuster CM (2006). Experience-dependent formation and recruitment of large vesicles from reserve pool. Neuron 50, 723-733

*       Comment in Nature Research Highlight: Neurotransmission: Gas for global tuning (Nature Reviews Neuroscience 10, 3 (January 2009) | doi:10.1038/nrn2562)

 

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Collaborators

Dr Flaviano Giorgini (University of Leicester)

Dr Ezio Rosato (University of Leicester)

Prof Ian Forsythe (University of Leicester)

Prof Linda Partridge (Max Planck Institute for Biology of Ageing)

Dr Guy Bewick (The University of Aberdeen)

 

 

Research Techniques

Electrophysiology

Whole-cell voltage and current clamp recordings in acute brain slices and cultured neurons (Double Patchstar System, Scientifica)

Drosophila CNS patch clamp recordings
Intracellular recordings at Drosophila larvae NMJs (DCC, dSEVC, TEVC)

 

Imaging

Calcium imaging using CCD
FM dye imaging
Multiphoton confocal microscopy (LSM 510 with Mai Tai Deep See)
Nitric oxide detection using fluorescent dyes/electrodes (WPI)

Biochemical assays

Immunocytochemistry, Western Blotting, PCR

Membership of Learned Societies

Physiological Society, UK

Society for Neuroscience, USA

 

Editorial Membership

Oxidative Medicine and Cellular Longevity

Cogent Biology

 

 

 

 

Steinert Group