Dr Mohammed A. Nassar

Dr Mohammed A. NassarLecturer
Department of Biomedical Science
The University of Sheffield
Western Bank
Sheffield S10 2TN
United Kingdom

Room: D07 Florey building
Telephone: +44 (0) 114 222 2392
Email: m.nassar@sheffield.ac.uk

Neuroscience


General

Brief career history

  • 2010-present: Lecturer, dept. of Biomedical Science, Sheffield University, UK.
  • 1999-2009: Senior postdoctoral Fellow, Molecular Nociception Group, Dept. of Biology, UCL, UK.
  • 1998-99: Postdoctoral Fellow (Wellcome Prize Fellowship), Wellcome Laboratory for Molecular Pharmacology, Dept. of Pharmacology, UCL, UK.
  • 1994-98: Postgraduate student (Wellcome Prize studentship), Wellcome Laboratory for Molecular Pharmacology, Dept. of Pharmacology, UCL, UK.
  • 1994: Research assistant in the Wellcome Laboratory for Molecular Pharmacology, Dept. of Pharmacology, UCL, UK.

Research interests

My research is focused on primary sensory neurons which are part of the peripheral nervous system (PNS). Sensory neurons convey sensory information from the both the internal (e.g. viscera, muscles and bones) and the external (skin) environments to the central nervous system (CNS).

Sensory neurons convey both innoxious and noxious stimuli. The latter is perceived in the CNA as pain. Inflammation and nerve injury sensitise sensory neurons which results in decreased pain thresholds. My research interest lies in investigating the molecular changes in sensory neurons that are associated with pathological pain.

This is important in order to identify potential targets for new, effective and specific analgesic drugs. My lab uses a variety of methods based on molecular biology, cellular biology and functional assays.

Professional activities

  • 2015: Postgraduate Certificate in Learning and Teaching from the University of Sheffield (Fellow of The Higher Education Academy, FHEA)

Full publications list

Research

General Aim: Elucidating the molecular mechanisms of sensitisation of sensory neurons in pain.

To achieve the general aim above, research in my lab is organised around three projects. The first project investigates the regulation of the plasma membrane pool of the channel Nav1.7. I was the first to reveal the crucial role of Nav1.7 in pain signalling (Nassar, PNAS 2004). Since then Nav1.7 has been shown to underlie three genetic pain disorders in humans; these are primary erthromyalgia, familial rectal pain and complete insensitivity to pain.

However, little is known about how the Nav1.7 surface pool is regulated to set pain thresholds and respond to changes in the environment (e.g. inflammation). Nav1.7 surface pool is determined by mechanisms controlling its transport to nerve terminals, insertion into and endocytosis from the membrane. Investigation of these processes may lead to new druggable targets for pain relief. We employ several approaches to investigate Nav1.7 trafficking, these include generation of GFP-tagged Nav1.7 channel, generation of reporter proteins containing parts of the Nav1.7 channel, super-resolution microscopy and a proteomic characterisation of proteins that interact with Nav1.7..

Figure 01

The second project evaluates the use of a VGSC channel opener or “agonist” called Veratridine and calcium imaging to provide a high throughput assay to assess the excitability of sensory neurons. We were the first to characterise Veratridine responses in cultured sensory neurons (Mohammed, Sci Rep 2017). We found that Veratridine produces distinct response-profiles in cultured sensory neurons that map to known functional neuronal subtypes.

Therefore, these response-profiles allow a simple identification of nociceptive neurons (pain sensing) and non-nociceptive neurons. Changes in the properties of these profiles reflect changes in the excitability of sensory neurons. We are currently investigating how Veratridine profiles can be used to assess the activity of two important sodium channels, Nav1.7 and Nav1.8, in sensory neurons. This may provide a biologically relevant yet high throughput assay to screen for blockers for these channels. This project involves the use of calcium imaging on cultured sensory neurons.

Figure 02

The third project aims to provide a new in vitro model of sensory neurons to replace the use of primary sensory neurons. Primary sensory neurons form rodents are the standard in vitro model used in pain research. However, the number of neurons that can be obtained from one animal is limited and is insufficient for molecular and biochemical experiments. Furthermore, sensory neurons cultures contain a variety of cell types including glia and fibroblasts, making it difficult to interpret data from biochemical studies.

Therefore, the generation of an immortal cell line from sensory neurons will lead to experiments being carried out that would not have been possible with primary cultures. Equally important, a cell line will replace the use of rodents to obtain primary cultures which will reduce the number of animals used in research. My lab has generated a DRG-derived cell line (MED17.11) that can be propagated in culture indefinitely (Doran et al, 2015). The cell line can be differentiated to express DRG markers. The project aims to improve the differentiation protocol to produce sensory neurons of an adult phenotype. This cell line may provide a new in vitro model that is useful for drug screens.

Figure 03

Teaching

Teaching experience:

  • 2015: Postgraduate Certificate in Learning and Teaching from the University of Sheffield (Fellow of The Higher Education Academy, FHEA)

Undergraduate and postgraduate taught modules

Level 1:

  • BMS109 Molecular Biology (Coordinator)

Level 2:

  • BMS235 Integrated Physiology and Pharmacology

Level 3:

  • BMS303 Molecular Physiology of Ion Channels, and Human Disease
  • BMS319/BMS6084 Pharmacological Techniques
  • BMS339 Patients as Educators Project
  • BMS369 Laboratory Research Project

Masters (MSc):

  • BMS6062 Molecular Physiology of Ion Channels and Human Disease
Opportunities

Postgraduate studentship opportunities

PhD Studentship starting October 2017

Application deadline: Friday 30 June 2017

Title: Elucidating the mechanisms of regulation of Nav1.7 channel in the plasma membrane of sensory neurons

Supervisor: Dr Mohammed Nassar

Funding: Competition funded project UK students only

Project Description

"Sensory neurons detect and transmit painful stimuli to the CNS. Inflammation and nerve injury sensitise sensory neurons which results in a decrease of pain thresholds. This can be due, at least in part, to an enhanced trafficking of voltage gated sodium channels to the plasma membrane which would result in increased excitability of sensory neurons. The VGSC subunit Nav1.7 has been shown to be crucial for pain signalling in mouse and human. Three genetic pain disorders have been mapped to Nav1.7 in humans; these are primary erthromyalgia, familial rectal pain and complete insensitivity to pain. However, little is known about how the Nav1.7 surface pool is regulated to set pain thresholds and respond to changes in the environment (e.g. inflammation). Nav1.7 surface pool is determined by mechanisms controlling its transport to nerve terminals, insertion into and endocytosis from the membrane. Investigation of these processes may lead to new druggable targets for pain relief. The aim of this project is to identify the contribution of the intracellular parts of Nav1.7 channel in regulation of its membrane expression.

Methods work plan:
We have fused the reporter protein GFP to the Nav1.7 channel. This allows us to use live imaging to track Nav1.7 localisation in sensory neurons. Nav1.7 on plasma membrane will be quantified in standard and inflammation-like conditions. Live imaging will be performed using the DeltaVision OMX super resolution system. Published reports and our preliminary research indicated that the N and C-termini of Nav1.7 may play a role in regulating its surface pool.

DNA engineering will be used to produce N and C-termini mutants of the GFP-tagged channel to assess their contribution. Once the role of N and C-termini has been assessed, proteins that interact with them will be identified by a mass spectrometry. The role of the identified proteins will be validated by knockdown approaches. Lentiviruses will be used to introduce DNA coding for recombinant proteins and knockdown microRNA into sensory neurons. The project will involve using molecular biology methods to generate fusion protein constructs, transfection of DNA into cell lines and primary DRG neurons, immunocytochemistry and Western blotting. Functional effect of transfected fusions on Nav1.7 will be assessed by calcium imaging and patch clamping.

Impact:
The proposed work will provide a better understanding of Nav1.7 regulation in sensory neurons and may provide insights into pathways relevant to pathological changes in chronic pain conditions. Moreover, results may prove relevant to other membrane proteins in DRG whose surface pool could co-regulated with Nav1.7 by same pathways to decrease pain thresholds.

References

  1. Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain. Nassar MA, Stirling LC, Forlani G, Baker MD, Matthews EA, Dickenson AH, Wood JN. Proc Natl Acad Sci U S A. 2004 Aug 24;101(34):12706-11.
  2. Veratridine produces distinct calcium response profiles in mouse Dorsal Root Ganglia neurons. Mohammed ZA, Doran C, Grundy D, Nassar MA. Sci Rep. 2017 Mar 24;7:
  3. Waxman SG, Merkies IS, Gerrits MM, Dib-Hajj SD, Lauria G, Cox JJ, Wood JN, Woods CG, Drenth JP, Faber CG. Sodium channel genes in pain-related disorders: phenotype-genotype associations and recommendations for clinical use. Lancet Neurol. 2014 Nov;13(11):1152-60.

The role of the intracellular parts of the Nav1.7 channel in its membrane expression

Sensory neurons detect and transmit painful stimuli to the CNS. Inflammation and nerve injury sensitise sensory neurons in vivo which results in a decrease of pain thresholds. This can be due, at least in part, to an enhanced trafficking of voltage gated sodium channels to the plasma membrane which would result in increased excitability of sensory neurons. The VGSC subunit Nav1.7 has been shown to be crucial for pain signaling in mouse and human.

Three genetic pain disorders have been mapped to Nav1.7 in humans; these are primary erthromyalgia, familial rectal pain and complete insensitivity to pain. However and despite its importance in pain signaling, little is known about Nav1.7 transport to nerve terminals, insertion into the membrane and endocytosis from the membrane. Investigation of these processes may lead to new druggable targets for pain relief. Drugs will be directed to inhibit insertion in membrane and increase endocytosis form membrane. Therefore, the aim of this project is to identify the contribution of the intracellular parts of Nav1.7 channel to its membrane expression.

The project will involve using molecular biology methods to generate fusion protein constructs, transfection of DNA into cell lines and primary DRG neurons, immunocytochemistry and Western blotting. Functional effect of transfected fusions on Nav1.7 will be assessed by calcium imaging and patch clamping.

For further details about this project and how to apply, see our PhD Opportunities page:

PhD Opportunities

Selected publications

Journal articles