international centre for neurotherapeutics

The new ICNT facility at Dublin City University was refurbished during 2004, with an occupancy date of 15th November. It comprises 500 m2 of laboratory and office space to house a team of up to 25 investigators. There are 12 laboratories with a range of support units, and a bridge linking the Research & Engineering and Science Buildings at the second floor level.

Molecular Neurobiology Laboratory

This suite – the backbone of the ICNT facility – is a large, open-plan area designed to encourage communication and technology transfer between researchers.

Projects to be carried out therein include:

1. In vivo imaging of the remodeling of motor nerve terminals induced by SNARE-cleaving botulinum neurotoxins (BoNTs) to reveal its effects when used therapeutically. High-sensitivity computer-controlled video microscopy is being used to perform repeated in vivo imaging of single identified end-plates in sternomastoid muscle of living mice, before and after treatment with different serotypes of BoNT. This unique strategy for investigating dynamics of the remodeling of motor nerve endings involves in vivo imaging of exo- and endo-cytosis using special dyes to reveal alterations in both the morphology of individual terminals and loci of synaptic vesicle turnover. Such investigations should not only establish important therapeutic properties of BoNT (e.g. duration of action, extent of nerve remodelling, effects of repeated injections), but could also uncover mechanistic insights into nerve recovery processes and their dysfunction in motor neuron diseases.
2. Investigation into the signalling underlying plasticity of nerve terminals and eventual retraction of those neurites, during remodelling of synapses following blockade of transmitter release by BoNT. The effects of inhibiting exocytosis with the toxins on cultured motor, autonomic and sensory neurones will be monitored at the level of RNA expression. Candidate genes are to be isolated using RNA array technology, comparing mRNA transcripts in control neurons and those treated with toxin. Altered expression of any gene will be verified in tissue sections of prenatal rat or mouse dorsal spinal cord, motor neuron-muscle co-cultures, and on sensory ganglia.
3. Elucidation of the molecular basis for the toxin’s cholinergic specificity and extended duration of action in the treatment of hyper-active autonomic nerves. Efforts are underway to solve the fascinating question of how BoNT type A alleviates symptoms of over-active cholinergic innervation of bladder, secretory glands and gastro-intestinal tract for such long periods. Modern biochemical, physiological and pharmacological techniques will be utilised to delineate cell biological and metabolic features that are responsible for its unique prolonged therapeutic effect in these tissues. Cholinergic pheno-typing of cultured post-ganglionic neurons will be exploited to reveal insight into their sensitivity to toxin serotypes, with the goal of engineering a variant of the protein displaying selectivity for autonomic rather than motor neurons.
4. Identification of the trafficking route(s) in neurons commandeered by BoNT for acceptor-mediated internalisation. High-resolution microscopy, coupled with immuno-cytochemical amplification to increase sensitivity, are being employed to monitor toxin uptake via endocytotic pathways. For this purpose, use of cells expressing mutated proteins (e.g. clathrin, adaptins, caveolin or Rabs), which are components of various endocytotic processes, should unveil the different trafficking mechanisms involved. Such new information, together with associated studies on the toxins’ membrane acceptors, will prove invaluable in targeting toxin variants to particular target cells for therapeutic purposes.
5. Deciphering the mechanisms by which BoNT can relieve chronic pain. Using cultured sensory neurons from rat trigeminal nucleus, the toxin’s ability to inhibit the release of transmitters that mediate pain is being assessed. Experimentation will focus on identifying populations of nociceptive nerves susceptible to BoNT, with the aim of pinpointing relevant pain pathways. The resultant findings should allow future targeting of toxins “tailored” to more selectively and effectively control the sensation of chronic pain.
6. Targeted delivery of drugs and genes to cholinergic neurons with a non-toxic mutant of BoNT. As distinct toxin domains mediate acceptor binding, internalisation and inhibitory protease activity, mutagenesis of its enzymic site has yielded a BoNT innocuous mutant (BoTIM), that retained all its other functional activities. Methodologies are to be refined for using recombinant BoTIM as a cholinergic-specific vehicle for targeted delivery of chemotherapeutics (e.g. to reverse neuronal deficiencies, defects or toxic insults) or therapeutic genes (for rescuing function in poisoned nerve terminals or correcting certain genetic disorders). Direct conjugation of drugs to BoTIM is being achieved through non-essential free thiols or via a cassette engineered into the vehicle. Other potential therapies entail packing the relevant gene into a suitable viral vector, ablating its tropism and re-targeting to cholinergic terminals by attachment to BoTIM, at the DNA level.

Molecular Biology Laboratory

Ongoing applications:

1. Development of 2nd and 3rd generations of BOTOX engineered to act on different neuron types for therapeutic application.
2. Design of delivery vehicles for drugs based on innocuous derivatives of botulinum and tetanus toxins.
3. Production of novel targeting agents for drug-delivery to the peripheral and central nervous systems.
4. Elucidation of the molecular basis for the exocytotic release of neurotransmitters.

Gene-Targeting Laboratory

This is a self-contained lab designed for the large-scale production of innocuous recombinant viral vectors. These will be used in the targeted delivery of therapeutic genes to the spinal cord in order to counteract the effect of toxins acting on the neuromuscular junction and to explore therapies for diseases of motor neurones.

Projects underway:

1. Generation of viral vectors for toxin therapy - these are being used to protect or rescue affected neurons from the deleterious effects of botulinum toxin.
2. Targeting of Adeno-associated and Lenti-viruses to spinal motor neurons to deliver therapeutic genes to ameliorate the effects of diseases like ALS, etc.
3. Creation of cell lines expressing different K+ channel gene combinations – the resultant oligomeric subtypes will provide novel and authentic targets for high-throughput robotic screening of drugs that can specifically interact with these channel variants.

Neuronal Systems Laboratory

This self-contained laboratory is built to high specifications for safe handling of risk group 2 biological agents and culturing of central and peripheral neurons.

A class II containment cabinet provides hazard protection to users and the valuable research materials. Dedicated equipment and facilities are provided for the manipulation and storage of dangerous substances. Stimulators, tissue baths, transducers, amplifiers and software-based trace recorders are used to monitor toxin-induced neuromuscular paralysis.

1. Primary culture of neurons derived from various tissues for comparative studies of the effects of botulinum toxin on motor, autonomic and sensory systems.
2. Quantification of the exocytosis of neurotransmitters and hormones by ELISA, radio-immuno/fluorescent assays, and analysis of the inhibition by Clostridial toxins.
3. Characterisation of the neuro-inhibitory actions of wild-type and engineered toxins on synaptic transmission, with a view to optimising forms for extending their clinical uses.

Neurotherapeutics Laboratory

This suite is specialised for research focussed on voltage-activated K+ channels in human brain, which mediate the propagation of nerve impulses and, indirectly, modulate synaptic transmission by regulating the excitability of nerve terminals.

These labs are well equipped for determining the subunit and oligomeric structures of native and recombinant K+ channels in neurones from both normal individuals and those with inherited disorders (e.g. Episodic Ataxia I), in relation to their functional characteristics.

Ongoing projects are:

1. Recombinant ‘recreation’ of the oligomeric subtypes of voltage-activated K+ channels isolated from human brain for biophysical and functional characterisation. Tetrameric K+ channels containing the requisite combination/ stoichiometries of a subunits are generated by tandem linkage of their genes, followed by expression of the proteins on the surface of mammalian cells in functional form. Structural features are related to the channels’ functional properties, assessed by ligand-binding, ion-flux and neurophysiological recordings in the Robotics and Electrophysiological facilities within ICNT.
2. Discovery and refinement of drugs to control neuronal excitability which act on novel and authentic targets – K+ channel variants. The exclusive availability here of oligomeric subtypes of voltage-activated (Kv1) K+ channels, together with detailed structural data on selective inhibitors – dendrotoxin polypeptides – are allowing us to screen numerous K+ channel binding drugs, already available, for selective action towards individual variants. This will be performed with the aid of an automatic robotic system enabling high-throughput screening of drugs from existing banks. ‘Hits’ are to be confirmed by detailed analysis of their inhibitory specificity, using patch-clamp recording and other functional assays. A similar approach is to be adopted to find a therapeutic agent capable of blocking a unique K+ channel found in the demyelinated axons from patients with Multiple Sclerosis, which could alleviate the symptoms.

Robotics Laboratory

An automated liquid handling system with a robotic micro-plate handler is being utilised to develop a high-throughput method for screening existing compounds for K+ channel blocking activity. This platform will entail using cells expressing not only normal K+ channels but also mutated forms associated with various diseases such as Multiple Sclerosis, Episodic Ataxia I and certain Attention Deficit Disorders. Efflux of Rb+ through these channels will be measured, using an atomic absorption spectrometer, with and without the various drugs.

Electrophysiology Unit

This contains two electrophysiological set-ups:

• Equipment for two-electrode voltage-clamp experiments.
• A state-of-the-art system for patch-clamp recordings (including the latest generation amplifier, Nikon fluorescent microscope, precise micro-manipulators etc.).

Work herein will initially be performed on isolated neurons (cell lines and primary cultures) and, eventually, on acute tissue slices. The ultimate goal of these projects is to devise interventions to control neuronal excitability in hyper- or hypo-active conditions:

1. Determination of the physiological properties of recombinant K+ channels mimicking those from normal and diseased tissues.
2. Documentation of the specificity of K+ channel blocking drugs for particular subtypes, indicated by biochemical and robotic screening.
3. Characterisation of the effects of recombinant botulinum toxins on synaptic transmission in different nerve types (e.g. motor, autonomic & sensory).

Microscopy/Imaging Room

This unit will provide expertise on microscopy to researchers working on several different projects:

Light and fluorescence microscopes:

Zeiss Axioskop FS – upright microscope equipped for bright field, phase contrast and epi-fluorescence, with standard and special water-immersion objectives for in vivo imaging.
Olympus IX70 – inverted microscope equipped for bright field, phase contrast and epi-fluorescence, and fitted with Marzhauser motorised stage controlled by Scope-Pro 5.0 software.

Imaging and image analysis:

Image capture is by the fast, sensitive Retiga Exi digital camera connected using firewire interface to a powerful computer system. Image processing, analysis and quantitation are carried out using the Image–Pro PLUS 5.1 image analysis software.

The expertise in microscopy/imaging and the aforementioned resources will not only be used for routine microscopical imaging of both endogenous and over-expressed proteins of interest, in live or fixed cells and tissue sections, but also for sophisticated repeated in vivo visualisation of transmitter release from single individual nerve-endings in live animals.

Equipment Room

This spacious utility area is located centrally within the ICNT for ease of access from all rooms. It also serves as a central storage area for communal equipment and consumables with plentiful freezer and refrigerated space, including a cold room fitted out with shelving and a work-bench. A weighing up/solution preparation area has been included complete with a supply of purified de-ionised water. Heavy equipment, such as large centrifuges, shakers and an autoclave are positioned here. There is also a central gas store, a fume cupboard and a safety shower.

Biohazard Suite

This purpose-built facility in the R&E building exceeds the rigorous specifications legislated for containment level 3, to ensure the safe-handling of Class II biological agents (including genetically-modified bacteria) and Clostridial neurotoxins (botulinum and tetanus). The suite is continuously supplied with clean, HEPA-filtered air and the extracted air passes through at least two HEPA filters to prevent escape of biological materials. The lab can only be entered through a lobby maintained under negative pressure, which provides isolation from the surrounding area. It is fully equipped and functions as a stand-alone lab. A Class I/III hybrid containment cabinet provides user protection that can be adjusted to provide maximum protection (in Class III mode) or increased flexibility and user comfort when using less hazardous materials (in Class I mode). An in situ autoclave is used to sterilise reagents, and all contaminated waste before removal from the contained facility.

In these facilities, native and recombinant botulinum toxin of different serotypes will be produced, especially forms genetically engineered to increase yield and optimise their properties for therapeutic purposes. Moreover, mutations and deletions are to be introduced to further restrict its action to specific targets, and modulate the duration of action. A specific protease cleavage site is being introduced between the heavy chain and light chain of the toxin which offers the advantage of a pro-form with much lower toxicity for safe handling which can be activated specifically when needed.