A survival factor for axons: roles in disease and downstream mechanism

Lead Research Organisation: Babraham Institute
Department Name: Babraham Bioscience Technologies

Abstract

Our nervous system cannot function without axons, the long ?wires? conducting electrical signals from one nerve cell (neuron) to another. Even if other parts of the neuron (the cell body and dendrites) survive, a neuron without an axon is functionally dead. Axons are very vulnerable because of their immense length (up to one metre in man) and their need to deliver essential components from cell bodies to all locations along their lengths using a sophisticated process known as ?axonal transport?. Consequently, axon degeneration makes critical contributions to symptoms in many neurodegenerative conditions, including multiple sclerosis, glaucoma, diabetic neuropathy, motor neuron disease and Alzheimer?s disease. Once lost, axons in our brain and spinal cord do not regenerate so it is essential to preserve them in ageing and disease.

We recently identified an enzyme (Nmnat2) as an axon survival factor using a neuronal culture system. We propose that failure to deliver Nmnat2 could be responsible for axon death in diseases where axonal transport fails. This is based on experiments where substituting with a similar but longer-lasting enzyme named WldS increases axon survival and alleviates disease. WldS is not present in people whereas Nmnat2 is, which makes this new development particularly exciting. Because we can now regulate the degenerative process by manipulating a single molecule that humans do have, we can make rapid progress in understanding this type of degeneration and working out the best way to block it pharmacologically.

To get the full picture, we also need to study this process in the context of a mammalian nervous system and its roles in neurodegenerative disorders. We will genetically modify mice to block or reduce the production of Nmnat2 in neurons. When its production stops altogether we expect that axons will die through a mechanism called ?Wallerian-like degeneration?, a pathway we have studied for many years and can test for using the WldS gene. When production of the proposed survival factor is reduced by around 50%, we expect that axons may initially survive but become more susceptible to other stresses such as neurotoxins, physical pressure, inherited defects that ?clog up? our axons and possibly even normal ageing. By testing whether axonal Nmnat2 levels are reduced in axonal transport disorders, and whether the degree of reduction is related to the severity of the disease, we aim to understand the molecular steps leading to axon degeneration and ultimately target them therapeutically.

Technical Summary

In neurodegenerative disorders axons typically degenerate before neuronal cell death. This sequence of events, and particularly the early loss of distal axons, is known as ?dying back? degeneration. The causes of axon degeneration include protein aggregation, inflammation, neurotoxicity and ischaemia, and many of these diverse stresses converge on a common degenerative pathway involving axonal transport impairment. Axonal transport is the bidirectional trafficking of molecules and organelles along axons for huge cellular distances. It is essential for axon survival but deficient in multiple sclerosis, glaucoma, motor neuron disease and many other disorders.

Despite the prevalence of axonal transport impairment, the specific molecular changes leading to axon degeneration are poorly understood. Cutting axons, which causes Wallerian degeneration, is a useful experimental model that can help identify the key molecular events. A mutant protein named Wallerian degeneration slow (WldS) delays Wallerian degeneration by tenfold and alleviates some ?dying back? disorders, showing that the mechanisms are related. Thus, axons do not die by passive wasting when isolated from cell bodies but by a specific and regulatable process.

WldS is an aberrant protein that occurs naturally in only one strain of mouse, so until now it has been largely unclear how we might use it to protect axons in human disease. Recently, we identified the NAD+ synthesising enzyme Nmnat2 as an endogenous regulator of the same pathway in primary neuronal cultures. Nmnat2 is an unstable protein, so if axonal transport fails to replenish it, continual protein turnover in axons takes Nmnat2 below a threshold level that triggers Wallerian degeneration.

Nmnat2 is now the key to understanding the degenerative mechanism and thereby identifying suitable steps to target pharmacologically, but for the full picture it must also be studied in vivo. We hypothesise that depleting Nmnat2 is sufficient to initiate Wallerian-like degeneration in vivo and that failure to deliver it to distal axons in some axonopathies is the direct cause of ?dying back? axon loss. We also hypothesise that Nmnat2 and WldS control a common downstream pathway, which we can activate very specifically by removing Nmnat2. Thus, we can now factor out the many non-specific consequences of cutting axons or of blocking axonal transport, and focus specifically on events leading to axon degeneration. This is a unique opportunity to move towards translation for axonal transport disorders and for significant progress in understanding how axon survival and degeneration are controlled at the molecular level.

Publications

10 25 50
 
Description BBSRC CASE studentship with AstraZeneca
Amount £100,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2020 
End 09/2024
 
Description BBSRC Industrial Partnership Award
Amount £934,853 (GBP)
Organisation AstraZeneca 
Department Astra Zeneca
Sector Private
Country United States
Start 02/2019 
End 02/2022
 
Description MRC Project Grant
Amount £835,237 (GBP)
Funding ID MR/N004582/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 02/2016 
End 01/2019
 
Description Sir Henry Wellcome Postdoctoral Fellowship (as sponsor of Dr. Andrea Loreto)
Amount £250,000 (GBP)
Funding ID 210904/Z/18/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2018 
End 10/2022
 
Title Nmnat2 gene trap mouse 
Description We have generated a novel mutant mouse line from a EUCOMM gene trap clone targeting the Nmnat2 gene. The gene trap is conditional such that it can be inactivated and then reactivated to assess the effects of Nmnat2 depletion in mature mice. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Initial experiments indicate that knock-down of Nmnat2 expression from the trapped allele is substantial or complete. We have bred heterozygotes with an active gene trap, and have found that homozygotes die just before, or at birth. We have shown that this reflects an axonal growth defect and we have rescued this defect with the WldS gene. We find evidence for compensatory changes that allow heterozygotes to develop normally and survive. The heterozygote mice are being aged to study whether Nmnat2 loss contributes to age-related axon loss. 
 
Description Coleman 
Organisation University of Cambridge
Department John van Geest Centre for Brain Repair
Country United Kingdom 
Sector Academic/University 
PI Contribution I contributed experimental data derived from muscle tension recordings from knockout mice with axonal and synaptic protection
Collaborator Contribution Characterisation of Sarm1/Nmnat-2 double knockout mice with axonal and neuromuscular synaptic protection.
Impact Gilley J, Ribchester RR, Coleman MP. Sarm1 Deletion, but Not Wld(S), Confers Lifelong Rescue in a Mouse Model of Severe Axonopathy. Cell Rep. 2017 Oct 3;21(1):10-16. doi: 10.1016/j.celrep.2017.09.027. PubMed PMID: 28978465; PubMed Central PMCID: PMC5640801.
Start Year 2016
 
Description Conditional knock-down of NMNAT2 in CNS neurons 
Organisation Baylor College of Medicine
Department Department of Pediatrics
Country United States 
Sector Academic/University 
PI Contribution Provided conditional gene trap mice
Collaborator Contribution They will perform the conditonal knock-down
Impact A colony has been established and experiments are in progress
Start Year 2013
 
Description Determination of NMN levels in degenerating nerves 
Organisation Marche Polytechnic University
Department Department of Molecular Pathology and Innovative Therapies
Country Italy 
Sector Academic/University 
PI Contribution We have provided the nerve tissues for the analysis
Collaborator Contribution Provides analytical techniques and expertise that would otherwise not be available to the group.
Impact This work is helping to establish the timing of NMN accumulation in degenerating nerves
Start Year 2010
 
Description Generation of mice with 75% reduction in NMNAT2 
Organisation University of Texas Southwestern Medical Center
Country United States 
Sector Academic/University 
PI Contribution Imported gene trap mice with partial knock-down of NMNAT2 from targeted allele and crossed with our own gene trap targeted mice to be used in experiments.
Collaborator Contribution Provided gene trap mice with partial knock-down of NMNAT2 from targeted allele.
Impact We have been monitoring effects of a 75% reduction in NMNAT2 expression during aging.
Start Year 2011
 
Description Café Scientifique presentation (Cambridge 2012) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Many people attended talk entitled "keeping your nerves".

Positive feedback about research
Year(s) Of Engagement Activity 2012
 
Description Cambridge Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Lots of interest in our axon transport exhibit

Exhibit has been used at multiple events since, including pint of science and school visits
Year(s) Of Engagement Activity 2015
 
Description Public discussion (Cambridge, UK 2012) 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Informed Q&A session with a panel of experts and members of the public about aging research

Useful feedback
Year(s) Of Engagement Activity 2012
 
Description Radio/podcast interview (BBC Radio Cambridgeshire, 2012) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Not quantifiable

Positive feedback from people who had heard the broadcast
Year(s) Of Engagement Activity 2012
 
Description School visits: 3 presentations to secondary school/6th formers 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Paper Presentation
Geographic Reach Local
Primary Audience Schools
Results and Impact Lay science presentation about the nervous system in general and axon survival specifically. Also dealt with issues including how basic science leads to unpredictable but useful outcomes (GFP, monoclonal antibodies, etc) and careers advice

Very positive feedback. Too early to know effect on exam results.
Year(s) Of Engagement Activity 2010,2011,2012
 
Description Talk at Hills Road Sixth Form College, Cambridge 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Presentation to 40 6th form students. Title: The nervous system: the life and death of cells inside your head.
Year(s) Of Engagement Activity 2018