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.
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.
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


Di Stefano M
(2015)
A rise in NAD precursor nicotinamide mononucleotide (NMN) after injury promotes axon degeneration.
in Cell death and differentiation

Di Stefano M
(2017)
NMN Deamidase Delays Wallerian Degeneration and Rescues Axonal Defects Caused by NMNAT2 Deficiency In Vivo.
in Current biology : CB


Fricker M
(2018)
Neuronal Cell Death.
in Physiological reviews

Gilley J
(2013)
Rescue of peripheral and CNS axon defects in mice lacking NMNAT2.
in The Journal of neuroscience : the official journal of the Society for Neuroscience

Gilley J
(2011)
Modelling early responses to neurodegenerative mutations in mice.
in Biochemical Society transactions

Gilley J
(2015)
Absence of SARM1 rescues development and survival of NMNAT2-deficient axons.
in Cell reports

Gilley J
(2017)
Sarm1 Deletion, but Not Wld, Confers Lifelong Rescue in a Mouse Model of Severe Axonopathy.
in Cell reports

Martino Carpi F
(2018)
Simultaneous quantification of nicotinamide mononucleotide and related pyridine compounds in mouse tissues by UHPLC-MS/MS
in Separation Science Plus
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 | Outreach Award |
Amount | $150,000 (USD) |
Organisation | Thompson Family Foundation Initiative |
Sector | Charity/Non Profit |
Country | United States |
Start | 10/2018 |
End | 09/2020 |
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 |