Nidogens are therapeutic targets for the prevention of tetanus Kinga Bercsenyi1, Nathalie Schmieg1, Barney Bryson2, Martin Wallace2, Paola Caccin3, Matthew Golding1, Giuseppe Zanotti3, Linda Greensmith2, Roswitha Nischt4 and Giampietro Schiavo2 1Molecular Neuropathobiology Laboratory, Cancer Research UK, London Research Institute, WC2A 3LY London, UK 2Sobell Department of Motor Neuroscience & Movement Disorders, University College London Institute of Neurology, WC1N 3BG London, UK 3Department of Biomedical Sciences, University of Padova, Padova, Italy 4Department of Dermatology, University of Cologne, Cologne, Germany N
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N
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AA
500
400
(%
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β-­‐III Tubulin
H
H
D
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300
**
**
200
NS
100
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1
1
AA
T+
N
1
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C
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C
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H
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1
1
1
T+
N
1
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H
C
H
C
H
T
T+
N
AA
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0
C
H CT
HC_Alexa 555 c
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First we tested the candidate pep<des for HCT binding in an ELISA (A). The top candidates were carried 500
**
**
forward for further tests, and the N1 pep<de showed the greatest promise as a poten<al blocking agent of
400
HCT binding. second panel in C. Both the N1 and N2 pep<de (origina<ng from nidogen-­‐2) can block the 300
interac<on between Nidogens and HCT (B). The addi<on of soluble exogenous nidogen-­‐1 facilitates HCT 200the N1 pep<de can block this ac<on (C,D). Scale bar: 50 μm. binding and Survival post-injection (h)
1
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1
1
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T+
N
1
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C
C
H
C
H
cc
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wild
wild
typetype
nidogen-1
nidogen-2
nidogen-1
nidogen-2
KO KO
KO KO
0.10
0.10
0.05
0.05
wild
wild
type
type
nidogen-2
nidogen-2
KO
KO
10
5
0
0
5
10 15 20
Int. intensity (HCT)
e
wild
type
wild
type
150
150
nidogen DKO
nidogen DKO
HCT+H T+
C
tubulin
tubulin
dd
***
***
15
H CT H T
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BTX
BTX
0.15
0.15
De
20
200
200
*** ***
*** ***
150
150
100
100
50 50
0 0
f
f
nidogen-1
nidogen-2
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typetypenidogen-1
nidogen-2
KO KO
KO KO
100
50
0
HCT binding
1
T+
N
wild type
nidogen-2 KO
60
40
**
**
100
50
0
DKO
nidogen nidogen
DKO
wild typewild type
TeNT+N1
TeNT+N1AA TeNT+N1
Nidogen-­‐2 and HcT colocalise at the neuromuscular junc<on (A) and there is a strong correla<on between Nidogen-­‐2 content and HcT binding. Nidogen-­‐2 KO motor neurons take up significantly less HcT, than their wild type counterparts (B). Nidogen-­‐1 and -­‐2 KO muscles do not bind HcT (C) and double knock-­‐out embryonic hindbrains show a significantly decreased HcT uptake (D). Scale bars: 20μm in A,B; 5μm in C and 0.5mm in D. 20
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Normalised muscle force
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TeNT
+DMSO
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80
TeNT+N1AA
Merge
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TeNT+DMSO
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Te
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BTX
HCHT T
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Non-treated
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HCTT
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H
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TeNT+N1AA
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TeNT+DMSO
nidogen-2
H CT
3. The N1 peptide blocks tetanus intoxication in vivo A
A
Nidogen 2
H CT
HCT binding
D
BTX
Int. intensity (nidogen-2)
C
A
HCT binding
Dd 4. Nidogen deOicient tissue does not bind tetanus toxin HCT binding
Cc C
EGF
****
H
G3
G3
LYs
EGFs
EGFs
T+
N
1
G2
G2
EGF
EGF
C
NIDO
G1
TG
TG
H
TM
T
B
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N1N
Aa
The N1 pep<de is located in the G2 domain of nidogen-­‐1 (A). 71% of the N1 pep<de surface is interac<ng with TeNT HC (B: N cyan, toxin orange). T h e G 2 d o m a i n o f nidogen-­‐1 fits into the same binding pocket, covering an overall area o f o v e r 1 1 0 0 Å ( C : nidogen-­‐1 cyan, toxin orange). And the two proteins have opposite electrosta<c surfaces at the binding site (D). NS
100
C
Bb H CT
a A
(%
2. The N1 peptide and nidogen 1 Oit into the same the binding pocket 0
Discussion 1.5
3
6
12
TeNT dose (ng/kg)
Na<ve TeNT loses its potency when preincubated with the N1 pep<de as mice injected with this mixture do not develop tetanic paralysis (A,B) due to maintained muscle force (C). Nidogen-­‐2 deficient mice are less sensi<ve to tetanus, they show a later onset, survive for longer (D) and they display botulism-­‐like symptoms, such as the silver eye syndrome. The iden<ty of the protein receptor of tetanus toxin has been a mystery in the past 2,000 years. We found a pep<de present in the G2 domain of nidogen-­‐1 that is not only able to bind tetanus toxin in vitro, but can also block tetanic paralysis in vivo. Once TeNT is taken up at the neuromuscular junc<on it is sequestered in the lumen of signalling endosomes while it’s transported back to the motor neuron cell body. We showed that the presence of nidogens is indispensable for TeNT uptake and when mice are deficient in nidogens, they are less sensi<ve to tetanus intoxica<on. The finding that nidogens are crucial for the binding of TeNT and that this interac<on can be blocked by a specific pep<de opens new ways to cure tetanus as well as showcasing a great example of a neurotropic toxin using the extracellular matrix to reach its target cells (5). References: 1.  Lalli G et al. The journey of tetanus and botulinum neurotoxins in neurons. Trends Microbiol. 2003 2.  Jayaraman et al. Common binding site for disialyllactose and tri-­‐pep<de in C-­‐fragment of TeNT. Proteins. 2005 3.  Deinhardt K et al. Rab5 and Rab7 control endocy<c sor<ng along the axonal retrograde transport pathway. Neuron. 2006 4.  Bercsenyi et al. The elusive compass of clostridial neurotoxins: deciding when and where to go? Curr. Op. Microbiol. 2013 5.  Bercsenyi et al. Nidogens are therapeu<c targets for the preven<on of tetanus. Science 2014 N
1
M
T+
H
CA
nidog
HA-HCT
C
H
1 D
1
AA
1
T+
N
H
C
T
C
H
Peptides
TB
S
O
AA
N
3
N
3
AA
N
1
N
1
AA
J
J
D
2
AA
D
A
D
D
e
nidogen-2
50
0
SO
250
150
0
50
N
N
2
2
1
Binding (%)
HA-HCT
50
100
C
N
1
D
M
nidogen-1
T
3
AA
SO
be
y
HCT
C
*
MW
(kDa)
150
C
Normalised absorbance
4
pt
A
5
B
ad
A
B
Em
A
Tetanus toxin (TeNT) is one of the three most poisonous substances on Earth. It binds to the neuromuscular junc<on with very high affinity, but its surface protein receptor is s<ll unknown. To exert its ac<on, upon entry into motor neurons, TeNT is transcytosed into inhibitory interneurons located in the spinal cord. By blocking their neurotransmiEer release, motor neurons become over-­‐excited causing spas<c paralysis, which is the landmark symptom of clinical tetanus1. Previous work have iden<fied a tri-­‐pep<de, which interacts with the TeNT binding domain2. We selected candidate receptors containing this mo<f from a cohort of proteins, which reside in the same endocy<c carriers as TeNT3. The corresponding pep<des containing this binding mo<f were tested for binding in vitro and ex vivo in primary motor neuron cultures and the we carried the top pep<de forward to test for a poten<al blocking effect in vivo. When na<ve TeNT is pre-­‐incubated with this pep<de and injected into the gastrocnemius muscle of mice, they do not develop tetanic paralysis. Nidogen, the protein from which the pep<de originates has a neuromuscular junc<on specific isoform and it is taken up together with TeNT into primary motor neurons. Mice lacking this protein are less sensi<ve to tetanus intoxica<on and show botulism-­‐like symptoms. Finding the protein receptor of TeNT will enable us to find a cure for the disease and it will pave the way to the use of the toxin as a carrier for targeted delivery of therapeu<cs into the central nervous system4. s
1. The N peptides are the top candidates for blocking tetanus Abstract