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The Elusive Compass of Clostridial Neurotoxins: Deciding When and Where to

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The Elusive Compass of Clostridial
Neurotoxins: Deciding When
and Where to Go?

Kinga Bercsenyi, Francesco Giribaldi

and Giampietro Schiavo

Abstract Axonal transport ensures long-range delivery of essential components

and signals between proximal and distal areas of the neuron, and it is crucial for
neuronal homeostasis and survival. Several pathogens and virulence factors use
this route to gain access to the central nervous system, exploiting the complex and
still poorly understood trafficking mechanisms that regulate the dynamics of their
cellular receptors. Studying the intracellular transport of neurotropic pathogens is
therefore instrumental to glean new insights into these important molecular events.
Botulinum (BoNT) and tetanus (TeNT) neurotoxins bind with high affinity to a
variety of neurons and are internalised by specialised endocytic pathways leading
to specific intracellular fates. Whereas BoNT trafficking is largely confined to the
neuromuscular junction, TeNT is internalised in signalling endosomes shared with
neurotrophins and their receptors, which are recruited to the fast axonal retrograde
transport pathway. Recently, important paradigms regarding the mechanisms by
which BoNT and TeNT interact with their cellular targets and are transported in
neurons have been challenged. In this review, we summarise new findings con-
cerning the uptake and intracellular trafficking of these neurotoxins, and discuss
their implications in terms of the physiological effects of BoNT and TeNT in the
central nervous system.

Keywords Axonal transport Botulinum neurotoxin Endocytosis motor neurons

Tetanus toxin

K. Bercsenyi
G. Schiavo (&)

Molecular NeuroPathobiology Laboratory, Cancer Research UK London Research Institute,
44 Lincoln’s Inn Fields, London WC2A 3LY, UK
e-mail: [email protected]
F. Giribaldi
Department of Experimental Medicine, Section of Pharmacology and Toxicology,
viale Cembrano 4, 16148 Genoa, Italy

A. Rummel and T. Binz (eds.), Botulinum Neurotoxins, Current Topics 91

in Microbiology and Immunology 364, DOI: 10.1007/978-3-642-33570-9_5,
Ó Springer-Verlag Berlin Heidelberg 2013
92 K. Bercsenyi et al.

Abbreviations
Ab Amyloid beta
AP2 Adaptor protein 2
BAR Bin–amphiphysin–rvs domain
BDNF Brain-derived neurotrophic factor
BoNT Botulinum neurotoxins
CAR Coxsackie- and adenovirus receptor
CAV2 Canine adenovirus 2
CCP Clathrin coated pits
CCV Clathrin coated vesicles
ChAT Choline acetyltransferase
CHO Chinese hamster ovary
CME Clathrin-mediated endocytosis
CNS Central nervous system
CNT Clostridial neurotoxins
DRG Dorsal root ganglia
ERK Extracellular signal-regulated kinase
GPI Glycosylphosphatidylinositol
HC Heavy chain
H CT Binding fragment of tetanus toxin
LC Light chain
Kidins220/ARMS Kinase-D-interacting substrate of
220 kDa/ankyrin-rich membrane spanning
MT Microtubules
NMJ Neuromuscular junction
NT Neurotrophins
p75NTR p75 neurotrophin receptor
PC12 Pheochromocytoma cells
PLCc1 Phospholipase Cc1
PrP Prion protein
PtdIns(4,5)P2 Phosphatidylinositol(4,5)bisphosphate
PV Poliovirus
SC Superior colliculus
SNAP-25 Synaptosomal associated protein of 25 kDa
SNARE Soluble NSF attachment protein receptor
SV2 Synaptic vesicle glycoprotein 2
SV40 Simian virus 40
Syt Synaptotagmin
TeNT Tetanus neurotoxin
Trk Tropomyosin-receptor-kinase
VAMP Vesicle associated membrane protein
Elusive Compass of Clostridial Neurotoxins 93

Contents

1 Introduction.......................................................................................................................... 93
2 Clostridial Neurotoxins are Multi-Domain Proteins .......................................................... 94
3 Binding of Clostridial Neurotoxins to the Cell Surface .................................................... 95
4 Clostridial Neurotoxin Endocytosis .................................................................................... 98
5 Fate Decision in the Trafficking of Clostridial Neurotoxins........................................... 100
6 Axonal Transport of Tetanus Neurotoxin ........................................................................ 101
7 Tetanus Toxin Shares Transport Compartments with Neurotrophins
and Their Receptors .......................................................................................................... 101
8 Shared Pathways with Pathogens ..................................................................................... 103
9 Botulinum Neurotoxins and Their Long-Range Transport Mechanisms ........................ 104
10 Future Perspectives............................................................................................................ 106
References................................................................................................................................ 107

1 Introduction

Efficient mechanisms ensuring the reliable exchange of information between cells

and their environment are essential for all organisms. The outer surface of the
plasma membrane provides a platform for a wide range of receptors, which sense
extracellular signals and inform cells about changes in their surroundings. To
ensure that cells receive relevant messages and are able to assemble suitable
physiological responses, both the lipid and protein compositions of the plasma
membrane must be tightly regulated. A main player in this homeostatic control is
the process of endocytosis, in which selective components of the plasma mem-
brane are internalised in a highly regulated fashion and undergo intracellular
trafficking leading to degradation, recycling or targeting to other membrane
compartments (McMahon and Boucrot 2011). The main roles of endocytosis
include nutrient uptake, receptor-mediated signalling and the turnover of mem-
brane components and receptors (Hoeller et al. 2005). Given the key physiological
role and evolutionary conservation of these mechanisms, numerous pathogens and
virulence factors, including clostridial neurotoxins (CNT) exploit selective endo-
cytic routes to gain access to host cells. Despite the plethora of different endocytic
cargoes, surprisingly little information is available regarding the molecular
mechanisms regulating the recruitment of ligand-receptor complexes to specific
carriers, their internalisation and transport and their ultimate fate. In light of this,
infectious agents represent invaluable tools shaped by millions of years of evo-
lution to study the molecular determinants of these membrane trafficking pathways
(Schiavo and van der Goot 2001).
The most extensively studied type of endocytosis is clathrin-mediated endocy-
tosis (CME). The list of proteins involved in the regulation of CME has been con-
tinuously growing, and as a result, a detailed view of the molecular mechanisms
controlling its progression is now available (McMahon and Boucrot 2011). Mech-
anistically, CME occurs via four main steps, the first of which is the nucleation of
94 K. Bercsenyi et al.

clathrin at specific membrane sites. This process is initiated by accessory and adaptor
proteins, such as adaptor protein 2 (AP2) and epsins (Henne et al. 2010; Jackson et al.
2010; Qualmann et al. 2011; Taylor et al. 2011). Following the recruitment of
membrane curvature-inducing proteins, clathrin-coated pits (CCPs) are formed.
Once the budding is completed, clathrin-coated vesicles (CCV) are clipped off the
membrane by dynamin, a GTPase involved in membrane remodelling (Harper et al.
2011; Ferguson and De Camilli 2012), and the clathrin basket is removed by cyto-
plasmic factors to form an uncoated vesicle (Bocking et al. 2011). Interestingly,
CME was shown to be the main, but not the only route taken by CNT to get access to
their target cells (Deinhardt et al. 2006a; Montal 2010).

2 Clostridial Neurotoxins are Multi-Domain Proteins

The CNT family comprises tetanus toxin (TeNT) and several related botulinum
neurotoxins (BoNT, serotypes A to G; Swaminathan 2011). TeNT and BoNT are
synthesised as single chain proteins of 150 kDa, which are cleaved by endogenous
or tissue proteases, resulting in a 50 kDa light chain (LC) and a 100 kDa heavy chain
(HC) linked via a disulphide bridge. The carboxy-terminal half of the HC is
responsible for the binding of CNT to the neuronal surface, whilst the amino-
terminal half is involved in the translocation of the LC through the endosomal
membrane (Montal 2010). The LC contains the active site of the neurotoxin and
displays a very specific metalloprotease activity (Schiavo et al. 2000). To date, the
synaptic SNARE (soluble NSF attachment protein receptor) proteins syntaxin-1,
SNAP-25 (synaptosomal-associated protein of 25 kDa) and VAMP-1–2 (vesicle-
associated membrane protein) (termed also synaptobrevin-1 and -2) are the only
identified substrates of TeNT and BoNT (McMahon et al. 1993; Schiavo et al. 2000),
in addition to VAMP-3, syntaxin-2 and syntaxin-3. Cleavage of these synaptic
SNARE proteins yields the persistent blockade of neurotransmitter release in
intoxicated neurons (Schiavo et al. 1992; Blasi et al. 1993a, b; Schiavo et al. 1993a,
b; Yamasaki et al. 1994).
CNT share their core structural characteristics (Fig. 1), cleave the same class of
intracellular substrates and are taken up by the same neurons (Schiavo et al. 2000).
However, once endocytosed, TeNT and BoNT are sorted to different membrane
trafficking pathways (Fig. 1). BoNT mainly target the neuromuscular junction
(NMJ) in vivo, and only a minor fraction of the toxin is transported back to the cell
body of the motor neuron (Restani et al. 2012a). In contrast, TeNT efficiently reaches
the inhibitory interneurons located in the spinal cord (Fig. 1). This distinct intra-
cellular trafficking leads to a different symptomatology of the pathologies caused by
BoNT and TeNT. Botulism, which is caused by BoNT, leads to flaccid paralysis,
whilst tetanus is characterised by a sustained spastic paralysis. It remains to be seen
whether or not this difference in sorting is due to specific receptor complexes for
TeNT and BoNT, which would target them to distinct endocytic compartments, or to
the recruitment of different sorting factors after internalisation.
Elusive Compass of Clostridial Neurotoxins 95

Fig. 1 Trafficking of botulinum and tetanus neurotoxins in neurons. Top. The three-domain
structure of BoNT/A (Lacy et al. 1998), which is shared with the other CNT, is characterized by a
50 kDa LC containing the active site (shaded, on the right) and a 100 kDa HC responsible for
membrane translocation (HN; shaded, centre) and binding to the neuronal surface (HC; unshaded,
on the left). CNT fragments can be expressed as recombinant proteins bearing specific short
domains that allow their labelling with fluorophores (tag) and other functional groups (Deinhardt
et al. 2006b). Due to its binding and sorting characteristics and its lack of toxicity, fluorescent
HCs can be used instead of the full length CNT to study binding, transport and transcytosis.
Middle. Schematic representation of CNT trafficking in a spinal cord motor neuron. TeNT (light
blue) and BoNT (red) specifically bind to neuronal membranes via receptor complex(es) formed
by polysialogangliosides (black dots) and synaptic proteins (blue and red bars). CNT are then
internalised and sorted into different intracellular compartments. Whilst BoNT remain mainly
localised to the NMJ, where they enter the synaptic vesicle recycling pathway, TeNT undergoes
fast axonal retrograde transport exploiting MT as tracks (grey) and cytoplasmic dynein as a main
molecular motor (red). Once it has reached the soma, TeNT is trancytosed into inhibitory
interneurons, where the LC reaches the cytoplasm and blocks neurotransmitter release. Bottom.
Mouse NMJ after intramuscular administration of HCT (red). HCT is internalised and transported
into the motor neuron shaft. The motor endplate has been counterstained with a-bungarotoxin
(green)

3 Binding of Clostridial Neurotoxins to the Cell Surface

Complex gangliosides were shown to serve as receptors for CNT. TeNT binds to
GT1b and GD1b, whereas BoNT bind to GT1b and GD1a (Habermann and Dreyer
1986; Schengrund et al. 1991; Yowler and Schengrund 2004). The affinity of CNT
to immobilised polysialogangliosides is in the high nanomolar range, whilst these
neurotoxins bind to synaptosomes and neurons with a much higher affinity, making
96 K. Bercsenyi et al.

their interactions with neuronal membrane almost irreversible (Habermann and

Dreyer 1986). The addition of exogenous polysialogangliosides to non-neuronal
cells, such as undifferentiated rat pheochromocytoma (PC12), renders them sen-
sitive to TeNT or BoNT/A (Marxen and Bigalke 1989; Marxen et al. 1990). In
agreement with this finding, neuraminidase treatment makes cells insensitive to
CNT by removing sialic acid residues from the plasma membrane (Bigalke et al.
1986; Marxen and Bigalke 1989). These results, together with the partial insen-
sitivity to CNT of mice lacking complex gangliosides (Kitamura et al. 1999, 2005;
Rummel et al. 2009), indicate that these lipids are essential components of
CNT receptor complexes. On the other hand, the relatively low affinity of poly-
sialogangliosides for TeNT and BoNT and the major inhibitory effect on binding
caused by pre-treatment of neurons with extracellular proteases, strongly suggests
the existence of a dual protein and lipid receptor for CNT (Montecucco 1986).
According to the dual receptor hypothesis (Montecucco 1986; Rummel et al.
2007), polysialogangliosides either favour the recruitment of TeNT and BoNT to
specific areas of the plasma membrane enriched in protein receptors, or maintain
these toxins in a preferred conformation for the receptor to bind.
The molecular identification of the protein receptors for CNT posed a challenge
to the field. Both binding and internalisation of BoNT were shown to be activity-
dependent (Black and Dolly 1986; Keller et al. 2004; Baldwin and Barbieri 2007)
suggesting that synaptic vesicle proteins might be involved in BoNT binding and
uptake. The intraluminal leaflet of the synaptic vesicle membrane becomes tran-
siently exposed to the extracellular medium upon fusion of synaptic vesicles with
the active zone during synaptic stimulation, potentially allowing the intravesicular
domains of synaptic vesicle proteins to interact with the binding domain of CNT
and act as their protein receptors. Pioneering work from Kozaki’s group has
demonstrated that BoNT/B binds simultaneously to polysialogangliosides and
synaptotagmin-I (Syt-I), the main calcium sensor for synaptic vesicle fusion, and
the closely related isoform synaptotagmin-II (Syt-II), with ten times higher affinity
than polysialogangliosides alone (Ochanda et al. 1986; Nishiki et al. 1994; 1996a).
However, direct evidence that the luminal domain of both Syt-I and Syt-II binds
BoNT/B and mediates the internalisation of the toxin was provided only a decade
later (Dong et al. 2003, 2007). Hippocampal neurons lacking Syt-I are insensitive
to both BoNT/B and BoNT/G, but can be rendered sensitive by overexpression of
Syt-I or -II (Dong et al. 2007). Similarly, the cytoplasm of PC12 and Chinese
hamster ovary (CHO) cells became accessible to BoNT/B upon the surface
expression of the intraluminal domain of Syt-I/II (Nishiki et al. 1996b; Pirazzini
et al. 2011). However, Syt are not universal protein receptors for BoNT, as only
BoNT/B, BoNT/G and the mosaic toxin BoNT/DC bind to Syt-I/II (Rummel et al.
2004, 2007; Peng et al. 2012). Based on the hypothesis that the luminal portion of
synaptic vesicle proteins may serve as protein receptor of the remaining BoNT
serotypes, two groups independently demonstrated that synaptic vesicle glyco-
protein 2 (SV2) acts as a protein receptor for BoNT/A (Dong et al. 2006; Mahrhold
et al. 2006) and several other serotypes, such as BoNT/E (Dong et al. 2008),
BoNT/F (Rummel et al. 2009) and BoNT/D (Peng et al. 2011).
Elusive Compass of Clostridial Neurotoxins 97

Fig. 2 Uptake of TeNT and BoNTs in cultured motor neurons. Synaptic vesicle exo/endocytosis
accounts for the majority of endocytic events at the presynaptic terminal and may involve
multiple clathrin-dependent step(s). Several BoNTs exploit this pathway for their internalization
into neurons by binding to the luminal domain of specific vesicle proteins, such as SV2 and Syt-I/
II. High affinity interaction to these proteins requires polysialogangliosides and may be regulated
by lipid microdomains. Once internalised into the synaptic vesicle lumen, acidification driven by
the vATPase triggers the translocation of LC into the nerve terminal cytoplasm that leads to the
cleavage of SNARE proteins essential for membrane fusion. This process determines the long-
lasting inhibition of neurotransmitter release at the NMJ (local effects). At very high doses, TeNT
enters this pathway and induces a flaccid paralysis similar to that caused by BoNT. In contrast, at
physiological doses TeNT exploits a pathway requiring lipid microdomains and the clathrin
machinery that is largely independent of synaptic vesicle exo/endocytosis. At the NMJ, TeNT
binds to a lipid-protein receptor complex containing polysialogangliosides such as GD1b, and is
then laterally sorted into clathrin-coated vesicles. During this sorting event, GD1b is excluded
from the toxin receptor complex (Deinhardt et al. 2006a). Internalisation of TeNT is dependent
on dynamin, AP-2 and AP180, but does not require epsin1. Once internalised, TeNT is targeted to
a stationary (or oscillating) early sorting compartment positive for the small GTPase Rab5, to
which other endocytic routes may converge. Some of these routes may be responsible for the
entry of specific BoNT serotypes (e.g. BoNT/A) to sorting endosomes, their targeting to the
axonal retrograde transport pathway and their transport together with TeNT and neurotrophin
receptor to the soma of motor neurons (long-range effects). Fast retrograde transport of these
organelles requires Rab7 activity. Question marks indicate molecular events that have not been
conclusively demonstrated to date and DN stands for dominant-negative mutants

Whilst it became clear that synaptic vesicle proteins play a role in the binding
and uptake of BoNT, independent lines of evidence suggested that at least a
fraction of these neurotoxins are internalised in an activity-independent fashion
(Verderio et al. 1999; Restani et al. 2012a). These findings strongly suggest that a
98 K. Bercsenyi et al.

proportion of BoNT is not internalised via synaptic vesicle recycling and may have
additional receptors and/or additional routes of entry at nerve terminals (Fig. 2).
Furthermore, BoNT/C has been shown to bind to both polysialogangliosides and
phospholipids (Tsukamoto et al. 2005, 2008; Kroken et al. 2011; Strotmeier et al.
2011; Zhang and Varnum 2012), suggesting that this serotype has a unique binding
modality to neuronal membranes.
The search for the protein receptor of TeNT has posed an even bigger challenge
than the identification of BoNT receptors. Whilst TeNT entry into hippocampal
neurons is stimulation-dependent (Matteoli et al. 1996; Blum et al. 2012), its inter-
nalisation into the NMJ and cultured motor neurons is largely independent (Schmitt
et al. 1981; Deinhardt et al. 2006a) or only partially modulated (Simpson 1985) by
synaptic activity. This remarkable difference suggests that TeNT may use a different
endocytic mechanism to be sorted in motor neurons from other neuronal types that
are blocked by this neurotoxin, such as inhibitory interneurons and hippocampal
neurons (Fig. 2). This unique intracellular sorting may couple TeNT to the retro-
grade transport route in motor neurons in contrast to BoNT, which are preferably
sorted to recycling synaptic vesicles at the NMJ. Early evidence suggested that TeNT
interacts with Thy-1, a glycosylphosphatidylinositol (GPI)-anchored glycoprotein,
in PC12 cells (Schiavo et al. 1991; Herreros et al. 2000). Interestingly, one or more
GPI-anchored proteins are involved in TeNT binding and intracellular activity, since
treatment of PC12 cells or neurons with a phosphatidylinositol-specific phospholi-
pase C prevents the TeNT-induced cleavage of VAMP-2 (Herreros et al. 2001;
Munro et al. 2001). Disruption of the integrity of membrane microdomains, which is
essential for GPI-anchored protein clustering, also prevented VAMP-2 cleavage by
TeNT (Herreros et al. 2001; Munro et al. 2001). However, Thy-1 is unlikely to be the
only protein receptor for TeNT since mice lacking Thy-1 are only slightly less
sensitive to TeNT intoxication than wild-type controls (Herreros et al. 2001). SV2
was also shown to bind TeNT in central neurons (Yeh et al. 2010). However, this
latter interaction has been recently disputed (Blum et al. 2012) and no evidence is
presently available validating this mechanism in motor neurons or inhibitory inter-
neurons. Last, but not least, the possibility that the interaction of TeNT with neuronal
and non-neuronal cells is mediated by the binding of polysialogangliosides to two
distinct sites of its binding domain (HCT) has been proposed (Fotinou et al. 2001;
Rummel et al. 2003; Chen et al. 2008, 2009). Interestingly, immobilised glycolipid
complexes have been shown to display higher affinity to HCT (Rinaldi et al. 2009),
suggesting that ganglioside cis interactions may have important modulatory roles in
the initial binding of TeNT to the neuronal membrane.

4 Clostridial Neurotoxin Endocytosis

Once CNT are bound to their surface receptors, a complex cascade of protein–
protein and protein–lipid interactions trigger the recruitment of clathrin and
adaptor proteins to the inner leaflet of the plasma membrane, which marks the
Elusive Compass of Clostridial Neurotoxins 99

onset of the endocytic process (Fig. 2). Although BoNT and TeNT are mainly
internalised by distinct pathways (synaptic vesicle recycling and synaptic vesi-
cle-independent clathrin-dependent endocytosis, respectively) (Deinhardt et al.
2006a; Montal 2010; Blum et al. 2012), several aspects of these mechanisms are
shared at the molecular level. One of the early events of these processes is the
recruitment of specific clathrin adaptors, which leads to the accumulation of
effector proteins altering membrane curvature at endocytic sites. A major
determinant of this process is the enrichment of specific lipids, such as phos-
phatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2), at these sites. This event is
likely to precede membrane curvature initiation as most of the accessory pro-
teins, such as AP2, bind to this lipid (Haucke 2005). Several factors mediating
membrane curvature possess a bin–amphiphysin–rvs (BAR) domain (Qualmann
et al. 2011), which enables them to initiate and maintain membrane curvature,
whilst other proteins lacking the BAR domain, such as epsins, shape membranes
by inserting an amphipathic helix into the inner leaflet of the lipid bilayer,
making it more accessible to clathrin (Ford et al. 2002; Hinrichsen et al. 2006).
Deinhardt et al. have shown that the internalisation of TeNT relies on the
canonical clathrin adaptors AP2 and AP180, but is independent of epsin1, since
the overexpression of an epsin1 mutant unable to bind PtdIns(4,5)P2 did not
affect the internalisation of the toxin, whilst completely abolished transferrin
uptake (Deinhardt et al. 2006a). Epsin1 was shown to target ubiquitinated
receptors to the late endosomal/lysosomal pathway (Le Roy and Wrana 2005),
suggesting that TeNT follows an intracellular trafficking route bypassing this
sorting step. Accordingly, TeNT is known to access an atypical endosomal
compartment in motor neurons, which is not acidified (Lalli et al. 2003a; Bo-
hnert and Schiavo 2005). Thus, this epsin1-independent endocytic route may
prevent the membrane insertion of HC and the subsequent translocation of
the active LC into the cytoplasm, a process that is triggered by low pH, allowing
the delivery of TeNT to axonal retrograde carriers in an intact form (Fig. 2). At
the same time, this specific sorting avoids the targeting of TeNT to lysosomes
and its degradation. As to which other membrane curvature protein(s) might act
as a replacement of epsin1 remains unknown.
Following the recruitment of clathrin adaptors, a clathrin basket is formed
around the pit and a new CCV is ready to be born. The last step in the biogenesis
of CCV is the recruitment of a member of the dynamin family, a large GTPase,
which upon GTP hydrolysis, drives the fission of the CCV from the plasma
membrane. Crucially, the uptake of both TeNT and BoNT/A is disrupted in the
presence of dynamin inhibitors (Deinhardt et al. 2006a; Harper et al. 2011) or by
overexpression of dynamin mutants (Deinhardt et al. 2006a) (Fig. 2). Electron
microscopy studies have confirmed that CNT are taken up into CCV (Black and
Dolly 1986; Deinhardt et al. 2006a), which undergo uncoating in the synaptic
cytoplasm.
100 K. Bercsenyi et al.

5 Fate Decision in the Trafficking of Clostridial Neurotoxins

Although several binding and endocytic determinants are shared by BoNT and
TeNT, the intracellular fate of these neurotoxins is largely distinct. In motor
neurons, TeNT is internalised together with neurotrophin receptors and their
ligands into transport endosomes (Fig. 2), which are characterised by near-neutral
pH and low degradative potential (Bohnert and Schiavo 2005), and are delivered to
the cell body. Upon arrival in the soma, TeNT is sorted to a transcytotic route and
gains access to inhibitory interneurons (Schiavo et al. 2000). In contrast, BoNT are
mostly taken up in synaptic vesicles and remain confined at distal synapses, such
as the NMJ. During reloading with neurotransmitters, the pH drop in the lumen of
synaptic vesicles determines the insertion of BoNT into the lipid bilayer
(Montecucco et al. 1986, 1989) which triggers the translocation of the LC into the
cytoplasm, where the disulphide bond is reduced and it can specifically cleave the
synaptic targets (Koriazova and Montal 2003; Fischer and Montal 2007; Fischer
et al. 2008; Montal 2010). However, recent evidence suggests that BoNT/A is also
retrogradely transported in several neuronal types, including hippocampal, tectal
and motor neurons (Antonucci et al. 2008; Restani et al. 2012a) and undergoes
transcytosis in the visual system (Restani et al. 2011, 2012b), mimicking at least in
part the behaviour of TeNT.
For its long-range transport, TeNT exploits a highly specialised trafficking
pathway shaped by strong evolutionary pressure. Eukaryotic cells are characterised
by a highly compartmentalised organisation and efficient communication between
different cellular areas is required to ensure cellular homeostasis. Transport over long
distances reaches its higher specialisation in neurons, where dendritic and axonal
compartments are in dynamic equilibrium via a network of highly regulated transport
routes powered by molecular motors (Hirokawa et al. 2010; Soo et al. 2011; Winckler
and Yap 2011). Eukaryotic cells, including neurons, rely on three superfamilies of
motor proteins: kinesins and dyneins transport their cargoes on microtubules (MT),
whereas myosins are F-actin-dependent (Stiess and Bradke 2011). MT and actin
microfilaments extend longitudinally within neurons, with MT mainly present in
axons and dendrites and actin microfilaments enriched at synaptic regions (Hirokawa
et al. 2010). Both cytoskeletal elements are characterised by a highly polarised
architecture. The plus, fast-growing end of MT is directed towards the periphery in
axons and distal dendrites, whereas the barbed, growing end of actin microfilaments
points to the plasma membrane in pre- and post-synaptic regions. Fast axonal and
dendritic transport is mainly MT-dependent and relies on kinesins and cytoplasmic
dynein for the distribution within different neuronal regions of a variety of cargoes,
which include organelles, proteins and RNAs (Hirokawa et al. 2010). Moreover,
modulation of axonal transport by varying the concentration and/or activity of
individual motors constitutes a reliable size-sensing mechanism in vitro and in vivo
(Rishal et al. 2012). In axons, fast axonal transport occurs both in the anterograde
(from cell body towards the periphery) and retrograde (from axonal tips to cell body)
directions by means of kinesins and cytoplasmic dynein, respectively (Vale et al.
Elusive Compass of Clostridial Neurotoxins 101

1985; Hirokawa et al. 2010). Myosins also participate in axonal transport in the
proximity of the synaptic regions or in areas where the distribution of MT is less
uniform (Langford 2002; Lalli et al. 2003b).

6 Axonal Transport of Tetanus Neurotoxin

Kinetic analysis of axonal transport assessed in primary motor neuron cultures using
either HCT or the directly labelled full-length neurotoxin revealed a complex speed
profile, which can be deconvolved in a trimodal Gaussian distribution with peaks at 1,
1.5 and 2.1 lm/s, well within the range of fast axonal transport (Lalli and Schiavo
2002; Hafezparast et al. 2003). Two different types of pleiomorphic organelles
responsible for the axonal retrograde transport of TeNT were identified: vesiculo-
tubular structures, that show a continuous retrograde movement and account for the
faster transport component, and round vesicles characterised by a more discontin-
uous and slower retrograde transport (Lalli et al. 2003a). In vivo characterisation of
HCT transport in the sciatic nerve or in sensory neurons confirmed this trimodal speed
distribution (Bilsland et al. 2010) (Fig. 4). Strikingly, the average velocities
observed in vivo were higher than those observed in vitro (Bilsland et al. 2010). This
is likely to be due to the extensive axon myelination occurring in motor neurons in the
sciatic nerve, which is known to stabilise MT, and/or differences between embryonic
(in vitro) and adult (in vivo) motor neurons (Bilsland et al. 2010). In addition to a
major role of kinesins, cytoplasmic dynein and MT, the retrograde transport of TeNT
also relies on myosin Va, an actin-associated motor protein. Whilst the fastest
component of the transport is accomplished almost totally by cytoplasmic dynein and
requires kinesins at equilibrium, the intermediate component is dependent on myosin
Va, as demonstrated by the transport impairments observed in myosin Va-null motor
neuron cultures (Lalli et al. 2003b). The requirement of both actin microfilaments
and MT for fast axonal transport is in line with the tight association occurring
between these two cytoskeletal elements seen by ultrastructural analysis (Bearer and
Reese 1999). Furthermore, it has been proposed that the recruitment of myosins, in
addition to cytoplasmic dynein, allows neurons to assure a continuous retrograde
movement of organelles also during transitions between different MT (Langford
2002; Lalli et al. 2003b).

7 Tetanus Toxin Shares Transport Compartments

with Neurotrophins and Their Receptors

Many different external stimuli rely on axonal retrograde transport for reliable and
fast signalling from distal synapses to the cell body. Activated receptor complexes
are sorted to transport organelles, called signalling endosomes (Fig. 3), that
102 K. Bercsenyi et al.

Receptors
Neurotrophin receptor (p75NTR)
Tropomyosin-receptor kinase (TrkA-C)
Axonal retrograde Poliovirus receptor-related protein 2
transport Coxsackievirus and adenovirus receptor
(CAR)

Cargoes
Neurotrophins (NGF, BDNF)
Tetanus toxin
Cytoplasmic dynein Botulinum neurotoxins
Poliovirus
Canine adenovirus 2 (CAV2)
Trks
Rab7
Motors and Regulators
Cytoplasmic dynein
Kidins220/ARMS neuro Dynactin
trophins Rab7
Rab5

CAV2
Additional components
Activated leukocyte cell adhesion
HC molecule (ALCAM)/CD166
p75NTR
Neuronal cell adhesion molecule (NCAM)
Kidins220/ARMS

Fig. 3 Schematic representation of a TeNT-positive signalling endosome. TeNT and its binding
fragment HCT exploit the retrograde transport machinery used by physiological cargoes, such as
neurotrophins (light blue), for their entry into the CNS. Once internalised together with its still
unknown receptor(s) (green), HCT enters signalling endosomes containing Trks, p75NTR and their
interacting protein Kidins220/ARMS (kinase-D-interacting substrate of 220 kDa/ankyrin-rich
membrane spanning; green). The progression and fast axonal transport of these signalling
endosomes are dependent on the sequential recruitment of the two small GTPases Rab5 (blue) and
Rab7 (green). Fast axonal retrograde transport of these signalling organelles relies on the MT-
dependent motor cytoplasmic dynein (blue) and its associated complex dynactin (grey). Signalling
endosomes contain several plasma membrane proteins, such as coxsackie- and adenovirus receptor
(CAR, dark red), which are known to bind viruses. As such, these organelles are exploited by several
viruses, such as CAV2 and poliovirus, to access the CNS. Additional components and cargoes of
these axonal carriers are listed on the right. An electron microscopy image of a signalling endosome
containing HCT (empty arrows) is shown on the top. Scale bar, 200 nm

undergo fast axonal transport together with their adaptors and downstream
signalling molecules, thus overcoming the limitations of signal transduction
mechanisms based uniquely on diffusion (Howe 2005). Neurotrophins (NT) and
their receptors, tropomyosin receptor kinase (Trk) and p75 neurotrophin receptor
(p75NTR), are among the best known examples of such retrogradely transported
signalling complexes (Schecterson and Bothwell 2010). NT interact with Trks and
p75NTR at distal sites and these activated receptor complexes are transported
towards the nucleus, where they promote gene expression events controlling dif-
ferentiation and survival in most types of neurons (Butowt and von Bartheld 2003;
Winckler and Yap 2011). It has recently been shown that TeNT exploits the
organelles used by NT receptor complexes for its journey from nerve terminals to
the soma. Experiments performed in primary spinal cord motor neurons using
fluorescently labelled HCT showed that TeNT shares retrograde carriers with NGF
Elusive Compass of Clostridial Neurotoxins 103

and p75NTR (Lalli and Schiavo 2002). A common route for TeNT and neurotrophin
receptors was further demonstrated by showing the presence in the same trans-
ported endosome of HCT, brain-derived neurotrophic factor (BDNF), p75NTR and
TrkB (Deinhardt et al. 2006b). This is a general mechanism, since it has been
shown both in primary motor and dorsal root ganglia (DRG) neurons. Progression
towards the cell body is dependent on Rab5 and Rab7, two small GTPases with
multiple roles in the endocytic pathway (Deinhardt et al. 2006b) (Fig. 3). Rab5 and
Rab7 act in a sequential manner: Rab5 is involved in the initial steps of the
internalisation process, whereas Rab7 is required for fast progression along axons
(Deinhardt et al. 2006a; Salinas et al. 2009). This unanticipated relationship
between the trafficking of TeNT and NT is further supported by independent lines
of evidence. BDNF was shown to increase the efficiency and kinetic of inter-
nalisation of HCT at the murine NMJ in a dose-dependent manner (Roux et al.
2006). Other neurotrophins, such as NT4, have similar effects on the localisation
and internalisation of TeNT but are less potent than BDNF (Roux et al. 2006). The
functional interaction between TeNT and NT is bidirectional, since both TeNT and
HCT activate TrkA and its downstream effectors extracellular signal-regulated
kinase 1/2 (ERK1/2) and phospholipase C c1 (PLCc1) in a dose-dependent manner
(Gil et al. 2000; Gil et al. 2001, 2003).

8 Shared Pathways with Pathogens

Neurotoxins, such as TeNT and to a lesser extent BoNT, are not the only exogenous
molecules gaining access to the CNS by exploiting the axonal retrograde transport
pathway. Many viruses, which are endocytosed by clathrin-dependent and -inde-
pendent mechanisms, rely on controlled acidification steps for their uncoating and
cytoplasmic entry (Salinas et al. 2010). Once released in the cytoplasm, these
virions are transported to the nucleus by binding directly motor proteins, such as
cytoplasmic dynein (Greber and Way 2006; Salinas et al. 2010). However, other
neurotrophic viruses, such as canine adenovirus 2 (CAV2), are retrogradely
transported together with their receptors, in TeNT-positive carriers containing
p75NTR (Salinas et al. 2009) (Fig. 3). Poliovirus (PV) is another example of a
neurotrophic virus that undergoes retrograde transport in a TeNT-positive com-
partment in primary motor neurons (Ohka et al. 2009). Interestingly, the kinetics of
these organelles seems to be controlled by the PV receptor, probably via its ability
to bind directly cytoplasmic dynein (Ohka et al. 2009). Other viruses resemble
TeNT dynamics in terms of their intracellular sorting and axonal trafficking.
Accordingly, the first phase of influenza virus transport is largely MT-dependent
with speed ranging between 1 and 4 lm/s and a pH of its carriers approaching
neutrality (Lakadamyali et al. 2003). The sequential involvement of Rab5 and Rab7
is a key feature of the sorting and retrograde transport of this virus as well as of
HIV (Vidricaire and Tremblay 2005), influenza virus H3N2 (Sieczkarski and
Whittaker 2003) and simian virus 40 (SV40) (Vonderheit and Helenius 2005).
104 K. Bercsenyi et al.

Similar to TeNT, SV40 and polyoma virus bind to neuronal receptors associated
with lipid rafts and are internalised by a cholesterol- and glycosphingolipid-
dependent mechanism (Smith et al. 2003). Although not essential for its infectivity,
p75NTR contributes to the binding, internalisation and axonal transport of rabies
virus and its subsequent transcytosis to second-order neurons (Ugolini 1995;
Tuffereau et al. 2007). In addition to viruses, amyloid beta (Ab), prion protein (PrP)
and toxic protein aggregates have been found to be associated with axonal carriers
and affect axonal transport. In particular, Ab, which has been implicated in
Alzheimer’s disease, was found to directly interact with p75NTR (Yaar et al. 1997)
and to impair axonal retrograde transport of BDNF in primary neurons (Poon et al.
2011). Similarly, a toxic PrP peptide has been shown to induce cell death via direct
activation of p75NTR signalling (Della-Bianca et al. 2001), whereas the full-length
protein undergoes fast axonal transport both in peripheral and central neurons
(Borchelt et al. 1994; Encalada et al. 2011). Altogether, these findings suggest that
the axonal retrograde transport route is a main gateway for the entry and spread into
the CNS of pathological agents and virulence factors.

9 Botulinum Neurotoxins and Their Long-Range Transport

Mechanisms

BoNT activity is mainly restricted to distal synapses, a feature that is exploited in

the many therapeutic applications of BoNT (Schiavo et al. 2000; Hackett and Kam
2007). In spite of the overwhelming evidence supporting this peripheral mecha-
nism of action, several reports suggest that BoNT might also have central effects in
humans and animal models. More than 50 years ago, the case of a patient affected
by botulism showing clear CNS effects was reported (Polley et al. 1965). Initially,
these central effects of BoNT were explained by synaptic plasticity changes
occurring between the central and the peripheral neuron of the network, after the
latter had been silenced by BoNT intoxication. However, the suggestion of a
possible, direct, central action of BoNT arose from experiments performed by
Wiegand and colleagues who detected a rise in radioactivity in the ventral horn of
the spinal cord after injection of radiolabelled BoNT/A in the cat gastrocnemius
muscle, both ipsilaterally and contralaterally to the site of administration (Wiegand
et al. 1976). Further evidence of axonal retrograde transport of BoNT/A and
BoNT/B was found in mouse muscle diaphragm after intraperitoneal administra-
tion or incubation in vitro with radiolabelled neurotoxins. Although internalisation
occurred with different efficiency for BoNT/A and B, both toxins were found in the
axoplasm of myelinated axons within vacuolar-like structures, thus suggesting the
entry of BoNT in a specific endocytic pathway (Black and Dolly 1986). Direct
evidence supporting the hypothesis that BoNT/A is retrogradely transported in an
active form and undergoes transcytosis in second-order neurons was provided by
the detection of BoNT/A-cleaved SNAP-25 in cholinergic synapses in rat retina
Elusive Compass of Clostridial Neurotoxins 105

after injection of the neurotoxin in the superior colliculus (SC) (Antonucci et al.
2008). No BoNT/A-cleaved SNAP-25 was detected in synapses of the retina when
this neurotoxin was injected in SC after MT depolymerisation, thus ruling out
diffusion-based spreading mechanisms. Supporting evidence for a long-range
action was also obtained by detecting changes in the activity of CA1 pyramidal
neurons after injection of BoNT/A into the contralateral hippocampus (Antonucci
et al. 2008).
Interestingly, transport (and transcytosis) of catalytically active BoNT/A occurs
not only in the retrograde direction, but also anterogradely. This unexpected
property of BoNT/A has been demonstrated by detecting the presence of neuro-
toxin-cleaved SNAP-25 in the SC after BoNT/A injection in the contralateral
retina (Restani et al. 2011).
Interestingly, different BoNT serotypes display distinct long-range effects in
vivo. Conversely to BoNT/A, BoNT/E seems unable to alter the activity of CA1
neurons after injection in the contralateral hippocampus (Antonucci et al. 2008), in
spite of overwhelming evidence demonstrating its silencing activity upon ipsilat-
eral injection (Costantin et al. 2005). This result suggests distinct sorting pathways
and/or distal kinetics for different BoNT serotypes (Antonucci et al. 2008; Caleo
and Schiavo 2009). This conclusion seems to be supported by overt differences in
the rate of SNAP-25 cleavage induced by BoNT/A and BoNT/E in sympathetic
neurons cultured in Campenot chambers (Lawrence et al. 2012). After distal
application of these neurotoxins, BoNT/A-cleaved SNAP-25 was readily detect-
able in cell bodies, whilst the BoNT/E truncated form of this synaptic protein was
only slightly detectable in the somas, despite a much higher quantity of BoNT/E
being used in this experiment (Lawrence et al. 2012). The documented short-lived
enzymatic activity of BoNT/E was suggested to be responsible for this lack of
detection (Lawrence et al. 2012).
However, direct evidence supporting a differential rate of transport of BoNT/
A and BoNT/E is still lacking. Experiments recently performed in our laboratory
filled this gap by analysing the kinetic properties of the axonal transport of these
serotypes once internalised in primary spinal cord motor neurons (Restani et al.
2012a). Full-length BoNT/A and BoNT/E, or their atoxic binding fragments
undergo axonal retrograde transport in non-acidic organelles with speed profiles
matching fast MT-dependent transport and largely overlapping with TeNT-
positive carriers in living motor neurons (Restani et al. 2012a). Our analysis
suggests that a lower transport efficiency of BoNT/E compared to BoNT/A may
contribute to the differential in vivo effects of these two serotypes (Caleo and
Schiavo 2009). Whilst BoNT/A undergoes fast and continuous retrograde
transport, BoNT/E-positive organelles show a discontinuous movement towards
the cell body, characterised by a higher frequency of pauses and short periods of
anterograde transport (Restani et al. 2012a). Altogether, these results demonstrate
that BoNT undergo axonal retrograde transport in neurons both in vitro and in
vivo.
106 K. Bercsenyi et al.

Fig. 4 Innovative strategies to monitor axonal transport in vitro and in vivo. Left panel.
Microfluidic chambers allow the physical separation of distal axons and synapses (bottom) from
cell bodies (top) in a variety of neuronal cultures, including motor neurons. HcT (red), added only
to the axonal side, is transported towards the cell bodies located in the top compartment. The two
compartments are separated by small microgrooves (10 lm width), which allow the passage of
axons, but not of cell bodies. These compartments are also microfluidically separated, a feature
that impedes the passive diffusion of ligands and drugs between the two sides of the device.
Motor neurons are stained for b-tubulin (blue). Scale bar, 500 lm. Right panel. Axonal transport
of HcT can be monitored in the intact sciatic nerve. After intramuscular injection in the mouse
hindlimb, fluorescent HcT (red) was found within axons of the sciatic nerve (top), which have
been stained for choline acetyl transferase (ChAT, green), a motor neuron marker, and myelin
(blue). This technique allows a quantitative kinetic analysis of axonal transport of HcT in vivo
(Bilsland et al. 2010). Still images from a confocal movie show an HCT-positive endosome
undergoing fast axonal retrograde transport (arrowheads). The cell body is out of view on the left.
Scale bar, 5 lm

10 Future Perspectives

In spite of the wealth of data presently available, there are important aspects of the
interaction of BoNT with the neuronal membrane that deserve further investiga-
tions. Among these the role of different isoforms of SV2 and Syt in the binding of
BoNT to distinct neuronal subtypes is presently unclear. Similarly, very little is
known about the role of specific posttranslational modifications on the affinity of
SV2 and Syt to BoNT and on their intracellular trafficking. It is also unclear
whether phospholipids and polysialogangliosides are sufficient to mediate high
affinity binding and internalisation of BoNT/C and other serotypes in neurons.
Elusive Compass of Clostridial Neurotoxins 107

Strikingly, the protein receptor(s) of TeNT at the NMJ is still unknown,

although SV2 seems to play a role in TeNT binding to interneurons (Yeh et al.
2010). Previous data suggest that GPI-anchored proteins play a role in TeNT
intoxication (Herreros et al. 2001; Munro et al. 2001), but it is unclear whether
their contribution is direct or mediated through changes in the clustering of
polysialogangliosides on the synaptic plasma membrane. Future experiments are
necessary to elucidate the nature of this high affinity TeNT receptor complex and
its putative role in axonal retrograde transport and intracellular sorting.
The quantitative analysis of the retrograde transport of different BoNT is still in
its infancy. To date, little is known about the molecular mechanisms controlling
this process, its modulators and additional cargoes sharing the same transport
organelles. As significant differences between the axonal transport of BoNT/A and
BoNT/E have recently been detected (Lawrence et al. 2012; Restani et al. 2012a),
the CNT field is now facing the exciting prospect of better understanding the
molecular determinants of this differential neuronal trafficking, and the relation-
ship between the 3D structure of these serotypes and their intracellular fate.
Furthermore, additional studies are necessary to assess whether other serotypes
might be transported as well. Novel in vitro approaches, such as compartmenta-
lized motor neuron cultures in microfluidic devices (Fig. 4) will be instrumental
for the analysis of the axonal transport of these neurotoxins and the definition of
the cellular machinery controlling their trafficking.
Last but not least, the molecular mechanism of transcytosis of TeNT from
motor neurons into interneurons is still poorly characterised, as well as the path-
way followed by BoNT/A for its transneuronal migration in the visual system
(Restani et al. 2011, 2012b).
These studies will not only enable a better characterisation of the CNT at
molecular level, but they will also shed light on fundamental processes essential
for neuronal homeostasis and survival, providing new potential strategies for the
delivery of therapeutics to the CNS.

Acknowledgments We thank Victoria Hill and the members of our laboratories for constructive
comments and critical reading of the manuscript. This work was supported by Cancer Research
UK (KB and GS). The authors have no conflicting financial interests.

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