Engineering a Stable Synaptogenic Extracellular Matrix

Laila Al-Alwan

, Markus Hellmund

, Rainer Haag

and Timothy Kennedy

Montreal Neurological Institute, McGill University, 3801 Univerity Street, Montreal, Canada

Chemistry and Biochemistry Institute, Freie University Berlin, Berlin, Germany

Keywords: Neural Biocompatibility, Synthetic Synapse, Neural Prosthetic, Biomimetic Functionalised Surfaces,

Polymer Chemistry, Synaptogenic Protein.

Abstract: Synapses are specialized sites of asymmetric cell – cell contact that mediate information transfer between

neurons and their targets. Many proteins involved in the recruitment, organization and maintenance of

synapses have been identified. Surprisingly, synaptic differentiation does not require a biological membrane

surface. Instead, synaptic specializations can form quickly at sites of neurite adhesion to microspheres

(beads) coated with synaptogenic proteins or even poly-lysine, a synthetic cationic polypeptide, raising the

possibility that functional hemi-synaptic connections could be formed onto designer engineered surfaces.

Previous studies examining the stability of synapses formed in brain onto poly-lysine coated beads found

they were unstable, degraded, and ultimately replaced by a glial scar. Here, we address the capacity of an

extreme biomimetic of poly-lysine, PGB50, a dendritic polyglycerol (dPG)-amine soft matter nanoparticle,

to enhance synapse formation in long-term cultures of rat cortical neurons. Microbeads coated with PGB50

exhibit substantially enhanced synaptogenesis and synapse stability compared to poly-lysine. We propose

that synaptogenic extracellular matrices could be used to engineer synaptogenic electrodes with enhanced

neural-compatibility, reducing glial scaring and inflammation, and allowing for bi-directional

communication with neurons through the formation of stable of synaptic specializations.

1 INTRODUCTION

Deficits due to neurodegeneration or injury-induced

brain diseases are, ultimately, a direct reflection of

the loss of functional synapses. Synapses are

specialized sites of asymmetric cell – cell contact

that mediate information transfer between neurons

and their targets. Many proteins involved in the

recruitment, organization, and maintenance of

synapses have been identified and the molecular

biology of synaptic adhesion is increasingly well

understood. Furthermore, neural activity can now be

read out to activate muscles or control the movement

of robotic limbs (Hochberg et al., 2012; van den

Brand et al., 2012). In spite of these advances,

contemporary microelectrodes, made of metal or

glass, present fundamentally invasive surfaces that

neural cells isolate by enclosing in a glial scar.

Synaptic specializations can assemble rapidly

following axon – dendrite contact. Surprisingly, the

formation of an active pre-synaptic terminal does not

require a biological post-synaptic membrane surface.

Instead, pre-synaptic specializations can form

quickly at sites of axonal adhesion to microspheres

(beads) coated with specific lipids or proteins,

including the synthetic poly-cationic polypeptide

poly-lysine (PLL) (Burry, 1982; Lucido et al., 2009;

Gopalakrishnan et al., 2010; Goldman et al., 2013;

Suarez et al., 2013).

PLL is a naturally occurring polymer that is

susceptible to degradation by several common

secreted proteases, including trypsin and cathepsins.

Consistent with this, presynaptic specializations

formed onto PLL coated beads in vivo were not

stable but completely degraded within two weeks,

replaced by an astrocytic glial scar that isolated the

bead (Burry, 1983, 1985). PDL, a protein

biomimetic enantiomer of PLL, was developed to

resist protease degradation, and thereby enhance its

utility as a cell culture substrate. Here, using long-

term cultures of embryonic rat cortical neurons we

address the capacity of an extreme biomimetic of

PLL, PGB50, an ~75 kDa dendritic polyglycerol

(dPG)-amine soft matter nanoparticle based on a

highly stable and biocompatible polyglycerol

scaffold (Hellmund et al., 2015), to enhance synapse

formation.

We propose that synaptogenic extracellular

matrices may be engineered to enhance biocompati-

Al-Alwan, L., Hellmund, M., Haag, R. and Kennedy, T.

Engineering a Stable Synaptogenic Extracellular Matrix.

In Extended Abstracts (NEUROTECHNIX 2016), pages 11-13

bility and promote the stable formation of synaptic

specializations onto manufactured surfaces in vivo.

Our goal is to engineer synaptogenic electrodes with

enhanced neural-compatibility that reduce glial

scaring and inflammation, and allow for bi-

directional communication with neurons.

2 METHODS

Cell cultures were prepared from cerebral cortex of

embryonic day 17–18 (E17–E18). Cells were plated

at high density (∼40,000 cells/cm

) and maintained

for 14-25 days in vitro (DIV) in Neurobasal medium

containing 1% B27, 2 mM glutamax and 0.5% N2.

Microspheres (7.3 μm polystyrene; Bangs Beads)

were washed 3× in PBS (sterile, pH 7.4) before use,

then incubated overnight with (50 μg/ml) PLL, PDL

and PGB50. Beads were then washed in PBS,

pelleted by centrifugation, 7 min at 6500 rpm, and

resuspended in culture medium before addition to

cultures. Control beads were treated similarly,

without coating. Beads were added to cultures at 11

DIV and maintained for an additional 3, 7 or 14 days

of incubation (DOI). All images were captured using

an Olympus FV1000 confocal microscope. At least

60 Beads were quantified per condition using

ImageJ. Corrected total cell fluorescence intensity

(CTCF) was calculated for both bead and neurite as:

Integrated Density - (Area of selected cell X Mean

fluorescence of background), and fold changes in

CTCF of bead/neurite plotted. Data are mean ±

SEM, Two-way ANOVA followed by a post hoc test

was used to calculate p values.

3 RESULTS

3.1 PGB50 Enhances Synapse

Formation

Using long-term cultures of embryonic rat cortical

neurons, we tested and compared the efficacy of

beads coated with PLL, PDL or PGB50 to induce

synapse formation and promote synapse stability.

Immunoreactive CTCF for synaptophysin and PSD-

95, marking pre- and post-synaptic specializations,

was significantly enhanced for PGB50-coated beads

when compared to Control, PLL- and PDL-coated

beads (Figures 1A and 1B). These results reveal

more effective induction of long-term stable

synaptic specializations by PGB-50 compared to

either PLL or PDL.

Figure 1: PGB50 Enhances Synapse Formation.

Quantification of corrected total cell fluorescence intensity

(CTCF) of A. The post-synaptic marker; PSD-95 and B.

The pre-synaptic marker; Synaptophysin. Neuronal

cultures were incubated with control, PLL-, PDL- or

PGB50-coated beads for 3, 7 and 14 days of incubation

(DOI). ***p < 0.001 vs. control,  p < 0.05,  p <

0.001 vs. PLL, xx p < 0.01, xxx p < 0.001 vs. PDL.

3.2 PGB50 Enhances Synaptic

Specialization

The formation of synaptic specializations, with a

post-synaptic bouton localized adjacent to its pre-

synaptic counterpart, is essential for synapse

formation, maturation and function. To test whether

PGB50, PLL and PDL promote local synapse

formation, we quantified the overlap of presynaptic

synaptophysin with postsynaptic PSD-95, within a

3D volume around the beads (voxels).

The overlap of PSD-95 and synaptophysin was

enhanced by all coatings compared to control beads

(Figure 2). PGB50-coated beads exhibited signify-

cantly higher numbers of voxels positive for both

Figure 2: PGB50 Enhances Synaptic Specialization.

Quantification of number of co-labeled voxels (N-Coloc)

of the post-synaptic marker; PSD-95 and the pre-synaptic

marker; Synaptophysin. Neuronal cultures were incubated

with control, PLL-, PDL- or PGB50- coated beads for 3, 7

and 14 days of incubation (DOI). ***p < 0.001 vs. control,

 p < 0.001 vs. PLL, x p < 0.05, xxx p < 0.001 vs.

PDL.

NEUROTECHNIX 2016 - 4th International Congress on Neurotechnology, Electronics and Informatics

PSD-95 and synaptophysin at all time points

examined. These results suggest a greater capacity

of PGB50 compared to PLL and PDL to initiate and

support the local formation of synapses.

4 DISCUSSION

Studies carried out in the 1980s, addressing the

function of synaptogenic poly-cationic polymers,

found that simple beads coated with PLL had the

capacity to direct the formation of presynaptic

specializations in vitro and in vivo, but the synapses

formed did not persist, and within a few days in vivo

were displaced by an astrocytic scar (Burry, 1983,

1985). Although these findings supported the idea

that non-neuronal surfaces, when decorated with the

“correct” chemical signals could induce the forma-

tion of synaptic specializations, the short lifetime of

the synapses formed was fundamentally problematic

for translational applications. Here, we provide evi-

dence for enhanced synapse formation and stability

induced by the dendritic polyglycerol PGB50, a

highly stable non-protein molecular biomimic of

poly-lysine.

Our ongoing studies aim to enhance the function

and stability of synapses formed onto modified

synaptogenic surfaces in vivo, and develop

approaches to stimulate and record from these

surfaces. We aim to promote the formation of

adhesive contacts by axons and dendrites that will be

inherently more stable and better positioned to

record neuronal activity than is currently possible

using conventional electrodes. Our findings suggest

that the hemi-synaptic specializations formed onto

the dendritic polyglycerol surface will in turn induce

synapse formation by adjacent axons and dendrites,

resulting in the development of a dense local web of

synaptic connections surrounding the electrode.

Ultimately such an implant would achieve functional

integration into the neuronal network. We envision

that such implanted synaptogenic interfaces would

be broadly applicable to extend the function of the

injured or diseased human nervous system.

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Engineering a Stable Synaptogenic Extracellular Matrix