Engineering mechanobiology: the bacterial exclusively-mechanosensitive ion channel MscL as a future tool for neuronal stimulation technology
Creators: name: SOLOPERTO, ALESSANDRO
Publisher: name: Universit� degli studi di Genova
Year: 2018

description: The development of novel approaches to stimulate neuronal circuits is crucial to
understand the physiology of neuronal networks, and to provide new strategies
to treat neurological disorders.
Nowadays, chemical, electrical or optical approaches are the main exploited
strategies to interrogate and dissect neuronal circuit functions. However,
although all these methods have contributed to achieve important insights into
neuroscience research field, they all present relevant limitations for their use in
in-vivo studies or clinical applications. For example, while chemical stimulation
does not require invasive surgical procedures, it is difficult to control the
pharmacokinetics and the spatial selectivity of the stimulus; electrical stimulation
provides high temporal bandwidth, but it has low spatial resolution and it
requires implantation of electrodes; optical stimulation provides subcellular
resolution but the low depth penetration in dense tissue still requires the invasive
insertion of stimulating probes.
Due to all these drawbacks, there is still a strong need to develop new
stimulation strategies to remotely activate neuronal circuits as deep as possible.
The development of remote stimulation techniques would allow the combination
of functional and behavioral studies, and the design of novel and minimally
invasive prosthetic approaches.
Alternative approaches to circumvent surgical implantation of probes include
transcranial electrical, thermal, magnetic, and ultrasound stimulation. Among
v
these methods, the use of magnetic and ultrasound (US) fields represents the
most promising vector to remotely convey information to the brain tissue. Both
magnetic and low-intensity US fields provide an efficient mean for delicate and
reversible alteration of cells and tissues through the generation of local
mechanical perturbations.
In this regard, advances in the mechanobiology research field have led to the
discovery, design and engineering of cellular transduction pathways to perform
stimulation of cellular activity. Furthermore, the use of US pressure fields is
attracting considerable interest due to its potential for the development of
miniaturized, portable and implantation-free US stimulation devices.
The purpose of my PhD research activity was the establishment of a novel
neuronal stimulation paradigm adding a cellular selectivity to the US stimulation
technology through the selective mechano-sensitization of neuronal cells, in
analogy to the well-established optogenetic approach. In order to achieve the
above mentioned goal, we propose the cellular overexpression of
mechanosensitive (MS) ion channels, which could then be gated upon the
application of an US generated pressure field. Therefore, we selected the bacterial
large conductance mechanosensitive ion channel (MscL), an exclusively-MS ion
channel, as ideal tool to develop a mechanogenetic approach. Indeed, the MscL
with its extensive characterization represents a malleable nano-valve that could
be further engineered with respect to channel sensitivity, conductance and gating
mechanism, in order to obtain the desired biophysical properties to achieve
reliable and efficient remote mechanical stimulation of neuronal activity.
In the first part of the work, we report the development of an engineered MscL
construct, called eMscL, to induce the heterologous expression of the bacterial
protein in rodent primary neuronal cultures. Furthermore, we report the
structural and functional characterization of neuronal cells expressing the eMscL
channel, at both single-cell and network levels, in order to show that the
functional expression of the engineered MscL channel induces an effective
vi
neuronal sensitization to mechanical stimulation, which does not affect the
physiological development of the neuronal itself.
In the second part of the work, we report the design and development of a water
tank-free ultrasound delivery system integrated to a custom inverted
fluorescence microscope, which allows the simultaneous US stimulation and
monitoring of neuronal network activity at single resolution.
Overall, this work represents the first development of a genetically mechanosensitized
neuronal in-vitro model. Moreover, the developed US delivery system
provides the platform to perform high-throughput and reliable investigation,
testing and calibration of the stimulation protocols.
In this respect, we propose, and envisage in the near future, the exploitation of
the engineered MscL ion channel as a mature tool for novel neuro-technological applications.