Annual-Report-2019

RECOMBINANT INTRABODIES AS MOLECULAR
TOOLS AND POTENTIAL THERAPEUTICS FOR
AMYOTROPHIC LATERAL SCLEROSIS (INTRABALS)
Life & Health Sciences 2019
48
Dr Denis Reis de Assis
LE STUDIUM Research Fellow
ARD 2020 BIOPHARMACEUTICALS Programme
PRESTIGE
From: Pontifical Catholic University of Rio
Grande do Sul - BR
In residence at: Imaging and Brain laboratory
(iBrain) - Tours
Nationality: Brazilian
Dates: March 2018 to March 2019
Dr Reis de Assis did his masters and PhD studying
brain energy metabolism. He showed that
metabolites accumulating in a fatty acid oxidation
disorder called MCAD deficiency decrease
the activity of the enzyme Na + , K + -ATPase,
Krebs cycle, the activities of the mitochondrial
respiratory chain complexes, creatine kinase
and cause lipid peroxidation. He did his first
post-doc in the neural stem cells field. During
his second post-doc, he clarified mechanisms
by which cell therapy is neuroprotective in a rat
model of epilepsy, methylprednisolone improves
aversive memory, neurotoxicity of venoms, and
by which a neuropeptide involved in appetites and
neuropsychiatric disorders acts in hippocampal
cells. He received hands-on training in iPS cells
and human neural progenitors. Currently, D Reis
de Assis studies the effects of a hallmark protein
in amyotrophic lateral sclerosis, TDP-43, on
calcium signalling and mitochondrial
bioenergetics.
Prof. Hélène Blasco
Host scientist
H. Blasco is professor and practitioner in the
Laboratory of Biochemistry and Molecular
Biology and in the team «Neurogenomics and
Neuronal physiopathology» of INSERM U1253
(CHU and University of Tours). She is specifically
working on Amyotrophic Lateral Sclerosis (ALS),
a neurodegenerative disease characterized by
degeneration of motor neurons that leads to a
progressive muscular paralysis. ALS diagnosis
is mostly based on clinical criteria that lack the
precision to establish a rapid diagnosis. Thus, her
research activity is focused on the development
of biomarkers, the understanding the aetiology
of the disease and the identification of new
neuroprotective agents. More recently, the team
is developing a therapeutic approach based
on intrabodies to target protein aggregates.
Thus, her current project is mainly to develop
biopharmaceuticals in ALS and to use pharmacometabolomics
to assist this development.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that
has no diagnostic marker, prognosis, nor an effective treatment. Numerous
physio-pathological mechanisms have been described for this disease, in
particular the aggregation of cytoplasmic TDP-43. Additionally, a “prionlike”
mechanism of the propagation of the pathology including TDP-43 has
been described. Much effort has been directed to therapeutic treatments
for ALS, but these efforts explore sparsely the potential of biomolecules.
The objective of this project is to target the protein aggregates containing
TDP-43 by a novel approach. We planned to characterise the therapeutic
sites of TDP-43 through fragments of antibodies synthesised by the cell,
termed intrabodies. The results of this project
will have applications not only for ALS but also
potentially for other neurological diseases,
such as dementias.
We have just obtained our first intrabodies
(about 6 clones). We are currently evaluating
several markers, including cellular respiration,
glycolysis, the endometabolome, as well as
calcium signaling and neurotransmission. We have obtained interesting
results and we have determined few robust and reproducible parameters
to test for intrabodies screening. We are currently testing the effects of
TDP-43 on synaptic neurotransmission by a technique called patch clamp,
which measures several electrophysiological parameters using primary
cultures of motor neurons from mice (Fig1 and Fig2). These experiments
have the potential of showing if TDP-43 overexpression could disturb
neurotransmission in motor neurons, which is a property exclusive of
excitable cells, such as neurons.
In addition, our preliminary results
suggest that overexpression of
TDP-43 in HEK293 cells do not
affect directly mitochondrial
respiration (Fig3), however it
provokes an increase in the basal
levels of intracellular calcium (Fig
4). Thus, we are currently testing
whether TDP-43 overexpression
also affects intramitochondrial
calcium levels, which are critical to
stimulate mitochondrial respiration. Our results are important, since there
is currently a disagreement among data from different research groups
regarding the effects of TDP-43 on the mitochondrial energy metabolism.
The collaboration with the industrial partner (still under negotiation) will
provide a library of small molecules that could mimic the activity of the
intrabodies with the advantage of an easier access to the brain and into
target cells. Therefore, we will test the competitive binding of these small
molecules against the intrabodies then will test their protective effects in
vitro in the models described previously.