Imaging the Second Brain
(December 7th, 2017) What causes Parkinson’s disease, why do neurons in the brain die? How could we diagnose the disease earlier? A group at KU Leuven in Belgium, counterintuitively, took a closer look at patients’ guts.
Many people are afraid of developing a neurodegenerative disease. In Parkinson’s disease (PD), for instance, the neurons that produce the neurotransmitter, dopamine, die off – the patients, thus, lose control over their movements, develop depression, sleeping problems or gastro-intestinal problems. As with many other diseases, early diagnosis is key to a better prognosis. Interestingly, in this context is the fact that gastro-intestinal problems occur before motor dysfunction and, therefore, potentially before most neurons in the brain are lost.
“We started this project based on the intriguing and increasing evidence that (maybe) the periphery could be looked at as the ‘starting point’ during neurodegeneration in PD,” recapitulates Carla Cirillo, who together with An-Sofie Desmet and group leader Pieter Vanden Berghe at KU Leuven, published the first functional analysis of live neurons in the gut of PD patients. They performed live calcium and mitochondrial imaging on biopsy samples. “We merged our expertise in live imaging and microscopy with the clinical part at the hospital, coordinated by Wim Vandenberghe, to have a privileged and translational point of view on the neurons in the gut of patients with PD. This match represents the ‘plus’ value of our study,” Carla Cirillo points out.
But why try to find an early PD biomarker in the gastrointestinal (GI) tract? This is because some hypothesise that the disease starts, not in the brain but in the gut – in neurons of the so-called enteric nervous system (ENS). Neurons of the ENS control the gut's function and are even called the second brain. An-Sofie explains that the typical aggregates of alpha-synuclein, normally found in the brain of PD patients, have also been detected in the GI tract. “This made researchers believe that the disease can start in the ENS and spread towards the brain,” she says. Moreover, it has been suggested that environmental factors contribute to PD development and, thus, it seems only logical that toxins entering the body through food or water consumption could affect the neurons there first.
Environmental factors, however, played no role during the Belgian researchers' study. “By recruiting couples we could exclude environmental factors, such as diet and environmental toxins, as these couples are all exposed to the same environment. This is not standard in this kind of study and to our knowledge, we are the first group that used this strategy,” An-Sofie shares.
In a next step, the scientists monitored the activity of ENS neurons in the biopsy samples, using calcium imaging. “Calcium is one of the most important cell regulators and its signalling is extremely useful to monitor cell activity,” explains Carla. “The technique is based on a fluorescent probe, which enters the neuron and, when in contact with calcium ions, generates a fluorescent signal. The intensity of the signal can be measured by microscopy and translated into the activity state of the neuron,” An-Sofie adds.
There is, however, one disadvantage: fresh biopsy samples are autofluorescent. The group decided, nevertheless, to use this difficult technique because they knew already from another study on patients with GI disorders that if the neurons are less functional, the signal is lower. “Our hypothesis was that if the neurons in the ENS of PD patients would be affected, the intensity increase after stimulation would be lower in neurons from PD patients compared to control subjects,” An-Sofie says. Surprisingly, this was not what they found. There was no significant difference between the neuronal activity of PD patients and their partners. “Personally, I would have expected lower neuronal firing in biopsies from PD patients,” Carla says.
So, how could these unexpected findings be explained? Perhaps all unhealthy neurons died and, therefore, do not react to stimulation anymore? To find out, the researchers counted the numbers of ganglia and neurons in the samples. “Already, other research groups have investigated neuronal loss in the ENS but until now there was no clear answer as there is some controversy in these results,” explains An-Sofie. In the Belgian researchers' samples, the number of neurons and ganglia in the PD patients and their healthy partners was basically the same. Carla Cirillo recalls, “We did not know what to expect and, actually, I was not surprised when it turned out that the number of neurons and ganglia was not different in the two cohorts of subjects. This means that, at least in the population of PD patients enrolled in our study, the enteric nervous system is not affected in its quantity of neurons.” Although this result crushed another of their hypotheses, they could be sure that all gut neurons are still functional.
What else did the group have up their experimental sleeves? For the project, Carla, An-Sofie and co. developed a new technique to measure mitochondrial activity in the ENS of PD patients. Looking at the small cell factories is interesting because mitochondria are known to play a role in PD. “We were able to visualise the mitochondria in living neurons and, here again, we did not find a difference between PD patients and control subjects. This approach was never used before, especially in combination with live imaging in human neurons,” An-Sofie says.
Although this study might sound like a lot of negative results only, it did actually show something: “Our live imaging techniques showed for the first time that the ENS was not functionally affected in PD patients. However, this was only done on a small subset of patients and, therefore, more research is necessary to understand the role of the ENS in PD,” explains An-Sofie Desmet. Her supervisor also points out that they should take their results with caution. “We have to admit that our study has a few limitations that deserve to be tackled in future studies. For example, it would be ideal to study a bigger population of PD patients and to focus on well-defined PD subtypes.”
For the moment, however, the group is focussing on other aspects of the ENS, from developmental biology to degeneration – everything that can be studied by microscopy. An-Sofie is crazy about the technique, “You can make such nice pictures from stainings that it should be sold as art!”
Besides An-Sofie, who is currently finishing her PhD, there are four PhD students and three postdocs in the group, coming from countries like China, Syria, Australia and Italy. An-Sofie herself is Belgian, she studied biomedical sciences at KU Leuven and then did an internship in the Laboratory for Enteric NeuroScience (LENS). “During my internship, I came across the hypothesis that the ENS could be the starting point for Parkinson’s disease and this hypothesis prompted me to continue in the field. However, I decided not to continue in academia after finishing my PhD and to start a job in industry,” Ann-Sofie admits.
Postdoc Carla Cirillo has the same plan. She is just finishing her postdoc in a hosting lab in France. “After that I may be leaving academia to find a job in industry or to re-orientate my expertise toward a new job experience,” she says. But she has enjoyed her years in academia, working on the gastrointestinal tract. “To me, the fun fact of the gastrointestinal tract is its fantastic resemblance to the brain - at least in my eyes!”
Photo: Pixabay/MabelAmber & Vanden Berghe group