The present work is part of an advanced project that points to characterize the molecular mechanisms of Dimethylfumarate (DMF), recently approved as a treatment for Multiple Sclerosis (MS). The DMF is supposed to exert anti-inflammatory effects, but its therapeutic action is unknown.
Our preliminary data showed that Ferritin uptake in microglia is greatly enhanced by DMF presence: as we proved, this phenomenon is due to a higher Transferrin Receptor expression on microglia surface caused by DMF. This observation could link the recent studies stating iron metabolism dysregulation in microglia of brains in sclerotic patients and the neuroprotective effects that DMF exercises: according to our conjectures, DMF would reestablish the damaged iron internalization by microglia, thus acting both as a neuroprotector and as a regulator of iron metabolism.
The action of this compound seems to be highly specific for Ferritin uptake in microglia: in the proposed research we will focus on the diffusion of ripples that are triggered only by the simultaneous presence of DMF and Ferritin. These ruffles are spherical waves, created by microglia, that spread on the surface of the culture dish from cells somas to a long distance. We think that these waves are due to a Calcium dependent phenomenon: our aim is to confirm this hypothesis.
To do so, we will analyze the dynamics of the ripples by detecting the Calcium levels in microglial cultures through the activity of fluorescent probes. This characterization will be done with a handmade innovative optical setup that we built in order to detect the collective dynamics of Calcium transients in a large area; an automatized software, that we wrote in Matlab, will recognize front waves and quantify their temporal behavior.
In this way, we will use multidisciplinary and complementary techniques to better understand the link between iron dysregulation in microglia and DMF anti-inflammatory properties.
In the first two parts of this project we established a direct correlation between DMF and iron metabolism: according to our data, DMF would be able to restore an impaired iron uptake in microglial cells by enhancing TfR1 expression on their surface and hence favoring Ferritin uptake. The internalization of this protein, mediated by this compound, is also distributed homogeneously in the cytoplasm, in a very different way to the classical one, reported in literature, in discrete vesicles. It is really important to notice that in literature there is no trace of the connection between DMF and iron: by further studying this phenomenon, we may be able to be the first ones to achieve this result.
In addition to this, we noticed that the Ferritin and DMF system acts as a biophysical switch that leads to the creation of spherical ripples propagating from microglial somas to their surroundings. These ripples and the massive Ferritin uptake with DMF are two highly related phenomena and are both probably dependent on Calcium, since the presence of Calcium chelator Bapta-AM turns off the protein internalization and the ruffles.
With the proposed research we will understand the role of these ripples in the action of DMF compound on iron uptake. Once again, it is remarkable to note that in literature there is no mention neither of these ripples nor of the connection between Calcium, DMF presence and iron uptake.
We will monitor Calcium transients in cultures both in presence and absence of DMF compound and Ferritin. We will use BV-2 cellular line or primary microglia polarized in different conditions (i.e. M0, M1 or M2 states).
We can rely on the use of a homemade optical setup, built in IIT CLNS laboratories, that we optimized to perform high resolution fluorescence Calcium imaging in large portions of the cultures. In details, a LED light source, whose focal plane is on the sample, excites the Calcium sensitive fluorophores (Fluo-4, Fura-2 or Oregon Green Bapta) loaded in the cells. The emission of the fluorescent molecules is imaged on the display of the computer through a sCMOS camera which allows high-speed readout (100 frames/s). The use of a low-power LED light minimizes the effects of fluorophores photo-bleaching and prevents the cells damage. During the experiment recording, the cells temperature is stabilized by a temperature controller and the release of chemical compounds (e.g. DMF or Ferritin) to the specimen is achieved through an automatized perfusion-aspiration system.
We synchronized LED light and camera recording with a Data AcQuisition (DAQ) board controlled by a Matlab code that we wrote by ourselves. The setup is also equipped with a 5x objective chosen to observe large areas (3.5 mm^2) of the cultures with high spatial resolution (1.3 micrometers), which is important in order to perform significant statistical analysis of a big number of cells.
Data will be collected as a series of frames caught by the camera and saved as Matlab files of matrices. Extracting the information from these images requires an extended analysis, but we built up an automated software (written in Matlab) that will facilitate this task. This software automatically detects front waves: in this way, it will be possible to produce quantitative measurements of ripples velocities, phases and phenomena related to their superposition (as beats or patterns formation). So, we will analyze the data by ourselves, without the manipulation of any external program.
The advantages of this experimental approach are multifold. Thanks to the wide field imaging, we will obtain the statistical properties of a big number of cells and, also, we will monitor the propagation of waves signals through a large area (thus having the behavior of collective dynamics). In addition to that, the fact of being home-made offers the possibility to change a single element according to the needs of the experiment. For example, we may change the field of view using different objectives, or we may use different fluorophores changing LED; other parameters (e.g. the recording speed, the size of the images, the LED power, etc.) can be adjusted to balance the image quality and a sufficient spatiotemporal resolution.
As can be noted, this research is based on a highly multidisciplinary approach: we will use notions and methods proper of biochemistry, biology and physics to deal with the serious medical problem of MS disease. Future perspectives of this work are aimed to deepening the molecular mechanisms of DMF as iron regulator for MS treatments.