Periodic Reporting for period 1 - GLUGLU (Deciphering the internalization profiles of mGlu receptors)
Okres sprawozdawczy: 2023-09-01 do 2025-08-31
The brain functions by processing electrical and chemical signals, thanks to the presence of brain cells: neurons, the primary functional units, and glial cells, which provide support to neurons. For these connections to function properly, brain cells rely on cell surface proteins that act as receptors to external stimuli, facilitating communication between neurons at junctions called synapses.
In this context, metabotropic glutamate (mGlu) receptors are membrane proteins widely expressed in the human brain. Their main role is to modulate synaptic transmission, and they have been implicated in diseases such as Alzheimer’s disease, schizophrenia, and Parkinson’s disease. However, to date, no drugs targeting these receptors are available on the market to treat neurological diseases—primarily due to limited efficacy observed in clinical trials.
One possible explanation is the dynamic nature of mGlu receptors. Although they are classified as cell surface proteins, they are highly mobile and can translocate inside the cell—a process known as internalization—in response to stimuli. This behaviour can affect their availability at the cell surface and, consequently, their response to drugs. This is particularly relevant for chronic neurological disorders, where sustained drug action is often required.
Despite over 30 years of research on mGlu receptors, their internalization properties have remained poorly understood. Do mGlu receptors internalize in response to stimuli in brain cells? If so, which stimuli? What cellular machinery do they use to internalize?
Answering these questions is critical for designing more effective drugs that can target mGlu receptors in chronic brain diseases.
GLUGLU aimed to address these questions by combining two cutting-edge techniques mastered by the host team: nanobodies and time-resolved Förster Resonance Energy Transfer (TR-FRET).First, nanobodies are antibody fragments derived from camelids. Due to their small size, they can access tight spaces and selectively bind to a specific protein—or even to a specific conformation of a protein. Second, TR-FRET is based on the transfer of energy between two fluorescent molecules when they are in close proximity.
Thanks to the use of selective nanobodies for mGlu receptors tagged with FRET-compatible fluorophores, GLUGLU revealed for the first time how mGlu receptors are internalized in brain cells, as well as the pathways involved. By uncovering the internalization mechanisms of mGlu receptors for the first time, GLUGLU addresses a relevant barrier in the design of effective and long-lasting brain-targeted therapies.
In this context, metabotropic glutamate (mGlu) receptors are membrane proteins widely expressed in the human brain. Their main role is to modulate synaptic transmission, and they have been implicated in diseases such as Alzheimer’s disease, schizophrenia, and Parkinson’s disease. However, to date, no drugs targeting these receptors are available on the market to treat neurological diseases—primarily due to limited efficacy observed in clinical trials.
One possible explanation is the dynamic nature of mGlu receptors. Although they are classified as cell surface proteins, they are highly mobile and can translocate inside the cell—a process known as internalization—in response to stimuli. This behaviour can affect their availability at the cell surface and, consequently, their response to drugs. This is particularly relevant for chronic neurological disorders, where sustained drug action is often required.
Despite over 30 years of research on mGlu receptors, their internalization properties have remained poorly understood. Do mGlu receptors internalize in response to stimuli in brain cells? If so, which stimuli? What cellular machinery do they use to internalize?
Answering these questions is critical for designing more effective drugs that can target mGlu receptors in chronic brain diseases.
GLUGLU aimed to address these questions by combining two cutting-edge techniques mastered by the host team: nanobodies and time-resolved Förster Resonance Energy Transfer (TR-FRET).First, nanobodies are antibody fragments derived from camelids. Due to their small size, they can access tight spaces and selectively bind to a specific protein—or even to a specific conformation of a protein. Second, TR-FRET is based on the transfer of energy between two fluorescent molecules when they are in close proximity.
Thanks to the use of selective nanobodies for mGlu receptors tagged with FRET-compatible fluorophores, GLUGLU revealed for the first time how mGlu receptors are internalized in brain cells, as well as the pathways involved. By uncovering the internalization mechanisms of mGlu receptors for the first time, GLUGLU addresses a relevant barrier in the design of effective and long-lasting brain-targeted therapies.
1. Evaluation of mGlu receptor internalization in immortalized cells for prototype validation. mGlu receptors were expressed in immortalized cells and labelled with a FRET donor to monitor their internalization kinetics in real time. All receptor variants were constitutively internalized over time in the absence of an activator. However, we found that, among the 19 mGlu receptor (8 homodimers and 11 heterodimers), only 4 underwent internalization in the presence of an activator.
2. Decoding mGlu receptor internalization mechanisms in immortalized cells. We investigated the role of internalization adaptors for other membrane proteins in mGlu receptor internalization. While canonical adaptors do contribute, the role played in mGlu receptor internalization is different to other proteins from the same family.
3. Validation of nanobodies as tools for mGlu receptor internalization in immortalized cells. We selected for neutral nanobodies (nanobodies without activity) that selectively target mGlu receptors, to label them and follow internalization kinetics in immortalized cells. Nanobodies emerged as valuable tools for investigating mGlu receptor internalization of untagged (native) receptors.
4. Investigation of endogenous mGlu receptor internalization kinetics in glial cells. We used the validated nanobodies to evaluate the internalization kinetics of mGlu receptors in glial cells precedent from rat cortex. We show that mGlu receptors are internalizing in native tissue, making internalization a process to consider when designing long-lasting drugs targeting mGlu receptors.
2. Decoding mGlu receptor internalization mechanisms in immortalized cells. We investigated the role of internalization adaptors for other membrane proteins in mGlu receptor internalization. While canonical adaptors do contribute, the role played in mGlu receptor internalization is different to other proteins from the same family.
3. Validation of nanobodies as tools for mGlu receptor internalization in immortalized cells. We selected for neutral nanobodies (nanobodies without activity) that selectively target mGlu receptors, to label them and follow internalization kinetics in immortalized cells. Nanobodies emerged as valuable tools for investigating mGlu receptor internalization of untagged (native) receptors.
4. Investigation of endogenous mGlu receptor internalization kinetics in glial cells. We used the validated nanobodies to evaluate the internalization kinetics of mGlu receptors in glial cells precedent from rat cortex. We show that mGlu receptors are internalizing in native tissue, making internalization a process to consider when designing long-lasting drugs targeting mGlu receptors.
The main achievement of GLUGLU was to identify the non-canonical internalization mechanism of mGlu receptors in immortalized cells for the first time. The identification of the role played by these adaptors in mGlu receptor internalization is key to understand how these receptors are being internalized. Also, GLUGLU combined the use of FRET and nanobodies to create a tool that allows to follow endogenous mGlu receptor internalization kinetics in brain cells, and that could be expanded to other receptors in the future. Thus, GLUGLU pioneered the identification of native mGlu receptor internalization in real time.
Knowing the internalization properties of membrane receptors, and in particular, of mGlu receptors, takes special relevance within the context of brain disorders, where protein expression is often altered. Still, future work must reveal all the internalization adaptors that participate in mGlu receptor internalization, in order to fully understand the internalization mechanisms of these particular receptors, not only in immortalized cells but also in brain cells. This knowledge will pave the way for the design of mGlu receptor targeted drugs for neurological disorders.
Knowing the internalization properties of membrane receptors, and in particular, of mGlu receptors, takes special relevance within the context of brain disorders, where protein expression is often altered. Still, future work must reveal all the internalization adaptors that participate in mGlu receptor internalization, in order to fully understand the internalization mechanisms of these particular receptors, not only in immortalized cells but also in brain cells. This knowledge will pave the way for the design of mGlu receptor targeted drugs for neurological disorders.