University of Queensland researchers have used super-resolution microscopy to observe key molecules at work inside living brain cells, further unravelling the puzzle of memory formation and the elusive causes of dementia.
UQ Queensland Brain Institute’s Clem Jones Centre for Ageing and Dementia Research Professors Frédéric Meunier and Jürgen Götz found a protein, Tau, involved in Alzheimer’s disease affects the organisation of the signalling protein Fyn, which plays a critical role in memory formation.
“One of the distinguishing features of Alzheimer’s disease is the tangles of Tau protein that form inside brain cells, but this is the first time anyone has demonstrated that Fyn nanoclustering is affected by Tau,” Professor Götz said.
Professor Meunier said single molecule imaging in living brain cells allowed unprecedented access to the organisation of key proteins in small nanoclusters that were not detectable previously.
“We have shown that Tau controls the Fyn nanoclustering in dendrites, where the communication between brain cells occurs,” Professor Meunier said.
“When Tau is mutated, Fyn makes aberrantly large clusters, thereby altering nerve signals and contributing to dysfunction of the synapse-junctions between nerve cells.”
The signalling protein Fyn moving and forming clusters in living brain cells – viewed using super-resolution microscopy. The image is credited to Meunier Lab, University of Queensland.
Professor Meunier’s team used the super-resolution single molecule imaging technique to see how Tau and its mutants control Fyn nanoclustering.
Professor Meunier went on to investigate a different mutant of Tau found in families with a very high risk of developing frontotemporal dementia and found that Fyn was over-clustered in the spines of dendrites.
“Imagine that you have clustering of Fyn, a signalling molecule, throughout your life; it’s going to give rise to an over-signalling problem — this could be one of the ways in which Fyn is toxic to cells,” he said.
“The spines of the dendrites are critical to how nerve cells communicate with each other and underpin memory and learning.”
Exactly what causes Alzheimer’s and other forms of dementia is still a mystery, but Fyn is linked to both the plaques of amyloid protein that form between brain cells, and tangles of Tau protein that form inside brain cells — two distinguishing features of Alzheimer’s disease.
“Super-resolution single molecule imaging gives us an unprecedented insights into what is happening in living nerve cells, with the aim of understanding the biology behind these complex and debilitating diseases,” Professor Meunier said.
Funding: The study was published in the journal eLife and supported by organisations including the Australian and Queensland governments, the National Health and Medical Research Council of Australia and the Australian Research Council.
Dendritic spines compartmentalize biochemical reactions that are critical for synaptic plasticity, which underpins memory and learning.
A myriad of signaling molecules acts in a spatiotemporally controlled manner to translate the information encoded in post-synaptic calcium influx into appropriate changes in synaptic strength during learning (Nishiyama and Yasuda, 2015).
A central signaling role in the spine is assumed by the tyrosine kinase Fyn, a member of the Src family, which is widely expressed throughout the brain (Ohnishi et al., 2011).
Fyn is myristoylated, a process that occurs co-translationally on free ribosomes, and is subsequently palmitoylated, which enhances the hydrophobicity of the molecule and membrane association (Sato et al., 2009).
The synaptic scaffolding protein PSD-95 is also palmitoylated and known to bind membrane-associated Fyn, recruiting it into the proximity of the GluN2B subunit of the NMDA receptor (NMDAR), thereby enhancing Fyn-mediated phosphorylation of GluN2B (Tezuka et al., 1999).
This results in an increased stability of the NMDAR complex at the synaptic membrane, which is critical for synaptic plasticity (Prybylowski et al., 2005).
Fyn acts as a molecular hub that interacts with multiple synaptic proteins and controls major signaling pathways (Nishiyama and Yasuda, 2015).
A functional role for Fyn in the dendritic compartment is underscored by the finding that Fyn knockout mice exhibit a decreased spine density in layer V pyramidal neurons (Morita et al., 2006), reduced axonal branching in the cerebellar cortex (Cioni et al., 2013), and deficits in long-term potentiation and spatial learning (Grant et al., 1992).
Fyn requires the microtubule-associated protein Tau, a protein that is implicated in neurodegenerative diseases including Alzheimer’s disease (AD) and frontotemporal dementia (FTD), for its efficient targeting to the dendritic compartment (Ittner et al., 2010; Xia et al., 2015).
Further, increased Tau expression (in particular that of mutant Tau forms found in FTD, such as P301L Tau) is associated with increased synaptic localization, not only of Tau but also of Fyn (Ittner et al., 2010; Xia et al., 2015; Hoover et al., 2010).
Fyn acts as a key mediator of two central molecules in AD, amyloid-β (Aβ) and Tau, which forms amyloid plaques and neurofibrillary tangles, respectively, with Aβ lying upstream of Tau in the pathocascade (Götz et al., 2001).
Aβ acts on Fyn via multiple receptors that are located on the plasma membrane, including the prion protein PrPc (Um et al., 2012; Larson et al., 2012; Um et al., 2013). Activated Fyn phosphorylates NMDARs and mediates interactions between NMDAR and PSD-95, which are required for Aβ-induced excitotoxicity (Um et al., 2012).
Fyn further mediates Aβ-induced local protein translation and the accumulation of Tau in the somatodendritic compartment by activating the ERK/rpS6 signaling pathway (Li and Götz, 2017). Fyn overexpression also accelerates cognitive impairment (Chin et al., 2005; Kaufman et al., 2015), whereas depleting Fyn or inhibiting its activity restores memory function and synaptic density in AD model mice (Chin et al., 2004).
How Fyn integrates these diverse signals in subcellular compartments such as spines is currently unknown.
It is difficult to conceive how individual Fyn molecules can act as a nexus that is capable of integrating such a variety of signals. Whether Fyn is spatiotemporally organized to mediate efficient signal transduction remains to be established.
The spatial organization of receptors and signaling molecules into nanodomains in biological membranes is emerging as an essential feature of cell signaling (Kusumi et al., 2012).
These nanodomains are formed by a combination of protein–protein, lipid–lipid, protein–lipid and cytoskeletal interactions (Milovanovic and Jahn, 2015; Goyette and Gaus, 2017; Padmanabhan et al., 2019), as well as by membrane-mediated forces (Johannes et al., 2018).
Consequently, these nanodomains concentrate various molecules in discrete areas, thereby facilitating efficient and robust processing of cellular information by regulating a complex series of biochemical reactions (Harding and Hancock, 2008).
Indeed, a recent study has demonstrated that ligand-induced CD36 receptor clustering promotes Fyn activation within these clusters in non-neuronal cells (Githaka et al., 2016). Whether Fyn concentrates in such nanodomains in the dendrites, and whether Tau regulates the nanoscale organization of Fyn, is not known but such mechanisms could underlie the pleiotropic roles that Fyn assumes in neuronal signaling.
The advent of super-resolution microscopy has paved the way for investigations of the nanoscale organization and dynamic behavior of receptors (Nair et al., 2013; Hoze et al., 2012), as well as for studies on their signaling (Lu et al., 2014), trafficking (Joensuu et al., 2016) and scaffolding molecules (Chamma et al., 2016).
Here, we used single-particle tracking photoactivated localization microscopy (sptPALM) to determine whether Tau controls the organization of Fyn in the somatodendritic compartment.
We found that dendritic Fyn displayed a nanocluster organization that is underpinned by multiple mobility states, and that Fyn mobility significantly decreased in dendrites with neuronal maturation.
In neurons from Tau knockout (Tau KO) mice, Fyn mobility increased in the dendritic shafts, an effect that was rescued by the re-expression of wildtype (WT) Tau. More importantly, pathological P301L mutant Tau, as found in familial FTD, but not a truncated form of Tau lacking the microtubule-binding domain (ΔTau), promoted the trapping of Fyn in the dendritic spines. Our study therefore reveals a complex interplay between Fyn and Tau in the somatodendritic compartment and points to a novel role of altered Fyn nanoclustering in causing synaptic dysfunction in disease.
University of Queensland
Jane Ilsley – University of Queensland
The image is credited to Meunier Lab, University of Queensland.
Original Research: Open access
“Frontotemporal dementia mutant Tau promotes aberrant Fyn nanoclustering in hippocampal dendritic spines”. Pranesh Padmanabhan, Ramón Martínez-Mármol, Di Xia, Jürgen Götz Is a corresponding author , Frédéric A Meunier Is a corresponding author.