In Alzheimer’s disease (AD), amyloid plaques and tau tangles are classic hallmark pathologies, but another important change that contributes to cognitive decline is the loss of synapses. Synapses are points of connection between neurons that support the transfer and receipt of information between them. They are essential for memory, learning, and controlling the body. When synapses are damaged or lost, as occurs in AD, communication between neurons is disrupted, and cognition deteriorates.
Synapse stability is in part regulated by specialized scaffolding proteins, which provide structure to the synapse and organize the molecules necessary for signaling and communication. One key group of synaptic proteins is kinases, enzymes that attach a chemical tag, called a phosphate group, to other proteins. This process, called phosphorylation, can alter how proteins behave. Scaffolding proteins help localize and position kinases close to their target proteins so that cellular signals are transmitted efficiently. Kinases can also phosphorylate scaffolding proteins themselves, further influencing synaptic structure and function. This tightly coordinated system of scaffolds, kinases, and phosphorylated proteins is essential for healthy brain activity. In AD, however, disruptions in kinase activity and scaffolding protein stability, which are potentially influenced by amyloid and tau pathology, undermine synaptic communication. Understanding these mechanisms may reveal new therapeutic targets for intervention in AD.
Dr. Newton and her collaborator, Dr. Kim Dore, are investigating how amyloid pathology contributes to synaptic dysfunction in AD, with a particular focus on protein kinase C alpha (PKCα), a member of the PKC family of enzymes, and two scaffolding proteins: PSD-93 and IQGAP1. Their work builds on previous work with Dr. Rudy Tanzi, Chair of the CureAlz Research Leadership Group, which revealed that rare genetic variations in PKCα contribute to AD risk. They found that increased PKCα activity impaired cognition in healthy mice and worsened synapse loss and memory problems in amyloid mouse models. To identify the underlying molecular mechanisms, Newton’s team identified the PKC⍺ proteome (i.e., the complete set of proteins phosphorylated by PKCα) in mice and cells. This analysis revealed numerous candidate proteins that could mediate PKCα’s negative effects on synapses. Scaffolding proteins emerged as a prominent category, with PSD-93 and IQGAP1 being particularly compelling targets. Supporting PSD-93’s relevance, Dr. Tanzi and the Alzheimer’s Genome Project identified a rare variant in the gene associated with AD. Based on these findings, Drs. Newton and Dore hypothesize that PSD-93 and IQGAP1 coordinate PKCα signaling at the synapse downstream of amyloid pathology, ultimately leading to synaptic and cognitive impairments.
They proposed three aims. In the first aim, they are establishing whether the scaffolding proteins IQGAP1 and PSD-93 directly bind to PKCα and other key synaptic signaling proteins. They are labeling IQGAP1, PSD-93, and PKC⍺ with fluorescent tags and monitoring their binding to each other in real time in live cultured cells. They are also using biochemical approaches (co-immunoprecipitation and peptide arrays) to identify which specific regions (or domains) of these proteins are key for their binding interactions with each other. Further, they are examining whether the interactions of PKCα with IQGAP1 or PSD-93 are sensitive to phosphorylation by PKCα. In the second aim, they are testing whether toxic forms of amyloid activate PKCα to increase its binding with—and subsequent phosphorylation of—IQGAP1 and PSD-93. They hypothesize that the specific phosphorylation of these and other scaffold proteins by PKCα alters the structure and function of the synapse. They are using advanced imaging methods and fluorescent tags (FRET-FLIM) to measure binding between the proteins in hippocampal neurons cultured from amyloid (APP/PS1) or healthy mice. In the third aim, they are evaluating how PKCα activation of scaffold proteins IQGAP1 and PSD-93 changes synaptic function by measuring electrical signals in brain slices from both control and amyloid mice. They predict that increased PKCα activity weakens synaptic communication and that this effect is further exacerbated by increasing amyloid levels.
In the first year of funding, the team made significant progress in understanding how PKCα facilitates the organization and movement of other proteins at synapses. Live-cell imaging, co-immunoprecipitation, and FRET-FLIM techniques to study how proteins interact with each other demonstrated that PKCα binds to IQGAP1. Notably, they found that this binding does not rely on PKCα’s ability to phosphorylate IQGAP1, and that a small-molecule inhibitor of multiple members of the PKC family of enzymes can enhance PKCα interaction with IQGAP1. The specific regions mediating their interaction were mapped and identified to the PDZ ligand of PKCα and the IQ domain of IQGAP1. Using neurons from amyloid-model mice (APP/PS1), they discovered that the PKC inhibitor increases the movement of both PKCα and IQGAP1 into dendritic spines—the parts of neurons that receive signals. Taken together, these experiments suggest that PKCα’s role in AD may not involve its enzymatic activity on its binding partners (ex., IQGAP1), but rather its cellular localization and its ability to scaffold other proteins, which is modulated by general PKC-isoform activity. They observed similar changes with another scaffolding protein, PSD95. They are currently developing tagged versions of PSD93 and modified forms of IQGAP1 to explore how phosphorylation alters these interactions. In Year 2, to uncover how PKCα influences synaptic communication, the team will use mass spectrometry and electrophysiology to identify additional binding partners and characterize their functional impact. These studies offer new insights into how changes in PKCα may contribute to Alzheimer’s-related brain dysfunction.
