Perineuronal nets (PNNs) are conspicuous neural extracellular matrix (ECM) structures that have garnered
significant interest over the last decade for the critical roles they play in neural developmental plasticity. These
complex macromolecular structures are implicated in an array of cognitive functions, and are altered in a variety
of neurological disorders. Despite the growing interest in PNN functions, the mechanisms by which they
modulate neural functions are poorly understood, because there are currently no tools or techniques to
manipulate PNNs specifically. We surmise that our inability to target and disrupt PNNs is primarily driven by a
lack of understanding of their molecular composition or structure. Our goal in this proposal is to conduct a
structure-function analysis of known PNN components as well as to identify proteins that anchor nets to neuronal
surfaces. Using a powerful combination of in vitro and in vivo approaches, we have obtained strong preliminary
data detailing how the newly identified PNN component receptor protein tyrosine phosphatase zeta (RPTP¿)
associates with tenascin-R (TNR) within PNNs at a molecular level. Furthermore, our data indicate that the
RPTP¿•TNR complex anchors PNNs to the neuronal cell surface via the GPI-linked protein contactin-1 (CNTN1),
which makes CNTN1 the first surface binding protein for PNNs ever identified. Our central hypothesis is that
there are a set of unique components and receptors of PNNs that nucleate PNNs and anchor them to specific
neuronal cell surfaces, thereby defining their unique structure and functions. The overall objective of this proposal
is to identify PNN-specific components and dissect the formation of PNNs through a unique combination of
proximity-labeling assays, protein-binding assays, and protein X-ray crystallography in order to create the tools
to target and manipulate these structures specifically and precisely. Our long-term goal is then to use these tools
to dissect PNN function in order to better understand disease pathogenesis and ultimately to target PNNs
therapeutically. Guided by our strong preliminary data, this proposal seeks to discover the unique components
that guide the assembly of PNNs by pursuing three non-overlapping specific aims: 1) defining the role of the
RPTP¿•TNR complex in anchoring PNNs to neuronal surfaces; 2) pursuing the biochemical and structural
characterization of interactions between ACAN, HAPLN1, and TNR; and 3) identifying cell surface receptors and
novel components of PNNs. The proposed work is significant because it will attempt to identify the key unique
components that contribute to the formation and thereby function of PNNs. Successful completion of the aims
will provide key insights and reagents to manipulate PNNs specifically and precisely and ultimately understand
their functional mechanisms. This approach is innovative because it brings together a novel combination of
physiological, biochemical and structural approaches to investigate these important macromolecular assemblies
in the central nervous system. Ultimately, the proposed work could be transformative for the field and lead to key
mechanistic insights into of PNN function in health and disease.