Identification of NO synthase and roles for NO in brain and skeletal muscle While a graduate student in Prof. Solomon Snyder's lab, Bredt discovered and characterized the family of enzymes that generate
nitric oxide (NO). Whereas a single measurement of endogenous NO had previously required complex and laborious methods, Bredt developed a simple, sensitive, and specific assay that monitored the conversion of [3H]
arginine to [3H]
citrulline. This assay enabled hundreds or thousands of daily measurements of endogenous NO. He first employed this assay to discover that endogenous NO mediates
glutamate-linked increases of
cyclic GMP in brain (
PNAS, 1989). He then biochemically isolated the biosynthetic enzyme, which he named
nitric oxide synthase (NOS). In addition, he determined that NOS is a
calmodulin-dependent enzyme (
PNAS, 1990), which explained how NO is generated rapidly following glutamate-mediated increases in synaptic calcium. Bredt performed studies that established NO as a diffusible neurotransmitter. He molecularly cloned and sequenced the first NOS cDNA (
Nature, 1991), which showed that the N-terminal half of NOS protein is unique and is separated by a calmodulin-binding domain from the C-terminal half, which resembles
cytochrome P-450 reductase (
Nature, 1991). This protein structure revealed how calmodulin regulates NOS enzyme activity and how
redox co-factors enable the complex enzymology of NOS. He
immunohistochemically mapped NOS distribution in brain and peripheral tissues (
Nature, 1990). He determined that neurons are the primary source of NO throughout the body. In the brain, he found that NOS found in neurons (nNOS) was enriched in specific neuronal populations, and often concentrated at
postsynaptic sites, such as
cerebellar glomerular synapses, where
glutamate receptors activate nNOS. In the
peripheral nervous system, he found nNOS enriched in non-
adrenergic, non-
cholinergic neurons that innervate gastrointestinal and vascular
smooth muscle (
Nature, 1990). These findings opened a gateway to investigating how NO participates in diverse physiology processes including aspects of
peristalsis and
penile erection (
Science, 1992). As a professor at UCSF, Bredt identified the therapeutic potential of modulating NO in brain and skeletal muscle. In brain, Bredt and his team demonstrated that nNOS is enriched at synapses owing to a "PDZ" domain in nNOS that associates with a similar PDZ domain in the synaptic scaffolding protein postsynaptic density 95kD (PSD-95) (
Cell, 1996). They showed that PSD-95 physically and functionally links nNOS with
NMDA-type
glutamate receptors at synapses. As
excitotoxic neuronal death associated with
cerebral ischemia involves excessive NO production downstream of
NMDA receptors (
PNAS, 1991), Bredt's research pointed to exploitation of the antagonism of the NMDA receptor/PSD-95/nNOS complex as a stroke treatment (
Journal of Biological Chemistry, 1999), which has shown promising clinical outcomes (
Nature Reviews Drug Discovery, 2020). In addition to finding nNOS in neurons, Bredt helped identify nNOS on skeletal muscle
sarcolemma (
Nature, 1994), and his team determined that nNOS in muscle associates with
dystrophin (
Cell, 1995). They discovered a selective loss of nNOS from skeletal muscle sarcolemma in patients with
Duchenne and Becker dystrophies, which involve dystrophin mutations (
Journal of Experimental Medicine, 1996). Bredt's team discovered the mechanism by which nNOS is lost from skeletal muscle sarcolemma by identifying that the
PDZ domain in nNOS binds to the PDZ in the dystrophin-associated protein
syntrophin (
Cell, 1996). Through collaboration, they determined the structural correlate to this unexpected PDZ-PDZ interaction (
Science, 1999). Today, restoration of NO bioactivity in muscular dystrophies remains a key therapeutic goal.
Protein scaffolding and palmitoylation underlying synaptic signaling and plasticity Bredt has made contributions to understanding the molecular organization and stability of
excitatory synapses. Biochemical studies by Bredt's team determined that
PSD-95 is amongst the most abundant
palmitoylated proteins in brain (
Neuron, 1998), and that palmitoylation localizes PSD-95 to synaptic sites (
Neuron, 1999). They found that synaptic function is regulated dynamically by palmitate cycling on PSD-95 (
Cell, 2002). They characterized a family of 24 palmitoyl-transferase enzymes and identified those responsible for regulating PSD-95 (
Neuron, 2004). They found that palmitoylated PSD-95 powerfully regulates maturation of excitatory synapses and enhances AMPA receptor clustering (
Science, 2000). These discoveries are central to current models of synaptic development, anatomy, and plasticity.
TARPs as auxiliary subunits for a neurotransmitter receptor Bredt, together with Prof. Roger Nicoll, discovered and characterized the first auxiliary subunits for mammalian glutamate neurotransmitter receptor. Bredt and Nicoll determined that a then recently discovered protein,
stargazin, links PSD-95 to the
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subclass of
glutamate receptors (
Nature, 2000). Stargazin is one member of a family of related proteins, which Bredt termed transmembrane AMPAR regulatory proteins (TARPs), that are differentially distributed in brain (
Journal of Cell Biology, 2003). Bredt and Nicoll found that stargazin and related TARPs regulate the synaptic targeting (
Nature, 2000), gating (
Nature, 2005), and pharmacology (
PNAS, 2006) of all AMPA receptors. TARPs not only mediate synaptic function but also bidirectionally regulate
synaptic plasticity (
Neuron, 2005). This led to the conceptual breakthrough that TARPs are AMPA receptor auxiliary subunits (
PNAS, 2005). This model was initially met with resistance, but it is now recognized that
ionotropic receptors for many neurotransmitters have auxiliary subunits.
NACHO as a chaperone for a mammalian neurotransmitter receptor Bredt's team at Janssen assessed whether analogous protein accessories might enable drug discovery for previously intractable and medically-important
nicotinic acetylcholine receptors (nAChRs). Whereas
cDNAs encoding nAChRs were discovered in the 1980s, most nAChR subtypes could not be expressed functionally in cell lines. In a study, Bredt's team used an innovative genome-wide cloning strategy to search systemically for neuronal proteins that could reconstitute the α7 nAChR subtype. They found that assembly of α7 nAChRs requires a novel
endoplasmic reticulum protein that Bredt named "nAChR regulator chaperone" (NACHO) (
Neuron, 2016), the first client-specific
chaperone for a mammalian neurotransmitter receptor family (
Cell Reports, 2017). His team found that NACHO engages N‐
glycosylation and
endoplasmic reticulum chaperone pathways for
Alpha-7 nicotinic receptor oligomerization and
membrane trafficking (
Cell Reports, 2020). By exploiting their genome-wide screening paradigm, they also found an array of other neuronal proteins including Bcl-2 (
Nature Communications, 2019),
spermidine/
spermine N1-acetyltransferase (
Nature Communications, 2020) and BARP (
Cell Reports, 2019) that conspire with NACHO for functional expression of diverse nACh receptors in brain and peripheral tissues. These discoveries are now enabling biochemical and pharmaceutical studies of limbic α6-containing receptors for psychiatric indications (
Cell Reports, 2019), sensory α6-containing receptors for chronic pain (
Journal of Clinical Investigation, 2020) and cochlear α9α10 receptors for auditory disorders (
PNAS, 2020). Taken together, Bredt's discoveries have illuminated unanticipated mediators and mechanisms for
neuronal communication. His conceptional advances in neurotransmitter receptor biology have translated into new approaches for treating neuromuscular, neurological, and neuropsychiatric disorders. His papers have been cited more than 75,000 times in the literature. ==Cassava Sciences==