Joshua M. Kaplan

Mutations in ACTN4, encoding α-actinin-4, cause familial focal segmental glomerulosclerosis

Focal and segmental glomerulosclerosis (FSGS) is a common, non-specific renal lesion. Although it is often secondary to other disorders, including HIV infection, obesity, hypertension and diabetes, FSGS also appears as an isolated, idiopathic condition. FSGS is characterized by increased urinary protein excretion and decreasing kidney function. Often, renal insufficiency in affected patients progresses to end-stage renal failure, a highly morbid state requiring either dialysis therapy or kidney transplantation. Here we present evidence implicating mutations in the gene encoding α-actinin-4 (ACTN4; ref. 2), an actin-filament crosslinking protein, as the cause of disease in three families with an autosomal dominant form of FSGS. In vitro, mutant α-actinin-4 binds filamentous actin (F-actin) more strongly than does wild-type α-actinin-4. Regulation of the actin cytoskeleton of glomerular podocytes may be altered in this group of patients. Our results have implications for understanding the role of the cytoskeleton in the pathophysiology of kidney disease and may lead to a better understanding of the genetic basis of susceptibility to kidney damage.

Systematic analysis of genes required for synapse structure and function

Chemical synapses are complex structures that mediate rapid intercellular signalling in the nervous system. Proteomic studies suggest that several hundred proteins will be found at synaptic specializations. Here we describe a systematic screen to identify genes required for the function or development of Caenorhabditis elegans neuromuscular junctions. A total of 185 genes were identified in an RNA interference screen for decreased acetylcholine secretion; 132 of these genes had not previously been implicated in synaptic transmission. Functional profiles for these genes were determined by comparing secretion defects observed after RNA interference under a variety of conditions. Hierarchical clustering identified groups of functionally related genes, including those involved in the synaptic vesicle cycle, neuropeptide signalling and responsiveness to phorbol esters. Twenty-four genes encoded proteins that were localized to presynaptic specializations. Loss-of-function mutations in 12 genes caused defects in presynaptic structure.

Facilitation of synaptic transmission by EGL-30 Gqalpha and EGL-8 PLCbeta: DAG binding to UNC-13 is required to stimulate acetylcholine release.

We show that neurotransmitter release at Caenorhabditis elegans neuromuscular junctions is facilitated by a presynaptic pathway composed of a Gqalpha (EGL-30), EGL-8 phospholipase Cbeta (PLCbeta), and the diacylglycerol- (DAG-) binding protein UNC-13. Activation of this pathway increased release of acetylcholine at neuromuscular junctions, whereas inactivation decreased release. Phorbol esters stimulated acetylcholine release, and this effect was blocked by a mutation that eliminates phorbol ester binding to UNC-13. Expression of a constitutively membrane-bound form of UNC-13 restored acetylcholine release to mutants lacking the egl-8 PLCbeta. Activation of this pathway with muscarinic agonists caused UNC-13 to accumulate in punctate structures in the ventral nerve cord. These results suggest that presynaptic DAG facilitates synaptic transmission and that part of this effect is mediated by UNC-13.

Modulation of serotonin-controlled behaviors by Go in Caenorhabditis elegans

Seven transmembrane receptors and their associated heterotrimeric guanine nucleotide-binding proteins (G proteins) have been proposed to play a key role in modulating the activities of neurons and muscles. The physiological function of the Caenorhabditis elegans G protein Go has been genetically characterized. Mutations in the goa-1 gene, which encodes an alpha subunit of Go (G alpha o), cause behavioral defects similar to those observed in mutants that lack the neurotransmitter serotonin (5-HT), and goa-1 mutants are partially resistant to exogenous 5-HT. Mutant animals that lack G alpha o and transgenic animals that overexpress G alpha o [goa-1(xs) animals] have reciprocal defects in locomotion, feeding, and egg laying behaviors. In normal animals, all of these behaviors are regulated by 5-HT. These results demonstrate that the level of Go activity is a critical determinant of several C. elegans behaviors and suggest that Go mediates many of the behavioral effects of 5-HT.

Serotonin Inhibition of Synaptic Transmission

We show that serotonin inhibits synaptic transmission at C. elegans neuromuscular junctions, and we describe a signaling pathway that mediates this effect. Release of acetylcholine from motor neurons was assayed by measuring the sensitivity of intact animals to the acetylcholinesterase inhibitor aldicarb. By this assay, exogenous serotonin inhibited acetylcholine release, whereas serotonin antagonists stimulated release. The effects of serotonin on synaptic transmission were mediated by GOA-1 (a Galpha0 subunit) and DGK-1 (a diacylglycerol [DAG] kinase), both of which act in the ventral cord motor neurons. Mutants lacking goa-1 G(alpha)0 accumulated abnormally high levels of the DAG-binding protein UNC-13 at motor neuron nerve terminals, suggesting that serotonin inhibits synaptic transmission by decreasing the abundance of UNC-13 at release sites.

Ubiquitin and AP180 Regulate the Abundance of GLR-1 Glutamate Receptors at Postsynaptic Elements in C. elegans

Regulated delivery and removal of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors (GluRs) from postsynaptic elements has been proposed as a mechanism for regulating synaptic strength. Here we test the role of ubiquitin in regulating synapses that contain a C. elegans GluR, GLR-1. GLR-1 receptors were ubiquitinated in vivo. Mutations that decreased ubiquitination of GLR-1 increased the abundance of GLR-1 at synapses and altered locomotion behavior in a manner that is consistent with increased synaptic strength. By contrast, overexpression of ubiquitin decreased the abundance of GLR-1 at synapses and decreased the density of GLR-1-containing synapses, and these effects were prevented by mutations in the unc-11 gene, which encodes a clathrin adaptin protein (AP180). These results suggest that ubiquitination of GLR-1 receptors regulates synaptic strength and the formation or stability of GLR-1-containing synapses.

LIN-10 Is a Shared Component of the Polarized Protein Localization Pathways in Neurons and Epithelia

We tested the model that neurons and epithelial cells use a shared mechanism for polarized protein sorting by comparing the pathways for localizing basolateral and postsynaptic proteins in C. elegans. GLR-1 glutamate receptors are localized to postsynaptic elements of central synapses and, when ectopically expressed, to basolateral membranes of epithelial cells. Proper localization of GLR-1 in both neurons and epithelia requires the PDZ protein LIN-10, defining LIN-10 as a shared component of the basolateral and postsynaptic localization pathways. Changing the GLR-1 carboxy-terminal sequence from a group I PDZ-binding consensus (-TAV) to a group II consensus (-FYV) restores GLR-1 synaptic localization in lin-10 mutants. Thus, these interneurons utilize at least two separate postsynaptic localization pathways.

PKC-1 regulates secretion of neuropeptides.

The secretion of neurotransmitters and neuropeptides is mediated by distinct organelles—synaptic vesicles (SVs) and dense-core vesicles (DCVs), respectively. Relatively little is known about the factors that differentially regulate SV and DCV secretion. Here we show that protein kinase C-1 (PKC-1), which is most similar to the vertebrate PKC η and ε isoforms, regulates exocytosis of DCVs in Caenorhabditis elegans motor neurons. Mutants lacking PCK-1 activity had delayed paralysis induced by the acetylcholinesterase inhibitor aldicarb, whereas mutants with increased PKC-1 activity had more rapid aldicarb-induced paralysis. Imaging and electrophysiological assays indicated that SV release occurred normally in pkc-1 mutants. By contrast, genetic analysis of aldicarb responses and imaging of fluorescently tagged neuropeptides indicated that mutants lacking PKC-1 had reduced neuropeptide secretion. Similar neuropeptide secretion defects were found in mutants lacking unc-31 (encoding the protein CAPS) or unc-13 (encoding Munc13). These results suggest that PKC-1 selectively regulates DCV release from neurons.

CaMKII regulates the density of central glutamatergic synapses in vivo.

Synaptic connections undergo a dynamic process of stabilization or elimination during development, and this process is thought to be critical in memory and learning and in establishing the specificity of synaptic connections. The type II calcium- and calmodulin-dependent protein kinase (CaMKII) has been proposed to be pivotal in regulating synaptic strength and in maturation of synapses during development. Here we describe how CaMKII regulates the formation of central glutamatergic synapses in Caenorhabditis elegans. During larval development, the density of ventral nerve cord synapses containing the GLR-1 glutamate receptor is held constant despite marked changes in neurite length. The coupling of synapse number to neurite length requires both CaMKII and voltage-gated calcium channels. CaMKII regulates GLR-1 by at least two distinct mechanisms: regulating transport of GLR-1 from cell bodies to neurites; and regulating the addition or maintenance of GLR-1 to postsynaptic elements.

The Anaphase-Promoting Complex Regulates the Abundance of GLR-1 Glutamate Receptors in the Ventral Nerve Cord of C. elegans

The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that targets key cell cycle regulatory proteins for degradation. Blockade of APC activity causes mitotic arrest. Recent evidence suggests that the APC may have roles outside the cell cycle. Several studies indicate that ubiquitin plays an important role in regulating synaptic strength. We previously showed that ubiquitin is directly conjugated to GLR-1, a C. elegans non-NMDA (N-methyl-D-aspartate) class glutamate receptor (GluR), resulting in its removal from synapses. By contrast, endocytosis of rodent AMPA GluRs is apparently regulated by ubiquitination of associated scaffolding proteins. Relatively little is known about the E3 ligases that mediate these effects. We examined the effects of perturbing APC function on postmitotic neurons in the nematode C. elegans. Temperature-sensitive mutations in APC subunits increased the abundance of GLR-1 in the ventral nerve cord. Mutations that block clathrin-mediated endocytosis blocked the effects of the APC mutations, suggesting that the APC regulates some aspect of GLR-1 recycling. Overexpression of ubiquitin decreased the density of GLR-1-containing synapses, and APC mutations blunted this effect. APC mutants had locomotion defects consistent with increased synaptic strength. This study defines a novel function for the APC in postmitotic neurons.