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Biomedical subjects

Thomas L Schwarz

Publications and source records attributed to Thomas L Schwarz.

18 recordsLinked to original sources
PubMed2006

Deletion of Kv4.2 gene eliminates dendritic A-type K+ current and enhances induction of long-term potentiation in hippocampal CA1 pyramidal neurons.

Dendritic, backpropagating action potentials (bAPs) facilitate the induction of Hebbian long-term potentiation (LTP). Although bAPs in distal dendrites of hippocampal CA1 pyramidal neurons are attenuated when propagating from the soma, their amplitude can be increased greatly via downregulation of dendritic A-type K+ currents. The channels that underlie these currents thus may represent a key regulatory component of the signaling pathways that lead to synaptic plasticity. We directly tested this hypothesis by using Kv4.2 knock-out mice. Deletion of the Kv4.2 gene and a loss of Kv4.2 protein resulted in a specific and near-complete elimination of A-type K+ currents from the apical dendrites of CA1 pyramidal neurons. The absence of dendritic Kv4.2-encoded A-type K+ currents led to an increase of bAP amplitude and an increase of concurrent Ca2+ influx. Furthermore, CA1 pyramidal neurons lacking dendritic A-type K+ currents from Kv4.2 knock-out mice exhibited a lower threshold than those of wild-type littermates for LTP induction with the use of a theta burst pairing protocol. LTP triggered with the use of a saturating protocol, on the other hand, remained indistinguishable between Kv4.2 knock-out and wild-type neurons. Our results support the hypothesis that dendritic A-type K+ channels, composed of Kv4.2 subunits, regulate action potential backpropagation and the induction of specific forms of synaptic plasticity.

Action Potentials↗
PubMed2006

Bchs, a BEACH domain protein, antagonizes Rab11 in synapse morphogenesis and other developmental events.

BEACH proteins, an evolutionarily conserved family characterized by the presence of a BEACH (Beige and Chédiak-Higashi) domain, have been implicated in membrane trafficking, but how they interact with the membrane trafficking machinery is unknown. Here we show that the Drosophila BEACH protein Bchs (Blue cheese) acts during development as an antagonist of Rab11, a small GTPase involved in vesicle trafficking. We find that reduction in, or loss of, bchs function restores viability and normal bristle development in animals with reduced rab11 function, while reductions in rab11 function exacerbate defects caused by bchs overexpression in the eye. Consistent with a role for Bchs in modulating Rab11-dependent trafficking, Bchs protein is associated with vesicles and extensively colocalized with Rab11 at the neuromuscular junction (NMJ). At the NMJ, we find that rab11 is important for synaptic morphogenesis, as reductions in rab11 function cause increases in bouton density and branching. These defects are also suppressed by loss of bchs. Taken together, these data identify Bchs as an antagonist of Rab11 during development and uncover a role for these regulators of vesicle trafficking in synaptic morphogenesis. This raises the interesting possibility that Bchs and other BEACH proteins may regulate vesicle traffic via interactions with Rab GTPases.

Alleles↗
PubMed2006

Axonal transport of mitochondria requires milton to recruit kinesin heavy chain and is light chain independent.

Mitochondria are distributed within cells to match local energy demands. We report that the microtubule-dependent transport of mitochondria depends on the ability of milton to act as an adaptor protein that can recruit the heavy chain of conventional kinesin-1 (kinesin heavy chain [KHC]) to mitochondria. Biochemical and genetic evidence demonstrate that kinesin recruitment and mitochondrial transport are independent of kinesin light chain (KLC); KLC antagonizes milton's association with KHC and is absent from milton-KHC complexes, and mitochondria are present in klc (-/-) photoreceptor axons. The recruitment of KHC to mitochondria is, in part, determined by the NH(2) terminus-splicing variant of milton. A direct interaction occurs between milton and miro, which is a mitochondrial Rho-like GTPase, and this interaction can influence the recruitment of milton to mitochondria. Thus, milton and miro are likely to form an essential protein complex that links KHC to mitochondria for light chain-independent, anterograde transport of mitochondria.

Animals↗
PubMed2006

The kv4.2 potassium channel subunit is required for pain plasticity.

A-type potassium currents are important determinants of neuronal excitability. In spinal cord dorsal horn neurons, A-type currents are modulated by extracellular signal-regulated kinases (ERKs), which mediate central sensitization during inflammatory pain. Here, we report that Kv4.2 mediates the majority of A-type current in dorsal horn neurons and is a critical site for modulation of neuronal excitability and nociceptive behaviors. Genetic elimination of Kv4.2 reduces A-type currents and increases excitability of dorsal horn neurons, resulting in enhanced sensitivity to tactile and thermal stimuli. Furthermore, ERK-mediated modulation of excitability in dorsal horn neurons and ERK-dependent forms of pain hypersensitivity are absent in Kv4.2(-/-) mice compared to wild-type littermates. Finally, mutational analysis of Kv4.2 indicates that S616 is the functionally relevant ERK phosphorylation site for modulation of Kv4.2-mediated currents in neurons. These results show that Kv4.2 is a downstream target of ERK in spinal cord and plays a crucial role in pain plasticity.

Action Potentials↗
PubMed2006

Altered synaptic development and active zone spacing in endocytosis mutants.

Many types of synapses have highly characteristic shapes and tightly regulated distributions of active zones, parameters that are important to the function of neuronal circuits. The development of terminal arborizations must therefore include mechanisms to regulate the spacing of terminals, the frequency of branching, and the distribution and density of release sites. At present, however, the mechanisms that control these features remain obscure. Here, we report the development of supernumerary or "satellite" boutons in a variety of endocytic mutants at the Drosophila neuromuscular junction. Mutants in endophilin, synaptojanin, dynamin, AP180, and synaptotagmin all show increases in supernumerary bouton structures. These satellite boutons contain releasable vesicles and normal complements of synaptic proteins that are correctly localized within terminals. Interestingly, however, synaptojanin terminals have more active zones per unit of surface area and more dense bodies (T-bars) within these active zones, which may in part compensate for reduced transmission per active zone. The altered structural development of the synapse is selectively encountered in endocytosis mutants and is not observed when synaptic transmission is reduced by mutations in glutamate receptors or when synaptic transmission is blocked by tetanus toxin. We propose that endocytosis plays a critical role in sculpting the structure of synapses, perhaps through the endocytosis of unknown regulatory signals that organize morphogenesis at synaptic terminals.

Acyltransferases↗
PubMed2006

The HSPGs Syndecan and Dallylike bind the receptor phosphatase LAR and exert distinct effects on synaptic development.

The formation and plasticity of synaptic connections rely on regulatory interactions between pre- and postsynaptic cells. We show that the Drosophila heparan sulfate proteoglycans (HSPGs) Syndecan (Sdc) and Dallylike (Dlp) are synaptic proteins necessary to control distinct aspects of synaptic biology. Sdc promotes the growth of presynaptic terminals, whereas Dlp regulates active zone form and function. Both Sdc and Dlp bind at high affinity to the protein tyrosine phosphatase LAR, a conserved receptor that controls both NMJ growth and active zone morphogenesis. These data and double mutant assays showing a requirement of LAR for actions of both HSPGs lead to a model in which presynaptic LAR is under complex control, with Sdc promoting and Dlp inhibiting LAR in order to control synapse morphogenesis and function.

Animals↗
PubMed2005

Targeted deletion of Kv4.2 eliminates I(to,f) and results in electrical and molecular remodeling, with no evidence of ventricular hypertrophy or myocardial dysfunction.

Previous studies have demonstrated a role for voltage-gated K+ (Kv) channel alpha subunits of the Kv4 subfamily in the generation of rapidly inactivating/recovering cardiac transient outward K+ current, I(to,f), channels. Biochemical studies suggest that mouse ventricular I(to,f) channels reflect the heteromeric assembly of Kv4.2 and Kv4.3 with the accessory subunits, KChIP2 and Kvbeta1, and that Kv4.2 is the primary determinant of regional differences in (mouse ventricular) I(to,f) densities. Interestingly, the phenotypic consequences of manipulating I(to,f) expression in different mouse models are distinct. In the experiments here, the effects of the targeted deletion of Kv4.2 (Kv4.2(-/-)) were examined. Unexpectedly, voltage-clamp recordings from Kv4.2(-/-) ventricular myocytes revealed that I(to,f) is eliminated. In addition, the slow transient outward K+ current, I(to,s), and the Kv1.4 protein (which encodes I(to,s)) are upregulated in Kv4.2(-/-) ventricles. Although Kv4.3 mRNA/protein expression is not measurably affected, KChIP2 expression is markedly reduced in Kv4.2(-/-) ventricles. Similar to Kv4.3, expression of Kvbeta1, as well as Kv1.5 and Kv2.1, is similar in wild-type and Kv4.2(-/-) ventricles. In addition, and in marked contrast to previous findings in mice expressing a truncated Kv4.2 transgene, the elimination I(to,f) in Kv4.2(-/-) mice does not result in ventricular hypertrophy. Taken together, these findings demonstrate not only an essential role for Kv4.2 in the generation of mouse ventricular I(to,f) channels but also that the loss of I(to,f) per se does not have overt pathophysiological consequences.

Animals↗
PubMed2005

A slowed classical pathway rather than kiss-and-run mediates endocytosis at synapses lacking synaptojanin and endophilin.

The extent to which a "kiss-and-run" mode of endocytosis contributes to synaptic-vesicle recycling remains controversial. The only genetic evidence for kiss-and-run at the synapse comes from mutations in the genes encoding synaptojanin and endophilin, proteins that together function to uncoat vesicles in classical clathrin-mediated endocytosis. Here we have characterized the endocytosis that persists in null alleles of Drosophila synaptojanin and endophilin. In response to high-frequency stimulation, the synaptic-vesicle pool can be reversibly depleted in these mutants. Recovery from this depletion is slow and indicates the persistence of an impaired form of classical endocytosis. Steady-state exocytosis rates reveal that endocytosis saturates in mutant neuromuscular terminals at approximately 80 vesicles/s, 10%-20% of the wild-type rate. Analyses of quantal size, FM1-43 loading, and dynamin function further demonstrate that, even in the absence of synaptojanin or endophilin, vesicles undergo full fusion and re-formation. Therefore, no genetic evidence remains to indicate that synaptic vesicles undergo kiss-and-run.

Acyltransferases↗
PubMed2005

Sec6 mutations and the Drosophila exocyst complex.

To allow a detailed analysis of exocyst function in multicellular organisms, we have generated sec6 mutants in Drosophila. We have used these mutations to compare the phenotypes of sec6 and sec5 in the ovary and nervous system, and we find them to be similar. We also find that Sec5 is mislocalized in sec6 mutants. Additionally, we have generated an epitope-tagged Sec8 that localized with Sec5 on oocyte membranes and was mislocalized in sec5 and sec6 germ-line clones. This construct further revealed a genetic interaction of sec8 and sec5. These data, taken together, provide new information about the organization of the exocyst complex and suggest that Sec5, Sec6 and Sec8 act as a complex, each member dependent on the others for proper localization and function.

Alleles↗
PubMed2003

The exocyst component Sec5 is required for membrane traffic and polarity in the Drosophila ovary.

The directed traffic of membrane proteins to the cell surface is crucial for many developmental events. We describe the role of Sec5, a member of the exocyst complex, in directed membrane traffic in the Drosophila oocyte. During oogenesis, we find that Sec5 localization undergoes dynamic changes, correlating with the sites at which it is required for the traffic of membrane proteins. Germline clones of sec5 possess defects in membrane addition and the posterior positioning of the oocyte. Additionally, the impaired membrane trafficking of Gurken, the secreted ligand for the EGF receptor, and Yolkless, the vitellogenin receptor, results in defects in dorsal patterning and egg size. However, we find the cytoskeleton to be correctly oriented. We conclude that Sec5 is required for directed membrane traffic, and consequently for the establishment of polarity within the developing oocyte.

Animals↗
PubMed2003

Mutations in the exocyst component Sec5 disrupt neuronal membrane traffic, but neurotransmitter release persists.

The exocyst (Sec6/8) complex is necessary for secretion in yeast and has been postulated to establish polarity by directing vesicle fusion to specific sites along the plasma membrane. The complex may also function in the nervous system, but its precise role is unknown. We have investigated exocyst function in Drosophila with mutations in one member of the complex, sec5. Null alleles die as growth-arrested larvae, whose neuromuscular junctions fail to expand. In culture, neurite outgrowth fails in sec5 mutants once maternal Sec5 is exhausted. Using a trafficking assay, we found impairments in the membrane addition of newly synthesized proteins. In contrast, synaptic vesicle fusion was not impaired. Thus, Sec5 differentiates between two forms of vesicle trafficking: trafficking for cell growth and membrane protein insertion depend on sec5, whereas transmitter secretion does not. In this regard, sec5 differs from the homologs of other yeast exocytosis genes that are required for both neuronal trafficking pathways.

Animals↗
PubMed2002

Axonal transport of mitochondria to synapses depends on milton, a novel Drosophila protein.

A protein required to localize mitochondria to Drosophila nerve terminals has been identified genetically. Photoreceptors mutant for milton show aberrant synaptic transmission despite normal phototransduction. Without Milton, synaptic terminals and axons lack mitochondria, although mitochondria are numerous in neuronal cell bodies. In contrast, synaptic vesicles continue to be transported to and concentrated at synapses. Milton protein is associated with mitochondria and is present primarily in axons and synapses. A likely explanation of the apparent trafficking defect is offered by the coimmunoprecipitation of Milton and kinesin heavy chain. Transfected into HEK293T cells, Milton induces a redistribution of mitochondria within the cell. We propose that Milton is a mitochondria-associated protein required for kinesin-mediated transport of mitochondria to nerve terminals.

Animals↗
PubMed2002

Distribution of rSlo Ca2+-activated K+ channels in rat astrocyte perivascular endfeet.

Evidence that Ca(2+)-activated K(+) (K(Ca)) channels play a role in cell volume changes and K(+) homeostasis led to a prediction that astrocytes would have K(Ca) channels near blood vessels in order to maintain K(+) homeostasis. Consistent with this thinking the present study demonstrates that rSlo K(Ca) channels are in glial cells of the adult rat central nervous system (CNS) and highly localized to specializations of astrocytes associated with the brain vasculature. Using confocal and thin-section electron microscopic immunolabeling methods the distribution of rSlo was examined in adult rat brain. Strong rSlo immunolabeling was present around the vasculature of most brain regions. Examination of dye-filled hippocampal astrocytes revealed rSlo immunolabeling polarized in astrocytic endfeet. Ultrastructural analysis confirmed that the rSlo staining was concentrated in astrocytic endfeet ensheathing capillaries as well as abutting the pia mater. Immunostaining within the endfeet was predominantly distributed at the plasma membrane directly adjacent to either the vascular basal lamina or the pial surface. The distribution of the aquaporin-4 (AQP-4) water channel was also examined using dye-filled hippocampal astrocytes. In confirmation of earlier reports, intense AQP-4 immunolabeling was generally observed at the perimeter of blood vessels, and coincided with perivascular endfeet and rSlo labeling. We propose that rSlo K(Ca) channels, with their sensitivity to membrane depolarization and intracellular calcium, play a role in the K(+) modulation of cerebral blood flow. Additional knowledge of the molecular and cellular machinery present at perivascular endfeet may provide insight into the structural and functional molecular elements responsible for the neuronal activity-dependent regulation of cerebral blood flow.

Animals↗
PubMed2002

Members of the synaptobrevin/vesicle-associated membrane protein (VAMP) family in Drosophila are functionally interchangeable in vivo for neurotransmitter release and cell viability.

Synaptobrevins or VAMPs are vesicle-associated membrane proteins, often called v-SNARES, that are important for vesicle transport and fusion at the plasma membrane. Drosophila has two characterized members of this gene family: synaptobrevin (syb) and neuronal synaptobrevin (n-syb). Mutant phenotypes and gene-expression patterns indicate that n-Syb is exclusively neuronal and required only for synaptic vesicle secretion, whereas Syb is ubiquitous and, as shown here, essential for cell viability. When the eye precursor cells were made homozygous for syb(-), the eye failed to develop. In contrast, n-syb(-) eye clones developed appropriately but failed to activate downstream neurons. To determine whether the two proteins are structurally specialized to accomplish these distinct in vivo functions, we have driven the expression of each gene in the absence of the other to look for phenotypic rescue. We find that expression of n-syb during eye development can rescue the cell lethality of the syb mutations, as can rat VAMP2 and cellubrevin. Expression of syb can restore synaptic transmission to n-syb mutants as assayed both by electroretinogram and recordings of excitatory junctional currents at the neuromuscular junction. Therefore, we find that Syb, which usually is not involved in synaptic function, can mediate Ca(2+)-triggered synaptic activity and that no particular specialization of the v-SNARE is required to differentiate synaptic exocytosis from other forms.

Animals↗
PubMed2002

Synaptotagmins I and IV promote transmitter release independently of Ca(2+) binding in the C(2)A domain.

At nerve terminals, a focal and transient increase in intracellular Ca(2+) triggers the fusion of neurotransmitter-filled vesicles with the plasma membrane. The most extensively studied candidate for the Ca(2+)-sensing trigger is synaptotagmin I, whose Ca(2+)-dependent interactions with acidic phospholipids and syntaxin have largely been ascribed to its C(2)A domain, although the C(2)B domain also binds Ca(2+) (refs 7, 8). Genetic tests of synaptotagmin I have been equivocal as to whether it is the Ca(2+)-sensing trigger of fusion. Synaptotagmin IV, a related isoform that does not bind Ca(2+) in the C(2)A domain, might be an inhibitor of release. We mutated an essential aspartate of the Ca(2+)-binding site of the synaptotagmin I C(2)A domain and expressed it in Drosophila lacking synaptotagmin I. Here we show that, despite the disruption of the binding site, the Ca(2+)-dependent properties of transmission were not altered. Similarly, we found that synaptotagmin IV could substitute for synaptotagmin I. We conclude that the C(2)A domain of synaptotagmin is not required for Ca(2+)-dependent synaptic transmission, and that synaptotagmin IV promotes rather than inhibits transmission.

Amino Acid Sequence↗