The pathways of mitophagy for quality control and clearance of mitochondria.

Selective autophagy of mitochondria, known as mitophagy, is an important mitochondrial quality control mechanism that eliminates damaged mitochondria. Mitophagy also mediates removal of mitochondria from developing erythrocytes, and contributes to maternal inheritance of mitochondrial DNA through the elimination of sperm-derived mitochondria. Recent studies have identified specific regulators of mitophagy that ensure selective sequestration of mitochondria as cargo. In yeast, the mitochondrial outer membrane protein autophagy-related gene 32 (ATG32) recruits the autophagic machinery to mitochondria, while mammalian Nix is required for degradation of erythrocyte mitochondria. The elimination of damaged mitochondria in mammals is mediated by a pathway comprised of PTEN-induced putative protein kinase 1 (PINK1) and the E3 ubiquitin ligase Parkin. PINK1 and Parkin accumulate on damaged mitochondria, promote their segregation from the mitochondrial network, and target these organelles for autophagic degradation in a process that requires Parkin-dependent ubiquitination of mitochondrial proteins. Here we will review recent advances in our understanding of the different pathways of mitophagy. In addition, we will discuss the relevance of these pathways in neurons where defects in mitophagy have been implicated in neurodegeneration.

Mitochondria are redistributed in Drosophila photoreceptors lacking milton, a kinesin-associated protein.

Photoreceptors are richly supplied with mitochondria, where they are required to meet the energetic demands, in the soma, of phototransduction and, in the terminal, of neurotransmitter release. Compromising the latter, we have made photoreceptors R1-R6 in Drosophila ommatidia homozygous for either of two alleles, milt(186) and milt(92), of milton in whole-eye mosaics. Such mutant photoreceptors fail to target mitochondria to their terminals. We show from quantitative electron microscopy (EM) that mitochondria are totally lacking at the terminal but nevertheless abundant and present throughout the soma, where their distribution differs from that of control ommatidia, however, being more heavily concentrated in the nuclear region. Mitochondria are sparse at the basalmost level of mutant ommatidia, and are lacking beneath the basement membrane, in the axons and terminals of these cells. The absence of mitochondria from R1-R6 terminals and concommitant reductions in synaptic vesicle packing density, previously reported, we show here are accompanied by reduced immunoreactivity to the photoreceptor transmitter histamine but not by any change in total head histamine content, as determined by high-performance liquid chromatography. Mutant terminals also contain vesicle profiles with a wider range of sizes. These two phenotypes suggest that the reduced availability of ATP when mutant terminals lack a mitochondrial supply compromises their ability to pump histamine into synaptic vesicles and perturbs membrane distribution within the terminal. In addition, a band of somata in the lamina cortex, at least some of which are postsynaptic neurons not homozygous for milton, also shows altered mitochondrial targeting, with abnormal clusters of mitochondria, as visualized by immunolabeling with anti-hsp and by serial EM. Within the lamina, terminals of mutant photoreceptors are penetrated by neighboring cells with invaginations that frequently contain mitochondria, suggesting that a mechanism exists for intercellular metabolic support. Our findings indicate the direct and compensatory responses in a population of neurons when mitochondria are not correctly targeted to their synaptic terminals.

Genetic manipulation of cardiac K(+) channel function in mice: what have we learned, and where do we go from here?

In the mammalian myocardium, potassium (K(+)) channels control resting potentials, action potential waveforms, automaticity, and refractory periods and, in most cardiac cells, multiple types of K(+) channels that subserve these functions are expressed. Molecular cloning has revealed the presence of a large number of K(+) channel pore forming (alpha) and accessory (beta) subunits in the heart, and considerable progress has been made recently in defining the relationships between expressed K(+) channel subunits and functional cardiac K(+) channels. To date, more than 20 mouse models with altered K(+) channel expression/functioning have been generated using dominant-negative transgenic and targeted gene deletion approaches. In several instances, the genetic manipulation of K(+) channel subunit expression has revealed the role of specific K(+) channel subunit subfamilies or individual K(+) channel subunit genes in the generation of myocardial K(+) channels. In other cases, however, the phenotypic consequences have been unexpected. This review summarizes what has been learned from the in situ genetic manipulation of cardiac K(+) channel functioning in the mouse, discusses the limitations of the models developed to date, and explores the likely directions of future research.

Absence of junctional glutamate receptor clusters in Drosophila mutants lacking spontaneous transmitter release.

Little is known about the functional significance of spontaneous miniature synaptic potentials, which are the result of vesicular exocytosis at nerve terminals. Here, by using Drosophila mutants with specific defects in presynaptic function, we found that glutamate receptors clustered normally at neuromuscular junctions of mutants that retained spontaneous transmitter secretion but had lost the ability to release transmitter in response to action potentials. In contrast, receptor clustering was defective in mutants in which both spontaneous and evoked vesicle exocytosis were absent. Thus, spontaneous vesicle exocytosis appears to be tightly linked to the clustering of glutamate receptors during development.

The consequences of disrupting cardiac inwardly rectifying K(+) current (I(K1)) as revealed by the targeted deletion of the murine Kir2.1 and Kir2.2 genes.

1. Ventricular myocytes demonstrate a steeply inwardly rectifying K(+) current termed I(K1). We investigated the molecular basis for murine I(K1) by removing the genes encoding Kir2.1 and Kir2.2. The physiological consequences of the loss of these genes were studied in newborn animals because mice lacking Kir2.1 have a cleft palate and die shortly after birth. 2. Kir2.1 (-/-) ventricular myocytes lack detectable I(K1) in whole-cell recordings in 4 mM external K(+). In 60 mM external K(+) a small, slower, residual current is observed. Thus Kir2.1 is the major determinant of I(K1). Sustained outward K(+) currents and Ba(2+) currents through L- and T-type channels were not significantly altered by the mutation. A 50 % reduction in I(K1) was observed in Kir2.2 (-/-) mice, raising the possibility that Kir2.2 can also contribute to the native I(K1). 3. Kir2.1 (-/-) myocytes showed significantly broader action potentials and more frequent spontaneous action potentials than wild-type myocytes. 4. In electrocardiograms of Kir2.1 (-/-) neonates, neither ectopic beats nor re-entry arrhythmias were observed. Thus the increased automaticity and prolonged action potential of the mutant ventricular myocytes were not sufficiently severe to disrupt the sinus pacing of the heart. The Kir2.1 (-/-) mice, however, had consistently slower heart rates and this phenotype is likely to arise indirectly from the influence of Kir2.1 outside the heart. 5. Thus Kir2.1 is the major component of murine I(K1) and the Kir2.1 (-/-) mouse provides a model in which the functional consequences of removing I(K1) can be studied at both cellular and organismal levels.

SNAP-24, a Drosophila SNAP-25 homologue on granule membranes, is a putative mediator of secretion and granule-granule fusion in salivary glands.

Fusion of vesicles with target membranes is dependent on the interaction of target (t) and vesicle (v) SNARE (soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor) proteins located on opposing membranes. For fusion at the plasma membrane, the t-SNARE SNAP-25 is essential. In Drosophila, the only known SNAP-25 isoform is specific to neuronal axons and synapses and additional t-SNAREs must exist that mediate both non-synaptic fusion in neurons and constitutive and regulated fusion in other cells. Here we report the identification and characterization of SNAP-24, a closely related Drosophila SNAP-25 homologue, that is expressed throughout development. The spatial distribution of SNAP-24 in the nervous system is punctate and, unlike SNAP-25, is not concentrated in synaptic regions. In vitro studies, however, show that SNAP-24 can form core complexes with syntaxin and both synaptic and non-synaptic v-SNAREs. High levels of SNAP-24 are found in larval salivary glands, where SNAP-24 localizes mainly to granule membranes rather than the plasma membrane. During glue secretion, the massive exocytotic event of these glands, SNAP-24 containing granules fuse with one another and the apical membrane, suggesting that glue secretion utilizes compound exocytosis and that SNAP-24 mediates secretion.

Targeted disruption of Kir2.1 and Kir2.2 genes reveals the essential role of the inwardly rectifying K(+) current in K(+)-mediated vasodilation.

The molecular bases of inwardly rectifying K(+) (Kir) currents and K(+)-induced dilations were examined in cerebral arteries of mice that lack the Kir2.1 and Kir2.2 genes. The complete absence of the open reading frame in animals homozygous for the targeted allele was confirmed. Kir2.1(-/-) animals die 8 to 12 hours after birth, apparently due to a complete cleft of the secondary palate. In contrast, Kir2.2(-/-) animals are viable and fertile. Kir currents were observed in cerebral artery myocytes isolated from control neonatal animals but were absent in myocytes from Kir2.1(-/-) animals. Voltage-dependent K(+) currents were similar in cells from neonatal control and Kir2.1(-/-) animals. An increase in the extracellular K(+) concentration from 6 to 15 mmol/L caused Ba(2+)-sensitive dilations in pressurized cerebral arteries from control and Kir2.2 mice. In contrast, arteries from Kir2.1(-/-) animals did not dilate when the extracellular K(+) concentration was increased to 15 mmol/L. In summary, Kir2.1 gene expression in arterial smooth muscle is required for Kir currents and K(+)-induced dilations in cerebral arteries.

Flamingo, a seven-pass transmembrane cadherin, regulates planar cell polarity under the control of Frizzled.

We identified a seven-pass transmembrane receptor of the cadherin superfamily, designated Flamingo (Fmi), localized at cell-cell boundaries in the Drosophila wing. In the absence of Fmi, planar polarity was distorted. Before morphological polarization of wing cells along the proximal-distal (P-D) axis, Fmi was redistributed predominantly to proximal and distal cell edges. This biased localization of Fmi appears to be driven by an imbalance of the activity of Frizzled (Fz) across the proximal/distal cell boundary. These results, together with phenotypes caused by ectopic expression of fz and fmi, suggest that cells acquire the P-D polarity by way of the Fz-dependent boundary localization of Fmi.

A genetic method for generating Drosophila eyes composed exclusively of mitotic clones of a single genotype.

The genetic analysis of a gene at a late developmental stage can be impeded if the gene is required at an earlier developmental stage. The construction of mosaic animals, particularly in Drosophila, has been a means to overcome this obstacle. However, the phenotypic analysis of mitotic clones is often complicated because standard methods for generating mitotic clones render mosaic tissues that are a composite of both mutant and phenotypically normal cells. We describe here a genetic method (called EGUF/hid) that uses both the GAL4/UAS and FLP/FRT systems to overcome this limitation for the Drosophila eye by producing genetically mosaic flies that are otherwise heterozygous but in which the eye is composed exclusively of cells homozygous for one of the five major chromosome arms. These eyes are nearly wild type in size, morphology, and physiology. Applications of this genetic method include phenotypic analysis of existing mutations and F(1) genetic screens to identify as yet unknown genes involved in the biology of the fly eye. We illustrate the utility of the method by applying it to lethal mutations in the synaptic transmission genes synaptotagmin and syntaxin.

Identification and localization of Ca(2+)-activated K+ channels in rat sciatic nerve.

To understand the physiology of Schwann cells and myelinated nerve, we have been engaged in identifying K+ channels in sciatic nerve and determining their subcellular localization. In the present study, we examined the slo family of Ca(2+)-activated K+ channels, a class of channel that had not previously been identified in myelinated nerve. We have determined that these channels are indeed expressed in peripheral nerve, and have cloned rat homologues of slo that are more than 95% identical to the murine slo. We found that sciatic nerve RNA contained numerous alternatively spliced variants of the slo homologue, as has been seen in other tissues. We raised a polyclonal antibody against a peptide from the carboxyl terminal of the channels. Immunocytochemistry revealed that the channel proteins are in Schwann cells and are associated with canaliculi that run along the outer surface of the cells. They are also relatively concentrated near the node of Ranvier in the Schwann cell outer membrane. This staining pattern is quite similar to what we previously reported for the voltage-dependent K+ channel Kv 1.5. We did not observe staining of axons or connective tissue in the nerve and so it seems likely that most or all of the splicing variants are located in the Schwann cells. The localization of these channels also suggests that they may participate in maintaining the resting potential of the Schwann cells during K+ buffering.

Genetic evidence for an equilibrium between docked and undocked vesicles.

By using the shibire mutation to block endocytosis in a temperature-dependent fashion, we have manipulated the number of synaptic vesicles in a nerve terminal and have observed a remarkable proportionality of the number of quanta released to the size of the total vesicle pool. In the experiments described below we determine that approximately 0.3% of the vesicle pool is released per stimulus. The data suggest that the pool of readily releasable docked vesicles does not represent the saturation of a limiting number of release sites, but instead represents a subset of vesicles that is in equilibrium with the larger pool of vesicles. Before presenting this data and the significance of the finding for the regulation of neurotransmission, we will briefly review the use of Drosophila genetics as a tool for dissecting synaptic transmission.

Selective effects of neuronal-synaptobrevin mutations on transmitter release evoked by sustained versus transient Ca2+ increases and by cAMP.

Synaptobrevin is a key constituent of the synaptic vesicle membrane. The neuronal-synaptobrevin (n-syb) gene in Drosophila is essential for nerve-evoked synaptic currents, but miniature excitatory synaptic currents (mESCs) remain even in the complete absence of this gene. To further characterize the defect in these mutants, we have examined conditions that stimulate secretion. Despite the inability of an action potential to trigger fusion, high K+ saline could increase the frequency of mESCs 4- to 17-fold in a Ca2+-dependent manner, and the rate of fusion approached 25% of that seen in wild-type synapses under the same conditions. Similarly, the mESC frequency in n-syb null mutants could be increased by a Ca2+ ionophore, A23187, and by black widow spider venom. Thus, the ability of the vesicles to fuse in response to sustained increases in cytosolic Ca2+ persisted in the absence of this protein. Tetanic stimulation could also increase the frequency of mESCs, particularly toward the end of a train and after the train of stimuli. In contrast, these mutants did not respond to an elevation of cAMP induced by an activator of adenylyl cyclase, forskolin, or a membrane-permeable analog of cAMP, dibutyryl cAMP, which in wild-type synapses causes a marked increase in the mESC frequency even in the absence of external Ca2+. These results are discussed in the context of models that invoke a special role for n-syb in coupling fusion to the transient, local changes in Ca2+ and an as yet unidentified target of cAMP.

Dynamic potassium channel distributions during axonal development prevent aberrant firing patterns.

The distribution and function of Shaker-related K+ channels were studied with immunofluorescence and electrophysiology in sciatic nerves of developing rats. At nodes of Ranvier, Na+ channel clustering occurred very early (postnatal days 1-3). Although K+ channels were not yet segregated at most of these sites, they were directly involved in action potential generation, reducing duration, and the refractory period. At approximately 1 week, K+ channel clusters were first seen but were within the nodal gap and in paranodes, and only later (weeks 2-4) were they shifted to juxtaparanodal regions. K+ channel function was most dramatic during this transition period, with block producing repetitive firing in response to single stimuli. As K+ channels were increasingly sequestered in juxtaparanodes, conduction became progressively insensitive to K+ channel block. Over the first 3 weeks, K+ channel clustering was often asymmetric, with channels exclusively in the distal paranode in approximately 40% of cases. A computational model suggested a mechanism for the firing patterns observed, and the results provide a role for K+ channels in the prevention of aberrant excitation as myelination proceeds during development.

Morphologically docked synaptic vesicles are reduced in synaptotagmin mutants of Drosophila.

Nerve terminal specializations include mechanisms for maintaining a subpopulation of vesicles in a docked, fusion-ready state. We have investigated the relationship between synaptotagmin and the number of morphologically docked vesicles by an electron microscopic analysis of Drosophila synaptotagmin (syt) mutants. The overall number of synaptic vesicles in a terminal was reduced, although each active zone continued to have a cluster of vesicles in its vicinity. In addition, there was an increase in the number of large vesicles near synapses. Examining the clusters, we found that the pool of synaptic vesicles immediately adjacent to the presynaptic membrane, the pool that includes the docked population, was reduced to 24 +/- 5% (means +/- SEM) of control in the sytnull mutation. To separate contributions of overall vesicle depletion and increased spontaneous release from direct effects of synaptotagmin on morphological docking, we examined syt mutants in an altered genetic background. Recombining syt alleles onto a second chromosome bearing an as yet uncharacterized mutation resulted in the expected decrease in evoked release but suppressed the increase in spontaneous release frequency. Motor nerve terminals in this genotype contained more synaptic vesicles than control, yet the number of vesicles immediately adjacent to the presynaptic membrane near active zones was still reduced (33 +/- 4% of control). Our findings demonstrate that there is a decrease in the number of morphologically docked vesicles seen in syt mutants. The decreases in docking and evoked release are independent of the increase in spontaneous release. These results support the hypothesis that synaptotagmin stabilizes the docked state.

Distinct requirements for evoked and spontaneous release of neurotransmitter are revealed by mutations in the Drosophila gene neuronal-synaptobrevin.

Two modes of vesicular release of transmitter occur at a synapse: spontaneous release in the absence of a stimulus and evoked release that is triggered by Ca2+ influx. These modes often have been presumed to represent the same exocytotic apparatus functioning at different rates in different Ca2+ concentrations. To investigate the mechanism of transmitter release, we have examined the role of synaptobrevin/VAMP, a protein involved in vesicular docking and/or fusion. We generated a series of mutations, including null mutations, in neuronal-synaptobrevin (n-syb), the neuronally expressed synaptobrevin gene in Drosophila. Mutant embryos completely lacking n-syb form morphologically normal neuromuscular junctions. Electrophysiological recordings from the neuromuscular junction of these mutants reveal that the excitatory synaptic current evoked by stimulation of the motor neuron is abolished entirely. However, spontaneous release of quanta from these terminals persists, although its rate is reduced by 75%. Thus, at least a portion of the spontaneous "minis" that are seen at the synapse can be generated by a protein complex that is distinct from that required for an evoked synaptic response.

Potassium channel distribution, clustering, and function in remyelinating rat axons.

The K+ channel alpha-subunits Kv1.1 and Kv1.2 and the cytoplasmic beta-subunit Kvbeta2 were detected by immunofluorescence microscopy and found to be colocalized at juxtaparanodes in normal adult rat sciatic nerve. After demyelination by intraneural injection of lysolecithin, and during remyelination, the subcellular distributions of Kv1.1, Kv1.2, and Kvbeta2 were reorganized. At 6 d postinjection (dpi), axons were stripped of myelin, and K+ channels were found to be dispersed across zones that extended into both nodal and internodal regions; a few days later they were undetectable. By 10 dpi, remyelination was underway, but Kv1.1 immunoreactivity was absent at newly forming nodes of Ranvier. By 14 dpi, K+ channels were detected but were in the nodal gap between Schwann cells. By 19 dpi, most new nodes had Kv1.1, Kv1.2, and Kvbeta2, which precisely colocalized. However, this nodal distribution was transient. By 24 dpi, the majority of K+ channels was clustered within paranodal regions of remyelinated axons, leaving a gap that overlapped with Na+ channel immunoreactivity. Inhibition of Schwann cell proliferation delayed both remyelination and the development of the K+ channel distributions described. Conduction studies indicate that neither 4-aminopyridine (4-AP) nor tetraethylammonium alters normal nerve conduction. However, during remyelination, 4-AP profoundly increased both compound action potential amplitude and duration. The level of this effect matched closely the nodal presence of these voltage-dependent K+ channels. Our results suggest that K+ channels may have a significant effect on conduction during remyelination and that Schwann cells are important in K+ channel redistribution and clustering.

The synaptic protein syntaxin1 is required for cellularization of Drosophila embryos.

Syntaxins are membrane proteins involved in vesicle trafficking and are required for the release of neurotransmitter at nerve terminals. The presence of syntaxins on target membranes has been hypothesized to confer specificity to targeting and fusion via interactions with complementary vesicle-associated proteins, the synaptobrevins or VAMPS. We have mutagenized syntaxin1 in Drosophila and have found that it links the mechanism of synaptic transmission to a distinct cell biological process: the cellularization of early embryos. This specialized form of cell division separates the 6,000 nuclei of the syncytial blastoderm into separate cells through the invagination of the surface membrane of the embryo. During this process, syntaxin1 protein is present on the newly forming lateral cell surfaces and invaginating cleavage furrows. This protein is derived both from maternal deposition of mRNA and protein and from early zygotic transcription. To analyze syntaxin1's role in early development, female germ line mosaics mutant for syntaxin1 expression were generated by mitotic recombination to reduce the maternal contribution. Visualizing the actin cytoskeleton and glycosylated surface proteins reveals that embryos with insufficient syntaxin1 have large acellular patches. The patches do not appear until cellularization begins, and the process fails entirely within these regions. These results provide genetic evidence that membrane trafficking is required for the cellularization of the syncytial blastoderm. We propose that the invagination of the surface membrane proceeds by the fusion of intracellular membrane vesicles with the surface. This reaction uses the same syntaxin1 protein as is required for neurotransmitter secretion at synapses. Thus, a single syntaxin can participate in trafficking steps that are functionally as distinct as synaptic transmission and cell division.

Complex gene organization of synaptic protein SNAP-25 in Drosophila melanogaster.

The evolutionarily conserved protein SNAP-25 (synaptosome-associated protein 25 kDa (kilodaltons)) is a component of the protein complex involved in the docking and/or fusion of synaptic vesicles in nerve terminals. We report here that the SNAP-25 gene (Snap) in the fruit fly Drosophila melanogaster has a complex organization with eight exons spanning more than 120 kb (kilobases). The exon boundaries coincide with those of the chicken SNAP-25 gene (Bark, 1993). Only a single exon 5 has been found in Drosophila, whereas human, rat, chicken, zebrafish and goldfish have two alternatively spliced versions of this exon. In situ hybridization and immunocytochemistry to whole mount embryos show that SNAP-25 mRNA and protein are detected in stage 14 and later developmental stages, and are mainly localized to the ventral nerve cord. Thus, Snap has an evolutionarily conserved and complex gene organization, and its onset of expression in Drosophila melanogaster correlates with a time in neuronal development when synapses begin to be formed and when other synapse-specific genes are switched on.

Anomalous mole fraction effect induced by mutation of the H5 pore region in the Shaker K+ channel.

Mutagenesis of the H5 region of the Shaker K+ channel has provided strong evidence that these amino acids form a major portion of the ionic pore. We have previously observed that a single-site mutation (T441S) in this region increased the apparent relative permeability of the channel to NH4+. We now report that this increased relative permeability to NH4+ is sensitive to small changes in external K+ in a pattern consistent with an anomalous mole fraction effect. The effect is not apparent in the wild-type channel. These findings, in combination with other studies showing effects of this particular mutation on the binding of tetraethylammonium and hydroxylamine, support the hypothesis that T441S alters the affinity of a putative ion binding site for NH4+ and ammonium derivatives. The mutation T441S alters ionic selectivity and reveals the multi-ion nature of the mutant Shaker K+ channel.