Search

mTOR is a highly conserved eukaryotic protein kinase that coordinates cell growth and metabolism and plays a critical role in cancer, immunity, and aging. It remains unclear how mTOR signaling in individual tissues contributes to whole-organism processes because mTOR inhibitors, like the natural product Rapamycin, are administered systemically and target multiple tissues simultaneously. We developed a chemical-genetic system, termed selecTOR, that restricts the activity of a Rapamycin analog to specific cell populations through targeted expression of a mutant FKBP12 protein. This analog has reduced affinity for its obligate binding partner FKBP12, which reduces its ability to inhibit mTOR in wild-type cells and tissues. Expression of the mutant FKBP12, which contains an expanded binding pocket, rescues the activity of this Rapamycin analog. Using this system, we show that selective mTOR inhibition can be achieved in S. cerevisiae and human cells, and we validate the utility of our system in an intact metazoan model organism by identifying the tissues responsible for a Rapamycin-induced developmental delay in Drosophila.Significance StatementmTOR plays a number of critical organismal roles, including in cell growth, development, immunity and aging, but dissecting the tissue-specific influences of mTOR has proven challenging. This work describes a simple system for identifying the specific tissues and cells responsible for the diverse functions of mTOR, and we show that our system can be used in organisms ranging from yeast to humans.

Mitochondria are essential organelles that provide cellular energy and buffer cytoplasmic calcium. At the same time they produce damaging reactive oxygen species and sequester pro-apoptotic factors. Hence, eukaryotes have evolved exquisite homeostatic processes that maintain mitochondrial integrity, or ultimately remove damaged organelles. This subject has garnered intense interest recently following the discovery that two Parkinson's disease genes, PINK1 and parkin, regulate mitochondrial degradation (mitophagy). The molecular details of PINK1/Parkin-induced mitophagy are emerging but much of our insight derives from work using cultured cells and potent mitochondrial toxins, raising questions about the physiological significance of these findings. Here we review the evidence supporting PINK1/Parkin mitophagy in vivo and its causative role in neurodegeneration, and outline outstanding questions for future investigations.

The destruction of mitochondria through macroautophagy (autophagy) has been recognised as a major route of mitochondrial protein degradation since its discovery more than 50 years ago, but fundamental questions remain unanswered. First, how much mitochondrial protein turnover occurs through auto-phagy? Mitochondrial proteins are also degraded by nonautophagic mechanisms, and the proportion of mitochondrial protein turnover that occurs through autophagy is still unknown. Second, does auto-phagy degrade mitochondrial proteins uniformly or selectively? Autophagy was originally thought to degrade all mitochondrial proteins at the same rate, but recent work suggests that mitochondrial autophagy may be protein selective. To investigate these questions, we used a proteomics-based approach in the fruit fly Drosophila melanogaster, comparing mitochondrial protein turnover rates in autophagy-deficient Atg7 mutants and controls. We found that ~35% of mitochondrial protein turnover occurred via autophagy. Similar analyses using parkin mutants revealed that parkin-dependent mitophagy accounted for ~25% of mitochondrial protein turnover, suggesting that most mitochondrial autophagy specifically eliminates dysfunctional mitochondria. We also found that our results were incompatible with uniform autophagic turnover of mitochondrial proteins and consistent with protein-selective autophagy. In particular, the autophagic turnover rates of individual mitochondrial proteins varied widely, and only a small amount of the variation could be attributed to tissue differences in mitochondrial composition and autophagy rate. Furthermore, analyses comparing autophagy-deficient and control human fibroblasts revealed diverse autophagy-dependent turnover rates even in homogeneous cells. In summary, our work indicates that autophagy acts selectively on mitochondrial proteins, and that most mitochondrial protein turnover occurs through non-autophagic processes. Abbreviations: Atg5: Autophagy-related 5 (Drosophila); ATG5: autophagy related 5 (human); Atg7: Autophagy-related 7 (Drosophila); ATG7: autophagy related 7 (human); DNA: deoxyribonucleic acid; ER: endoplasmic reticulum; GFP: green fluorescent protein; MS: mass spectrometry; park: parkin (Drosophila); Pink1: PTEN-induced putative kinase 1 (Drosophila); PINK1: PTEN-induced kinase 1 (human); PRKN: parkin RBR E3 ubiquitin protein ligase (human); RNA: ribonucleic acid; SD: standard deviation; Ub: ubiquitin/ubiquitinated; WT: wild-type; YME1L: YME1 like ATPase (Drosophila); YME1L1: YME1 like 1 ATPase (human)

The m-AAA proteases play a critical role in the proteostasis of inner mitochondrial membrane proteins, and mutations in the genes encoding these proteases cause severe incurable neurological diseases. To further explore the biological role of the m-AAA proteases and the pathological consequences of their deficiency, we used a genetic approach in the fruit fly Drosophila melanogaster to inactivate the ATPase family gene 3-like 2 (AFG3L2) gene, which encodes a critical component of the m-AAA proteases. We found that null alleles of Drosophila AFG3L2 die early in development, but partial inactivation of AFG3L2 using RNAi allowed survival to the late pupal and adult stages of development. Flies with partial inactivation of AFG3L2 exhibited behavioral defects, neurodegeneration, accumulation of unfolded mitochondrial proteins, and diminished respiratory chain (RC) activity. Further work revealed that the reduced RC activity was primarily a consequence of severely diminished mitochondrial transcription and translation. These defects were accompanied by activation of the mitochondrial unfolded protein response (mito-UPR) and autophagy. Overexpression of mito-UPR components partially rescued the AFG3L2-deficient phenotypes, indicating that protein aggregation partly accounts for the defects of AFG3L2-deficient animals. Our work suggests that strategies designed to activate mitochondrial stress pathways and mitochondrial gene expression could be therapeutic in the diseases caused by mutations in AFG3L2., Author summary Mitochondria produce virtually all of the cellular energy through the actions of the respiratory chain (RC) complexes. However, mitochondria also present a quality control challenge for the eukaryotic cell. In particular, biogenesis of the RC complexes depends on the coordinated expression of nuclear and mitochondrially encoded subunits and an imbalance in this process can cause protein aggregation. Moreover, the RC complexes produce highly damaging reactive oxygen species as a side product of their activity. The Mitochondrial AAA+ family of proteases are believed to provide the first line of defense against these insults. The importance of this protease family is best exemplified by the severe neurodegenerative diseases that are caused by mutations in their respective genes. To better understand the biological roles of the AAA+ proteases, and the physiological consequences of their inactivation we used a genetic approach in Drosophila to study the Afg3l2 AAA+ protease. Partial inactivation of the AFG3L2 gene resulted in mitochondrial functional deficits, shortened lifespan, and neurodegeneration. Unexpectedly, we found that severely diminished mitochondrial gene expression, including transcription, and translation, primarily accounts for these phenotypes. The defects in gene expression appear to be caused by a failure in mitochondrial ribosome maturation and assembly, and possibly, sequestration of regulatory factors into inactive aggregates. Our work indicates Afg3l2 plays critical roles in degrading unfolded mitochondrial proteins and regulating mitochondrial gene expression, and that strategies to address these matters could be therapeutic in the diseases caused by mutations in the AFG3L2 gene.

Mutations in the glucosylceramidase beta (GBA) gene are strongly associated with neurodegenerative diseases marked by protein aggregation. GBA encodes the lysosomal enzyme glucocerebrosidase, which breaks down glucosylceramide. A common explanation for the link between GBA mutations and protein aggregation is that lysosomal accumulation of glucosylceramide causes impaired autophagy. We tested this hypothesis directly by measuring protein turnover and abundance in Drosophila mutants with deletions in the GBA ortholog Gba1b. Proteomic analyses revealed that known autophagy substrates, which had severely impaired turnover in autophagy-deficient Atg7 mutants, showed little to no overall slowing of turnover or increase in abundance in Gba1b mutants. Likewise, Gba1b mutants did not have the marked impairment of mitochondrial protein turnover seen in mitophagy-deficient parkin mutants. Proteasome activity, microautophagy, and endocytic degradation also appeared unaffected in Gba1b mutants. However, we found striking changes in the turnover and abundance of proteins associated with extracellular vesicles (EVs), which have been proposed as vehicles for the spread of protein aggregates in neurodegenerative disease. These changes were specific to Gba1b mutants and did not represent an acceleration of normal aging. Western blotting of isolated EVs confirmed the increased abundance of EV proteins in Gba1b mutants, and nanoparticle tracking analysis revealed that Gba1b mutants had six times as many EVs as controls. Genetic perturbations of EV production in Gba1b mutants suppressed protein aggregation, demonstrating that the increase in EV abundance contributed to the accumulation of protein aggregates. Together, our findings indicate that glucocerebrosidase deficiency causes pathogenic changes in EV metabolism and may promote the spread of protein aggregates through extracellular vesicles., Author summary Mutations in the GBA gene, which encodes the enzyme glucocerebrosidase, are common and increase the risk of Parkinson disease. A widely accepted explanation for the increased risk is that the fatty substance normally broken down by glucocerebrosidase builds up in the lysosome, which is the cell’s recycling center, until the cell can no longer get rid of damaged parts. At that point, proteins that should be destroyed in the lysosome form large clumps (aggregates) throughout the cell. We used mutant fruit flies without glucocerebrosidase to test this theory, and we were surprised to see no evidence that the lysosome was failing. The destruction of proteins usually recycled by the lysosome was not slowed down in the mutant flies. Instead, we saw evidence that the mutants’ cells might be producing too many extracellular vesicles, tiny spheres that transport cargo and messages from cell to cell. Some researchers have also suggested that extracellular vesicles carry the protein aggregates that spread between cells as Parkinson disease get worse. Our study supports this idea. It suggests that increased spread of aggregates through extracellular vesicles, rather than failure of the lysosome, might explain why GBA mutations increase the risk of neurodegenerative disease.

Mutations in the glucosidase, beta, acid (GBA1) gene cause Gaucher’s disease, and are the most common genetic risk factor for Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) excluding variants of low penetrance. Because α-synuclein-containing neuronal aggregates are a defining feature of PD and DLB, it is widely believed that mutations in GBA1 act by enhancing α-synuclein toxicity. To explore this hypothesis, we deleted the Drosophila GBA1 homolog, dGBA1b, and compared the phenotypes of dGBA1b mutants in the presence and absence of α-synuclein expression. Homozygous dGBA1b mutants exhibit shortened lifespan, locomotor and memory deficits, neurodegeneration, and dramatically increased accumulation of ubiquitinated protein aggregates that are normally degraded through an autophagic mechanism. Ectopic expression of human α-synuclein in dGBA1b mutants resulted in a mild enhancement of dopaminergic neuron loss and increased α-synuclein aggregation relative to controls. However, α-synuclein expression did not substantially enhance other dGBA1b mutant phenotypes. Our findings indicate that dGBA1b plays an important role in the metabolism of protein aggregates, but that the deleterious consequences of mutations in dGBA1b are largely independent of α-synuclein. Future work with dGBA1b mutants should reveal the mechanism by which mutations in dGBA1b lead to accumulation of protein aggregates, and the potential influence of this protein aggregation on neuronal integrity., Author Summary Mutations in the glucosidase, beta, acid (GBA1) gene cause Gaucher’s disease (GD), a lysosomal storage disease that includes neurodegenerative phenotypes. Recently, mutations in GBA1 were identified as the strongest genetic risk factor for Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), which are neurodegenerative conditions characterized by intraneuronal protein aggregates containing α-synuclein. To explore how GBA1 mutations lead to neurodegeneration in GD, PD and DLB, we developed a novel invertebrate model of GBA1 insufficiency by deleting the Drosophila GBA1 homolog, dGBA1b. We found that dGBA1b mutants have multiple phenotypes consistent with neuronal dysfunction as seen in PD, DLB and GD, and dramatically increased protein aggregation that is normally cleared through an autophagic mechanism. dGBA1b mutants expressing human α-synuclein in dopaminergic neurons led to dopaminergic neuron loss and α-synuclein aggregation. However, α-synuclein expression had minimal effect on dGBA1b mutant phenotypes, suggesting that the deleterious consequences of glucocerebrosidase deficiency are independent of α-synuclein. These findings significantly contribute to our understanding of the role of GBA1 mutations in the pathogenesis of PD, DLB, and GD, and further studies using this model should elucidate mechanisms underlying these diseases.

Loss-of-function mutations of the parkin gene, which encodes a ubiquitin-protein ligase, are a common cause of autosomal recessive juvenile parkinsonism (ARJP). Previous work has led to the identification of a number of Parkin substrates that implicate specific pathways in ARJP pathogenesis, including endoplasmic reticulum (ER) stress and cell cycle activation. To test the involvement of previously implicated pathways, as well as to identify novel pathways in ARJP pathogenesis, we are using genetic and genomic approaches to study Parkin function in the fruit fly Drosophila melanogaster. In previous work, we demonstrated that Drosophila parkin null mutants exhibit mitochondrial pathology and flight muscle degeneration. To further explore the mechanisms responsible for pathology in parkin mutants, we analyzed the transcriptional alterations that occur during muscle degeneration and performed a genetic screen for parkin modifiers. Results of these studies indicate that oxidative stress response components are induced in parkin mutants and that loss-of-function mutations in oxidative stress components enhance the parkin mutant phenotypes. Genes involved in the innate immune response are also induced in parkin mutants. In contrast, our studies did not reveal evidence for cell cycle or ER stress pathway induction in parkin mutants. These results suggest that oxidative stress and/or inflammation may play a fundamental role in the etiology of ARJP.

Senescent and damaged mitochondria undergo selective mitophagic elimination through mechanisms requiring two Parkinson’s disease factors, the mitochondrial kinase PINK1 and the cytosolic ubiquitin ligase Parkin. The nature of the PINK-Parkin interaction and identity of key factors directing Parkin to damaged mitochondria are unknown. We show that the mitochondrial outer membrane GTPase mitofusin (Mfn) 2 mediates Parkin recruitment to damaged mitochondria. Parkin bound to Mfn2 in a PINK1-dependent manner; PINK1 phosphorylated Mfn2 and promoted its Parkin-mediated ubiqitination. Ablation of Mfn2 in mouse cardiac myocytes prevented depolarization-induced translocation of Parkin to the mitochondria and suppressed mitophagy. Accumulation of morphologically and functionally abnormal mitochondria induced respiratory dysfunction in Mfn2-deficient mouse embryonic fibroblasts and cardiomyocytes, and in Parkin-deficient Drosophila heart tubes, causing dilated cardiomyopathy. Thus, Mfn2 functions as a mitochondrial receptor for Parkin, and is required for quality control of cardiac mitochondria.

The accumulation of somatic mitochondrial DNA (mtDNA) mutations is implicated in aging and common diseases of the elderly, including cancer and neurodegenerative disease. However, the mechanisms that influence the frequency of somatic mtDNA mutations are poorly understood. To develop a simple invertebrate model system to address this matter, we used the Random Mutation Capture (RMC) assay to characterize the age-dependent frequency and distribution of mtDNA mutations in the fruit fly Drosophila melanogaster. Because oxidative stress is a major suspect in the age-dependent accumulation of somatic mtDNA mutations, we also used the RMC assay to explore the influence of oxidative stress on the somatic mtDNA mutation frequency. We found that many of the features associated with mtDNA mutations in vertebrates are conserved in Drosophila, including a comparable somatic mtDNA mutation frequency (∼10−5), an increased frequency of mtDNA mutations with age, and a prevalence of transition mutations. Only a small fraction of the mtDNA mutations detected in young or old animals were G∶C to T∶A transversions, a signature of oxidative damage, and loss-of-function mutations in the mitochondrial superoxide dismutase, Sod2, had no detectable influence on the somatic mtDNA mutation frequency. Moreover, a loss-of-function mutation in Ogg1, which encodes a DNA repair enzyme that removes oxidatively damaged deoxyguanosine residues (8-hydroxy-2′-deoxyguanosine), did not significantly influence the somatic mtDNA mutation frequency of Sod2 mutants. Together, these findings indicate that oxidative stress is not a major cause of somatic mtDNA mutations. Our data instead suggests that somatic mtDNA mutations arise primarily from errors that occur during mtDNA replication. Further studies using Drosophila should aid in the identification of factors that influence the frequency of somatic mtDNA mutations., Author Summary Mitochondria are the evolutionary remnants of bacteria that were acquired by the cells of our ancestors more than a billion years ago and now produce virtually all of the cellular energy. Due to their bacterial ancestry, mitochondria have their own genomes, which encode some of the machinery responsible for producing energy. These genes occasionally acquire mutations—irreversible alterations that can adversely affect the energy production machinery. The accumulation of mitochondrial DNA (mtDNA) mutations is thought to cause aging and common age-related diseases, but we know little about the factors that influence the frequency of these mutations. Our study tested whether fruit flies would serve as a good animal model to study this problem. We found that flies accumulate mtDNA mutations in a pattern similar to that of humans. We then used flies to test the long-standing theory that toxic free radicals, chemical byproducts of energy production, cause mtDNA mutations to accumulate. Our data do not support this hypothesis, and instead suggest that rare errors associated with duplicating mitochondrial genomes are primarily responsible for mtDNA mutations. In sum we demonstrate that Drosophila serves as a tractable genetic model to investigate the mechanisms that influence the frequency of somatic mtDNA mutations.

The PINK1–Parkin pathway plays a critical role in mitochondrial quality control by selectively targeting damaged mitochondria for autophagy. In this issue, Tanaka et al. (2010. J. Cell Biol. doi: 10.1083/jcb.201007013) demonstrate that the AAA-type ATPase p97 acts downstream of PINK1 and Parkin to segregate fusion-incompetent mitochondria for turnover. p97 acts by targeting the mitochondrial fusion-promoting factor mitofusin for degradation through an endoplasmic reticulum–associated degradation (ERAD)-like mechanism.

The Purkinje cell degeneration (pcd) mouse is a recessive model of neurodegeneration, involving cerebellum and retina. Purkinje cell death in pcd is dramatic, as >99% of Purkinje neurons are lost in three weeks. Loss-of-function of Nna1 causes pcd, and Nna1 is a highly conserved zinc carboxypeptidase. To determine the basis of pcd, we implemented a two-pronged approach, combining characterization of loss-of-function phenotypes of the Drosophila Nna1 orthologue (NnaD) with proteomics analysis of pcd mice. Reduced NnaD function yielded larval lethality, with survivors displaying phenotypes that mirror disease in pcd. Quantitative proteomics revealed expression alterations for glycolytic and oxidative phosphorylation enzymes. Nna proteins localize to mitochondria, loss of NnaD / Nna1 produces mitochondrial abnormalities, and pcd mice display altered proteolytic processing of Nna1 interacting proteins. Our studies indicate that Nna1 loss-of-function results in altered bioenergetics and mitochondrial dysfunction, and suggest that pcd shares pathogenic features with neurodegenerative disorders such as Parkinson's disease.

Significant insight into the mechanisms that contribute to dopaminergic neurodegeneration in Parkinson disease has been gained from the analysis of genes linked to rare heritable forms of parkinsonism such as PINK1 and parkin, loss-of-function mutations of which cause autosomal recessive parkinsonism. PINK1 encodes a mitochondrially targeted Ser/Thr kinase and parkin encodes a ubiquitin-protein ligase. Functional studies of PINK1 and Parkin in animal and cellular model systems have shown that both proteins play important roles in maintaining mitochondrial integrity. Genetic studies of PINK1 and Parkin orthologs in flies have shown that PINK1 acts upstream from Parkin in a common pathway that appears to regulate mitochondrial morphology. Mitochondrial morphology is regulated by mitochondrial fission and fusion-promoting proteins, and is important in a variety of contexts, including mitochondrial trafficking and mitochondrial quality control. In particular, mitochondrial fission appears to promote the segregation of terminally dysfunctional mitochondria for degradation in the lysosome through a process termed mitophagy. Recent work has shown that Parkin promotes the degradation of dysfunctional mitochondria in vertebrate cell culture. Here we postulate a model whereby the PINK1/Parkin pathway regulates mitochondrial dynamics in an effort to promote the turnover of damaged mitochondria.

Loss-of-function mutations in the PTEN-induced kinase 1 ( PINK1 ) or parkin genes, which encode a mitochondrially localized serine/threonine kinase and a ubiquitin-protein ligase, respectively, result in recessive familial forms of Parkinsonism. Genetic studies in Drosophila indicate that PINK1 acts upstream of Parkin in a common pathway that influences mitochondrial integrity in a subset of tissues, including flight muscle and dopaminergic neurons. The mechanism by which PINK1 and Parkin influence mitochondrial integrity is currently unknown, although mutations in the PINK1 and parkin genes result in enlarged or swollen mitochondria, suggesting a possible regulatory role for the PINK1/Parkin pathway in mitochondrial morphology. To address this hypothesis, we examined the influence of genetic alterations affecting the machinery that governs mitochondrial morphology on the PINK1 and parkin mutant phenotypes. We report that heterozygous loss-of-function mutations of drp1 , which encodes a key mitochondrial fission-promoting component, are largely lethal in a PINK1 or parkin mutant background. Conversely, the flight muscle degeneration and mitochondrial morphological alterations that result from mutations in PINK1 and parkin are strongly suppressed by increased drp1 gene dosage and by heterozygous loss-of-function mutations affecting the mitochondrial fusion-promoting factors OPA1 and Mfn2. Finally, we find that an eye phenotype associated with increased PINK1/Parkin pathway activity is suppressed by perturbations that reduce mitochondrial fission and enhanced by perturbations that reduce mitochondrial fusion. Our studies suggest that the PINK1/Parkin pathway promotes mitochondrial fission and that the loss of mitochondrial and tissue integrity in PINK1 and parkin mutants derives from reduced mitochondrial fission.

Mutations resulting in the expansion of a polyglutamine tract in the protein ataxin-2 give rise to the neurodegenerative disorders spinocerebellar ataxia type 2 and Parkinson's disease. The normal cellular function of ataxin-2 and the mechanism by which polyglutamine expansion of ataxin-2 causes neurodegeneration are unknown. Here, we demonstrate that ataxin-2 and its Drosophila homolog, ATX2, assemble with polyribosomes and poly(A)-binding protein (PABP), a key regulator of mRNA translation. The assembly of ATX2 with polyribosomes is mediated independently by two distinct evolutionarily conserved regions of ATX2: an N-terminal Lsm/Lsm-associated domain (LsmAD), found in proteins that function in nuclear RNA processing and mRNA decay, and a PAM2 motif, found in proteins that interact physically with PABP. We further show that the PAM2 motif mediates a physical interaction of ATX2 with PABP in addition to promoting ATX2 assembly with polyribosomes. Our results suggest a model in which ATX2 binds mRNA directly through its Lsm/LsmAD domain and indirectly via binding PABP that is itself directly bound to mRNA. These findings, coupled with work on other ataxin-2 family members, suggest that ATX2 plays a direct role in translational regulation. Our results raise the possibility that polyglutamine expansions within ataxin-2 cause neurodegeneration by interfering with the translational regulation of particular mRNAs.

Despite the prevalence and severity of Parkinson's disease (PD), little is known about the molecular etiology of this disease, and preventative and disease-modifying therapies remain elusive. Recently, linkage studies have begun to identify single-gene mutations that are responsible for rare, heritable forms of PD, which offer an opportunity to gain insight into the molecular mechanisms of this disorder through the creation and analysis of appropriate animal models. One model system that is tractable for these studies is the fruit fly, Drosophila melanogaster. Analysis of several Drosophila models of PD has revealed some surprising insights into the pathogenesis of PD and begun to highlight potential treatment strategies.

An autosomal recessive juvenile-onset form of Parkinson's disease (AR-JP) is caused by loss-of-function mutations of the parkin gene, which encodes a ubiquitin-protein ligase. Three recent reports demonstrate that parkin can protect neurons from diverse cellular insults, including alpha-synuclein toxicity, proteasomal dysfunction, Pael-R accumulation, and kainate-induced excitotoxicity. These findings suggest a central role for parkin in maintaining dopaminergic neuronal integrity and strengthen the link between AR-JP and the more common sporadic form of Parkinson's disease.

Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disorder caused by the expansion of a CAG repeat encoding a polyglutamine tract in ataxin-2, the SCA2 gene product. The normal cellular function of ataxin-2 and the mechanism by which polyglutamine expansion of ataxin-2 causes neurodegeneration remain unknown. In this study we have used genetic and molecular approaches to investigate the function of a Drosophila homolog of the SCA2 gene (Datx2). Like human ataxin-2, Datx2 is found throughout development in a variety of tissue types and localizes to the cytoplasm. Mutations that reduce Datx2 activity or transgenic overexpression of Datx2 result in female sterility, aberrant sensory bristle morphology, loss or degeneration of tissues, and lethality. These phenotypes appear to result from actin filament formation defects occurring downstream of actin synthesis. Further studies demonstrate that Datx2 does not assemble with actin filaments, suggesting that the role of Datx2 in actin filament formation is indirect. These results indicate that Datx2 is a dosage-sensitive regulator of actin filament formation. Given that loss of cytoskeleton-dependent dendritic structure defines an early event in SCA2 pathogenesis, our findings suggest the possibility that dysregulation of actin cytoskeletal structure resulting from altered ataxin-2 activity is responsible for neurodegeneration in SCA2.

Ca 2+ influx into the presynaptic terminal triggers fusion of synaptic vesicles with the plasma membrane and release of neurotransmitters. A decade of intensive study of the synaptic vesicle protein synaptotagmin I has led to the general belief that this polypeptide functions as a Ca 2+ sensor in this process. Previous biochemical work on synaptotagmin I has centered on its two Ca 2+ -binding C 2 domains, designated C 2 A and C 2 B, which mediate the phospholipid- and effector-protein-binding interactions believed to be essential to its function. Recent tests of this hypothesis reveal that surprisingly, the C 2 A-mediated interactions are fully dispensable in vivo . However, at least some of the C 2 B-mediated interactions appear crucial to synaptotagmin I function.

Summary Mutations in VCP cause multisystem degeneration impacting the nervous system, muscle, and/or bone. Patients may present with ALS, Parkinsonism, frontotemporal dementia, myopathy, Paget's disease, or a combination of these. The disease mechanism is unknown. We developed a Drosophila model of VCP mutation-dependent degeneration. The phenotype is reminiscent of PINK1 and parkin mutants, including a pronounced mitochondrial defect. Indeed, VCP interacts genetically with the PINK1/parkin pathway in vivo. Paradoxically, VCP complements PINK1 deficiency but not parkin deficiency. The basis of this paradox is resolved by mechanistic studies in vitro showing that VCP recruitment to damaged mitochondria requires Parkin-mediated ubiquitination of mitochondrial targets. VCP recruitment coincides temporally with mitochondrial fission, and VCP is required for proteasome-dependent degradation of Mitofusins in vitro and in vivo. Further, VCP and its adaptor Npl4/Ufd1 are required for clearance of damaged mitochondria via the PINK1/Parkin pathway, and this is impaired by pathogenic mutations in VCP.

Neurons are the pedestals of human consciousness, yet from a cell biological view they are little more than specialized epithelia. Indeed, as highlighted in the “Neuronal Development and Degeneration” Minisymposium, neurons inhabit conventional cell biology at an extreme: in their diversity of cell types, the specificity of their cell–cell junctions and cell–cell signaling, their degree of asymmetric polarization, their rate and volume of membrane traffic, the size and stability of their cytoskeletal assemblies, and their metabolic activity and ion flux. This extreme lifestyle requires extreme upkeep and, although neurons can be extreme in their longevity, if any of the above processes are compromised then neurodegenerative diseases can ensue. This session highlighted what can be learned by studying cell biology at the extremes, beginning with neuronal development and ending in studies of neurodegenerative disease.