Glucocerebrosidase deficiency promotes protein aggregation through dysregulation of extracellular vesicles.

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.

The PINK1-Parkin pathway promotes both mitophagy and selective respiratory chain turnover in vivo.

The accumulation of damaged mitochondria has been proposed as a key factor in aging and the pathogenesis of many common age-related diseases, including Parkinson disease (PD). Recently, in vitro studies of the PD-related proteins Parkin and PINK1 have found that these factors act in a common pathway to promote the selective autophagic degradation of damaged mitochondria (mitophagy). However, whether Parkin and PINK1 promote mitophagy under normal physiological conditions in vivo is unknown. To address this question, we used a proteomic approach in Drosophila to compare the rates of mitochondrial protein turnover in parkin mutants, PINK1 mutants, and control flies. We found that parkin null mutants showed a significant overall slowing of mitochondrial protein turnover, similar to but less severe than the slowing seen in autophagy-deficient Atg7 mutants, consistent with the model that Parkin acts upstream of Atg7 to promote mitophagy. By contrast, the turnover of many mitochondrial respiratory chain (RC) subunits showed greater impairment in parkin than Atg7 mutants, and RC turnover was also selectively impaired in PINK1 mutants. Our findings show that the PINK1-Parkin pathway promotes mitophagy in vivo and, unexpectedly, also promotes selective turnover of mitochondrial RC subunits. Failure to degrade damaged RC proteins could account for the RC deficits seen in many PD patients and may play an important role in PD pathogenesis.

Topograph, a software platform for precursor enrichment corrected global protein turnover measurements.

Defects in protein turnover have been implicated in a broad range of diseases, but current proteomics methods of measuring protein turnover are limited by the software tools available. Conventional methods require indirect approaches to differentiate newly synthesized protein when synthesized from partially labeled precursor pools. To address this, we have developed Topograph, a software platform which calculates the fraction of peptides that are from newly synthesized proteins and their turnover rates. A unique feature of Topograph is the ability to calculate amino acid precursor pool enrichment levels which allows for accurate calculations when the precursor pool is not fully labeled, and the approach used by Topograph is applicable regardless of the stable isotope label used. We validate the Topograph algorithms using data acquired from a mouse labeling experiment and demonstrate the influence that precursor pool corrections can have on protein turnover measurements.

A Drosophila model for mechanistic investigation of tau protein spread.

Brain protein aggregates are a hallmark of neurodegenerative disease. Previous work indicates that specific protein components of these aggregates are toxic, including tau in Alzheimer's disease and related tauopathies. Increasing evidence also indicates that these toxic proteins traffic between cells in a prion-like fashion, thereby spreading pathology from one brain region to another. However, the mechanisms involved in trafficking are poorly understood. We therefore developed a transgenic Drosophila model to facilitate rapid evaluation of candidate tau trafficking modifiers. Our model uses the bipartite Q system to drive co-expression of tau and GFP in the fly eye. We find age-dependent tau spread into the brain, represented by detection of tau, but not GFP in the brain. We also found that tau trafficking was attenuated upon inhibition of the endocytic factor dynamin or the kinase glycogen synthase kinase-3ß ( GSK-3ß ). Further work revealed that dynamin promotes tau uptake in recipient tissues, whereas GSK-3ß appears to promote tau spread via direct phosphorylation of tau. Our robust and flexible system will promote the identification of tau trafficking components involved in the pathogenesis of neurodegenerative diseases. SUMMARY STATEMENT: The trafficking of toxic proteins in neurodegenerative disease is well-known but poorly understood. Our model will allow rapid and new insight into molecular mechanisms underlying this process.

Glucocerebrosidase deficiency leads to neuropathology via cellular immune activation.

Mutations in GBA (glucosylceramidase beta), which encodes the lysosomal enzyme glucocerebrosidase (GCase), are the strongest genetic risk factor for the neurodegenerative disorders Parkinson's disease (PD) and Lewy body dementia. Recent work has suggested that neuroinflammation may be an important factor in the risk conferred by GBA mutations. We therefore systematically tested the contributions of immune-related genes to neuropathology in a Drosophila model of GCase deficiency. We identified target immune factors via RNA-Seq and proteomics on heads from GCase-deficient flies, which revealed both increased abundance of humoral factors and increased macrophage activation. We then manipulated the identified immune factors and measured their effect on head protein aggregates, a hallmark of neurodegenerative disease. Genetic ablation of humoral (secreted) immune factors did not suppress the development of protein aggregation. By contrast, re-expressing Gba1b in activated macrophages suppressed head protein aggregation in Gba1b mutants and rescued their lifespan and behavioral deficits. Moreover, reducing the GCase substrate glucosylceramide in activated macrophages also ameliorated Gba1b mutant phenotypes. Taken together, our findings show that glucosylceramide accumulation due to GCase deficiency leads to macrophage activation, which in turn promotes the development of neuropathology.

Slowed Protein Turnover in Aging Drosophila Reflects a Shift in Cellular Priorities.

The accumulation of protein aggregates and dysfunctional organelles as organisms age has led to the hypothesis that aging involves general breakdown of protein quality control. We tested this hypothesis using a proteomic and informatic approach in the fruit fly Drosophila melanogaster. Turnover of most proteins was markedly slower in old flies. However, ribosomal and proteasomal proteins maintained high turnover rates, suggesting that the observed slowdowns in protein turnover might not be due to a global failure of quality control. As protein turnover reflects the balance of protein synthesis and degradation, we investigated whether decreases in synthesis or decreases in degradation would best explain the observed slowdowns in protein turnover. We found that while many individual proteins in old flies showed slower turnover due to decreased degradation, an approximately equal number showed slower turnover due to decreased synthesis, and enrichment analyses revealed that translation machinery itself was less abundant. Mitochondrial complex I subunits and glycolytic enzymes were decreased in abundance as well, and proteins involved in glutamine-dependent anaplerosis were increased, suggesting that old flies modify energy production to limit oxidative damage. Together, our findings suggest that age-related proteostasis changes in Drosophila represent a coordinated adaptation rather than a system collapse.

Autophagy accounts for approximately one-third of mitochondrial protein turnover and is protein selective.

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).

Preliminary evidence for sensitive periods in the effect of childhood sexual abuse on regional brain development.

Volumetric MRI scans from 26 women with repeated episodes of childhood sexual abuse and 17 healthy female comparison subjects (ages 18-22 years) were analyzed for sensitive period effects on hippocampal and amygdala volume, frontal cortex gray matter volume and corpus callosum area. Hippocampal volume was reduced in association with childhood sexual abuse at ages 3-5 years and ages 11-13 years. Corpus callosum was reduced with childhood sexual abuse at ages 9-10 years, and frontal cortex was attenuated in subjects with childhood sexual abuse at ages 14-16 years. Brain regions have unique windows of vulnerability to the effects of traumatic stress.

Lon protease inactivation in Drosophila causes unfolded protein stress and inhibition of mitochondrial translation.

Mitochondrial dysfunction is a frequent participant in common diseases and a principal suspect in aging. To combat mitochondrial dysfunction, eukaryotes have evolved a large repertoire of quality control mechanisms. One such mechanism involves the selective degradation of damaged or misfolded mitochondrial proteins by mitochondrial resident proteases, including proteases of the ATPase Associated with diverse cellular Activities (AAA+) family. The importance of the AAA+ family of mitochondrial proteases is exemplified by the fact that mutations that impair their functions cause a variety of human diseases, yet our knowledge of the cellular responses to their inactivation is limited. To address this matter, we created and characterized flies with complete or partial inactivation of the Drosophila matrix-localized AAA+ protease Lon. We found that a Lon null allele confers early larval lethality and that severely reducing Lon expression using RNAi results in shortened lifespan, locomotor impairment, and respiratory defects specific to respiratory chain complexes that contain mitochondrially encoded subunits. The respiratory chain defects of Lon knockdown (Lon KD ) flies appeared to result from severely reduced translation of mitochondrially encoded genes. This translational defect was not a consequence of reduced mitochondrial transcription, as evidenced by the fact that mitochondrial transcripts were elevated in abundance in Lon KD flies. Rather, the translational defect of Lon KD flies appeared to be derived from sequestration of mitochondrially encoded transcripts in highly dense ribonucleoparticles. The translational defect of Lon KD flies was also accompanied by a substantial increase in unfolded mitochondrial proteins. Together, our findings suggest that the accumulation of unfolded mitochondrial proteins triggers a stress response that culminates in the inhibition of mitochondrial translation. Our work provides a foundation to explore the underlying molecular mechanisms.

Elevated expression of 5-HT1B receptors in nucleus accumbens efferents sensitizes animals to cocaine.

Although the effects of psychostimulants on brain dopamine systems are well recognized, the direct actions of cocaine on serotonin systems also appear to be important to its addictive properties. For example, serotonin actions at 5-HT1B receptors in the ventral tegmental area (VTA) modulate cocaine-induced dopamine release in the nucleus accumbens (NAcc) and alter the rewarding and stimulant properties of cocaine. However, the mechanisms of these effects have been unclear, because several neuron types in VTA express 5-HT1B receptors. One possibility is that 5-HT1B receptors on the terminals of GABAergic projections from NAcc to VTA inhibit local GABA release, thereby disinhibiting VTA neurons. We tested this hypothesis directly by using viral-mediated gene transfer to overexpress 5-HT1B receptors in NAcc projections to VTA. A viral vector containing either epitope hemagglutinin-tagged 5-HT1B and green fluorescent protein (HA1B-GFP) cassettes or green fluorescent protein cassette alone (GFP-only) was injected into the NAcc shell, which sends projections to the VTA. HA1B-GFP injection induced elevated expression of 5-HT1B receptors in neuronal fibers in VTA and increased cocaine-induced locomotor hyperactivity without affecting baseline locomotion. Overexpression of 5-HT1B receptors also shifted the dose-response curve for cocaine-conditioned place preference to the left, indicating alterations in the rewarding effects of cocaine. Thus, increased expression of 5-HT1B receptors in NAcc efferents, probably in the terminals of medium spiny neurons projecting to the VTA, may contribute to psychomotor sensitization and offer an important target for regulating the addictive effects of cocaine.

Increased expression of 5-HT1B receptor in dorsal raphe nucleus decreases fear-potentiated startle in a stress dependent manner.

5-HT(1B) autoreceptors regulate serotonin release from terminals of dorsal raphe nucleus (DRN) projections. Due to postsynaptic 5-HT(1B) receptors in DRN terminal fields, it has not previously been possible to manipulate 5-HT(1B) autoreceptor activity without also changing 5-HT(1B) heteroreceptor activity. We have developed a viral gene transfer strategy to express epitope-tagged 5-HT(1B) and green fluorescent protein in vivo, allowing us to increase 5-HT(1B) expression in DRN neurons. We have shown that increased 5-HT(1B) autoreceptor expression reduced anxiety in unstressed animals but increased anxiety following inescapable stress. These findings suggest that effects of increased 5-HT(1B) autoreceptor expression are dependent on stress context. To better understand the mechanisms underlying these observations, we have used fear-potentiated startle (FPS). FPS is especially sensitive to the activity of the amygdala, which shares reciprocal connections with DRN. In the absence of an inescapable stressor, increased 5-HT(1B) autoreceptor expression attenuated FPS response compared with animals injected with a virus expressing only green fluorescent protein. Administration of the 5-HT(1B) antagonist SB224289 (5 mg/kg i.p.) before startle testing blocked the effects of increased 5-HT(1B) autoreceptor expression. Since SB224289 had no effect on FPS in the absence of viral gene transfer, these results suggest that the antagonist reversed the behavioral effects of increased 5-HT(1B) autoreceptor expression through blockade of transgenic receptors. When tested 24 h following water-restraint stress, animals with increased 5-HT(1B) autoreceptors demonstrated restoration of robust FPS response. These results extend our previous studies and suggest explanations for the complex relationship between 5-HT(1B) autoreceptor expression, stress, and anxiety behavior.

5-HT1B receptor mRNA levels in dorsal raphe nucleus: inverse association with anxiety behavior in the elevated plus maze.

Serotonergic neurons in the dorsal raphe nucleus, the major source of forebrain serotonin projections, synthesize a terminal autoreceptor that inhibits serotonin release-the 5-HT(1B) autoreceptor. Overexpression of this autoreceptor is hypothesized to contribute to anxiety. Antidepressants decrease (while learned helplessness increases) 5-HT(1B) mRNA in dorsal raphe neurons, and viral-mediated overexpression of 5-HT(1B) here increases anxiety behavior after stress. However, 5-HT(1B) mRNA levels in dorsal raphe are substantially elevated in unstressed rats in two models of stress resistance. Thus, the role of dorsal raphe 5-HT(1B) autoreceptors in anxiety is complex. Therefore, we tested whether different stressors differentially affect dorsal raphe 5-HT(1B) mRNA [via in situ hybridization histochemistry] and anxiety behavior (using the elevated plus maze). Rats were assigned to a stressor (either forced swim, water restraint, dry restraint, or electric tail shock) or a control condition, then were tested and sacrificed 24 h later. Overall, controls exhibited less anxiety than stressed rats as indicated by a higher ratio of open arm to total arm entries (OTR). The stressors did not differentially affect the OTR, nor did any alter dorsal raphe 5-HT(1B) mRNA levels. There was, however, a significant positive correlation between the OTR and 5HT(1B) mRNA intensity in controls (r=.64; P=.006), but not in stressed rats (r=.16, P=.36), providing further evidence that elevated dorsal raphe 5-HT(1B) levels are associated with reduced anxiety in animals that have not been exposed to stress.

The mitochondrial fusion-promoting factor mitofusin is a substrate of the PINK1/parkin pathway.

Loss-of-function mutations in the PINK1 or parkin genes result in recessive heritable forms of parkinsonism. Genetic studies of Drosophila orthologs of PINK1 and parkin indicate that PINK1, a mitochondrially targeted serine/threonine kinase, acts upstream of Parkin, a cytosolic ubiquitin-protein ligase, to promote mitochondrial fragmentation, although the molecular mechanisms by which the PINK1/Parkin pathway promotes mitochondrial fragmentation are unknown. We tested the hypothesis that PINK1 and Parkin promote mitochondrial fragmentation by targeting core components of the mitochondrial morphogenesis machinery for ubiquitination. We report that the steady-state abundance of the mitochondrial fusion-promoting factor Mitofusin (dMfn) is inversely correlated with the activity of PINK1 and Parkin in Drosophila. We further report that dMfn is ubiquitinated in a PINK1- and Parkin-dependent fashion and that dMfn co-immunoprecipitates with Parkin. By contrast, perturbations of PINK1 or Parkin did not influence the steady-state abundance of the mitochondrial fission-promoting factor Drp1 or the mitochondrial fusion-promoting factor Opa1, or the subcellular distribution of Drp1. Our findings suggest that dMfn is a direct substrate of the PINK1/Parkin pathway and that the mitochondrial morphological alterations and tissue degeneration phenotypes that derive from mutations in PINK1 and parkin result at least in part from reduced ubiquitin-mediated turnover of dMfn.