methods-to-characterize-spontaneous-startle-induced-locomotion

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of <em>Drosophila</em>

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, ...

Colby College

Defects in the endosomal-lysosomal pathway have been implicated in a number of neurodegenerative disorders. A key step in the endocytic regulation of transmembrane proteins occurs in a subset of late-endosomal compartments known as multivesicular bodies (MVBs), whose formation is controlled by endosomal sorting complex required for transport (ESCRT). The roles of ESCRT in dendritic maintenance and neurodegeneration remain unknown. Here, we show that mSnf7-2, a key component of ESCRT-III, is highly expressed in most mammalian neurons. Loss of mSnf7-2 in mature cortical neurons caused retraction of dendrites and neuronal cell loss. mSnf7-2 binds to CHMP2B, another ESCRT-III subunit, in which a rare dominant mutation is associated with frontotemporal dementia linked to chromosome 3 (FTD3). Ectopic expression of the mutant protein CHMP2B(Intron5) also caused dendritic retraction prior to neurodegeneration. CHMP2B(Intron5) was associated more avidly than CHMP2B(WT) with mSnf7-2, resulting in sequestration of mSnf7-2 in ubiquitin-positive late-endosomal vesicles in cortical neurons. Moreover, loss of mSnf7-2 or CHMP2B(Intron5) expression caused the accumulation of autophagosomes in cortical neurons and flies. These findings indicate that ESCRT-III dysfunction is associated with the autophagy pathway, suggesting a novel neurodegeneration mechanism that may have important implications for understanding FTD and other age-dependent neurodegenerative diseases.

ESCRT-III dysfunction causes autophagosome accumulation and neurodegeneration.

Frontotemporal dementia (FTD) is the most common form of dementia before 60 years of age. Rare pathogenic mutations in CHMP2B, which encodes a component of the endosomal sorting complex required for transport (ESCRT-III), are associated with FTD linked to chromosome 3 (FTD3). Animal models of FTD3 have not yet been reported, and what signaling pathways are misregulated by mutant CHMP2B in vivo is unknown. Here we report the establishment of a Drosophila model of FTD3 and show the genetic interactions between mutant CHMP2B and other components of ESCRT. Through an unbiased genome-wide screen, we identified 29 modifier loci and found that serpin5 (Spn5), a largely uncharacterized serine protease inhibitor, suppresses the melanization phenotype induced by mutant CHMP2B in the fly eye. We also found that Spn5 is a negative regulator of the Toll pathway and functions extracellularly, likely by blocking the proteolytic activation of Spaetzle, the Toll receptor ligand. Moreover, Spn5 inhibited activation of the Toll pathway by mutant CHMP2B. Our findings identify Spn5 as a regulator of the Toll pathway and CHMP2B toxicity and show that the Toll pathway is a major signaling pathway misregulated by mutant CHMP2B in vivo. This fly model will be useful to further dissect genetic pathways that are potentially relevant to the pathogenesis and treatment of FTD.

Genetic screen identifies serpin5 as a regulator of the toll pathway and CHMP2B toxicity associated with frontotemporal dementia.

Alcohol consumption causes disruptions in a variety of daily rhythms, including the sleep-wake cycle. Few studies have explored the effect of alcohol exposure only during developmental stages preceding maturation of the adult circadian clock, and none have examined the effects of alcohol on clock function in Drosophila. This study investigates developmental and behavioral correlates between larval ethanol exposure and the adult circadian clock in Drosophila melanogaster, a well-established model for studying circadian rhythms and effects of ethanol exposure. We reared Drosophila larvae on 0%, 10%, or 20% ethanol-supplemented food and assessed effects upon eclosion and the free-running period of the circadian rhythm of locomotor activity. We observed a dose-dependent effect of ethanol on period, with higher doses resulting in shorter periods. We also identified the third larval instar stage as a critical time for the developmental effects of 10% ethanol on circadian period. These results demonstrate that developmental ethanol exposure causes sustainable shortening of the adult free-running period in Drosophila melanogaster, even after adult exposure to ethanol is terminated, and suggests that the third instar is a sensitive time for this effect.

Larval ethanol exposure alters adult circadian free-running locomotor activity rhythm in Drosophila melanogaster.

Alcohol consumption causes disruptions in a variety of daily rhythms, including the circadian free-running rhythm. A previous study conducted in our laboratories has shown that larval ethanol exposure alters the free-running period in adult Canton-S Drosophila melanogaster. Few studies, however, have explored the effect of alcohol exposure on organisms exhibiting circadian periods radically different than (normal) 24-h. We reared Canton-S, period long, and period short Drosophila melanogaster larvae on 10%-ethanol supplemented food, and assessed their adult free-running locomotor activity and period transcript at ZT 12. We demonstrate that in Canton-S larval ethanol exposure shortens the adult free-running locomotor activity but does not significantly alter period mRNA levels at ZT 12. Period long mutants exposed to larval ethanol had significantly shortened adult free-running locomotor activity rhythms and decreased period mRNA levels, while period short mutants lengthened their free-running rhythm and showed increased period mRNA levels at ZT 12 after being exposed to larval ethanol. These results indicate that the effects of ethanol on the circadian clock might depend upon the baseline circadian period of the organism or that period mutant gene expression is sensitive to developmental ethanol treatment.

Larval ethanol exposure alters free-running circadian rhythm and per Locus transcription in adult D. melanogaster period mutants.

Neurite growth requires neurite extension and retraction, which are associated with protein degradation. Autophagy is a conserved bulk degradation pathway that regulates several cellular processes. However, little is known about autophagic regulation during early neurite growth. In this study, we investigated whether autophagy was involved in early neurite growth and how it regulated neurite growth in primary cortical neurons. Components of autophagy were expressed and autophagy was activated during early neurite growth. Interestingly, inhibition of autophagy by atg7 small interfering RNA (siRNA) caused elongation of axons, while activation of autophagy by rapamycin suppressed axon growth. Surprisingly, inhibition of autophagy reduced the protein level of RhoA. Moreover, expression of RhoA suppressed axon overelongation mediated by autophagy inhibition, whereas inhibition of the RhoA signaling pathway by Y-27632 recovered rapamycin-mediated suppression of axon growth. Interestingly, hnRNP-Q1, which negatively regulates RhoA, accumulated in autophagy-deficient neurons, while its protein level was reduced by autophagy activation. Overall, our study suggests that autophagy negatively regulates axon extension via the RhoA-ROCK pathway by regulating hnRNP-Q1 in primary cortical neurons. Therefore, autophagy might serve as a fine-tuning mechanism to regulate early axon extension.

Autophagy negatively regulates early axon growth in cortical neurons.

Endosomal sorting complexes required for transport (ESCRTs) mediate sorting of ubiquitinated membrane proteins into multivesicular bodies en route to lysosomes for degradation. A mutation in CHMP2B (CHMP2B(Intron5), an ESCRT-III component) that is associated with a hereditary form of frontotemporal dementia (FTD3) disrupts the endosomal-lysosomal pathway and causes accumulation of autophagosomes and multilamellar structures. We previously demonstrated that expression of CHMP2B(Intron5) in the Drosophila eye using GMR-Gal4 causes misregulation of the Toll receptor pathway. Here, we show that ectopic expression of CHMP2B(Intron5) using eyeless-Gal4 (ey>CHMP2B(Intron5)), a driver with different spatiotemporal expression attributes than GMR-Gal4 in the Drosophila eye, causes eye deformities when compared to expression of wild-type CHMP2B (CHMP2B(WT)) and the Drosophila homologue of CHMP2B (CG4618). In addition, ey>CHMP2B(Intron5) flies showed defects in photoreceptor cell patterning and phototactic behavior. Furthermore, ey>CHMP2B(Intron5) flies showed accumulation of Notch in enlarged endosomes and up-regulation of Notch activity. Partial loss of Notch activity in ey>CHMP2B(Intron5) flies significantly rescued eye deformities, photoreceptor patterning defect, and phototactic behavior defect, indicating that these defects are primarily due to Notch misregulation. These results demonstrate that CHMP2B(Intron5) preferentially affects different receptor signaling pathways in a cellular and developmental context-dependent manner.

Expression of mutant CHMP2B, an ESCRT-III component involved in frontotemporal dementia, causes eye deformities due to Notch misregulation in Drosophila.

Article	Year
ESCRT-III dysfunction causes autophagosome accumulation and neurodegeneration. Current biology : CB\| PubMed ID: 17683935	2007
Genetic screen identifies serpin5 as a regulator of the toll pathway and CHMP2B toxicity associated with frontotemporal dementia. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19581577	2009
Larval ethanol exposure alters adult circadian free-running locomotor activity rhythm in Drosophila melanogaster. Chronobiology international\| PubMed ID: 22217104	2012
Larval ethanol exposure alters free-running circadian rhythm and per Locus transcription in adult D. melanogaster period mutants. Behavioural brain research\| PubMed ID: 23219838	2013
Autophagy negatively regulates early axon growth in cortical neurons. Molecular and cellular biology\| PubMed ID: 23918799	2013
Expression of mutant CHMP2B, an ESCRT-III component involved in frontotemporal dementia, causes eye deformities due to Notch misregulation in Drosophila. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 24158394	2014

S. Tariq Ahmad

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