S. Tariq Ahmad

ESCRT-III Dysfunction Causes Autophagosome Accumulation and Neurodegeneration

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.

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

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.

Autophagy Negatively Regulates Early Axon Growth in Cortical Neurons

ABSTRACT 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.

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

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 Adult Circadian Free-Running Locomotor Activity Rhythm in Drosophila melanogaster

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. (Author correspondence: jseggio@bridgew.edu)

Spearmint ( l -carvone) Oil and Wintergreen (methyl salicylate) Oil Emulsion is an Effective Immersion Anesthetic of Fishes

Abstract This study evaluates the effects of a spearmint (/-carvone) and wintergreen oil (methyl salicylate) emulsion (CMSE) on age 1 landlocked Atlantic salmon Salmo salar sebago (hereafter salmon). Salmon were immersed in either 257 µl/L CMSE or 75 mg/L tricaine methanesulfonate (MS-222) to induce anesthesia (stage 4), useful for emersion and noninvasive husbandry procedures, and then salmon were recovered in fresh water. Induction was quicker in the CMSE group; however, recovery was quicker in the MS-222 group. A second experiment was conducted in which salmon were immersed in 257 µl/L CMSE for 8.5 min, or 75 mg/L MS-222 for 8.5 min in order to compare electrocardiographs during deeper anesthesia (stage 5) between salmon continuously immersed in CMSE to those continuously immersed in MS-222. Because salmon remained sedated longer after CMSE exposure than after MS-222 exposure, a third group of salmon was immersed in 257 µl/L CMSE for just 2.5 min before undergoing the 6-min electrocardiograph procedure...

The role of CHMP2BIntron5 in autophagy and frontotemporal dementia

Charged multivesicular body protein 2B (CHMP2B) - a component of the endosomal complex required for transport-III (ESCRT-III) - is responsible for the vital membrane deformation functions in autophagy and endolysosomal trafficking. A dominant mutation in CHMP2B (CHMP2BIntron5) is associated with a subset of heritable frontotemporal dementia - frontotemporal dementia linked to chromosome 3 (FTD-3). ESCRT-III recruits Vps4, an AAA-ATPase that abscises the membrane during various cellular processes including autophagy and intraluminal vesicle formation. CHMP2BIntron5 results in a C-terminus truncation removing an important Vps4 binding site as well as eliminating the normal autoinhibitory resting state of CHMP2B. CHMP2B is expressed in most cell types but seems to be especially vital for proper neuronal function. CHMP2BIntron5-mediated phenotypes include misregulation of transmembrane receptors, accumulation of multilamellar structures, abnormal lysosomal morphology, down regulation of a brain-specific micro RNA (miRNA-124), abnormal dendritic spine morphology, decrease in dendritic arborization, and cell death. Currently, transgenic-fly,-mouse, and -human cell lines are being used to better understand the diverse phenotypes and develop therapeutic approaches for the CHMP2BIntron5-induced FTD-3. This article is part of a Special Issue entitled SI:Autophagy.

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

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 (CHMP2BIntron5, 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 CHMP2BIntron5 in the Drosophila eye using GMR‐Gal4 causes misregulation of the Toll receptor pathway. Here, we show that ectopic expression of CHMP2BIntron5 using eyeless‐Gal4 (ey>CHMP2BIntron5), 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 (CHMP2BWT) and the Drosophila homologue of CHMP2B (CG4618). In addition, ey>CHMP2BIntron5 flies showed defects in photoreceptor cell patterning and phototactic behavior. Furthermore, ey>CHMP2BIntron5 flies showed accumulation of Notch in enlarged endosomes and up‐regulation of Notch activity. Partial loss of Notch activity in ey>CHMP2BIntron5 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 CHMP2BIntron5 preferentially affects different receptor signaling pathways in a cellular and developmental context‐dependent manner.—Cheruiyot, A., Lee, J‐A., Gao, F‐B., Ahmad, S. T. Expression of mutant CHMP2B, an ESCRT‐III component involved in frontotemporal dementia, causes eye deformities due to Notch misregulation in Drosophila. FASEB J. 28, 667–675 (2014). www.fasebj.org

Mutations in the circadian gene period alter behavioral and biochemical responses to ethanol in Drosophila

Clock genes, such as period, which maintain an organisms circadian rhythm, can have profound effects on metabolic activity, including ethanol metabolism. In turn, ethanol exposure has been shown in Drosophila and mammals to cause disruptions of the circadian rhythm. Previous studies from our labs have shown that larval ethanol exposure disrupted the free-running period and period expression of Drosophila. In addition, a recent study has shown that arrhythmic flies show no tolerance to ethanol exposure. As such, Drosophila period mutants, which have either a shorter than wild-type free-running period (perS) or a longer one (perL), may also exhibit altered responses to ethanol due to their intrinsic circadian differences. In this study, we tested the initial sensitivity and tolerance of ethanol exposure on Canton-S, perS, and perL, and then measured their Alcohol Dehydrogenase (ADH) and body ethanol levels. We showed that perL flies had slower sedation rate, longer recovery from ethanol sedation, and generated higher tolerance for sedation upon repeated ethanol exposure compared to Canton-S wild-type flies. Furthermore, perL flies had lower ADH activity and had a slower ethanol clearance compared to wild-type flies. The findings of this study suggest that period mutations influence ethanol induced behavior and ethanol metabolism in Drosophila and that flies with longer circadian periods are more sensitive to ethanol exposure.

Molecular Mechanisms Underlying the Role of Autophagy in Neurodegenerative Diseases

Autophagy is a highly regulated process that promotes vital cellular homeostasis by allowing bulk non-specific degradation of the cytoplasmic contents, mainly damaged and/or surplus organelles and proteins. Autophagy is ubiquitous in eukaryotes, highly conserved from yeast to mammals, and occurs in all mammalian tissues. Historically, autophagy was characterized as the coping response to limited energy resources (starvation), to generate additional biomolecular raw materials. However, research in the past two decades has demonstrated the indispensible roles of autophagy in eukaryotic physiology and pathology with respect to wide-ranging processes such as development, differentiation, aging, immunity, cancer biology, and neurodegenerative disorders. In this chapter, we will provide an overview of the types of autophagy and mechanisms of the autophagy pathway followed by a discussion of the current understanding of the role of autophagy in neuronal physiology, pathology of neurodegenerative disorders, and potential therapeutic approaches.