TER94 , the Drosophila Homologue of the ALS- related VCP gene, In uences Lifespan and Leads to a Decline in Motor Function


 Background: Valosin-Containing Protein (VCP) is an essential AAA+ ATPase with diverse functions within the cell. Mutations in the VCP gene have been detected in patients with familial amyotrophic lateral sclerosis (ALS). The aim of this study is to create a novel model of human neurodegenerative disease in Drosophila melanogaster by altering the expression of TER94, the Drosophila orthologue of the human VCP gene. TER94 expression was altered in all neurons, the dopaminergic neurons and in the motor neurons, with longevity and locomotor function assessed over time. Altered TER94 expression in combination with the altered expression of known Parkinson Disease (PD) genes was examined to investigate potential interactions.Results: Inhibition of TER94 altered median lifespan in a manner dependent upon the transgene selected for use and the tissue-specific expression directed by the Gal4 transgene selected. Locomotor ability was significantly reduced in all cases of TER94 inhibition tested. The inhibition of TER94 by two TER94-RNAi inhibitory transgenes, in the motor neurons via D42-Gal4 lead to increases in median lifespan, with one inhibitory transgene generating a slightly reduced lifespan. Inhibition of TER94 in the dopaminergic neurons resulted in a severe reduction in lifespan. The co-inhibition of TER94 and parkin in the neurons resulted in a major decline in lifespan by approximately 30%. While the inhibition of TER94 and the co-expression of alpha-synuclein in the neurons resulted in an increase in lifespan by approximately 28%. Conclusions: The inhibition of TER94 in the motor neurons is an interesting model of ALS, due to the small, but reduced lifespan coupled with a strong decline in locomotor function. The inhibition of TER94 in the dopaminergic neurons is a potential model of ALS, due to the reduction of both lifespan and locomotor function over time. The co-inhibition of TER94 and parkin in the neurons provides a promising novel model of neurodegenerative disease, displaying a great reduction in lifespan and in locomotor ability over time.


Background
The neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS), one of the most common adultonset motor neuron diseases, is characterized by the progressive loss of upper and lower motor neurons from the spinal cord, brain stem and motor cortex: ALS progression eventually leads to muscle weakness and atrophy (11). To date, at least three primary ALS genes have been identi ed: superoxide dismutase 1 (SOD1), Fused in Sarcoma (FUS) and TAR DNA Binding Protein (TARDBP) (1,10). Aside from the wellcharacterized disease-causing genes, there are several other, less well-established ALS-linked genes. Valosin-containing protein (VCP), also known as Transitional endoplasmic reticulum 94 or TER94 in Drosophila melanogaster, is an ALS-related gene which encodes the enzyme Valosin-Containing Protein, an essential AAA + ATPase. In the cell, VCP is ubiquitously expressed in the endoplasmic reticulum, mitochondria and nucleus, and associated with diverse functions in processes such as mitophagy, autophagy, and Ubiquitin Proteasome System (UPS) (3,8,10). Other functions of VCP include involvement with ER-associated protein degradation and DNA repair (3,10). In mitophagy, VCP is required for the turnover of mitochondrial outer membrane proteins (12), and is a direct component in the PINK1/parkinmediated process of mitophagy (6,12). In autophagy, VCP is heavily involved in the initiation phase and in the maturation of autophagosomes (5). An absence of VCP has been known to disturb both the aggregation of misfolded proteins, referred to as an aggresome, along with the degradation of proteins (5). Not only is the VCP gene associated with ALS, it has been linked to other diseases such as early onset Paget disease, Frontal Lobe Dementia (FTD) (8,9), and more recently in Parkinson Disease (PD) (9).
Through whole-exome sequencing, mutations in the VCP gene have been linked to patients with familial ALS (4), with mutations in VCP accounting for approximately 1 to 2% of familial ALS cases, and the demonstration that VCP mutations can result in impaired autophagy (10). Dominant pathogenic mutations of VCP alter the amino-terminal domain, with others that lead to changes within the ATPase domains, strongly in uence mitochondrial function (14). Similar to human VCP, the protein encoded by Drosophila TER94 has associations with various speci c proteins including the product of Cabeza (Cas), the Drosophila orthologue of the signi cant ALS gene FUS, where it functions as a modulator of motor neuron degeneration (2). Similar to human VCP, Drosophila TER94 regulates the Notch signalling pathway, which is critical in tissue development and homeostasis. Impairment of Notch signalling has been known to lead to various diseases, particularly neurodegenerative diseases (7). Furthermore, in the y, TER94 interacts with Drosophila clueless (clu) through PINK1/parkin-dependent mitophagy, where clu functions with VCP and parkin to degrade and promote the clearance of dysfunctional mitochondria (14). As VCP has prominent roles in autophagic processes, particularly in the stages of initiation, impairment in this gene, can have detrimental in uence upon such pathways. Moreover, as VCP is a known ALS-related gene that has an active involvement with the process of PINK1-parkin-mediated mitophagy, it is bene cial to study this ALS-associated gene in light of a potential relationship to Parkinson Disease. Although studies into the role of VCP during neurodegeneration have been conducted, the mechanisms by which mutations in VCP contribute to the progression of disease must be investigated. In these experiments, TER94 was inhibited in the motor neurons directed by the D42-Gal4 transgene, in the dopaminergic neurons using the tyrosine hydroxylase (TH-Gal4) transgene, and in a wider range of neurons that include the dopaminergic through the ddc-Gal4 transgenes (ddc-Gal4 HL4.3D and ddc-Gal4 HL4.36 ). It was predicted that inhibition of the TER94 gene in Drosophila would decrease median lifespan and impair locomotor ability, as the loss-of-function mutations in the orthologous VCP gene have been observed in ALS patients. The main objective was to create a VCP-based ALS model in D. melanogaster by altering the expression of the orthologous TER94 gene.

Results
Inhibition of TER94 in the motor neurons in uences longevity and reduces locomotor ability The inhibition of TER94 via the UAS-TER94-RNAi GL00448 transgene through D42-Gal4 resulted in a slight reduction in median lifespan compared to the control UAS-lacZ median lifespan of 70 days, inhibition of TER94 via TER94-RNAi GL00448 lead to a median lifespan of 68 days (Fig. 1a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 8 weeks the TER94-RNAi GL00448 ies lost their ability to climb at week 4 (Fig. 1b). The inhibition of TER94 via the UAS-TER94-RNAi HMS00656 transgene through D42-Gal4 resulted in a signi cant increase in median lifespan, compared to the control UAS-lacZ median lifespan of 70 days, inhibition of TER94 via TER94-RNAi HMS00656 lead to a median lifespan of 80 days (Fig. 1a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 8 weeks the TER94-RNAi HMS00656 ies lost their ability to climb at week 3 (Fig. 1b). The inhibition of TER94 via UAS-TER94-RNAi JF03402 transgene through D42-Gal4 resulted in a signi cant increase in median lifespan compared to the control UAS-lacZ median lifespan of 70 days, inhibition of TER94 via TER94-RNAi JF03402 lead to a median lifespan of 78 days (Fig. 1a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 8 weeks the TER94-RNAi JF03402 ies lost their ability to climb at week 3 (Fig. 1b).
Inhibition of TER94 in the dopaminergic neurons reduces both longevity and locomotor ability The inhibition of TER94 via UAS-TER94-RNAi GL00448 through TH-Gal4 resulted in a signi cant reduction in median lifespan compared to the control UAS-lacZ median lifespan of 82 days, inhibition of TER94 via TER94-RNAi GL00448 lead to a median lifespan of 54 days (Fig. 2a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 8 weeks the TER94-RNAi GL00448 ies lost their ability to climb at week 4 ( Fig. 2b). The inhibition of TER94 via UAS-TER94-RNAi HMS00656 through TH-Gal4 resulted in a signi cant reduction in median lifespan compared to the control UAS-lacZ median lifespan of 82 days, inhibition of TER94 via TER94-RNAi HMS00656 lead to a median lifespan of 46 days (Fig. 2a) (Fig. 2a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 8 weeks the TER94-RNAi HMS00656 ies lost their ability to climb at week 4 ( Fig. 2b).
Inhibition of TER94 in the ddc-Gal4 HL4.3D -expressing neurons has a minimal impact longevity, and reduces locomotor ability The inhibition of TER94 via the UAS-TER94-RNAi GL00448 transgene though ddc-Gal4 HL4.3D resulted in a slight change in median lifespan compared to the control UAS-lacZ median lifespan of 70 days, inhibition of TER94 via TER94-RNAi GL00448 lead to a median lifespan of 68 days (Fig. 3a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 8 weeks the TER94-RNAi GL00448 ies lost their ability to climb at week 3 (Fig. 3b). The inhibition of TER94 via the UAS-TER94-RNAi HMS00656 transgene though ddc-Gal4 HL4.3D resulted in a slight increase in median lifespan compared to the control UAS-lacZ median lifespan of 70 days, inhibition of TER94 via TER94-RNAi HMS00656 lead to a median lifespan of 74 days (Fig. 3a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 8 weeks the TER94-RNAi HMS00656 ies lost their ability to climb at week 4 ( Fig. 3b).
Inhibition of TER94 and parkin in the ddc-Gal4 HL4.3D -expressing neurons reduces longevity and locomotor ability The co-inhibition of TER94 via UAS-TER94-RNAi GL00448 transgene and parkin through ddc-Gal4 HL4.3Dexpressing neurons resulted in a large reduction in median lifespan (by ~ 30%). Compared to the control UAS-lacZ median lifespan of 84 days, inhibition of TER94 via TER94-RNAi GL00448 lead to a median lifespan of 58 days (Fig. 4a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 5 weeks the TER94-RNAi GL00448 ies lost their ability to climb at week 3 ( Fig. 4b).
Inhibition of TER94 in the ddc-Gal4 HL4.36 -expressing neurons increases longevity, and reduces locomotor ability The inhibition of TER94 via the UAS-TER94-RNAi GL00448 though ddc-Gal4 HL4.36 resulted in a signi cant increase in median lifespan compared to the control UAS-lacZ median lifespan of 79 days, inhibition of TER94 via TER94-RNAi GL00448 lead to a median lifespan of 88 days (Fig. 5a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 5 weeks the TER94-RNAi GL00448 ies lost their ability to climb also at week 4, however their climbing ability was drastically poorer (Fig. 5b). The inhibition of TER94 via UAS-TER94-RNAi HMS00656 though ddc-Gal4 HL4.36 resulted in a signi cant increase in median lifespan compared to the control UAS-lacZ median lifespan of 79 days, inhibition of TER94 via TER94-RNAi HMS00656 lead to a median lifespan of 90 days (Fig. 5a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 5 weeks the TER94-RNAi HMS00656 ies lost their ability to climb also at week 4, however their climbing ability was drastically poorer (Fig. 5b). The inhibition of TER94 via the UAS-TER94-RNAi JF03402 transgene though ddc-Gal4 HL4.36 resulted in a signi cant reduction in median lifespan compared to the control UAS-lacZ median lifespan of 79 days, inhibition of TER94 via TER94-RNAi JF03402 lead to a median lifespan of 44 days (Fig. 5a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 5 weeks the TER94-RNAi JF03402 ies lost their ability to climb also at week 4, however their climbing ability was drastically poorer (Fig. 5b).
Inhibition of TER94 and the co-expression of alpha-synuclein in the ddc-Gal4 HL4.36 -expressing neurons increases longevity, and reduces locomotor ability The inhibition of TER94 via the UAS-TER94-RNAi GL00448 transgene and the co-expression of alphasynuclein through ddc-Gal4 HL4.36 -expressing neurons resulted in a signi cant reduction in median lifespan compared to the control UAS-lacZ median lifespan of 86 days, inhibition of TER94 via TER94-RNAi GL00448 lead to a median lifespan of 80 days (Fig. 6a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 4 weeks the TER94-RNAi GL00448 ies lost their ability to climb at week 3 (Fig. 6b).The inhibition of TER94 via the UAS-TER94-RNAi JF03402 transgene and the co-expression of alpha-synuclein through ddc-Gal4 HL4.36expressing neurons resulted in a signi cant reduction in median lifespan compared to the control UAS-lacZ median lifespan of 86 days, inhibition of TER94 via TER94-RNAi JF03402 lead to a median lifespan of 48 days (Fig. 6a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 4 weeks the TER94-RNAi JF03402 ies lost their ability to climb at week 2 (Fig. 6b).The inhibition of TER94 via the UAS-TER94-RNAi HMS00656 transgene and the coexpression of alpha-synuclein through ddc-Gal4 HL4.36 -expressing neurons resulted in a large increase in median lifespan (by ~ 28%). Compared to the control UAS-lacZ median lifespan of 86 days, inhibition of TER94 via TER94-RNAi HMS00656 lead to a median lifespan of 110 days (Fig. 6a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 4 weeks the TER94-RNAi HMS00656 ies lost their ability to climb at week 3 (Fig. 4b).

Discussion
In

Conclusions
The inhibition of TER94 under some conditions can alter median lifespan coupled with greatly reduced motor function. As VCP mutations are known to exist in ALS patients, consequences of TER94 reduction in ies supports the hypothesis that inhibition of the human VCP gene may contribute to alterations to a subcellular mechanism that leads to the pathology associated with ALS. Interesting, the inhibition of TER94 in the motor neurons can result in a slightly increased median lifespan accompanied by a reduction in motor function over time. The inhibition of TER94 in the dopaminergic neurons lead to a severe reduction in lifespan and motor function, which suggests that the inhibition of TER94 in the dopaminergic neurons may yield a model of PD. Although the inhibition of TER94 through UAS-TER94-RNAi GL00448 in the ddc-Gal4-expressing neurons did not signi cantly change lifespan, however the coinhibition of parkin lead to a severe reduction in lifespan and motor function. These ndings suggest that the co-inhibition of TER94 and parkin in the neuron may be a promising model of neurodegenerative disease. The inhibition of TER94 through UAS-TER94-RNAi HMS00656 in the ddc-Gal4-expressing neurons lead to a signi cant increase in lifespan, while the co-expression of alpha-synuclein lead to an increase in lifespan, followed by a reduction in motor function. These phenotypes suggest a clear synergistic effect between the two gene activities.

Drosophila Stocks and Culture
The . Drosophila melanogaster was maintained on a standard media comprised of 65 g/L cornmeal, 50 ml/L fancy grade molasses, 10 g/L yeast and 5.5 g/L agar which was then treated with 2.5 ml propionic acid and 5 ml of 0.1 g/ml methylparaben. This mixture was then allowed to solidify at the bottom of vials and stored in 4 to 6º C until use. Stocks were stored at room temperature (~ 21º C), while crosses and experiments were performed at 25º C.

Longevity Assays
The survival of Drosophila was analyzed to examine the lifespan of experimental ies in comparison to control ies. Critical class male progeny were collected daily and placed in vials with fresh medium in cohorts of twenty ies or less to avoid over-crowding. A sample size of approximately three hundred males was collected in total and stored at 25º C for the duration of the experiment. The ies were scored every two days to examine if any death had occurred. A y was considered dead when no movement was observed. Males were transferred onto fresh media every four days to obtain a healthy environment. Graphpad Prism 8 (Graphpad Software Inc.) was used to analyze longevity data, and survival curves were analyzed and compared using the Log-rank (Mantel-Cox) test, with a P-value less than or equal to 0.05 with Bonferroni correction being considered statistically signi cant.

Locomotor Assays
Approximately seventy critical class male progeny to be collected from crosses between female D42-Gal4, TH-Gal4, ddc-Gal4 HL4.3D , ddc-Gal4 HL4.36 , ddc-Gal4 HL4.36 ;UAS-alpha-synuclein and ddc-Gal4 HL4.3D ;parkin-RNAi ies and male, UAS-TER94-RNAi GL00448 , UAS-TER94-RNAi HMS00656 , UAS-TER94-RNAi JF03402 , and UAS-lacZ ies. This assay was used to measure the ability of ies to climb up a narrow glass tube over the course of their lifespan, with fty male ies from each genotype being evaluated once every seven days, beginning at the seventh day post-eclosion. Critical class males were maintained in vials with ten ies per vial, stored at 25º C, and placed on new medium once per week throughout the experiment. Climbing analysis followed the standard protocol outlined by our laboratory (Todd & Staveley, 2004). Graphpad Prism 8 (Graphpad Software Inc.) was used to analyze the data, and to generate climbing curves tted using non-linear regression. 95% con dence intervals were used to test for signi cance with the curves considered to be signi cantly different if P < 0.05. Altered expression of TER94 directed through the D42-Gal4 transgene affects longevity and climbing ability. A: Longevity assay of Drosophila melanogaster males displaying altered TER94 expression in the motor neurons. Longevity is depicted by percent survival. Signi cance is P <0.05 using the log-rank test with Bonferroni correction. Error bars represent standard error of the mean.

Figure 2
Altered expression of TER94 directed through the D42-Gal4 transgene affects longevity and climbing ability. B: Locomotor assay of D. melanogaster males displaying altered TER94 expression in the motor neurons. Climbing ability was determined by a nonlinear curve t (CI=95%). Error bars indicate standard error of the mean.

Figure 3
Altered expression of TER94 directed through the TH-Gal4 transgene affects longevity and climbing ability. A: Longevity assay of Drosophila melanogaster males displaying altered TER94 expression in the dopaminergic neurons. Longevity is depicted by percent survival. Signi cance is P <0.05 using the logrank test with Bonferroni correction. Error bars represent standard error of the mean.   Altered expression of TER94 directed through the ddc-Gal4HL4.3D transgene affects longevity and climbing ability. A: Longevity assay of Drosophila melanogaster males displaying altered TER94 expression in the neurons. Longevity is depicted by percent survival. Signi cance is P <0.05 using the logrank test with Bonferroni correction. Error bars represent standard error of the mean.

Figure 6
Altered expression of TER94 directed through the ddc-Gal4HL4.3D transgene affects longevity and climbing ability. B: Locomotor assay of D. melanogaster males displaying altered TER94 expression in the neurons. Climbing ability was determined by a nonlinear curve t (CI=95%). Error bars indicate standard error of the mean. Altered expression of TER94 and parkin directed through the ddc-Gal4HL4.3D transgene affects longevity and climbing ability. A: Longevity assay of Drosophila melanogaster males displaying altered TER94 and parkin expression in the neurons. Longevity is depicted by percent survival. Signi cance is P <0.05 using the log-rank test with Bonferroni correction. Error bars represent standard error of the mean.   Altered expression of TER94 directed through the ddc-Gal4HL4.36 transgene affects longevity and climbing ability. A: Longevity assay of Drosophila melanogaster males displaying altered TER94 expression in the neurons. Longevity is depicted by percent survival. Signi cance is P <0.05 using the logrank test with Bonferroni correction. Error bars represent standard error of the mean.

Figure 10
Altered expression of TER94 directed through the ddc-Gal4HL4.36 transgene affects longevity and climbing ability. B: Locomotor assay of D. melanogaster males displaying altered TER94 expression in the neurons. Climbing ability was determined by a nonlinear curve t (CI=95%). Error bars indicate standard error of the mean.

Figure 11
Altered expression of TER94 and the expression of alpha-synuclein directed through the ddc-Gal4HL4.36 transgene affects longevity and climbing ability. A: Longevity assay of Drosophila melanogaster males displaying the expression of alpha-synuclein and the altered TER94 expression in the neurons. Longevity is depicted by percent survival. Signi cance is P <0.05 using the log-rank test with Bonferroni correction.
Error bars represent standard error of the mean.