Name: measuring glucose uptake in drosophila models of tdp-43 proteinopathy
Uploaded: 2021-08-03T15:00:00
Description: Watch this Scientific Journal Video about Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy at JoVE.com

We thank Stefanie Schirmeier and Takeshi Iwatsubo for providing Drosophila strains. We also thank Patricia Jansma for assisting with imaging in the Marley Imaging Core at the University of Arizona. This work was funded by National Institutes of Health NIH NS091299, NS115514 (to DCZ), HHMI Gilliam Fellowship (to EM) and the Undergraduate Biology Research Program (to HB).

The UAS FLII12Pglu-700&#181;&#948;6 transgenic flies were reported in Volkenhoff et al.10 and kindly provided by Dr. S. Schirmeier. The UAS TDP-43G298S transgenic lines were kindly provided by Dr. T. Iwatsubo13. Recombinant Drosophila lines harboring both UAS FLII12Pglu-700&#181;&#948;6 and UAS TDP-43 transgenes were generated in the Zarnescu laboratory using standard genetic approaches and reported in Manzo et al.9. D42 GAL4 was ...

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The technique described in detail here can be applied to measure glucose uptake in a specific cell type of interest in live Drosophila using FLII12Pglu-700&#181;&#948;6, a FRET based sensor which can detect changes in glucose levels to a millimolar range10,11,12. This sensor has been previously used in conjunction with the UAS-GAL4 system to target its expression to specific cell types, including neurons<sup class="xref...

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder that is currently incurable. ALS affects upper and lower motor neurons leading to loss of motor coordination, irreversible paralysis, respiratory failure, and eventual death within 2-5 years of diagnosis1. ALS is associated with metabolic defects such as weight loss, dyslipidemia, and hypermetabolism (reviewed in2); however, it remains unclear how these alterations in metabolism relate to motor neuron degeneration. A common denominator in ALS and related neurodegenerative diseases is TDP-43, a nucleic acid binding protein involved in several steps of RNA processing3,4,5. Although mutations in TDP-43 only affect 3%-5% of patients, wild-type TDP-43 protein is found within cytoplasmic aggregates in &#62;97% of ALS cases (reviewed in6). This pathology was modeled in Drosophila by overexpression of human wildtype or mutant TDP-43 (G298S) in motor neurons, which recapitulates multiple aspects of ALS, including cytoplasmic inclusions, locomotor dysfunction and reduced lifespan7,8. Using these models, it was recently reported that TDP-43 proteinopathy causes a significant increase in pyruvate levels and phosphofructokinase (PFK) mRNA, the rate limiting enzyme of glycolysis9. Similar increases in PFK transcripts were found in patient-derived motor neurons and spinal cords, suggesting that glycolysis is upregulated in the context of TDP-43 proteinopathy. Interestingly, further increase in glycolysis using dietary and genetic manipulations mitigated several ALS phenotypes such as locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy, consistent with a compensatory, neuroprotective mechanism in degenerating motor neurons.
To further probe changes in glycolysis and measure glucose uptake in Drosophila models of TDP-43 proteinopathy, a previously reported genetically encoded FRET-based sensor FLII12Pglu-700&#181;&#948;610 was expressed in motor neurons specifically using the UAS-GAL4 expression system. The FLII12Pglu-700&#181;&#948;6 glucose sensor uses resonance energy transfer between two variants of green fluorescent protein, cyan and yellow fluorescent proteins (CFP and YFP) to detect glucose at the cellular level. It consists of a bacterial glucose binding domain from the E. coli MglB gene fused to CFP and YFP at opposite ends of the molecule. When bound to a glucose molecule, the sensor undergoes a conformational change bringing CFP and YFP closer together and allowing FRET to occur, which can then be used to quantify intracellular glucose levels10,11,12 (Figure 1). Here, we show how the FLII12Pglu-700&#181;&#948;6 sensor can be used to determine changes in glucose uptake caused by TDP-43 proteinopathy in motor neurons. The experiments described here show that overexpression of an ALS-associated mutant, TDP-43G298S, in motor neurons causes a significant increase in glucose uptake compared to controls. This approach can be used in other types of ALS (e.g., SOD1, C9orf72, etc.) and/or other cell types (e.g., glia, muscles) to determine changes in glucose uptake associated with neurodegeneration.

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			<td>35 mm tissue culture dishes</td>
			<td>Sigma Aldrich</td>
			<td>CLS430165</td>
			<td></td>
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			<td>40X water immersion lens</td>
			<td>Zeiss</td>
			<td>440090</td>
			<td>dippable, N.A. 0.8</td>
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			<td>dissection scissors</td>
			<td>Roboz</td>
			<td>RS-5618</td>
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			<td>Dumont #5 forceps</td>
			<td>VWR</td>
			<td>100189-236</td>
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			<td>Dumont #55 forceps</td>
			<td>VWR</td>
			<td>100189-244</td>
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			<td>Minutien pins</td>
			<td>Fine Science tools</td>
			<td>26002-10</td>
			<td>used for dissections</td>
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			<td>SYLGARD 184 Silicone Elastomer Kit</td>
			<td>Dow</td>
			<td>1317318</td>
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			<td>Zeiss LSM880 NLO upright multiphoton/confocal microscope</td>
			<td>Zeiss</td>
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measuring glucose uptake in drosophila models of tdp-43 proteinopathy

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy that recapitulates several features of ALS including cytoplasmic inclusions, locomotor dysfunction, and reduced lifespan, we recently identified broad ranging metabolic deficits. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700&#181;&#948;6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700&#181;&#948;6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43G298S, an ALS causing variant. Here, we show how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700&#181;&#948;6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.

Image acquisition of the glucose sensor in the ventral nerve cord (VNC), ex vivo 
To determine differences in glucose uptake in a Drosophila model of ALS based on TDP-43, a genetically encoded FRET-based glucose sensor was used. The sensor comprised CFP and YFP fused to the glucose binding domain from the E. coli MglB gene. Glucose binding elicits a conformational change, which can be detected by fluorescence resonance energy transfer (FRET) between the CF...

Watch this Scientific Journal Video about Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy at JoVE.com

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Glucose uptake is increased in Drosophila motor neurons affected by TAR DNA binding protein (TDP-43) proteinopathy, as indicated by a FRET-based, genetically encoded glucose sensor.

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical ...

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