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 ...

<ol>
	<li>Ingre, C., Roos, P. M., Piehl, F., Kamel, F., Fang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+for+amyotrophic+lateral+sclerosis.">Risk factors for amyotrophic lateral sclerosis.</a> Clinical Epidemiology. 7, 181-193 (2015).</li><li>Dupuis, L., Pradat, P. F., Ludolph, A. C., Loeffler, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+metabolism+in+amyotrophic+lateral+sclerosis.">Energy metabolism in amyotrophic lateral sclerosis.</a> The Lancet Neurology. 10 (1), 75-82 (2011).</li><li>Buratti, E., Baralle, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+functional+implications+of+the+RNA+binding+properties+of+nuclear+factor+TDP-43,+a+novel+splicing+regulator+of+CFTR+exon+9.">Characterization and functional implications of the RNA binding properties of nuclear factor TDP-43, a novel splicing regulator of CFTR exon 9.</a> The Journal of Biological Chemistry. 276 (39), 36337-36343 (2001).</li><li>Polymenidou, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+pre-mRNA+depletion+and+RNA+missplicing+contribute+to+neuronal+vulnerability+from+loss+of+TDP-43.">Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43.</a> Nature Neuroscience. 14 (4), 459-468 (2011).</li><li>Tollervey, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+the+RNA+targets+and+position-dependent+splicing+regulation+by+TDP-43.">Characterizing the RNA targets and position-dependent splicing regulation by TDP-43.</a> Nature Neuroscience. 14 (4), 452-458 (2011).</li><li>Ling, S. C., Polymenidou, M., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Converging+mechanisms+in+ALS+and+FTD:+disrupted+RNA+and+protein+homeostasis.">Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis.</a> Neuron. 79 (3), 416-438 (2013).</li><li>Estes, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wild-type+and+A315T+mutant+TDP-43+exert+differential+neurotoxicity+in+a+Drosophila+model+of+ALS.">Wild-type and A315T mutant TDP-43 exert differential neurotoxicity in a Drosophila model of ALS.</a> Human Molecular Genetics. 20 (12), 2308-2321 (2011).</li><li>Estes, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+neurons+and+glia+exhibit+specific+individualized+responses+to+TDP-43+expression+in+a+Drosophila+model+of+amyotrophic+lateral+sclerosis.">Motor neurons and glia exhibit specific individualized responses to TDP-43 expression in a Drosophila model of amyotrophic lateral sclerosis.</a> Disease Models &amp; Mechanisms. 6 (3), 721-733 (2013).</li><li>Manzo, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycolysis+upregulation+is+neuroprotective+as+a+compensatory+mechanism+in+ALS.">Glycolysis upregulation is neuroprotective as a compensatory mechanism in ALS.</a> eLife. 8, 45114 (2019).</li><li>Volkenhoff, A., Hirrlinger, J., Kappel, J. M., Klambt, C., Schirmeier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+using+a+FRET+glucose+sensor+reveals+glucose+delivery+to+all+cell+types+in+the+Drosophila+brain.">Live imaging using a FRET glucose sensor reveals glucose delivery to all cell types in the Drosophila brain.</a> Journal of Insect Physiology. 106, 55-64 (2018).</li><li>Fehr, M., Lalonde, S., Lager, I., Wolff, M. W., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+the+dynamics+of+glucose+uptake+in+the+cytosol+of+COS-7+cells+by+fluorescent+nanosensors.">In vivo imaging of the dynamics of glucose uptake in the cytosol of COS-7 cells by fluorescent nanosensors.</a> The Journal of Biological Chemistry. 278 (21), 19127-19133 (2003).</li><li>Takanaga, H., Chaudhuri, B., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GLUT1+and+GLUT9+as+major+contributors+to+glucose+influx+in+HepG2+cells+identified+by+a+high+sensitivity+intramolecular+FRET+glucose+sensor.">GLUT1 and GLUT9 as major contributors to glucose influx in HepG2 cells identified by a high sensitivity intramolecular FRET glucose sensor.</a> Biochimedica et Biophysica Acta. 1778 (4), 1091-1099 (2008).</li><li>Ihara, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+binding+mediates+neurotoxicity+in+the+transgenic+Drosophila+model+of+TDP-43+proteinopathy.">RNA binding mediates neurotoxicity in the transgenic Drosophila model of TDP-43 proteinopathy.</a> Human Molecular Genetics. 22 (22), 4474-4484 (2013).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118 (2), 401-415 (1993).</li><li>Diaz-Garcia, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+vivo+imaging+of+neuronal+glucose+concentrations+with+a+genetically+encoded+fluorescence+lifetime+sensor.">Quantitative in vivo imaging of neuronal glucose concentrations with a genetically encoded fluorescence lifetime sensor.</a> Journal of Neuroscience Research. 97 (8), 946-960 (2019).</li><li>Mergenthaler, P., Lindauer, U., Dienel, G. A., Meisel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sugar+for+the+brain:+the+role+of+glucose+in+physiological+and+pathological+brain+function.">Sugar for the brain: the role of glucose in physiological and pathological brain function.</a> Trends in Neurosciences. 36 (10), 587-597 (2013).</li><li>Lu, B., Vogel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+models+of+neurodegenerative+diseases.">Drosophila models of neurodegenerative diseases.</a> Annual Review of Pathology. 4, 315-342 (2009).</li></ol>

The authors declare no conflicts of interest.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm tissue culture dishes</td>
			<td>Sigma Aldrich</td>
			<td>CLS430165</td>
			<td></td>
		</tr>
		<tr>
			<td>40X water immersion lens</td>
			<td>Zeiss</td>
			<td>440090</td>
			<td>dippable, N.A. 0.8</td>
		</tr>
		<tr>
			<td>dissection scissors</td>
			<td>Roboz</td>
			<td>RS-5618</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 forceps</td>
			<td>VWR</td>
			<td>100189-236</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #55 forceps</td>
			<td>VWR</td>
			<td>100189-244</td>
			<td></td>
		</tr>
		<tr>
			<td>Minutien pins</td>
			<td>Fine Science tools</td>
			<td>26002-10</td>
			<td>used for dissections</td>
		</tr>
		<tr>
			<td>SYLGARD 184 Silicone Elastomer Kit</td>
			<td>Dow</td>
			<td>1317318</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM880 NLO upright multiphoton/confocal microscope</td>
			<td>Zeiss</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

This technique is significant because it can measure glucose uptake in specific cells in live Drosophila tissues. The FRET-based sensor provides a direct measure of changes in intracellular glucose levels. This technique allows researchers to evaluate changes in glucose levels in real time, within living motor neurons from ALS models versus controls.A challenging aspect is imaging the same set of neurons after changing the buffer. Leaving the sample on the microscope helps ensure that the VMC moves as little as possible. Before beginning the dissections turn on the imaging microscope and lasers.Then collect a wandering third instar larva, rinse it with double distilled water and place it in a drop of HL3 buffer on a previously prepared elastomer lined dish. Next under a dissecting microscope, use a pair of forceps to pin the anterior and posterior ends of the larva, dorsal side up, by carefully stretching the larva length-wise with insect pins. Using a pair of angled Iris scissors, make an incision just above the posterior pin.Then make a vertical cut from the incision towards the anterior end of the larva. After adding a few drops of HL3 buffer if needed, remove the trachea and the rest of the floating organs without disturbing the central nervous system. Then pin the flaps, stretching the body wall to expose the central nervous system while keeping the neuromuscular system intact.For image acquisition, use upright confocal microscope with a 40 times water immersion lens. Then select the 405 nanometer laser to excite the CFP. Next, optimize the acquisition parameters such as scan speed, average, objective, zoom pinhole size, and spatial resolution.Adjust the gain such that the signal is in the optimal dynamic range and use the same parameters for all genotypes. To image motor neurons within the ventral nerve cord, place the silicone dish with the dissected sample under the lens and lower the lens so that it comes in contact with the trehalose sucrose HL3 buffer. Ensure that the lens is completely submerged in the buffer and add more buffer if required.Next.Using the CFP and FRET channels, manually select an optical selection consisting of at least six motor neurons in focus located along the ventral nerve cord midline. Then acquire images every 10 seconds for 10 minutes. These images represent the baseline.For glucose stimulation, remove the trehalose sucrose containing HL3 buffer using a Pasteur pipette and add five millimolar glucose supplemented HL3 buffer without moving the ventral nerve cord. Then acquire images every 10 seconds for another 10 minutes. These images represent the stimulation phase.Save the images as CZI files or any file type supported by the imaging software with a file name including date, genetic background, experimental condition and channels used. Reuse the parameters to image all the genotypes. For image processing open the Fiji-ImageJ imaging software, then open the CZI files, choose view stack with hyper stack and set color mode to gray scale and select split channels into separate windows.Next.Process the images using drift correction plug-ins, turbo reg and stack reg. Each channel is corrected separately by selecting stack reg under plug-ins and then choosing transformation to translation. Manually choose the regions of interest or ROIs by tracing all motor neurons that remain in focus over the entire time series.Then use the ROI manager to save each motor neuron outline. Use the multi measure function to measure the mean gray value in each ROI at each time point. Then copy the ROIs traced from the first channel to the second channel for the pre stimulation image.For data analysis, calculate the FRET by CFP ratio using the mean gray values for the FRET and CFP channels. Changes in the FRET by CFP ratios reflect alterations in the intracellular glucose concentration. Then plot FRET by CFP ratios for each ROI and time point versus time.A genetically encoded FRET-based glucose sensor was used to measure glucose uptake in motor neurons. When glucose is not bound to the sensor, CFP excitation causes CFP emission only. And when glucose binds, the YFP signal can be detected due to FRET occurring between CFP and YFP.Shown here are images of FRET and CFP signals in glucose sensor controls in TDP-43 mutant neurons under baseline and stimulation conditions. A representative motor neuron is outlined in each image as an example of ROI. Upon glucose stimulation, control motor neurons expressing glucose sensors, exhibit a small increase in glucose uptake while neurons expressing TDP-43 showed a significantly higher glucose uptake.Further FRET by CFP ratios of TDP-43 mutant motor neurons were normalized to glucose sensor controls at each time point. Under baseline conditions, there was no difference between the genotypes. However, upon glucose stimulation, a rapid and significant increase in glucose uptake was observed in TDP-43 mutants compared to the control.In a bar graph representation, glucose stimulation in neurons expressing the glucose sensor demonstrated a small but significant increase in FRET by CFP ratio compared to the baseline. In contrast neurons expressing TDP-43 showed a significant increase in ratio upon stimulation compared to the baseline. The net difference in FRET by CFP ratios between motor neurons expressing TDP-43 and glucose sensor is around 10.9%Time management is critical for this procedure so that the neurons do not die over the course of imaging.It's important to image immediately following dissections to avoid this. This technique highlighted the importance of glucose metabolism in neurons and has led our group to further investigate the role of glycolysis in motor neuron regeneration.

measuring-glucose-uptake-in-drosophila-models-of-tdp-43-proteinopathy

Research

JoVE Journal

Neuroscience

2.6K visualizações.  University of Arizona, Tucson. Esta técnica é significativa porque pode medir a absorção de glicose em células específicas em tecidos Drosophila vivos. O sensor baseado em FRET fornece uma medida direta das alterações nos níveis de glicose intracelular. Essa técnica permite que os pesquisadores avaliem mudanças nos níveis de glicose em tempo real, dentro de neurônios motores vivos de modelos de ELA versus controles.Um aspecto desafiador é a imagem do mesmo conjunto de neurônios depois de mudar o buffe...