It is widely known that ALS patients display gross metabolic dysregulation described as hypermetabolism (for a recent review, see Joardar et al., 2017). However, a major challenge in the field has been to understand how these clinical observations relate to metabolic changes at the cellular level. Here, we take advantage of cell type specific genetic tools available in Drosophila to pinpoint how specific metabolic changes in neurons and glia relate to disease progression. Using metabolic profiling we first found that the neuronal expression of TDP-43 increases the abundance of pyruvate, the end product of glycolysis. Notably, this alteration is consistent with metabolite changes in plasma from ALS patients (Lawton et al., 2012) and suggest an increase in glycolysis. Substantiating this scenario is transcriptional profiling of ALS spinal cords and patient derived iPSC motor neurons showing that just as in flies, PFK, the rate limiting step in glycolysis, is significantly upregulated (Figure 2). Interestingly, metabolic profiling in flies also suggests increased glycolytic input into the pentose phosphate pathway (Supplementary file 1), which provides a mechanism for countering oxidative stress via increased NADPH production (Kruger and von Schaewen, 2003). Supporting this possibility are our findings that G6PD, the rate limiting enzyme of the pentose phosphate pathway is upregulated in fly and human ALS ventral and spinal cords, respectively (Figure 2). Oxidative stress is a well-established ALS manifestation (Barber and Shaw, 2010) and the most recent FDA approved drug for treating ALS patients, Radicava, is aimed at countering this aspect of the disease (Cruz, 2018). Collectively, molecular and metabolic alterations identified in the fly validate in patient derived motor neurons and spinal cords, albeit the magnitude of the changes found in human ALS tissues is often more dramatic and, at some level, more comparable with mutant TDP-43 dependent phenotypes in flies. These findings highlight the predictive power of the fly model and its relevance to studying disease pathomechanisms in humans.

Using a genetically encoded glucose sensor we show that TDP-43G298S expressing motor neurons have increased capacity to import glucose (Figure 3). This is consistent with increased glycolysis and its end product, pyruvate. It is unclear why despite finding that pyruvate is also increased in motor neurons overexpressing TDP-43WT, we could not detect higher glucose uptake than in controls, although this is consistent with PFK transcript levels trending up, yet not reaching significance in our data set (Figure 2). A possible explanation is that while our model is based on TDP-43 expression specifically in motor neurons or glia, the metabolite profiling was performed on whole larvae and captured non-cell autonomous changes, occurring in cells other than motor neurons. Nevertheless, taken together our data show that there is a clear dysregulation of glycolysis in TDP-43 expressing Drosophila and patient tissue samples. Alterations in glucose metabolism may also be a feature of multiple ALS types. Indeed, pre-symptomatic SOD1 mutant mice have been shown to have high glucose levels in spinal cords; however, glucose levels gradually decrease as disease symptoms progress (Miyazaki et al., 2012). One question that remained is whether alterations in glucose metabolism are compensatory or causative.

In an effort to address this question, we first increased glucose availability in Drosophila by raising the dietary concentration of glucose (5–10 X higher than normal levels in standard fly food), which mitigates locomotor defects and increases lifespan in ALS flies while making wild-type larvae sluggish (Figure 3). Interestingly, a high sugar diet only mitigates locomotor defects when TDP-43 is expressed in motor neurons or glia but not muscles, indicating that the primary metabolic defect lies in the nervous system and that muscles do not benefit from increased glucose availability (Figure 4 and Figure 4—figure supplements 2 and 5). This is somewhat surprising given that muscles normally rely on glycolysis for ATP production but is also consistent with findings that in ALS, muscles switch to primarily using lipids and not glucose for energy production (Palamiuc et al., 2015).

This systemic approach, however, does not provide information on which cells specifically benefit from increased glucose availability. To answer this question, we intervened genetically by overexpressing human glucose transporters GLUT-3 or GLUT-4 in motor neurons, glia or muscles (Figures 4 and 5 and Figure 4—figure supplements 2 and 5). These experiments showed that both GLUT-3 and GLUT-4 rescued locomotor dysfunction of TDP-43 expressing larvae in the nervous system, but not in muscles (Figure 4 and Figure 4—figure supplements 2 and 5) confirming our findings from high sugar feedings. Furthermore, our findings that overexpression of either GLUT-3 or GLUT-4 can help mitigate specific ALS-like phenotypes in flies are consistent with recent reports that both GLUT-3 and GLUT-4 are required at synapses to regulate activity (Ashrafi et al., 2017; Ferreira et al., 2011). Together, our dietary intervention and GLUT-3/4 overexpression experiments indicate that increased glucose availability in motor neurons and glia confers protection in models of TDP-43 proteinopathy, in a cell type dependent manner.

A possible explanation for why increased glucose availability improves defects caused by TDP-43 proteinopathy is that cells are actively consuming glucose to increase glycolytic flux. A recent study using vertebrate cell lines suggests that glycolytic flux depends on four key steps. They include: (1) glucose uptake by glucose transporters, (2) hexokinase levels, (3) PFK levels (and associated enzymes), and (4) lactate export, but not other enzymes or steps involved in glycolysis (Tanner et al., 2018). The co-over-expression of PFK mitigates TDP-43 defects in flies, and is consistent with our GLUT-3 over-expression experiments (Figure 6). Surprisingly, PFK transcript levels were higher in human ALS post-mortem tissues compared to controls, however protein levels in total tissue homogenates were not significantly increased despite trending upwards (data not shown). Nevertheless, our findings of increased PFK mRNA levels in flies and human tissues, and the genetic interaction experiments support the notion that glycolysis is increased in ALS as a compensatory mechanism (see Figure 7 for model).

The fact that neurons would compensate deficits in cellular energetics through increasing glycolysis makes sense in the light of well-established defects in mitochondrial function. TDP-43 has been previously shown to localize to the inner mitochondrial membrane of mitochondria (Wang et al., 2016), and it has been shown to cause abnormally small mitochondria in Drosophila (Khalil et al., 2017). Consistent with the importance of glycolysis in neurodegeneration, there are reports of SNPs within PFK that are present in ALS patients but not in healthy controls (Rouillard et al., 2016; Xie et al., 2014). Our findings that glycolysis is increased as a compensatory mechanism and this confers neuroprotection is consistent with findings from a small clinical trial showing that a high caloric high carbohydrate diet improves patient outcome (Wills et al., 2014) and also that patients with diabetes have a later onset of disease (Kioumourtzoglou et al., 2015). While upregulation of glycolysis decreases the dependence of the organism for ATP derived from mitochondria, it is also possible that increased glycolysis is improving organismal health through a different mechanism. For example, pyruvate has been previously shown to protect mitochondria from oxidative stress (Wang et al., 2007). Future studies should focus on the link between glycolysis, oxidative stress and mitochondria in ALS.