Larval locomotor defects were previously found in most mutant larvae lines (Section 11). We thus tested if Metformin was also able to rescue this phenotype. After 3 days of 10mM Metformin treatment, CG5599 and CG7430 mutant larvae demonstrated
more straight locomotor behavior than untreated ones (Figure 17A.). Larval travelled length was also rescued in mutant lines treated with Metformin but not average larval speed, suggesting that Metformin could recover mutants discontinuous mobility.
(Student’s t test; p < 0.001.) (Figure 17B.).
Discussion
Here, we generated a novel model of MSUD in D. melanogaster, then validated it by comparing molecular, electrophysiological and behavioral phenotypes of mutant flies with other animal models/ humans.
Knock out flies for the CG1673 (BCAT), CG5599 (E2 component) and CG7430
(E3 component) genes showed severe developmental problems. Homozygous flies were unable to eclose from pupae in knockout lines under normal feeding conditions.
This is similar in some respects to the human form of MSUD, with consumption of protein potentially leading to lethality in infants11. On the other hand, CG8199 (E1α component) and CG17691 (E1β component) mutants were homozygous viable at all developmental stages. Both mutants were E1 component knockouts however; as both subunits serve the same function, it is potentially unsurprising that inactivation of only one of these genes has little or no effect on the biological phenotype.
MSUD causes not only metabolic problems but also central nervous system disorders. In humans, MSUD patients have been shown to suffer from both white matter and neuronal injuries in magnetic resonance imaging studies; this includes extensive brain edema and pathological changes in the basal ganglia60,61. Neuronal defects are therefore a significant risk for MSUD patients, with even the most effective available treatment (liver transplantation) only proven effective for preventing acute crises and
unable to reverse psychiatric disease62. Similar to this phenotype, we found the appearance of vacuoles in brain paraffin sections of mutant flies. Furthermore, eye morphology changes revealed severe injury traits in mutant larvae, particularly CG5599 and CG7430 mutants. This phenotype became more serious when intense light was supplied for 7 and 14 days, suggesting the absence of these two subunits led to
neurodegeneration.
Additionally, analysis of our ERG data on photoreceptor function lent credence to the morphological data. Reduction of ERG depolarization amplitudes and absence of on- and off- transients resulted in neuronal defects. These data indicate that CG5599 and CG7430 could play a critical role in the normal function of neurons.
High BCAA levels are often found in obese, insulin-resistant states and in Type 2 Diabetes patients35. BCAAs thus became as a biomarker to detect type 2 diabetes.
However, the mechanism of how BCAAs elevated in obesity individual is still unclear.
There is a hypothesis to explain increased plasma BCAA levels to insulin resistance linked to activation of mTORC1: excessive nutrients lead to increase plasma level of leucine which together with insulin activate mTORC1 and S6K1. Persistent activation leads to serine phosphorylation of IRS-1 and IRS-2, which interferes with signalling and might target IRS1 for proteolysis via a proteasomal pathway. The resulting insulin resistance increases demand on insulin to expenditure overmuch glucose. Long-term
demand for insulin secretion, ultimately fail to produce sufficient quantities of insulin and lead to the T2DM18(Appendix v.).
In adipose tissue, obesity was associated with declines in BCATm (homologous to CG1673) and BCKD E1α (homologous to CG8199) protein concentrations in
ob/ob mice and Zucker fatty rats37,63. However, we found BCAT (CG1673) and two
BCKDH (CG5599 and CG7430) mutants were associated with increasing lipid droplet size in fat body which is a sign od obesity. Due to the absence of CG1673, CG5599 and CG7430 in adipocytes, we hypothesized that elevated BCAAs activated dTOR, which
downregulated autophagy and protein/lipid synthesis. Therefore, decline of protein and lipid turnover rate made excessive lipid storage in fat body. We would also like to investigate whether elevated BCAAs regulate insulin resistant and hyperglycemia in the MSUD Drosophila model in the future.
In order to investigate autophagy in MSUD mutants, we used an Atg8a marker and Lysotracker to monitor the progress of autophagy and formation of lysosomes. Here, we found that Atg8a marker expression was markedly decreased in mutant lines, suggesting that autophagy was downregulated. We hypothesize that activation of dTOR resulted in an inhibition of autophagy. Misregulation of autophagy possibly represents protein degradation imbalances in the fat body and mitochondria homeostasis in muscle.
Alteration of muscular, neural, and olfactory systems should affect locomotor
behavior. Analysis of mutant mobility could therefore help to increase our understanding of the exact effects of MSUD. In human cases, twitching of limbs has been reported to happen in two month old infants64. In our model all mutants, apart from CG17691 mutants, demonstrated reduced mobility compared to controls. This includes not only the distance they were able to travel within a set time limit but also
their average speed. Their paths also tended to be highly irregularity, which is suggestive of damage to muscles and neurons.
It is increasingly clear that altered mitochondrial dynamics also underlie the pathology of many degenerative diseases65. Thus, understanding mitochondrial distribution, shape, and dynamics in all cell types should help to develop treatment regimens targeting different tissues66. We observed mitochondria in larval fat bodies and muscles and found that mitochondria in CG1673 mutant were smaller than in controls, whilst CG5599 and CG7430 mutant mitochondria were more dispersed.
Consistent with mitochondria in muscles, we found that the number of mitochondria decreased in mutant fat bodies and mitochondrial clusters appeared in CG5599 and CG7430 mutants, implying that this abnormal distribution contributed to functional
disorders. These results suggest that future research into mitochondrial function in mutants could be highly promising, particularly if done in conjunction with detection of TCA cycle substrate levels (which are linked to energy production).
Current treatments for MSUD are not satisfactory and require new approaches to combat this disease. A major hurdle in developing new treatments has been the lack of a suitable widespread animal model. Drosophila melanogaster is a well-studied, genetically pliable model organism that has proven useful for understanding molecular mechanisms of human diseases. Recently, scientists had been able to
establish live organism drug screening protocols based chemical screenings in Drosophila67. Though the value of flies in terms of human disease may be unclear, Drosophila and human cell had been found to often share the same molecular
mechanisms. This includes actin and microtubule poisons, inhibitors of DNA topoisomerases, kinases, and phosphatases, alkylating agents and modulators of membrane channels67. Thus small compounds identified for their activity in flies may show similar efficacy in mammals, whilst also being much quicker and easier to identify.
Our preliminary testing of the antidiabetic drug, Metformin (which has been previously used in mice models of MSUD) was able to rescued several mutant larvae phenotypes to the WT state46. In our findings, 10mM Metformin treatment was able to rescue BCAA levels and locomotor behavior, though mutant pupae from several lines were still unable to eclose. Testing of a range of concentrations is still required to
determine optimal dosage levels however, as well as determination of the most efficient treatment length.
Based on this newly established MUSD model, more metabolic processes and mechanism can be investigated and be understood. Taken together, our data illustrates how defects in BCAA metabolic processes can disrupt nervous system development
and function, and establishes D. melanogaster mutants as a model to better understand MSUD.
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Figures
Figure 1. The branched-chain amino acid (BCAA) metabolic pathway BCAAs are degraded first by BCAT and then the BCKDH complex, which is
comprised of three components (E1, E2 and E3). Intermediate products then go to the TCA cycle and produce energy. BCKDH complex is the rate-limiting step that is responsible for BCAA metabolism8. The absence of any individual subunit should cause MSUD. CG number represents the Drosophila homolog.
Figure2. Generation of five mutant lines via CRISPR/Cas9
(A, C, E) w1118 Drosophila melanogaster were injected with constructs containing a knock-in cassette (attPX-RFP cassette) by insertion of two gRNAs complementary to the five metabolic genes (editing by homology dependent repair). The cassette
contained attPX, two STOP codons and 3xP3-RFP. (B,F,D) Mutants were validated by checking for RFP fluorescence (WellGenetics Inc.).
Figure 3. Quantification of BCAA accumulation by mass spectrometry.
(A-C) Leucine, isoleucine and valine levels were increased in mutant flies (N=25 for all groups) compared to controls as quantified by LC-MS. Each experiment was repeated three times; p values were calculated using a Student’s t test. *p<0.05, **p <
0.01, ***p < 0.001, ****p<0.0001, n.s. indicates not statistically significant compared to WT; error bars indicate standard deviation.
Figure 4. Pupae eclosion rate.
Comparison of eclosion rates across control, CG1673, CG8199, CG17691, CG5599 and CG7430 lines. CG1673 and CG5599 mutant were found to be homozygous lethal, while CG7430 mutants are embryonic lethal. CG8199 and CG17691 mutants only show slight, but significant, decreases in eclosion rate. Chi square tests were used to test significant differences between mutants and controls (* p<0.01; *** p<0.0002).
Three biological repeats were conducted (Total N=300 for each group); error bars indicate standard deviation. CG1673△/Y p value< 2.2x10^-16; CG8199△/CG8199△ p value< 4.0x10^-14; CG17691△/CG17691△ p value< 0.002; CG5599△/Y p value<
B r a in p a r a f f in s e c t i o n
Figure 5. Flies brain paraffin section revealed neuron defects phenotype.
(A-F) H&E stained seven-day-old fly brains from w1118, CG1673Δ/+, CG8199Δ /CG8199Δ, CG17691Δ/CG17691Δ, CG5599Δ/+ and CG7430Δ/+. (G) Quantification of the number of vacuoles for each line, based on brains per genotype (N=3). p values were calculated using a Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001. n.s.
indicates not statistically significant; error bars indicate standard deviation.
G
rhabdomeres/ommatidium
Figure 6. BCKDH RNAi lines demonstrate neuronal degeneration.
(A) Immunostaining of Drosophila photoreceptors after exposure under constant light stimulation for 7 or 14 days (N=5). Rhabdomeres were stained by phalloidin (green) and neuronal membrane was stained by Na+/K+‐ATPase (magenta). (B) Quantification of number of rhabdomeres in (A). N=15. p values were calculated using a Student’s t test. *p<0.05, **p < 0.01, ***p < 0.001, ****p<0.0001 compared to WT; error bars
indicate standard deviation.
A
B
Figure 7. BCKDH RNAi lines demonstrate neuronal dysfunction.
(A) Neuronal function as measured by ERG for control flies and CG1673, CG8199 CG17691, CG5599 and CG7430 RNAi lines. Sample ERG traces showed striking decrease in the depolarization amplitude for CG5599 and CG7430 knockdowns after 7- and 14-days constant light stimulation compared to WT. A sample size of flies eyes (N=10) was used for each group. (B) Quantification of depolarization amplitudes in (A). p values were calculated using Student’s t test. **p < 0.01, ***p
< 0.001, ****p<0.0001; error bars indicate standard deviation.
A B
Figure 8. BCKDH gene expression distribution across tissues. (A) BCKDH gene expression in head, thorax and fat body (N=20, 17, 5 respectively; three biological repeats) of wild type Drosophila (w1118) quantified by qPCR mRNA abundance was normalized to rp49. Mean ± SD abundance of thorax and fat body is relative to CG1673.
(B) Gene expression in head, thorax and fat body (N=20, 17, 5 respectively; three biological repeats) of w1118; error bars indicate standard deviation. GAPDH served as the internal standard for normalization.
B
A
Figure 9. BCKDH genes are involved in regulation of lipid size in the Drosophila
Figure 9. BCKDH genes are involved in regulation of lipid size in the Drosophila