Increased vesicular glutamate transporter expression causes excitotoxic neurodegeneration

Abstract

Increases in vesicular glutamate transporter (VGLUT) levels are observed after a variety of insults including hypoxic injury, stress, methamphetamine treatment, and in genetic seizure models. Such overexpression can cause an increase in the amount of glutamate released from each vesicle, but it is unknown whether this is sufficient to induce excitotoxic neurodegeneration. Here we show that overexpression of the Drosophila vesicular glutamate transporter (DVGLUT) leads to excess glutamate release, with some vesicles releasing several times the normal amount of glutamate. Increased DVGLUT expression also leads to an age-dependent loss of motor function and shortened lifespan, accompanied by a progressive neurodegeneration in the postsynaptic targets of the DVGLUT-overexpressing neurons. The early onset lethality, behavioral deficits, and neuronal pathology require overexpression of a functional DVGLUT transgene. Thus overexpression of DVGLUT is sufficient to generate excitotoxic neuropathological phenotypes and therefore reducing VGLUT levels after nervous system injury or stress may mitigate further damage.

Figure 1

Large spontaneous events occur in muscles of animals overexpressing DVGLUT in motoneurons. A. Intracellular voltage recording in an animal overexpressing DVGLUT showing numerous large events (>4 mV), in the absence of extracellular Ca²⁺ and in the presence of 1 μM TTX and after cutting the motoneuron axon. B. Sample trace from a control muscle. C. Large events are much more frequent in larvae overexpressing DVGLUT (p<0.0001, Student’s t-test, n=12 cells with 70 events for each genotype). D. The largest 2% of events is larger in larvae overexpressing DVGLUT (p<0.0001, Student’s t-test, n=12 cells with 70 events for each genotype).

Figure 2

Overexpression of DVGLUT leads to decreased lifespan and progressive motor deficits. A. Survival over a two-week period was significantly decreased in male flies overexpressing DVGLUT compared with flies overexpressing GFP (control). B. Flies overexpressing DVGLUT in glutamatergic neurons exhibit progressive motor impairment. The time it took the fastest fly to climb 16 cm (labeled first fly) and the time it took half of the flies (labeled fifth fly) to climb 16 cm were measured to obtain both the group best and a measure of the population average, respectively. Flies were measured on 2, 8, and 30 days after eclosing.

Figure 3

Pronounced neurodegeneration occurs in flies overexpressing DVGLUT in glutamatergic neurons. A–D. 1 μm plastic sections stained with toluidine blue and basic fuchsin. Horizontal sections of controls (A and C) and mutants (B and D) at 2 days (A and B) and 8 days (C and D). Holes in neuropil regions are apparent in 2 day-old mutants (B) and become more severe at 8 days (D). Scale bar represents 50 μm. E1-3. Thin confocal stack from a whole-mount adult brain demonstrating holes in the medial inferior lateral protocerebrum (milpr) by immunostaining for postsynaptic marker Dlg (E1). GFP fluorescence from GFP:DVGLUT (E2) shows that holes in Dlg staining are filled by presynaptic GFP. E3 is the merged image. Scale bar represents 10 μm. F1-3. Single confocal section of the antennal lobe in a control stained with anti-Dlg (F1). GFP fluorescence from mCD8::GFP (F2) shows the expression pattern of dvglut^CNSIII-Gal4 in the antennal lobe. Large holes in the antennal lobe glomeruli are not seen opposite GFP-positive terminals. F3 shows the merged image. G1-3. Single confocal section from the antennal lobe of a fly overexpressing GFP:DVGLUT with dvglut^CNSIII-Gal4 stained with anti-Dlg (G1). Holes in Dlg staining within glomeruli (arrowhead) colocalize with enlarged GFP-positive presynaptic terminals (G2, green in merged image), indicating loss of postsynaptic targets of DVGLUT-expressing terminals. G3 shows the merged image. Scale bar represents 20 μm (for F and G). H and I. Or83b-Gal4 expression of GFP:DVGLUT in olfactory receptor neurons (ORNs) does not kill ORNs or cause degeneration in the antennal lobe. H. Robust GFP signal from GFP:DVGLUT is seen in the antennal lobe glomeruli and in the axons of the antennal nerve (asterisk), indicating that ORN axons and terminals are intact when expressing GFP:DVGLUT (2D maximum projection of confocal stack). I. Projection of a thin confocal stack stained with anti-Dlg (magenta) and with GFP fluorescence in green. Insets show a single confocal section of the boxed area with uniform Dlg staining, indicating lack of degeneration of postsynaptic ORN targets. Scale bar in I represents 20 μm and in the inset represents 5 μm.

Figure 4

Phenotypes of DVGLUT overexpression in glutamatergic neurons requires DVGLUT function. Overexpression of the mutant DVGLUT protein containing a single amino acid change at position 470 from alanine to valine (A470V) is significantly less deleterious than overexpression of wild type DVGLUT protein. A. Large spontaneous event frequency is not significantly increased in flies overexpressing DVGLUT A470V (p>0.06 between control and A470V; n=20 cells in WT, n=37 cells for DVGLUT overexpression, and n=15 cells for A470V overexpression). B. Expression of DVGLUT A470V does not reduce survival rate. C and D. Expression of DVGLUT A470V does not significantly decrease climbing ability at 2 or 8 days after eclosion, although a slight difference can be seen after 30 days. The time for the first fly to climb 16 cm is shown in C, and the time for 5 of 10 flies to climb 16 cm is presented in D, used as a indication of the best and average times, respectively. E and F. Plastic sections of adult brains from 2 days old (E) and 8 day old (F) flies overexpressing DVGLUT A470V stained with basic fucshin and toluidine blue. Notice the low occurrence of vacuolar degeneration compared with figure 3B and 3D. Scale bar represents 50 μm.