Two new reports this week show what can happen when neurotransmitters get out of whack, with ramifications for Alzheimer disease and Parkinson disease.
First, a paper in the September 7 Neuron describes a new mouse model of cholinergic dysfunction, with some intriguing memory and learning problems that are reminiscent of Alzheimer disease. The report, from Marc Caron of Duke University in Durham, North Carolina; Marco Prado of the Universidade Federal de Minas Gerais, in Belo Horizonte, Brazil; and colleagues, shows that mice with reduced expression of the vesicular acetylcholine transporter (VAChT) have moderately decreased cholinergic tone in the CNS. Behaviorally, the animals display notable defects in recognizing familiar objects and animals, similar to the cognitive problems seen with Alzheimer disease. The deficiency in social memory was reversed by treating the mice with cholinesterase inhibitors, the same strategy used for treating cognitive symptoms in AD. Besides demonstrating an important role for VAChT in maintaining normal acetylcholine turnover, the mice provide a useful model for investigating new treatments for the cholinergic dysfunction of AD and other diseases.
A second report in the September 6 Journal of Neuroscience shows that α-synuclein, which can cause inherited Parkinson disease when mutated or overexpressed, increases cytosolic dopamine in neurons, adding support to the idea that it is dopamine itself that causes the most damage in Parkinson disease (see ARF related news story).
In the first report, the researchers applied a knockdown strategy to understand the role of VAChT in acetylcholine neurotransmission. VAChT is required to fill storage vesicles with ACh for release at synapses, and first author Vania Prado and colleagues reasoned that knocking it out completely would be lethal. Instead, they created a knockdown allele by disrupting the 5’ untranslated region of the gene, which caused a partial reduction in the levels of VAChT mRNA. The result in heterozygous mice was a roughly 40 percent reduction in VAChT protein; homozygotes showed a 65 percent decrease.
The animals were viable, but they had lower ACh release at neuromuscular junctions, which was attributed to a decreased vesicular content of ACh. The ramifications were severe for homozygotes: they showed significant impairments in muscle strength, an inability to navigate the rotorod, and very little physical endurance on a treadmill. In contrast, the heterozygotes took longer than normal mice to learn the rotorod, but eventually achieved the same proficiency as their wild-type littermates. Apparently, the mice tolerated a moderate decrease in VAChT at the neuromuscular junctions without motor problems, but below a certain threshold the deficiency was disabling.
The muscular problems of the homozygotes prevented them being tested for behavioral problems, but the heterozygote mice provided an opportunity to study the contribution of central nervous system acetylcholine to complex behaviors. To do this, the researchers first confirmed that cholinergic tone was reduced in the heterozygotes. Microdialysis measurements in the frontal cortex and striatum of living animals showed extracellular ACh levels reduced by one-third and stimulated release dampened. Total brain ACh was actually increased, but the reduction in VAChT resulted in a smaller releasable pool.
The heterozygous animals performed just like their normal littermates in an avoidance test, where they had to learn and remember not to step down onto an electrified platform. The results show that this hippocampal-dependent learning and memory pathway is preserved in animals despite their poor cholinergic tone. But in a different test, of object recognition, the heterozygotes did more poorly at remembering familiar objects 1.5 or 24 hours after training. When the “object” was another animal, they also failed to react to it as familiar. Since mice recognize each other based on odor, the investigators ruled out that the heterozygotes had olfactory problems, and concluded that the failure of social recognition was a true cognitive defect. This memory defect mimics some symptoms of AD, and interestingly it was reversed by increasing ACh with cholinesterase inhibitors. This shows that the memory effects were due to reduced ACh and not some developmental effects of lowered VAChT. The cholinesterase inhibitors had no effect on the behavior of wild-type mice.
“Our observations support the notion that reduced cholinergic tone in AD mouse models can indeed cause deficits in social memory,” the authors write. Future studies using these mice, they say, may help to understand the contributions of cholinergic decline to the behavioral changes that accompany CNS pathologies. In addition, they note that their results suggest a decrease in vesicular transporter expression is less tolerated than decreases in the ACh synthetic enzyme choline acetyltransferase, which is widely used to measure cholinergic deficits in AD.
On the basic research side, the work demonstrates another site of regulation of neurotransmission, this one at the presynaptic level of ACh loading into vesicles. This point is explored in an accompanying preview by Thomas Hnasko and Robert Edwards from the University of California, San Francisco.
In the second paper, the pathological effects of α-synuclein are tied to a case of too much neurotransmitter rather than too little. In this case, the neurotransmitter is dopamine, which can cause oxidative damage when it builds up in the cytosol. Under normal conditions, cytosolic dopamine contributes only a small fraction of total cellular dopamine, most of which is sequestered in vesicles. To measure just the cytosolic pool, first author Eugene Mosharov utilized intracellular patch electrochemistry. He found that in PC12 cells, cytosolic dopamine is below the limit of detection by this technique, but treating the cell with L-DOPA produced detectable signals. Treatment of PC12 cells that overexpressed wild-type or mutant α-synuclein (A30P or A53T) caused a greater increase in cytosolic dopamine than cells without the proteins, with the mutants having the biggest effect. To make sure that the result was not confined to L-DOPA-treated cells, the researchers looked at mouse adrenal chromaffin cells which had detectable basal levels of cytosolic dopamine. They found that cells derived from transgenic mice expressing the α-synuclein A30P mutant (but not wild-type α-synuclein) showed a twofold increase in cytosolic dopamine concentration.
What could account for the increase? The researchers checked levels of key proteins involved in catecholamine metabolism, but none of the changes explained the effects. Based on previous observations that α-synuclein could increase vesicle permeability, they looked at the protein’s effect on isolated chromaffin granules. Treatment of vesicles with purified α-synuclein, either mutant or wild-type, induced proton leakage from the vesicles. The collapse of the protein gradient across the vesicle membrane would be expected to decrease dopamine uptake into vesicles. Consistent with the effects on cytosolic dopamine in cells, the mutant proteins had a greater effect than wild-type on vesicle permeability.
If synuclein causes a dopamine leak in cells, this could account for the selective toxicity of the protein for dopaminergic neurons. Increasing dopamine in cells creates oxidative stress, a property not shared by other neurotransmitters which might be liberated by synuclein in other kinds of neurons.—Pat McCaffrey