11 Sep 2006

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

Comments

1. • Matthew LaVoie
     Ann Romney Center for Neurologic Diseases, BWH and HMS
   • Posted: 14 Sep 2006

   Dopaminergic cells represent the major class of neurons lost in Parkinson disease. The unique ability of dopamine and its reactive metabolites to foster oxidative stress and protein modification has led many researchers to focus on the potential role of the dopamine molecule as an intraneuronal toxin. Previous work has implicated dopamine in the aggregation of α-synuclein (Conway et al., 2001), the neurotoxicity associated with α-synuclein overexpression (Xu et al., 2002), and the aggregation and inactivation of parkin (LaVoie et al., 2005). It has been demonstrated in vitro that α-synuclein aggregates may facilitate leakage of small molecules out of synthetic vesicles, perhaps in some pore-forming conformation (Volles et al., 2001). The implications of this model are that dopaminergic neurons might not efficiently sequester cytosolic dopamine in the presence of increased or mutant α-synuclein. This could lead to increased cytosolic dopamine levels and perhaps oxidative stress, selectively within dopamine neurons. However, the direct demonstration of altered dopamine homeostasis was not feasible until the recent advances made by Dave Sulzer’s group at Columbia. Eugene Mosharov, a postdoc in the Sulzer lab, developed a technique, referred to as intracellular patch electrochemistry (IPE), designed to estimate intracellular concentrations of catecholamines (Mosharov et al., 2003). This clever merging of patch-clamp electrophysiological techniques and carbon fiber electrochemistry allows for reliable determinations of intracellular (cytosolic) catecholamine levels, as well as the tracking of dynamic changes in dopamine levels in response to pharmacologic or stressful stimuli. In the past, this data could only have been estimated, indirectly, by dopamine turnover rates measured from ground tissue.

   The Sulzer group have now turned their attention to matters relevant to Parkinson disease. Familiar with the hypothesis that α-synuclein might permeabilize vesicles and alter cytoplasmic dopamine, they took on the admirable challenge of testing this hypothesis not only in living cell cultures, but also using in vivo models of α-synuclein overexpression. Mosharov et al. (2006) report that overexpression of α-synuclein in dopaminergic PC12 cells is associated with significant increases in cytosolic catecholamines. This effect could not be explained by changes in the enzymes responsible for dopamine synthesis or metabolism, pointing toward a homeostatic alteration.

   The authors were also able to show similar changes in peripheral, dopamine-producing tissues in α-synuclein transgenic mice, and replicate earlier findings with recombinant α-synuclein protein with respect to α-synuclein-induced proton leakage across chromaffin granules. Surprisingly, however, the wild-type transgenic α-synuclein mice did not show altered catecholamine levels despite expression levels of α-synuclein that were equal to or greater than the A30P mice. Therefore, not all aspects of the paper fully support the hypothesis; however, the authors provide a very balanced discussion highlighting important consistencies as well as caveats.

   It should be noted that in the PC12 cell culture experiments, both the A30P and A53T variants had greater effects on cytosolic catecholamine levels than did wild-type α-synuclein. This trend is consistent with the aggregation rates of these α-synuclein isoforms (Conway et al., 1998), as well as their reported ability to permeabilize synthetic vesicles in vitro (Volles and Lansbury, 2002). This aspect of the current paper strongly suggests that the authors have the story right. They have demonstrated an interesting property of α-synuclein in cultured cells with broad implications as to the redox status specifically within dopaminergic neurons with respect to α-synuclein levels. Overproduction or decreased turnover of wild-type or mutant α-synuclein might increase cytosolic dopamine, inducing oxidative stress, as well as the covalent modification of thiol-dependent proteins such as parkin (LaVoie et al., 2005), resulting in cell death.

   While support for the dopamine hypothesis in Parkinson disease certainly appears to be gaining some momentum, it should also be remembered that several non-dopaminergic nuclei are affected in this disorder. Therefore, many other factors are likely involved, and hopefully the near future will uncover important clues as to what other molecular events may participate in the neurodegenerative process within the parkinsonism brain.

   References:

   Conway KA, Rochet JC, Bieganski RM, Lansbury PT. Kinetic stabilization of the alpha-synuclein protofibril by a dopamine-alpha-synuclein adduct. Science. 2001 Nov 9;294(5545):1346-9. PubMed.

   Xu J, Kao SY, Lee FJ, Song W, Jin LW, Yankner BA. Dopamine-dependent neurotoxicity of alpha-synuclein: a mechanism for selective neurodegeneration in Parkinson disease. Nat Med. 2002 Jun;8(6):600-6. PubMed.

   LaVoie MJ, Ostaszewski BL, Weihofen A, Schlossmacher MG, Selkoe DJ. Dopamine covalently modifies and functionally inactivates parkin. Nat Med. 2005 Nov;11(11):1214-21. PubMed.

   Volles MJ, Lee SJ, Rochet JC, Shtilerman MD, Ding TT, Kessler JC, Lansbury PT. Vesicle permeabilization by protofibrillar alpha-synuclein: implications for the pathogenesis and treatment of Parkinson's disease. Biochemistry. 2001 Jul 3;40(26):7812-9. PubMed.

   Mosharov EV, Gong LW, Khanna B, Sulzer D, Lindau M. Intracellular patch electrochemistry: regulation of cytosolic catecholamines in chromaffin cells. J Neurosci. 2003 Jul 2;23(13):5835-45. PubMed.

   Conway KA, Harper JD, Lansbury PT. Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease. Nat Med. 1998 Nov;4(11):1318-20. PubMed.

   Volles MJ, Lansbury PT. Vesicle permeabilization by protofibrillar alpha-synuclein is sensitive to Parkinson's disease-linked mutations and occurs by a pore-like mechanism. Biochemistry. 2002 Apr 9;41(14):4595-602. PubMed.

   View all comments by Matthew LaVoie
2. • Mary Reid
     �
   • Posted: 19 Sep 2006

   Speaking of α-synuclein, Kim and colleagues (1) report that Dyrk1A, a dual-specificity tyrosine-regulated kinase can phosphorylate α-synuclein and that aggregates formed by phosphorylated α-synuclein are more neurotoxic compared with aggregates composed of unmodified wild-type α-synuclein. Increased Dyrk1A immunoreactivity has been found in the cytoplasm and nuclei of scattered neurons of the neocortex, entorhinal cortex, and hippocampus in AD, DS, and Pick Disease (2).

   It would be interesting to see whether there was a reduction in Lewy bodies with the use of the Dyrk1A inhibitor - (-)-epigallocatechin gallate, (EGCG), a polyphenol found in green tea. Perhaps this may explain the neuroprotective effect of EGCG in Parkinson models.

   References:

   Kim EJ, Sung JY, Lee HJ, Rhim H, Hasegawa M, Iwatsubo T, Min do, Kim J, Paik SR, Chung KC. Dyrk1A phosphorylates alpha-synuclein and enhances intracellular inclusion formation. J Biol Chem. 2006 Nov 3;281(44):33250-7. PubMed.

   Ferrer I, Barrachina M, Puig B, Martínez de Lagrán M, Martí E, Avila J, Dierssen M. Constitutive Dyrk1A is abnormally expressed in Alzheimer disease, Down syndrome, Pick disease, and related transgenic models. Neurobiol Dis. 2005 Nov;20(2):392-400. PubMed.

   View all comments by Mary Reid

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References

News Citations

1. Dopamine Renders α-Synuclein Toxic to Neurons 22 Nov 2002

Further Reading

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