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Selective K-ATP channel-dependent loss of pacemaking in vulnerable nigrostriatal dopamine neurons by α-synuclein aggregates

Poonam Thakur, Kelvin Luk, Jochen Roeper

Preprint posted on November 15, 2019 https://www.biorxiv.org/content/10.1101/842344v1

Why do alpha-synuclein aggregates affect specific neuron populations in Parkinson’s disease? a-synuclein fibrils stop pacemaking firing in PD-vulnerable midbrain dopamine neurons but not resistant neurons; K-ATP channels play a key role.

Selected by Jessica Xie

Categories: neuroscience

Background

One curious commonality of various neurodegenerative diseases is the issue of selective vulnerability of particular populations of neurons (Fu et al. 2018). In Parkinson’s disease (PD), there is near-total loss of dopaminergic (DA) neurons in the lateral substantia nigra (SN) of the midbrain, while DA neurons in the medial SN and ventral tegmental area (VTA) are significantly less affected (Surmeier et al. 2017; Kordower et al. 2013). Huntington’s disease initially affects striatal medium spiny neurons projecting to the external globus pallidus, while sparing those projecting to the internal globus pallidus. Alzheimer’s disease notably impacts the hippocampus and entorhinal cortex but not motor and sensory cortex; ALS too differentially affects various motor neuron populations.

This feature of selective neuronal vulnerability is an intriguing one, not just in terms of biology (what differentiates subtypes of neurons?), but also for its potential therapeutic significance: discovering what underlies the vulnerability—or conversely, resistance—of certain neuronal subtypes to the disease-causing process would be a major step towards understanding the disease, and consequently developing strategies for its treatment or prevention.

How then to identify the key factors distinguishing neuronal subtypes? Several previous studies in the field of PD have compared expression profiles of SN and VTA and proposed potential area-specific markers (notable mentions include GIRK2 and CALB1), but to date there are no definitive molecular markers of SN or VTA regional identity (Anderegg et al. 2015; Brichta et al. 2014). Some electrophysiological differences are better established—for example, pacemaking in SN DA neurons is understood to be driven by voltage-gated calcium channels, and pacemaking in VTA DA neurons by voltage-gated sodium channels (Chan et al. 2007). Additionally, the Roeper lab has previously identified activation of K-ATP channels in SN but not VTA, and suggested that this could be responsible for selective SN DA neuronal death (Liss et al. 2005).

In addition to selective neuronal death, many neurodegenerative diseases are also associated with aberrant protein aggregation in affected regions—PD, for example, is characterized by Lewy body aggregates composed primarily of the a-synuclein protein. While it is widely accepted that a-synuclein is critically involved in PD pathogenesis, how exactly is unclear. It has been suggested that the process of a-synuclein aggregation and/or Lewy body formation may be stressful and consequently toxic, but could fibrils themselves be acutely toxic? Are pathogenic fibrillar forms of a-synuclein preferentially produced in the vulnerable region of the midbrain, or are these a-synuclein fibrils only toxic to certain midbrain neurons, but not others—and if so, what molecular differences underlie this disparity?

This study by Thakur et al. provides the tempting beginnings of a potential resolution to these important outstanding issues in the field of PD, with the exciting finding that a-synuclein fibrils cause dramatic loss of pacemaking firing in regions vulnerable in PD, while having a much smaller effect in other neighboring, resistant regions—and that K-ATP channels could potentially be the reason why.

Key findings

The authors began by performing retrograde tracing in order to label subpopulations of neurons within the mouse midbrain, taking advantage of the fact that selectively vulnerable and resistant midbrain neuron populations can be anatomically identified. Specifically, they injected fluorescent beads into the dorsolateral striatum, innervated by lateral SN DA neuron populations most vulnerable in PD. They also made injections into the dorsomedial striatum and ventral striatum (nucleus accumbens), labeling less affected midbrain DA neurons in the medial SN and VTA.

Next, the authors applied a-synuclein fibrils intracellularly to these labeled neurons, by performing whole-cell patch clamp electrophysiology with a-synuclein fibrils added to the internal recording solution (which theoretically equilibrates with cellular contents). While a-synuclein fibrils are generally applied extracellularly (in cell culture media or through in vivo injections) and as a chronic treatment lasting days to months (Luk et al. 2012; Volpicelli-Daley et al. 2011), this relatively novel experimental paradigm—intracellular application of a-synuclein fibrils—resulted in a remarkable finding: a-synuclein fibrils markedly and rapidly disrupted the endogenous pacemaking activity of midbrain DA neurons, causing a complete loss of pacemaking firing activity in a majority (65%) of lateral SN neurons within 5 minutes. Moreover, this deficit was specific to the PD-vulnerable midbrain DA neuron population, namely lateral SN neurons projecting to dorsolateral striatum. In contrast, medial SN and VTA neurons innervating dorsomedial and ventral striatum were significantly less affected.

Thakur et al. found that intracellular addition of a-synuclein fibrils cause a rapid loss of electrical pacemaking activity in lateral substantia nigra dopaminergic neurons projecting to the dorsolateral striatum (DLS-lSN), the subpopulation that is most vulnerable in Parkinson’s disease. In contrast, dopaminergic populations less affected in PD—such as neighboring medial substantia nigra neurons that innervate dorsomedial striatum (DMS-mSN)—are also less affected by a-synuclein fibrils.

 

The authors additionally showed that monomeric forms of a-synuclein had a reduced effect on the pacemaking activity of lateral SN DA neurons, consistent with many previous studies showing greater pathological effects of fibrillar forms of a-synuclein relative to monomers. Furthermore, extracellular application of a-synuclein fibrils also acutely interfered with pacemaking activity, confirming that this was not an experimental artifact of intracellular a-synuclein fibril application.

But what mechanisms might underlie this regional vulnerability? The authors show that bath application of a K-ATP channel inhibitor, glibenclamide, partially protected against the detrimental effect of a-synuclein fibrils, suggesting a possible mechanism that also nicely corroborates their previous work on SN K-ATP channels (Liss et al. 2005).

Questions for the authors:

  1. I was intrigued by your experimental choice to look at the acute effect of a-syn fibril application, as most studies seem to go chronic/long-term. Any additional backstory to share behind your decision to investigate the acute effect on an ephys feature?
  2. I’m also very impressed by how fast and dramatic the reported finding was, especially given that most PD/a-syn fibril phenotypes have been quite slow/subtle (e.g. takes weeks for some cell death). Upon first read I’d initially assumed it must be because of intracellular application, then realized that extracellular fibrils also had a rapid effect on timescale of seconds. I’m wondering if this could be a potential clue as to mechanism: might you speculate if it could be a-syn fibril binding to extracellular region of protein (K-ATP channel maybe?), some rapid protein uptake/import, or something else?
  3. Finally, could you speculate how loss of pacemaking activity might lead to neuronal death/neurodegeneration?

References

  • Anderegg, A., Poulin, J.F., & Awatramani, R. (2015). Molecular heterogeneity of midbrain dopaminergic neurons—Moving toward single cell resolution. FEBS letters, 589(24 Pt A), 3714-26. https://doi.org/10.1016/j.febslet.2015.10.022
  • Brichta, L., & Greengard, P. (2014). Molecular determinants of selective dopaminergic vulnerability in Parkinson’s disease: an update. Frontiers in neuroanatomy, 8, 152. https://doi.org/10.3389/fnana.2014.00152
  • Chan, C.S., Guzman, J.N., Ilijic, E., Mercer, J.N., Rick, C., Tkatch, T., … & Surmeier, D.J. (2007). ‘Rejuvenation’ protects neurons in mouse models of Parkinson’s disease. Nature, 447(7148), 1081-6. https://doi.org/10.1038/nature05865
  • Fu, H., Hardy, J., & Duff, K.E. (2018). Selective vulnerability in neurodegenerative diseases. Nature neuroscience, 21(10), 1350-8. https://doi.org/10.1038/s41593-018-0221-2
  • Kordower, J.H., Olanow, C.W., Dodiya, H.B., Chu, Y., Beach, T.G., Adler, C.H., … & Bartus, R.T. (2013). Disease duration and the integrity of the nigrostriatal system in Parkinson’s disease. Brain : a journal of neurology, 136(Pt 8), 2419-31. https://doi.org/10.1093/brain/awt192
  • Liss, B., Haeckel, O., Wildmann, J., Miki, T., Seino, S., & Roeper, J. (2005). K-ATP channels promote the differential degeneration of dopaminergic midbrain neurons. Nature neuroscience, 8(12), 1742-51. https://doi.org/10.1038/nn1570
  • Luk, K.C., Kehm, V., Carroll, J., Zhang, B., O’Brien, P., Trojanowski, J.Q., & Lee, V.M. (2012). Pathological α-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice. Science (New York, N.Y.), 338(6109), 949-53. https://doi.org/10.1126/science.1227157
  • Surmeier, D.J., Obeso, J.A., & Halliday, G.M. (2017). Selective neuronal vulnerability in Parkinson disease. Nature reviews. Neuroscience, 18(2), 101-13. https://doi.org/10.1038/nrn.2016.178
  • Volpicelli-Daley, L.A., Luk, K.C., Patel, T.P., Tanik, S.A., Riddle, D.M., Stieber, A., … & Lee, V.M. (2011). Exogenous α-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death. Neuron, 72(1), 57-71. https://doi.org/10.1016/j.neuron.2011.08.033

 

Posted on: 18th December 2019

doi: https://doi.org/10.1242/prelights.15883

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  • Author's response

    Dr. Poonam Thakur shared

    Q1- Phenotype such as cell death or appearance of aggregation takes longer time to manifest as has been shown by several studies looking at chronic effects of fibrils on neurons. However, neurons fire action potentials at a much shorter time scale of milliseconds. Therefore, an electrophysiological approach allows us to look at the phenotype not easily possible by cell biology or molecular biology approach. Using an acute exposure, we were trying to look for the very early changes that might take place in the neurons as soon as they come in contact with alpha-synuclein fibrils either extracellularly or intracellularly. In future, we will continue to investigate this phenomenon further using a PD animal model and see if K-ATP channel also plays a role in mediating any long-term changes in action potential firing of vulnerable neurons.

    Q2- The exact mechanism of alpha-synuclein entry into the neurons is a topic of immense interest. However, at the moment, we do not know if alpha-synuclein fibrils directly bind to K-ATP channels. Further investigations using biophysical techniques might help to reveal this possibility.

    Q3- Once again, it is hard to speculate the mechanism of cell death (that takes place in a much longer time scale) based on loss of pacemaking (taking place in a very short time scale). K-ATP channel activation can lead to different consequences in-vitro in comparison to in-vivo based on context (1). However, to put it simply, there is a possibility that lack of firing over long term might cause the neuron to be discarded from the circuits in accordance to “use it or lose it” principle of neuronal plasticity.

    1. Duda J, Pötschke C, Liss B. Converging roles of ion channels, calcium, metabolic stress, and activity pattern of Substantia nigra dopaminergic neurons in health and Parkinson’s disease. Journal of Neurochemistry. Blackwell Publishing Ltd; 2016. p. 156–78.

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