Synaptic location is a determinant of the detrimental effects of α-synuclein pathology to glutamatergic transmission in the basolateral amygdala

  1. Liqiang Chen
  2. Chetan Nagaraja
  3. Samuel Daniels
  4. Zoe A Fisk
  5. Rachel Dvorak
  6. Lindsay Meyerdirk
  7. Jennifer A Steiner
  8. Martha L Escobar Galvis
  9. Michael X Henderson
  10. Maxime WC Rousseaux
  11. Patrik Brundin
  12. Hong-Yuan Chu

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    Evaluation Summary:

    The manuscript by Nagaraja et al. examines the synapse-specificity of alpha-synuclein aggregation and corresponding circuit dysfunction in the amygdala. Using confocal microscopy and slice electrophysiology, along with alpha-synuclein knockout mice and preformed fibrils, the authors demonstrate that cortico-amygdala, but not thalamo-amygdala, inputs are more vulnerable to alpha-synuclein aggregation and corresponding synaptic dysfunction. This has important implications for the etiology of psychiatric deficits that are common in Parkinson's disease.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

The presynaptic protein α-synuclein (αSyn) has been suggested to be involved in the pathogenesis of Parkinson’s disease (PD). In PD, the amygdala is prone to develop insoluble αSyn aggregates, and it has been suggested that circuit dysfunction involving the amygdala contributes to the psychiatric symptoms. Yet, how αSyn aggregates affect amygdala function is unknown. In this study, we examined αSyn in glutamatergic axon terminals and the impact of its aggregation on glutamatergic transmission in the basolateral amygdala (BLA). We found that αSyn is primarily present in the vesicular glutamate transporter 1-expressing (vGluT1 + ) terminals in the mouse BLA, which is consistent with higher levels of αSyn expression in vGluT1 + glutamatergic neurons in the cerebral cortex relative to the vGluT2 + glutamatergic neurons in the thalamus. We found that αSyn aggregation selectively decreased the cortico-BLA, but not the thalamo-BLA, transmission; and that cortico-BLA synapses displayed enhanced short-term depression upon repetitive stimulation. In addition, using confocal microscopy, we found that vGluT1 + axon terminals exhibited decreased levels of soluble αSyn, which suggests that lower levels of soluble αSyn might underlie the enhanced short-term depression of cortico-BLA synapses. In agreement with this idea, we found that cortico-BLA synaptic depression was also enhanced in αSyn knockout mice. In conclusion, both basal and dynamic cortico-BLA transmission were disrupted by abnormal aggregation of αSyn and these changes might be relevant to the perturbed cortical control of the amygdala that has been suggested to play a role in psychiatric symptoms in PD.

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  1. Version published to 10.7554/elife.78055 on eLife
  2. Version published to 10.1101/2022.02.20.481207v3 on bioRxiv
  3. eLife

    Author Response

    Reviewer #1 (Public Review):

    Psychiatric symptoms in Parkinson's disease are debilitating, but there are few treatments that effectively reduce these symptoms long-term. The mechanisms that cause psychiatric symptoms in Parkinson's disease are unknown. However, it has been known for decades that abnormal alpha-synuclein is found in the amygdala, a brain region important for the control of emotions. Nagaraj et. al. present an article in which they attempt to characterize the differences in α-synuclein colocalization with vGluT1+ compared to vGluT2+ terminals in the BLA of a PFF mouse model. They successfully demonstrate convincing data that points to the preferential association of α-synuclein with vGluT1+ puncta and not vGluT2+ puncta. The authors also demonstrate that PFFs promote short-term depression of cortico-BLA synapses in response to repetitive stimuli which does not occur in vGluT2+ terminals.

    Clearly differentiating the association of α-synuclein with different glutamatergic terminals and cortical or thalamic projections, and the subsequent effect of abnormal α-synuclein and how it affects transmission in the BLA is novel and points to mechanisms of differential vulnerability to inclusions in different neuronal bodies.

    This study is one of the first to use electrophysiology to show that abnormal alpha-synuclein contributes to defects in the amygdala in Parkinson's disease. The study also pinpoints cortical-amygdala projections as the culprit in amygdala dysfunction. Therefore this study has a major impact in the field by determining how abnormal amygdala function caused by pathologic alpha-synuclein can potentially cause psychiatric symptoms in Parkinson's disease.

    The main weakness of the study is the lack of mechanism. Although the authors attempt to show that loss of synuclein in mice injected with PFFs is responsible for the amygdala defects, the data are insufficient to make this conclusion.

    We thank the reviewer for the comments on the importance of this work and suggestions for improvement. We agree with the reviewer that the initial submission was descriptive, thus we have performed additional experiments and analyses that have allowed us to reveal potential mechanisms underlying the changes in synaptic strength and plasticity, as outlined below.

    Reviewer #2 (Public Review):

    The data presented are clear and of high quality. The conclusion that alpha-synuclein aggregation and corresponding synaptic dysfunction preferentially occurs in vGluT1 expressing cortical inputs (as opposed to vGluT2 expressing thalamic inputs) to the BLA is convincing, but a few additional clarifications and experiments would greatly help describe the mechanism of synapse dysfunction. Overall this manuscript provides helpful insight into the circuit dysfunctions that may contribute to non-motor psychiatric symptoms that commonly occur in Parkinson's disease.

    1. The BLA is a relatively large structure, and the labeled terminal fields of cortical and thalamic inputs (figure 2) don't show matching patterns. It would be helpful to clarify where in the BLA recordings were made (and where high mag images in figure 1 were taken from).

    In the present study, immunohistochemistry (Figures 1 and 5) and electrophysiology (Figure 3, 4, and 6) data were collected from the medial part of the anterior basolateral amygdala (BLAam), where heavy αSyn pathology can be found (Figure 3B). This subregion of BLA also receives both cortical and thalamic inputs (Figure 3I, M). Images from similar rostrocaudal sections have been updated as representative images in the related figures.

    1. The short-term plasticity experiments shown in figure 4 are informative, but by themselves don't necessarily rule out post-synaptic mechanisms of adaptation. Since the mobilization of synaptic vesicles is likely involved, it would be helpful to also look at the effect of PFFs on release probability using pared pulse ratios.

    To address the Reviewer’s comments, we performed additional experiments/analyses, as outlined below:

    • To assess postsynaptic adaptations at cortico-BLA synapses, we analyzed AMPA/NMDA ratio and AMPA receptor rectification index (Figure 4 K-M). Interestingly, we detected a reduced AMPA/NMDA ratio and an enhanced inward rectification of AMPA receptors at cortico-BLA synapses in slices from PFFs-injected mice. These data suggest an overall reduction of postsynaptic AMPA receptor function and an increased relative contribution of GluA2-lacking AMPA receptors to cortico-BLA transmission in PFFs-injected mice. These data and discussion are now included in the last paragraph of Page 17.

    • To assess changes in the initial release probability of SVs, we stimulated cortico-BLA synapses using paired pulses of electric (20 Hz) and optogenetic (10 Hz) pulses. Our data showed no change in the ratio of EPSC2/EPSC1 between groups using either approach (Figure 4D-G), suggesting that the initial release probability was not altered in PFFs-injected mice versus controls. Thus, we propose that the development of αSyn pathology could affect SV mobility, leading to a slower refilling of the active zone from reserve pool, which is then revealed by prolonged repetitive stimulation (Figure 6).

    • We assessed the quantal release at cortico-BLA synapses by replacing Ca2+ with Sr2+. We detected a decreased frequency of Sr2+-induced, optogenetically-evoked cortico-BLA EPSCs in slices from PFFs-injected mice (Figure 4H, I). Together with the unaltered density of cortico-BLA axon terminals and the initial release probability, we proposed that αSyn pathology decreases the number of release sites at axon terminals. Testing of this hypothesis warrants further studies using electron microscope or expansion microscope techniques.

    • Last, we quantified the density of vGluT1 in the BLA and did not detect change in vGluT1 density between groups (Figure 4A-C), suggesting no degeneration of cortical axon terminals (as mentioned by Reviewer 3 below).

    Together, we conclude that both pre- and post-synaptic alterations contribute to the altered basal and dynamic cortico-BLA connection as αSyn pathology develops.

    1. PPFs reduce cortico-BLA EPSC amplitudes but not thalamo-EPSC amplitudes in response to single electrical and optogenetic stimuli (figure 2). In figure 4, however, the starting amplitudes appear to be similar (at least in the exemplar traces). I'm assuming this is because stimulus intensities were adjusted to achieve a similar starting point? If so, are differences in short-term plasticity also observed if similar stimulus intensities are used?

    As noted by the reviewer, stimulation intensity was adjusted to evoke 200-300 pA the 1st EPSCs for these experiments.

    In a subset of experiments, we delivered the same intensity of electric stimulation to slices from control and PFFs-injected mice and repeated the repetitive stimulating experiments. We observed very similar faster and stronger suppression of cortico-BLA EPSCs in slices from PFFs injected mice (e.g., EPSC200/EPSC1, control = 0.350.14, n = 5 cells; PFFs = 0.200.04, n = 6 cells). We would like to point out that under such conditions, EPSCs from PFFs-injected mice showed smaller initial amplitudes and the subsequent EPSCs decayed quickly to noise level, making the quantification of later EPSCs less meaningful. Thus, these data were not included in the revised manuscript.

    Reviewer #3 (Public Review):

    1. In this manuscript, the authors try to address whether glutamatergic axonal terminals are differentially impacted by a-syn aggregation, a key pathology seen in Parkinson's disease. Using a-syn PFF injection, and a-syn KO mice, the authors show a few interesting findings: 1. After a-syn PFF injection in the BLA, the strength of the cortical inputs was selectively reduced, while leaving thalamic inputs unaffected. 2. There is an interesting parallel finding on the release probability of cortical glutamatergic synaptic transmission after a-syn PFF injection and in the a-syn KO mice. The key findings are interesting, showing selective vulnerability of glutamatergic synapses, in which vGluT1+ terminals are more profoundly affected by a-syn PFF or loss of function.

    The authors thank this reviewer for highlighting the importance of this work and the thoughtful comments.

    1. However, mechanistically, the authors implied that a-syn PFF induced aggregation sequesters soluble a-syn, acting more similar to a-syn KO conditions. This does seem to be plausible for the enhancement of release probability. But, what would be responsible for the reduction of cortico-BLA synaptic transmission? Previous studies showed that there was no neurodegeneration one month after a-syn PFF injections in the cortex.

    In the revised manuscript, we have now included additional experiments/analyses and further clarified potential mechanisms underlying the impaired basal and dynamic cortico-BLA transmission in the PFFs model. Please see above responses to the point #2 of the response to Reviewer 1 as well as the point #2 of the response to Reviewer 2.

    1. Do the authors imply that some vGluT1+ terminals are lost after a-syn PFF injection? The authors did not quantify the number of vGluT1+ puncta in the BLA after a-syn PFF injections.

    We apologize for the confusing descriptions in the initial submission. We did not detect a reduction of vGluT1 density in PFFs-injected mice at 1 mpi (Figure 4A-C), indicating no degeneration of vGluT1+ cortical terminals.

    1. The authors also used intrastriatal a-syn PFF injection as a comparison. However, the data were not shown in the manuscript. Because striatum also receives convergent cortical and thalamic inputs, it would strengthen the conclusion if the authors systematically investigate corticostriatal vs thalmostriatal terminals in parallel.

    In the present work, we injected αSyn PFFs into the striatum to induce αSyn aggregation in the BLA through the seeding process and assessed its impact on synaptic transmission in the BLA. This intrastriatal PFFs model induces heavy pS129 pathology in the BLA and avoids local inflammatory responses at the injection site.

    Systematically study the corticostriatal and thalamostriatal transmission in the context of αSyn aggregation and PD pathophysiology would be the next follow-up experiment to do. However, it is worthy to note that PFFs injections into the striatum is likely to trigger local inflammation and chronic microglia activation, which may confound the conclusion on striatal circuitry changes. We sincerely request to leave this question for future studies, since the present work was initially designed to understand the impact of αSyn pathology to amygdala function, which may be relevant to the biology of psychiatric defects in PD.

  4. Version published to 10.1101/2022.02.20.481207v2 on bioRxiv
  5. eLife

    Evaluation Summary:

    The manuscript by Nagaraja et al. examines the synapse-specificity of alpha-synuclein aggregation and corresponding circuit dysfunction in the amygdala. Using confocal microscopy and slice electrophysiology, along with alpha-synuclein knockout mice and preformed fibrils, the authors demonstrate that cortico-amygdala, but not thalamo-amygdala, inputs are more vulnerable to alpha-synuclein aggregation and corresponding synaptic dysfunction. This has important implications for the etiology of psychiatric deficits that are common in Parkinson's disease.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  6. eLife

    Reviewer #1 (Public Review):

    Psychiatric symptoms in Parkinson's disease are debilitating, but there are few treatments that effectively reduce these symptoms long-term. The mechanisms that cause psychiatric symptoms in Parkinson's disease are unknown. However, it has been known for decades that abnormal alpha-synuclein is found in the amygdala, a brain region important for the control of emotions. Nagaraj et. al. present an article in which they attempt to characterize the differences in α-synuclein colocalization with vGluT1+ compared to vGluT2+ terminals in the BLA of a PFF mouse model. They successfully demonstrate convincing data that points to the preferential association of α-synuclein with vGluT1+ puncta and not vGluT2+ puncta. The authors also demonstrate that PFFs promote short-term depression of cortico-BLA synapses in response to repetitive stimuli which does not occur in vGluT2+ terminals.

    Clearly differentiating the association of α-synuclein with different glutamatergic terminals and cortical or thalamic projections, and the subsequent effect of abnormal α-synuclein and how it affects transmission in the BLA is novel and points to mechanisms of differential vulnerability to inclusions in different neuronal bodies.

    This study is one of the first to use electrophysiology to show that abnormal alpha-synuclein contributes to defects in the amygdala in Parkinson's disease. The study also pinpoints cortical-amygdala projections as the culprit in amygdala dysfunction. Therefore this study has a major impact in the field by determining how abnormal amygdala function caused by pathologic alpha-synuclein can potentially cause psychiatric symptoms in Parkinson's disease.

    The main weakness of the study is the lack of mechanism. Although the authors attempt to show that loss of synuclein in mice injected with PFFs is responsible for the amygdala defects, the data are insufficient to make this conclusion.

  7. eLife

    Reviewer #2 (Public Review):

    The data presented are clear and of high quality. The conclusion that alpha-synuclein aggregation and corresponding synaptic dysfunction preferentially occurs in vGluT1 expressing cortical inputs (as opposed to vGluT2 expressing thalamic inputs) to the BLA is convincing, but a few additional clarifications and experiments would greatly help describe the mechanism of synapse dysfunction. Overall this manuscript provides helpful insight into the circuit dysfunctions that may contribute to non-motor psychiatric symptoms that commonly occur in Parkinson's disease.

    1. The BLA is a relatively large structure, and the labeled terminal fields of cortical and thalamic inputs (figure 2) don't show matching patterns. It would be helpful to clarify where in the BLA recordings were made (and where high mag images in figure 1 were taken from).
    2. The short-term plasticity experiments shown in figure 4 are informative, but by themselves don't necessarily rule out post-synaptic mechanisms of adaptation. Since the mobilization of synaptic vesicles is likely involved, it would be helpful to also look at the effect of PFFs on release probability using pared pulse ratios.
    3. PPFs reduce cortico-BLA EPSC amplitudes but not thalamo-EPSC amplitudes in response to single electrical and optogenetic stimuli (figure 2). In figure 4, however, the starting amplitudes appear to be similar (at least in the exemplar traces). I'm assuming this is because stimulus intensities were adjusted to achieve a similar starting point? If so, are differences in short-term plasticity also observed if similar stimulus intensities are used?

  8. eLife

    Reviewer #3 (Public Review):

    In this manuscript, the authors try to address whether glutamatergic axonal terminals are differentially impacted by a-syn aggregation, a key pathology seen in Parkinson's disease. Using a-syn PFF injection, and a-syn KO mice, the authors show a few interesting findings: 1. After a-syn PFF injection in the BLA, the strength of the cortical inputs was selectively reduced, while leaving thalamic inputs unaffected. 2. There is an interesting parallel finding on the release probability of cortical glutamatergic synaptic transmission after a-syn PFF injection and in the a-syn KO mice. The key findings are interesting, showing selective vulnerability of glutamatergic synapses, in which vGluT1+ terminals are more profoundly affected by a-syn PFF or loss of function.

    However, mechanistically, the authors implied that a-syn PFF induced aggregation sequesters soluble a-syn, acting more similar to a-syn KO conditions. This does seem to be plausible for the enhancement of release probability. But, what would be responsible for the reduction of cortico-BLA synaptic transmission? Previous studies showed that there was no neurodegeneration one month after a-syn PFF injections in the cortex.

    Do the authors imply that some vGluT1+ terminals are lost after a-syn PFF injection? The authors did not quantify the number of vGluT1+ puncta in the BLA after a-syn PFF injections.

    The authors also used intrastriatal a-syn PFF injection as a comparison. However, the data were not shown in the manuscript. Because striatum also receives convergent cortical and thalamic inputs, it would strengthen the conclusion if the authors systematically investigate corticostriatal vs thalmostriatal terminals in parallel.

  9. Version published to 10.1101/2022.02.20.481207v1 on bioRxiv