Synaptic deregulation of cholinergic projection neurons causes olfactory dysfunction across 5 fly Parkinsonism models

Reviewer #1 (Public Review):

This is a fantastic, comprehensive, timely, and landmark pan-species work that demonstrates the convergence of multiple familial PD mutations onto a synaptic program. It is extremely well written and I have only a few comments that do not require additional data collection.

Major Comments:

(1) In the functional experiments performing calcium imaging on projection neurons I could not find a count of cell bodies across conditions. Since the loss of OPNs could explain the reduced calcium signal, this is a critical control to perform. A differential abundance test on the single-cell data would also suffice here and be easy for the authors to perform with their existing data.

(2) One of the authors' conclusions is that cholinergic neurons and the olfactory system are acutely impacted by these PD mutations. However, I wonder if this is the case:
a. Most Drosophila excitatory neurons are cholinergic and only a subpopulation appear to be dysregulated by these mutations. The authors point out that visual neurons also have many DEGs, couldn't the visual system also be dysregulated in these flies? Is there something special about these cholinergic neurons versus other cholinergic neurons in the fly brain? I wonder if they can leverage their nice dataset to say something about vulnerability.
b. As far as I can tell, the cross-species analysis of DEGs (Figure 3) is agnostic to neuronal cell type, although the conclusion seems to suggest only cholinergic neurons were contrasted. Is this correct? Could you please clarify this in the text as it's an important detail. If not, Have the authors tried comparing only cholinergic neuron DEGs across species? That would lend strength to their specificity argument. The results for the NBM are impressive. Could the authors add more detail to the main text here about other regions to the main text?
c. Uniquely within the human data, are cholinergic neurons more dysregulated than others? I understand this is not an early timepoint but would still be useful to discuss.
d. In the discussion, the authors say that olfactory neurons are uniquely poised to be dysregulated as they are large and have high activity. Is this really true compared to other circuits? I didn't find the references convincing and I am not sure this has been borne out in electron microscopy reconstructions for anatomy.

Reviewer #2 (Public Review):

Summary:

Pech et al selected 5 Parkinson's disease-causing genes, and generated multiple Drosophila lines by replacing the Drosophila lrrk, rab39, auxilin (aux), synaptojanin (synj), and Pink1 genes with wild-type and pathogenic mutant human or Drosophila cDNA sequences. First, the authors performed a panel of assays to characterize the phenotypes of the models mentioned above. Next, by using single-cell RNA-seq and comparing fly data with human postmortem tissue data, the authors identified multiple cell clusters being commonly dysregulated in these models, highlighting the olfactory projection neurons. Next, by using selective expression of Ca2+-sensor GCaMP3 in the OPN, the authors confirmed the synaptic impairment in these models, which was further strengthened by olfactory performance defects.

Strengths:

The authors overall investigated the functionality of PD-related mutations at endogenous levels and found a very interesting shared pathway through single-cell analysis, more importantly, they performed nice follow-up work using multiple assays.

Weaknesses:

While the authors state this is a new collection of five familial PD knock-in models, the AuxR927G model has been published and carefully characterized in Jacquemyn et al., 2023. ERG has been performed for Aux R927G in Jacquemyn et al., 2023, but the findings are different from what's shown in Figure 1b and Supplementary Figure 1d, which the authors should try to explain. Moreover, according to the authors, the hPINK1control was the expression of human PINK1 with UAS-hPINK1 and nsyb-Gal4 due to technical obstacles. Having PINK1 WT being an overexpression model, makes it difficult to explain PINK1 mutant phenotypes. It will be strengthened if the authors use UAS-hPINK1 and nsyb-Gal4 (or maybe ubiquitous Gal4) to rescue hPink1L347P and hPink1P399L phenotypes. In addition, although the authors picked these models targeting different biology/ pathways, however, Aux and Synj both act in related steps of Clathrin-mediated endocytosis, with LRRK2 being their accessory regulatory proteins. Therefore, is the data set more favorable in identifying synaptic-related defects?

GH146-GAL4+ PNs are derived from three neuroblast lineages, producing both cholinergic and GABAergic inhibitory PNs (Li et al, 2017). Therefore, OPN neurons have more than "cholinergic projection neurons". How do we know from single-cell data that cholinergic neurons were more vulnerable across 5 models?

In Figure 1b, the authors assumed that locomotion defects were caused by dopaminergic neuron dysfunction. However, to better support it, the author should perform rescue experiments using dopaminergic neuron-specific Gal4 drivers. Otherwise, the authors may consider staining DA neurons and performing cell counting. Furthermore, the authors stated in the discussion, that "We now place cholinergic failure firmly ahead of dopaminergic system failure in flies", which feels rushed and insufficient to draw such a conclusion, especially given no experimental evidence was provided, particularly related to DA neuron dysfunction, in this manuscript.

It is interesting to see that different familial PD mutations converge onto synapses. The authors have suggested that different mechanisms may be involved directly through regulating synaptic functions, or indirectly through mitochondria or transport. It will be improved if the authors extend their analysis on Figure 3, and better utilize their single-cell data to dissect the mechanisms. For example, for all the candidates listed in Figure 3C, are they all altered in the same direction across 5 models?

While this approach is carefully performed, the authors should state in the discussions the strengths and the caveats of the current strategy. For example, what kind of knowledge have we gained by introducing these mutations at an endogenous locus? Are there any caveats of having scRNAseq at day 5 only but being compared with postmortem human disease tissue?

Reviewer #3 (Public Review):

Summary:

This study investigates the cellular and molecular events leading to hyposmia, an early dysfunction in Parkinson's disease (PD), which develops up to 10 years prior to motor symptoms. The authors use five Drosophila knock-in models of familial PD genes (LRRK2, RAB39B, PINK1, DNAJC6 (Aux), and SYNJ1 (Synj)), three expressing human genes and two Drosophila genes with equivalent mutations.

The authors carry out single-cell RNA sequencing of young fly brains and single-nucleus RNA sequencing of human brain samples. The authors found that cholinergic olfactory projection neurons (OPN) were consistently affected across the fly models, showing synaptic dysfunction before the onset of motor deficits, known to be associated with dopaminergic neuron (DAN) dysfunction.

Single-cell RNA sequencing revealed significant transcriptional deregulation of synaptic genes in OPNs across all five fly PD models. This synaptic dysfunction was confirmed by impaired calcium signalling and morphological changes in synaptic OPN terminals. Furthermore, these young PD flies exhibited olfactory behavioural deficits that were rescued by selective expression of wild-type genes in OPNs.

Single-nucleus RNA sequencing of post-mortem brain samples from PD patients with LRRK2 risk mutations revealed similar synaptic gene deregulation in cholinergic neurons, particularly in the nucleus basalis of Meynert (NBM). Gene ontology analysis highlighted enrichment for processes related to presynaptic function, protein homeostasis, RNA regulation, and mitochondrial function.

This study provides compelling evidence for the early and primary involvement of cholinergic dysfunction in PD pathogenesis, preceding the canonical DAN degeneration. The convergence of familial PD mutations on synaptic dysfunction in cholinergic projection neurons suggests a common mechanism contributing to early non-motor symptoms like hyposmia. The authors also emphasise the potential of targeting cholinergic neurons for early diagnosis and intervention in PD.

Strengths:

This study presents a novel approach, combining multiple mutants to identify salient disease mechanisms. The quality of the data and analysis is of a high standard, providing compelling evidence for the role of OPN neurons in olfactory dysfunction in PD. The comprehensive single-cell RNA sequencing data from both flies and humans is a valuable resource for the research community. The identification of consistent impairments in cholinergic olfactory neurons, at early disease stages, is a powerful finding that highlights the convergent nature of PD progression. The comparison between fly models and human patients' brains provides strong evidence of the conservation of molecular mechanisms of disease, which can be built upon in further studies using flies to prove causal relationships between the defects described here and neurodegeneration.

The identification of specific neurons involved in olfactory dysfunction opens up potential avenues for diagnostic and therapeutic interventions.

Weaknesses:

The causal relationship between early olfactory dysfunction and later motor symptoms in PD remains unclear. It is also uncertain whether this early defect contributes to neurodegeneration or is simply a reflection of the sensitivity of olfactory neurons to cellular impairments. The study does not investigate whether the observed early olfactory impairment in flies leads to later DAN deficits. Additionally, the single-cell RNA sequencing analysis reveals several affected neuronal populations that are not further explored. The main weakness of the paper is the lack of conclusive evidence linking early olfactory dysfunction to later disease progression. The rationale behind the selection of specific mutants and neuronal populations for further analysis could be better qualified.