Pathogenic LRRK2 causes age-dependent and region-specific deficits in ciliation, innervation and viability of cholinergic neurons

Reviewer #1 (Public review):

Summary:

This study represents valuable insight into the potential contribution of ciliation deficits and cholinergic neuron survival in an etiologically appropriate Parkinson's disease mouse model. The evidence presented is convincing, employing a validated methodology to assess measures across multiple brain regions and time points, with adequate observation numbers. Similarities between some of the data here and human patients further validate the model, and the study provides numerous avenues to aid future advances.

Strengths:

Overall, this study presents a thorough analysis of ciliary defects and cell loss in cholinergic neurons throughout the brain in the LRRK2 G2019S knockin mouse model of Parkinson's disease. The authors aimed to characterize ciliary defects in areas not only implicated in PD but also in cholinergic neuron function. Additionally, they repeated measures across age and sex, presenting a body of work that is more readily translatable to human disease states. The strengths of the paper included the breadth of brain regions tested and additional mechanistic contributions of LRRK2 that may correlate to their observed phenotypes. The study conveys to the reader the ciliary phenotype observed in all the cholinergic neurons assessed throughout the brains of knock-in LRRK2 mutant mice. Importantly, the pattern of changes is, in some instances, strikingly similar to PD, which strengthens the case for construct and face validation of the G2019S knock-in mouse model. Future investigations of the physiological and behavioural correlates/consequences of these changes will inform ongoing and, as yet untried, therapeutic intervention attempts.

Weaknesses:

At times, the claims are only partially substantiated by how the data are presented (e.g., inappropriate statistics within an age (t-tests, not ANOVA) and a lack of comparison between ages (despite referring to the progress of a phenotype). More appropriate statistical analyses and revisions to the data presentation are required to substantiate basic and more 'progressive' conclusions. Further, distributing the central claim over 10 figures dilutes the impact, many of which could be compressed into a couple of single figures (e.g., cell counts in all regions and ciliation). Also, a summary graphic showing the brain regions affected by ciliation alterations and cell loss at young, middle, and old age in the GS mice would be hugely beneficial. This peer would like to see more discussion of how the observed changes would impact circuit-level function and more speculation of the underlying mechanisms leading to the deficits. Minor changes to the abstract and introduction (to include more detail in the rationale and supporting evidence) are recommended, as summaries of existing literature are vague and could flow better between one statement and the next.

Reviewer #2 (Public review):

Summary:

LRRK2 has previously been shown to affect cilia formation and stability both in vitro and in vivo, in striatal cholinergic interneurons, in both transgenic mice and in human post-mortem brain samples from subjects carrying one of the LRRK2 pathogenic mutations: G2019S. In the current study, Brahmia and colleagues have conducted a comprehensive assessment of G2019S knock-in mice to address some gaps in the field, specifically: extending analysis to additional cholinergic neurons across 3 time points and determining the functional consequences of the ciliation deficits. They find that primary cilia are lost in all cholinergic neurons, with basal forebrain cholinergic neurons displaying an early onset (in 4-5-month-old mice) compared with other regions. They also show early dystrophic changes in cholinergic axons derived from basal forebrain and brainstem cholinergic neurons and age-dependent cholinergic cell loss in select forebrain and brainstem nuclei.

Strengths:

This is a comprehensive and careful analysis of ciliary deficits and their downstream consequences, which we assume are deficits in innervation and cell loss.

Weaknesses:

This study is observational and does not address the underlying mechanisms. The data on pRab12, although downstream of LRRK2, does not clearly address this and, instead, raises more questions than answers: e.g., is there really differentiation from Rab10 and its phosphorylation or is it primarily due to the limitations of pRab10 antibodies with regards to the lack of suitability of this antibody in mouse brain sections (could immunoblots on brain punches have been performed to overcome this?). Are Rab10, Rab12, and LRRK2 expressed at different levels in the vulnerable cell types? Plenty of recent high-quality single-cell/single nuclear RNA-seq data could have been used to address such a fundamental question. LRRK2 small molecule inhibitors are available and progressing in the clinic. They could/should have been used to demonstrate the LRRK2 dependence, reversibility, and timing of therapeutic intervention. The authors suggest that the mouse data mirror (and potentially explain) the cholinergic loss in PD patient brains, but this is not measured in the current work (the authors do acknowledge this limitation and suggest that this is an important further study). There are some recent human data (Khan et al 2024 PMID: 38293195, BioRxiv, which the authors cite) showing loss of primary cilia and cholinergic neurons in sporadic PD (no evidence of aberrant LRRK2 activity) and, interestingly, this is not further exacerbated in G2019S carriers, which may suggest a more complex underlying mechanism.

Reviewer #3 (Public review):

Summary:

The authors described cilia deficits, phospho-Rab12 accumulation, dystrophic axons in cholinergic neurons, and loss of the cholinergic neurons in the mouse brains of G2019S-LRRK2 knock-in mice, a preclinical animal model for Parkinson's disease. They showed that the above changes associated with cholinergic neurons are age-dependent and region-specific. The observation is interesting considering the neuron-type-specific effect of the LRRK2-G2019S in mice.

Strengths:

The observations are important and show neuron type-specific effects of the PD mutation of LRRK2 relevant to PD pathologies.

Weaknesses:

The authors may over-interpret the data, and the study may lack mechanistic investigation.