Selective Inactivation of Leucine-Rich Repeat Kinase 1 and 2 in Dopaminergic Neurons Leads to Age-Dependent Loss of Midbrain Dopaminergic Neurons

Core Concepts

Selective inactivation of leucine-rich repeat kinase 1 and 2 (LRRK1 and LRRK2) in dopaminergic neurons leads to age-dependent loss of midbrain dopaminergic neurons, demonstrating the intrinsic importance of LRRK proteins in the survival of dopaminergic neurons.

Abstract

The study investigates the intrinsic role of LRRK1 and LRRK2 in the survival of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). The authors first generated and characterized floxed LRRK1 and LRRK2 mice, as well as confirmed the specificity of Cre-mediated recombination driven by the dopamine transporter-Cre (DAT-Cre) knockin allele in DA neurons. They then crossed the floxed LRRK1 and LRRK2 mice with DAT-Cre mice to generate DA neuron-specific LRRK conditional double knockout (cDKO) mice. The key findings are: DA neuron-specific LRRK cDKO mice exhibit normal mortality and body weight, unlike the previously reported germline LRRK double knockout mice. LRRK cDKO mice develop age-dependent, progressive loss of DA neurons in the SNpc, starting at 20 months of age. The loss of DA neurons in LRRK cDKO mice is accompanied by increased apoptosis and elevated microgliosis in the SNpc, as well as decreased dopaminergic terminals in the striatum. Quantitative electron microscopy analysis showed no significant difference in the number and area of electron-dense vacuoles in the SNpc between LRRK cDKO and control mice, in contrast to the age-dependent increases observed in germline LRRK double knockout mice. LRRK cDKO mice exhibit impaired motor coordination at 10 months of age, preceding the loss of DA neurons. These findings provide unequivocal evidence for the intrinsic importance of LRRK proteins in the survival of DA neurons, and suggest that LRRK2 mutations may impair this crucial physiological function, leading to the loss of DA neurons in Parkinson's disease.

Stats

The number of TH+ DA neurons in the SNpc of LRRK cDKO mice is similar to controls at 15 months (Control: 10,077 ± 310, cDKO: 10,000 ± 141), but is significantly reduced at 20 months (Control: 10,244 ± 220, cDKO: 8,948 ± 273, p = 0.0041) and further decreased at 24 months (Control: 9,675 ± 232, cDKO: 8,188 ± 452, p = 0.0010). The number of NeuN+ neurons in the SNpc of LRRK cDKO mice at 24 months is significantly lower than controls (Control: 21,907 ± 469, cDKO: 17,923 ± 813, p = 0.0006). The number of active Caspase-3+/TH+ apoptotic DA neurons in the SNpc of LRRK cDKO mice at 24 months is significantly higher than controls (Control: 157 ± 8, cDKO: 323 ± 38, p = 0.0004). The TH immunoreactivity in the striatum of LRRK cDKO mice is reduced by 19% compared to controls at 24 months (p = 0.0215). The number of Iba1+ microglia in the SNpc of LRRK cDKO mice is significantly increased compared to controls at 15 months (Control: 1,737 ± 83, cDKO: 2,541 ± 193, p = 0.0017), 20 months (Control: 2,426 ± 68, cDKO: 3,639 ± 127, p < 0.0001), and 24 months (Control: 2,640 ± 187, cDKO: 4,089 ± 100, p < 0.0001).

Quotes

"These findings provide the unequivocal evidence for the importance of LRRK in DA neurons and raise the possibility that LRRK2 mutations may impair this crucial physiological function, leading to the loss of DA neurons in Parkinson's disease." "While DA neuron-restricted LRRK cDKO mice of both sexes exhibit normal mortality and body weight, they develop age-dependent loss of DA neurons in the SNpc, as demonstrated by the progressive reduction of TH+ DA neurons or NeuN+ neurons in the SNpc of cDKO mice at the ages of 20 and 24 months."

Key Insights Distilled From

Cell autonomous role of leucine-rich repeat kinase in protection of dopaminergic neuron survival

by Kang,J., Hua... at www.biorxiv.org 10-10-2023

https://www.biorxiv.org/content/10.1101/2023.10.06.561293v5

Deeper Inquiries

What are the potential mechanisms by which LRRK1 and LRRK2 deficiency specifically in dopaminergic neurons leads to their age-dependent degeneration

The age-dependent degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) due to LRRK1 and LRRK2 deficiency in this study may be attributed to several potential mechanisms. Firstly, LRRK proteins are known to play essential roles in the autophagy-lysosomal pathway, which is crucial for the clearance of damaged organelles and proteins in neurons. Deficiency of LRRK1 and LRRK2 in dopaminergic neurons could disrupt this pathway, leading to the accumulation of toxic protein aggregates and dysfunctional organelles, ultimately resulting in neuronal degeneration. Secondly, LRRK proteins have been implicated in regulating inflammatory responses in microglia, the brain's immune cells. Microglial activation and neuroinflammation are known contributors to neurodegeneration, and the enhanced microgliosis observed in the SNpc of LRRK cDKO mice could exacerbate neuronal damage. Additionally, LRRK proteins have been linked to mitochondrial function and oxidative stress, both of which are critical factors in neuronal health. Deficiency of LRRK1 and LRRK2 may impair mitochondrial function and increase oxidative stress, leading to neuronal dysfunction and death. Overall, the age-dependent degeneration of dopaminergic neurons in LRRK cDKO mice likely results from a combination of disrupted autophagy, increased inflammation, mitochondrial dysfunction, and oxidative stress.

How do the findings in this study contrast with the lack of dopaminergic neuron loss observed in previous studies using LRRK2 knockout or knock-in mouse models

The findings in this study contrast with the lack of dopaminergic neuron loss observed in previous studies using LRRK2 knockout or knock-in mouse models in several key ways. Firstly, the selective inactivation of both LRRK1 and LRRK2 in dopaminergic neurons in the current study resulted in age-dependent degeneration of these neurons, whereas germline deletions of LRRK1 and LRRK2 did not lead to dopaminergic neuron loss. This suggests that the cell-autonomous role of LRRK proteins in dopaminergic neuron survival is crucial and may not be compensated for by other mechanisms in germline knockout models. Secondly, the presence of enhanced microgliosis in the SNpc of LRRK cDKO mice indicates a potential role of microglia in mediating dopaminergic neuron degeneration, which was not observed in previous LRRK knockout models. This suggests that the interaction between LRRK proteins in dopaminergic neurons and microglia may be critical for neuronal health. Additionally, the age-dependent loss of dopaminergic terminals in the striatum of LRRK cDKO mice further highlights the specific impact of LRRK deficiency in dopaminergic neurons on their function and connectivity, which was not explored in previous models.

Given the intrinsic importance of LRRK proteins in dopaminergic neuron survival, how might this knowledge inform the development of therapeutic strategies for Parkinson's disease targeting LRRK2

The intrinsic importance of LRRK proteins in dopaminergic neuron survival, as demonstrated in this study, provides valuable insights for the development of therapeutic strategies for Parkinson's disease targeting LRRK2. Understanding the specific mechanisms by which LRRK1 and LRRK2 deficiency leads to dopaminergic neuron degeneration can guide the design of targeted therapies aimed at restoring autophagy-lysosomal function, reducing neuroinflammation, improving mitochondrial health, and mitigating oxidative stress in dopaminergic neurons. Therapeutic approaches that focus on enhancing autophagy, modulating microglial activation, promoting mitochondrial function, and reducing oxidative stress in dopaminergic neurons may hold promise for slowing or halting the progression of Parkinson's disease in individuals with LRRK2 mutations. Additionally, the age-dependent nature of dopaminergic neuron loss in LRRK cDKO mice underscores the importance of early intervention strategies that target LRRK2 dysfunction to preserve dopaminergic neuron viability and function. By leveraging the insights gained from this study, researchers and clinicians can develop more targeted and effective therapies for Parkinson's disease that address the specific role of LRRK proteins in dopaminergic neuron survival.