Motor Impairments and Dopaminergic Defects Caused by Loss of Leucine-Rich Repeat Kinase Function in Mice

Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease (PD), but the pathogenic mechanism underlying LRRK2 mutations remains unresolved. In this study, we investigate the consequence of inactivation of LRRK2 and its functional homolog LRRK1 in male and female mice up to 25 months of age using behavioral, neurochemical, neuropathological, and ultrastructural analyses. We report that LRRK1 and LRRK2 double knock-out (LRRK DKO) mice exhibit impaired motor coordination at 12 months of age before the onset of dopaminergic neuron loss in the substantia nigra (SNpc). Moreover, LRRK DKO mice develop age-dependent, progressive loss of dopaminergic terminals in the striatum. Evoked dopamine (DA) release measured by fast-scan cyclic voltammetry in the dorsal striatum is also reduced in the absence of LRRK. Furthermore, LRRK DKO mice at 20-25 months of age show substantial loss of dopaminergic neurons in the SNpc. The surviving SNpc neurons in LRRK DKO mice at 25 months of age accumulate large numbers of autophagic and autolysosomal vacuoles and are accompanied with microgliosis. Surprisingly, the cerebral cortex is unaffected, as shown by normal cortical volume and neuron number as well as unchanged number of apoptotic cells and microglia in LRRK DKO mice at 25 months. These findings show that loss of LRRK function causes impairments in motor coordination, degeneration of dopaminergic terminals, reduction of evoked DA release, and selective loss of dopaminergic neurons in the SNpc, indicating that LRRK DKO mice are unique models for better understanding dopaminergic neurodegeneration in PD.

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

“…Importantly, LRRK cDKO mice develop age-dependent DA neurodegeneration, as evidenced by progressive reductions of DA neurons in the SNpc at the age of 20-24 months ( Figure 3 ) and increases of apoptotic DA neurons ( Figure 4 ), whereas the number of noradrenergic neurons in the LC is uncaged, consistent with the lack of Cre-mediated recombination in the LC by DAT-Cre ( Figure 5 ). Interestingly, quantitative EM analysis showed similar numbers of electron-dense vacuoles in the SNpc of LRRK cDKO and control mice even at 25 months of age ( Figure 6 ), in contrast to dramatic, age-dependent increases of vacuoles in the SNpc of LRRK DKO mice (30, 31). Lastly, DA neurodegeneration in the SNpc of LRRK cDKO mice is accompanied with elevated microgliosis ( Figure 7 ).…”

“…Histological analyses were performed as described previously (30, 31, 68). Briefly, for DAB-derived TH-immunohistochemistry, coronal sections were deparaffinized, alcohol-dehydrated, and then subjected to permeabilization with a solution containing 0.1% Triton X-100 in TBS followed by antigen retrieval for 5 min in 10 mM sodium citrate buffer, pH 6.0.…”

“…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 20 and 24 months of age. Interestingly, quantitative electron microscopy analysis showed a similar number of electron-dense vacuoles in SNpc neurons of LRRK cDKO mice relative to controls, in contrast to age-dependent, dramatic increases of vacuoles in SNpc neurons of germline LRRK DKO mice (30, 31). Moreover, DA neurodegeneration is accompanied with increases of apoptosis and elevated microgliosis in the SNpc of LRRK cDKO mice.…”

“…Mice were perfused with PBS containing 0.25 g/L heparin and 5 g/L procaine followed by a fixative solution containing 2.5% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer was quantified. We previously calculated the diameter of electron-dense autophagic/lysosomal vacuoles by measuring the longest side manually (30,31). In the current study, we used Feret's Diameter (82) to accurately measure the longest distance between any of two points in the electron-dense autophagic/lysosomal vacuoles, resulting in a higher number of electron-dense vacuoles quantified.…”

Molecular Pathways Involved in LRRK2-Linked Parkinson’s Disease: A Systematic Review

“…These mutations lead to gain-of-function mechanisms, where kinase activity of LRRK2 will increase to phosphorylate a group of Rab proteins in different subcellular localities, thus affecting downstream pathways to drive PD pathogenesis [ 14 ]. Interestingly, in in vitro and in vivo studies, knocking out LRRK2 recapitulated aspects of PD pathogenesis such as dopaminergic neuron loss, accumulation of α-Syn, impairment of protein degradation, and dysregulation of autophagy [ 15 , 16 , 17 , 18 ]. However, there is a lack of evidence of phenotypic impact, as there was no increase in the risk of PD in humans carrying loss of function variants of LRRK2 [ 19 ].…”