R1441C mutation in LRRK2 impairs dopaminergic neurotransmission in mice

Abstract

Dominantly inherited mutations in leucine-rich repeat kinase 2 (LRRK2) are a common genetic cause of Parkinson's disease (PD). The importance of the R1441 residue in the pathogenesis is highlighted by the identification of three distinct missense mutations. To investigate the pathogenic mechanism underlying LRRK2 dysfunction, we generated a knockin (KI) mouse in which the R1441C mutation is expressed under the control of the endogenous regulatory elements. Homozygous R1441C KI mice appear grossly normal and exhibit no dopaminergic (DA) neurodegeneration or alterations in steady-state levels of striatal dopamine up to 2 years of age. However, these KI mice show reductions in amphetamine (AMPH)-induced locomotor activity and stimulated catecholamine release in cultured chromaffin cells. The introduction of the R1441C mutation also impairs dopamine D2 receptor function, as suggested by decreased responses of KI mice in locomotor activity to the inhibitory effect of a D2 receptor agonist, quinpirole. Furthermore, the firing of nigral neurons in R1441C KI mice show reduced sensitivity to suppression induced by quinpirole, dopamine, or AMPH. Together, our data suggest that the R1441C mutation in LRRK2 impairs stimulated dopamine neurotransmission and D2 receptor function, which may represent pathogenic precursors preceding dopaminergic degeneration in PD brains.

Fig. 1.

Normal DA neurons and striatal dopamine levels in R1441C KI mice. (A) Targeting strategy. The R1441C mutation was introduced into exon 31. The locations of the 5′ and 3′ probes used for Southern blotting are indicated. Restriction sites: N, NcoI; S, SpeI, Bs, BssSI; Ba, BamHI are indicated. (B) Southern blotting of F2 mice using the 5′ probe shows the germ-line transmission of the KI allele. Tail genomic DNAs were digested with SpeI. The 13.7-kb band represents the WT allele, whereas the 5.3-kb band represents the KI allele. (C) Northern blotting of total RNAs from KI/KI brains. A 406-bp cDNA fragment spanning exons 1–5 of LRRK2 was used as a probe. (D) Western blotting of brain samples indicates normal protein levels of LRRK2 in KI/KI mice. The specificity of our LRRK2 antibody is confirmed by the absence of LRRK2 in the brain of our two distinct lines of LRRK2 KO mice, KO1 and KO2. Antibodies specific for valosin containing protein (VCP) or spectrin were used to normalize the amounts of protein in each lane for KI or for KO samples, respectively. (E) TH staining of DA neurons in the SNpc. A series of 16-μm coronal sections from 4 KI/KI and 4 WT littermate (+/+) mice at ≈2 years of age were stained with a TH antibody. The representative comparable sections are shown. (Lower) Higher magnification views of the boxed areas. (F) Quantification of TH-immunoreactive neurons in the SNpc by unbiased stereological counting. (G) Levels of striatal DA measured by HPLC in the 2-year-old mice (n = 6 each genotype). Data in all panels are expressed as mean ± SEM.

Fig. 2.

Reduced response to AMPH in locomotor activity of R1441C KI mice. (A) Spontaneous activity of KI/KI (n = 14) and +/+ (n = 11) mice at 1 year of age in the open field during 1-h test. Two infrared light arrays measured horizontal movements, and one array measured vertical movements (rearing on hind legs). Repeated sequential breakings of the same beam were scored as occurrences of stereotyped behaviors (scratching, grooming, etc.). (B) Rotarod test. KI/KI and +/+ mice show similar latencies to fall off an accelerating rotating rod. The average time before falling off the rod is shown for each of three consecutive trials. (C) KI/KI and +/+ mice show similar acoustic startle responses at each decibel of loud noise tested. Each mouse was tested 10 times at each decibel level in a semirandom order with a 30-s interstimulus interval, and the startle response was averaged and normalized to body mass. Body mass does not differ between the genotypes. (D) AMPH injection (i.p., 2.0 mg/kg body weight) dramatically stimulates the locomotor activity of WT (+/+), but not that of KI/KI mice during the 1-h test (#, P < 0.05; ns, not significant). The numbers shown in the bars indicate the numbers of mice used for each experiment. Data in all panels are expressed as mean ± SEM.

Fig. 3.

Reduced catecholamine release in cultured chromaffin cells from R1441C KI mice. (A) Expression of LRRK2 mRNAs, shown by RT-PCR, in medulla of adrenal glands, which is composed mainly of chromaffin cells. (B) The representative traces for high-K⁺-stimulated catecholamine release from KI/KI and +/+ controls. (C) Reduced total catecholamine released per cell, quantal size, and numbers of vesicles released per cell in chromaffin cells of KI/KI mice. The number of cells recorded (Left) and mice used (Right) is indicated in the parenthesis. Data in all panels are expressed as mean ± SEM. Asterisk denotes statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Fig. 4.

Reduced inhibition of locomotor activity by quinpirole in R1441C KI mice. Intraperitoneal injections of low (0.05 mg/kg body weight) or high (1.0 mg/kg body weight) dose of quinpirole, a D2 receptor agonist, result in significant inhibition of locomotor activity in both KI/KI and +/+ mice in a dose-dependent manner (#, P < 0.05; ##, P < 0.01). However, the extent of the inhibition in KI/KI mice is smaller for both horizontal and vertical movements, compared with the control (*, P < 0.05). The number shown in the bar indicates the number of mice used for each experiment. Data in all panels are expressed as mean ± SEM.

Fig. 5.

Decreased sensitivity to inhibition of firing induced by DA, quinpirole, and AMPH in R1441C KI nigral neurons. (A) Sample traces showing the comparable spontaneous, rhythmic action potentials recorded in DA neurons from +/+ (Left) and KI/KI (Right) mice. Firing activity is shown at different time scales marked by an arrow. (B) In midbrain slices from +/+ mice (left trace), bath-application of DA (100 μM, 45 s) hyperpolarizes the cell membrane and blocks the firing activity. On DA washout, the membrane potential slowly recovers, and the action potential discharge returns to control levels. DA-induced firing cessation is significantly shorter in nigral neurons of KI/KI mice (right trace). (C and D) Sample traces showing the reduced responses in firing of nigral neurons in KI/KI mice to bath-application of quinpirole (10 μM, 2 min) (C) or AMPH (100 μM, 2 min) (D). The arrow in D indicates a 10-min interruption in the trace of +/+ nigral neurons.