Long-Term Exposure to PFE-360 in the AAV-α-Synuclein Rat Model: Findings and Implications

Abstract

Parkinson's disease (PD) is a progressive neurodegenerative disorder associated with impaired motor function and several non-motor symptoms, with no available disease modifying treatment. Intracellular accumulation of pathological α-synuclein inclusions is a hallmark of idiopathic PD, whereas, dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with familial PD that is clinically indistinguishable from idiopathic PD. Recent evidence supports the hypothesis that an increase in LRRK2 kinase activity is associated with the development of not only familial LRRK2 PD, but also idiopathic PD. Previous reports have shown preclinical effects of LRRK2 modulation on α-synuclein-induced neuropathology. Increased subthalamic nucleus (STN) burst firing in preclinical neurotoxin models and PD patients is hypothesized to be causally involved in the development of the motor deficit in PD. To study a potential pathophysiological relationship between α-synuclein pathology and LRRK2 kinase activity in PD, we investigated the effect of chronic LRRK2 inhibition in an AAV-α-synuclein overexpression rat model. In this study, we report that chronic LRRK2 inhibition using PFE-360 only induced a marginal effect on motor function. In addition, the aberrant STN burst firing and associated neurodegenerative processes induced by α-synuclein overexpression model remained unaffected by chronic LRRK2 inhibition. Our findings do not strongly support LRRK2 inhibition for the treatment of PD. Therefore, the reported beneficial effects of LRRK2 inhibition in similar α-synuclein overexpression rodent models must be considered with prudence and additional studies are warranted in alternative α-synuclein-based models.

Keywords: AAV-α-synuclein; LRRK2; PFE-360; Parkinson’s disease; subthalamic nucleus; α-synuclein.

Figure 1.

Chronic PFE-360 treatment slightly normalizes motor function but not STN burst firing induced by AAV-α-synuclein overexpression. A, Motor symmetry assessed nine weeks after intracerebral viral inoculation of AAV-α-synuclein or CTRL (empty AAV-vector). The test was performed 1–2 h after dosing (one-way ANOVA, p < 0.001; CTRL veh and PFE-360 N = 6, α-syn veh and PFE-360 N = 5). B, Locomotor activity measured 5–7 h after dosing revealed a non-significant trend for a decreased activity in α-synuclein overexpressing rats in both treatment groups compared to CTRL rats. PFE-360 treatment did not have any adverse effect on locomotion in the CTRL group (one-way ANOVA, p = 0.51; CTRL veh and PFE-360, N = 6, α-syn veh and PFE-360, N = 5). C, Representative action potential and spike-trains from single unit recordings of putative glutamatergic neuron in the STN. The spike-trains are representative examples of regular (top), irregular (middle), and bursty (bottom) firing patterns. D, CV ISI is increased by α-synuclein overexpression in both treatment groups (Kruskal–Wallis test, p < 0.001). E, Proportional distribution of regular, irregular and bursty neurons (χ² = 22.28, df = 6, p = 0.0011) shows increased proportion of bursty neurons in α-synuclein overexpressing rats in both treatment groups. F, The average firing rate of STN neurons is not significantly different between groups (Kruskal–Wallis test, p = 0.11). D–F, N: number of animals; n: number of neurons; CTRL veh (N = 5, n = 98), α-synuclein veh (N = 6, n = 99), CTRL PFE-360 (N = 6, n = 90), and α-synuclein PFE-360 (N = 6, n = 113). Tukey’s multiple comparison is represented by the lines; *p < 0.05, **p < 0.01, ***p < 0.001. For multiple χ² tests in (E) the Bonferroni-corrected p value was used, *p < 0.0125 (four comparisons: significance level, p = 0.0125).

Figure 2.

Striatal LRRK2 expression is not changed by PFE-360 treatment despite full LRRK2 inhibition. A, Western blot analysis of total LRRK2 and phosphorylated LRRK2-Ser935 (LRRK2-pSer935) after 10- to 12-week chronic PFE-360 dosing. The tissues were collected 6–8 h after last dosing. i = ipsilateral to the viral injection; c = contralateral to the viral injection. B, Quantification of Western blottings shows that chronic PFE-360 treatment did not affect LRRK2 expression in CTRL rats compared to vehicle treatment. In α-syn rats, PFE-360 treatment significantly lowered LRRK2 expression compared to vehicle treatment, although each α-syn group did not differ significantly from their respective CTRL group (one-way ANOVA, p = 0.0032). C, The LRRK2-pS935 to total LRRK2 ratio was used as a measure of target engagement. At full inhibition, the ratio is typically <0.1 and was achieved in both CTRL and α-syn rats treated with PFE-360 (one-way ANOVA, p < 0.001). D, Unbound PFE-360 brain concentrations 6–8 h after dosing. IC₅₀ of PFE-360 was previously calculated to 2.3 nM giving theoretical 99% LRRK2 inhibition at 23 nM (unpaired t test, p = 0.85). CTRL veh, CTRL PFE-360 and α-syn veh; N = 5, α-syn PFE-360 N = 6. Tukey’s multiple comparison is represented by the lines; **p < 0.01, ***p < 0.001.

Figure 3.

Striatal neurodegenerative processes and α-synuclein phosphorylation are not halted by chronic LRRK2 inhibition. A, Western blot analysis of tyrosine hydroxylase (TH) and hwt α-synuclein (hwt α-syn; left) as well as phosphorylation of α-synuclein-S129 (α-syn pS129; right). i = ipsilateral to the viral injection; c = contralateral to the viral injection. B, Quantification of TH expression from ipsilateral and contralateral striatum normalized to STEP46 and presented as percentage of contralateral striatum shows a significant loss of TH expression in α-syn overexpressing rats in both treatment groups (one-way ANOVA, p < 0.001). C, The expression level of hwt α-synuclein normalized to STEP46 is not significantly different between groups (unpaired t test, p = 0.22). D, The α-synuclein-S129 phosphorylation from ipsilateral and contralateral striatum normalized to STEP46 and presented as percentage of contralateral striatum is not significantly different between groups (one-way ANOVA, p = 0.076). CTRL veh, CTRL PFE-360 and α-syn veh; N = 5, α-syn PFE-360 N = 6. Tukey’s multiple comparison is represented by the lines; ***p < 0.001.