ASL expression in ALDH1A1+ neurons in the substantia nigra metabolically contributes to neurodegenerative phenotype

Abstract

Argininosuccinate lyase (ASL) is essential for the NO-dependent regulation of tyrosine hydroxylase (TH) and thus for catecholamine production. Using a conditional mouse model with loss of ASL in catecholamine neurons, we demonstrate that ASL is expressed in dopaminergic neurons in the substantia nigra pars compacta, including the ALDH1A1 + subpopulation that is pivotal for the pathogenesis of Parkinson disease (PD). Neuronal loss of ASL results in catecholamine deficiency, in accumulation and formation of tyrosine aggregates, in elevation of α-synuclein, and phenotypically in motor and cognitive deficits. NO supplementation rescues the formation of aggregates as well as the motor deficiencies. Our data point to a potential metabolic link between accumulations of tyrosine and seeding of pathological aggregates in neurons as initiators for the pathological processes involved in neurodegeneration. Hence, interventions in tyrosine metabolism via regulation of NO levels may be therapeutic beneficial for the treatment of catecholamine-related neurodegenerative disorders.

Conflict of interest statement

The authors declare no conflict of interest.

© 2021. The Author(s).

Figures

Fig. 1

ASL co-localizes with TH and ALDH1A1 in the SNc, and its deficiency is associated with decreased TH expression and catecholamine synthesis. A VTA and SNcM dopaminergic regions are demonstrated in a scheme of coronal midbrain section (bregma -3.65 mm) (top panel left) and by TH staining (top panel middle, green) (Scale bar = 500 µm). Magnification of the box area indicates TH (top panel right, green), ALDH1A1 (lower panel left, cyan), ASL (lower panel middle, red), and their co-staining (lower panel right). Dashed lines differentiate the SNcM and VTA subregions (Scale bar = 250 µm). Arrows point to representative cells co-expressing the three proteins. B Quantification of TH mRNA in the SNcM and VTA regions of Aslf/f; TH Cre+/− and in Aslf/f control mice as measured by RT-PCR with specific TaqMan probes (n = 7 mice in each group). C, D Quantification of TH immunostaining of TH- ALDH1A1+ neurons in the SNc medial (SNcM) (C) and striatum (D) of Aslf/f; TH Cre+/− and Aslf/f control mice (n = 5). E Measurement of SNcM catecholamine levels shows a reduction in both dopamine (left) and norepinephrine (right) in Aslf/f; TH Cre+/− as compared to Aslf/f control mice (n ≥ 9 mice in each group). SNCD-SNc dorsal, PBP-parabrachial pigmented nucleus. Data represent mean ± s.e.m. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns not significant)

Fig. 2

ASL deficiency results in motor dysfunctions in both humans and rodents. A–H Gait analysis in Aslf/f;TH Cre+/− and Aslf/f control mice. Dynamic paw parameters included: A Cadence. B Run duration. C Maximum variation. D Swing speed. E Stride length F Duty cycle. G Step cycle. H Paws support. (n≥ 10). I Pie charts demonstrating the percentage of subjects with tremors in ASLD (left part) and ASS1D (right part) patients with or without documented episode of HA. Data represent mean ± s.e.m. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns not significant)

Fig. 3

Adult Aslf/f; TH Cre+/− mice display cognitive deficits that can be partially rescued with NO donors. A Adult Aslf/f control group exhibit strong preference to the target quarter on probe day (left panel) and two weeks later (right panel), in comparison to adult Aslf/f; TH Cre+/− mice. Acute treatment with NO donor did not rescue the performance of Aslf/f; TH Cre+/− mice. The dashed line indicates the time expected by chance. B Adult control mice trained for the fear conditioning paradigm and tested for demonstrating freezing responses to context (left panel) and cue (tone) challenges (right panel), froze significantly more than adult Aslf/f;TH Cre+/− mice. Acute treatment with NO donor rescues the performance of Aslf/f;TH Cre+/− mice. C Two weeks following the first fear-conditioning assessment, mice were tested again and showed the same trend in both the context (left panel) and cue (right panel) tests. (n ≥8). Data represent mean ± s.e.m. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns not significant)

Fig. 4

ASL deficiency in human patients associates with tyrosine accumulation and α-synuclein aggregation. A Tyrosine levels in the CSF (left panel) and plasma (right panel) of ASLD patients underwent liver transplantation. Dashed lines indicate the normal level range (n = 8 for CSF, n = 4 for plasma) (p < 0.02 for CSF, p = 0.56 for plasma). B SH-SY5Y neurons were stained with anti-tyrosine aggregates antibodies and visualized using confocal microscopy. Representative staining of DAPI (blue) and anti-tyrosine staining (red) are shown (Scale bars = 10 µm). A representative 3D volume reconstruction of the Z-series with XZ-slice projection staining of control shGFP neurons (left panel) and shASL neurons (middle panel) (Interval between individual Z-stack serial images = 0.5 µm). Right panel: quantification of the fluorescence intensity of the anti-tyrosine aggregates staining of shGFP and shASL neurons supplemented with or without NO donors (n ≥ 14) (One-way ANOVA with Bonferroni). C Measurements of neuronal cell viability following the addition of the XTT reagent (n = 4). D Quantification of α-synuclein protein levels from SH-SY5Y neuronal cells (n = 4). Lower panel: representative western blot for α- synuclein levels. E ASL is located close to a PD locus variant identified by recent GWAS studies from the iPDGC Locus Browser v1.5 (59). The lead single nucleotide polymorphism (SNP) at this locus is rs76949143 (purple). Recombination rate peaks are marked in blue, and variants are colored by their r2 linkage disequilibrium values. Data represent mean ± s.e.m. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns not significant). F A summary model of our findings demonstrating that ASL loss leads to decreased TH activity, subsequently causing decreased catecholamine (CA) levels and accumulation of tyrosine which together with elevation of α- synuclein may predispose to aggregate formation