Glucocerebrosidase haploinsufficiency in A53T α-synuclein mice impacts disease onset and course

Abstract

Mutations in GBA1 encountered in Gaucher disease are a leading risk factor for Parkinson disease and associated Lewy body disorders. Many GBA1 mutation carriers, especially those with severe or null GBA1 alleles, have earlier and more progressive parkinsonism. To model the effect of partial glucocerebrosidase deficiency on neurological progression in vivo, mice with a human A53T α-synuclein (SNCA^A53T) transgene were crossed with heterozygous null gba mice (gba^+/−). Survival analysis of 84 mice showed that in gba^+/−//SNCA^A53T hemizygotes and homozygotes, the symptom onset was significantly earlier than in gba^+/+//SNCA^A53T mice (p-values 0.023–0.0030), with exacerbated disease progression (p-value < 0.0001). Over-expression of SNCA^A53T had no effect on glucocerebrosidase levels or activity. Immunoblotting demonstrated that gba haploinsufficiency did not lead to increased levels of either monomeric SNCA or insoluble high molecular weight SNCA in this model. Immunohistochemical analyses demonstrated that the abundance and distribution of SNCA pathology was also unaltered by gba haploinsufficiency. Thus, while the underlying mechanism is not clear, this model shows that gba deficiency impacts the age of onset and disease duration in aged SNCA^A53T mice, providing a valuable resource to identify modifiers, pathways and possible moonlighting roles of glucocerebrosidase in Parkinson pathogenesis.

1. Introduction

Parkinson disease (PD), a common neurodegenerative disorder associated with aging, is characterized pathologically by intraneuronal Lewy bodies and the loss of dopaminergic neurons in the substantia nigra [1]. Clinically, patients manifest with resting tremors, bradykinesia, rigidity and postural instability [2], as well as non-motor symptoms including cognitive impairment [3]. Identifying genetic contributions to Parkinson disease has illuminated molecular mechanisms underlying disease pathogenesis [4,5], particularly the gene encoding alpha-synuclein (SNCA), a major component of Lewy bodies. The Ala53Thr (A53T) mutation in SNCA, initially identified in rare families with Parkinson disease [6] increases its propensity to aggregate [7,8].

The genetic landscape of Parkinson disease now includes nearly thirty genes [9]. Heterozygous mutations in GBA1, the gene mutated in Gaucher disease, encoding the lysosomal enzyme glucocerebrosidase (GCase, EC 3.2.1.45) are the most common genetic risk factor for Parkinson disease as well as dementia with Lewy bodies, often associated with an earlier onset and more severe cognitive and non-motor symptoms [10,11]. Severe GBA1 mutations, such as loss of function mutations c.84insG and IVS2+1G > A, confer a higher risk for Parkinson disease and a more progressive course compared to milder mutations like N409S (N370S) [12,13]. Many groups have investigated the relationship between SNCA levels and GCase deficiency or inhibition, but only a few have utilized animal models [14–17], including mice treated with the GCase inhibitor conduritol-β-epoxide (CBE) [16,18]. To further investigate the link between GBA1 mutations and Parkinson disease, we sought to develop an in vivo mouse model recapitulating the accelerated Parkinson disease course observed in loss-of-function GBA1 mutation carriers, by crossing a SNCA^A53T transgenic mouse that develops severe motor impairment and neuronal SNCA inclusions [19], with heterozygotes for a functionally null gba allele [20]. While homozygous null gba mice die perinatally, heterozygotes, with 40–50% of wildtype GCase enzyme activity, develop normally [20], and mimic patients with Parkinsonism carrying loss-of-function GBA1 mutations. Unlike the CBE treated models, these mice age naturally, enabling the analysis of factors associated with aging.

2. Materials and methods

2.1. The generation of gba^+/−//SNCA^A53T mice

In this study, gba^+/− mice [20] were crossed with mice with a human SNCA^A53T transgene [19]. gba^+/−//SNCA^A53T homozygotes and hemizygotes, along with their gba^+/− and gba^+/+//SNCA^A53T controls, were followed for two years. All housing and breeding of mice were performed under NHGRI Animal Care and Use Committee-approved protocols.

2.1.1. Mouse background

gba knock-out (gba^tm1Nsb; maintained as heterozygotes) and transgenic SNCA^A53T (Tg(Prnp-SNCA*A53T)83Vle) mice were originally on mixed backgrounds (C57BL/6 X FVB/N and C57BL/C3H, respectively). Both lines were backcrossed against the C57Bl/6 J (B6) background, with matings being informed by genetic markers. The Mouse Medium Density (MD) Linkage panel (1449 SNPs; Illumina) was used to define blocks of heterozygosity. At each subsequent generation, pups were genotyped for the presence of the mutation (gba−) or transgene (SNCA^A53T) and were assessed using progressively more focused panels of microsatellite markers within these heterozygous blocks. Markers, first chosen at intervals of < 5 Mb within each heterozygous block, were selected at decreased intervals as heterozygosity diminished with each generation. The two male pups with the highest levels of homozygosity were mated to B6 females at each generation. This process was repeated in excess of ten generations for gba^tm1Nsb, and five generations for Tg(Prnp-SNCA*A53T) mice, at which time the Y chromosome was fixed by mating a female mutant/transgenic pup with a B6 male mouse.

2.1.2. Mouse genotyping

Genomic DNA was prepared from tail biopsies as previously described [21]. The SNCA primers used for PCR amplification to confirm the presence of human SNCA, and neo primer set used to screen for the gba null allele are shown below.

• SNCA-F 5′-TGC CTG TGG ATC CTG ACA AT-3′.

• SNCA-R 5′-GGG GAG GGG CAA ACA ACA GA -3′.

• NEO-1F 5′-ACA GAC AAT CGG CTG CTC TGA TGC -3′.

• NEO-2R 5′-CTC GTC AAG AAG GCG ATA GAA GGC-3′.

2.1.3. Evaluation of SNCA copy number

To distinguish hemizygous from homozygous SNCA^A53T mice, transgene copy-number was evaluated using TaqMan (applied Biosystems, Foster City, CA) Copy Number real-time PCR assays. Two FAM-labeled probes were used to measure the human SNCA in genomic DNA: Hs05969230_cn in exon 2 and Hs02236645_cn in exon 7. The mouse Tfrc gene assay (VIC-labeled; 4,458,366) was used as an endogenous control. Assays were performed according to the manufacturer's directions, using 20 ng of genomic DNA. Reactions were run on a StepOnePlus Real-Time PCR system and data were analyzed using the StepOne Software v2.3. Relative quantitation was calculated using the 2^−ΔΔCT method. Heterozygote and homozygote controls were included in each panel of reactions.

2.1.4. Phenotyping of mice

Six groups of mice were followed: wildtype controls (gba^+/+), gba null heterozygotes (gba^+/−), SNCA^A53T hemizygotes with and without the null gba allele ((gba^+/−//SNCA^A53T) and (gba^+/+//SNCA^A53T)) and SNCA^A53T homozygotes with and without the null gba allele ((gba^+/−//SNCA^A53T/A53T) (gba^+/+//SNCA^A53T/A53T)). An equal number of male and female mice were included in this study. Weight was recorded weekly from age 6 months. Once a 13% drop in weight was observed, measurements continued at least biweekly. Mice were monitored for gait abnormalities, hunching or arching of the back, abnormal grooming, and urinary retention, and symptoms were recorded or filmed. Mice were euthanized when total limb paralysis and/or bladder dysfunction developed, together with the analogous aged-matched controls.

2.2. Tissue collection and processing

2.2.1. Murine tissues

Mice were deeply anesthetized and perfused with PBS, followed by 70% ethanol/150 mM NaCl. Brains and spinal cords were dissected out and further fixed in 70% ethanol/150 mM NaCl followed by paraffin infiltration [19] for imaging. For RNA and protein studies, brain and other tissues were collected and immediately snap-frozen in liquid N_2.

2.2.2. Human brain samples

Total protein from five cortical brain samples collected from autopsies performed at the NIH Clinical Center or from the Massachusetts General Hospital Brain Bank was extracted in RIPA buffer. Samples included a normal control, two patients with type 1 Gaucher disease (genotype N370S/N370S), one with and one without Parkinson disease, a c.84insG heterozygote with parkinsonism, and a patient with Parkinson disease without GBA1 mutations. Each subject was male and above age 55 years. Samples were processed as described for mouse brain samples.

2.3. Protein extraction and evaluation

2.3.1. Protein extraction

Total protein was extracted from forebrain, midbrain and total mouse brain samples using two different extraction buffers: 1:10 (w/v) citrate-phosphate buffer (pH 5.4, 0.25%) Triton X-100 and protease inhibitor cocktail) and RIPA buffer (Thermo Scientific, Waltham, MA). Samples were homogenized on ice, sonicated for 10 s, and centrifuged at 5000 rpm for 10 min.

2.3.2. Glucocerebrosidase levels

The lysates in citrate-phosphate buffer were used for GCase activity and immunoblotting using a fluorescent activity-based probe specific for GCase (MDW933) [22]. 15 μg of midbrain and forebrain lysate and 1.2 μM imiglucerase (Genzyme, Cambridge, MA), used as a control, were incubated with 100 nM of the GCase-specific MDW933 fluorescent probe at 37° C for 90 min and run on a 4–20% Criterion: TGX™ gel (Bio-Rad laboratories). The results were analyzed using an excitation wavelight of 488 nm and emission of 520 nm to measure the fluorescent signal in the gel [23].

2.3.3. Separation into soluble and insoluble protein

Brain lysates in RIPA buffer were used for western immunoblotting for monomeric SNCA, and other lysosomal proteins. Extracted protein was also fractionated into soluble and insoluble forms. The entire brain from symptomatic SNCA^A53T mice with and without gba^+/−, aged-matched non-symptomatic mice and control mice (wt/wt, and wt/gba^+/−) were homogenized in 5 volumes of Triton X-100 buffer (1% Triton X-100, 125 mM NaCl, 20 mM NaCl, 5 mM EDTA, 1.5 mM MgCl_2, 10% glycerol) supplemented with protease and phosphatase inhibitors (Thermo Scientific) using a glass Teflon Dounce homogenizer. The brain lysates were centrifuged at 100,000 g for 90 min at 4°. The collected supernatant was considered the soluble fraction. The pellet was extracted in 2.5 volumes of 2% SDS, 50 mM Tris-HCl, 125 mM NaCl, 1 mM EDTA and protease and phosphatase inhibitors, followed by sonication with a probe sonicator at 40% amplitude, and then boiled for 15 mins. The re-suspended pellet was centrifuged at 25000 g for 25 min at 25 °C. The supernatant was considered the insoluble fraction. The protein concentration was determined by BCA assay (ThermoScientific).

2.3.4. Western blotting and antibodies

The SDS-soluble and insoluble fractions and whole lysate from brain samples from symptomatic and control mice were run on Western immunoblots to evaluate levels of monomeric, soluble and HMW SNCA. GCase levels were also compared to samples from age-matched, pre-symptomatic and control mice. Twenty μg of protein was incubated at 70 °C with DTT for 10 min. Protein was loaded onto BioRad Criterion TGX precast gels (Hercules, CA) and run for 45 min at 200 V. Protein was transferred using the BioRad Turbo Transfer system with the default mixed molecular weight setting onto a PVDF membrane. The membrane was dried between filter paper for at least 30 min, rehydrated in methanol and blocked before probing. SNCA (1:1000, SC-7011, S129 and SNCA-211) (Santa Cruz, CA) primary antibodies were incubated for 1 h in 5% milk following 1 h of blocking. Secondary antibodies were prepared in 1:3000 dilutions in 5% milk. HRP conjugate beta-actin (ab20272) (abcam, Cambridge, England) was incubated for 1 h in 5% BSA following 1 h of blocking. Westerns were developed using ECL reagent and imaged. Antibodies used included antibody to Cathepsin-D, (Abcam, ab 52,832, 1/1000); Cathepsin-B (Abcam, ab 137662, 1/1000); Cathepsin-L (Abcam, ab 6314, 1/1000); LAMP1 (Abcam, ab 25630, 1/1000); GCase (Sigma, G4171, 1/1000) (St. Louis, MO) and LIMP-2 (Santa Cruz, SC 133127, 1/1000).

2.4. Enzymatic activity

2.4.1. Glucocerebrosidase activity

GCase activity was measured using a standard fluorometric assay [24]. Protein extracts were incubated for 15 min at 37 °C with 100 μM CBE (Sigma Aldrich) to subtract non-specific GCase activity. Assay buffer, composed of 10 mM 4-methylumbelliferyl-β-D-glucopyranoside (Sigma Aldrich) in pH 5.4 citrate-phosphate buffer, was added to CBE-treated and untreated samples and incubated for 1.5 h at 37 °C. The reaction was terminated with stop solution (1 M NaOH and 1 M glycine), and measured fluorescence was normalized to protein concentrations measured with a Pierce BCA protein assay kit (Thermo Scientific).

2.4.2. Cathepsin-D activity assay

Cathepsin D (CAT D, (EC 3.4.23.5) activity was measured using a fluorogenic CAT D substrate (MCA-GKPILFFRLK(Dnp)-D-R-NH2) (Calbiochem, #219360) (Long beach, CA). Briefly, 7 μg brain samples from wt/wt, gba^+/− and SNCA^A53T hemizygotes with and without the null gba allele were homogenized in buffer (50 mM sodium acetate with 20 mM sodium chloride at pH = 5) and freeze-thawed four times to generate lysates. The lysates were incubated with the substrate at a final concentration of 20 μM in buffer. Reactions were performed in polypropylene, 384-well flat-bottom microplates (781,209, Greiner Bio-one) (Monroe, North Carolina) containing 50 μL buffer incubated at 25 °C using a microplate reader (Tecan Infinite M200 Pro). For CAT-D absorbance (excitation and emission wavelengths at 328 and 393 nm, respectively) was recorded as a function of time.

2.5. Quantification of glucosylsphingosine and glucosylceramide

Glycosphingolipids (glucosyl- and galactosyl-sphingosines, glucosyl-and galactosyl-ceramides) were extracted from mouse forebrain and midbrain regions. Tissues, homogenized with PBS buffer. N, N-di-methyl-galactosylsphingosine and galactosylceramide (d18:1 8:0), were used as internal standards prior to the glycosphingolipid extraction. Quantification of glucosylsphingosines and glucosylceramides was performed using the API 4000 LC-MS-MS system (Applied Biosystems) with a Varian Metasil AQ C-18 column. Isomer separation of glucosylsphingolipids was performed using a Supelco HILIC column. A positive ion electrospray method using MRM was utilized for both analyses [25]. Data were normalized to the total protein content in samples, and was analyzed by a Student's two-tailed t-test (represented as mean ± SEM).

2.6. Pathological assessments

2.6.1. Evaluation of SNCA inclusion pathology

To evaluate the extent and distribution of SNCA inclusion pathology, brains from both symptomatic and control mice were processed and assessed by immunocytochemistry and immunofluorescence. Tissue sections were stained with: anti-pSer129 SNCA antibody 81A [26], anti-pSer129 SNCA antibody EP1536Y (Abcam), anti-SNCA antibody Syn 506 [27,28] anti-p62/sequestrome-1 (SQSTM1; Proteintech) (Chicago, IL) and anti-tyrosine hydroxylase antibody (TH; Millipore) (Fisher Scientific). Quantification of SNCA pathology burden area was performed as described [29]. Briefly, tissue sections stained with EP1536Y were loaded into ImageScope TM software and the burden was calculated by dividing the number of positive pixels by the total area. The images were randomized, coded, and analyzed blinded to the experimental conditions. Two-tailed t-tests and one-way analysis of variance (ANOVA) with post-hoc Dunnett's multiple comparison tests were performed in GraphPad Prism v5.03 software.

Quantification of HMW SNCA in the insoluble protein fraction (from 37 to 66 kDa) was performed using image J and were normalized to levels of monomeric SNCA.

2.6.2. Evaluation of proteinase K resistant inclusions

Proteinase K (PK) digestion was performed on paraffin embedded brain sections from symptomatic mice using a modified protocol [30]. Sections were rehydrated and subsequently digested with 50 μg/ml PK (Roche Molecular Biochemicals, Mannheim, Germany) in PK buffer (10 mM Tris-HCl, pH 7.8, 100 mM NaCl, 0.1% Nonidet-P40) for 1 h at 55 °C. Following this, immunohistochemical staining using the anti-pSer 129 antibody EP1536Y (Abcam) was conducted as previously described [29], with the PK digestion in lieu of steam based antigen exposure.

2.7. RNA evaluations

2.7.1. RNA extraction

RNA was extracted from frozen midbrain samples from five symptomatic gba^+/−//SNCA^A53T/A53T and five gba^+/+//SNCA^A53T/A53T mice and age-matched controls using TriZol (Fischer Scientific).

2.7.2. Quantitative gene expression analysis

RNA levels were measured using the following probes, FAM-labeled probes, Hs01103383 m1(SNCA), Mm00484705_g1 (gba), Mm00484700_m1(gba, Mm00515586_m1(ctsd), and Mm00446977_ m1(scarb2) (Applied Biosystem) were used to quantify the exogenous human SNCA and four other related genes in total mouse mRNA. The mouse Tfrc gene assay (VIC-labeled; Mm0044194_m1) was used as an endogenous control for these experiments. Total RNA (2 μg) was reverse transcribed using the Applied Biosystem High Capacity cDNA Reverse Transcription Kit (Invtrogen) following the manufacturer's instructions. For the duplex, real-time assay, 1X master mixes were prepared for each gene probe with water, 20X Tfrc- endogenous control probe, 20XFAM labeled gene probe, and 2X TaqMan Universal PCR Master Mix (Invitrogen). In a 96-well plate, 2 μL of cDNA and 18 μL of master mix were plated in triplicate. The assay was run on the StepOnePlus Real-Time PCR system and the data analyzed with StepOne Software v2.3 via the 2^−ΔΔCT method.

2.8. Statistical analysis

Statistical analyses were performed using SAS statistics software version 9.14, and the Type I Error rate for statistical significance level was set at P < 0.05. The length of lifespan in gba^+/+//SNCA^A53T and gba^+/−//SNCA^A53T mice was evaluated using Kaplan-Meier survival analysis. The severity of symptoms was analyzed by comparing the interval between initial weight loss and when symptomatic animals required euthanasia. A Student's t-test was used to evaluate mean differences in the time intervals between the groups, reporting the observed p-value. Statistical analysis of the protein data was performed using GraphPad prism 5 software, *p < 0.05, **p < 0.001, ***p < 0.0001 and ****p < 0.00001. Data were evaluated as mean ± SEM.t-test or ANOVA were used to compare means of different groups of samples.

3. Results

3.1. The symptom onset and progression is faster in SNCA^A53T mice with gba haploinsufficiency

gba^+/− and SNCA^A53T lines were backcrossed to B6 mice to reduce the effect of genetic background. The two models were then crossed, aged, and over 200 mice with genotypes including gba^+/−//SNCA^A53T and gba^+/−//SNCA^A53T/A53T and their controls (wt/wt, gba^+/−, gba^+/+//SNCA^A53T and gba^+/+//SNCA^A53T/A53T) were followed for two years, monitoring their weight and the onset and progression of motor symptoms. In symptomatic mice, weight loss of over 13% was the first symptom, followed by arching of the back, impaired axial rotation, limb paralysis and bladder dysfunction. Survival analysis of 84 symptomatic mice, showed that gba^+/−//SNCA^A53T hemizygous and homozygous mice exhibited symptoms on average 9.76 weeks and 4.04 weeks earlier than gba^+/+//SNCA^A53T hemizygous and homozygous controls, respectively (p < 0.023, 0.0030), (Fig. 1A, B, Table 1). The rate of progression between initial onset of symptoms until euthanasia was 2.4 times more rapid in gba^+/−SNCA^A53T/A53T mice (p < 0.0001) and 4.1 times more rapid in gba^+/−SNCA^A53T hemizygotes (p < 0.0001) compared to gba^+/+//SNCA^A53T mice (Fig. 1, Table 2).

Fig. 1. Symptom severity and lethality in the different groups of mice.

Kaplan-Meier plot depicting age at death for 84 mice with different genotypes. Mice were sacrificed when they were unable to move (A) SNCA^A53T homozygotes (B) SNCA^A53T hemizygotes. Red (solid) line indicates mice with wt gba and blue (dashed) line mice with a null gba allele (gba^+/−).

SE= standard error and CI= confidence interval (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1.

Age of mice when euthanasia was required.

Genotype	Sample size	Mean age (month)	SE	95% CI	P-value
SNCAA53T homozygotes
gba+/+//SNCAA53T/A53T	13	11.65	0.22	11.19, 12.10	< 0.0030
gba+/−//SNCAA53T/A53T	14	10.64	0.21	10.20, 11.08
SNCAA53T hemizygotes
gba+/+//SNCAA53T/wt	45	18.64	0.53	17.59, 19.79	< 0.0230
gba+/−//SNCAA53T/wt	12	16.20	0.91	14.39, 18.02

SE = standard error and CI = confidence interval.

Table 2.

Interval of symptoms progression.

Genotype	Sample size	Time to euthanize (day)	SE	95% CI	P-value
SNCAA53T homozygotes
gba+/+//SNCAA53T/A53T	13	1.962	0.1831	1.563, 2.361	< 0.0001
gba+/−//SNCAA53T/A53T	14	0.7857	0.1358	0.4923, 1.079
SNCAA53T hemizygotes
gba+/+//SNCAA53T/wt	45	3.667	0.206	3.246, 4.088	< 0.0001
gba+/−//SNCAA53T/wt	12	0.889	0.266	0.4923, 1.079

SE = standard error and CI = confidence interval.

3.2. Differences in protein and RNA expression in mice with and without gba haploinsufficiency

To investigate the effect of over-expression of SNCA^A53T on GCase and other related proteins, we evaluated GCase activity, RNA expression and protein levels. Symptomatic mice were compared to littermates with the same genotype without overt symptoms (referred to as pre-symptomatic). Western blots of forebrain and midbrain lysates (N = 5) demonstrated an approximate 50% reduction in GCase levels in mice with a gba^+/− genoptype compared to gba^+/+ (Fig. 2A–D). However, there were no significant differences in GCase levels in symptomatic gba^+/−//SNCA^A53T and gba^+/+//SNCA^A53T mice compared to gba^+/− and wt/wt controls, respectively (Fig. 2A–D). RNA quantification showed no influence of exogenous SNCA^A53T on gba expression (Supplementary Fig. 1). GCase activity, measured in forebrain and midbrain samples, matched the corresponding GCase levels (Fig. 2E). Quantitative RNA assays for prosaposin, (psap), the GCase activator saposin-C, and scarb-2, encoding LIMP-2, which transports GCase from the ER to lysosomes, were unaffected by SNCA^A53T over-expression (Supplementary Fig. 1). LIMP-2 levels Fig. 2J, K) were similar in all groups. SNCA expression in both gba^+/+//SNCA^A53T and gba^+/−//SNCA^A53T mice was 40-fold higher than in controls (Supplementary Fig. 1). We next evaluated LAMP1 (a member of the LAMP complex reflecting lysomal density) and specific cathepsins (CATs) involved in SNCA degradation. Protein levels of LAMP1 (Fig. 2F, I) and CAT-B (Fig.2K) and L (Fig. 2L) in all mice were the same as wildtype. No differences were observed in either RNA expression of CAT-D (Supplementary Fig. 1) or cleaved CAT-D species among the different genotypes (N = 3) (Fig. 2F–H). CAT-D activity, was similar in midbrain and whole brain in symptomatic gba^+/+//SNCA^A53T and gba^+/−//SNCA^A53T mice (Supplementary Fig. 2).

Fig. 2. Evaluation of GCase and related genes.

(A, C) Western blot showing levels of GCase in forebrain (A) and midbrain (C) samples from SNCA^A53T heterozygotes with and without the null gba allele using the MDW933 probe. (B, D) Quantification of GCase levels in mouse brain samples. (B) Forebrain, (N = 5 for each genotype) and (D) midbrain (N = 3 for each genotype). (E) Glucocerebrosidase activity in forebrain and midbrain samples (N = 3 for each genotype). (F) Western blot analyses showing levels of pro-CAT-D with cleaved forms and LAMP1 in whole brain samples. (G, H, I) Quantification (N = 3) of heavy chain (G) and cleaved (H) CAT-D and LAMP1 (I). (J) Western blot showing levels of LIMP-2, CAT-B and CAT-L. (K, L, M) Quantification of Western blots (N = 3) for CAT-B (K), CAT-L (L) and LIMP-2 (M). Graph Pad Prism 7 was used to analyze the data. Statistical significance was determined by comparing the means of different groups using t-test or one-way ANOVA, *p < 0.05, **p < 0.001.

3.3. Evaluation of human SNCA^A53T over-expression in murine brain samples

To determine whether the over-expression of human SNCA^A53T contributed to the earlier symptom onset and severity in gba^+/−//SNCA^A53T mice, we analyzed levels of monomeric, soluble and insoluble SNCA in brain from mice with the different genotypes.

3.3.1. Evaluation of monomeric SNCA

SNCA^A53T mice with and without gba haploinsufficiency had similar levels of the monomeric SNCA in protein lysates from forebrain, midbrain and whole brain (Fig. 3A–F). Levels were also comparable in symptomatic and age-matched pre-symptomatic mice (Fig. 3E, F). Quantification of endogenous SNCA in controls (wt, gba^+/−), and exogenous SNCA^A53T in mice with and without gba^+/−, differed significantly (p < 0.0001), but there was no effect of the null gba allele on levels of monomeric SNCA (Fig. 3F).

Fig. 3. Analysis of human SNCA in mouse brain.

Western blot of monomeric SNCA in lysates from forebrain (A, B) and midbrain (C, D) in symptomatic SNCA^A53T mice with and without gba^+/− and controls (wt/wt & gba^+/−) (N = 2, except, for gba^+/−//SNCA^A53T where N = 3). (E, F) Western blot and quantification of monomeric SNCA in whole brain homogenates. Age-matched pre-symptomatic (~) mice are also shown (N = 3). (G) Monomeric SNCA in the soluble protein fraction. Blot is also probed with antibody 211 which is specific for human SNCA. (H) Quantification of soluble monomeric SNCA (N = 3). (I) Insoluble SNCA, showing both the monomeric and HMW forms. (J) Quantification of insoluble monomeric SNCA (N = 3). (K) Semi-quantification of HMW SNCA (quantifying SNCA between 37 and 66 kDa) in symptomatic mice with and without the null gba allele, N = 3. (L, M, N) Immunoblotting of monomeric and HMW forms of SNCA in human cortex from: 1-normal control; 2-patient with type 1 Gaucher disease; 3-patient with type 1 Gaucher disease and Parkinson disease; 4- c.84insG carrier with Parkinson disease and 5-patient with Parkinson disease without GBA1 mutations. β-actin was used as a loading control. (L) Monomeric SNCA in human whole brain lysates. (M, N) Monomeric and high molecular weight SNCA. (M) Soluble protein fraction. (N) Insoluble protein fraction.

3.3.2. Analysis of soluble and insoluble SNCA

Levels of monomeric SNCA in the soluble (Fig. 3G, H) and in the insoluble brain fractions from SNCA^A53T hemizygotes (Fig. 3I, J) were significantly higher in symptomatic mice than in pre-symptomatic mice (p < 0.05–0.001). To exclusively evaluate human SNCA, we used the human SNCA211 antibody, which also showed higher levels of SNCA once mice became symptomatic, regardless of genotype (Fig. 3I). However, we consistently observed less aggregated SNCA in symptomatic gba^+/−//SNCA^A53T mice compared to gba^+/+//SNCA^A53Tmice, (p < 0.05) (N = 3), (Fig. 3I, K).

3.4. Evaluation of fractionated SNCA in human brain samples

To determine whether levels of high molecular weight (HMW) SNCA in this mouse accurately reflected SNCA in human brain, we evaluated SNCA levels in cortex from a control, a patient with idiopathic Parkinson disease, patients with Gaucher disease with and without Parkinson disease, and a c.84insG Gaucher carrier with parkinsonism (analogous to murine genotype gba^+//). Total monomeric SNCA was clearly present in all cases, but was lowest in the idiopathic Parkinson disease sample (Fig. 3L). However, blotting the soluble and insoluble fractions (Fig. 3M, N) suggested that this patient had more of the HMW and oligomeric forms of SNCA than those with GBA1 mutations.

3.5. Assessment of SNCA pathology

To assess the presence and distribution of SNCA inclusion pathology, symptomatic homozygous and hemizygous SNCA^A53T mice with and without the null gba allele were analyzed by immunocytochemistry and immunofluorescence analyses with antibodies EP1536Y and 81A to SNCA phosphorylated at Ser129 (a marker of SNCA inclusion pathology) [31], anti-SNCA antibody Syn506 (preferentially detects SNCA in pathological inclusions) [28] or anti-p62/sequestosome (an autophagic adaptor and ubiquitin-like protein) (Fig. 4A). Quantification of SNCA inclusion density in the pons of symptomatic mice (N = 17; 4–5 per genotype) (Fig. 4B), assessed by a blinded observer, showed no significant effect of the gba^+/− allele on the density of SNCA pathology. Like mice with wildtype gba[19], SNCA^A53T mice with a gba^+/− allele had SNCA inclusions in the midbrain, but not in TH-positive neurons (Fig. 4C). p62, SNCA and tyrosine hydroxylase (TH) levels in midbrain from symptomatic SNCA^A53T mice, evaluated by western blotting, showed no effect of the gba^+/− allele (N = 3) (Fig. 4D–G). All symptomatic mice analyzed had widespread SNCA inclusion pathology in the brainstem, midbrain, hypothalamus and spinal cord regions, with more sparse pathology in the cerebellum and motor cortex (Fig. 4H, Supplementary Fig. 3A, B). Pre-symptomatic mice were also evaluated, but no SNCA inclusions were observed (Supplementary Fig. 3D). Paraffin embedded brain sections of brainstem and midbrain from symptomatic mice with and without the gba^+/− allele (N = 3) were digested with proteinase K and stained for pSer129 SNCA. Proteinase K resistant inclusions were seen in a similar density and distribution in both groups (Supplementary Fig. 3C).

Fig. 4. Assessment of SNCA pathology.

(A) Representative images of immunofluorescence analysis depicting SNCA inclusion pathology in the pons of symptomatic SNCA^A53T hemizygote (top two panels) or SNCA^A53T homozygote (lower two panels) mice with gba^+/+or gba^+/− using anti-p62 antibody and Syn 506. Merged images counterstained with 4,6-diamidino-2-phenylindole (DAPI) are shown (right). (B) Quantitative analysis of SNCA burden in the pons using antibody Syn 506. (C) Representative images of immunofluorescence analysis depicting SNCA inclusion pathology in the midbrain of symptomatic SNCA^A53T hemizygous or homozygous mice with gba^+/+or gba^+/− using anti-TH and Syn 506. (D) Immunoblotting of three proteins, p62, SNCA and TH, evaluated by immunofluorescence and their quantification (E, F, G), N = 3. (H) Immunohistochemical analysis depicting pathology in the hypothalamus, spinal cord, cerebellum and motor cortex in symptomatic SNCA^A53T hemizygous or SNCA^A53T homozygous mice with gba^+/+or gba^+/− using antibodies to pSer129 α-syn (EP1536Y or 81A). Scale bar 50 μm.

3.6. Glucosylsphingosine and glucosylceramide levels in murine brain samples

Mass spectrometry was performed to evaluate whether the null gba allele in SNCA^A53T mice impacted brain glucosylsphingosine or glucosylceramide levels. No significant differences in lipids levels were noted, as expected in heterozygote animals (N = 5) (Fig. 5).

Fig. 5. Analyses of glucosylceramide and glucosyl-sphingosine in mouse brain samples.

Glucosylceramide (GlcCer) isoforms, analyzed in forebrain (A) and midbrain (B) of control and SNCA^A53T mice with and without the gba null allele; N = 5.

Glucosylsphingosine (GlcSph) levels in forebrain (C) and midbrain (D) of controls and SNCA^A53T mice with and without the gba null allele; N = 5. Graph Pad Prism 7 was used to analyze the data. Statistical significance was determined by comparing the means of different groups using t-test or one -way ANOVA.

4. Discussion

Identifying the mechanistic basis for the association between GBA1 and the synucleinopathies continues to be a challenge, and additional pathways, genes and/or epigenetics may play a role [9,32]. An appropriate animal model would greatly facilitate our understanding of this complicated relationship. Previous attempts at modeling GBA1-associated parkinsonism in the mouse used the inhibitor CBE, which inactivates GCase [16,18], but fails to represent the natural development of Parkinson disease, an age-related disorder. Studying L483P (L444P) mice over-expressing SNCA^A53T, it was shown that the mutated GCase interfered with degradation and caused accumulation of SNCA in cultured primary neurons, although the mice had few symptoms [15]. By deleting one gba allele in a transgenic mouse overexpressing SNCA^A53T, we sought to create a model that, upon aging, developed SNCA aggregates in neurons. This in vivo model with gba haploinsufficiency mimics the situation encountered in carriers of functionally null mutations, like c.84insG, IVS2+1G > A or recombinant alleles, who develop parkinsonism. The aging mice developed symptoms gradually, as occurs in patients with parkinsonism. Survival analysis of 84 mice with different genotypes (Fig. 1A, B and Tables 1 and 2), demonstrated that absence of one gba allele in SNCA^A53T homozygotes and hemizygotes resulted in earlier and more severe symptoms. Lipidomic analyses of mouse brain samples showed no increase in glucosylsphingosine or glucosylceramide, indicating that the more severe clinical course was not due to substrate storage. Symptomatic gba^+/−//SNCA^A53T and gba^+/− mice showed similar GCase levels and activity in midbrain and forebrain, (Fig. 2A–E). These findings differ from other mouse models where overexpression of SNCA resulted in reduced GCase levels and activity [17]. Studies in neuronal models and iPSC-derived neurons from a patient with SNCA triplication also indicated that excess SNCA inhibited GCase activity, while subsequent substrate accumulation enhanced SNCA aggregation [33,34]. In our mice, over-expressed SNCA^A53T did not have a similar impact on GCase or its substrates.

Surprisingly, immunoblotting suggested that gba^+/−//SNCA^A53T mice have less insoluble HMW and oligomeric forms of SNCA [35] than mice without a null gba allele (Fig. 3I, K). Furthermore, the distribution of SNCA pathology and abundance of SNCA aggregates throughout the neuroaxis were evaluated in multiple brain region, revealing that results in symptomatic SNCA^A53T homozygotes and hemizygotes with and without the null gba allele were not significantly different(Fig. 4H). There were also indications of less HMW SNCA in brain autopsy samples from patients with GBA1-associated parkinsonism (Fig. 3L–N) [36]. These results emphasize the need to evaluate both monomeric and aggregated forms of SNCA in studies of human and mouse brain, as the level of monomeric SNCA does not reflect the actual pathology. Furthermore, factors modifying SNCA such as phosphorylation, ubiquitination, alternative splicing [37,38] could contribute to the altered forms of SNCA observed. Levels of the different species of CAT-D, a protein thought to reduce SNCA aggregation [39], as well as CAT-D activity at two timepoints, did not differ in gba^+/−//SNCA^A53T mice (Fig. 2D, E). Thus, the impact of mutant GCase in Parkinson pathogenesis may not be directly related to its enzymatic activity or that of CAT-D. In the aging brain, other factors such as oxidative stress, variation in RNA expression, protein modifications and epigenetics may alter pathways involving GCase and SNCA levels or function.

Our finding that gba haploinsufficiency accelerates disease onset and exacerbates symptom progression in this mouse corresponds to the clinical observation of an earlier age of Parkinson disease onset and, at times, more progressive course in carriers of GBA1 mutations. However, the basis for these findings remains enigmatic. Since gba haploinsufficency did not result in significantly enhanced SNCA levels or abundance of SNCA pathology in terminal mice, another, yet unknown secondary function of GCase protein in distinct neuronal populations may initiate or contribute to the earlier and more severe symptoms in gba^+/−//SNCA^A53T mice. This is supported by a study of dopaminergic neurons from aged mice with a conditional GBA1 knock-out, which had no motor dysfunction and no midbrain accumulation of endogenous SNCA [40]. It has been proposed that deficient GCase promotes cellular cascades contributing to earlier Parkinson pathogenesis and synaptic dysfunction [16]. It is also possible that GCase can influence the formation of prion-like SNCA amyloid seeds that initiate inclusion formation and/or the subsequent prion-like spread of SNCA inclusion pathology [31,41] or it's cell-to-cell transmission as previously suggested [42]. Further cellular and molecular evaluations using this model may facilitate the identification of modifier genes, or other factors impacting the association between GBA1 and parkinsonism or reveal a moonlighting role for GCase in neuronal cells.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ymgme.2017.11.001.

Abbreviations

CAT

cathepsin

CBE

conduritol-β-epoxide

ER

endoplasmic reticulum

gba^+/−

heterozygous null mouse gba allele

GCase

glucocerebrosidase

GD

Gaucher disease

HMW

high molecular weight

LAMP2

lysosomal membrane protein 1

LIMP2

lysosomal integral membrane protein 2

PD

Parkinson disease

SNCA

α-synuclein

SNCA^A53T

mutant human A53T α-synuclein

TH

tyrosine hydroxylase