Species: Mouse
Genes: Gba1
Modification: Gba1: Knock-In
Disease Relevance: Parkinson's Disease
Strain Name: Gba1^tm4Ggb
Genetic Background: C57BL6, but may be mixed with 129Sv or FVB depending on the cross.
Availability: N/A

This knock-in (KI) mouse model was generated by introducing a c.A1366T point mutation into exon 9 of the mouse Gba1 (acid β-glucosidase) gene resulting in a D409V substitution (Xu et al., 2003). Gba1 encodes the lipid-degrading lysosomal enzyme glucocerebrosidase (GCase), and mutations in the human gene are a common risk factor for Parkinson’s disease (PD) and are present in up to 10% of patients (Farfel-Becker et al., 2019). Although the D409V mutation, specifically, is rare in Parkinson’s disease, this mutation leads to reduced GCase activity, which can be associated with α-synuclein aggregation.

These mice have been studied as homozygotes (D409V/D409V), heterozygotes (D409V/wild-type), and compound heterozygotes (D409V/null), the findings for which are outlined as appropriate below.

Homozygote KI mice were viable and fertile, had grossly normal behavior, and survived over 66 weeks of age (Xu et al., 2003). GCase activity was significantly lower in KI mouse liver, spleen, brain, lung, and skin fibroblasts versus that in wild-type mice: ~20-25% of wild-type levels in the brain and <10% in the other tissues (Xu et al., 2003; Sardi et al., 2011). Glucosylceramide accumulated in liver, lung, and spleen, but not brain, tissue at 6.5 months of age in D409V/null mice, and to a lesser extent in homozygote KI mice (Xu et al., 2003). In contrast, progressive glucosylsphingosine accumulation was observed in the hippocampus, cerebral cortex, and cerebellum of homozygous KI mice, starting at 2 months of age (Sardi et al., 2011). Heterozygous KI mice did not exhibit glucosylsphingosine accumulation, and although glucocerebrosidase activity was lower than that in wild-type mice, it was higher than that in homozygous KI mice (Sardi et al., 2011). In another study, the brains of D409V/null mice displayed progressive glucosylsphingosine accumulation, becoming significantly greater than in wild-type controls starting at 3 months of age; glucosylceramide levels were greater than that in controls starting at 6 months of age (Dai et al., 2016). At 3 months of age in D409V/null mice, large numbers of storage cells positive for Mac-3 appeared in the lung and a few appeared in the liver (Xu et al., 2003; Xu et al., 2011).

Ratios of spleen and liver weights to body weight in D409V/null mice showed differences from wild-type controls that were dependent on sex (Dai et al., 2016). Namely, female D409V/null mice showed enlarged spleen and liver ratios compared to wild-type controls as early as 6 months of age, while these differences were only apparent in males at around 2 years of age. In contrast, homozygous KI mice did not display altered ratios of spleen- or liver-to-body weight at 4 or 8 months of age, although these data were not segregated by sex (Zhao et al., 2023).

Motor Behavior | Non-Motor Behavior |Neuropathology | Neuroinflammation | Immune Response | Endocytic Pathway | Gene Expression |Modification Details

Motor Behavior
Using the open-field test at 12 months of age in homozygous KI mice, no differences were observed in distance travelled compared to wild-type mice (Sardi et al., 2011).

Gait analysis revealed that D409V/null mice had greater front- and hindpaw widths compared to wild-type mice, and this difference became apparent earlier in females than in males (3 versus 12 months of age; Dai et al., 2016). Female D409V/null mice also had a greater stride length than wild-type controls as early as 3 months of age, a difference that was not observed in males. In contrast, homozygous KI mice did not display any gait abnormalities at 8 months of age (Zhao et al., 2023).

Non-Motor Behavior
Memory impairment was observed in 6-month-old homozygous KI mice versus wild-type controls based on the novel object recognition and contextual fear-conditioning tests (Sardi et al., 2011). Novel object recognition was also impaired at 12 months of age. However, anxiety-like behavior, as assessed by the open-field test (time spent in center), did not differ between wild-type and homozygous KI mice. In contrast to homozygous KI mice, heterozygotes did not exhibit memory deficits based on the novel object recognition test at 6 months of age. In another study, novel object recognition deficits appeared as early as at 4 months of age in homozygous KI mice, relative to wild-type controls (Sardi et al., 2013).

In D409V/null mice, memory was impaired at 9 and 12 months of age, as assessed by a lowered reduction of activity on a repeated open-field test compared with wild-type controls, indicating abnormal habituation (Dai et al., 2016). Emotional memory was also impaired at 12 months of age in D409V/null mice, based on a conditioned fear test, and this KI-related dysfunction appeared to be specific to male mice. Anxiety- and compulsive-like behaviors were assessed using the marble burying test, and while both male and female D409V/null mice showed differences in marble burying behavior compared to wild-type mice (starting at 6 months of age), females showed more severe abnormalities, especially at older ages (12 and 18 months).

Neuropathology
Homozygous KI mice (6- and 12-month-old but not 2-month-old) exhibited progressive α-synuclein accumulation and α-synuclein aggregation (unmasked by proteinase K) in the hippocampus, a finding that was absent in wild-type control mice (Sardi et al., 2011). Aggregates in homozygous KI mice also contained ubiquitin and were primarily located in neurites. In a separate study, homozygous KI mice had increased α-synuclein in the forebrain at 12 months, but not 6 months, of age compared to control littermates (Cullen et al., 2011). In D409V/null mice, insoluble α-synuclein aggregates were also found in the cortex to a greater extent than in wild-type controls at 6 months of age (Dai et al., 2016). Another study found that α-synuclein aggregation and ubiquitin immunoreactivity was higher in homozygote KI mice than wild-type mice as early as 4 months of age (Sardi et al., 2013). Heterozygous KI mice also contained α-synuclein/ubiquitin aggregates at 6 months of age, although to a lesser extent than that observed in homozygous mice (Sardi et al., 2011). Progressive tau aggregation was also observed in the hippocampus, starting at 4 months of age in homozygous KI mice (Sardi et al., 2013). A separate study also found that α-synuclein aggregation as well as tau and ubiquitin immunoreactivity was observed in heterozygous KI mice at 10 months of age (Viel et al., 2021).

Neurodegeneration in the hippocampus, as revealed by Fluoro-Jade C, did not differ between homozygous KI and wild-type mice at 12 months of age (Sardi et al., 2011). Moreover, no overt nigrostriatal loss was observed in 12-month-old homozygous KI mice, based on immunostaining of tyrosine hydroxylase in the striatum and substantia nigra.

Neuroinflammation
GFAP, Iba-1, and CD68 immunostaining in the hippocampus of 12-month-old homozygous KI mice did not differ from that seen in wild-type mice, suggestive of no inflammatory response (Sardi et al., 2011). CD68 staining also did not differ in the brains of D409V/null mice versus wild-type mice at 24 months of age (Dai et al., 2016).

Immune Response
In 24- to 28-week-old D409V/null mice, greater numbers of macrophages and CD4+ T cells were found in the liver, spleen, and lung, but not in the bone marrow, compared to wild-type mice (Pandey et al., 2012). The number of dendritic cells, on the other hand, was increased in all of those tissues. The number of CD8+ T cells did not differ in any tissue examined. Levels of cytokines (IFNγ IL-12p40, TNFα, IL-17A/F, IL-6, TGBβ; but not IL-4) were also increased in the serum of D409V/null mice.

Macrophages and dendritic cells isolated from lungs of D409V/null mice expressed higher levels of the CXCL9 chemokine than wild-type controls (Magnusen et al., 2021). Moreover, blood-derived CD3+ T cells (as well as CD4+ and CD8+ T cells specifically) had higher levels of CXCR3 as compared to wild-type mice. This indicates that the CXCL9/CXCR3 axis may be heightened in this mouse model, potentially leading to enhanced recruitment of T cells. Indeed, in another study, the sera of D409V/null mice was found to contain increased levels of many other chemokines and induce chemotaxis of several types of immune cells (Pandey et al., 2014).

Levels of complement component C5a—involved in the differentiation and activation of CD4+ T cells—were elevated in the sera of 20-to 24-week-old D409V/null mice compared to wild-type mice (Pandey et al., 2017). C5aR1 expression was also increased in dendritic cells and macrophages from the liver, spleen, and lung.

Endocytic Pathway
Levels of beclin, but not other markers of autophagy or lysosomes (Lamp2, p62/SQSTM1, LC3B), were decreased in hippocampal lysates from 12-month-old KI mice (Sardi et al., 2011).

Gene Expression
The expression of many genes was dysregulated, as detected using microarrays, in samples from lung and liver of D409V/null mice across time (4, 12, 18, and 28 weeks of age) compared to wild-type controls (Xu et al., 2011). Notably, macrophage activation and immune response genes were preferentially altered in lung tissue of KI mice. In another study, gene expression differences detected by mRNA-Seq and microarrays were also observed in the spleen (Dasgupta et al., 2013).

Modification Details

This KI mouse model was generated by introducing a c.A1366T point mutation into exon 9 of the mouse Gba1 gene to create a D409V substitution in the lipid-degrading lysosomal enzyme glucocerebrosidase (Xu et al., 2003).

Phenotype Characterization

Q&A with Model Creator

Q&A with Ying Sun

What would you say are the unique advantages of this model?

• D409V/D409V and D409V/null models produce viable mice that can live up to two years. Both male and female mice are fertile, making it easy to maintain the colony.
• Both models develop varying degrees of phenotypes that can be used for studies on Gaucher disease (GD) and Parkinson’s disease, as well as for therapeutic studies for of these conditions.
• Both models have been characterized for their neurological phenotypes, brain pathology, and α-synuclein pathology.
• Reduced gene dosage in the D409V/null model results in an earlier phenotype and faster disease progression in both the viscera and central nervous system compared to that in the D409V/D409V model.

What do you think this model is best used for?

• Due to the low GCase activity in D409V/D409V mice and their mild phenotype, this model can be transformed into a severe GD model through chemical induction, e.g., Conduritol B Epoxide (CBE), a specific covalent β-glycosidase enzyme inhibitor, for testing new therapies that are not GCase-based.
• Studying the interplay of modifiers on modulating GD and GBA1 mutation-associated Parkinson’s disease.
• The D409V/null model, with its earlier phenotype and faster disease progression in the viscera, is a suitable preclinical animal model for evaluating treatment for GD.

What caveats are associated with this model?

• In the D409V/D409V model, the mild phenotype and delayed disease progression (often until the second year), will make the study costly and challenging in assessing disease phenotypes.
• In the D409V/null model, the mild phenotype in the central nervous system and the delayed disease progression until 6 months of age will result in higher costs for studying the central nervous system phenotype.

Anything else useful or particular about this model you think our readers would like to know?

Choose appropriate controls, such as strain background, age, and sex, and use reliable reagents and protocols for your study. 

COMMENTS / QUESTIONS

1. • Matthew M J Farrer
     University of Florida
   • Posted: 08 Jul 2011
   • Paper: CNS expression of glucocerebrosidase corrects alpha-synuclein pathology and memory in a mouse model of Gaucher-related synucleinopathy.

   Pablo Sardi and colleagues did an excellent job describing the GBA
   mouse model; however, the data/experimental design could not decipher the central
   conundrum, i.e., whether GBA mutations act through a loss of function or a
   toxic gain of function. The authors speculate that glucocerebrosidase replacement may be a
   strategy for the treatment of α-synucleinopathies. This is tautology in the
   paper, but with merit for select patients where GBA mutations can be
   ascribed as a major contributor to disease.

   Sardi et al. did not address the mechanism in the context of past gene discovery, or the new synthesis emerging. Readers may be aware that we recently identified pathogenic mutations in VPS35, a
   central retromer component, in late-onset Parkinson's disease. The paper by
   Vilarino-Guell and colleagues is embargoed in American Journal of Human
   Genetics until 14 July 2011. My reason for mentioning it briefly is that the
   retromer is required to recycle mannose-6-phosphate receptors (MPR) that are
   necessary to traffic lysosomal enzymes, including glucocerebrosidase, from
   the Golgi complex to an acidified (pre)lysosomal compartment.

   Of note, the endosome Rab7L1 (which we postulate explains the PARK16 GWAS signal) is also
   required for proper MPR trafficking and for retromer localization and
   function. Dynactin and tau have also been directly implicated in retromer
   formation and parkinsonism. α-synuclein has been shown to more generally
   disrupt cellular Rab homeostasis, in a dose-dependent manner, and may affect
   multiple trafficking steps between the ER and Golgi. Likewise, LRRK2 GTPase
   and kinase signalling/scaffolding functions are most consistent with its
   complex being a master regulator of membrane protein trafficking.

   Many specific details need to be resolved, but GBA, VPS35, Rab7L1, DCTN1, MAPT, SNCA, and LRRK2—indeed most of the major
   genes identified in late-onset parkinsonism (to date)—now elucidate an
   overlapping biologic network. The phenomenology of selective vulnerability,
   variable expressivity, and penetrance may be addressed using a similar
   framework.

   Ultimately, successful neuroprotective therapeutics for neurodegenerative
   disorders will result from a combination of genetic insight and model
   development, and Pablo Sardi's work nicely illustrates the approach.

   View all comments by Matthew M J Farrer
2. • Valerie Cullen
     Link Medicine Corporation
   • Posted: 08 Jul 2011
   • Paper: CNS expression of glucocerebrosidase corrects alpha-synuclein pathology and memory in a mouse model of Gaucher-related synucleinopathy.

   This PNAS paper represents the latest in a recent flurry of papers (Xu et al., 2010; Cullen et al., 2011; Mazzulli et al., 2011) examining the biochemical and mechanistic links between GBA mutations and synuclein mismetabolism. In the current paper, Sardi et al. find that mice carrying two copies of the D409V mutation in GBA exhibit progressive mismetabolism of synuclein and generalized ubiquitinopathy.

   We made the same observation in these mice in our recent Annals of Neurology paper (Cullen et al., 2011), where we showed an age-dependent increase in the synuclein content of the membrane fraction (containing within it the lysosomal compartment) and ubiquitin staining. Like Sardi et al., we also observed that mice carrying only one copy of the D409V mutation had subtle changes in synuclein levels, supporting the notion of a gain-of-toxic function as at least one aspect of the mechanistic interplay between GBA and synuclein.

   We also showed an accumulation of synuclein in simple cell models when D409V or the similar variant, D409H, was overexpressed. Importantly, when we overexpressed wild-type GBA in cells, we observed a significant reduction in cellular synuclein levels. This was the case in both PC12 cells, which were transfected with GBA, and in HEK293 cells, which experienced a more robust increase in GBA expression and activity due to viral overexpression.

   Sardi et al. have now extended our observations into animals by showing that viral overexpression of wild-type GBA into rodent brain can reduce the synuclein accumulation and memory deficits caused by GBA mutation. Thus, as discussed in our Annals Neurology paper (Cullen et al., 2011), and as demonstrated by Sardi et al., increasing the brain's GBA content may be a viable therapeutic strategy to explore further. Perhaps eventually, a combination approach may be used, with GBA expression utilized to combat loss of enzyme function, and GBA chaperoning and/or lysosomal support (see, e.g., our results with isofagomine and rapamycin) utilized to combat a gain of toxic function of mutant GBA.

   References:

   Xu YH, Sun Y, Ran H, Quinn B, Witte D, Grabowski GA. Accumulation and distribution of α-synuclein and ubiquitin in the CNS of Gaucher disease mouse models. Mol Genet Metab. 2011 Apr;102(4):436-47. Epub 2010 Dec 31 PubMed.

   Cullen V, Sardi SP, Ng J, Xu YH, Sun Y, Tomlinson JJ, Kolodziej P, Kahn I, Saftig P, Woulfe J, Rochet JC, Glicksman MA, Cheng SH, Grabowski GA, Shihabuddin LS, Schlossmacher MG. Acid β-glucosidase mutants linked to Gaucher disease, Parkinson disease, and Lewy body dementia alter α-synuclein processing. Ann Neurol. 2011 Jun;69(6):940-53. Epub 2011 Apr 6 PubMed.

   View all comments by Valerie Cullen
3. • Joseph Mazzulli
     Harvard Medical School
   • Posted: 08 Jul 2011
   • Paper: CNS expression of glucocerebrosidase corrects alpha-synuclein pathology and memory in a mouse model of Gaucher-related synucleinopathy.

   The paper by Sardi et al., 2011, provides further important in-vivo evidence of a mechanistic link between Gaucher’s disease and α-synuclein processing. The demonstration that accumulation of α-synuclein and behavioral deficits in Gaucher's disease mice can be ameliorated by increasing glucocerebrosidase levels suggests that this lysosomal enzyme may be an important therapeutic target for the treatment of synucleinopathies. It will be of future interest to determine whether enhancing glucocerebrosidase function, either through adenoviral-mediated glucocerebrosidase expression or administration of pharmacological chaperones, has the ability to reverse or clear the accumulation of α-synuclein in aged (12-month-old) Gaucher's mice that are symptomatic.

   The recent exciting paper by Lim et al. (Lim et al., 2011), which demonstrates that behavioral deficits and pathology induced by α-synuclein overexpression can be reversed, suggests that methods that enhance the clearance of α-synuclein may provide therapeutic benefit even after symptoms are apparent. Augmentation of glucocerebrosidase function appears to be one such option that may accelerate the clearance of α-synuclein and prevent further disease progression in Parkinson's disease and other synucleinopathies.

   References:

   Lim Y, Kehm VM, Lee EB, Soper JH, Li C, Trojanowski JQ, Lee VM. α-Syn suppression reverses synaptic and memory defects in a mouse model of dementia with Lewy bodies. J Neurosci. 2011 Jul 6;31(27):10076-87. PubMed.

   View all comments by Joseph Mazzulli

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References

Paper Citations

1. Xu YH, Quinn B, Witte D, Grabowski GA. Viable mouse models of acid beta-glucosidase deficiency: the defect in Gaucher disease. Am J Pathol. 2003 Nov;163(5):2093-101. PubMed.
2. Farfel-Becker T, Do J, Tayebi N, Sidransky E. Can GBA1-Associated Parkinson Disease Be Modeled in the Mouse?. Trends Neurosci. 2019 Sep;42(9):631-643. Epub 2019 Jul 6 PubMed.
3. Sardi SP, Clarke J, Kinnecom C, Tamsett TJ, Li L, Stanek LM, Passini MA, Grabowski GA, Schlossmacher MG, Sidman RL, Cheng SH, Shihabuddin LS. CNS expression of glucocerebrosidase corrects alpha-synuclein pathology and memory in a mouse model of Gaucher-related synucleinopathy. Proc Natl Acad Sci U S A. 2011 Jul 19;108(29):12101-6. Epub 2011 Jul 5 PubMed.
5. Xu YH, Jia L, Quinn B, Zamzow M, Stringer K, Aronow B, Sun Y, Zhang W, Setchell KD, Grabowski GA. Global gene expression profile progression in Gaucher disease mouse models. BMC Genomics. 2011 Jan 11;12:20. PubMed.
6. Zhao X, Lin Y, Liou B, Fu W, Jian J, Fannie V, Zhang W, Setchell KD, Grabowski GA, Sun Y, Liu CJ. PGRN deficiency exacerbates, whereas a brain penetrant PGRN derivative protects, GBA1 mutation-associated pathologies and diseases. Proc Natl Acad Sci U S A. 2023 Jan 3;120(1):e2210442120. Epub 2022 Dec 27 PubMed.
7. Sardi SP, Clarke J, Viel C, Chan M, Tamsett TJ, Treleaven CM, Bu J, Sweet L, Passini MA, Dodge JC, Yu WH, Sidman RL, Cheng SH, Shihabuddin LS. Augmenting CNS glucocerebrosidase activity as a therapeutic strategy for parkinsonism and other Gaucher-related synucleinopathies. Proc Natl Acad Sci U S A. 2013 Feb 26;110(9):3537-42. Epub 2013 Jan 7 PubMed.
8. Cullen V, Sardi SP, Ng J, Xu YH, Sun Y, Tomlinson JJ, Kolodziej P, Kahn I, Saftig P, Woulfe J, Rochet JC, Glicksman MA, Cheng SH, Grabowski GA, Shihabuddin LS, Schlossmacher MG. Acid β-glucosidase mutants linked to Gaucher disease, Parkinson disease, and Lewy body dementia alter α-synuclein processing. Ann Neurol. 2011 Jun;69(6):940-53. Epub 2011 Apr 6 PubMed.
9. Viel C, Clarke J, Kayatekin C, Richards AM, Chiang MS, Park H, Wang B, Shihabuddin LS, Sardi SP. Preclinical pharmacology of glucosylceramide synthase inhibitor venglustat in a GBA-related synucleinopathy model. Sci Rep. 2021 Oct 22;11(1):20945. PubMed.
10. Pandey MK, Rani R, Zhang W, Setchell K, Grabowski GA. Immunological cell type characterization and Th1-Th17 cytokine production in a mouse model of Gaucher disease. Mol Genet Metab. 2012 Jul;106(3):310-22. Epub 2012 Apr 30 PubMed.
11. Magnusen AF, Rani R, McKay MA, Hatton SL, Nyamajenjere TC, Magnusen DN, Köhl J, Grabowski GA, Pandey MK. C-X-C Motif Chemokine Ligand 9 and Its CXCR3 Receptor Are the Salt and Pepper for T Cells Trafficking in a Mouse Model of Gaucher Disease. Int J Mol Sci. 2021 Nov 24;22(23) PubMed.
12. Pandey MK, Jabre NA, Xu YH, Zhang W, Setchell KD, Grabowski GA. Gaucher disease: chemotactic factors and immunological cell invasion in a mouse model. Mol Genet Metab. 2014 Feb;111(2):163-71. Epub 2013 Sep 10 PubMed.
13. Pandey MK, Burrow TA, Rani R, Martin LJ, Witte D, Setchell KD, Mckay MA, Magnusen AF, Zhang W, Liou B, Köhl J, Grabowski GA. Complement drives glucosylceramide accumulation and tissue inflammation in Gaucher disease. Nature. 2017 Mar 2;543(7643):108-112. Epub 2017 Feb 22 PubMed.
14. Dasgupta N, Xu YH, Oh S, Sun Y, Jia L, Keddache M, Grabowski GA. Gaucher disease: transcriptome analyses using microarray or mRNA sequencing in a Gba1 mutant mouse model treated with velaglucerase alfa or imiglucerase. PLoS One. 2013;8(10):e74912. Epub 2013 Oct 4 PubMed.

Further Reading