Species: Mouse
Genes: Gba1
Modification: Gba1: Knock-In
Disease Relevance: Parkinson's Disease
Strain Name: B6;129S4-Gba1^tm1Rlp/Mmnc
Genetic Background: C57BL/6 and 129S4/SvJae
Availability: Available through the Mutant Mouse Regional Resource Centres (MMRRC), Stock# 000117-UNC, Cryopreserved.

Summary

This is a knock-in (KI) mouse model carrying a L444P mutation in the endogenous Gba (glucocerebrosidase [GC] or acid β-glucosidase) gene (Liu et al., 1998). The GBA1 protein is the lipid-degrading lysosomal enzyme glucocerebrosidase (GCase), and mutations in the human GBA1 gene are a common risk factor for Parkinson’s disease (Farfel-Becker et al., 2019). The L444P variant represents approximately one-third of all GBA variants linked to Parkinson's disease.

Overall Health | Neuropathology | Motor Behavior | Non-motor Behavior | Mitochondrial Abnormalities | Cellular Processes

Overall Health
Homozygous L444P KI mice have a compromised skin barrier, characterized by abnormally red and wrinkled skin and excessive transepidermal water loss. Homozygous KI mice die early if optimal breeding and husbandry conditions are not followed. The type of bedding used is important for promoting survival, with wood chips being better than highly absorbent materials (i.e., avoid recycled paper or corn cobs). The second factor to promote survival of homozygous L444P KI mice is to generate them using homozygous L444P KI breeding pairs—this is proposed to enhance survival by removing competition with healthy wild-type or heterozygous littermates (Mizukami et al., 2002). Despite these measures, only about half of homozygous L444P KI mice survive past weaning. Moreover, starting around 50 days of age, body weight of homozygous KI mice plateaus, and sits around 15% less than that of wild-type controls. In addition, the liver and spleen are enlarged in KI compared with control mice. The majority of studies cited below use heterozygous KI mice.

GC enzyme activity in the liver, spleen, lung, brain, and skin of homozygous KI mice was found to be about 15-20% of that seen in wild-type mice (Liu et al., 1998; Mizukami et al., 2002). GC enzyme activity was also reduced (by 30-40%) in heterozygous KI adult mice compared to wild-type controls in the ventral midbrain and hippocampus (Yun et al., 2018; Li et al., 2019). In aged heterozygous KI mice (24 months of age), GC enzyme activity was reduced (~30%) in the midbrain, cortex, and striatum compared to wild-type controls (Migdalska-Richards et al., 2017). And finally, in 3-month-old heterozygous KI mice, reductions of ~20-30% in GC enzyme activity were observed in the cortex, striatum, hippocampus, and midbrain (Mahoney-Crane et al., 2023). No reductions were observed in hippocampal GC protein.

Glucosylceramide accumulation was observed only in the epidermis of 1-day-old homozygous KI mice, and not in the brain or liver (Liu et al., 1998); glucosylceramide levels in the liver, spleen, and brain also did not differ between control and homozygous KI mice at 2 months to over 1 year of age (Mizukami et al., 2002). In another study of heterozygous L444P KI mice, glucosylceramide levels did not differ from wild-type controls in the forebrain at 3, 6, 9, or 12 months of age (Mahoney-Crane et al., 2023). However, glucosylsphingosine levels were increased in the forebrains of KI mice at 6, 9, and 12 months, but not at 3 months of age. In addition, plasma levels of glucosylsphingosine, but not of glucosylceramide, were increased in heterozygous KI mice at the time of death.

The hematological profile of homozygous L444P KI mice was perturbed compared with wild-type controls. For instance, white blood cell count, red blood cell count, hemoglobin, hematocrit, and total cholesterol were significantly reduced, although platelet levels were unchanged, and liver damage was indicated by a significant increase in the liver enzymes aspartate aminotransferase and alanine aminotransferase (Mizukami et al., 2002).

Histologic examination of the skin in homozygous L444P KI mice revealed a compromised skin barrier, with the stratum corneum and epidermis being thickened compared with wild-type controls (Liu et al., 1998; Mizukami et al., 2002). Other histologic abnormalities indicative of systemic inflammation (i.e., immune cell infiltration) were observed in KI mice, including in the liver, as early as 1 month of age, as well as in the spleen and lung. In addition, elevated mRNA levels of TNF-α (in the liver) and IL-1β (in the cervical lymph nodes) were observed. Lymphadenitis was also observed at 2 months of age throughout the body (Mizukami et al., 2002).

Neuropathology
Alpha-synuclein levels are increased in the ventral midbrain in heterozygous L444P KI mice at 8 months of age, as detected by western blot, compared to wild-type controls (Yun et al., 2018). Alpha-synuclein also accumulates in the hippocampus of heterozygous KI mice (Li et al., 2019). In contrast, in hippocampal lysates from 3-month-old heterozygous KI mice, no differences were observed in total synuclein, but soluble phosphorylated α-synuclein was reduced in comparison to wild-type controls (Mahoney-Crane et al., 2023). Using immunohistochemistry, an increase in α-synuclein staining was also observed in 24-month-old heterozygous L444P KI mice compared to wild-type controls in the olfactory bulb, hippocampus, secondary motor cortex, striatum, and substantia nigra (Migdalska-Richards et al., 2017). However, no proteinase K–resistant aggregates or changes in S129 phosphorylation of α-synuclein were observed in any of the brain regions analyzed in these aged KI mice.

Other aspects of neural structure and function in the nigrostriatal pathway seem intact based on a comparison of 8-month-old heterozygous L444P KI mice to wild-type controls that were treated with saline via an intraperitoneal injection (Yun et al., 2018). For instance, the number of Nissl-stained or tyrosine hydroxylase (TH)-positive neurons in the substantia nigra pars compacta did not differ between heterozygous L444P KI mice and wild-type controls following saline injection, nor did the density of TH-immunopositive fibers in the striatum.

Levels of dopamine, DOPAC (3,4-dihydroxyphenylacetic acid), HVA (homovanillic acid), or their ratio (to assess dopamine turnover) were also similar in saline-treated heterozygote KI and wild-type mice at 8 months of age (Yun et al., 2018).

Astrocyte activation, as assessed by GFAP (glial fibrillary acidic protein) staining in the substantia nigra, also did not differ between saline-treated heterozygous and wild-type animals at 8 months of age (Yun et al., 2018). In aged, 24-month-old heterozygous L444P KI mice, no differences were found in TH or GFAP staining compared to wild-type control mice (Migdalska-Richards et al., 2017). Staining for Iba1 (ionized calcium binding adaptor molecule 1) to detect microglia was increased in heterozygous KI mice only in the granule cell layer of the olfactory bulb.

Injection of the dopaminergic mitochondrial neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) led to neuropathology in both wild-type and heterozygous KI mice at 8 months of age. The effects were more severe in KI animals, however, including loss of TH- or Nissl-stained neurons, reductions in dopamine, DOPAC, and HVA, as well as increased GFAP staining (Yun et al., 2018). 

Levels of synaptic and neuronal proteins were evaluated in hippocampal lysates of 3-month-old heterozygous KI mice, and presynaptic vGLUT1 was reduced, while Homer1 and Tuj1 levels were unchanged compared with wild-type controls (Mahoney-Crane et al., 2023). In addition, no differences were observed between genotypes in the synaptic vesicle SNARE proteins VAMP2, syntaxin1a, or SNAP-25.

Motor Behavior
The open-field test was conducted in 3-month-old heterozygous KI mice to measure overall motor behavior. Compared with wild-type controls, no differences were observed in average velocity, total distance traveled, or the time spent in the center (Mahoney-Crane et al., 2023). The pole test was used to assess basal ganglia motor activity, and while KI mice had a faster total descent time, there were no differences in turnaround time or the time descending the pole compared to wild-type mice.

In another study, the pole and grip strength tests were used to assess motor behavior in 8-month-old heterozygous L444P KI mice and wild-type controls (following intraperitoneal saline injection), and no differences were found in time to reach the base of the pole. However, KI mice performed worse on both tests compared to wild-type mice following the injection of MPTP, indicating that the L444P KI mutation exacerbates motor pathology (Yun et al., 2018).

At 24 months of age, untreated, heterozygous L444P KI mice still did not exhibit deficits in pole test function compared with wild-type controls (Migdalska-Richards et al., 2017).

Non-Motor Behavior
In 3-month-old heterozygous L444P KI mice, fear conditioning was used to measure associative learning, and impairments were observed in contextual but not cued fear conditioning settings compared with wild-type mice (Mahoney-Crane et al., 2023).

At 24 months of age, heterozygous KI mice did not exhibit deficits in olfaction (as measured by the buried pellet test) or cognition (as measured by the novel object recognition test) compared with wild-type controls (Migdalska-Richards et al., 2017).

Mitochondrial Abnormalities
In the ventral midbrain, mitochondria were smaller, as detected by transmission electron microscopy, and less mitochondrial DNA (CYTB and COX) was detected in heterozygous L444P KI mice at 8 months of age compared to control wild-type mice (Yun et al., 2018). Moreover, mitochondrial function was perturbed, as assessed in primary cortical neurons cultured from heterozygous KI mice: reactive oxygen species generation was increased, mitochondrial complex I enzyme activity was decreased, and oxygen consumption rate was decreased. In another study, primary hippocampal neurons exhibited greater total mitochondrial content and a lower baseline mitochondrial membrane potential, among other functional impairments (Li et al., 2019). In vivo, this study also demonstrated increased mitochondrial content in the hippocampus of heterozygous KI mice compared with wild-type controls based on western blotting and immunostaining of mitochondrial membrane proteins.

Cellular Processes
In primary cortical neurons from heterozygous L444P KI mice, protaeosome activity (20S) was decreased and levels of proteins involved in autophagy (SQSTM1/p62 and LC3A/B-II) were also perturbed (Yun et al., 2018). Mitophagy deficits were also observed in primary hippocampal neurons from heterozygous L444P KI mice on a background of a transgenic mouse model expressing mitochondrial-targeted Keima that allows the measurement of mitophagic flux (Li et al., 2019). This study further confirmed that mitophagy was impaired based on immunostaining of mitochondrial proteins in the hippocampus and western blotting of brain tissue samples from heterozygous L444P KI mice. Additional experiments further identified impairments in basal autophagy and lysosomal degradation in heterozygous L444P KI mice.

In contrast, another study did not find differences in various proteins (as measured by western blotting of brainstem, midbrain, or striatal tissue) involved in autophagy, lysosomal functioning, and endoplasmic reticulum stress in 24-month-old heterozygous L444P KI mice compared to wild-type controls (Migdalska-Richards et al., 2017).

Phenotype Characterization

Q&A with Model Creator

Q&A with Richard Proia

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

This model carries the GBA L444P variant, which represents approximately one-third of all GBA variants linked to Parkinson's disease.

What do you think this model is best used for?

It is best used for studying the effect of GBA L444P modifiers in Parkinson's disease and Gaucher disease.

What caveats are associated with this model?

Generating viable homozygous Gba L444P mice as a model for Gaucher disease is challenging.

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

The Gba L444P mice were derived through an insertional gene targeting procedure, resulting in a partial duplication of the Gba locus. The presence of this partial duplication should be taken into account when devising genotyping procedures.

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References

Paper Citations

1. Liu Y, Suzuki K, Reed JD, Grinberg A, Westphal H, Hoffmann A, Döring T, Sandhoff K, Proia RL. Mice with type 2 and 3 Gaucher disease point mutations generated by a single insertion mutagenesis procedure. Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2503-8. 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. Mizukami H, Mi Y, Wada R, Kono M, Yamashita T, Liu Y, Werth N, Sandhoff R, Sandhoff K, Proia RL. Systemic inflammation in glucocerebrosidase-deficient mice with minimal glucosylceramide storage. J Clin Invest. 2002 May;109(9):1215-21. PubMed.
4. Yun SP, Kim D, Kim S, Kim S, Karuppagounder SS, Kwon SH, Lee S, Kam TI, Lee S, Ham S, Park JH, Dawson VL, Dawson TM, Lee Y, Ko HS. α-Synuclein accumulation and GBA deficiency due to L444P GBA mutation contributes to MPTP-induced parkinsonism. Mol Neurodegener. 2018 Jan 8;13(1):1. PubMed.
5. Li H, Ham A, Ma TC, Kuo SH, Kanter E, Kim D, Ko HS, Quan Y, Sardi SP, Li A, Arancio O, Kang UJ, Sulzer D, Tang G. Mitochondrial dysfunction and mitophagy defect triggered by heterozygous GBA mutations. Autophagy. 2019 Jan;15(1):113-130. Epub 2018 Oct 12 PubMed.
6. Migdalska-Richards A, Wegrzynowicz M, Rusconi R, Deangeli G, Di Monte DA, Spillantini MG, Schapira AH. The L444P Gba1 mutation enhances alpha-synuclein induced loss of nigral dopaminergic neurons in mice. Brain. 2017 Oct 1;140(10):2706-2721. PubMed.
7. Mahoney-Crane CL, Viswanathan M, Russell D, Curtiss RA, Freire J, Bobba SS, Coyle SD, Kandebo M, Yao L, Wan BL, Hatcher NG, Smith SM, Marcus JN, Volpicelli-Daley LA. Neuronopathic GBA1L444P Mutation Accelerates Glucosylsphingosine Levels and Formation of Hippocampal Alpha-Synuclein Inclusions. J Neurosci. 2023 Jan 18;43(3):501-521. Epub 2022 Dec 7 PubMed.

External Citations

1. Mutant Mouse Regional Resource Centres (MMRRC), Stock# 000117-UNC

Further Reading