Species: Mouse
Genes: Vps35
Modification: Vps35: Knock-In
Disease Relevance: Parkinson's Disease
Strain Name: B6.Cg-Vps35^tm1.1Mjff/J
Genetic Background: C57BL/6J
Availability: Available through The Jackson Laboratory, Stock# 023409, Cryopreserved.

Modification Details:

This constitutive KI mouse model expresses the g.85,263,520G>A missense mutation in exon 15 of Vps35, which leads to the mutated (D620N) protein (Cataldi et al., 2018). The model was generated to drive mutant protein expression at physiological levels and avoid the confounds of random insertion. This KI model was created and maintained in C57Bl/6J mice (The Jackson Laboratory).

Summary

The VPS35 p.D620N missense mutation (causing an aspartate to asparagine substitution) has been linked to dominantly inherited, late-onset parkinsonism (Vilariño-Güell et al., 2011; Zimprich et al., 2011, Jul 2011 news). The Vps35 gene encodes vacuolar protein sorting 35 (VPS35), a component of the “retromer” that can enhance or restrict intracellular pathogen growth as part of the innate immune response (Elwell and Engel, 2018). VPS35 is considered a core part of the cargo-recognition machinery, necessary for endosomal membrane-associated protein trafficking (Seaman, 2021). This constitutive knock-in (KI) mouse model was generated by introducing a G to A nucleotide mutation in exon 15 of Vps35 (p.D620N) of C57Bl/6J mice (Cataldi et al., 2018). Vps35 p.D620N induces a gain-of-function to constitutively activate LRRK2 kinase to regulate vesicular trafficking (Mir et al., 2018; Bu et al., 2023).

Vps35 p.D620N KI (VKI) mice exhibit profound alterations in the dopaminergic system, but no nigral neuronal loss at younger ages. Homozygous VKI mice are grossly normal, viable, breed well, and do not show evidence of overt distress or locomotor dysfunction (Cataldi et al., 2018; Niu et al., 2021). They also exhibit normal survival up to at least 24 months of age (Chen et al., 2019). Although body weight does not differ between VKI and wild-type mice at younger ages (3-10 months), decreased body weight is observed in males, but not females, at older ages (13-15 months) (Cataldi et al., 2018; Niu et al., 2021).

Protein and mRNA levels of VPS35 in cerebellar tissue are unchanged in heterozygous and homozygous VKI mice versus wild-type mice (Cataldi et al., 2018; Chen et al., 2019). Overexpression of Vps35 is neurotoxic (Munsie et al., 2015), but in these animals, the 1:1:1 stoichiometry between VPS26:VPS29:VPS35 is preserved.

The data described below are reflective of homozygous VKI mice, unless otherwise specified. Nevertheless, Vps35 p.D620N manifests as an autosomal dominant disease (heterozygous trait) and no human homozygotes have ever been observed (Matt Farrer, personal communication, March 2024).

Motor Behavior | Non-motor Behavior | Neuropathology | Dopamine Release | Other Cellular Processes (Autophagy, Mitochondrial Function) | Intracellular Trafficking | Related Strains

Motor Behavior

On the open-field test, 3-month-old VKI mice performed similar to wild-type littermates, showing comparable measurements for distance traveled, moving time, and center path ratio (Cataldi et al., 2018). Slightly older—6- to 10-month-old—VKI mice also did not show perturbations on the open-field test, but at 14 months of age reductions in speed and distance travelled were observed compared to wild-type control mice (Niu et al., 2021). Rearing activity, measured in the cylinder test, and accelerating Rotarod performances were similar between genotypes at 3 months of age (Cataldi et al., 2018). Rotarod and grip strength did not differ between genotypes from 6 to 14 months of age (Niu et al., 2021). The beam walking test was used to assess fine motor coordination and balance, and 14-month-old VKI mice took longer than wild-type mice to cross the beam, while 6- and 10-month-old mice did not differ on this measure. In yet another study, VKI mice from age 3 months to 12 to 13 months also did not exhibit significant differences from wild-type mice on the open-field test, Rotarod, or gait analysis (Chen et al., 2019).

VKI mice show a significant increase in amphetamine-induced hyperlocomotion in an open field chamber compared to Vps35 haploinsufficient mice, which is abolished by a 7-day treatment with a LRRK2 kinase inhibitor (Bu et al., 2023).

Non-motor Behavior

No differences were observed between wild-type and VKI mice on the buried pellet test to measure olfactory function (Niu et al., 2021). In addition, no differences between genotypes were observed in stool frequency or stool water content, suggesting gastrointestinal function was intact. These assessments were conducted on mice aged 6, 10, and 14 months. In unpublished findings, deficits have been observed, however, in elevated plus maze performance starting at 3 months of age that may reflect changes in mood (e.g., anxiety, apathy) and/or cognition (Matt Farrer, personal communication, March 2024).

Neuropathology

Tyrosine hydroxylase (TH) staining of nigral terminals in the dorsolateral striatum did not differ (intensity or number) between VKI and wild-type mice at 3 months of age (Cataldi et al., 2018). Also in 6- and 10-month-old VKI mice, no TH staining–based differences were observed in the fibers of the striatum or the neurons of the substantia nigra pars compacta (SNpc) compared with wild-type mice (Niu et al., 2021). TH was also measured in striatal tissue by western blotting, and similar findings were found between VKI and wild-type mice at 3 months of age (Cataldi et al., 2018). These data suggest there is no dopaminergic neurodegeneration in younger VKI mice. In contrast, in older mice (15- to 16-month-olds), loss of TH-positive neurons was observed in the SNpc and a loss of TH-positive nerve terminals was observed in the striatum (Niu et al., 2021). In line with the above studies, another set of experiments revealed that the number of neurons in the SNpc did not differ between VKI and wild-type mice at 3 months of age, but at 13 months of age, there was a significant loss of TH-positive and Nissl-stained neurons, suggesting age-related neurodegenerative processes (Chen et al., 2019). However, in contrast to one finding above, older mice (13 months of age) in this study exhibited a similar density of TH-positive striatal nerve terminals, and dopaminergic neuron morphology was grossly normal, as assessed based on the soma and neurite projections (Chen et al., 2019). Closer examination of axonal degeneration at 13 months of age using immunostaining for amyloid precursor protein and staining with Gallyas silver, however, uncovered significant neurite degeneration, not only in the striatum and substantia nigra, but also more widely across the brain, in the hippocampus, cerebellum, and brainstem.

SNpc dopaminergic neuron function was assessed electrophysiologically in 3-month-old heterozygous VKI mice, and no differences were observed versus wild-type mice in basal pace-making activity (Bu et al., 2023).

Levels of proteins involved in corticostriatal synaptic transmission (GluA1 subunit of the AMPA receptor, D2-type dopamine receptor [D2R], and NMDA receptor) were similar in striatal samples in 3-month-old VKI mice versus wild-type mice (Kadgien et al., 2021). Moreover, association of these proteins with VPS35 was also similar in the same samples, based on co-immunoprecipitation experiments.

With regard to α-synuclein, no differences were observed in puncta density or distribution from the SNpc of VKI mice versus wild-type mice at 3 months of age. Western blotting of striatal tissue also showed similar levels of α-synuclein between VKI and wild-type mice (Cataldi et al., 2018). No pathological α-synuclein observations were seen throughout the brain in VKI mice aged 13 months (Chen et al., 2019). At 15 to 16 months of age, however, increased somatic α-synuclein immunoreactivity was found in the SNpc of VKI versus wild-type mice (Niu et al., 2021). Moreover, in the ventral midbrain of older (15- to 16-month-old) VKI mice, increases in α-synuclein oligomers and aggregated α-synuclein were observed in western blots.

Tau neuropathology has also been observed in older (13-month-old) VKI mice using AT8 and MC-1 antibodies (Chen et al., 2019). This comprised abnormal phosphorylation, conformation, and localization of tau, as detected by immunostaining in the cortex, hippocampus, cerebellum, brainstem, and ventral midbrain, and to a lesser extent in the striatum. Tau neuropathology was further confirmed in TH-positive cells within the substantia nigra, and this was present even as early as at 3 months of age. However, there was little evidence for tau-positive neurofibrillary tangles, inclusions, or neuritic pathology containing fibrillar forms of tau (Chen et al., 2019). In contrast, in another study that also relied on AT8 and MC-1 immunostaining, no tau pathology was observed in 15- to 16-month-old mice in the SNpc or cortex (Niu et al., 2021). Authors note that these contradictory findings may be due to slight variations in the animals’ genetic backgrounds, rederivation procedures, or experimental settings.

In 15- to 16-month-old VKI mice, increased GFAP immunostaining (a marker of astrogliosis) was observed in the SNpc, but not in the striatum (Niu et al., 2021). Western blotting, however, did reveal an increase in GFAP in the striatum, as well as in the ventral midbrain. Younger (6-, 10-, and 13-month-old) VKI mice did not exhibit astrogliosis in the SNpc or striatum (Niu et al., 2021; Chen et al., 2019).

No differences in microgliosis were observed between VKI and wild-type mice in the SNpc or the striatum at any age assessed (6, 10, 13, and 15-16 months of age), as detected by Iba-1 immunostaining (Niu et al., 2021; Chen et al., 2019).

Dopamine Release

Nigrostriatal dopamine release, as detected in the dorsolateral striatum from acute brain slices, was measured in 3-month-old mice using fast scan cyclic voltammetry (FSCV; Cataldi et al., 2018). VKI mice exhibited increased evoked dopamine release, and slower dopamine re-uptake. Another study supported these findings, also using FSCV, in acute striatal slices: VKI mice exhibited enhanced peak amplitude in dopamine release and prolonged reuptake kinetics (Bu et al., 2023).

Dopamine neurotransmission was further measured by in vivo microdialysis (Cataldi et al., 2018). Basal levels of dopamine and its metabolites in the dorsolateral striatum did not differ from wild-type mice at 3 months of age, but the ratio of metabolites (3,4-dihydroxyphenylacetic acid [DOPAC] plus homovanillic acid [HVA]/dopamine) was higher in the dialysate, pointing to increased dopamine turnover in VKI mice. Nevertheless, levels of dopamine and its metabolites, as measured by HPLC of homogenized striatal tissue, were similar between VKI and wild-type mice. This was true in another study as well, in animals up to 14 to 15 months of age and in other tissues, namely, the prefrontal cortex and olfactory bulb (Chen et al., 2019). In contrast, in even older mice, aged 15 to 16 months, levels of dopamine in the striatum were reduced in VKI compared to wild-type mice, as measured by HPLC (Niu et al., 2021). The levels of the dopamine metabolites DOPAC and HVA, however, did not differ significantly.

Vesicular monoamine transporter 2 (VMAT2) protein levels were increased and dopamine transporter (DAT) protein levels were decreased in striatal brain of 3-month-old VKI mice (as measured by western blot), indicating a perturbation of synaptic proteins needed for dopamine packaging and reuptake (Cataldi et al., 2018; Bu et al., 2023). Decreases in DAT were confirmed by immunohistochemistry, although those for VMAT2 (using H-V008, Phoenix Pharmaceuticals) were not. However, VMAT2 staining in brain sections is notoriously unreliable for many antibody reagents (Matt Farrer, personal communication, March 2024). Nonetheless, synaptic connections appear largely intact, as levels of post-synaptic scaffold protein 95 were normal.

LRRK2 kinase inhibitor treatment normalized DAT levels and FSCV phenotypes (e.g., dopamine reuptake) in acute striatal slices from VKI mice, suggesting that the D620N mutation is gain-of-function for LRRK2 kinase activity (Bu et al., 2023).

Other Cellular Processes (Autophagy, Mitochondrial Function)

Autophagy marker Lamp2a, but not Cathepsin D, was decreased (as measured by western blot) in the ventral midbrain (Niu et al., 2021). In addition, in the SNpc of 14-month-old VKI mice, but not in wild-type mice, autophagic vacuoles were found in dystrophic myelinated axons by electron microscopy. Lipofuscin and lysosome densities were also elevated in the SNpc of 14-month-old VKI mice versus wild-type mice.

Mitochondrial structure, as assessed by electron microscopy, was perturbed in VKI mice at 14 months, but not at 3 months, of age (Niu et al., 2021). Specifically, mitochondria were shorter, rounder, and smaller than those in wild-type mice. Mitochondrial fragmentation was also indicated by reductions in length, size, and aspect ratio.

Mitochondrial function was also impacted in older (15-month-old) mice: the oxygen consumption rate was reduced in VKI compared to control mice (Niu et al., 2021). In addition, expression of DLP1, which is involved in mitochondrial fission, was perturbed in VKI mice compared with control mice at 15 months of age. Although overall DLP1 levels did not change in the ventral midbrain or striatum, co-immunoprecipitation revealed an increased DLP1-VPS35 interaction in the whole brain, and mitochondrial fractions from the ventral midbrain exhibited decreased oligomeric DLP1 complexes and increased monomeric DLP1.

Intracellular Trafficking

Phosphorylation of key proteins involved in endosomal, lysosomal, and Golgi trafficking were examined in striatal tissue of 3-month-old VKI mice (Bu et al., 2023). Western blot analysis revealed increased phosphorylation of Rab10 (pThr73; also supported by Kadgien et al., 2021), Rab12 (pSer106), and Rab29 (pThr71). Other lysosomal markers were also perturbed in striatal samples from VKI mice: lysosomal associated protein-1 (LAMP1) was increased and Rab Interacting Lysosomal Protein Like 1 (RILPL1) was decreased. However, levels of the classic retromer cargo cation-independent mannose-phosphate receptor (CI-MPR) were unchanged in VKI mice compared with wild-type mice.

Subunit assembly with other retromer complex members (e.g., Vps26 and Vps29) was not affected in the brains of 3-month-old VKI mice, but interaction was reduced with the Wiskott–Aldrich syndrome protein and SCAR homolog (WASH) complex member family with sequence similarity 21 (FAM21; Kadgien et al., 2021; Chen et al., 2019).

Related Strains

floxΔneo Vps35 (B6.Cg-Vps35tm1.2Mjff/J). This is the floxed, inducible version of the constitutive knock-in B6.Cg-Vps35tm1.1Mjff/J strain described above. This strain can be crossed with a Cre recombinase–expressing mouse line to generate conditional expression of the Vps35 p.D620N mutation. However, this floxed allele is silenced, and as homozygous knock-out of Vps35 is embryonic lethal, doubly-heterozygous matings will not breed with expected Mendelian ratios (Matt Farrer, personal communication, March 2024). Available through The Jackson Laboratory, Stock# 021807, Available cryopreserved and as a frozen embryo.

Phenotype Characterization

Q&A with Model Creator

Expert/Creator Q&A with Matt Farrer

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

The retromer core trio of VPS26:VPS29:VPS35 must be maintained in a 1:1:1 stoichiometry. This is critically important in neurons, as overexpression of VPS35 is more neurotoxic than the point mutation alone (i.e., resulting in ~50% of synapse loss in primary cortical cultures; Munsie et al., 2015). Hence, this Vps35 p.D620N knock-in model is physiologically faithful to the molecular dysfunction observed in the human condition.

What do you think this model is best used for?

It is best used for molecular studies of VPS35 p.D620N mutant gene dysfunction, perhaps the most prominent of which is LRRK2 kinase activation.  Nevertheless, most aspects of the pathophysiology of Parkinson’s disease can be explored in this model, as the human phenotype of VPS35 p.D620N largely recapitulates typical late-onset Parkinson’s disease (Struhal et al., 2014). The model may be particularly well suited to test “‘on-" and ‘“off-target” effects of LRRK2 kinase inhibitors to assess their optimal dosing and efficacy.  

What caveats are associated with this model?

No caveats but some cautions:
1) As this is a knock-in model predicated by a single nucleotide change in the mouse genome, it is important to routinely backcross these animals to new C57BL/6J mice when breeding from The Jackson Laboratory (at least every year). This will ensure the strain background remains consistent with the founders and with other laboratories.
2) As retromer and LRRK2 are implicated in intracellular innate immunity the animals housing conditions should be well- documented, and wild-type and mutant animals should be well-matched, whether raised in germ-free, barrier microisolator caging, or open caging.
3) The mice, especially males, put on less weight with age, and their body mass is an important consideration in behavioral testing.

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

VPS35 p.D620N is one of the most penetrant monogenic causes of mid/late-onset parkinsonism.  However, these mice should be considered a model of gene dysfunction, not a model of Parkinson’s disease.  If relevant to the human condition, any genotype-specific phenotypes will be subtle/chronic, because they are physiologic. Genotype-specific differences may need to be evoked with secondary challenges (e.g., with stimulants such as amphetamine; see Bu et al., 2023), environmental toxins such as lipopolysaccharide, or genetic crosses (e.g., for aging, to the PolG mutator mice).

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References

News Citations

1. Sorting Out Parkinson’s: Exome Sequencing Points to Recycling Defect 26 Jul 2011

Paper Citations

1. Vilariño-Güell C, Wider C, Ross OA, Dachsel JC, Kachergus JM, Lincoln SJ, Soto-Ortolaza AI, Cobb SA, Wilhoite GJ, Bacon JA, Behrouz B, Melrose HL, Hentati E, Puschmann A, Evans DM, Conibear E, Wasserman WW, Aasly JO, Burkhard PR, Djaldetti R, Ghika J, Hentati F, Krygowska-Wajs A, Lynch T, Melamed E, Rajput A, Rajput AH, Solida A, Wu RM, Uitti RJ, Wszolek ZK, Vingerhoets F, Farrer MJ. VPS35 mutations in Parkinson disease. Am J Hum Genet. 2011 Jul 15;89(1):162-7. PubMed.
2. Zimprich A, Benet-Pagès A, Struhal W, Graf E, Eck SH, Offman MN, Haubenberger D, Spielberger S, Schulte EC, Lichtner P, Rossle SC, Klopp N, Wolf E, Seppi K, Pirker W, Presslauer S, Mollenhauer B, Katzenschlager R, Foki T, Hotzy C, Reinthaler E, Harutyunyan A, Kralovics R, Peters A, Zimprich F, Brücke T, Poewe W, Auff E, Trenkwalder C, Rost B, Ransmayr G, Winkelmann J, Meitinger T, Strom TM. A mutation in VPS35, encoding a subunit of the retromer complex, causes late-onset Parkinson disease. Am J Hum Genet. 2011 Jul 15;89(1):168-75. PubMed.
3. Elwell C, Engel J. Emerging Role of Retromer in Modulating Pathogen Growth. Trends Microbiol. 2018 Sep;26(9):769-780. Epub 2018 Apr 24 PubMed.
4. Seaman MN. The Retromer Complex: From Genesis to Revelations. Trends Biochem Sci. 2021 Jul;46(7):608-620. Epub 2021 Jan 29 PubMed.
5. Cataldi S, Follett J, Fox JD, Tatarnikov I, Kadgien C, Gustavsson EK, Khinda J, Milnerwood AJ, Farrer MJ. Altered dopamine release and monoamine transporters in Vps35 p.D620N knock-in mice. NPJ Parkinsons Dis. 2018;4:27. Epub 2018 Aug 21 PubMed.
6. Mir R, Tonelli F, Lis P, Macartney T, Polinski NK, Martinez TN, Chou MY, Howden AJ, König T, Hotzy C, Milenkovic I, Brücke T, Zimprich A, Sammler E, Alessi DR. The Parkinson's disease VPS35[D620N] mutation enhances LRRK2-mediated Rab protein phosphorylation in mouse and human. Biochem J. 2018 Jun 6;475(11):1861-1883. PubMed.
7. Bu M, Follett J, Deng I, Tatarnikov I, Wall S, Guenther D, Maczis M, Wimsatt G, Milnerwood A, Moehle MS, Khoshbouei H, Farrer MJ. Inhibition of LRRK2 kinase activity rescues deficits in striatal dopamine physiology in VPS35 p.D620N knock-in mice. NPJ Parkinsons Dis. 2023 Dec 18;9(1):167. PubMed.
8. Niu M, Zhao F, Bondelid K, Siedlak SL, Torres S, Fujioka H, Wang W, Liu J, Zhu X. VPS35 D620N knockin mice recapitulate cardinal features of Parkinson's disease. Aging Cell. 2021 May;20(5):e13347. Epub 2021 Mar 21 PubMed.
9. Chen X, Kordich JK, Williams ET, Levine N, Cole-Strauss A, Marshall L, Labrie V, Ma J, Lipton JW, Moore DJ. Parkinson's disease-linked D620N VPS35 knockin mice manifest tau neuropathology and dopaminergic neurodegeneration. Proc Natl Acad Sci U S A. 2019 Mar 19;116(12):5765-5774. Epub 2019 Mar 6 PubMed. Correction.
10. Munsie LN, Milnerwood AJ, Seibler P, Beccano-Kelly DA, Tatarnikov I, Khinda J, Volta M, Kadgien C, Cao LP, Tapia L, Klein C, Farrer MJ. Retromer-dependent neurotransmitter receptor trafficking to synapses is altered by the Parkinson's disease VPS35 mutation p.D620N. Hum Mol Genet. 2015 Mar 15;24(6):1691-703. Epub 2014 Nov 21 PubMed.
11. Kadgien CA, Kamesh A, Milnerwood AJ. Endosomal traffic and glutamate synapse activity are increased in VPS35 D620N mutant knock-in mouse neurons, and resistant to LRRK2 kinase inhibition. Mol Brain. 2021 Sep 16;14(1):143. PubMed.
12. Struhal W, Presslauer S, Spielberger S, Zimprich A, Auff E, Bruecke T, Poewe W, Ransmayr G, Austrian VPS-35 Investigators Team. VPS35 Parkinson's disease phenotype resembles the sporadic disease. J Neural Transm (Vienna). 2014 Jul;121(7):755-9. Epub 2014 Feb 21 PubMed.

External Citations

1. The Jackson Laboratory, Stock# 021807
2. The Jackson Laboratory, Stock# 023409
3. The Jackson Laboratory

Further Reading