Species: Mouse
Genes: Atp13a2
Modification: Atp13a2: Knock-Out
Disease Relevance: Parkinson's Disease
Strain Name: Atp13a2^tm1.1Wtd/J
Genetic Background: Mixed C57BL6/129
Availability: Available through The Jackson Laboratory, Stock# 028387, Cryopreserved.

Summary

This transgenic mouse model expresses a floxed ATPase type 13A2 (Atp13a2) gene, with loxP sites flanking exons 2 to 3 (The Jackson Laboratory). The ATP13A2 protein is a transmembrane lysosomal ATPase that serves to maintain intracellular cation homeostasis. When crossed to a strain expressing Cre recombinase, the Cre will excise a portion of the Atp13a2 gene, leading to the generation of a premature stop codon in exon 4 that abolishes gene expression (Kett et al., 2015).

This mouse model can be crossed with a germline-expressing (Rosa-Cre) “Cre-deletor” mouse line to first generate heterozygote knock-out mice, and later full knock-out mice, referred to here as Atp13a2 null mice (Kett et al., 2015). Germline Atp13a2 null mice are viable and fertile, and only develop movement deficits later in life. Atp13a2-flox mice can also be crossed to other Cre lines, or have recombination induced from viral (AAV) delivery of Cre recombinase to specific brain regions, which may obviate potential compensatory pathways associated with germline deletion.

Motor Behavior
In the tail suspension test, germline Atp13a2 null mice demonstrated abnormal clasping positions in comparison to wild-type control mice at 18 months of age, while this abnormal behavior was not observed at 12 months of age (Kett et al., 2015).

On the open-field test, Atp13a2 null mice had decreased spontaneous horizontal movement, but did not exhibit differences in number of rears, based on testing conducted at 9, 12, 15, and 18 months of age (Kett et al., 2015).

Motor function testing on the balance beam and Rotarod did not reveal any differences between wild-type and Atp13a2 null mice at 9, 12, 15, or 18 months of age (Kett et al., 2015).

Neuropathology
No differences were observed between 18-month-old Atp13a2 null and wild-type mice in dopaminergic (by tyrosine hydroxylase [TH]-staining) or Nissl-stained cell numbers in the substantia nigra pars compacta (SNpc; Kett et al., 2015). TH levels (by western blot) also did not differ by genotype in the striatum or midbrain in 18-month-old mice.

In contrast to germline null mice, neuropathology was observed when Atp13a2 KO was induced in adulthood in the ventral midbrain of young adult (2- to 6-month-old) Atp13a2-flox mice via unilateral delivery of an AAV-Cre into the SNpc (Erb et al., 2024). Namely, a progressive loss of striatal dopaminergic nerve terminals was observed 3 and 10 months after delivery of the AAV-Cre versus the contralateral side or versus control mice that received AAV-GFP. Moreover, 10 months after delivery of the AAV-Cre, dopaminergic neuronal degeneration was also observed in the SN compared to the contralateral side. Since parvalbumin- and GAD67-positive neuron numbers did not differ between the ipsilateral and contralateral sides of the SN, it appears that dopaminergic neurons are particularly vulnerable to Atp13a2 KO.

In 18-month-old germline Atp13a2 null mice, gliosis (as measured by GFAP immunoreactivity and western blot) was increased compared to wild-type controls across brain regions, including in the cortex, striatum, hippocampus, cerebellum, thalamus, and midbrain (Kett et al., 2015). This gliosis worsened with age, and was already present at 1 month of age in the cortex. However, in midbrain tissue, TH and GFAP staining did not colocalize. In contrast to germline Atp13a2 KO mice, in mice with selective Atp13a2 KO via AAV-Cre delivery in adulthood in the ventral midbrain, reactive astrogliosis and microglial activation are also present in the SN, but only transiently at 3 months after viral injection, and not at 10 months (Erb et al., 2024).

In 18-month-old Atp13a2 null mice, α-synuclein pathology was absent in the cortex and midbrain (Kett et al., 2015). There was no evidence of α-synuclein-positive aggregation or changes in α-synuclein levels or the phosphorylation of synuclein at serine 129 compared to wild-type mice. There was also no difference between null and wild-type mice in the amount of endogenous α-synuclein present in lysosomes. Alpha-synuclein pathology was also absent in the SN of mice with selective Atp13a2 KO that was induced by AAV-Cre delivery in adulthood into the ventral midbrain (Erb et al., 2024).

Impairments in Cellular Homeostasis
Lipofuscin deposits were detected using electron microscopy in the cortex, cerebellum, hippocampus, and striatum of 12-month-old germline Atp13a2 null mice, and these deposits were closely associated with lipid droplets (Kett et al., 2015). Compared to wild-type mice, Atp13a2 null animals had larger and more numerous lipid droplets. These findings were age-dependent, as there were no genotype differences in lipid droplet or lipofuscin presence at 1 month of age. In addition, bis(monoacylglycero)phosphate (BMP), but no other lipid species, was present at increased levels in the cortex of 18-month-old Atp13a2 null versus wild-type mice; BMP is found exclusively in late endosomes and lysosomes.

Lysosomal markers LAMP1 and LAMP2 were elevated in the cortex and cerebellum, as detected by immunostaining and western blot in 18-month-old Atp13a2 null mice versus wild-type controls (Kett et al., 2015). A time-course experiment revealed that LAMP1 was elevated in the cortex of null mice starting at 6 months of age. Moreover, LAMP2 immunoreactivity was increased in TH-positive neurons of the SNpC in 18-month-old null mice versus wild-type controls. In mice with selective Atp13a2 KO via AAV-Cre delivery in adulthood into the ventral midbrain, LAMP2-positive vesicles accumulated in the SN (Erb et al., 2024).

Ubiquitin-positive aggregates were detected within neurons of the cortex, hippocampus, and SNpc of Atp13a2 null mice, but not in astrocytes, microglia, nor SNpc dopaminergic cells (Kett et al., 2015).

Defects in autophagy function were also detected in Atp13a2 null mice. Levels of p62 (an autophagy substrate) were elevated in the striatum, cerebellum, and midbrain, but not in the cortex or hippocampus, of 18-month-old null mice compared to controls, as assessed by western blot analysis (Kett et al., 2015). Nonetheless, p62-positive protein aggregates were detected in the cortex with immunohistochemistry. p62-positive inclusions and nuclear localization of transcription factor E3 (TFE3) were also observed in the SN of mice with selective Atp13a2 KO in the ventral midbrain via AAV-Cre delivery in adulthood, pointing to lysosomal activity deficits and stress (Erb et al., 2024).

LC3-II levels, which are a proxy for autophagy induction, did not differ by genotype, as assessed by western blotting in 18-month-old Atp13a2 null mice (Kett et al., 2015). However, endolysosomal trafficking was perturbed, as detected by deficits in cathepsin D processing in the cortex and cerebellum of older (18-month-old), but not younger (9-month-old), null mice compared to wild-type controls. Cathepsin B and cathepsin L maturation were not affected, suggesting a selective impairment of secondary lysosomes (i.e., autolysosomes). Moreover, cathepsin D was reduced in both lysosomes involved in macroautophagy and those involved in chaperone-mediated autophagy in 18-month-old null mice, and data further indicated that this reduction was due to defects in trafficking the protein to lysosomes.

No differences in the structure of the trans-Golgi network were observed between 12-month-old Atp13a2 null and wild-type mice, using electron microscopy (Kett et al., 2015).

Modification Details

In this floxed mouse model, one loxP site is present upstream exon 2 and the second loxP site is present downstream exon 3 of the Atp13a2 gene (The Jackson Laboratory). This mouse was generated using homologous recombination of a targeting vector electroporated into B6/129 embryonic stem cells, with the aid of an FRT-flanked neomycin resistance cassette that was subsequently removed.

Phenotype Characterization

COMMENTS / QUESTIONS

No Available Comments

Make a comment or submit a question

To make a comment you must login or register.

References

Paper Citations

1. Kett LR, Stiller B, Bernath MM, Tasset I, Blesa J, Jackson-Lewis V, Chan RB, Zhou B, Di Paolo G, Przedborski S, Cuervo AM, Dauer WT. α-Synuclein-independent histopathological and motor deficits in mice lacking the endolysosomal Parkinsonism protein Atp13a2. J Neurosci. 2015 Apr 8;35(14):5724-42. PubMed.
2. Erb ML, Sipple K, Levine N, Chen X, Moore DJ. Adult-onset deletion of ATP13A2 in mice induces progressive nigrostriatal pathway dopaminergic degeneration and lysosomal abnormalities. NPJ Parkinsons Dis. 2024 Jul 20;10(1):133. PubMed.

External Citations

1. The Jackson Laboratory
2. The Jackson Laboratory, Stock# 028387

Further Reading

No Available Further Reading