Impaired mitochondrial accumulation and Lewy pathology in neuron-specific FBXO7-deficient mice

Parkinson’s disease, the second most common neurodegenerative disorder, is characterized by the loss of nigrostriatal dopamine neurons. FBXO7 (F-box protein only 7) (PARK15) mutations cause early-onset Parkinson’s disease. FBXO7 is a subunit of the SCF (SKP1/cullin-1/F-box protein) E3 ubiquitin ligase complex, but its neuronal relevance and function have not been elucidated. To determine its function in neurons, we generated neuronal cell-specific FBXO7 conditional knockout mice (FBXO7flox/flox: Nestin-Cre) by crossing previously characterized FBXO7 floxed mice (FBXO7flox/flox) with Nestin-Cre mice (Nestin-Cre). The resultant Fbxo7flox/flox: Nestin-Cre mice showed juvenile motor dysfunction, including hindlimb defects and decreased numbers of dopaminergic neurons. Fragmented mitochondria were observed in dopaminergic and cortical neurons. Furthermore, p62- and synuclein-positive Lewy body-like aggregates were identified in neurons. Our findings highlight the unexpected role of the homeostatic level of p62, which is regulated by a non-autophagic system that includes the ubiquitin–proteasome system, in controlling intracellular inclusion body formation. These data indicate that the pathologic processes associated with the proteolytic and mitochondrial degradation systems play a crucial role in the pathogenesis of PD.


Main text
Autosomal recessive mutations in the FBXO7 (PARK15) gene are involved in a juvenile form of Parkinsonism with heterogenic phenotypes characterized by either a classic Parkinson's disease (PD) phenotype, pyramidal tract signs only, or by a combination of Parkinsonism and pyramidal signs [1,2]. FBXO7 is expressed in various types of tissues, including the gray and white matter of the brain [3].
To determine the function of FBXO7 in vivo, we generated FBXO7 flox/flox mice. A targeting vector was constructed using 5.0-and 3-kb DNA fragments as the 5′ and 3′ homologous sequences, respectively (Fig. 1A). The linearized targeting vector was transfected into C57BL/6 embryonic stem (ES) cells. Selected clones were screened for homologous recombination by Southern blotting. Using the 5′ external probe and a probe specific for the neo sequence, we confirmed that the clones carried the desired homologous recombination. ES cells derived from these clones were injected into C57BL/6 embryos. Chimeric offspring were crossed with C57BL/6 mice to obtain germline transmission, which was confirmed by Southern blot analysis with the 5′ (Fig. 1B upper panel) and 3′ (Fig. 1B lower panel) probes. Heterozygous mice were then interbred to obtain homozygous knockout and wild-type control mice. All animals were kept in a pathogen-and odor-free environment, which was maintained under a 12-h light/dark cycle at ambient temperature. Procedures were approved by the Animal Experimental Committee of the Juntendo University Graduate School Open Access *Correspondence: nhattori@juntendo.ac.jp of Medicine and performed in accordance with the guidelines of the National Institutes of Health and the Juntendo University Graduate School of Medicine. Next, we generated neuronal cell-specific FBXO7 flox/flox : Nestin-Cre mice by crossing the previously characterized FBXO7 flox/ flox mice with FBXO7 flox/flox : Nestin-Cre mice harboring the Cre recombinase-coding sequence downstream of a characterized fragment of the Nestin promoter, and confirmed that FBXO7 protein levels were decreased in the whole brain of Cre-expressing mice (Fig. 1C).
FBOX7 flox/flox : Nestin-Cre mice were viable at birth and indistinguishable in appearance from their littermates, but experienced gradually increasing weight loss (Fig. 1D, E) and had a markedly lower survival rate after birth (Fig. 1F). FBXO7 flox/flox : Nestin-Cre mice began to show impairment in motor coordination tasks and motor behavioral deficits, as determined by the footprint test (Fig. 1G) and runway test (Fig. 1H). We first conducted the footprint test, because a short stride is a characteristic of PD, including in patients with PARK15 mutations. FBXO7 flox/flox : Nestin-Cre mice had a shorter stride than FBXO7 flox/flox mice (Fig. 1G). Regarding the runway test, while FBXO7 flox/flox mice exhibited well-coordinated movement and almost no slips of the forepaw or Fig. 1 A Schematic representation of the targeting vector and the targeted allele of the FBXO7 gene. The 3.2-kb region of the mouse FBXO7 gene, including exons 3-4, was followed by an FRT-flanked PGK-neo expression cassette in the opposite transcriptional orientation. In the targeting construct, a 5.0-kb 5′ fragment and a 3.0-kb 3′ fragment were used as the long and short homologous arms, respectively. B Southern blot analysis of genomic DNA from ES cells that had undergone homologous recombination. Genomic DNA was digested with EcoRV and hybridized with the 5′ probe or 3′ probe. Upper panel: Bands detected by the 5′ probe. Lower panel: Bands detected by the 3′ probe. C Immunoblot of anti-FBXO7 antibody (Millipore #ABN1038) and actin (Millipore #MAB1501). Lanes 1-3: whole-brain tissues of 3-week-old FBXO7 flox/flox mice; lanes 4-6: whole-brain tissues of 3-week-old FBXO7 flox/flox : Nestin-Cre mice. The genotypes of Nestin-Cre mice were determined by PCR using two primers: 5′-TTT GCC TGC ATT ACC GGT CGA TGC AAC-3′ and 5′-TGC CCC TGT TTC ACT ATC CAG GTT ACG GA-3′; these permitted the detection of the 1000-bp Nestin-Cre-targeted allele (lower panel). D 3-week-old FBXO7 flox/flox mice (left) and 3-week-old FBXO7 flox/flox : Nestin-Cre mice (right). Scale bars: 1 cm. E Body weight of mice 2 or 3 weeks of age (2-week-old FBXO7 flox/flox mice and FBXO7 flox/flox : Nestin-Cre mice, n = 10; 3-week-old FBXO7 flox/flox mice and FBXO7 flox/flox : Nestin-Cre mice, n = 10). Data are presented as means ± SE (error bars); **p < 0.01 (significance was evaluated using Student's t-test). hindpaw from the beam, FBXO7 flox/flox : Nestin-Cre mice could hardly move on the beam and slipped frequently (Fig. 1H). In particular, the hindpaws of FBXO7 flox/flox : Nestin-Cre mice often slipped off the beam (Fig. 1I). Gait disturbance progressed, and by the terminal stage, the majority of affected mice could hardly move. Mitochondrial damage and dysfunction in the substantia nigra have been previously reported in patients with sporadic PD [4,5]. These observations support the notion that aberrant mitochondrial function is a critical contributor to pathological neuronal degeneration. Accumulating knowledge regarding PINK1 and Parkin, both of which are associated with mitochondria, has increased our understanding of mitochondrial quality control [6,7].
FBXO7 and Parkin are interaction partners. FBXO7 is recruited to damaged mitochondria, and is required for the successful recruitment of Parkin. FBXO7 also binds to PINK1, and PINK1, Parkin, and FBXO7 act in concert to control the events leading to mitophagy. Interestingly, the expression of human FBXO7 in a Parkin mutant fly model rescues its phenotype, which is characterized by mitochondrial disruption and locomotor defects [8].
To characterize the damaged mitochondria in FBXO7 flox/flox : Nestin-Cre mice, we performed immunohistological (Fig. 1J) and ultrastructural (Fig. 1K) analysis of cortical and dopaminergic neurons of 3-week-old mice. We observed small, round, fragmented mitochondria in these neurons in FBXO7 flox/flox : Nestin-Cre mice but not in FBXO7 flox/flox mice. Precise quantification revealed that the mitochondrial area was reduced in cortical and dopaminergic cells (Fig. 1L). Together, these observations suggest that mitochondrial fragmentation might be facilitated in FBXO7-deficient mice. In order for damaged mitochondria to be degraded by autophagy, they must be segregated by fission [9]. Our in vivo results are reasonable if FBXO7-mediated mitophagy contributes to mitochondrial degradation systems.
Finally, to identify the relationship between our model mice and PD, we conducted immunohistological and blot analysis in the brain. Interestingly, in FBXO7 flox/flox : Nestin-Cre mice we identified p62-positive aggregates (Fig. 1O) that exhibited a significant increase in p62 levels (Fig. 1P) and synuclein colocalization (Fig. 1Q). These pathologies were identified in the dopaminergic neurons (Fig. 1R), including internal neurons (Fig. 1R arrows). Because p62 aggregates were not seen in endothelial cells (Fig. 1S) or microglia (Fig. 1T), FBXO7 flox/flox : Nestin-Cre mice were thought to demonstrate a neuron-specific phenotype. Further research is needed, but our results suggest that loss of FBXO7 may affect p62 and synuclein proteolysis, and impaired mitochondria elimination.