Central nervous system dysfunction in a mouse model of FA2H deficiency

Abstract

Fatty acid 2-hydroxylase (FA2H) is responsible for the synthesis of myelin galactolipids containing hydroxy fatty acid (hFA) as the N-acyl chain. Mutations in the FA2H gene cause leukodystrophy, spastic paraplegia, and neurodegeneration with brain iron accumulation. Using the Cre-lox system, we developed two types of mouse mutants, Fa2h(-/-) mice (Fa2h deleted in all cells by germline deletion) and Fa2h(flox/flox) Cnp1-Cre mice (Fa2h deleted only in oligodendrocytes and Schwann cells). We found significant demyelination, profound axonal loss, and abnormally enlarged axons in the CNS of Fa2h(-/-) mice at 12 months of age, while structure and function of peripheral nerves were largely unaffected. Fa2h(-/-) mice also exhibited histological and functional disruption in the cerebellum at 12 months of age. In a time course study, significant deterioration of cerebellar function was first detected at 7 months of age. Further behavioral assessments in water T-maze and Morris water maze tasks revealed significant deficits in spatial learning and memory at 4 months of age. These data suggest that various regions of the CNS are functionally compromised in young adult Fa2h(-/-) mice. The cerebellar deficits in 12-month-old Fa2h(flox/flox) Cnp1-Cre mice were indistinguishable from Fa2h(-/-) mice, indicating that these phenotypes likely stem from the lack of myelin hFA-galactolipids. In contrast, Fa2h(flox/flox) Cnp1-Cre mice did not show reduced performance in water maze tasks, indicating that oligodendrocytes are not involved in the learning and memory deficits found in Fa2h(-/-) mice. These findings provide the first evidence that FA2H has an important function outside of oligodendrocytes in the CNS.

Fig. 1. Deletion of exons 5 and 6 of Fa2h eliminates hFA-galactolipids in the nervous system

(A) The targeting vector and the Fa2h^flox and Fa2h⁻ alleles. Exons 5 and 6 (E5, E6) of the Fa2h^flox allele were successfully excised in vivo by Cre recombinase. Note that the diagram is not to scale. (B) PCR-genotyping. The agarose gel on the left shows the presence or absence of the Fa2h⁺ allele amplified by primers 1 and 2. The agarose gel on the right shows the presence or absence of the Fa2h⁻ allele amplified by primers 5 and 6. I.C., internal control. (C) HPTLC analysis of myelin galactosylceramide. Alkaline-resistant lipids were analyzed by HPTLC. Lipids were extracted from brain (2 mg protein equivalent per lane) and sciatic nerve (100 mg protein equivalent per lane) from 23-day-old males. GalCer, galactosylceramide. (D) Analysis of spinal cord lipids by mass spectrometry. Total lipids were extracted from spinal cord tissue of 14-month-old male Fa2h^+/− mice (WT, n=2, black bars) and Fa2h^−/− mice (Null, n=2, white bars). Asterisks (*) denote below detectable levels. The data shown are the combined levels of the four groups of galactolipids [non-hydroxy (nFA) and hydroxy (hFA) galactosylceramide and sulfatide], each including various molecular species with different N-acyl chains. The values for individual molecular species are shown in Supplementary Table 1.

Fig. 2. Pathological findings in the optic nerves of Fa2h-null mice

Images from semi-thin (A, B) and thin (C, D) sections of optic nerves from 12-month-old Fa2h^−/− (A, C) and Fa2h^+/+ (B, D) mice are shown. (A) A region of a nerve with markedly reduced numbers of myelinated axons as well as many vacuoles (v) and enlarged bulbous axons (b). (B) A similar region of a normal nerve. Note the dense packing of myelinated axons that range from 0.5 to 3 microns in diameter. (C) An electron micrograph of a similarly affected region as in (A), showing the fine structure of vacuoles (v), demyelinated axons (asterisks), and two bulbous axons (b) that are filled with dense bodies, lysosomes, and mitochondria. (D) An electron micrograph of a similar region of a normal nerve. Scale bar: 10 μm in panels A and B, 1 μm in panels B and D.

Fig. 3. Pathological findings in the Fa2h-null mice

These are images of semi-thin sections of the ventral funiculus of the cervical spinal cord (A, B) and tibial nerves (C, D) from 12 month old Fa2h^−/− mice (A, C) or Fa2h^+/+ mice (B, D). The spinal cord of the Fa2h^−/− mice contains demyelinated axons (asterisks), as well as vacuoles (v) and enlarged bulbous axons (b) filled with dense bodies, lysosomes, and mitochondria (not visible at this magnification). The sciatic nerve of the Fa2h^−/− mice has a few remyelinated axons (r). Scale bars: 10 μm.

Fig. 4. Cerebellum histology

(A) Overall cerebellum histology (size and gross morphology) of 3- or 12-month-old male Fa2h^−/− (Null) and control mice (WT) did not differ from one another (H&E, 4x magnification). (B) LFB staining of the cerebellum. While 3-month-old Fa2h^−/− and control mice were identical, 12-month-old Fa2h^−/− mice exhibited significantly reduced staining relative to control littermates (4x magnification). (C) LFB staining of 12-month-old Fa2h^+/flox Cnp1-Cre (+/flox) and Fa2h^flox/flox Cnp1-Cre (flox/flox) littermates (4x magnification). (D) Calbindin immunostaining of the cerebellum. Fa2h^−/− mice have altered size and distribution of Purkinje neurons relative to control mice as evidenced by an irregular pattern of calbindin-immunoreactive Purkinje neurons (denoted by black arrows). Left and center panels, 4x magnification; right panels, 20x magnification of the black rectangles in center panels. (E) Calbindin immunostaining of the cerebellum of Fa2h^+/flox Cnp1-Cre (+/flox) and Fa2h^flox/flox Cnp1-Cre mice (flox/flox). The black arrows denote areas with disrupted Purkinje neurons. Left panels, 4x magnification; right panels, 20x magnification of the black rectangles in left panels.

Fig. 5. Behavioral analyses reveal motor deficits in Fa2h deficient mice

(A) General motor activity of 12-month-old male Fa2h^−/− mice (Null, n=5, grey bars) and their littermate controls (WT, n=5, black bars). The control littermates included Fa2h^+/+ and Fa2h^+/− mice, whose performances were indistinguishable from one another (p>0.05). *, p<0.05; **, p<0.01. (B) Motor coordination and motor learning assessed using an accelerating rotarod device. Grey squares/lines, Fa2h^−/− mice (n=5); black squares/lines, control littermates (n=5). (C&D) Motor deficits in 12-month-old Fa2h^flox/flox Cnp1-Cre mice (flox/flox, n=7, grey bars/ squares/lines) were practically identical to Fa2h^−/− mice. They showed reduced general ambulation (panel C; *, p<0.05; **, p<0.01) and impaired rotarod performance (panel D, p<0.05) in comparison to Fa2h^+/flox Cnp1-Cre mice (+/flox, n=5, black bars/squares/lines) at all speeds greater than 4 rpm. All data are presented as mean±standard error. (E) Motor deficits emerge at 7 months of age. A separate group of male Fa2h^−/− mice (n=4, grey squares/lines) and control mice (n=6, black squares/lines) were tested monthly for rotarod performance. The control group included age-matched Fa2h^+/+ and Fa2h^+/− mice, whose performances were indistinguishable from one another (p>0.05). All data are presented as mean±standard error.

Fig. 6. Learning and memory deficits are present in Fa2h^−/− mice but not in Fa2h^flox/flox Cnp1-Cre mice

(A) 3-month-old male Fa2h^+/+ mice [black bars (+/+), n=8] and Fa2h^−/− mice [grey bars (−/−), n=8] were evaluated for their performance on a water T-maze task. No differences were seen between the genotypes on their ability to locate and swim to a hidden platform (Initial, p>0.05). After initial training, the platform was placed in the opposite arm. Fa2h^+/+ mice took significantly fewer sessions to learn the new platform location (Reverse; *, p< 0.015) than Fa2h^−/− mice. All data are presented as mean+standard error. (B) A separate group of 4-month-old Fa2h^+/+ mice [black bars (+/+), n=8] and Fa2h^−/− mice [grey bars (−/−), n=5] were trained to swim to a hidden platform within a Morris water maze. Fa2h^{−/ −} mice took significantly more sessions to learn the location of the platform compared to Fa2h^+/+ mice, (Initial; *, p<0.05). Once learning was achieved, the platform was moved to a new location and training was repeated. Again, Fa2h^{−/ −} mice took significantly more sessions to learn the new location of the platform (Reverse; *, p<0.05). (C) Water T-maze test was performed with male Fa2h^+/flox Cnp1-Cre mice [hatched bars (+/f), n=10] and Fa2h^flox/flox Cnp1-Cre mice [white bars (f/f), n=6]. No significant difference was observed between the two groups for both Initial (p>0.05) and Reverse (p>0.05). (D) Morris water maze test was performed with male Fa2h^+/flox Cnp1-Cre mice [hatched bars (+/f), n=10] and Fa2h^flox/flox Cnp1-Cre mice [white bars (f/f), n=6]. No significant difference was observed between the two groups for both Initial (p>0.05) and Reverse (p>0.05). (E) TLC analysis of brain galactolipids from Fa2h^+/flox Cnp1-Cre (+/f) and Fa2h^flox/flox Cnp1-Cre (f/f) mice. Total brain lipids were extracted from two 10-week-old Fa2h^flox/flox Cnp1-Cre mice and their respective littermates. Lipids were separated on a HPTLC plate along with GalCer standards and visualized by primuline staining and UV illumination. (F) Mass spectrometry analysis of brain galactolipids from 3-month-old Fa2h deficient mice and normal littermates. Individual lipids (12 distinct GalCer and 11 distinct hFA-GalCer) in whole brain lipid extracts obtained from Fa2h^+/−, Fa2h^{−/ −}, Fa2h^+/flox Cnp1-Cre, and Fa2h^flox/flox Cnp1-Cre mice (n=2 for each genotype) were measured by LC/MS/MS. The mean and range of the total GalCer (nFA) and hFA-GalCer (hFA) are shown. The values for individual molecular species in each animal are included in Supplementary Table 2.