Uncovering the Complex Cerebellar Phenotype in Peroxisomal Multifunctional Protein-2 Deficiency

Similar to mitochondria and lysosomes, peroxisomes are present in all human cells, except erythrocytes. Patients with peroxisomal dysfunction exhibit a variety of neurological symptoms ranging from severe brain malformations at birth to blindness in adulthood, underlining the primordial role of these organelles in the central nervous system. In recent years, adolescent and adult patients that displayed profound ataxia and cerebellar atrophy as the primary pathologies were found to have a defect in peroxisomal proteins. A subset of patients with this ‘mild peroxisomal phenotype’ suffer from multifunctional protein-2 (MFP2) deficiency. Being the central enzyme of the peroxisomal β-oxidation pathway, this protein has a crucial function in metabolizing certain lipid species.

Cerebellar problems also occur in global Mfp2 and conditional Nestin-Mfp2 knockout mice, with deletion of MFP2 from all neural cells. Both models show early-onset gait abnormalities and an impaired balance. On a histological level, Purkinje cell (PC) axonal swellings evolved in cerebellar white matter loss, PC degeneration and cerebellar atrophy. However, several questions were unsolved: (1) is there a developmental or degenerative origin of cerebellar deterioration, (2) at which age do the first (histo) pathological events occur in the cerebellum, (3) how do these contribute to the early-onset ataxic phenotype and (4) how are they related to disturbances in the peroxisomal β-oxidation pathway? The present study aimed to elucidate the mechanisms underlying the uncoordinated phenotype and cerebellar deterioration in MFP2 deficiency. As Nestin-Mfp2 knockout mice constitute a valuable mouse model to study cerebellar degeneration due to peroxisomal β-oxidation defects, this model was further exploited. Moreover, we studied cerebellar pathology in a newly generated mouse model of MFP2 deficiency, in which the enzyme was specifically deleted in the PCs (L7-Mfp2-/-) at an early postnatal age. In this way, we could unravel how MFP2 influences the only output neurons of the cerebellar cortex and whether a cell autonomous process contributes to cerebellar deterioration in MFP2 defciency.

We analyzed the function and structure of PCs in young Nestin-Mfp2-/- mice and found that their juvenile motor phenotype is caused by deviations in the electrical properties of these neurons. The decreases in PC spontaneous firing frequency and excitability are possibly related to their increased spine number and the altered, elongated shape of these spines. Although these morphological changes are considered rather mild, together they can significantly increase the neuronal surface and thereby affect the PC membrane capacitance. Moreover, we were able to show developmental problems of glutamatergic axons (climbing fibers and parallel fibers) innervating the PCs that most likely also contribute to the early-onset motor phenotype of Nestin-Mfp2-/- mice. Finally, the presence of multiple swellings on PC axons proves disturbances in PC axonal transport in young knockout mice.

With our PC specific MFP2 deficient mouse model (L7-Mfp2-/-) we prove for the first time that the deletion of MFP2 from neurons can also result in a postnatal and degenerative cerebellar pathology, besides the developmental problems mentioned above. PCs situated in the anterior lobules of L7-Mfp2 knockout mice seem more vulnerable to degeneration as they disappeared first, while neurons located in lobule X appear rather resistant to cell death. Strikingly, we also noticed that MFP2 deficient PCs vanished in a striped, zebra-like fashion suggesting the specific vulnerability of neurons expressing certain molecular markers. The exact mechanisms underlying PC degeneration in L7-Mfp2-/- mice remain to be elucidated, but interim results point in the direction of the autophagy process. Similar to Nestin-Mfp2-/- mice, PC axonal swellings were detected as the earliest histopathological sign in L7-Mfp2-/- cerebella, and therefore they can be considered as the primary sign of PC discontent in MFP2 deficiency. Finally, we excluded that the early-onset motor phenotype of constitutive Mfp2 and Nestin-Mfp2 knockout mice originates from MFP2 deletion from PCs only as motor problems in L7-Mfp2-/- mice initiated at a later time point.

In conclusion, our findings provide new insights in the cerebellar pathogenesis of patients diagnosed with a mild variant of MFP2 deficiency. The precise molecular mechanisms underlying PC dysfunction and degeneration remain however unsolved. There is no doubt that in the future more patients with ataxias of an unknown origin will be pinpointed to be caused by peroxisomal dysfunction.