We have demonstrated that the murine Pank2 protein is mainly located in the mitochondrial inter-membrane space, which is compatible with its role in CoA metabolism and with its regulation in the presence of CoA at concentrations found in mitochondrial matrix (10
). Further investigation using, for instance, immuno-electronmicroscopy is needed to unequivocally establish this sub-mitochondrial localization.
This observation makes the Pank2−/−
mouse model a promising system to study pathophysiology of human PKAN. In fact, although the mouse model does not recapitulate the clinical and neuropathological features of the human condition (7
), it could serve as a system in which to interrogate the basic defect in mitochondrial function.
We did not detect the deficiency in the enzymatic activity of any mitochondrial respiratory chain complex in different tissues of Pank2−/−
mice. However, recent observations indicate that mitochondrial respiration is largely carried out by the fraction of respiratory chain complexes that assemble together in supercomplexes forming functionally active units, called respirasomes (15
). Thus, the spectrophotometric measurement of each respiratory complex activity does not necessarily reflect the mitochondrial functional capacity in vivo
. Microscale oxygraphy could overcome this limitation and in fact, using this approach, we detected a modification in the respiratory profile of mitochondria derived from Pank2−/−
when compared with Pank2+/+
brains. Highly significant differences of OCR-B, -ADP, -O and –F were obtained by statistical analysis of either the single values or as overall values of Pank2+/+
mitochondria, expressed as z
-scores (Fig. ). Together, these results indicate that the absence of the Pank2 protein leads to a global failure of the mitochondrial bioenergetic performance without affecting the function of any single respiratory chain complex.
An interesting result of our study, strictly correlated with the absence of Pank2 function, was the demonstration of alteration in mitochondrial membrane potential in neurons derived from sciatic nerve and hair bulge stem cells of adult mice. In addition, the same alteration was also present in neonatal hippocampal neurons, suggesting the presence of defective mitochondria in Pank2−/− mice since birth.
These results were confirmed by electron microscopy analysis on cultured neurons derived from Pank2−/− mice, in which aberrant mitochondria with remodelled cristae were present. Moreover, peripheral and CNSs examination of Pank2−/− mice showed the presence of swollen mitochondria with amorphous electron-dense inclusions and dysmorphic cristae.
Recently, the characterization of a PKAN Drosophila
model demonstrated that impaired function of pantothenate kinase induced a neurodegenerative phenotype with mitochondrial dysfunction, decreased levels of CoA, increased protein oxidation and reduced lifespan (17
JC1 staining and electron microscopic analysis revealed that, in contrast to WT flies, mitochondria of dPANK/fbl mutants showed an alteration in the transmembrane potential, were swollen and presented with altered cristae and ruptured membranes (18
). These observations are in agreement with our results in the nervous system of Pank2−/−
mice in which mitochondria are severely damaged.
In search for mechanisms underlying the alteration of mitochondria, we found a reduced level of ATP in Pank2−/− mice. Interestingly, we found that state 3 activity, which represent the maximum respiration rate in the presence of ADP, is significantly decreased in mitochondria isolated from Pank2−/− mice. It is tempting to speculate that the reduction in the respiration could be attributable to the severe alteration of the cristae structure, which could prevent the respirasomes from remaining functionally active.
We also investigated the presence of oxidative damage in brains, but we did not find significant differences in oxidative damage at least in 6-month-old Pank2−/−
mice. As demonstrated by other studies, mitochondrial membrane damage contributes to the pathogenesis of many neurodegenerative diseases (19
Interestingly enough, a recent investigation of a KO mouse model for the Pla2g6
gene, which is defective in a different but related form of NBIA, revealed the presence of collapsed mitochondria with degenerated inner membranes (21
gene encodes a group VIA calcium-independent phospholipase A2, an esterase that hydrolyzes the sn-2 ester bond in phospholipids to yield free fatty acids and lysophospholipids and which is involved in cardiolipin remodelling (22
). Analysis of phospholipids and fatty acids revealed differences between KO and WT mice. The vulnerability of mitochondrial inner membranes in Pla2g6
KO mice might be attributable to increased production of reactive oxygen species (ROS) (23
) and a rich content of polyunsaturated fatty acids that can readily be peroxidized, such as linoleic acid in cardiolipin (22
Alteration in cholesterol and lipid metabolism was also recently demonstrated by a metabolomics investigation in a group of PKAN patients (24
). In this case, however, the alteration was mainly due to a defective synthesis and not, as in the case of PLA2G6, to the absence of a catabolic enzyme.
Irrespective of the anabolic (PANK2) or catabolic (PLA2G6) role of these two mitochondrial proteins, a common culprit in the pathogenesis of both neurodegenerative diseases could be an altered lipid metabolism (25
). Further characterization of the lipid profile in Pank2−/−
mice is in progress to understand if and how alteration of this metabolic pathway could be responsible for the observed mitochondrial membranes modifications.
We demonstrated insufficient energy production and severe mitochondrial dysfunction in the brain and peripheral nerve of Pank2−/−
mice, without increased oxidative stress or signs of neurodegeneration. It is possible that mice can better tolerate alterations in bioenergetics metabolism without suffering any overt clinical manifestations because of the presence of compensatory mechanisms. Few examples of mouse models of mitochondrial disorders, which display a biochemical phenotype but do not present any clinical signs typical of the human pathology, are described in the literature (26
Although we clearly demonstrated mitochondrial dysfunction in Pank2−/− mice, we remain uncertain why neither neurological signs typical of PKAN nor iron accumulation occur in mice. However, our findings suggest that there would be value in investigating bioenergetic competence and ultra-structural abnormalities of mitochondria in PKAN patients.