Several arguments support the view that the G877R mutation is harmful for the ATP13A2 protein function. The glycine 877 lies in a stretch of five amino acids that have not only been conserved in the evolution of ATP13A2 homologues until the yeasts but are also invariably present in all members of the ATPase P5 protein family (Fig. ), suggesting a crucial role for the protein function. Furthermore, the G877R mutation replaces a small non-polar amino acid (glycine) with a larger polar one (arginine). The large cytoplasmic loop located between the M4 and M5 transmembrane domains of the ATP13A2 protein (Fig. ) contains the catalytic autophosphorylation domain (P domain, including an N-terminal and C-terminal part) and the nucleotide binding domain (N domain, necessary for ATP binding) [9
]. The presence of an arginine in position 877 places a net positive charge next to the highly conserved negatively charged aspartic acid in position 878, in a very important motif within the C-terminal part of the P domain (Fig. ).
Structural studies of a similar P-type cationic pump, the calcium ATPase of skeletal muscle sarcoplasmic reticulum (SERCA1), revealed that the conserved aspartic acid corresponding to Asp-878 in ATP13A2 is one of the most critical residues for the ATP hydrolysis and is spatially very close to the aspartic acid residue (Asp351, corresponding to Asp513 in ATP13A2) which provides the autophosphorylation site [14
]. Thus, the G877R mutation is very likely to interfere with the ATPase and autophosphorylation activity of ATP13A2, necessary for the function as cationic pump. Taken together, these considerations argue strongly that the G877R mutation is deleterious for the ATP13A2 function and disease-causing when present in homozygous state.
The R481C mutation replaces a highly conserved arginine residue in the FBXO7 protein, within the so called R(Ar)DP motif (where Ar indicates any aromatic amino acid) in the C-terminal proline-rich region of the protein (Fig. ) [15
]. The function of the R(Ar)DP motif remains undetermined, but the proline-rich region is important for binding of the known FBXO7 substrates [15
]. The mutation might therefore also be deleterious for the FBXO7 function. However, being found as a single heterozygous mutation in the two affected brothers and their unaffected mother, its significance for the disease causation remains unclear. This could be a coincidental finding, or the mutation could act as a disease modifier, or play some pathogenic role, together with still unknown mutations in other genes.
Different research lines point to an important role for the lysosomes in the pathogenesis of common, late-onset PD. Lysosomes are important for the degradation of the alpha-synuclein protein [18
], and heterozygous mutations in the GBA
gene, encoding the lysosomal enzyme glucocerebrosidase, are an important risk factor for PD [19
]. Intriguingly, the ATP13A2 mRNA is highly expressed in the brain, particularly in substantia nigra, and it might be up-regulated in the brain of patients with the common late-onset idiopathic PD [3
]. Moreover, the ATP13A2 protein has been recently identified as a potent modifier of the toxicity induced by alpha-synuclein in animal models of PD [10
]. On the other hand, manganese, one possible substrate of ATP13A2, is a well-known cause of toxic parkinsonism in humans [20
]. Despite the rarity of PARK9 mutations, the ATP13A2 protein might therefore offer clues for understanding the pathogenesis of the common, late-onset forms of PD, linking genetic (alpha-synuclein), and environmental (manganese) factors in the disease etiology.
The clinical phenotype associated with PARK9
mutations remains poorly defined, as only few patients with clear disease-causing genotypes have been reported. Some phenotypic variability has been described (i.e., variable presence and degrees of dementia, behavior disorders, visual hallucinations, l
-dopa responsiveness, and disease progression). Our proband resembles indeed the previously reported PARK9 cases, and his phenotype is compatible with the clinical diagnosis of KRS. The sub-continuous mini-myoclonus present in the lower facial muscles is reminiscent of the facial–faucial–finger mini-myoclonus described in the original KRS patients [2
] and other PARK9 cases [3
]. Remarkably, the younger brother, apart from a moderate cognitive deficit, remains otherwise asymptomatic at the age of 31 years, and only the neurological examination revealed a mild pyramidal–extra-pyramidal involvement. This marked phenotypic variability has not been previously described in patients with identical PARK9 mutations. It could be the result of the action of genetic or environmental modifiers, or both. An aggravating effect for the perinatal brain sufferance in the proband could not be excluded; however, the severity of his phenotype is similar to that in the other PARK9 patients, and therefore, a protective effect in the proband’s brother appears more likely. Mutations in the other known genes for early-onset PD (apart from the FBXO7
heterozygous mutation) were excluded in the proband, and therefore they cannot play a role as aggravating factors. Environmental factors could include the role of some cations, according to the evidence that the ATP13A2 protein is involved in their transport from the cytosol to the lysosomal lumen [10
]. However, the blood levels of manganese, cadmium, nickel, and selenium were normal in both sibs. Furthermore, the siblings have been living with their parents in the same place, being likely exposed to the same environment.
In both sibs, the brain MRI showed diffuse brain atrophy, more marked in the proband, and the DaTSCAN SPECT showed marked nigrostriatal dopaminergic defects, again more marked in the proband, and similar to those reported in advanced idiopathic PD. The neurophysiologic investigation confirmed the involvement of the pyramidal tract and showed the normality of the large myelinated peripheral fibers and of the somatosensory system.
Recently, hypointense signals in the basal ganglia in MRI T2*-weighted scans, suggesting iron accumulation, have been reported in a patient with homozygous PARK9 mutation [6
]. Based on this finding, it has been suggested to classify PARK9 within the neurodegenerations with brain iron accumulation [6
]. The MRI studies in other previously reported PARK9 cases did not include T2*-weighted scans [2
] and were therefore inconclusive concerning the iron accumulation. Using a comprehensive MRI protocol that includes T2*-weighted scans, we did not find evidence of metal accumulation in the basal ganglia of both sibs reported here. Brain metal accumulation seems therefore not a constant feature in PARK9, even after three decades of disease course. Perhaps brain metal accumulation occurs in the cases with ATP13A2
mutations leading to the most severe loss of protein function, such as that present in the patient reported elsewhere [6
] (p.Thr367ArgfsX29, causing frameshift and protein truncation), while in some patients with missense mutations a residual protein function is retained and brain metal accumulation does not occur.
In conclusion, this family, the third reported with homozygous PARK9 mutations and the first with mutations in two genes for atypical juvenile parkinsonism, illustrates that PARK9-linked disease might display wide intra-familial clinical variability and present with milder phenotypes, suggesting the existence of strong, still unknown, modifiers.