In the present study we report, for the first time, four cases of NA showing choreic manifestations and acanthocytosis with a novel alteration in the expression of the erythrocyte membrane proteins: a 4.1R protein deficiency. Structural modification of membrane proteins has rarely been observed in NA; only a few cases of autosomal recessive ChAc with abnormalities of the major integral membrane protein band 3 have been reported [1
]. Moreover, ChAc has never been associated with 4.1R protein alterations.
Acanthocytosis and abnormalities of erythrocyte membrane proteins were revealed in all of the patients. The erythrocyte membrane defect was due to a decreased content of 4.1R protein, a multifunctional skeletal protein necessary for membrane stability and flexibility. The 4.1R protein content was significantly lower in patients (3.40 ± 0.42) than in controls (4.41 ± 0.40, P < 0.0001), reflecting weakened interactions of the cytoskeleton with the membrane. In three patients the 4.1R protein defect involved horizontal cytoskeleton interactions as shown by the increased content of spectrin dimers and was indicative of an impairment of the dimer self-association into tetramers; in a patient with a decrease in protein band 3, the 4.1R protein defect might impair the vertical interactions with integral membrane proteins by affecting the skeletal attachment to membrane.
The discovery of the new specific erythrocyte membrane protein defect, an alteration of 4.1R protein, explains the morphological changes in acanthocytosis and can provide indications regarding the disease protein function. It remains unknown whether genetic defect(s) could be responsible for the two distinct phenotypes: acanthocytosis and hyperkinesia. Interestingly, acanthocytosis and the 4.1R protein defect were also observed in other members of the families showing no neurological symptoms. If phenotypes observed in the families are due to genetic defect(s), one possible explanation could be that unaffected members might have a single mutation with a normal allele, i.e. they might be in a heterozygous state, which leads to the phenotype of acanthocytosis only. Mutations in both alleles or in combination with other genetic modifier(s) would be required for the two distinct phenotypes. Identification of genetic defect(s) in these families would reveal the underlying mechanism(s) of both phenotypes.
None of the patients presented the clinical or biochemical abnormalities typically observed in either abetalipoproteinaemia or McLeod syndrome [1
It is known that 4.1R protein is selectively expressed in haematopoietic tissues and in specific neuronal populations [3
]. Erythroid 4.1R protein is an 80-kDa skeletal protein required for structural organization and maintenance of the RBC cytoskeleton [11
]. 4.1R protein interacts with spectrin and actin by strengthening the skeletal network and stabilizes the spectrin-actin complexes through the spectrin-actin binding (SAB) domain. Furthermore, the 4.1R N-terminal domain mediates the attachment of the underlying cytoskeleton to the overlying lipid bilayer through interactions with integral membrane proteins such as band 3 and glycophorin C [3
]. Abnormalities of 4.1R protein are associated with congenital RBC defects leading to severe membrane fragmentation and hereditary elliptocytosis [3
Four genes encoding 4.1 proteins are known to be expressed in the brain. The corresponding proteins are known as 4.1R, 4.1G, 4.1B, and 4.1N. 4.1 proteins are highly conserved and retain the same fundamental organization of domains [4
]. High levels of 4.1R were discretely localized in granule cells in the cerebellum and dentate gyrus [19
]. 4.1R protein is also selectively localized in central neurons interacting with the intermediate filament proteins of post-synaptic densities [7
]. Recently, it has been postulated that 4.1 proteins play an essential role in synaptic plasticity by delivering specific subunits of glutamate AMPA receptors in central synapses [6
]. In line with this hypothesis 4.1R protein-null mice, as well as the abnormal morphology and lowered membrane stability of RBC, have specific deficits in movements, coordination, and balance [19
], therefore highlighting a connection between 4.1R protein deficiency and human neurodegenerative syndromes.
The present observation suggests a likely association between a defect of 4.1R protein and the expression of hyperkinetic disorders caused by a possible abnormal glutamatergic transmission in the basal ganglia [21
]. Pre- and/or post-synaptic modulation of glutamate AMPA receptor-mediated transmission, by facilitating glutamate release from corticostriatal terminals or AMPA receptor currents with ampakines [22
], might prove beneficial in treating motor disorders associated with 4.1R alterations.
Our sequence analyses did not show any disease-associated mutations or polymorphisms in the ChAc. If the decreased expression of 4.1R protein is due to genetic defect(s), two possible explanations might be considered for the results of our genetic study. Firstly, there might be other genetic defect(s) in the ChAc gene including the regulatory region, which was not examined in our analysis. Secondly, there might be further as yet unknown genetic defect(s) or further locus heterogeneity in NA. However, previous papers have revealed little evidence of further locus heterogeneity in neuroacanthocytosis [10
We have demonstrated that expression of 4.1R protein in erythrocytes is decreased in patients with atypical neuroacanthocytosis, but there is no evidence of decreased expression of the protein in the brain because we did not examine post-mortem brains of patients. Analyses of 4.1R-deficient mice showed lower membrane stability of RBC, movement abnormalities, abnormal morphology and decreased expression of the protein in the brain nucleus/tissues including granule cells and dentate gyrus [24
]. The functional effect(s) of decreased expression of 4.1R protein in the brain remains unknown. A previous study revealed that 4.1R proteins play an essential role in synaptic plasticity by delivering specific subunits of glutamate AMPA receptors into central synapses [25
]. The altered metabolism of glutamate AMPA might lead to dysfunction of neurotransmission in the basal ganglia and hyperkinetic movements in neuroacanthocytosis.
It also remains unknown whether the expression pattern of protein in erythrocytes might reflect their expression patterns in the brain. An investigation into the expression of alpha-synuclein in lymphocytes from PARK1-patients demonstrated that decreased expression of alpha-synuclein correlates with the severity of the clinical phenotype [26
], suggesting that expression patterns of protein in peripheral tissue might not be independent from those in the central nervous system. Investigation of expression of 4.1R protein in post-mortem brains of patients with neuroacanthocytosis is a prerequisite for validation of decreased expression of 4.1R protein and understanding biological mechanism(s) for hyperkinesia in NA.
In conclusion, our study demonstrates decreased expression of 4.1R protein in the erythrocytes, which might reflect decreased expression of this protein in the brain resulting in hyperkinetic movements in neuroacanthocytosis.