Hornerin deposits in neuronal intranuclear inclusion disease: direct identification of proteins with compositionally biased regions in inclusions.

Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disorder, characterized by the presence of eosinophilic inclusions (NIIs) within nuclei of central and peripheral nervous system cells. This study aims to identify the components of NIIs, which have been difficult to analyze directly due to their insolubility. In order to establish a method to directly identify the components of NIIs, we first analyzed the huntingtin inclusion-rich fraction obtained from the brains of Huntington disease model mice. Although the sequence with expanded polyglutamine could not be identified by liquid-chromatography mass spectrometry, amino acid analysis revealed that glutamine of the huntingtin inclusion-rich fraction increased significantly. This is compatible with the calculated amino acid content of the transgene product. Therefore, we applied this method to analyze the NIIs of diseased human brains, which may have proteins with compositionally biased regions, and identified a serine-rich protein called hornerin. Since the analyzed NII-rich fraction was also serine-rich, we suggested hornerin as a major component of the NIIs. A specific distribution of hornerin in NIID was also investigated by Matrix-assisted laser desorption/ionization imaging mass spectrometry and immunofluorescence. Finally, we confirmed a variant of hornerin by whole-exome sequencing and DNA sequencing. This study suggests that hornerin may be related to the pathological process of this NIID, and the direct analysis of NIIs, especially by amino acid analysis using the NII-rich fractions, would contribute to a deeper understanding of the disease pathogenesis.

Neuronal intranuclear inclusion disease is genetically heterogeneous.

Neuronal intranuclear inclusion disease (NIID) is a clinically heterogeneous neurodegenerative condition characterized by pathological intranuclear eosinophilic inclusions. A CGG repeat expansion in NOTCH2NLC was recently identified to be associated with NIID in patients of Japanese descent. We screened pathologically confirmed European NIID, cases of neurodegenerative disease with intranuclear inclusions and applied in silico-based screening using whole-genome sequencing data from 20 536 participants in the 100 000 Genomes Project. We identified a single European case harbouring the pathogenic repeat expansion with a distinct haplotype structure. Thus, we propose new diagnostic criteria as European NIID represents a distinct disease entity from East Asian cases.

ApoE epsilon3-haplotype modulates Alzheimer beta-amyloid deposition in the brain.

ApoE epsilon4 allele increases the risk of late-onset Alzheimer disease (AD) as well as the amount of beta-amyloid deposition in the brain. Because half of AD patients do not have ApoE epsilon4, it is important to search for other determinants of ApoE that modify AD risk. We tested whether the haplotype background of the most common ApoE allele, epsilon3, influences brain amyloid deposition or the risk of neuropathologically verified AD in a population-based sample of elderly Finns. To exclude the effects of ApoE protein polymorphism we focused these analyses on subjects homozygous for epsilon3. Haplotypes were defined using polymorphisms at positions - 491 and -219 of the ApoE promoter and at position +113 of intron-1. We found that epsilon3-haplotypes containing the promoter allele -219T were associated with reduced amyloid deposition and reduced risk of neuropathologically verified AD as compared to epsilon3-haplotypes containing -219G. The functional polymorphism(s) responsible for the haplotypic difference remains to be identified. These results indicate that there is significant allelic variation in the ApoE gene region, which modulates brain amyloid deposition and AD risk, independent of the ApoE protein polymorphism.

Cathepsin D-deficient Drosophila recapitulate the key features of neuronal ceroid lipofuscinoses.

Neuronal ceroid lipofuscinoses (NCLs) are a group of lysosomal storage disorders characterized pathologically by neuronal accumulation of autofluorescent storage material and neurodegeneration. An ovine NCL form is caused by a recessive point mutation in the cathepsin D gene, which encodes a lysosomal aspartyl protease. This mutation results in typical NCL pathology with neurodegeneration and characteristic neuronal storage material. We have generated a Drosophila NCL model by inactivating the conserved Drosophila cathepsin D homolog. We report here that cathepsin D mutant flies exhibit the key features of NCLs. They show progressive neuronal accumulation of autofluorescent storage inclusions, which are also positive for periodic acid Schiff and luxol fast blue stains. Ultrastructurally, the storage material is composed of membrane-bound granular electron-dense material, similar to the granular osmiophilic deposits found in the human infantile and ovine congenital NCL forms. In addition, cathepsin D mutant flies show modest age-dependent neurodegeneration. Our results suggest that the metabolic pathway leading to NCL pathology is highly conserved during evolution, and that cathepsin D mutant flies can be used to study the pathogenesis of NCLs.

High-resolution magic angle spinning and 1H magnetic resonance spectroscopy reveal significantly altered neuronal metabolite profiles in CLN1 but not in CLN3.

The neuronal ceroid lipofuscinoses (NCLs) are among the most severe inherited progressive neurodegenerative disorders of children. The purpose of this study was to compare the in vivo 1.5-T 1H magnetic resonance (MR) and ex vivo 14.3-T high-resolution (HR) magic angle spinning (MAS) 1H MR brain spectra of patients with infantile (CLN1) and juvenile (CLN3) types of NCL, to obtain detailed information about the alterations in the neuronal metabolite profiles in these diseases and to test the suitability of the ex vivo HR MAS (1)H MRS technique in analysis of autopsy brain tissue. Ex vivo spectra from CLN1 autopsy brain tissue (n = 9) significantly differed from those of the control (n = 9) and CLN3 (n = 5) groups, although no differences were found between the CLN3 and the control groups. Principal component analysis of ex vivo data showed that decreased levels of N-acetylaspartate (NAA), gamma-aminobutyric acid (GABA), glutamine, and glutamate as well as increased levels of inositols characterized the CLN1 spectra. Also, the intensity ratio of lipid methylene/methyl protons was decreased in spectra of CLN1 brain tissue compared with CLN3 and control brain tissue. In concordance with the ex vivo data, the in vivo spectra of late-stage patients with CLN1 (n = 3) revealed a dramatic decrease of NAA and a proportional increase of myo-inositol and lipids compared with control subjects. Again, the spectra of patients with CLN3 (n = 13) did not differ from those of controls (n = 15). In conclusion, the ex vivo and in vivo spectroscopic findings were in good agreement within all analyzed groups and revealed significant alterations in metabolite profiles in CLN1 brain tissue but not in CLN3 compared with controls. Furthermore, HR MAS 1H MR spectra facilitated refined detection of neuronal metabolites, including GABA, and composition of lipids in the autopsy brain tissue of NCL patients.