Non-allelic recombination between homologous repetitive elements contributes to evolution and human genetic disorders. Here, we combine short- and long-DNA read sequencing of repeat elements with a ...new bioinformatics pipeline to show that somatic recombination of Alu and L1 elements is widespread in the human genome. Our analysis uncovers tissue-specific non-allelic homologous recombination hallmarks; moreover, we find that centromeres and cancer-associated genes are enriched for retroelements that may act as recombination hotspots. We compare recombination profiles in human-induced pluripotent stem cells and differentiated neurons and find that the neuron-specific recombination of repeat elements accompanies chromatin changes during cell-fate determination. Finally, we report that somatic recombination profiles are altered in Parkinson’s and Alzheimer’s disease, suggesting a link between retroelement recombination and genomic instability in neurodegeneration. This work highlights a significant contribution of the somatic recombination of repeat elements to genomic diversity in health and disease.
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•Somatic recombination of Alu and L1 elements is widespread in the human genome•Somatic recombination events of Alu and L1 elements exhibit tissue-specific hallmarks•Neuronal differentiation of iPSCs is accompanied by changes in recombination profiles•Somatic recombination profiles are altered in Parkinson’s and Alzheimer’s diseases
Large-scale analysis of human tissue samples unveils the tissue-specific somatic recombination of repeat elements that is widespread and contributes to the genomic diversity underpinning human development and disease.
The propagation of conformational strains by templated seeding is central to the prion concept. Seeded assembly of α‐synuclein into filaments is believed to underlie the prion‐like spreading of ...protein inclusions in a number of human neurodegenerative diseases, including Parkinson's disease, dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). We previously determined the atomic structures of α‐synuclein filaments from the putamen of five individuals with MSA. Here, we used filament preparations from three of these brains for the in vitro seeded assembly of recombinant human α‐synuclein. We find that the structures of the seeded assemblies differ from those of the seeds, suggesting that additional, as yet unknown, factors play a role in the propagation of the seeds. Identification of these factors will be essential for understanding the prion‐like spreading of α‐synuclein proteinopathies.
The assembly of certain proteins into amyloids underlies multiple neurodegenerative diseases. The spreading of these assemblies through the brain is thought to occur through a prion‐like mechanism. We used filaments extracted from multiple system atrophy brains to seed recombinant α‐synuclein. The resulting structures differ from those of the seeds, indicating that seeded assembly does not necessarily replicate the seed structures.
MicroRNAs (miRNAs) are attractive molecules to utilize as one of the blood-based biomarkers for neurodegenerative disorders such as Alzheimer's disease (AD) because miRNAs are relatively stable in ...biofluid, including serum or plasma. To determine blood miRNA biomarkers for AD with next-generation sequencing genome-wide, we first surveyed 45 serum samples. These came from 27 AD patients and 18 controls (discovery set) that underwent autopsy within two weeks after their serum sampling and were neuropathologically diagnosed. We found that three miRNAs, hsa-miR-501-3p, hsa-let-7f-5p, and hsa-miR-26b-5p, were significantly deregulated between the AD samples and the controls. The deregulation for hsa-miR-501-3p was further confirmed by quantitative reverse transcription polymerase chain reaction (PCR) in a validation set composed of 36 clinically diagnosed AD patients and 22 age-matched cognitively normal controls with a sensitivity and specificity of 53% and 100%, respectively (area under the curve = 0.82). Serum hsa-miR-501-3p levels were downregulated in AD patients, and its lower levels significantly correlated with lower Mini-Mental State Examination scores. Contrary to its serum levels, we found that hsa-miR-501-3p was remarkably upregulated in the same donors' AD brains obtained at autopsy from the discovery set. The hsa-miR-501-3p overexpression in cultured cells, which mimicked the hsa-miR-501-3p upregulation in the AD brains, induced significant downregulation of 128 genes that overrepresented the Gene Ontology terms, DNA replication, and the mitotic cell cycle. Our results suggest that hsa-miR-501-3p is a novel serum biomarker that presumably corresponds to pathological events occurring in AD brains.
Introduction/Aims
Reliable neurophysiological markers in amyotrophic lateral sclerosis (ALS) are of great interest. The compound muscle action potential (CMAP) amplitude has been a conventional ...marker, although it is greatly influenced by the electrode position. We propose the far‐field potential of the CMAP (FFP‐CMAP) as a new neurophysiological marker in ALS.
Methods
Patients with ALS and age‐matched healthy controls were enrolled. We used a proximal reference (pref) in addition to the conventional distal reference (dref). Routine CMAP was recorded from the belly‐dref lead and FFP‐CMAP from the dref‐pref lead for the ulnar and tibial nerves. Multiple point stimulation motor unit number estimation (MUNE) was also examined in the ulnar nerve. Inter‐rater reproducibility was evaluated by two examiners, and some patients were followed up every 3 mo for 1 y.
Results
We tested 17 patients with ALS and 10 controls. The amplitudes of routine CMAP and FFP‐CMAP in the ulnar and tibial nerves, and hypothenar MUNE value in the ulnar nerve were significantly decreased in ALS compared to controls. Ulnar FFP‐CMAP achieved the highest inter‐rater intraclass correlation coefficient (ICC) value (0.942) when compared with routine CMAP (0.880) and MUNE (0.839). The tibial FFP‐CMAP had a higher ICC value (0.986) than the routine CMAP (0.697). In this way, the FFP‐CMAP showed high inter‐rater reproducibility because its shape was not much influenced by the electrode position. During 1‐y follow‐up, decline of CMAP, FFP, and MUNE showed significant correlations with the Amyotrophic Lateral Sclerosis Functional Rating Scale ‐ Revised (ALSFRS‐R).
Discussion
The FFP‐CMAP shows promise as a reliable marker for ALS.
See article on pages 237–239 in this issue
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease characterized by progressive muscle weakness due to degeneration of lower motor neurons in the anterior horn of the ...spinal cord. We analyzed autopsy findings of a male patient with SMA type 2 who survived until 61 years of age. Genetic analysis revealed a homozygous deletion of the survival motor neuron (SMN) gene 1 (SMN1) exon 7, confirming the diagnosis of SMA. Results of further analyses indicated that the patient had two copies of the genuine SMN gene 2 (SMN2) and one copy of a hybrid gene containing SMN2 exon 7 and SMN1 exon 8. Pathological examination revealed moderate neuronal loss of the anterior horn and appearance of heterotopic neurons in the lateral funiculus, whereas a few achromatic neurons were notably localized in the anterior horn of the lumbar segment. Microdysgenesis as a consequence of migration disturbance was found in the white matter of the frontal lobe, postulating the possibility of the maldevelopment of the nervous system.
The β‐site amyloid precursor protein cleaving enzyme‐1 (BACE1), an essential protease for the generation of amyloid‐β (Aβ) peptide, is a major drug target for Alzheimer's disease (AD). However, there ...is a concern that inhibiting BACE1 could also affect several physiological functions. Here, we show that BACE1 is modified with bisecting N‐acetylglucosamine (GlcNAc), a sugar modification highly expressed in brain, and demonstrate that AD patients have higher levels of bisecting GlcNAc on BACE1. Analysis of knockout mice lacking the biosynthetic enzyme for bisecting GlcNAc, GnT‐III (Mgat3), revealed that cleavage of Aβ‐precursor protein (APP) by BACE1 is reduced in these mice, resulting in a decrease in Aβ plaques and improved cognitive function. The lack of this modification directs BACE1 to late endosomes/lysosomes where it is less colocalized with APP, leading to accelerated lysosomal degradation. Notably, other BACE1 substrates, CHL1 and contactin‐2, are normally cleaved in GnT‐III‐deficient mice, suggesting that the effect of bisecting GlcNAc on BACE1 is selective to APP. Considering that GnT‐III‐deficient mice remain healthy, GnT‐III may be a novel and promising drug target for AD therapeutics.
Synopsis
Bisecting GlcNAc modification of BACE1 by GnT‐III is a novel regulator of BACE1 stability. Loss of GnT‐III curbs BACE1‐mediated Aβ plaque generation. Hence, the bisecting GlcNAc biosynthesis pathway is a promising new target for AD therapy.
Alzheimer's disease (AD) patients present high levels of bisecting GlcNAc modifications on BACE1.
Loss of bisecting GlcNAc reduces BACE1‐mediated Aβ generation, leading to amelioration of AD pathology.
Loss of bisecting GlcNAc reduces BACE1–APP localization.
The enzyme responsible for the biosynthesis of bisecting GlcNAc, GnT‐III, is therefore a potential new drug target for AD.
Bisecting GlcNAc modification of BACE1 by GnT‐III is a novel regulator of BACE1 stability. Loss of GnT‐III curbs BACE1‐mediated Aβ plaque generation. Hence, the bisecting GlcNAc biosynthesis pathway is a promising new target for AD therapy.
Abtract
Tau is a microtubule‐associated protein (MAP) that is localized to the axon. In Alzheimer's disease (AD), the distribution of tau undergoes a remarkable alteration, leading to the formation ...of tau inclusions in the somatodendritic compartment. While the abnormal aggregated tau has been extensively studied in human patient tissues and animal models of AD, how normal tau localizes to the axon, which would be the foundation to understand how the mis‐localization occurs, has not been well studied due to the poor detectability of normal unaggregated tau in vivo. Therefore, we developed immunohistochemical techniques that can detect normal mouse and human tau in brain tissues with high sensitivity. Using these techniques, we demonstrate the global distribution of tau in the mouse brain and confirmed that normal tau is exclusively localized to the axonal compartment in vivo. Interestingly, tau antibodies strongly labeled nonmyelinated axons such as hippocampal mossy fibers, while white matters generally exhibited low levels of immunoreactivity. Furthermore, mouse tau is highly expressed not only in neurons but also in oligodendrocytes. With super resolution imaging using the stimulated‐depletion microscopy, axonal tau appeared punctate rather than fibrous, indicating that tau decorates microtubules sparsely. Co‐labeling with presynaptic and postsynaptic markers revealed that normal tau is not localized to synapses but sparsely distributes in the axon. Taken together, this study reports novel antibodies to investigate the localization and mis‐localization of tau in vivo and novel findings of normal tau localization in the mouse brain.
Using newly developed antibodies, the physiological distribution of microtubule‐associated protein tau in the mouse brain was visualized at tissue, cellular, and super‐resolution levels. Tau is abundantly distributed in nonmyelinated axons or cell bodies of oligodendrocytes. Super resolution imaging also showed the distribution of tau on axonal microtubules for the first time.
The pathological hallmark of the majority of amyotrophic lateral sclerosis (ALS) cases is the mislocalization and aggregation of TAR DNA-binding protein 43 (TDP-43), an RNA-binding protein. Several ...studies have attributed disease processes of ALS to abnormal RNA metabolism. However, dysregulated biogenesis of RNA, especially non-coding RNA (ncRNA), is poorly understood. To resolve it, RNA-Seq, biochemical, and immunohistochemical analyses were performed on the pyramidal tract of the medulla oblongata of sporadic ALS (sALS) and control postmortem brain samples. Here, we report perturbation of ncRNA biogenesis in PIWI-interacting RNA (piRNA) in several sALS brain samples associated with TDP-43 pathology. In addition, we confirmed the dysregulation of two PIWI homologs, PIWI-like-mediated gene silencing 1 (PIWIL1) and PIWIL4, which bind to piRNAs to regulate their expression. PIWIL1 was mislocalized and co-localized with TDP-43 in motor neurons of sporadic ALS lumbar cords. Our results imply that dysregulation of piRNA, PIWIL1, and PIWIL4 is linked to pathogenesis of ALS. Based on these results, piRNAs and PIWI proteins are potential diagnostic biomarkers and therapeutic targets of ALS.
Amyloid β (Aβ) deposition in the brain is an early and invariable feature of Alzheimer's disease (AD). The Aβ peptides are composed of about 40 amino acids and are generated from amyloid precursor ...proteins (APP), by β- and γ-secretases. The distribution of individual Aβ peptides in the brains of aged people, and those suffering from AD and cerebral amyloid angiopathy (CAA), is not fully characterized. We employed the matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) to illustrate the spatial distribution of a broad range of Aβ species in human autopsied brains. With technical advancements such as formic acid pretreatment of frozen autopsied brain samples, we have: i) demonstrated that Aβ1-42 and Aβ1-43 were selectively deposited in senile plaques while full-length Aβ peptides such as Aβ1-36, 1-37, 1-38, 1-39, 1-40, and Aβ1-41 were deposited in leptomeningeal blood vessels. ii) Visualized distinct depositions of N-terminal truncated Aβ40 and Aβ42, including pyroglutamate modified at Glu-3 (N3pE), only with IMS for the first time. iii) Demonstrated that one single amino acid alteration at the C-terminus between Aβ1-42 and Aβ1-41 results in profound changes in their distribution pattern. In vitro, this can be attributed to the difference in the self-aggregation ability amongst Aβ1-40, Aβ1-41, and Aβ1-42. These observations were further confirmed with immunohistochemistry (IHC), using the newly developed anti-Aβ1-41 antibody. Here, distinct depositions of truncated and/or modified C- and N-terminal fragments of Aβs in AD and CAA brains with MALDI-IMS were visualized in a spacio-temporal specific manner. Specifically, Aβ1-41 was detected both with MALDI-IMS and IHC suggesting that a single amino acid alteration at the C-terminus of Aβ results in drastic distribution changes. These results suggest that MALDI-IMS could be used as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD and CAA.
Many pathological proteins related to neurodegenerative diseases are misfolded, aggregating to form amyloid fibrils during pathogenesis. One of the pathological proteins, alpha-synuclein (α-syn), ...accumulates in the brains of Parkinson disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), which are designated as synucleinopathies. Recently, structural properties of abnormal accumulated proteins are suggested to determine the disease phenotype. However, the biochemical and structural characteristics of those accumulated proteins are still poorly understood. We previously reported the sequence and seed-structure-dependent polymorphic fibrils of α-syn and the polymorphism was identified by proteinase K-resistant cores determined by mass spectrometry (MS) analysis. In this study, we applied this method to analyze α-syn aggregates of MSA and DLB. To perform MS analysis on proteinase K-resistant cores, we first performed amplification of α-syn aggregates by seeding reaction and protein misfolding cyclic amplification (PMCA) to obtain a sufficient amount of aggregates. Using SDS insoluble fraction of the disease brain, we successfully amplified enough α-syn aggregates for MS analysis. We differentiated between mouse and human α-syn aggregates by MS analysis on proteinase K-resistant cores of the aggregates before and after amplification. The results suggest that structural properties of amplified α-syn fibrils are preserved after PMCA and these methods can be applicable in the study of pathological proteins of the neurodegenerative disorders.
•Phosphorylated α-synuclein in SDS-insoluble fraction could be used as a seed.•α-synuclein aggregates were amplified by protein misfolding cyclic amplification.•Protease-resistant cores of amplified aggregates were identified by MALDI-TOF-MS.•The method can be applied to the study on misfolded proteins of neurodegeneration.
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