Brain Protein Aging and Dementia Control

Home > Groups and Members > Public Offering Members of Projects Research (2017-2018)

Public Offering Members of Projects Research (2017-2018)

Name Masahiro Ono
Affiliation / Position Kyoto University, Graduate School of Pharmaceutical Sciences
Associate Professor
Research Project Development of tau imaging with SPECT
Research Outline In Alzheimer’s disease brains, senile plaques composed of beta-amyloid protein and neurofibrillary tangles composed of tau protein are observed. It is generally accepted that the deposition of neurofibrillary tangles in the brain are closely related to the progression of Alzheimer’s disease compared with senile plaques. In this study, therefore, we aim to develop novel SPECT imaging probes targeting tau aggregates. We have previously found that benzimidazopyridine (BIP) may be a useful scaffold for the development of tau imaging probes as it showed highly selective binding affinity for tau aggregates and favorable pharmacokinetics in the brain (Ono et al., Sci Rep, 6, 34197, 2016). In the present study, we plan to design and synthesize a new series of BIP derivatives with various substituted groups and optimize the chemical structure for clinically useful SPECT imaging probes targeting tau aggregates.
Name Mizuta Kotaro
Affiliation / Position Kyoto University
Assistant Professor
Research Project Long-term visualization of functional breakdown processes of neural circuits in Alzheimer’s disease
Research Outline Breakdown of neural circuits as a result of deposition of pathogenic proteins leads to the decline of cognitive functions in Alzheimer’s disease (AD). One of early symptom of AD patients is dysfunction of spatial memory, where hippocampus plays an essential role. The precise process leading to functional breakdown progresses in the hippocampal neural circuits of AD has not been elucidated due to the technical difficulty in longitudinally monitoring hippocampal neuronal activities during pathogenesis. In this study, we will image activity of more than 1000 hippocampal neurons in awake AD model mice undergoing learning tasks over many months. For this purpose, we will combine two-photon calcium imaging of fluorescent calcium sensor protein G-CaMP7 and a virtual reality system for head-fixed mice. We aim to elucidate the entire process of AD pathology that may consist of abnormal neuronal activity, generation and deposition of aged brain proteins, and functional circuit breakdown in hippocampal microcircuits of AD model mice performing virtual recognition tasks.
Name Hiroaki Adachi
Affiliation / Position Department of Neurology, University of Occupational and Environmental Health School of Medicine
Research Project Functions of autophagy-related molecules with the aim of developing therapies for neurodegenarative diseases
Research Outline Neurodegenerative diseases (NDDs), including Alzheimer’s disease, frontotemporal dementia and polyglutamine diseases are a group of intractable diseases that significantly affect human health. To date, the pathogenesis of NDDs is still poorly understood and effective disease-modifying therapies for NDDs have not been established. NDDs share the common morphological characteristic of the deposition of abnormal proteins in the nervous system, including neurons. Autophagy is one of the major processes by which damaged organelles and abnormal proteins are removed from cells. Autophagy has been found to be involved in the pathogenesis of NDDs, and the regulation of autophagy may become a therapeutic strategy for NDDs. In this study, we investigate molecular mechanisms that regulate the decrease of pathogenic species by autophagy and aim to elucidate how this removal process contributes to ameliorate the pathology of NDDs. We also elucidate the role of dynactin1 and its related molecules in the fusion of autophagosomes with lysosomes and the removal of abnormal proteins.
Name Mitsunori Fukuda
Affiliation / Position Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University
Research Project Analysis of the molecular basis of Rab activation during brain protein aging
Research Outline Rab small GTPases are intracellular membrane trafficking proteins that are conserved in all eukaryotes. Approximately 60 Rab isoforms are present in humans and mice, and as the name indicates (ras genes in RAt Brain), most Rab isoforms are abundantly expressed in brain. Although mutations in certain Rab isoforms or their regulators have been shown to cause neurological disorders, how these molecules regulate intracellular membrane traffic or neuronal functions is poorly understood. In our laboratory, we have been systematically investigating the function of Rabs and their regulators in neurite outgrowth and axon/dendrite morphogenesis. During the course of the investigation, we recently found that ALS2 and C9ORF72 are functionally related to novel Rab-mediated membrane trafficking. Interestingly, mutations in ALS2 or C9ORF72 are known to cause amyotrophic lateral sclerosis (ALS), but the function(s) of their normal proteins in brain remains largely unknown. In this study, we would like to reveal the molecular mechanism by which ALS2 and C9ORF72 regulate intracellular membrane traffic, including autophagic pathways, especially focusing on their target Rabs, and to understand the association between defects in membrane traffic and brain protein aging that causes ALS.
Name Takafumi Hasegawa
Affiliation / Position Division of Neurology, Department of Neuroscience & Sensory Organs, Tohoku University Graduate School of Medicine
Associate Professor
Research Project Disease-modifying strategies targeting internalization of abnormal protein aggregates in synucleinopathy
Research Outline Propagation of abnormal protein aggregates via a prion-like mechanism has recently aroused special attention as a new paradigm for the pathogenesis of neurodegenerative diseases including Parkinson’s disease (PD). However, it should be kept in mind that the molecular mechanisms underlying the spreading of prionoid may differ depending on the biochemical nature of the protein aggregate, level of cellular stress, or the cell-type. We have elucidated complex mechanisms involved in membrane trafficking responsible for the uptake, secretion and degradation of alpha-synuclein (aS), a major culprit in PD. To further expand our knowledge and open up new avenue for disease-modifying therapy, we will investigate the protective effect of leading compounds such as endocytotic inhibitors on transcellualr spreads of aS in vivo. Furthermore, we will conduct comprehensive search for cell surface receptors that ease the entry of aS into neuronal and/or glial cells using brain-derived membrane protein library (MPL) and mass-spectrometric analysis. Looking ahead, we will also verify the usefulness of aS and Tau PET imaging as the tool for the surrogate marker in clinical trials in PD.
Name Tadafumi Hashimoto
Affiliation / Position Department of Neuropathology, the University of Tokyo
Associate Professor
Research Project Elucidation of the mechanisms of cell-to-cell transmission and neurotoxicity of FUS.
Research Outline Cell-to-cell transmission of disease-related proteins in neurodegenerative disorders is widely recognized, however, molecular mechanisms underlying the cell-to-cell transmission and the formation of inclusion bodies are still unclear. FUS has been identified as a causative protein in amyotrophic lateral sclerosis (ALS). Here, we will investigate the molecular mechanisms underlying the cell-to-cell transmission of FUS and the neurodegeneration induced by FUS. To address these topics, we have generated a human FUS overexpressing Drosophila melanogaster, which exhibited neurodegeneration in the retina. Moreover, we will develop a novel cellular sensor cells which detect cell-to-cell transmission of FUS using a bi-molecular complementation assay. Furthermore, we will develop a novel in vivo system which can visualize the cell-to-cell transmission from donor neurons to recipient neurons using a bi-cistronic expression vector. By the use of these novel models, we will elucidate the mechanism of cell-to-cell transmission of FUS, and answer the question whether the inhibition of cell-to-cell transmission of FUS suppresses the formation of inclusion bodies and the neurodegeneration induced by FUS.
Name Kazuki Tainaka
Affiliation / Position Brain Research Institute, Niigata University
Specially Appointed Professor
Research Project Analysis of brain protein aging by 3D immunohistochemistry
Research Outline In the aging-related neurodegenerative diseases, comprehensive analysis of pathological molecules (e.g. Tau, α-synuclein, TDP-43, prion) in human brain tissue would accelerate our understanding of molecular bases in brain protein aging. In this research, we aim to establish a novel 3D neuropathology by developing a highly efficient clearing protocol for human brain tissue and combining with a rapid 3D imaging using light-sheet fluorescence microscopy. We also develop an efficient photobleaching method for autofluorescence in the aged human brain tissues. Futhermore, we establish various kinds of specific chemical staining protocols and immunohistochemical protocols to visualize brain structure and pathological molecules.
Name Kaoru Yamada
Affiliation / Position The University of Tokyo, Graduate School of Medicine
Assistant professor
Research Project Elucidation of regulatory mechanisms for extracellular levels of tau that triggers intercellular spreading of tau pathology.
Research Outline Intracellular accumulation of misfolded tau protein as neurofibrillary tangles characterizes a set of neurodegenerative diseases called tauopathies. Despite the recognition that tau contributes to neuronal dysfunction, there is no clear understanding how tau pathology is developed in the diseases. Accumulating evidence from in vivo and cell culture studies have suggested that tau pathology propagates between cells via extracellular space. This transcellular spreading of tau pathology suggests new pathological mechanisms in which pathological tau species in a form of aggregates or fibrils are first released into the extracellular space and then taken up by neighboring neurons and induce further aggregation. This concept leads to the idea that interfering the processes involved in tau spreading could be a potential therapeutic strategy against tauopathies. In this study, we investigate molecular mechanisms that regulate the extracellular levels of tau species and aim to elucidate how this release process contributes to intercellular propagation of tau pathology.
Name Hideaki Morishita
Affiliation / Position Graduate School and Faculty of Medicine, The University of Tokyo
Assistant Professor
Research Project Dissection of the mechanisms by which autophagy suppresses neurodegeneration
Research Outline Autophagy is a major intracellular degradation system in which the autophagosome sequesters abnormal proteins and organelles and degrade them via fusion with the lysosome. Mutations in autophagy-related (ATG) genes have recently been identified in humans and shown to cause neurodegenerative disorders such as dementia and Parkinsonism. However, it has been unclear when and where autophagy is activated in the brain and why neurodegeneration occurs in autophagy-deficient brain. In this study, we reveal the spatiotemporal activity of autophagy in the brain using mice and zebrafish expressing a novel autophagic flux probe that we have recently developed. Furthermore, we generate several ATG-deficient zebrafish and mice and investigate the process and mechanisms underlying the development of neurodegeneration.
Name Hideaki Matsui
Affiliation / Position Department of Neuroscience of disease (in Brain Research Institute), Center for Transdisciplinary Research, Niigata University
Associate Professor
Research Project Aging of alpha-synuclein resulting in Parkinson’s disease
Research Outline Parkinson’s disease (PD) is strongly associated with aging. We have found that short-lived Africa killifish, which shows marked aging phenotypes, discloses PD-like pathology.

To elucidate the pathological mechanisms of PD for developing therapeutic agents, we will answer following three questions by analyzing small “fish” including Africa killifish, representative animal model “mouse”, and “human” samples.

I. Why PD is initiated at enteric nerves?
II. How we stop the progression of PD?
III. What is the mechanism of cell death in PD?

We will elucidate the mechanisms of PD initiation, progression and cell death by using various models and human samples, and clarify the strong relation between aging and PD pathogenesis.

Name Hiromitsu TANAKA
Affiliation / Position Department of Biophysics, Kyoto University Graduate School of Science
Assistant Professor
Research Project Imaging abnormal localization of glutamate receptors caused by Aβ oligomers
Research Outline We have succeeded in formation of
postsynaptic-like membrane (PSLM) directly on the glass surface in hippocampal neuronal culture by coating the glass with a kind of presynaptic adhesion molecule Neurexin, and performed live-cell imaging of glutamate receptor tagged by fluorescent protein with total internal reflection fluorescence microscopy. This method has enabled us to record the location and movement of glutamate receptors around PSLM with a high signal to noise ratio. Using this new method, we aim to elucidate the abnormal localization of AMAPA-type ionotropic glutamate receptors caused by amyloid beta (Aβ) oligomers during synaptic plasticity. This study would contribute the understanding of pathogenic mechanism of Alzheimer’s disease and molecular mechanism of learning and memory.
Name Ryosuke Takahashi
Affiliation / Position Department of Neurology, Kyoto University Graduate School of Medicie
Professor and Chair
Research Project New animal model of Parkinson’s disease based on a-synuclein propagation hypothesis
Research Outline Recent studies revealed that injection of a-synuclein preformed fibrils (a-Syn PFFs) into animal brains leads to formation of Lewy body-related pathology and its cell-to-cell propagation through neural networks. This has enabled creation of new generation PD models by injection of a-Syn PFFs into olfactory bulbs or gastrointestinal tracts and the striatum: The former may recapitulate the very early phase and latter nigral dopamine neuronal death accompanied by Lewy body formation. We have generated α-Syn(A53T) BAC Tg mouse, in which the physiological expression pattern of human α-Syn is maintained. We have also found that α-Syn propagation is dramatically accelerated in our mutant mouse model. In this project, we will establish a new generation PD model based on the combination of α-Syn Tg mouse and α-Syn injection. Moreover, we will elucidate PD pathophysiology and perform drug screening using these models.
Name Yoshitaka Nagai
Affiliation / Position Department of Neurotherapeutics, Osaka University Graduate School of Medicine
Research Project Maintenance of ribostasis against abnormal RNA accumulation by RNA-binding proteins
Research Outline In noncoding repeat expansion disorders, including C9orf72-linked amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD) and several types of spinocerebellar ataxia (SCA), abnormally expanded repeat RNAs are accumulated with sequestered RNA-binding proteins (RBPs) as RNA foci in affected tissues. However, a functional crosstalk between expanded repeat RNAs and RBPs has remained unclear. We recently discovered a novel role of TDP-43 as an RNA chaperone for UGGAA repeat RNA, which suppresses both RNA foci formation and repeat-associated translation, and subsequent neurodegeneration in an SCA31 Drosophila model (Ishiguro et al. Neuron 2017). In this study, we will elucidate mechanisms maintaining ribostasis against abnormal RNA accumulation by RBPs.
Name Suehiro Sakaguchi
Affiliation / Position Division of Molecular Neurobiology, The Institute foe Enzyme Research, Tokushima University
Research Project Identification of non-proteinaceous agents causing prion diseases and elucidation of their pathogenecity
Research Outline Conformational conversion of the cellular isoform of prion protein, designated PrPC, a membrane glycoprotein abundantly expressed on the cell surface of neurons, into the proteinase-resistant isoform of prion protein, PrPSc, is a pivotal pathogenic event in prion disease. Infectious cases account for less than 1% of human prion diseases. Hereditary cases linked to specific mutations in the PrP gene cover about 10%. Most cases are those of sporadic Creutzfeldt-Jakob disease with unknown etiologies.
An in vitro technique to induce the conversion of PrPC into PrPSc has been established. Using the in vitro conversion system, RNA and lipid molecules have a potential to induce the conversion of PrPC into PrPSc. These results suggest that agents other than prion might induce the conversion of PrPC into PrPSc. In this study, I will focus on identification of such agents causing the conversion of PrPC into PrPSc and elucidation of the conversion mechanism induced by the agents.
Name Kanae Ando
Affiliation / Position Department of Biological Sciences, Tokyo Metropolitan University
Associate Professor
Research Project Tau aggregation and toxicity in a Drosophila model
Research Outline Microtubule-associated protein tau accumulates in the brain of patients suffering from a group of neurodegenerative diseases called tauopathies, and tau accumulation is thought to contribute to disease pathogenesis. Tau assembles into various forms of aggregates including dimer, oligomer and fibrils in disease brains. However, the link between aggregation and toxicity is not fully understood. To tackle this problem, here we use Drosophila as a simple and powerful animal model. We will create transgenic fly lines to express mutant forms of human tau that are prone to form particular forms of aggregation, and compare the degree of neurodegeneration induced by those forms of tau. Degradation mechanisms of those tau will be also investigated. This study will shed light on causal link between tau aggregation and its toxicity and accumulation, which will further our understanding of disease pathogenesis and may lead to novel therapy for tauopathies.
Name Etsuro Ohta
Affiliation / Position Kitasato University School of Allied Health Sciences
Assistant professor
Research Project Analysis of the neurodegenerative mechanism through tau in disease-specific iPS cells
Research Outline Patients with Leucine-Rich-Repeat-Kinase 2 (LRRK2) mutation that is causal molecules of autosomal dominant hereditary form Parkinson’s disease (PD), exhibit clinical features indistinguishable from those of patients with sporadic PD. In addition, it has been reported that PD patient who has LRRK2 mutations cause dementia frequently. So, it is thought that LRRK2 has some kind of influences on the onset of dementia. Recently, we have reported induced pluripotent stem cells (iPSC) derived from patient with I2020T LRRK2 in the Sagamihara family (I2020T LRRK2-iPSC) replicate to some extent the pathologic phenotype evident in the brain of PD patients. In this study, to elucidate the onset mechanism of PD and dementia by 3R-Tau and 4R-Tau, we will analyze newly established gene corrected-iPSC from I2020T LRRK2-iPSC PD patient iPSC. Furthermore, to elucidate the neurodegeneration through Tau by mutant LRRK2, we will analyze microRNA-based conversion of human fibroblasts into subtype-specific neurons and iPSC-neurospheres-injected mice.
Name Yoshiaki Furukawa
Affiliation / Position Department of Chemistry, Keio University
Associate Professor
Research Project Pathogenic roles of misfolded proteins in neurodegenerative diseases
Research Outline Molecular mechanisms causing neurodegenerative diseases remain largely unknown. Most of the cases are sporadic, making it difficult to understand why and how the diseases occur. In my project, I will examine the mechanism in which transmission of misfolded proteins regulates the cause/progression of neurodegenerative diseases. Given that the protein misfolding is a part of the aging process, the ability of aged proteins to be transmitted in a prion-like manner could play important roles in the late-onset neurodegenerative diseases.
Name Masaki FUKATA
Affiliation / Position National Institute for Physiological Sciences
Research Project Role of the LGI1-mediated protein network in aging and dementia
Research Outline We have been working on the regulatory mechanisms for AMPA receptor-mediated synaptic transmission by an epilepsy-related LGI1-ADAM22 protein complex, as synaptic functions are assumed to be impaired at the earliest stage of dementia. LGI1 is a human epilepsy-related gene and one of the autoantigens for autoimmune limbic encephalitis, which is characterized by memory loss and seizures. So far, we have found that (1) secreted LGI1 functions as a ligand for ADAM22 and ADAM23 transmembrane proteins and regulates AMPA receptor-mediated synaptic transmission; (2) the human mutations of LGI1 or ADAM22 cause defects in LGI1 secretion or LGI1-ADAM22-binding; and (3) LGI1 autoantibodies neutralize LGI1-ADAM22 interaction, reducing synaptic AMPA receptors. Here, we will (1) examine the LGI1 expression and distribution in the brain of the aged brain, (2) clarify the molecular mechanisms of the LGI1-ADAM22 pathway, and (3) develop the LGI1 imaging in the brain.
Name Kozo Hamada
Affiliation / Position RIKEN/BSI
Research scientist
Research Project Role of endoplasmic reticulum calcium in protein aging and its toxic function
Research Outline One of the common symptoms in neurodegenerative diseases is abnormal regulation of conformations, oligomers, and fibrous structures of causative proteins, which we call as “protein aging”, but its cellular mechanism and gain of toxic function remains elusive. In this study, we focus on the “endoplasmic reticulum (ER) calcium” responsible for various cellular processes including synaptic plasticity and ER stress. We found that proteins causative for neurodegenerations could modulate calcium signaling mediated by the ER-resident calcium channel. Our next step is to investigate the molecular mechanism of the modulation and we will explore the relationship between ER calcium and protein aging.
Name Kozo Kaibuchi
Affiliation / Position Department of Cell Pharmacology, Graduate school of Medicine, Nagoya University
Research Project Proteomic approach for molecular pathogenesis underlying neurodegenerative disorders
Research Outline Dementia is a common neurocognitive disorder with large social, economic, and medical implications. One of the well-known Dementia is Alzheimer’s disease, which is characterized in clinical features by senile plaques and neurofibrillary tangles. However, the molecular pathogenesis underlying these features remains to be fully elucidated. We recently developed novel phosphoproteomic techniques, such as KISS and KIOSS methods, to comprehensively understand the cell signaling in cells and animals (Amano et al. J Cell Biol, 209, 2015; Nagai et al. Neuron, 89, 2016). In this research project, we identify the pathogenic signaling in the iPS cells derived from the patients with neurocognitive disorder using the proteomic approaches and photoactivated tools.
Name Hideki Mochizuki
Affiliation / Position Department of Neurology, Graduate School of Medicine, Osaka University
Research Project Establishment of a new PD mouse model using neurons differentiated from iPS cells derived from Parkinson disease patients
Research Outline Parkinson’s disease (PD) is the second most common neurodegenerative disease in Japan. Propagation hypothesis has been proposed as the pathological mechanism of PD. The structure of alpha synuclein changes in neurons and forms aggregates. Then, the α-synuclein aggregate propagates neurons and leads to neuronal cell death. We will develop a mouse model using patient-derived ips cells, not the conventional method using recombinant protein derived E. coli. By transplanting the neural cells differentiated from iPS cells derived from PD patients into the mouse brain, the intracellular environment associated with PD development is reproduced in the mouse brain. The purpose of this study is to elucidate the pathology of PD using this new PD model mouse.
Name Keiji Uchiyama
Affiliation / Position Division of Molecular Neurology, Institute for Advanced Medical Sciences, Tokushima University
Associate Professor
Research Project The analysis of the mechanism for membrane trafficking of aged brain protein involved in its degradation
Research Outline A fundamental event in the pathogenesis of prion disease is the conversion of cellular prion protein (PrPC) into abnormally folded isoform (PrPSc) which is the infectious causative agent of disease and accumulated with the progression of disease. To understand the pathogenesis of disease and develop a therapeutic approach, it is crucial to elucidate such mechanisms. PrPC mostly localized at the cell surface via its C-terminal glycosylphosphatidylinositol anchor. On the other hand, in our previous studies, the majority of PrPSc is observed intracellularly and widely distributed in post-Golgi compartments. We have identified a new PrP-binding protein, Sortilin and shown that Sortilin functions as a sorting receptor for PrP to the lysosomal degradation pathway and its dysfunction causes delayed degradation and excessive accumulation of PrPSc. However, it is unclear why the trafficking of PrPSc to the plasma membrane is restrained and it accumulates in recycling compartment. At present, we have identified a novel anti-prion compound which can reduce PrPSc level in prion-infected cells. This compound induced redistribution of intracellular PrPSc to the plasma membrane and PrPSc was subsequently internalized and degraded. In this study, we focused on such PrPSc-redistribution and aim to identify molecular machineries involved in the trafficking of PrPSc to the plasma membrane. Furthermore, we validate its anti-prion activity in vivo.
Name Yohei Okada
Affiliation / Position Aichi Medical University
Associate Professor
Research Project Analysis of pre-aggregation early pathology of neurodegenerative disorders in iPSC-derived neurons
Research Outline Disease specific human iPSCs, established from patients’ somatic cells, may provide valuable disease models by patient-derived cells, and could faithfully recapitulate disease onset and progression when differentiated into the cells affected by the disease. In this study, focusing on the abnormal protein accumulations that could cause neurodegeneration in neurological disorders, we would perform detailed analysis of the differentiation/maturation processes of iPSCs derived from patients of neurodegenerative disorders, and clarify underlying pathogenesis of disease onset and early disease progression, as well as the process of abnormal protein accumulation and neurodegeneration. By these analyses, we would especially focus on the early pathophysiology observed prior to abnormal protein accumulations to identify novel therapeutic targets for early diseases.
Name Koichi Kato
Affiliation / Position National Center of Neurology and Psychiatry, Integraive Brain Imaging Center
Section Chief
Research Project Development of PET tracers for imaging of CNS myelin
Research Outline Currently, we are trying to develop the PET tracers which image CNS myelin quantitatively with high sensitivity.
Dysfunction of myelination has been observed in dementia such as Alzheimer’s disease. Therefore quantitative imaging of CNS myelin change by PET could be an alternative bio-maker for differential diagnosis of dementia. In addition activated microglia participate in demyelinating lesions. Microglia possess both neurotoxic (M1) and neuroprotective (M2) properties and discrimination of M1/M2 polarization become a target for both evaluation of therapeutic efficacy and assessment of therapy.
We consider that specific PET imaging tracers enable rigorous imaging diagnosis and therapeutic evaluation for dementia. Visualization of immune response of microglia provides us novel therapeutic guidelines and should be therefore significant issue to overcome dementia.
Name Wado Akamatsu
Affiliation / Position Center for Genomic and Regenerative Medicine, Juntendo University Graduate School of Medicine
Project Professor
Research Project iPS cell-based modeling of pathogenic brain protein aging
Research Outline α-synuclein (α-syn) aggregation, mislocalization and accumulation are involved in the pathogenesis of neurodegenerative diseases. In this project, we develop a method to quantify abnormalα-syn in iPSC-derived neurons as a tool for disease research and drug screening. Further, we develop a method to model late-onset neurodegenerative diseases within several weeks of in vitro cultivation. Using these technologies, patient-specific iPSCs and genome-edited iPSCs, we try to establish a highly stable and reliable model of pathogenic brain protein aging.