Publications by authors named "Kenneth S Kosik"

158 Publications

High-content image-based analysis and proteomic profiling identifies Tau phosphorylation inhibitors in a human iPSC-derived glutamatergic neuronal model of tauopathy.

Sci Rep 2021 08 23;11(1):17029. Epub 2021 Aug 23.

Department of Neurology, Chemical Neurobiology Laboratory, Center for Genomic Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02114, USA.

Mutations in MAPT (microtubule-associated protein tau) cause frontotemporal dementia (FTD). MAPT mutations are associated with abnormal tau phosphorylation levels and accumulation of misfolded tau protein that can propagate between neurons ultimately leading to cell death (tauopathy). Recently, a p.A152T tau variant was identified as a risk factor for FTD, Alzheimer's disease, and synucleinopathies. Here we used induced pluripotent stem cells (iPSC) from a patient carrying this p.A152T variant to create a robust, functional cellular assay system for probing pathophysiological tau accumulation and phosphorylation. Using stably transduced iPSC-derived neural progenitor cells engineered to enable inducible expression of the pro-neural transcription factor Neurogenin 2 (Ngn2), we generated disease-relevant, cortical-like glutamatergic neurons in a scalable, high-throughput screening compatible format. Utilizing automated confocal microscopy, and an advanced image-processing pipeline optimized for analysis of morphologically complex human neuronal cultures, we report quantitative, subcellular localization-specific effects of multiple kinase inhibitors on tau, including ones under clinical investigation not previously reported to affect tau phosphorylation. These results demonstrate the potential for using patient iPSC-derived ex vivo models of tauopathy as genetically accurate, disease-relevant systems to probe tau biochemistry and support the discovery of novel therapeutics for tauopathies.
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http://dx.doi.org/10.1038/s41598-021-96227-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8382845PMC
August 2021

Extracellular detection of neuronal coupling.

Sci Rep 2021 07 19;11(1):14733. Epub 2021 Jul 19.

Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA, USA.

We developed a method to non-invasively detect synaptic relationships among neurons from in vitro networks. Our method uses microelectrode arrays on which neurons are cultured and from which propagation of extracellular action potentials (eAPs) in single axons are recorded at multiple electrodes. Detecting eAP propagation bypasses ambiguity introduced by spike sorting. Our methods identify short latency spiking relationships between neurons with properties expected of synaptically coupled neurons, namely they were recapitulated by direct stimulation and were sensitive to changing the number of active synaptic sites. Our methods enabled us to assemble a functional subset of neuronal connectivity in our cultures.
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http://dx.doi.org/10.1038/s41598-021-94282-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8289866PMC
July 2021

Liquid-liquid phase separation of Tau by self and complex coacervation.

Protein Sci 2021 Jul 19;30(7):1393-1407. Epub 2021 May 19.

Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, California, USA.

The liquid-liquid phase separation (LLPS) of Tau has been postulated to play a role in modulating the aggregation property of Tau, a process known to be critically associated with the pathology of a broad range of neurodegenerative diseases including Alzheimer's Disease. Tau can undergo LLPS by homotypic interaction through self-coacervation (SC) or by heterotypic association through complex-coacervation (CC) between Tau and binding partners such as RNA. What is unclear is in what way the formation mechanisms for self and complex coacervation of Tau are similar or different, and the addition of a binding partner to Tau alters the properties of LLPS and Tau. A combination of in vitro experimental and computational study reveals that the primary driving force for both Tau CC and SC is electrostatic interactions between Tau-RNA or Tau-Tau macromolecules. The liquid condensates formed by the complex coacervation of Tau and RNA have distinctly higher micro-viscosity and greater thermal stability than that formed by the SC of Tau. Our study shows that subtle changes in solution conditions, including molecular crowding and the presence of binding partners, can lead to the formation of different types of Tau condensates with distinct micro-viscosity that can coexist as persistent and immiscible entities in solution. We speculate that the formation, rheological properties and stability of Tau droplets can be readily tuned by cellular factors, and that liquid condensation of Tau can alter the conformational equilibrium of Tau.
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http://dx.doi.org/10.1002/pro.4101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8197434PMC
July 2021

The Multi-Partner Consortium to Expand Dementia Research in Latin America (ReDLat): Driving Multicentric Research and Implementation Science.

Front Neurol 2021 11;12:631722. Epub 2021 Mar 11.

Cognitive Neuroscience Center, Universidad de San Andrés, Buenos Aires, Argentina.

Dementia is becoming increasingly prevalent in Latin America, contrasting with stable or declining rates in North America and Europe. This scenario places unprecedented clinical, social, and economic burden upon patients, families, and health systems. The challenges prove particularly pressing for conditions with highly specific diagnostic and management demands, such as frontotemporal dementia. Here we introduce a research and networking initiative designed to tackle these ensuing hurdles, the Multi-partner consortium to expand dementia research in Latin America (ReDLat). First, we present ReDLat's regional research framework, aimed at identifying the unique genetic, social, and economic factors driving the presentation of frontotemporal dementia and Alzheimer's disease in Latin America relative to the US. We describe ongoing ReDLat studies in various fields and ongoing research extensions. Then, we introduce actions coordinated by ReDLat and the Latin America and Caribbean Consortium on Dementia (LAC-CD) to develop culturally appropriate diagnostic tools, regional visibility and capacity building, diplomatic coordination in local priority areas, and a knowledge-to-action framework toward a regional action plan. Together, these research and networking initiatives will help to establish strong cross-national bonds, support the implementation of regional dementia plans, enhance health systems' infrastructure, and increase translational research collaborations across the continent.
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http://dx.doi.org/10.3389/fneur.2021.631722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7992978PMC
March 2021

Patterns of neuronal Rhes as a novel hallmark of tauopathies.

Acta Neuropathol 2021 05 7;141(5):651-666. Epub 2021 Mar 7.

Memory and Aging Center, Weill Institute for Neurosciences, University of California, San Francisco, 675 Nelson Rising Lane, Box 1207, San Francisco, 94158, CA, USA.

The farnesyltransferase inhibitor, Lonafarnib, reduces tau inclusions and associated atrophy in familial tauopathy models through activation of autophagy, mediated by the inhibition of farnesylation of the Ras GTPase, Rhes. While hinting at a role of Rhes in tau aggregation, it is unclear how translatable these results are for sporadic forms of tauopathy. We examined histological slides of allocortex and neocortex from multiple postmortem cases in five different tauopathies, FTLD-TDP, and healthy controls using immunofluorescence for Rhes, several tau post-translational modifications, and phospho-TDP-43. Single nucleus RNA data suggest that Rhes is found in all cortical neuron subpopulations but not in glia. Histologic investigation showed that nearly all neurons in control brains display a pattern of diffuse cytoplasmic Rhes positivity. However, in the presence of abnormal tau, but not abnormal TDP-43, the patterns of neuronal cytoplasmic Rhes tend to present as either punctiform or entirely absent. This observation reinforces the relevance of findings that link Rhes changes and tau pathology from the in vivo and in vitro models of tauopathy. The results here support a potential clinical application of Lonafarnib to tauopathies.
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http://dx.doi.org/10.1007/s00401-021-02279-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8418783PMC
May 2021

Dementia in Latin America: Paving the way toward a regional action plan.

Alzheimers Dement 2021 02 20;17(2):295-313. Epub 2020 Nov 20.

Hospital Geral de Fortaleza, University of Fortaleza, Brazil.

Across Latin American and Caribbean countries (LACs), the fight against dementia faces pressing challenges, such as heterogeneity, diversity, political instability, and socioeconomic disparities. These can be addressed more effectively in a collaborative setting that fosters open exchange of knowledge. In this work, the Latin American and Caribbean Consortium on Dementia (LAC-CD) proposes an agenda for integration to deliver a Knowledge to Action Framework (KtAF). First, we summarize evidence-based strategies (epidemiology, genetics, biomarkers, clinical trials, nonpharmacological interventions, networking, and translational research) and align them to current global strategies to translate regional knowledge into transformative actions. Then we characterize key sources of complexity (genetic isolates, admixture in populations, environmental factors, and barriers to effective interventions), map them to the above challenges, and provide the basic mosaics of knowledge toward a KtAF. Finally, we describe strategies supporting the knowledge creation stage that underpins the translational impact of KtAF.
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http://dx.doi.org/10.1002/alz.12202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7984223PMC
February 2021

Comparison of Severe Acute Respiratory Syndrome Coronavirus 2 Screening Using Reverse Transcriptase-Quantitative Polymerase Chain Reaction or CRISPR-Based Assays in Asymptomatic College Students.

JAMA Netw Open 2021 02 1;4(2):e2037129. Epub 2021 Feb 1.

Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara.

Importance: The reopening of colleges and universities in the US during the coronavirus disease 2019 (COVID-19) pandemic is a significant public health challenge. The development of accessible and practical approaches for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in the college population is paramount for deploying recurrent surveillance testing as an essential strategy for virus detection, containment, and mitigation.

Objective: To determine the prevalence of SARS-CoV-2 in asymptomatic participants in a university community by using CREST (Cas13-based, rugged, equitable, scalable testing), a CRISPR-based test developed for accessible and large-scale viral screening.

Design, Setting, And Participants: For this cohort study, a total of 1808 asymptomatic participants were screened for SARS-CoV-2 using a CRISPR-based assay and a point-of-reference reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) test. Viral prevalence in self-collected oropharyngeal swab samples collected from May 28 to June 11, 2020, and from June 23 to July 2, 2020, was evaluated.

Exposures: Testing for SARS-CoV-2.

Main Outcomes And Measures: SARS-CoV-2 status, viral load, and demographic information of the study participants were collected.

Results: Among the 1808 participants (mean [SD] age, 27.3 [11.0] years; 955 [52.8%] female), 732 underwent testing from May to early June (mean [SD] age, 28.4 [11.7] years; 392 [53.6%] female). All test results in this cohort were negative. In contrast, 1076 participants underwent testing from late June to early July (mean [SD] age, 26.6 [10.5] years; 563 [52.3%] female), with 9 positive results by RT-qPCR. Eight of these positive samples were detected by the CRISPR-based assay and confirmed by Clinical Laboratory Improvement Amendments-certified diagnostic testing. The mean (SD) age of the positive cases was 21.7 (3.3) years; all 8 individuals self-identified as students. These metrics showed that a CRISPR-based assay was effective at capturing positive SARS-CoV-2 cases in this student population. Notably, the viral loads detected in these asymptomatic cases resemble those seen in clinical samples, highlighting the potential of covert viral transmission. The shift in viral prevalence coincided with the relaxation of stay-at-home measures.

Conclusions And Relevance: These findings reveal a shift in SARS-CoV-2 prevalence in a young and asymptomatic population and uncover the leading edge of a local outbreak that coincided with rising case counts in the surrounding county and the state of California. The concordance between CRISPR-based and RT-qPCR testing suggests that CRISPR-based assays are reliable and offer alternative options for surveillance testing and detection of SARS-CoV-2 outbreaks, as is required to resume operations in higher-education institutions in the US and abroad.
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http://dx.doi.org/10.1001/jamanetworkopen.2020.37129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7879237PMC
February 2021

miR-142-3p regulates cortical oligodendrocyte gene co-expression networks associated with tauopathy.

Hum Mol Genet 2021 03;30(1):103-118

Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 USA.

Oligodendrocytes exist in a heterogenous state and are implicated in multiple neuropsychiatric diseases including dementia. Cortical oligodendrocytes are a glial population uniquely positioned to play a key role in neurodegeneration by synchronizing circuit connectivity but molecular pathways specific to this role are lacking. We utilized oligodendrocyte-specific translating ribosome affinity purification and RNA-seq (TRAP-seq) to transcriptionally profile adult mature oligodendrocytes from different regions of the central nervous system. Weighted gene co-expression network analysis reveals distinct region-specific gene networks. Two of these mature myelinating oligodendrocyte gene networks uniquely define cortical oligodendrocytes and differentially regulate cortical myelination (M8) and synaptic signaling (M4). These two cortical oligodendrocyte gene networks are enriched for genes associated with dementia including MAPT and include multiple gene targets of the regulatory microRNA, miR-142-3p. Using a combination of TRAP-qPCR, miR-142-3p overexpression in vitro, and miR-142-null mice, we show that miR-142-3p negatively regulates cortical myelination. In rTg4510 tau-overexpressing mice, cortical myelination is compromised, and tau-mediated neurodegeneration is associated with gene co-expression networks that recapitulate both the M8 and M4 cortical oligodendrocyte gene networks identified from normal cortex. We further demonstrate overlapping gene networks in mature oligodendrocytes present in normal cortex, rTg4510 and miR-142-null mice, and existing datasets from human tauopathies to provide evidence for a critical role of miR-142-3p-regulated cortical myelination and oligodendrocyte-mediated synaptic signaling in neurodegeneration.
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http://dx.doi.org/10.1093/hmg/ddaa252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8496370PMC
March 2021

A Scalable, Easy-to-Deploy Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material.

J Clin Microbiol 2021 03 19;59(4). Epub 2021 Mar 19.

University of California, Santa Barbara, Department of Molecular, Cellular, and Developmental Biology, Santa Barbara, California, USA

The COVID-19 pandemic has created massive demand for widespread, distributed tools for detecting SARS-CoV-2 genetic material. The hurdles to scalable testing include reagent and instrument accessibility, availability of highly trained personnel, and large upfront investment. Here, we showcase an orthogonal pipeline we call CREST (Cas13-based, rugged, equitable, scalable testing) that addresses some of these hurdles. Specifically, CREST pairs commonplace and reliable biochemical methods (PCR) with low-cost instrumentation, without sacrificing detection sensitivity. By taking advantage of simple fluorescence visualizers, CREST allows a binary interpretation of results. CREST may provide a point-of-care solution to increase the distribution of COVID-19 surveillance.
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http://dx.doi.org/10.1128/JCM.02402-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092748PMC
March 2021

A Fast and Accessible Method for the Isolation of RNA, DNA, and Protein To Facilitate the Detection of SARS-CoV-2.

J Clin Microbiol 2021 03 19;59(4). Epub 2021 Mar 19.

Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, Santa Barbara, California, USA

Management of the coronavirus disease 2019 (COVID-19) pandemic requires widespread testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A main limitation for widespread SARS-CoV-2 testing is the global shortage of essential supplies, among them RNA extraction kits. The need for commercial RNA extraction kits places a bottleneck on tests that detect SARS-CoV-2 genetic material, including PCR-based reference tests. Here, we propose an alternative method we call PEARL (recipitation-nhanced nalyte etrieva) that addresses this limitation. PEARL uses a lysis solution that disrupts cell membranes and viral envelopes while simultaneously providing conditions suitable for alcohol-based precipitation of RNA, DNA, and proteins. PEARL is a fast, low-cost, and simple method that uses common laboratory reagents and offers performance comparable to that of commercial RNA extraction kits. PEARL offers an alternative method to isolate host and pathogen nucleic acids and proteins to streamline the detection of DNA and RNA viruses, including SARS-CoV-2.
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http://dx.doi.org/10.1128/JCM.02403-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092744PMC
March 2021

The impact of SARS-CoV-2 in dementia across Latin America: A call for an urgent regional plan and coordinated response.

Alzheimers Dement (N Y) 2020 23;6(1):e12092. Epub 2020 Nov 23.

Global Brain Health Institute (GBHI) University of California San Francisco (UCSF) San Francisco California USA.

The SARS-CoV-2 global pandemic will disproportionately impact countries with weak economies and vulnerable populations including people with dementia. Latin American and Caribbean countries (LACs) are burdened with unstable economic development, fragile health systems, massive economic disparities, and a high prevalence of dementia. Here, we underscore the selective impact of SARS-CoV-2 on dementia among LACs, the specific strain on health systems devoted to dementia, and the subsequent effect of increasing inequalities among those with dementia in the region. Implementation of best practices for mitigation and containment faces particularly steep challenges in LACs. Based upon our consideration of these issues, we urgently call for a coordinated action plan, including the development of inexpensive mass testing and multilevel regional coordination for dementia care and related actions. Brain health diplomacy should lead to a shared and escalated response across the region, coordinating leadership, and triangulation between governments and international multilateral networks.
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http://dx.doi.org/10.1002/trc2.12092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7683959PMC
November 2020

Liquid-Liquid Phase Separation of Tau Driven by Hydrophobic Interaction Facilitates Fibrillization of Tau.

J Mol Biol 2021 01 3;433(2):166731. Epub 2020 Dec 3.

Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, United States; Department of Chemical Engineering, University of California, Santa Barbara, CA 93106, United States. Electronic address:

Amyloid aggregation of tau protein is implicated in neurodegenerative diseases, yet its facilitating factors are poorly understood. Recently, tau has been shown to undergo liquid liquid phase separation (LLPS) both in vivo and in vitro. LLPS was shown to facilitate tau amyloid aggregation in certain cases, while being independent of aggregation in other cases. It is therefore important to understand the differentiating properties that resolve this apparent conflict. We report on a model system of hydrophobically driven LLPS induced by high salt concentration (LLPS-HS), and compare it to electrostatically driven LLPS represented by tau-RNA/heparin complex coacervation (LLPS-ED). We show that LLPS-HS promotes tau protein dehydration, undergoes maturation and directly leads to canonical tau fibrils, while LLPS-ED is reversible, remains hydrated and does not promote amyloid aggregation. We show that the nature of the interaction driving tau condensation is a differentiating factor between aggregation-prone and aggregation-independent LLPS.
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http://dx.doi.org/10.1016/j.jmb.2020.166731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855949PMC
January 2021

Tau PTM Profiles Identify Patient Heterogeneity and Stages of Alzheimer's Disease.

Cell 2020 12 13;183(6):1699-1713.e13. Epub 2020 Nov 13.

F.M. Kirby Neurobiology Center, Department of Neurobiology, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

To elucidate the role of Tau isoforms and post-translational modification (PTM) stoichiometry in Alzheimer's disease (AD), we generated a high-resolution quantitative proteomics map of 95 PTMs on multiple isoforms of Tau isolated from postmortem human tissue from 49 AD and 42 control subjects. Although Tau PTM maps reveal heterogeneity across subjects, a subset of PTMs display high occupancy and frequency for AD, suggesting importance in disease. Unsupervised analyses indicate that PTMs occur in an ordered manner, leading to Tau aggregation. The processive addition and minimal set of PTMs associated with seeding activity was further defined by analysis of size-fractionated Tau. To summarize, features in the Tau protein critical for disease intervention at different stages of disease are identified, including enrichment of 0N and 4R isoforms, underrepresentation of the C terminus, an increase in negative charge in the proline-rich region (PRR), and a decrease in positive charge in the microtubule binding domain (MBD).
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http://dx.doi.org/10.1016/j.cell.2020.10.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8168922PMC
December 2020

The proline-rich domain promotes Tau liquid-liquid phase separation in cells.

J Cell Biol 2020 11;219(11)

Department of Molecular, Cell and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA.

Tau protein in vitro can undergo liquid-liquid phase separation (LLPS); however, observations of this phase transition in living cells are limited. To investigate protein state transitions in living cells, we attached Cry2 to Tau and studied the contribution of each domain that drives the Tau cluster in living cells. Surprisingly, the proline-rich domain (PRD), not the microtubule binding domain (MTBD), drives LLPS and does so under the control of its phosphorylation state. Readily observable, PRD-derived cytoplasmic condensates underwent fusion and fluorescence recovery after photobleaching consistent with the PRD LLPS in vitro. Simulations demonstrated that the charge properties of the PRD predicted phase separation. Tau PRD formed heterotypic condensates with EB1, a regulator of plus-end microtubule dynamic instability. The specific domain properties of the MTBD and PRD serve distinct but mutually complementary roles that use LLPS in a cellular context to implement emergent functionalities that scale their relationship from binding α-beta tubulin heterodimers to the larger proportions of microtubules.
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http://dx.doi.org/10.1083/jcb.202006054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594490PMC
November 2020

COVID-19 in older people with cognitive impairment in Latin America.

Lancet Neurol 2020 09;19(9):719-721

Neuroscience Research Institute and Department of Molecular, Cellular, and Developmental Biology, University of California Santa Barbara, Santa Barbara, CA, USA.

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http://dx.doi.org/10.1016/S1474-4422(20)30270-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434452PMC
September 2020

LRP1 is a master regulator of tau uptake and spread.

Nature 2020 04 1;580(7803):381-385. Epub 2020 Apr 1.

Neuroscience Research Institute, Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA, USA.

The spread of protein aggregates during disease progression is a common theme underlying many neurodegenerative diseases. The microtubule-associated protein tau has a central role in the pathogenesis of several forms of dementia known as tauopathies-including Alzheimer's disease, frontotemporal dementia and chronic traumatic encephalopathy. Progression of these diseases is characterized by the sequential spread and deposition of protein aggregates in a predictable pattern that correlates with clinical severity. This observation and complementary experimental studies have suggested that tau can spread in a prion-like manner, by passing to naive cells in which it templates misfolding and aggregation. However, although the propagation of tau has been extensively studied, the underlying cellular mechanisms remain poorly understood. Here we show that the low-density lipoprotein receptor-related protein 1 (LRP1) controls the endocytosis of tau and its subsequent spread. Knockdown of LRP1 significantly reduced tau uptake in H4 neuroglioma cells and in induced pluripotent stem cell-derived neurons. The interaction between tau and LRP1 is mediated by lysine residues in the microtubule-binding repeat region of tau. Furthermore, downregulation of LRP1 in an in vivo mouse model of tau spread was found to effectively reduce the propagation of tau between neurons. Our results identify LRP1 as a key regulator of tau spread in the brain, and therefore a potential target for the treatment of diseases that involve tau spread and aggregation.
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http://dx.doi.org/10.1038/s41586-020-2156-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687380PMC
April 2020

Tau Condensates.

Adv Exp Med Biol 2019 ;1184:327-339

Biomolecular Science and Engineering, University of California, Santa Barbara, CA, USA.

Many proteins, particularly those that are intrinsically disordered and carry charges have a tendency to undergo liquid liquid phase separation (LLPS). Phase separation is a widespread mechanism by which cells concentrate a set of proteins to perform molecular reactions, and appear to compartmentalize molecular functions. Among the intrinsically disordered proteins are a subset that tend to form solid inclusions in cells and contribute to the pathology of several neurodegenerative diseases. Among this subset is the tau protein, a critically important inclusion in a class of conditions known as the tauopathies, which include Alzheimer's disease. Tau in neurons strongly and selectively associates with RNA species, most notably tRNA with a nanomolar dissociation constant. Furthermore, tau and RNA, under charge matching conditions, undergo LLPS in a process known as complex coacervation. Tau-RNA LLPS is reversible, and can persist for more than 15 h without subsequent fibrilization, although after longer time periods β-sheet content can be detected by thioflavin T. These findings suggest that LLPS tau droplets or condensates can be placed on a pathway to fibrillization and be arrested by solidification or dissolve into a soluble state, depending on the condition at hand, suggesting a regulatory and physiological role for the phase separated state of tau.
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http://dx.doi.org/10.1007/978-981-32-9358-8_24DOI Listing
April 2020

Microglial microRNAs mediate sex-specific responses to tau pathology.

Nat Neurosci 2020 02 23;23(2):167-171. Epub 2019 Dec 23.

Neuroscience Graduate Program, University of California, San Francisco, San Francisco, CA, USA.

Sex is a key modifier of neurological disease outcomes. Microglia are implicated in neurological diseases and modulated by microRNAs, but it is unknown whether microglial microRNAs have sex-specific influences on disease. We show in mice that microglial microRNA expression differs in males and females and that loss of microRNAs leads to sex-specific changes in the microglial transcriptome and tau pathology. These findings suggest that microglial microRNAs influence tau pathogenesis in a sex-specific manner.
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http://dx.doi.org/10.1038/s41593-019-0560-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7394069PMC
February 2020

Resistance to autosomal dominant Alzheimer's disease in an APOE3 Christchurch homozygote: a case report.

Nat Med 2019 11 4;25(11):1680-1683. Epub 2019 Nov 4.

The Banner Alzheimer's Institute, Phoenix, AZ, USA.

We identified a PSEN1 (presenilin 1) mutation carrier from the world's largest autosomal dominant Alzheimer's disease kindred, who did not develop mild cognitive impairment until her seventies, three decades after the expected age of clinical onset. The individual had two copies of the APOE3 Christchurch (R136S) mutation, unusually high brain amyloid levels and limited tau and neurodegenerative measurements. Our findings have implications for the role of APOE in the pathogenesis, treatment and prevention of Alzheimer's disease.
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http://dx.doi.org/10.1038/s41591-019-0611-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6898984PMC
November 2019

A Comprehensive Resource for Induced Pluripotent Stem Cells from Patients with Primary Tauopathies.

Stem Cell Reports 2019 11 17;13(5):939-955. Epub 2019 Oct 17.

Department of Molecular Cellular and Developmental Biology, Neuroscience Research Institute, Biomolecular Science and Engineering Program, University of California, Santa Barbara, Santa Barbara, CA, USA.

Primary tauopathies are characterized neuropathologically by inclusions containing abnormal forms of the microtubule-associated protein tau (MAPT) and clinically by diverse neuropsychiatric, cognitive, and motor impairments. Autosomal dominant mutations in the MAPT gene cause heterogeneous forms of frontotemporal lobar degeneration with tauopathy (FTLD-Tau). Common and rare variants in the MAPT gene increase the risk for sporadic FTLD-Tau, including progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). We generated a collection of fibroblasts from 140 MAPT mutation/risk variant carriers, PSP, CBD, and cognitively normal controls; 31 induced pluripotent stem cell (iPSC) lines from MAPT mutation carriers, non-carrier family members, and autopsy-confirmed PSP patients; 33 genome engineered iPSCs that were corrected or mutagenized; and forebrain neural progenitor cells (NPCs). Here, we present a resource of fibroblasts, iPSCs, and NPCs with comprehensive clinical histories that can be accessed by the scientific community for disease modeling and development of novel therapeutics for tauopathies.
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http://dx.doi.org/10.1016/j.stemcr.2019.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895712PMC
November 2019

Control over single-cell distribution of G1 lengths by WNT governs pluripotency.

PLoS Biol 2019 09 26;17(9):e3000453. Epub 2019 Sep 26.

Department of Molecular, Cellular, and Developmental Biology, Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, California, United States of America.

The link between single-cell variation and population-level fate choices lacks a mechanistic explanation despite extensive observations of gene expression and epigenetic variation among individual cells. Here, we found that single human embryonic stem cells (hESCs) have different and biased differentiation potentials toward either neuroectoderm or mesendoderm depending on their G1 lengths before the onset of differentiation. Single-cell variation in G1 length operates in a dynamic equilibrium that establishes a G1 length probability distribution for a population of hESCs and predicts differentiation outcome toward neuroectoderm or mesendoderm lineages. Although sister stem cells generally share G1 lengths, a variable proportion of cells have asymmetric G1 lengths, which maintains the population dispersion. Environmental Wingless-INT (WNT) levels can control the G1 length distribution, apparently as a means of priming the fate of hESC populations once they undergo differentiation. As a downstream mechanism, global 5-hydroxymethylcytosine levels are regulated by G1 length and thereby link G1 length to differentiation outcomes of hESCs. Overall, our findings suggest that intrapopulation heterogeneity in G1 length underlies the pluripotent differentiation potential of stem cell populations.
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http://dx.doi.org/10.1371/journal.pbio.3000453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6782112PMC
September 2019

Pathogenic Tau Impairs Axon Initial Segment Plasticity and Excitability Homeostasis.

Neuron 2019 11 18;104(3):458-470.e5. Epub 2019 Sep 18.

Gladstone Institute of Neurological Disease, San Francisco, CA 94158, USA; Department of Neurology, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:

Dysregulation of neuronal excitability underlies the pathogenesis of tauopathies, including frontotemporal dementia (FTD) with tau inclusions. A majority of FTD-causing tau mutations are located in the microtubule-binding domain, but how these mutations alter neuronal excitability is largely unknown. Here, using CRISPR/Cas9-based gene editing in human pluripotent stem cell (iPSC)-derived neurons and isogenic controls, we show that the FTD-causing V337M tau mutation impairs activity-dependent plasticity of the cytoskeleton in the axon initial segment (AIS). Extracellular recordings by multi-electrode arrays (MEAs) revealed that the V337M tau mutation in human neurons leads to an abnormal increase in neuronal activity in response to chronic depolarization. Stochastic optical reconstruction microscopy of human neurons with this mutation showed that AIS plasticity is impaired by the abnormal accumulation of end-binding protein 3 (EB3) in the AIS submembrane region. These findings expand our understanding of how FTD-causing tau mutations dysregulate components of the neuronal cytoskeleton, leading to network dysfunction.
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http://dx.doi.org/10.1016/j.neuron.2019.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880876PMC
November 2019

In vitro validation of in silico identified inhibitory interactions.

J Neurosci Methods 2019 06 6;321:39-48. Epub 2019 Apr 6.

Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA; Helen Wills Neuroscience Institute and Redwood Center for Theoretical Neuroscience, University of California, Berkeley, CA, USA. Electronic address:

Background: Understanding how neuronal signals propagate in local network is an important step in understanding information processing. As a result, spike trains recorded with multi-electrode arrays (MEAs) have been widely used to study the function of neural networks. Studying the dynamics of neuronal networks requires the identification of both excitatory and inhibitory connections. The detection of excitatory relationships can robustly be inferred by characterizing the statistical relationships of neural spike trains. However, the identification of inhibitory relationships is more difficult: distinguishing endogenous low firing rates from active inhibition is not obvious.

New Method: In this paper, we propose an in silico interventional procedure that makes predictions about the effect of stimulating or inhibiting single neurons on other neurons, and thereby gives the ability to accurately identify inhibitory effects.

Comparison: To experimentally test these predictions, we have developed a Neural Circuit Probe (NCP) that delivers drugs transiently and reversibly on individually identified neurons to assess their contributions to the neural circuit behavior.

Results: Using the NCP, putative inhibitory connections identified by the in silico procedure were validated through in vitro interventional experiments.

Conclusions: Together, these results demonstrate how detailed microcircuitry can be inferred from statistical models derived from neurophysiology data.
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http://dx.doi.org/10.1016/j.jneumeth.2019.04.002DOI Listing
June 2019

Narrow equilibrium window for complex coacervation of tau and RNA under cellular conditions.

Elife 2019 04 5;8. Epub 2019 Apr 5.

Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, United States.

The mechanism that leads to liquid-liquid phase separation (LLPS) of the tau protein, whose pathological aggregation is implicated in neurodegenerative disorders, is not well understood. Establishing a phase diagram that delineates the boundaries of phase co-existence is key to understanding whether LLPS is an equilibrium or intermediate state. We demonstrate that tau and RNA reversibly form complex coacervates. While the equilibrium phase diagram can be fit to an analytical theory, a more advanced model is investigated through field theoretic simulations (FTS) that provided direct insight into the thermodynamic driving forces of tau LLPS. Together, experiment and simulation reveal that tau-RNA LLPS is stable within a narrow equilibrium window near physiological conditions over experimentally tunable parameters including temperature, salt and tau concentrations, and is entropy-driven. Guided by our phase diagram, we show that tau can be driven toward LLPS under live cell coculturing conditions with rationally chosen experimental parameters.
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http://dx.doi.org/10.7554/eLife.42571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450672PMC
April 2019

A farnesyltransferase inhibitor activates lysosomes and reduces tau pathology in mice with tauopathy.

Sci Transl Med 2019 03;11(485)

Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA 93106, USA.

Tau inclusions are a shared feature of many neurodegenerative diseases, among them frontotemporal dementia caused by tau mutations. Treatment approaches for these conditions include targeting posttranslational modifications of tau proteins, maintaining a steady-state amount of tau, and preventing its tendency to aggregate. We discovered a new regulatory pathway for tau degradation that operates through the farnesylated protein, Rhes, a GTPase in the Ras family. Here, we show that treatment with the farnesyltransferase inhibitor lonafarnib reduced Rhes and decreased brain atrophy, tau inclusions, tau sumoylation, and tau ubiquitination in the rTg4510 mouse model of tauopathy. In addition, lonafarnib treatment attenuated behavioral abnormalities in rTg4510 mice and reduced microgliosis in mouse brain. Direct reduction of Rhes in the rTg4510 mouse by siRNA reproduced the results observed with lonafarnib treatment. The mechanism of lonafarnib action mediated by Rhes to reduce tau pathology was shown to operate through activation of lysosomes. We finally showed in mouse brain and in human induced pluripotent stem cell-derived neurons a normal developmental increase in Rhes that was initially suppressed by tau mutations. The known safety of lonafarnib revealed in human clinical trials for cancer suggests that this drug could be repurposed for treating tauopathies.
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http://dx.doi.org/10.1126/scitranslmed.aat3005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961212PMC
March 2019

iPSCs-derived nerve-like cells from familial Alzheimer's disease PSEN 1 E280A reveal increased amyloid-beta levels and loss of the Y chromosome.

Neurosci Lett 2019 06 20;703:111-118. Epub 2019 Mar 20.

Neuroscience Research Group, Medical Research Institute, Faculty of Medicine, University of Antioquia (UdeA), Calle 70 No. 52-21, and Calle 62 # 52-59, Building 1, Room 412, SIU Medellin, Colombia. Electronic address:

Alzheimer's disease (AD) is a progressive, degenerative disorder that mainly results in memory loss and a cognitive disorder. Although the cause of AD is still unknown, a minor percentage of AD cases are produced by genetic mutations in the presenilin-1 (PSEN1) gene. Differentiated neuronal cells derived from induced pluripotent stem cells (iPSCs) of patients can recapitulate key pathological features of AD in vitro; however, iPSCs studies focused on the p.E280 A mutation, which afflicts the largest family in the world with familial AD, have not been carried out yet. Although a link between the loss of the Y (LOY) chromosome in peripheral blood cells and risk for AD has been reported, LOY-associated phenotype has not been previously studied in PSEN1 E280 A carriers. Here, we report the reprogramming of fibroblast cells into iPSCs from a familial AD patient with the PSEN1 E280 A mutation, followed by neuronal differentiation into neural precursor cells (NPCs), and the differentiation of NPCs into differentiated neurons that lacked a Y chromosome. Although the PSEN1 E280 A iPSCs and NPCs were successfully obtained, after 8 days of differentiation, PSEN1 E280 A differentiated neurons massively died reflected by release and/ or activation of death markers, and failed to reach complete neural differentiation compared to PSEN 1 wild type cells.
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http://dx.doi.org/10.1016/j.neulet.2019.03.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845654PMC
June 2019

Non-contact monitoring of extra-cellular field potentials with a multi-electrode array.

Lab Chip 2019 04;19(8):1448-1457

Neuroscience Research Institute, University of California Santa Barbara, Santa Barbara, CA 93106, USA.

Developing tools to enable non-invasive, high-throughput electrophysiology measurements of large functional-networks of electrogenic cells used as in vitro disease models for the heart and brain remains an outstanding challenge for preclinical drug discovery, where failures are costly and can prove to be fatal during clinical trials. Here we demonstrate, for the first time, that it is possible to perform non-contact monitoring of extra-cellular field potentials with a multi-electrode array (MEA). To do this preliminary demonstration we built a prototype with a custom mechanical stage to micro-position cells grown on conventional glass coverslips over the recording surface of a MEA sensor. The prototype can monitor extra-cellular fields generated by multi-cellular networks in a non-contact configuration, enabling a single MEA sensor to probe different cultures in succession, without fouling or degrading its sensitive electronic surface. This first demonstration with easy to culture cardiomyocyte cells and a prototype device points to the exciting possibility for instrument development leading to more efficient and cost-effective drug screening paradigms for cardiovascular and neurological diseases.
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http://dx.doi.org/10.1039/c8lc00984hDOI Listing
April 2019

Genetic origin of a large family with a novel PSEN1 mutation (Ile416Thr).

Alzheimers Dement 2019 05 10;15(5):709-719. Epub 2019 Feb 10.

Grupo de Neurociencias de Antioquia, School of Medicine, Universidad de Antioquia, Medellín, Antioquia, Colombia.

Introduction: A small percentage of Alzheimer's disease (AD) cases are caused by genetic mutations with autosomal dominant inheritance. We report a family with a novel variant in PSEN1.

Methods: We performed clinical and genetic evaluation of 93 related individuals from a Colombian admixed population. 31 individuals had whole-genome sequencing.

Results: Genetic analysis revealed a missense variant in PSEN1 (NM_000021.3: c.1247T>C p.Ile416Thr), which originated on an African haplotype and segregated with AD logarithm of the odds score of 6. Their clinical phenotype is similar to sporadic AD except for earlier age at onset: the mean age at onset for mild cognitive impairment was 47.6 years (standard deviation 5.83) and for dementia 51.6 years (standard deviation 5.03).

Discussion: Ile416Thr is a novel pathogenic variant that causes AD in the sixth decade of life. The history of the region that included slave importation and admixtures within a confined geographic locale represents a "mini-population bottleneck" and subsequent emergence of a rare dominant mutation.
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http://dx.doi.org/10.1016/j.jalz.2018.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511480PMC
May 2019

Cell biology in support of neurological research: 2018 highlights.

Authors:
Kenneth S Kosik

Lancet Neurol 2019 Jan;18(1):19-20

Department of Molecular, Cellular and Developmental Biology, Neuroscience Research Institute, University of California at Santa Barbara, Santa Barbara, CA 93106-5060, USA. Electronic address:

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http://dx.doi.org/10.1016/S1474-4422(18)30462-9DOI Listing
January 2019

Cofactors are essential constituents of stable and seeding-active tau fibrils.

Proc Natl Acad Sci U S A 2018 12 11;115(52):13234-13239. Epub 2018 Dec 11.

Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106;

Amyloid fibrils are cross-β-rich aggregates that are exceptionally stable forms of protein assembly. Accumulation of tau amyloid fibrils is involved in many neurodegenerative diseases, including Alzheimer's disease (AD). Heparin-induced aggregates have been widely used and assumed to be a good tau amyloid fibril model for most biophysical studies. Here we show that mature fibrils made of 4R tau variants, prepared with heparin or RNA, spontaneously depolymerize and release monomers when their cofactors are removed. We demonstrate that the cross-β-sheet assembly formed in vitro with polyanion addition is unstable at room temperature. We furthermore demonstrate high seeding capacity with transgenic AD mouse brain-extracted tau fibrils in vitro that, however, is exhausted after one generation, while supplementation with RNA cofactors resulted in sustained seeding over multiple generations. We suggest that tau fibrils formed in brains are supported by unknown cofactors and inhere higher-quality packing, as reflected in a more distinct conformational arrangement in the mouse fibril-seeded, compared with heparin-induced, tau fibrils. Our study suggests that the role of cofactors in tauopathies is a worthy focus of future studies, as they may be viable targets for diagnosis and therapeutics.
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http://dx.doi.org/10.1073/pnas.1810058115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310788PMC
December 2018
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