Axodendritische Verteilung der Isoformen von Tau in primären neuronalen Zellkulturmodellen der Demenz

Tau ist ein neuronales Protein, welches in kausalen Zusammenhang mit der Alzheimer Demenzerkrankung (AD), der Frontotemporalen Demenz (FTD), und anderen Tauopathien gebracht wird. Als Mikrotubuli-Assoziertes Protein (MAP) ist Tau vorwiegend in axonalen Fortsätzen von Neuronen lokalisiert. Im pathologischen Verlauf der AD wird Tau aber auch in die dendritischen Fortsätze von Neuronen umverteilt. Die polare Verteilung von Tau wird somit aufgehoben. Tau kommt im Zentralen Nervensystem von Erwachsenen in 6 Isoformen vor, die sich durch die An- oder Abwesenheit von 2 N-terminalen Einschüben (N) oder der 2. (von insgesamt 4) Repeat-Einheit (R) unterscheiden. Der Einfluss dieser 6 Isoformen von Tau auf den Prozess der pathologischen Fehlverteilung ist nicht bekannt. In dieser Arbeit wurde daher die physiologische und die pathologische Verteilung der unterschiedlichen Tau-Isoformen untersucht. Als Modell wurde ein definiertes Modell der neuronalen Zellpolarität, primäre Nagetiervorderhirnneurone, verwendet. Primäre Neurone sind ein etabliertes Modellsystem für die Untersuchung von Tau im Kontext der neuronalen Entwicklung, sowie für die Untersuchung neuronaler Krankheitsprozesse. Primäre Neuronen zu kultivieren ist jedoch aufwendig und erfordert meist die Verwendung von kostenintensiven und patent-geschützten Zellkulturmedien unbekannter Zusammensetzung. Im ersten Kapitel wird daher eine kostengünstige und simplifizierte Prozedur zum Kultivieren von primären Neuronen beschrieben, basierend auf einer mittlerweile kommerziell erhältlichen Variante eines neuronalen Zellkultursupplement (NS21) mit bekannter Zusammensetzung. Weiterhin werden Techniken für die Fixierung und Immunfluoreszenzfärbung beschrieben, die für die Untersuchung der Verteilung von Tau in primären Neuronen notwendig sind. Primäre Neurone sind mit nicht-viralen Vektoren schwierig zu transfizieren und empfind-lich gegenüber zytoskelettalen Manipulationen und Lebend-Zell-Beobachtung, insbeson-dere nach mehrwöchiger Kultivierung. In einem zweiten Kapitel wird daher eine einfache nicht-virale Transfektionsmethode beschrieben, die es ermöglicht Tau in allen neuronalen Entwicklungsstufen in ähnlich niedrigen Mengen wie endogenes Tau zu exprimieren. Mithilfe der o. g. Methoden, insbesondere Transfektion der unterschiedlichen Tau-Isoformen des Menschen und der Maus in primären Neuronen, wurde schließlich in einem dritten Kapitel das axodendritische Verteilungsmuster von Tau untersucht. Es zeigte sich, dass die Tau Diffusionsbarriere (TDB), lokalisiert inmitten des Axon-Initialen-Segments (AIS), sowohl die retrograde als auch die anterograde Ausbreitung von Tau kontrolliert. Während die Tau-Isoformen ohne N-terminale Inserts effizient in das Axon geleitet werden, verbleibt die längste Tau-Isoform (2N4R-Tau) teilweise im Zellsoma und Dendriten, wo sie das Wachstum von Dendriten und dendritischen Dornen beschleunigt. Die TDB (lokalisiert im AIS) wurde durch Knockdown von AIS-Komponenten (ankyrin G, EB1), oder Überexpression einer AD-assozierten Kinase, Glykogen-Synthase-Kinase-3-beta (GSK3beta), gestört. Mithilfe von hochauflösender Nanoskopie und Lebendzellbeobachtung konnte gezeigt werden, dass die Mikrotubuli im AIS sehr dynamisch sind, eine axonale Besonderheit essentiell für die Funktion der TDB. Pathomechanistische Veränderungen im AIS nach Exposition mit Amyloid-beta (der AD-auslösende Faktor) waren Aktivierung von Cofilin und f-Aktin Umstrukturierung (beides wichtige Regulatoren des Aktin basierten Zytoskeletts), sowie eine reduzierte Dynamik des Mikrotubuli-Zellskelettsystems. Gleichzeitig brach die AIS/TDB Verteilungsfunktion zusammen, was zu AD-ähnlicher Fehlverteilung von Tau führte. Insgesamt wurden in der vorliegenden Arbeit Methoden für die Kultivierung und Transfektion von primären Neuronen entwickelt, mittels welcher dann gezeigt wurde, dass die unterschiedlichen Tau Isoformen von Mensch und Maus sich sowohl untereinander, als auch in AD-ähnlichem Stress im axodendritischen Verteilungsmuster unterscheiden. Die Tau-Isoformen beschleunigen in unterschiedlichem Ausmaß das Dendriten- und dendritisches Dornenwachstum. Weiterhin hängt die differenzielle axodendritische Verteilung der Tau Isoformen von der Integrität der TDB und des AIS ab. Zukünftige Forschung wird die spezielle Verteilung und die dendritischen Effekte insbesondere von 2N4R-Tau, welches derzeit bevorzugt für Mausmodelle eingesetzt wird, in Betracht ziehen müssen.Tau isoforms show differential axodendritic distributions in primary neuronal cell culture models of dementia The protein Tau is a neuronal protein associated with Alzheimer Disease (AD), Frontotemporal Dementia (FTD) and many other neurological diseases summarized as Tauopathies. Tau is a Microtubule Associated Protein (MAP) present predominantly in axons, but becomes mislocalized in pathological settings. In the human central nervous system (CNS), Tau exists in 6 splice variants, differentiated by the presence or absence of the second of four repeats or of 2 N-terminal inserts. The contribution of these Tau isoforms to axodendritic mislocalization in neuropsychiatric disease is understudied. Here we investigate the physiological sorting and pathological missorting of the different isoforms of Tau in primary rodent forebrain neurons. This cell model is an established model of neuronal cell polarity. Primary neurons have proven to be an invaluable tool for the investigation of Tau and neuronal cell polarity in the context of neuronal development and neurodegeneration. Culturing neurons, however, is time consuming and requires multiple feeding steps and media exchanges, and either the use of proprietary media supplements or tedious preparation of complex media. In a first step we describe and define a relatively cheap and easy cell culture procedure based on a commercially available neuronal culture supplement with 21 ingredents (NS21) of known composition, as well as basic fixation techniques. Also, mature primary neurons are notoriously difficult to transfect with nonviral vectors and are very sensitive both to cytoskeletal manipulation and to imaging. Thus, in a second step, we developed and described a simple nonviral transfection method enabling transfection of Tau to achieve expression levels comparable to endogenous Tau. Finally, using the above described methods such as expression of different isoforms of human and mouse Tau in primary neurons, we investigated the sorting behavior of Tau. We found that the Tau diffusion barrier (TDB), located within the axon initial segment (AIS), controls not only retrograde but also anterograde traffic of Tau. Tau isoforms without the N-terminal inserts were sorted efficiently into the axon. However, the longest isoform (2N4R-Tau) was partially retained in cell bodies and dendrites, where it accelerated spine and dendrite growth. The TDB was impaired when AIS components (ankyrin G, EB1) were knocked down or when glycogen synthase kinase-3-beta (GSK3beta; an AD-associated kinase tethered to the AIS) was overexpressed. Using superresolution nanoscopy and live-cell imaging, we observed that microtubules within the AIS appeared highly dynamic, a feature essential for the TDB. Pathomechanistically, amyloid-beta insult caused cofilin activation, f-actin remodeling and subsequent impaired microtubule dynamics in the AIS. Concomitantly, the AIS/TDB sorting function failed, causing AD-like Tau missorting. In summary, we provide evidence that the Tau isoforms differ in physiological and pathological axodendritic sorting, and that the axodendritic distribution of Tau depends on AIS integrity and maintenance of microtubule dynamics. Current mouse models mainly expressing only human 2N4R-Tau isoform will have to be reevaluated due to the particular distribution and function of this usually very little expressed isoform

A perspective on human cell models for POLG-spectrum disorders: advantages and disadvantages of CRISPR-Cas-based vs. patient-derived iPSC models

Neurogenetic diseases represent a broad group of diseases with variable genetic causes and clinical manifestations. Among these, polymerase-gamma (POLG)-spectrum disorders are relatively frequent with an estimated disease frequency of similar to 1:10.000. Also, mutations in the POLG gene are by far the most important cause for mitochondriopathy. POLG-spectrum disorders usually result in progressive loss of brain function and may involve severe and deadly encephalopathy, seizures, and neuromuscular disease, as well as cardiac and hepatic failure in some cases. Onset of disease may range from birth to late adulthood, and disease duration ranges from weeks in severe cases to decades. There is no curative treatment; current animal models do not faithfully recapitulate human disease, complicating preclinical therapeutic studies. Human-based preclinical model systems must be developed to understand the human disease mechanisms and develop therapeutic approaches. In this review, we provide an overview of the current approaches to model neurogenetic disorders in a human cellular and neuronal environment with a focus on POLG-spectrum disorders. We discuss the necessity of using neuronal cells and the advantages and pitfalls of currently available cell model approaches, namely (i) CRISPR-based (i. e., genetically engineered) and induced pluripotent stem cell (iPSC) (i. e., stem cell like)-derived neuronal models and (ii) the reprogramming of patient-derived cells into iPSCs and derived neurons. Despite the fact that cell models are by definition in vitro systems incapable of recapitulating all aspects of human disease, they are still the reasonable point of start to discover disease mechanisms and develop therapeutic approaches to treat neurogenetic diseases

SH-SY5Y-derived neurons: a human neuronal model system for investigating TAU sorting and neuronal subtype-specific TAU vulnerability

The microtubule-associated protein (MAP) TAU is mainly sorted into the axon of healthy brain neurons. Somatodendritic missorting of TAU is a pathological hallmark of many neurodegenerative diseases, including Alzheimer's disease (AD). Cause, consequence and (patho)physiological mechanisms of TAU sorting and missorting are understudied, in part also because of the lack of readily available human neuronal model systems. The human neuroblastoma cell line SH-SY5Y is widely used for studying TAU physiology and TAU-related pathology in AD and related tauopathies. SH-SY5Y cells can be differentiated into neuron-like cells (SH-SY5Y-derived neurons) using various substances. This review evaluates whether SH-SY5Y-derived neurons are a suitable model for (i) investigating intracellular TAU sorting in general, and (ii) with respect to neuron subtype-specific TAU vulnerability. (I) SH-SY5Y-derived neurons show pronounced axodendritic polarity, high levels of axonally localized TAU protein, expression of all six human brain isoforms and TAU phosphorylation similar to the human brain. As SH-SY5Y cells are highly proliferative and readily accessible for genetic engineering, stable transgene integration and leading-edge genome editing are feasible. (II) SH-SY5Y-derived neurons display features of subcortical neurons early affected in many tauopathies. This allows analyzing brain region-specific differences in TAU physiology, also in the context of differential vulnerability to TAU pathology. However, several limitations should be considered when using SH-SY5Y-derived neurons, e.g., the lack of clearly defined neuronal subtypes, or the difficulty of mimicking age-related tauopathy risk factors in vitro. In brief, this review discusses the suitability of SH-SY5Y-derived neurons for investigating TAU (mis)sorting mechanisms and neuron-specific TAU vulnerability in disease paradigms

A mitochondria cluster at the proximal axon initial segment controls axodendritic TAU trafficking in rodent primary and human iPSC-derived neurons

Loss of neuronal polarity and missorting of the axonal microtubule-associated-protein TAU are hallmarks of Alzheimer's disease (AD) and related tauopathies. Impairment of mitochondrial function is causative for various mitochondriopathies, but the role of mitochondria in tauopathies and in axonal TAU-sorting is unclear. The axon-initial-segment (AIS) is vital for maintaining neuronal polarity, action potential generation, and-here important-TAU-sorting. Here, we investigate the role of mitochondria in the AIS for maintenance of TAU cellular polarity. Using not only global and local mitochondria impairment via inhibitors of the respiratory chain and a locally activatable protonophore/uncoupler, but also live-cell-imaging and photoconversion methods, we specifically tracked and selectively impaired mitochondria in the AIS in primary mouse and human iPSC-derived forebrain/cortical neurons, and assessed somatic presence of TAU. Global application of mitochondrial toxins efficiently induced tauopathy-like TAU-missorting, indicating involvement of mitochondria in TAU-polarity. Mitochondria show a biased distribution within the AIS, with a proximal cluster and relative absence in the central AIS. The mitochondria of this cluster are largely immobile and only sparsely participate in axonal mitochondria-trafficking. Locally constricted impairment of the AIS-mitochondria-cluster leads to detectable increases of somatic TAU, reminiscent of AD-like TAU-missorting. Mechanistically, mitochondrial impairment sufficient to induce TAU-missorting results in decreases of calcium oscillation but increases in baseline calcium, yet chelating intracellular calcium did not prevent mitochondrial impairment-induced TAU-missorting. Stabilizing microtubules via taxol prevented TAU-missorting, hinting towards a role for impaired microtubule dynamics in mitochondrial-dysfunction-induced TAU-missorting. We provide evidence that the mitochondrial distribution within the proximal axon is biased towards the proximal AIS and that proper function of this newly described mitochondrial cluster may be essential for the maintenance of TAU polarity. Mitochondrial impairment may be an upstream event in and therapeutic target for AD/tauopathy

Differential Effects of the Six Human TAU Isoforms: Somatic Retention of 2N-TAU and Increased Microtubule Number Induced by 4R-TAU

In the adult human brain, six isoforms of the microtubule-associated protein TAU are expressed, which result from alternative splicing of exons 2, 3, and 10 of the MAPT gene. These isoforms differ in the number of N-terminal inserts (0N, 1N, 2N) and C-terminal repeat domains (3R or 4R) and are differentially expressed depending on the brain region and developmental stage. Although all TAU isoforms can aggregate and form neurofibrillary tangles, some tauopathies, such as Pick's disease and progressive supranuclear palsy, are characterized by the accumulation of specific TAU isoforms. The influence of the individual TAU isoforms in a cellular context, however, is understudied. In this report, we investigated the subcellular localization of the human-specific TAU isoforms in primary mouse neurons and analyzed TAU isoform-specific effects on cell area and microtubule dynamics in human SH-SY5Y neuroblastoma cells. Our results show that 2N-TAU isoforms are particularly retained from axonal sorting and that axonal enrichment is independent of the number of repeat domains, but that the additional repeat domain of 4R-TAU isoforms results in a general reduction of cell size and an increase of microtubule counts in cells expressing these specific isoforms. Our study points out that individual TAU isoforms may influence microtubule dynamics differentially both by different sorting patterns and by direct effects on microtubule dynamics

AAV-based gene therapy approaches for genetic forms of tauopathies and related neurogenetic disorders

Tauopathies comprise a spectrum of genetic and sporadic neurodegenerative diseases mainly characterized by the presence of hyperphosphorylated TAU protein aggregations in neurons or glia. Gene therapy, in particular adeno-associated virus (AAV)-based, is an effective medical approach for difficult-to-treat genetic diseases for which there are no convincing traditional therapies, such as tauopathies. Employing AAV-based gene therapy to treat, in particular, genetic tauopathies has many potential therapeutic benefits, but also drawbacks which need to be addressed in order to successfully and efficiently adapt this still unconventional therapy for the various types of tauopathies. In this Viewpoint, we briefly introduce some potentially treatable tauopathies, classify them according to their etiology, and discuss the potential advantages and possible problems of AAV-based gene therapy. Finally, we outline a future vision for the application of this promising therapeutic approach for genetic and sporadic tauopathies

Genetic forms of tauopathies: inherited causes and implications of Alzheimer's disease-like TAU pathology in primary and secondary tauopathies

Tauopathies are a heterogeneous group of neurologic diseases characterized by pathological axodendritic distribution, ectopic expression, and/or phosphorylation and aggregation of the microtubule-associated protein TAU, encoded by the gene MAPT. Neuronal dysfunction, dementia, and neurodegeneration are common features of these often detrimental diseases. A neurodegenerative disease is considered a primary tauopathy when MAPT mutations/haplotypes are its primary cause and/or TAU is the main pathological feature. In case TAU pathology is observed but superimposed by another pathological hallmark, the condition is classified as a secondary tauopathy. In some tauopathies (e.g. MAPT-associated frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Alzheimer's disease (AD)) TAU is recognized as a significant pathogenic driver of the disease. In many secondary tauopathies, including Parkinson's disease (PD) and Huntington's disease (HD), TAU is suggested to contribute to the development of dementia, but in others (e.g. Niemann-Pick disease (NPC)) TAU may only be a bystander. The genetic and pathological mechanisms underlying TAU pathology are often not fully understood. In this review, the genetic predispositions and variants associated with both primary and secondary tauopathies are examined in detail, assessing evidence for the role of TAU in these conditions. We highlight less common genetic forms of tauopathies to increase awareness for these disorders and the involvement of TAU in their pathology. This approach not only contributes to a deeper understanding of these conditions but may also lay the groundwork for potential TAU-based therapeutic interventions for various tauopathies

Microtubule affinity regulating kinase activity in living neurons was examined by a genetically encoded fluorescence resonance energy transfer/fluorescence lifetime imaging-based biosensor: inhibitors with therapeutic potential

BACKGROUND: Deregulation of the protein kinase MARK has been linked to Alzheimer disease. - RESULTS: Mark-specific inhibitors and a biosensor are identified. - CONCLUSION: The inhibitors and the biosensor are tools to provide new insights into the role of MARK during polarity establishment and maintenance of neurons. - SIGNIFICANCE: The inhibitors might possess therapeutic potential by interfering with abnormal Tau phosphorylation in Alzheimer disease.Protein kinases of the microtubule affinity regulating kinase (MARK)/Par-1 family play important roles in the establishment of cellular polarity, cell cycle control, and intracellular signal transduction. Disturbance of their function is linked to cancer and brain diseases, e.g. lissencephaly and Alzheimer disease. To understand the biological role of MARK family kinases, we searched for specific inhibitors and a biosensor for MARK activity. A screen of the ChemBioNet library containing ∼18,000 substances yielded several compounds with inhibitory activity in the low micromolar range and capable of inhibiting MARK activity in cultured cells and primary neurons, as judged by MARK-dependent phosphorylation of microtubule-associated proteins and its consequences for microtubule integrity. Four of the compounds share a 9-oxo-9H-acridin-10-yl structure as a basis that will serve as a lead for optimization of inhibition efficiency. To test these inhibitors, we developed a cellular biosensor for MARK activity based on a MARK target sequence attached to the 14-3-3 scaffold protein and linked to enhanced cyan or teal and yellow fluorescent protein as FRET donor and acceptor pairs. Transfection of the teal/yellow fluorescent protein sensor into neurons and imaging by fluorescence lifetime imaging revealed that MARK was particularly active in the axons and growth cones of differentiating neurons