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Microglia play vital roles in embryonic and post-natal development, homeostasis, and pathogen defence in the central nervous system. Human induced pluripotent stem cell (hiPSC)-based methods have emerged as an important source for the study of human microglia in vitro. Classical approaches to differentiate hiPSCs into microglia suffer from limitations including extended culture periods, consistency, and efficiency. More recently, forward programming has arisen as a promising alternative for the manufacture of bulk quantities of human microglia. This review provides a comprehensive assessment of published forward programming protocols that are based on forced expression of key lineage transcription factors (TFs). We focus on the choice of reprogramming factors, transgene delivery methods, and medium composition, which impact induction kinetics and the resulting microglia phenotype., Competing Interests: Declaration of interests M.P. is inventor of a patent for a protocol of microglia generation., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Optical coherence tomography angiography (OCT-A) provides detailed visualization of the perfusion of the vascular network of the eye. While in other forms of dementia, such as Alzheimer's disease and mild cognitive impairment, reduced retinal perfusion was frequently reported, data of patients with frontotemporal dementia (FTD) are lacking., Objective: Retinal and optic nerve head perfusion was evaluated in patients with FTD with OCT-A. Quantitative OCT-A metrics were analyzed and correlated with clinical markers and vascular cerebral lesions in FTD patients., Methods: OCT-A was performed in 18 eyes of 18 patients with FTD and 18 eyes of 18 healthy participants using RTVue XR Avanti with AngioVue. In addition, patients underwent a detailed ophthalmological, neurological, and neuropsychological examination, cerebral magnetic resonance imaging (MRI), and lumbar puncture., Results: The flow density in the optic nerve head (ONH) and in the superficial capillary plexus (SCP) of the macula of patients was significantly lower compared to that of healthy controls ( p < 0.001). Similarly, the VD in the deep capillary plexus (DCP) of the macula of patients was significantly lower compared to that of healthy controls ( p < 0.001). There was no significant correlation between the flow density data, white matter lesions in brain MRI, cognitive deficits, and cerebrospinal fluid markers of dementia., Conclusions: Patients with FTD showed a reduced flow density in the ONH, and in the superficial and deep retinal capillary plexus of the macula, when compared with that of healthy controls. Quantitative analyses of retinal perfusion using OCT-A may therefore help in the diagnosis and monitoring of FTD. Larger and longitudinal studies are necessary to evaluate if OCT-A is a suitable biomarker for patients with FTD.

Hyperexcitability is associated with neuronal dysfunction, cellular death, and consequently neurodegeneration. Redox disbalance can contribute to hyperexcitation and increased reactive oxygen species (ROS) levels are observed in various neurological diseases. NOX4 is an NADPH oxidase known to produce ROS and might have a regulating function during oxidative stress. We, therefore, aimed to determine the role of NOX4 on neuronal firing, hyperexcitability, and hyperexcitability-induced changes in neural network function. Using a multidimensional approach of an in vivo model of hyperexcitability, proteomic analysis, and cellular function analysis of ROS, mitochondrial integrity, and calcium levels, we demonstrate that NOX4 is neuroprotective by regulating ROS and calcium homeostasis and thereby preventing hyperexcitability and consequently neuronal death. These results implicate NOX4 as a potential redox regulator that is beneficial in hyperexcitability and thereby might have an important role in neurodegeneration., (© 2023. The Author(s).)

Tauopathies are a major type of proteinopathies underlying neurodegenerative diseases. Mutations in the tau-encoding MAPT-gene lead to hereditary cases of frontotemporal lobar degeneration (FTLD)-tau, which span a wide phenotypic and pathological spectrum. Some of these mutations, such as the N279K mutation, result in a shift of the physiological 3R/4R ratio towards the more aggregation prone 4R isoform. Other mutations such as V337M cause a decrease in the in vitro affinity of tau to microtubules and a reduced ability to promote microtubule assembly. Whether both mutations address similar downstream signalling cascades remains unclear but is important for potential rescue strategies. Here, we developed a novel and optimised forward programming protocol for the rapid and highly efficient production of pure cultures of glutamatergic cortical neurons from hiPSCs. We apply this protocol to delineate mechanisms of neurodegeneration in an FTLD-tau hiPSC-model consisting of MAPT N279K - or MAPT V337M -mutants and wild-type or isogenic controls. The resulting cortical neurons express MAPT-genotype-dependent dominant proteome clusters regulating apoptosis, ROS homeostasis and mitochondrial function. Related pathways are significantly upregulated in MAPT N279K neurons but not in MAPT V337M neurons or controls. Live cell imaging demonstrates that both MAPT mutations affect excitability of membranes as reflected in spontaneous and stimulus evoked calcium signals when compared to controls, albeit more pronounced in MAPT N279K neurons. These spontaneous calcium oscillations in MAPT N279K neurons triggered mitochondrial hyperpolarisation and fission leading to mitochondrial ROS production, but also ROS production through NOX2 acting together to induce cell death. Importantly, we found that these mechanisms are MAPT mutation-specific and were observed in MAPT N279K neurons, but not in MAPT V337M neurons, supporting a pathological role of the 4R tau isoform in redox disbalance and highlighting MAPT-mutation specific clinicopathological-genetic correlations, which may inform rescue strategies in different MAPT mutations., Competing Interests: Declaration of competing interest LK: none; AS: A patent application for the optimised forward programming protocol has been filed and submitted. CBS: none. LG: none. TK: none. AE: none. PD: none. MN: none. GS: none. SGM: none. HRS: none. HW: none. SK: none. MP: A patent application for the optimised forward programming protocol has been filed and submitted., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

N -Methyl- D -aspartate receptors (NMDARs) are central for learning and information processing in the brain. Dysfunction of NMDARs can play a key role in the pathogenesis of neurodegeneration and drug addiction. The development of selective NMDAR modulators represents a promising strategy to target these diseases. Among such modulating compounds are ifenprodil and its 3-benzazepine derivatives. Classically, the effects of these NMDAR modulators have been tested by techniques like two-electrode voltage clamp (TEVC), patch clamp, or fluorescence-based assays. However, testing their functional effects in complex human systems requires more advanced approaches. Here, we established a human induced pluripotent stem cell-derived (hiPSC-derived) neural cell system and proved its eligibility as a test system for investigating NMDAR modulators and pharmaceutical effects on human neurons., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

Hyperexcitability-induced neuronal damage plays a role both in epilepsy as well as in inflammatory brain diseases such as multiple sclerosis (MS) and as such represents an important disease pathway which potentially can be targeted to mitigate neuronal damage. Dimethyl fumarate (DMF) and its pharmacologically active metabolite monomethyl fumarate (MMF) are FDA-approved therapeutics for MS, which can induce immunosuppressive and antioxidant pathways, and their neuroprotective capacity has been demonstrated in other preclinical neurological disease models before. In this study, we used an unbiased proteomic approach to identify potential new targets upon the treatment of MMF in glio-neuronal hippocampal cultures. MMF treatment results in induction of antioxidative (HMOX1, NQO1) and anaplerotic metabolic (GAPDH, PC) pathways, which correlated with reduction in ROS production, increased mitochondrial NADH-redox index and decreased NADH pool, independent of glutathione levels. Additionally, MMF reduced glycolytic capacity indicating individual intra-cellular metabolic programs within different cell types. Furthermore, we demonstrate a neuroprotective effect of MMF upon hyperexcitability in vitro (low magnesium model), where MMF prevents glio-neuronal death via reduced ROS production. These results highlight MMF as a potential new therapeutic opportunity in hyperexcitability-induced neurodegeneration., Competing Interests: Declaration of competing interest SK, HW and TB received research funding from Biogen. HW receives speaker honoraria and travel support from Biogen and is acting as a paid consultant for Biogen., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Microglia, the resident immune cells of the central nervous system (CNS), are derived from yolk-sac macrophages that populate the developing CNS during early embryonic development. Once established, the microglia population is self-maintained throughout life by local proliferation. As a scalable source of microglia-like cells (MGLs), we here present a forward programming protocol for their generation from human pluripotent stem cells (hPSCs). The transient overexpression of PU.1 and C/EBPβ in hPSCs led to a homogenous population of mature microglia within 16 d. MGLs met microglia characteristics on a morphological, transcriptional, and functional level. MGLs facilitated the investigation of a human tauopathy model in cortical neuron-microglia cocultures, revealing a secondary dystrophic microglia phenotype. Single-cell RNA sequencing of microglia integrated into hPSC-derived cortical brain organoids demonstrated a shift of microglia signatures toward a more-developmental in vivo-like phenotype, inducing intercellular interactions promoting neurogenesis and arborization. Taken together, our microglia forward programming platform represents a tool for both reductionist studies in monocultures and complex coculture systems, including 3D brain organoids for the study of cellular interactions in healthy or diseased environments.

Background: Alzheimer's disease (AD) is the most common cause of dementia. The number of people affected will increase dramatically in the coming decades due to the demographic change. Causal pharmacological approaches have not been available to date. The monoclonal anti-amyloid beta antibody aducanumab was recently approved for the treatment of AD in the USA but was rejected in Europe in December 2021 by the European Medicines Agency (EMA)., Objective: This review presents the background and rationale for amyloid beta-directed treatment approaches in AD. The focus is on passive immunization with monoclonal anti-amyloid beta antibodies., Data Situation: There are four monoclonal anti-amyloid beta antibodies in an advanced stage of clinical development. Evidence of a clear and significant reduction of the cerebral amyloid load was found for all of them. In the case of aducanumab this has already led to approval by the U.S. Food and Drug Administration (FDA). In the USA donanemab, gantenerumab and lecanemab have received the status of a so-called breakthrough therapy and are expected to go through an accelerated approval process by the FDA in the next 1-2 years., Conclusion: Anti-amyloid antibodies represent the first cause-based, disease-modifying therapy for AD approved in the USA. Compared to the near-complete removal of cerebral amyloid plaques, the magnitude of the clinical effect is smaller and the benefit for patients is currently subject to controversial discussions. Nonetheless, the new treatment option represents an important step in the development of effective treatment. Future strategies for the treatment of AD will likely aim at a multimodal concept with different molecular targets. A prerequisite for all effective disease-modifying therapies will be an early biomarker-based diagnosis prior to the onset of a dementia-type syndrome., (© 2022. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Background and Objectives: Despite detection of autoantibodies, anti-IgLON5 disease was historically considered a tau-associated neurodegenerative disease, with limited treatment options and detrimental consequences for the patients. Observations in increasing case numbers hint toward underlying inflammatory mechanisms that, early detection provided, open a valuable window of opportunity for therapeutic intervention. We aimed to further substantiate this view by studying the CSF of patients with anti-IgLON5., Methods: We identified 11 patients with anti-IgLON5 from our database and compared clinical, MRI, and CSF findings with a cohort of 20 patients with progressive supranuclear palsy (PSP) (as a noninflammatory tauopathy) and 22 patients with functional neurologic disorder., Results: Patients with anti-IgLON5 show inflammatory changes in routine CSF analysis, an increase in B-lymphocyte frequency, and the presence of plasma cells in comparison to the PSP-control group and functional neurologic disease controls. Patients with intrathecal plasma cells showed a clinical response to rituximab., Discussion: Our findings indicate the importance of inflammatory mechanisms, in particular in early and acute anti-IgLON5 cases, which may support the use of immune-suppressive treatments in these cases. The main limitation of the study is the small number of cases due to the rarity of the disease., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Background and Purpose: Steroid-responsive encephalopathy associated with autoimmune thyroiditis (SREAT) is a rare condition defined by encephalopathy with acute or subacute onset, the presence of serum anti-thyroid antibodies, and reasonable exclusion of alternative causes. Despite having strong response towards corticosteroid treatment, some patients exhibit a chronic-relapsing course and require long-term immunosuppression. Markers for early identification of those patients are still absent. Thus, we aimed to characterise clinical as well as laboratory parameters of our local SREAT cohort., Methods: We retrospectively evaluated a cohort of 22 SREAT patients treated in our hospital from January 2014., Results: A total of 14 patients with a monophasic disease course and eight patients with multiple relapses were identified. Neither baseline characteristics nor routine cerebrospinal fluid (CSF) parameters were able to distinguish between those patient groups. Flow cytometry following initial relapse therapy showed treatment-resistant sequestration of activated CD4+ T cells in patients with a relapsing disease course, whereas other lymphocyte subsets showed uniform changes. Such changes were also present in long-term follow-up CSF examination., Conclusion: Our findings indicate a potential biomarker for risk stratification in patients with SREAT. Currently, it remains unclear whether the observed two phenotypes are different spectra of SREAT or represent separate diseases in terms of pathophysiology., (© 2020 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

Background: Due to the demographic change dementia is a common and dramatically increasing reason for medical presentations. In approximately 8% of cases dementia occurs before the age of 65 years. The psychosocial and economic consequences are often severe, particularly in younger patients. Clinicians face major diagnostic challenges. A rapid diagnosis is crucial for patient counselling and management., Objective: This review article presents the special features of dementia in younger people, the most important underlying diseases and a rational clinical diagnostic approach., Methods: Narrative review. The literature search was carried out in PubMed., Results: The differential diagnostic spectrum of dementia in younger people under the age of 65 years is very broad. The most common causes are Alzheimer's disease with typical or atypical clinical presentations and frontotemporal lobar degeneration. The younger the age of onset, the higher the proportion of treatable and potentially reversible causes of dementia., Conclusion: The diagnostics of primary neurodegenerative diseases have continuously improved, especially due to the availability of an increasing number of clinical, molecular and imaging biomarkers. Nevertheless, in order to avoid unnecessary and burdensome examinations, the diagnostic work-up of young onset dementia must be hypothesis-driven, i.e. following a precise clinical syndromic classification of the symptoms.

Microglia play an essential role for central nervous system (CNS) development and homeostasis and have been implicated in the onset, progression, and clearance of numerous diseases affecting the CNS. Previous in vitro research on human microglia was restricted to post-mortem brain tissue-derived microglia, with limited availability and lack of scalability. Recently, the first protocols for the generation of microglia from human pluripotent stem cells have become available, thus enabling the implementation of powerful platforms for disease modeling, drug testing, and studies on cell transplantation. Here we give a detailed and comprehensive overview of the protocols available for generating microglia from human pluripotent stem cells, highlighting the advantages, drawbacks, and operability and placing them into the context of current knowledge of human embryonic development. We review novel insights into microglia biology and the role of microglia in neurological diseases as drawn from the new methods and provide an outlook for future lines of research involving human pluripotent stem cell-derived microglia.

Objectives: Frontotemporal dementia (FTD) is a heterogeneous clinical syndrome linked to diverse types of underlying neuropathology. Diagnosis is mainly based on clinical presentation and accurate prediction of underlying neuropathology remains difficult., Methods: We present a large cohort of patients with FTD spectrum diseases (n=84). All patients were thoroughly characterised by cerebrospinal fluid (CSF) Alzheimer's disease (AD) biomarkers, neuroimaging, neuropsychological testing and standardised apraxia screening., Results: A potential AD pathology was found in 43% of patients with FTD. CSF AD biomarker levels positively correlated with AD-typical apraxia scores in patients with FTD. The discriminative power of apraxia test results indicative of AD pathology was high (sensitivity: 90%, specificity: 66%)., Conclusions: Apraxia is common in neurodegenerative dementias but under-represented in clinical workup and diagnostic criteria. Standardised apraxia screening may serve as bedside test to objectify an AD-typical apraxia profile as an early and robust sign of AD pathology in patients with FTD., Competing Interests: Competing interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. MP, VJ and AJ have nothing to disclose. HW receives honoraria for acting as a member of Scientific Advisory Boards and as consultant for Biogen, Evgen, MedDay Pharmaceuticals, Merck Serono, Novartis, Roche Pharma and Sanofi-Genzyme, as well as speaker honoraria and travel support from Alexion, Biogen, Cognomed, F Hoffmann-La Roche, Gemeinnützige Hertie-Stiftung, Merck Serono, Novartis, Roche Pharma, Sanofi- Genzyme, TEVA and WebMD Global. HW is acting as a paid consultant for AbbVie, Actelion, Biogen, IGES, Novartis, Roche, Sanofi-Genzyme and the Swiss Multiple Sclerosis Society. His research is funded by the German Ministry for Education and Research (BMBF), Deutsche Forschungsgesellschaft (DFG), Else Kröner Fresenius Foundation, Fresenius Foundation, Hertie Foundation, NRW Ministry of Education and Research, Interdisciplinary Center for Clinical Studies (IZKF) Muenster and RE Children’s Foundation, Biogen, GlaxoSmithKline, Roche Pharma and Sanofi-Genzyme. SGM has received honoraria for lecturing, travel expenses for attending meetings and financial research support from Almirall, Bayer Health Care, Biogen, Diamed, Fresenius Medical Care, Genzyme, Merck Serono, Novartis, Novo Nordisk, ONO Pharma, Roche, Sanofi-Aventis and Teva. TD has received speaker honoraria, consultancy fees and travel expenses from Genzyme, Shire, Sanofi Aventis, Novartis, Actelion Pharmaceuticals and Amicus, research support from Genzyme, Shire, Amicus and Actelion Pharmaceuticals, and educational grants from Novartis, Roche and Biogen., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: Although patients with Parkinson's disease (PD) often suffer from oropharyngeal dysphagia, knowledge about the underlying pathophysiological mechanisms is limited. Substance P (SP) is a localization-independent neurotransmitter of the entire nervous system. Reduced levels of SP were found in saliva of patients with impaired cough reflex and in advanced stages of PD. The aim of the study was to investigate SP in PD patients in order to gain further insights into the underlying pathophysiology of PD-related dysphagia and to evaluate the potential of SP as a biomarker for early dysphagia. Methods: Flexible endoscopic evaluation of swallowing (FEES) was used to objectively assess pharyngeal swallowing function. From a cohort of 105 consecutive PD patients 20 subjects were recruited: in 10 of them pharyngeal dysphagia was excluded by FEES, the other 10 subjects showed signs of early pharyngeal dysphagia defined as hypopharyngeal sensory deficit with mild to moderate vallecular residues after swallowing solid consistencies. Analysis of the Substance P level in saliva of the 20 included PD patients was performed in the clinical on state condition by ELISA-type immunoassay. Significant differences were calculated by using the Mann-Whitney test. Results: Twenty PD patients with a mean age of 69.5 ± 12.5 years (8 female) were included in the study. No significant differences were found regarding gender, age, UPDRS III, Hoehn and Yahr stage, disease duration, and Levodopa equivalent dose between the non-dysphagic and dysphagic subjects. Dysphagia was mainly characterized by unrecognized residues in the valleculae without any aspiration risk for all of the tested consistencies in FEES and was thereby scored as mild in all cases. Saliva SP concentrations were significantly lower in PD patients with pharyngeal dysphagia compared to those with a normal pharyngeal swallowing function (9,644 vs. 17,591 pg/mL; p = 0.001). Conclusion: Reduced saliva SP concentrations may predict early pharyngeal swallowing dysfunction in PD patients. This finding supports the hypothesis that an impaired SP mediated neurotransmission has a significant impact for the development of dysphagia in PD patients. Larger studies are needed to confirm SP as a clinical useful biomarker for early detection of PD-related dysphagia.

Background: Parkinson's disease (PD) is a common neurodegenerative disorder. The contribution of the immune system to its pathogenesis remains incompletely understood. Methods: In this study, we performed comprehensive immune cell profiling in the cerebrospinal fluid (CSF) and peripheral blood (PB) of PD patients. Ten PD patients were diagnosed according to brain bank criteria and underwent detailed clinical examination, magnetic resonance imaging, PB and CSF immune cell profiling by multiparameter flow cytometry, and cytokine and chemokine measurements by bead-based arrays. Thirteen healthy elderly volunteers served as control population. Results: The proportions of activated T-lymphocytes and non-classical monocytes in the CSF were increased in patients with PD compared to the control group. In accordance, we found increased levels of the pro-inflammatory cytokines IL-2, IL-6 and TNFα and of the monocyte chemoattractant protein 1 (MCP-1) in the CSF of the included PD patients. Conclusions: Our data provide novel evidence for a response of the innate and adaptive immune system in the central nervous system of patients with PD.

Background: Niemann-Pick disease type C (NP-C) is a rare, progressive neurodegenerative disease caused by mutations in the NPC1 or the NPC2 gene. Neurocognitive deficits are common in NP-C, particularly in patients with the adolescent/adult-onset form. As a disease-specific therapy is available, it is important to distinguish clinically between the cognitive profiles in NP-C and primary dementia (e.g., early Alzheimer's disease; eAD)., Methods: In a prospective observational study, we directly compared the neurocognitive profiles of patients with confirmed NP-C (n = 7) and eAD (n = 15). All patients underwent neurocognitive assessment using dementia screening tests (mini-mental status examination [MMSE] and frontal assessment battery [FAB]) and an extensive battery of tests assessing verbal memory, visuoconstructive abilities, visual memory, executive functions and verbal fluency., Results: Overall cognitive impairment (MMSE) was significantly greater in eAD vs. NP-C (p = 0.010). The frequency of patients classified as cognitively 'impaired' was also significantly greater in eAD vs. NP-C (p = 0.025). Patients with NP-C showed relatively preserved verbal memory, but frequent impairment in visual memory, visuoconstruction, executive functions and in particular, verbal fluency. In the eAD group, a wider profile of more frequent and more severe neurocognitive deficits was seen, primarily featuring severe verbal and visual memory deficits along with major executive impairment. Delayed verbal memory recall was a particularly strong distinguishing factor between the two groups., Conclusion: A combination of detailed yet easy-to-apply neurocognitive tests assessing verbal memory, executive functions and verbal fluency may help distinguish NP-C cases from those with primary dementia due to eAD.

The difficulties involved in conditionally perturbing complex gene expression networks represent major challenges toward defining the mechanisms controlling human development, physiology, and disease. We developed an OPTimized inducible KnockDown (OPTiKD) platform that addresses the limitations of previous approaches by allowing streamlined, tightly-controlled, and potent loss-of-function experiments for both single and multiple genes. The method relies on single-step genetic engineering of the AAVS1 genomic safe harbor with an optimized tetracycline-responsive cassette driving one or more inducible short hairpin RNAs (shRNAs). OPTiKD provides homogeneous, dose-responsive, and reversible gene knockdown. When implemented in human pluripotent stem cells (hPSCs), the approach can be then applied to a broad range of hPSC-derived mature cell lineages that include neurons, cardiomyocytes, and hepatocytes. Generation of OPTiKD hPSCs in commonly used culture conditions is simple (plasmid based), rapid (two weeks), and highly efficient (>95%). Overall, this method facilitates the functional annotation of the human genome in health and disease. © 2018 by John Wiley & Sons, Inc., (Copyright © 2018 John Wiley & Sons, Inc.)

Background Hemicrania continua (HC) -like headaches have been rarely reported as symptomatic headaches, including cases secondary to cervical artery dissection. Case series We present five cases of HC-like headaches following cervical artery dissection, in three cases with specific indomethacin response. In two cases, comorbidity of fibromuscular dysplasia (FMD) was noted. Conclusion Carotid artery dissection may result in an HC-like headache syndrome. A specific response to indomethacin does not rule out dissection as underlying pathology. Screening for extracranial manifestations of FMD should be considered, especially in middle-aged females.

Frontotemporal dementia (FTD) is a heterogeneous neurodegenerative disorder. The contribution of the immune system to its pathogenesis remains incompletely understood. In this study, we performed comprehensive immune cell profiling in the cerebrospinal fluid (CSF) and peripheral blood of patients with FTD. Thirty-two patients with behavioral variant frontotemporal dementia and 25 patients with primary progressive aphasia were included and compared to 14 healthy elderly controls. All patients underwent neuropsychological examination, magnetic resonance imaging, voxel-based diffusion tensor imaging, and peripheral blood and CSF immune cell profiling by multiparameter flow cytometry. The percentage of CSF monocytes was significantly increased specifically in patients with primary progressive aphasia. The proportion of monocytes in the CSF of the total FTD patient group directly correlated with semantic language impairment and microstructural temporal lesions. Increased intrathecal numbers of monocytes suggest a specific response of the innate immune system in a subset of patients with FTD. The findings are of clinical relevance since monocyte levels in the CSF were correlated with typical neuropsychological deficits and microstructural patterns of temporal degeneration., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Due to suboptimal sensitivity and specificity of structural and molecular neuroimaging tools, the diagnosis of behavioral variant frontotemporal dementia (bvFTD) remains challenging., Objective: Investigation of the sensitivity of diffusion tensor imaging (DTI) and fluorodeoxyglucose positron emission tomography (FDG-PET) to detect cerebral alterations in early stages of bvFTD despite inconspicuous conventional MRI., Methods: Thirty patients with early stages of bvFTD underwent a detailed neuropsychological examination, cerebral 3T MRI with DTI analysis, and FDG-PET. After 12 months of follow-up, all patients finally fulfilled the diagnosis of bvFTD. Individual FDG-PET data analyses showed that 20 patients exhibited a "typical" pattern for bvFTD with bifrontal and/or temporal hypometabolism (bvFTD/PET+), and that 10 patients showed a "non-typical"/normal pattern (bvFTD/PET-). DTI data were compared with 42 healthy controls in an individual and voxel-based group analysis. To examine the clinical relevance of the findings, associations between pathologically altered voxels of DTI or FDG-PET results and behavioral symptoms were estimated by linear regression analyses., Results: DTI voxel-based group analyses revealed microstructural degeneration in bifrontal and bitemporal areas in bvFTD/PET+ and bvFTD/PET- groups. However, when comparing the sensitivity of individual DTI data analysis with FDG-PET, DTI appeared to be less sensitive. Neuropsychological symptoms were considerably related to neurodegeneration within frontotemporal areas identified by DTI and FDG-PET., Conclusion: DTI seems to be an interesting tool for detection of functionally relevant neurodegenerative alterations in early stages of bvFTD, even in bvFTD/PET- patients. However, at a single subject level, it seems to be less sensitive than FDG-PET. Thus, improvement of individual DTI analysis is necessary.

The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids in vivo and demonstrate that ECOs self-organize into bile duct-like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded in vitro.

Alzheimer's disease is the most common cause of dementia. Over the last three decades, research has advanced dramatically and provided a detailed understanding of the molecular events underlying the pathogenesis of Alzheimer's disease. In parallel, assays for the detection of biomarkers that reflect the typical Alzheimer's disease-associated pathology have been developed and validated in myriads of clinical studies. Such biomarkers complement clinical diagnosis and improve diagnostic accuracy. The use of biomarkers will become even more important with the advent of disease-modifying therapies. Such therapies will likely be most beneficial when administered early in the disease course. Here, we summarise the development of the core Alzheimer's disease cerebrospinal fluid biomarkers: amyloid-β and tau. We provide an overview of their role in cellular physiology and Alzheimer's disease pathology, and embed their development as cerebrospinal fluid biomarkers into the historical context of Alzheimer's disease research. Finally, we summarise recommendations for their use in clinical practice, and outline perspectives for novel cerebrospinal fluid candidate biomarkers., Competing Interests: MP declares no conflict of interest. SGM has received honoraria for lecturing, travel expenses for attending meetings and financial research support from Almirall, Bayer Health Care, Biogen, Diamed, Fresenius Medical Care, Genzyme, Merck Serono, Novartis, Novo Nordisk, ONO Pharma, Roche, Sanofi-Aventis and Teva. TD received honorariums and travel expenses from Genzyme, Shire, Bristol-Myers Squibb, Boehringer-Ingelheim Pharma, Sanofi Aventis, Eisai, Novartis, Bayer Vital, Merz Pharma, Actelion, Amicus for serving as a speaker and consultant. TD received research support from Genzyme, Shire, Amicus and Actelion. For conducting of studies on dementia, TD received grants from Novartis, Merz Pharma, Roche and Biogen.

The isolation or in vitro derivation of many human cell types remains challenging and inefficient. Direct conversion of human pluripotent stem cells (hPSCs) by forced expression of transcription factors provides a potential alternative. However, deficient inducible gene expression in hPSCs has compromised efficiencies of forward programming approaches. We have systematically optimized inducible gene expression in hPSCs using a dual genomic safe harbor gene-targeting strategy. This approach provides a powerful platform for the generation of human cell types by forward programming. We report robust and deterministic reprogramming of hPSCs into neurons and functional skeletal myocytes. Finally, we present a forward programming strategy for rapid and highly efficient generation of human oligodendrocytes., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Inducible loss of gene function experiments are necessary to uncover mechanisms underlying development, physiology and disease. However, current methods are complex, lack robustness and do not work in multiple cell types. Here we address these limitations by developing single-step optimized inducible gene knockdown or knockout (sOPTiKD or sOPTiKO) platforms. These are based on genetic engineering of human genomic safe harbors combined with an improved tetracycline-inducible system and CRISPR/Cas9 technology. We exemplify the efficacy of these methods in human pluripotent stem cells (hPSCs), and show that generation of sOPTiKD/KO hPSCs is simple, rapid and allows tightly controlled individual or multiplexed gene knockdown or knockout in hPSCs and in a wide variety of differentiated cells. Finally, we illustrate the general applicability of this approach by investigating the function of transcription factors (OCT4 and T), cell cycle regulators (cyclin D family members) and epigenetic modifiers (DPY30). Overall, sOPTiKD and sOPTiKO provide a unique opportunity for functional analyses in multiple cell types relevant for the study of human development., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

Although various types of ion channels are known to have an impact on human T cell effector functions, their exact mechanisms of influence are still poorly understood. The patch clamp technique is a well-established method for the investigation of ion channels in neurons and T cells. However, small cell sizes and limited selectivity of pharmacological blockers restrict the value of this experimental approach. Building a realistic T cell computer model therefore can help to overcome these kinds of limitations as well as reduce the overall experimental effort. The computer model introduced here was fed off ion channel parameters from literature and new experimental data. It is capable of simulating the electrophysiological behaviour of resting and activated human CD4(+) T cells under basal conditions and during extracellular acidification. The latter allows for the very first time to assess the electrophysiological consequences of tissue acidosis accompanying most forms of inflammation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Key Points: During the behavioural states of sleep and wakefulness thalamocortical relay neurons fire action potentials in high frequency bursts or tonic sequences, respectively. The modulation of specific K(+) channel types, termed TASK and TREK, allows these neurons to switch between the two modes of activity. In this study we show that the signalling lipids phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG), which are components of their membrane environment, switch on and shut off TREK and TASK channels, respectively. These channel modulations contribute to a better understanding of the molecular basis of the effects of neurotransmitters such as ACh which are released by the brainstem arousal system. The present report introduces PIP2 and DAG as new elements of signal transduction in the thalamus. The activity of two-pore domain potassium channels (K2P ) regulates the excitability and firing modes of thalamocortical (TC) neurons. In particular, the inhibition of two-pore domain weakly inwardly rectifying K(+) channel (TWIK)-related acid-sensitive K(+) (TASK) channels and TWIK-related K(+) (TREK) channels, as a consequence of the stimulation of muscarinic ACh receptors (MAChRs) which are coupled to phosphoinositide-specific phospholipase C (PLCβ), induces a shift from burst to tonic firing. By using a whole cell patch-clamp approach, the contribution of the membrane-bound second messenger molecules phosphatidylinositol 4,5-bisphosphate (PIP2 ) and diacylglycerol (DAG) acting downstream of PLCβ was probed. The standing outward current (ISO ) was used to monitor the current through TASK and TREK channels in TC neurons. By exploiting different manoeuvres to change the intracellular PIP2 level in TC neurons, we here show that the scavenging of PIP2 (by neomycin) results in an increased muscarinic effect on ISO whereas increased availability of PIP2 (inclusion to the patch pipette; histone-based carrier) decreased muscarinic signalling. The degree of muscarinic inhibition specifically depends on phosphatidylinositol phosphate (PIP) and PIP2 but no other phospholipids (phosphatidic acid, phosphatidylserine). The use of specific blockers revealed that PIP2 is targeting TREK but not TASK channels. Furthermore, we demonstrate that the inhibition of TASK channels is induced by the application of the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Under current clamp conditions the activation of MAChRs and PLCβ as well as the application of OAG resulted in membrane depolarization, while PIP2 application via histone carrier induced a hyperpolarization. These results demonstrate a differential role of PIP2 and DAG in K2P channel modulation in native neurons which allows a fine-tuned inhibition of TREK (via PIP2 depletion) and TASK (via DAG) channels following MAChR stimulation., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

Background: Hemifacial spasm is a common movement disorder. Differential diagnosis relies on clinical examination and is often difficult. The Babinski-2 sign is an underrecognized physical sign specifically found in patients with hemifacial spasm, although its prevalence and usefulness are a matter of debate., Methods: We examined 35 patients with hemifacial spasm prospectively for the presence of the Babinski-2 sign. We evaluated its correlation with severity of hemifacial spasm, concomitant facial nerve paralysis, and response to botulinum toxin. Twelve patients with blepharospasm served as the control population., Results: The data for the Babinski-2 sign demonstrated high prevalence (86%), high specificity (100%), and high interrater reliability (92%)., Conclusions: Increased awareness of the Babinski-2 sign may aid diagnosis and potentially prompt earlier initiation of appropriate treatment. © 2013 Movement Disorder Society., (Copyright © 2013 Movement Disorder Society.)

Background: Epileptic seizures are well known sequelae of patients with stroke but only little is known about the different risk factors and about the influence of the different types of stroke including sinus thrombosis and bleedings on developing such seizures. Further, the association of post-stroke seizures and conventional vascular risk factors has not been evaluated to date., Methods: We performed a cohort study on a sample of 593 consecutive patients with different types of cerebrovascular events. In 421 patients, sufficient data were obtained in a personal interview over a mean observation period of 30 months. Data regarding the clinical history were recorded from the patients' charts., Results: The total prevalence of epileptic seizures was 11.6%, the total annual risk was 4.6%. We detected the following significant risk factors: younger age at stroke; higher NIH stroke scale score; any coagulopathy. TIA was found significantly less frequent as a cause of seizures as compared to infarction, bleeding, and sinus thrombosis. Patients with bleeding (14.3%) and with sinus thrombosis (16.3%) were significantly more frequent in the seizure group than in the non-seizure group (6.7% and 1.6%, respectively). The location of stroke, including cortical versus subcortical, did not influence the risk of seizures. The majority of patients developed secondary generalized seizures (57.1%). In adjusted analyses, the two major risk factors for post-stroke epilepsy were a higher NIH stroke scale and a sinus thrombosis as the initial cerebrovascular event. Common lifestyle, vascular, and metabolic risk factors of stroke and for dementia were not associated with the development of seizures., Conclusions: In conclusion, our data show that epileptic seizures occur in particular after major strokes and in sinus thrombosis. Interestingly, conventional vascular risk factors were not associated with the occurrence of post-stroke seizures. Considering the risk for seizures after certain types of cerebrovascular events might help to early identify patients for anticonvulsive treatment. In the future, it should be investigated whether these patients might benefit from pre-emptive anticonvulsant treatment., (Copyright © 2013 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

Modulation of the standing outward current (I (SO)) by muscarinic acetylcholine (ACh) receptor (MAChR) stimulation is fundamental for the state-dependent change in activity mode of thalamocortical relay (TC) neurons. Here, we probe the contribution of MAChR subtypes, G proteins, phospholipase C (PLC), and two pore domain K(+) (K(2P)) channels to this signaling cascade. By the use of spadin and A293 as specific blockers, we identify TWIK-related K(+) (TREK)-1 channel as new targets and confirm TWIK-related acid-sensitve K(+) (TASK)-1 channels as known effectors of muscarinic signaling in TC neurons. These findings were confirmed using a high affinity blocker of TASK-3 and TREK-1, namely, tetrahexylammonium chloride. It was found that the effect of muscarinic stimulation was inhibited by M(1)AChR-(pirenzepine, MT-7) and M(3)AChR-specific (4-DAMP) antagonists, phosphoinositide-specific PLCβ (PI-PLC) inhibitors (U73122, ET-18-OCH(3)), but not the phosphatidylcholine-specific PLC (PC-PLC) blocker D609. By comparison, depleting guanosine-5'-triphosphate (GTP) in the intracellular milieu nearly completely abolished the effect of MAChR stimulation. The block of TASK and TREK channels was accompanied by a reduction of the muscarinic effect on I (SO). Current-clamp recordings revealed a membrane depolarization following MAChR stimulation, which was sufficient to switch TC neurons from burst to tonic firing under control conditions but not during block of M(1)AChR/M(3)AChR and in the absence of intracellular GTP. These findings point to a critical role of G proteins and PLC as well as TASK and TREK channels in the muscarinic modulation of thalamic activity modes.

Objective: Reducing health and economic burdens from diagnostic delay of psychogenic nonepileptic seizures (PNES) requires prompt referral for video electroencephalography (VEEG) monitoring, the diagnostic gold standard. Practitioners make VEEG referrals when semiology suggests PNES, although few semiological signs are supported by well-designed studies, and most VEEG studies neglect to concurrently measure how accurately seizure witnesses can ascertain semiology. In this study, we estimate the value of eyewitness-reported and video-documented semiology for predicting PNES, and we measure accuracy of eyewitness reports., Methods: We prospectively interviewed eyewitnesses of seizures in patients referred for VEEG monitoring, to inquire about 48 putative PNES and ES signs. Multiple, EEG-blinded, epileptologists independently evaluated seizure videos and documented the presence/absence of signs. We used generalized estimating equations to identify reliable video-documented PNES and ES signs, and we compared eyewitness reports with video findings to assess how accurately signs are reported. We used logistic regression to determine whether eyewitness reports could predict VEEG-ascertained seizure type., Results: We analyzed 120 seizures (36 PNES, 84 ES) from 35 consecutive subjects. Of 45 video-documented signs, only 3 PNES signs ("preserved awareness," "eye flutter," and "bystanders can intensify or alleviate") and 3 ES signs ("abrupt onset," "eye-opening/widening," and postictal "confusion/sleep") were significant and reliable indicators of seizure type. Eyewitness reports of these 6 signs were inaccurate and not statistically different from guessing. Consequentially, eyewitness reports of signs did not predict VEEG-ascertained diagnosis. We validated our findings in a second, prospective cohort of 36 consecutive subjects., Interpretation: We identified 6 semiological signs that reliably distinguish PNES and ES, and found that eyewitness reports of these signs are unreliable. We offer suggestions to improve the accuracy of eyewitness reports., (Copyright © 2011 American Neurological Association.)

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels are the molecular substrate of the hyperpolarization-activated inward current (I(h)). Because the developmental profile of HCN channels in the thalamus is not well understood, we combined electrophysiological, molecular, immunohistochemical, EEG recordings in vivo, and computer modeling techniques to examine HCN gene expression and I(h) properties in rat thalamocortical relay (TC) neurons in the dorsal part of the lateral geniculate nucleus and the functional consequence of this maturation. Recordings of TC neurons revealed an approximate sixfold increase in I(h) density between postnatal day 3 (P3) and P106, which was accompanied by significantly altered current kinetics, cAMP sensitivity, and steady-state activation properties. Quantification on tissue levels revealed a significant developmental decrease in cAMP. Consequently the block of basal adenylyl cyclase activity was accompanied by a hyperpolarizing shift of the I(h) activation curve in young but not adult rats. Quantitative analyses of HCN channel isoforms revealed a steady increase of mRNA and protein expression levels of HCN1, HCN2, and HCN4 with reduced relative abundance of HCN4. Computer modeling in a simplified thalamic network indicated that the occurrence of rhythmic delta activity, which was present in the EEG at P12, differentially depended on I(h) conductance and modulation by cAMP at different developmental states. These data indicate that the developmental increase in I(h) density results from increased expression of three HCN channel isoforms and that isoform composition and intracellular cAMP levels interact in determining I(h) properties to enable progressive maturation of rhythmic slow-wave sleep activity patterns.

By combining electrophysiological, immunohistochemical, and computer modeling techniques, we examined the effects of halothane on the standing outward current (I (SO)) and the hyperpolarization-activated current (I (h)) in rat thalamocortical relay (TC) neurons of the dorsal lateral geniculate nucleus (dLGN). Hyperpolarizing voltage steps elicited an instantaneous current component (I (i)) followed by a slower time-dependent current that represented I (h). Halothane reduced I (h) by shifting the voltage dependency of activation toward more negative potentials and by reducing the maximal conductance. Moreover, halothane augmented I (i) and I (SO). During the blockade of I (h) through Cs+, the current-voltage relationship of the halothane-sensitive current closely resembled the properties of a current through members of the TWIK-related acid-sensitive K+ (TASK) channel family (I (TASK)). Computer simulations in a single-compartment TC neuron model demonstrated that the modulation of I (h) and I (TASK) is sufficient to explain the halothane-induced hyperpolarization of the membrane potential observed in current clamp recordings. Immunohistochemical staining revealed protein expression of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel proteins HCN1, HCN2, and HCN4. Together with the dual effect of halothane on I (h) properties, these results suggest that I (h) in TC neurons critically depends on HCN1/HCN2 heterodimers. It is concluded that the reciprocal modulation of I (h) and I (TASK) is an important mechanism of halothane action in the thalamus.