Forward programming human pluripotent stem cells into microglia.

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.

Quantitative Analysis of Retinal Perfusion in Patients with Frontotemporal Dementia Using Optical Coherence Tomography Angiography.

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.

NOX4-derived ROS are neuroprotective by balancing intracellular calcium stores.

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.

A novel NMDA receptor test model based on hiPSC-derived neural cells.

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.

MAPT genotype-dependent mitochondrial aberration and ROS production trigger dysfunction and death in cortical neurons of patients with hereditary FTLD.

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 MAPTN279K- or MAPTV337M-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 MAPTN279K neurons but not in MAPTV337M 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 MAPTN279K neurons. These spontaneous calcium oscillations in MAPTN279K 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 MAPTN279K neurons, but not in MAPTV337M 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.

MMF induces antioxidative and anaplerotic pathways and is neuroprotective in hyperexcitability in vitro.

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.

Deterministic programming of human pluripotent stem cells into microglia facilitates studying their role in health and disease.

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/EBPß 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.

[Causal treatment of Alzheimer's disease: amyloid antibodies]. / Kausale Therapie der Alzheimer-Krankheit: Amyloidantikörper.

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.

Increased Intrathecal B and Plasma Cells in Patients With Anti-IgLON5 Disease: A Case Series.

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.

Patients with a relapsing course of steroid-responsive encephalopathy associated with autoimmune thyroiditis exhibit persistent intrathecal CD4+ T-cell activation.

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.

[Young onset dementia]. / Früh beginnende Demenzen.

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.

Generating microglia from human pluripotent stem cells: novel in vitro models for the study of neurodegeneration.

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.

Substance P Saliva Reduction Predicts Pharyngeal Dysphagia in Parkinson's Disease.

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.

Apraxia screening predicts Alzheimer pathology in frontotemporal dementia.

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.

Distinguishing neurocognitive deficits in adult patients with NP-C from early onset Alzheimer's dementia.

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.

Diagnostic Value of Diffusion Tensor Imaging and Positron Emission Tomography in Early Stages of Frontotemporal Dementia.

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.

Conditional Manipulation of Gene Function in Human Cells with Optimized Inducible shRNA.

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.

Immune Cell Activation in the Cerebrospinal Fluid of Patients With Parkinson's Disease.

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.

Hemicrania continua in carotid artery dissection - symptomatic cases or linked pathophysiology?

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.