Endogenous recapitulation of Alzheimer's disease neuropathology through human 3D direct neuronal reprogramming.

Alzheimer's disease (AD) is a neurodegenerative disorder that primarily affects elderly individuals, and is characterized by hallmark neuronal pathologies including extracellular amyloid-ß (Aß) plaque deposition, intracellular tau tangles, and neuronal death. However, recapitulating these age-associated neuronal pathologies in patient-derived neurons has remained a significant challenge, especially for late-onset AD (LOAD), the most common form of the disorder. Here, we applied the high efficiency microRNA-mediated direct neuronal reprogramming of fibroblasts from AD patients to generate cortical neurons in three-dimensional (3D) Matrigel and self-assembled neuronal spheroids. Our findings indicate that neurons and spheroids reprogrammed from both autosomal dominant AD (ADAD) and LOAD patients exhibited AD-like phenotypes linked to neurons, including extracellular Aß deposition, dystrophic neurites with hyperphosphorylated, K63-ubiquitin-positive, seed-competent tau, and spontaneous neuronal death in culture. Moreover, treatment with ß- or γ-secretase inhibitors in LOAD patient-derived neurons and spheroids before Aß deposit formation significantly lowered Aß deposition, as well as tauopathy and neurodegeneration. However, the same treatment after the cells already formed Aß deposits only had a mild effect. Additionally, inhibiting the synthesis of age-associated retrotransposable elements (RTEs) by treating LOAD neurons and spheroids with the reverse transcriptase inhibitor, lamivudine, alleviated AD neuropathology. Overall, our results demonstrate that direct neuronal reprogramming of AD patient fibroblasts in a 3D environment can capture age-related neuropathology and reflect the interplay between Aß accumulation, tau dysregulation, and neuronal death. Moreover, miRNA-based 3D neuronal conversion provides a human-relevant AD model that can be used to identify compounds that can potentially ameliorate AD-associated pathologies and neurodegeneration.

Modeling Brain Pathology of Niemann-Pick Disease Type C Using Patient-Derived Neurons.

BACKGROUND: Niemann-Pick disease type C (NPC) is a rare autosomal-recessive lysosomal storage disease that is also associated with progressive neurodegeneration. NPC shares many pathological features with Alzheimer's disease, including neurofibrillary tangles, axonal spheroids, ß-amyloid deposition, and dystrophic neurites. Here, we examined if these pathological features could be detected in induced pluripotent stem cell (iPSC)-derived neurons from NPC patients. METHODS: Brain tissues from 8 NPC patients and 5 controls were analyzed for histopathological and biochemical markers of pathology. To model disease in culture, iPSCs from NPC patients and controls were differentiated into cortical neurons. RESULTS: We found hyperphosphorylated tau, altered processing of amyloid precursor protein, and increased Aß42 in NPC postmortem brains and in iPSC-derived cortical neurons from NPC patients. CONCLUSION: Our findings demonstrated that the main pathogenic phenotypes typically found in NPC brains were also observed in patient-derived neurons, providing a useful model for further mechanistic and therapeutic studies of NPC. © 2021 International Parkinson and Movement Disorder Society.

The Therapeutic and Diagnostic Potential of Amyloid ß Oligomers Selective Antibodies to Treat Alzheimer's Disease.

Improvements have been made in the diagnosis of Alzheimer's disease (AD), manifesting mostly in the development of in vivo imaging methods that allow for the detection of pathological changes in AD by magnetic resonance imaging (MRI) and positron emission tomography (PET) scans. Many of these imaging methods, however, use agents that probe amyloid fibrils and plaques-species that do not correlate well with disease progression and are not present at the earliest stages of the disease. Amyloid ß oligomers (AßOs), rather, are now widely accepted as the Aß species most germane to AD onset and progression. Here we report evidence further supporting the role of AßOs as pathological instigators of AD and introduce promising anti-AßO diagnostic probes capable of distinguishing the 5xFAD mouse model from wild type mice by PET and MRI. In a developmental study, Aß oligomers in 5xFAD mice were found to appear at 3 months of age, just prior to the onset of memory dysfunction, and spread as memory worsened. The increase of AßOs is prominent in the subiculum and correlates with concomitant development of reactive astrocytosis. The impact of these AßOs on memory is in harmony with findings that intraventricular injection of synthetic AßOs into wild type mice induced hippocampal dependent memory dysfunction within 24 h. Compelling support for the conclusion that endogenous AßOs cause memory loss was found in experiments showing that intranasal inoculation of AßO-selective antibodies into 5xFAD mice completely restored memory function, measured 30-40 days post-inoculation. These antibodies, which were modified to give MRI and PET imaging probes, were able to distinguish 5xFAD mice from wild type littermates. These results provide strong support for the role of AßOs in instigating memory loss and salient AD neuropathology, and they demonstrate that AßO selective antibodies have potential both for therapeutics and for diagnostics.

Early abolition of cough reflex predicts mortality in deeply sedated brain-injured patients.

BACKGROUND: Deep sedation may hamper the detection of neurological deterioration in brain-injured patients. Impaired brainstem reflexes within the first 24 h of deep sedation are associated with increased mortality in non-brain-injured patients. Our objective was to confirm this association in brain-injured patients. METHODS: This was an observational prospective multicenter cohort study involving four neuro-intensive care units. We included acute brain-injured patients requiring deep sedation, defined by a Richmond Assessment Sedation Scale (RASS) < -3. Neurological assessment was performed at day 1 and included pupillary diameter, pupillary light, corneal and cough reflexes, and grimace and motor response to noxious stimuli. Pre-sedation Glasgow Coma Scale (GCS) and Simplified Acute Physiology Score (SAPS-II) were collected, as well as the cause of death in the Intensive Care Unit (ICU). RESULTS: A total of 137 brain-injured patients were recruited, including 70 (51%) traumatic brain-injured patients, 40 (29%) vascular (subarachnoid hemorrhage or intracerebral hemorrhage). Thirty patients (22%) died in the ICU. At day 1, the corneal (OR 2.69, p = 0.034) and cough reflexes (OR 5.12, p = 0.0003) were more frequently abolished in patients that died in the ICU. In a multivariate analysis, abolished cough reflex was associated with ICU mortality after adjustment to pre-sedation GCS, SAPS-II, RASS (OR: 5.19, 95% CI [1.92-14.1], p = 0.001) or dose of sedatives (OR: 8.89, 95% CI [2.64-30.0], p = 0.0004). CONCLUSION: Early (day 1) cough reflex abolition is an independent predictor of mortality in deeply sedated brain-injured patients. Abolished cough reflex likely reflects a brainstem dysfunction that might result from the combination of primary and secondary neuro-inflammatory cerebral insults revealed and/or worsened by sedation.

Astrocytes Protect Human Dopaminergic Neurons from α-Synuclein Accumulation and Propagation.

The pathologic hallmark of Parkinson's disease is the accumulation of α-synuclein-containing Lewy bodies/neurites almost exclusively in neurons, and rarely in glial cells. However, emerging evidence suggests that glia such as astrocytes play an important role in the development of α-synuclein pathology. Using induced pluripotent stem-derived dopaminergic neurons and astrocytes from healthy subjects and patients carrying mutations in lysosomal ATP13A2, a monogenic form of synucleinopathy, we found that astrocytes rapidly internalized α-synuclein, and exhibited higher lysosomal degradation rates compared with neurons. Moreover, coculturing astrocytes and neurons led to decreased accumulation of α-synuclein in neurons and consequently diminished interneuronal transfer of α-synuclein. These protective functions of astrocytes were attenuated by ATP13A2 deficiency, suggesting that the loss of ATP13A2 function in astrocytes at least partially contributes to neuronal α-synuclein pathology. Together, our results highlight the importance of lysosomal function in astrocytes in the pathogenesis of synucleinopathies.SIGNIFICANCE STATEMENT While most neurodegenerative disorders are characterized by the accumulation of aggregated mutant proteins exclusively in neurons, the contribution of glial cells in this process remains poorly explored. Here, we demonstrate that astrocytes contribute to the removal of extracellular α-synuclein and that disruption of this pathway caused by mutations in the Parkinson's disease-linked gene ATP13A2 result in α-synuclein accumulation in human dopaminergic neurons. We found that astrocytes also protect neurons from α-synuclein propagation, whereas ATP13A2 deficiency in astrocytes compromises this protective function. These results highlight astrocyte-mediated α-synuclein clearance as a potential therapeutic target in disorders characterized by the accumulation of α-synuclein, including Parkinson's disease.

Progranulin mutations result in impaired processing of prosaposin and reduced glucocerebrosidase activity.

Frontotemporal dementia (FTD) is a common neurogenerative disorder characterized by progressive degeneration in the frontal and temporal lobes. Heterozygous mutations in the gene encoding progranulin (PGRN) are a common genetic cause of FTD. Recently, PGRN has emerged as an important regulator of lysosomal function. Here, we examine the impact of PGRN mutations on the processing of full-length prosaposin to individual saposins, which are critical regulators of lysosomal sphingolipid metabolism. Using FTD-PGRN patient-derived cortical neurons differentiated from induced pluripotent stem cells, as well as post-mortem tissue from patients with FTLD-PGRN, we show that PGRN haploinsufficiency results in impaired processing of prosaposin to saposin C, a critical activator of the lysosomal enzyme glucocerebrosidase (GCase). Additionally, we found that PGRN mutant neurons had reduced lysosomal GCase activity, lipid accumulation and increased insoluble α-synuclein relative to isogenic controls. Importantly, reduced GCase activity in PGRN mutant neurons is rescued by treatment with saposin C. Together, these findings suggest that reduced GCase activity due to impaired processing of prosaposin may contribute to pathogenesis of FTD resulting from PGRN mutations.

Fluctuations in cell density alter protein markers of multiple cellular compartments, confounding experimental outcomes.

The life cycle of cultured proliferating cells is characterized by fluctuations in cell population density induced by periodic subculturing. This leads to corresponding changes in micro- and macroenvironment of the cells, accompanied by altered cellular metabolism, growth rate and locomotion. Studying cell density-dependent morphological, physiological and biochemical fluctuations is relevant for understanding basic cellular mechanisms and for uncovering the intrinsic variation of commonly used tissue culture experimental models. Using multiple cell lines, we found that expression levels of the autophagic markers p62 and LC3II, and lysosomal enzyme cathepsin D were altered in highly confluent cells as a consequence of nutrient depletion and cell crowding, which led to inactivation of the mTOR signaling pathway. Furthermore, both Lamp1 and active focal adhesion kinase (FAK) were reduced in high-density cells, while chemical inhibition or deletion of FAK led to alterations in lysosomal and autophagic proteins, as well as in the mTOR signaling. This was accompanied by alterations in the Hippo signaling pathway, while cell cycle checkpoint regulator p-cdc2 remained unaffected in at least one studied cell line. On the other hand, allometric scaling of cellular compartments in growing cell populations resulted in biochemically detectable changes in the plasma membrane proteins Na+K+-ATPase and cadherin, and nuclear proteins HDAC1 and Lamin B1. Finally, we demonstrate how treatment-induced changes in cell density and corresponding modulation of susceptible proteins may lead to ambiguous experimental outcomes, or erroneous interpretation of cell culture data. Together, our data emphasize the need to recognize cell density as an important experimental variable in order to improve scientific rigor of cell culture-based studies.

Progranulin-mediated deficiency of cathepsin D results in FTD and NCL-like phenotypes in neurons derived from FTD patients.

Frontotemporal dementia (FTD) encompasses a group of neurodegenerative disorders characterized by cognitive and behavioral impairments. Heterozygous mutations in progranulin (PGRN) cause familial FTD and result in decreased PGRN expression, while homozygous mutations result in complete loss of PGRN expression and lead to the neurodegenerative lysosomal storage disorder neuronal ceroid lipofuscinosis (NCL). However, how dose-dependent PGRN mutations contribute to these two different diseases is not well understood. Using iPSC-derived human cortical neurons from FTD patients harboring PGRN mutations, we demonstrate that PGRN mutant neurons exhibit decreased nuclear TDP-43 and increased insoluble TDP-43, as well as enlarged electron-dense vesicles, lipofuscin accumulation, fingerprint-like profiles and granular osmiophilic deposits, suggesting that both FTD and NCL-like pathology are present in PGRN patient neurons as compared to isogenic controls. PGRN mutant neurons also show impaired lysosomal proteolysis and decreased activity of the lysosomal enzyme cathepsin D. Furthermore, we find that PGRN interacts with cathepsin D, and that PGRN increases the activity of cathepsin D but not cathepsins B or L. Finally, we show that granulin E, a cleavage product of PGRN, is sufficient to increase cathepsin D activity. This functional relationship between PGRN and cathepsin D provides a possible explanation for overlapping NCL-like pathology observed in patients with mutations in PGRN or CTSD, the gene encoding cathepsin D. Together, our work identifies PGRN as an activator of lysosomal cathepsin D activity, and suggests that decreased cathepsin D activity due to loss of PGRN contributes to both FTD and NCL pathology in a dose-dependent manner.

Growth Inhibition and DNA Damage Induced by X-Phenols in Yeast: A Quantitative Structure-Activity Relationship Study.

Phenolic compounds and their derivatives are ubiquitous constituents of numerous synthetic and natural chemicals that exist in the environment. Their toxicity is mostly attributed to their hydrophobicity and/or the formation of free radicals. In a continuation of the study of phenolic toxicity in a systematic manner, we have examined the biological responses of Saccharomyces cerevisiae to a series of mostly monosubstituted phenols utilizing a quantitative structure-activity relationship (QSAR) approach. The biological end points included a growth assay that determines the levels of growth inhibition induced by the phenols as well as a yeast deletion (DEL) assay that assesses the ability of X-phenols to induce DNA damage or DNA breaks. The QSAR analysis of cell growth patterns determined by IC50 and IC80 values indicates that toxicity is delineated by a hydrophobic, parabolic model. The DEL assay was then utilized to detect genomic deletions in yeast. The increase in the genotoxicity was enhanced by the electrophilicity of the phenolic substituents that were strong electron donors as well as by minimal hydrophobicity. The electrophilicities are represented by Brown's sigma plus values that are a variant of the Hammett sigma constants. A few mutant strains of genes involved in DNA repair were separately exposed to 2,6-di-tert-butyl-4-methyl-phenol (BHT) and butylated hydroxy anisole (BHA). They were subsequently screened for growth phenotypes. BHA-induced growth defects in most of the DNA repair null mutant strains, whereas BHT was unresponsive.

Avaliação do foto-eletrocardiograma como ferramenta de segunda opinião formativa / Evaluation of the photo-electrocardiogram as a tool for formative second opinion

Objetivos: Avaliar o foto-eletrocardiograma (foto-ECG), como uma ferramenta de segunda opinião formativa a distância.Métodos: Cinquenta eletrocardiogramas (ECGs) em papel milimetrado foram fotografados duas vezes, a primeira utilizando-se uma câmera digital Canon, na resolução 0,3 megapixel, e a segunda com um celular Nokia com câmera acoplada, na resolução 2,0 megapixels, gerando 100 foto-ECGs. Por meio de estudos-piloto, definiu-se o método de aquisição das imagens. Os 100 foto-ECGs foram randomizados, criptografados e enviados a um cardiologista remoto por e-mail; enquanto os 50 ECGs em papel milimetrado lhe foram entregues pessoalmente, sem randomização. Sexo e idade foram as únicas informações dos pacientes disponibilizadas ao especialista.Resultados: A análise dos dados demonstrou divergência em 14 dos 50 laudos (28%) na comparação dos ECGs originais com os foto-ECGs adquiridos pela câmera Canon e de 13 dos 50 laudos (26%) entre os laudos dos ECGs originais e dos foto-ECGs capturados pelo celular Nokia. Houve concordância considerável (Kappa=0,356) entre as interpretações de foto-ECGs e ECGs em papel, tanto para o celular Nokia quanto para a câmera Canon.Conclusões: A concordância entre o foto-ECG e os traçados originais demonstrou que o método descrito nesse estudo tem potencial de ser utilizado como uma ferramenta de auxílio à prática clínica, desde que a obtenção dos foto-ECGs seja adaptada de forma a melhorar as imagens dos exames. Apenas com concordância boa a ótima em relação aos ECGs originais, o foto-ECG possibilitará a segunda opinião formativa a distância, conferindo melhores opções diagnósticas e terapêuticas...

Aims: This study aimed to evaluate the photo-electrocardiogram (photo-ECG), as an alternative tool to enable remote formative second opinion in cardiology.Methods: Fifty paper electrocardiograms (ECGs) were photographed two times, the first using a Canon digital camera, 0.3 megapixel resolution, and the second using a Nokia mobile phone integrated camera, 2.0 megapixel resolution, resulting in 100 Photo-ECGs. A pilot study was responsible for determining the acquisition method. The 100 Photo-ECGs were randomized, encrypted and sent by e-mail to a remote cardiologist, while the 50 paper ECGs were delivered to him in person, without randomization. Gender and age were the only patient information made available to the specialist.Results: Data analysis demonstrated a disagreement in 14 of 50 interpretations (28%) when comparing paper ECGs to the Canon camera photo-ECGs and in 13 of 50 interpretations (26%) when comparing paper ECGs to the Nokia camera photo-ECGs. The Kappa test revealed a fair agreement (Kappa=0.356) between interpretations when comparing the original ECGs to their respective photo-ECGs for both camera devices.Conclusion: The concordance between photo-ECGs and original tracings demonstrated that the method described herein has the potential for use as a tool to assist clinical practice, provided that the acquisition of photo-ECGs is adapted so as to improve exam images. Only with good to very good concordance between the original ECGs and photo-ECGs will remote formative second opinion be possible, giving better diagnostic and therapeutic options...

Cleland's and Grayson's ligaments of the hand: a morphometrical investigation.

The cutaneous ligaments of the human digits are delicate functional structures essential for normal skin stability during digital movements. These ligaments extend bilaterally between the phalanx and the finger dermis, either posteriorly (Cleland's ligaments) or anteriorly (Grayson's ligaments) to the digital neurovascular bundles. We have performed a series of detailed anatomical dissections of the human digits so as to investigate morphometrically Cleland's and Grayson's ligaments and their topographic arrangements. Data were statistically compared between fingers, respecting both side (left or right) and sex, in an attempt to clarify some of the morphologic variations of these structures. The cutaneous ligaments of the human digits have been analyzed bilaterally both in 30 fixed cadavers (300 adult human digits) and in 10 nonfixed human cadaveric digits. A computerized morphometrical investigation of the human digits and their Cleland's and Grayson's ligaments has been performed and the resulting quantitative data have been statistically assessed, comparing groups according to finger, phalanx, side (left or right hands), and sex. The ratio between the origin and insertion (O:I) of these ligaments indicate a divergent arrangement of fibers, with values varying from 0.52 to 0.84, depending on the phalanx and finger analyzed. Our morphometrical data provide normal reference values, mainly for Grayson's ligaments, that can be useful in the comparison with the respective measurements obtained in Dupuytren's disease. Morphological bases are also provided, which may be relevant either in computerized tomography or magnetic resonance imaging involving the hand region and in their application in surgical procedures of the human hand.