bims-axbals 2024-12-08 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2024–12–08
seventeen papers selected by
TJ Krzystek, ALS Therapy Development Institute

Endo-IP and lyso-IP toolkit for endolysosomal profiling of human-induced neurons.
Human iPSC-derived myelinating organoids and globoid cells to study Krabbe disease.
CK1δ/ε-mediated TDP-43 phosphorylation contributes to early motor neuron disease toxicity in amyotrophic lateral sclerosis.
Temporal control of acute protein aggregate turnover by UBE3C and NRF1-dependent proteasomal pathways.
Shared and distinct changes in the molecular cargo of extracellular vesicles in different neurodegenerative diseases.
RuvBL1/2 reduce toxic dipeptide repeat protein burden in multiple models of C9orf72-ALS/FTD.
C9orf72-linked arginine-rich dipeptide repeats aggravate pathological phase separation of G3BP1.
Efficient derivation of functional astrocytes from human induced pluripotent stem cells (hiPSCs).
Unravelling neuronal and glial differences in ceramide composition, synthesis, and sensitivity to toxicity.
Trehalose extricates impaired mitochondrial and autophagy dysregulation in patient iPSC-derived macular corneal dystrophy disease model.
Generate and Analyze Three-Dimensional Dendritic Spine Morphology Datasets With SpineTool Software.
Linking glycosphingolipid metabolism to disease-related changes in the plasma membrane proteome.
Frontotemporal Dementia.
Cytoplasmic Aggregates of Splicing Factor Proline-Glutamine Rich Disrupt Nucleocytoplasmic Transport and Induce Persistent Stress Granules.
Calcium connects lysosomal damage to stress granule formation.
Using magnetic resonance relaxometry to evaluate the safety and quality of induced pluripotent stem cell-derived spinal cord progenitor cells.
Microfluidics in neural extracellular vesicles characterization for early Alzheimer's disease diagnosis.

Proc Natl Acad Sci U S A. 2024 Dec 24. 121(52): e2419079121

Endo-IP and lyso-IP toolkit for endolysosomal profiling of human-induced neurons.

Frances V Hundley, Miguel A Gonzalez-Lozano, Lena M Gottschalk, Aslan N K Cook, Jiuchun Zhang, Joao A Paulo, J Wade Harper.

  Plasma membrane protein degradation and recycling are regulated by the endolysosomal system, wherein endocytic vesicles bud from the plasma membrane into the cytoplasm and mature into endosomes and then degradative lysosomes. As such, the endolysosomal system plays a critical role in determining the abundance of proteins on the cell surface and influencing cellular identity and function. Highly polarized cells, like neurons, rely on the endolysosomal system for axonal and dendritic specialization and synaptic compartmentalization. The importance of this system to neuronal function is reflected by the prevalence of risk variants in components of the system in several neurodegenerative diseases, ranging from Parkinson's to Alzheimer's disease. Nevertheless, our understanding of endocytic cargo and core endolysosomal machinery in neurons is limited, in part due to technical limitations. Here, we develop a toolkit for capturing EEA1-positive endosomes (termed Endo-IP) and TMEM192-positive lysosomes (termed Lyso-IP) in stem cell-derived induced neurons (iNeurons). We demonstrate its utility by revealing the endolysosomal protein landscapes for stem cells and cortical-like iNeurons, and profiling endosomes in response to potassium-mediated neuronal depolarization. Through global profiling of endocytic cargo, we identify hundreds of transmembrane proteins, including neurogenesis and synaptic proteins, as well as endocytic cargo with predicted SNX17 or SNX27 recognition motifs. By contrast, parallel lysosome profiling reveals a simpler protein repertoire, reflecting in part temporally controlled recycling or degradation for many endocytic targets. This system will facilitate mechanistic interrogation of endolysosomal components found as risk factors in neurodegenerative disease.

Keywords:  endosome; iNeuron; lysosome; proteomics; stem cells

DOI:  https://doi.org/10.1073/pnas.2419079121
PLoS One. 2024 ;19(12): e0314858

Human iPSC-derived myelinating organoids and globoid cells to study Krabbe disease.

Lisa Marie P Evans, Joseph Gawron, Fraser J Sim, M Laura Feltri, Leandro N Marziali.

Krabbe disease (Kd) is a lysosomal storage disorder (LSD) caused by the deficiency of the lysosomal galactosylceramidase (GALC) which cleaves the myelin enriched lipid galactosylceramide (GalCer). Accumulated GalCer is catabolized into the cytotoxic lipid psychosine that causes myelinating cells death and demyelination which recruits microglia/macrophages that fail to digest myelin debris and become globoid cells. Here, to understand the pathological mechanisms of Kd, we used induced pluripotent stem cells (iPSCs) from Kd patients to produce myelinating organoids and microglia. We show that Kd organoids have no obvious defects in neurogenesis, astrogenesis, and oligodendrogenesis but manifest early myelination defects. Specifically, Kd organoids showed shorter but a similar number of myelin internodes than Controls at the peak of myelination and a reduced number and shorter internodes at a later time point. Interestingly, myelin is affected in the absence of autophagy and mTOR pathway dysregulation, suggesting lack of lysosomal dysfunction which makes this organoid model a very valuable tool to study the early events that drive demyelination in Kd. Kd iPSC-derived microglia show a marginal rate of globoid cell formation under normal culture conditions that is drastically increased upon GalCer feeding. Under normal culture conditions, Kd microglia show a minor LAMP1 content decrease and a slight increase in the autophagy protein LC3B. Upon GalCer feeding, Kd cells show accumulation of autophagy proteins and strong LAMP1 reduction that at a later time point are reverted showing the compensatory capabilities of globoid cells. Altogether, this supports the value of our cultures as tools to study the mechanisms that drive globoid cell formation and the compensatory mechanism in play to overcome GalCer accumulation in Kd.

DOI: https://doi.org/10.1371/journal.pone.0314858
Acta Neuropathol Commun. 2024 Dec 04. 12(1): 187

CK1δ/ε-mediated TDP-43 phosphorylation contributes to early motor neuron disease toxicity in amyotrophic lateral sclerosis.

Vivian I Ko, Kailee Ong, Deborah Y Kwon, Xueying Li, Alicia Pietrasiewicz, James S Harvey, Mukesh Lulla, Guruharsha Bhat, Don W Cleveland, John M Ravits.

  Hyperphosphorylated TDP-43 aggregates in the cytoplasm of motor neurons is a neuropathological signature of amyotrophic lateral sclerosis (ALS). These aggregates have been proposed to possess a toxic disease driving role in ALS pathogenesis and progression, however, the contribution of phosphorylation to TDP-43 aggregation and ALS disease mechanisms remains poorly understood. We've previously shown that CK1δ and CK1ε phosphorylate TDP-43 at disease relevant sites, and that genetic reduction and chemical inhibition could reduce phosphorylated TDP-43 (pTDP-43) levels in cellular models. In this study, we advanced our findings into the hTDP-43-ΔNLS in vivo mouse model of ALS and TDP-43 proteinopathy. This mouse model possesses robust disease-relevant features of ALS, including TDP-43 nuclear depletion, cytoplasmic pTDP-43 accumulation, motor behavior deficits, and shortened survival. We tested the effect of homozygous genetic deletion of Csnk1e in the hTDP-43-ΔNLS mouse model and observed a delay in the formation of pTDP-43 without significant ultimate rescue of TDP-43 proteinopathy or disease progression. Homozygous genetic deletion of Csnk1d is lethal in mice, and we were unable to test the role of CK1δ alone. We then targeted both CK1δ and CK1ε kinases by way of CK1δ/ε-selective PF-05236216 inhibitor in the hTDP-43-ΔNLS mouse model, reasoning that inhibiting CK1ε alone would be insufficient as shown by our Csnk1e knockout mouse model study. Treated mice demonstrated reduced TDP-43 phosphorylation, lowered Nf-L levels, and improved survival in the intermediate stages. The soluble TDP-43 may have been more amenable to the inhibitor treatment than insoluble TDP-43. However, the treatments did not result in improved functional measurements or in overall survival. Our results demonstrate that phosphorylation contributes to neuronal toxicity and suggest CK1δ/ε inhibition in combination with other therapies targeting TDP-43 pathology could potentially provide therapeutic benefit in ALS.

Keywords:  Amyotrophic lateral sclerosis; Casein kinase 1 delta; Casein kinase 1 epsilon; Kinase inhibitors; Phosphorylation; TAR DNA-binding protein (TDP-43)

DOI:  https://doi.org/10.1186/s40478-024-01902-z
Proc Natl Acad Sci U S A. 2024 Dec 10. 121(50): e2417390121

Temporal control of acute protein aggregate turnover by UBE3C and NRF1-dependent proteasomal pathways.

Kelsey L Hickey, Alexandra Panov, Enya Miguel Whelan, Tillman Schäfer, Arda Mizrak, Ron R Kopito, Wolfgang Baumeister, Rubén Fernández-Busnadiego, J Wade Harper.

  A hallmark of neurodegenerative diseases (NDs) is the progressive loss of proteostasis, leading to the accumulation of misfolded proteins or protein aggregates, with subsequent cytotoxicity. To combat this toxicity, cells have evolved degradation pathways (ubiquitin-proteasome system and autophagy) that detect and degrade misfolded proteins. However, studying the underlying cellular pathways and mechanisms has remained a challenge, as formation of many types of protein aggregates is asynchronous, with individual cells displaying distinct kinetics, thereby hindering rigorous time-course studies. Here, we merge a kinetically tractable and synchronous agDD-GFP system for aggregate formation with targeted gene knockdowns, to uncover degradation mechanisms used in response to acute aggregate formation. We find that agDD-GFP forms amorphous aggregates by cryo-electron tomography at both early and late stages of aggregate formation. Aggregate turnover occurs in a proteasome-dependent mechanism in a manner that is dictated by cellular aggregate burden, with no evidence of the involvement of autophagy. Lower levels of misfolded agDD-GFP, enriched in oligomers, utilizes UBE3C-dependent proteasomal degradation in a pathway that is independent of RPN13 ubiquitylation by UBE3C. Higher aggregate burden activates the NRF1 transcription factor to increase proteasome subunit transcription and subsequent degradation capacity of cells. Loss or gain of NRF1 function alters the turnover of agDD-GFP under conditions of high aggregate burden. Together, these results define the role of UBE3C in degradation of this class of misfolded aggregation-prone proteins and reveals a role for NRF1 in proteostasis control in response to widespread protein aggregation.

Keywords:  protein aggregates; protein quality control; protein turnover; ubiquitin-proteasome system

DOI:  https://doi.org/10.1073/pnas.2417390121
Cell Mol Life Sci. 2024 Dec 03. 81(1): 479

Shared and distinct changes in the molecular cargo of extracellular vesicles in different neurodegenerative diseases.

Anna F Wiersema, Alyssa Rennenberg, Grace Smith, Suzy Varderidou-Minasian, R Jeroen Pasterkamp.

  Neurodegenerative disorders such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD) affect millions of people worldwide. Curative treatment for these neurodegenerative disorders is still lacking and therefore a further understanding of their cause and progression is urgently needed. Extracellular vesicles (EVs) are nanosized vesicles loaded with cargo, such as proteins and miRNAs, that are released by cells and play an important role in intercellular communication. Intercellular communication through EVs can contribute to the spread of pathological proteins, such as amyloid-beta and tau, or cause pathogenesis through other mechanisms. In addition, EVs may serve as potential biomarkers for diagnosis and for monitoring disease progression. In this review, we summarize and discuss recent advances in our understanding of the role of EVs in AD, ALS an PD with an emphasis on dysregulated cargo in each disease. We highlight shared dysregulated cargo between these diseases, discuss underlying pathways, and outline future implications for therapeutic strategies.

Keywords:  Alzheimer’s disease; Amyotrophic lateral sclerosis; Extracellular vesicles; Neurodegeneration; Parkinson’s disease

DOI:  https://doi.org/10.1007/s00018-024-05522-7
Life Sci Alliance. 2025 Feb;pii: e202402757. [Epub ahead of print]8(2):

RuvBL1/2 reduce toxic dipeptide repeat protein burden in multiple models of C9orf72-ALS/FTD.

Christopher P Webster, Bradley Hall, Olivia M Crossley, Dana Dauletalina, Marianne King, Ya-Hui Lin, Lydia M Castelli, Zih-Liang Yang, Ian Coldicott, Ergita Kyrgiou-Balli, Adrian Higginbottom, Laura Ferraiuolo, Kurt J De Vos, Guillaume M Hautbergue, Pamela J Shaw, Ryan Jh West, Mimoun Azzouz.

A G4C2 hexanucleotide repeat expansion in C9orf72 is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Bidirectional transcription and subsequent repeat-associated non-AUG (RAN) translation of sense and antisense transcripts leads to the formation of five dipeptide repeat (DPR) proteins. These DPRs are toxic in a wide range of cell and animal models. Therefore, decreasing RAN-DPRs may be of therapeutic benefit in the context of C9ALS/FTD. In this study, we found that C9ALS/FTD patients have reduced expression of the AAA+ family members RuvBL1 and RuvBL2, which have both been implicated in aggregate clearance. We report that overexpression of RuvBL1, but to a greater extent RuvBL2, reduced C9orf72-associated DPRs in a range of in vitro systems including cell lines, primary neurons from the C9-500 transgenic mouse model, and patient-derived iPSC motor neurons. In vivo, we further demonstrated that RuvBL2 overexpression and consequent DPR reduction in our Drosophila model was sufficient to rescue a number of DPR-related motor phenotypes. Thus, modulating RuvBL levels to reduce DPRs may be of therapeutic potential in C9ALS/FTD.

DOI: https://doi.org/10.26508/lsa.202402757
Proc Natl Acad Sci U S A. 2024 Dec 10. 121(50): e2402847121

C9orf72-linked arginine-rich dipeptide repeats aggravate pathological phase separation of G3BP1.

Margot Van Nerom, Junaid Ahmed, Tamas Lazar, Attila Meszaros, Quentin Galand, Wim De Malsche, Joris Van Lindt, Rita Pancsa, Dominique Maes, Peter Tompa.

  The toxic effects of C9orf72-derived arginine-rich dipeptide repeats (R-DPRs) on cellular stress granules in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia remain unclear at the molecular level. Stress granules are formed through the switch of Ras GTPase-activating protein-binding protein 1 (G3BP1) by RNA from a closed inactive state to an open activated state, driving the formation of the organelle by liquid-liquid phase separation (LLPS). We show that R-DPRs bind G3BP1 a thousand times stronger than RNA and initiate LLPS much more effectively. Their pathogenic effect is underscored by the slow transition of R-DPR-G3BP1 droplets to aggregated, ThS-positive states that can recruit ALS-linked proteins hnRNPA1, hnRNPA2, and TDP-43. Deletion constructs and molecular simulations show that R-DPR binding and LLPS are mediated via the negatively charged intrinsically disordered region 1 (IDR1) of the protein, allosterically regulated by its positively charged IDR3. Bioinformatic analyses point to the strong mechanistic parallels of these effects with the interaction of R-DPRs with nucleolar nucleophosmin 1 (NPM1) and underscore that R-DPRs interact with many other similar nucleolar and stress-granule proteins, extending the underlying mechanism of R-DPR toxicity in cells. Our results also highlight characteristic differences between the two R-DPRs, poly-GR and poly-PR, and suggest that the primary pathological target of poly-GR is not NPM1 in nucleoli, but G3BP1 in stress granules in affected cells.

Keywords:  G3BP1; liquid–liquid phase separation; neurodegenerative disease; repeat-expansion disease; stress granules

DOI:  https://doi.org/10.1073/pnas.2402847121
PLoS One. 2024 ;19(12): e0313514

Efficient derivation of functional astrocytes from human induced pluripotent stem cells (hiPSCs).

Balazs Szeky, Veronika Jurakova, Eliska Fouskova, Anita Feher, Melinda Zana, Vivien Reka Karl, Janos Farkas, Maria Bodi-Jakus, Martina Zapletalova, Shashank Pandey, Radek Kucera, Jan Lochman, Andras Dinnyes.

Astrocytes are specialized glial cell types of the central nervous system (CNS) with remarkably high abundance, morphological and functional diversity. Astrocytes maintain neural metabolic support, synapse regulation, blood-brain barrier integrity and immunological homeostasis through intricate interactions with other cells, including neurons, microglia, pericytes and lymphocytes. Due to their extensive intercellular crosstalks, astrocytes are also implicated in the pathogenesis of CNS disorders, such as ALS (amyotrophic lateral sclerosis), Parkinson's disease and Alzheimer's disease. Despite the critical importance of astrocytes in neurodegeneration and neuroinflammation are recognized, the lack of suitable in vitro systems limits their availability for modeling human brain pathologies. Here, we report the time-efficient, reproducible generation of astrocytes from human induced pluripotent stem cells (hiPSCs). Our hiPSC-derived astrocytes expressed characteristic astrocyte markers, such as GFAP, S100b, ALDH1L1 and AQP4. Furthermore, hiPSC-derived astrocytes displayed spontaneous calcium transients and responded to inflammatory stimuli by the secretion of type A1 and type A2 astrocyte-related cytokines.

DOI: https://doi.org/10.1371/journal.pone.0313514
Commun Biol. 2024 Nov 30. 7(1): 1597

Unravelling neuronal and glial differences in ceramide composition, synthesis, and sensitivity to toxicity.

John J McInnis, Disha Sood, Lilu Guo, Michael R Dufault, Mariana Garcia, Rachel Passaro, Grace Gao, Bailin Zhang, James C Dodge.

Ceramides are lipids that play vital roles in complex lipid synthesis, membrane function, and cell signaling. Disrupted ceramide homeostasis is implicated in cell-death and several neurologic diseases. Ceramides are often analyzed in tissue, but this approach fails to resolve cell-type differences in ceramide homeostasis that are likely essential to understanding cell and non-cell autonomous contributions to neurodegeneration. We show that human iPSC-derived neurons and glia differ in their rate of ceramide synthesis, ceramide isoform composition, and responses to altered ceramide levels. RNA-sequencing of cells treated to increase or decrease ceramides revealed connections to inflammation, ER stress, and apoptosis. Moreover, introducing labeled sphinganine showed that glia readily synthesize ceramide de novo and that neurons are relatively more sensitive to ceramide toxicity. Our findings provide a framework for understanding neurologic diseases with sphingolipid alternations and insights into designing therapeutics that target ceramide for treating them.

DOI: https://doi.org/10.1038/s42003-024-07231-0
Stem Cell Res Ther. 2024 Dec 05. 15(1): 464

Trehalose extricates impaired mitochondrial and autophagy dysregulation in patient iPSC-derived macular corneal dystrophy disease model.

Divyani Nayak, Shivapriya Shivakumar, Rohit Shetty, K N Prashanthi, Arkasubhra Ghosh, Nallathambi Jeyabalan, Koushik Chakrabarty.

   BACKGROUND: Patient-derived induced pluripotent stem cell (iPSCs) represents a powerful tool for elucidating the underlying disease mechanisms. Macular corneal dystrophy (MCD) is an intractable and progressive bilateral corneal disease affecting the corneal stroma due to mutation/s in carbohydrate sulfotransferase 6 (CHST6) gene. The underlying molecular mechanisms leading to MCD are unclear due to a lack of human contextual model and limited access to affected corneal stromal keratocytes (CSKs) from MCD patients. This has restricted the current treatment option for MCD to restorative corneal transplantation thereby lending itself to the use of iPSCs.
METHODS: induced pluripotent stem cells (iPSCs) were generated from two MCD patients and a healthy participant by senai virus based reprogramming of the peripheral mononuclear blood cells (PBMCs). The iPSCs were characterized based on the expression of pluripotent markers and formation of embryoid bodies possessing tri-lineage potential. Directed differentiation of the iPSCs to corneal stromal keratocytes (CSKs) was done via intermediate induction of neural crest cells. The iCSKs were characterized by immunocytochemistry and qPCR. Proteostat staining of the iCSKs was done to validate the disease phenotype invitro. Expression of autophagy markers in the iCSKs and JC staining were visualized by immunochemistry and live-cell imaging in trehalose treated iCSKs.
RESULTS: We show that the MCD iPSC-derived CSKs (MCDiCSKs) exhibits impaired autophagy assessed by the profiles of autophagy-associated proteins (LAMP1, LC3II/I, p62 and Beclin-1) and mitochondrial membrane potential. Significantly higher protein aggregates in MCDiCSKs was seen compared with the control, which could be rescued upon autophagy modulation. Hence, we treated MCD-iCSKs with trehalose (autophagy inducer) and showed that it protects MCD-iCSKs from mitochondrial dysfunction and maintains autophagic degradation.
CONCLUSION: Our study highlights the possible pathological mechanisms involved in MCD. We found trehalose ameliorate the impaired mitochondrial and autophagy dysregulation in patient iPSC-derived macular corneal dystrophy disease model, which could be a potential alternative for MCD management.

Keywords:  Autophagy; Corneal stromal cells; Differentiation; Macular corneal dystrophy; Mitochondrial damage; Pluripotent stem cells

DOI:  https://doi.org/10.1186/s13287-024-04016-4
Curr Protoc. 2024 Dec;4(12): e70061

Generate and Analyze Three-Dimensional Dendritic Spine Morphology Datasets With SpineTool Software.

Anita Ustinova, Ekaterina Volkova, Anastasiya Rakovskaya, Daria Smirnova, Olesya Korovina, Ekaterina Pchitskaya.

  Dendritic spine morphology is associated with the current state of the synapse and neuron, and changes during synaptic plasticity in response to stimulus. At the same time, dendritic spine alterations are reported during various neurodegenerative and neurodevelopmental disorders and other brain states. Accurate and informative analysis of spine shape has an urgent need for studying the synaptic processes and molecular pathways in normal and pathological conditions, and for testing synapto-protective strategies during preclinical studies. Primary neuronal cultures enable high quality imaging of dendritic spines and offer a wide spectrum of accessible experimental manipulations. This article outlines the protocol for isolating, culturing, fluorescent labeling, and imaging of mouse primary hippocampal neurons by three-dimensional (3D) confocal microscopy in a normal state and in conditions of low amyloid toxicity-an in vitro model of Alzheimer's disease. An alternate protocol describes the neuronal morphology analysis using the EGFP expressing neurons in line-M transgenic mouse brain slices. Since the dendritic spines are relatively small structures lying close to the confocal microscope resolution limit, their proper segmentation on the images is challenging. This protocol highlights the image-preprocessing steps, including generation of theoretical point spread function and deconvolution, which enhances resolution and removes noise, thereby enhancing the 3D spine reconstruction results. SpineTool, an open source Python-based script, enables 3D segmentation of dendrites and spines and numerical metric calculation, including key measures, such as spine length, volume, and surface area, with a new feature, the chord length distribution histogram, improving clustering results. SpineTool supports both manual and machine learning spine classification (i.e., mushroom, thin, stubby, filopodia) and automated clustering using k-means and DBSCAN methods. This protocol provides detailed instructions for using SpineTool to analyze and classify dendritic spines in control and experimental groups, enhancing our understanding of spine morphology across different experimental conditions. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Obtaining 3D confocal dendritic spine images of hippocampal neuronal culture in normal state and conditions of low amyloid toxicity Alternate Protocol: Obtaining confocal dendritic spine images of mice hippocampal neurons from fixed brain slices Support Protocol: Post-processing deconvolution of confocal images Basic Protocol 2: Segmentation of dendritic spines with SpineTool Basic Protocol 3: Spine dataset preparation using SpineTool Basic Protocol 4: Clustering of dendritic spines with SpineTool Basic Protocol 5: Machine classification of dendritic spines with SpineTool.

Keywords:  classification; clustering; dendritic spines; segmentation; software

DOI:  https://doi.org/10.1002/cpz1.70061
Biochem Soc Trans. 2024 Dec 06. pii: BST20240315. [Epub ahead of print]

Linking glycosphingolipid metabolism to disease-related changes in the plasma membrane proteome.

Holly Monkhouse, Janet E Deane.

  Glycosphingolipids (GSLs) are vital components of the plasma membrane (PM), where they play crucial roles in cell function. GSLs form specialised membrane microdomains that organise lipids and proteins into functional platforms for cell adhesion and signalling. GSLs can also influence the function of membrane proteins and receptors, via direct protein-lipid interactions thereby affecting cell differentiation, proliferation, and apoptosis. Research into GSL-related diseases has primarily focussed on lysosomal storage disorders, where defective enzymes lead to the accumulation of GSLs within lysosomes, causing cellular dysfunction and disease. However, recent studies are uncovering the broader cellular impact of GSL imbalances including on a range of organelles and cellular compartments such as the mitochondria, endoplasmic reticulum and PM. In this review we describe the mechanisms by which GSL imbalances can influence the PM protein composition and explore examples of the changes that have been observed in the PM proteome upon GSL metabolic disruption. Identifying and understanding these changes to the PM protein composition will enable a more complete understanding of lysosomal storage diseases and provide new insights into the pathogenesis of other GSL-related diseases, including cancer and neurodegenerative disorders.

Keywords:  glycosphingolipid; lysosomal storage disease; trafficking; vacuolar protein sorting proteins

DOI:  https://doi.org/10.1042/BST20240315
Continuum (Minneap Minn). 2024 Dec 01. 30(6): 1642-1672

Frontotemporal Dementia.

David Glenn Clark.

OBJECTIVE: This article discusses frontotemporal dementia (FTD) syndromes using a simplified framework of three core syndromes, including details on their pathology and unique genetic variations.
LATEST DEVELOPMENTS: FTD includes at least seven major clinical syndromes. The three core syndromes are behavioral variant FTD and two forms of progressive aphasia, commonly referred to as the nonfluent variant and semantic variant of primary progressive aphasia. Clinical features reflect the involvement of major functional brain networks. Derangements of three proteins account for nearly all underlying pathology for FTD syndromes: transactive response DNA-binding protein 43 (TDP-43) (approximately 50% of cases), MAPT (45% of cases), and FUS (5% of cases). The clinical presentation and imaging provide clues to the underlying pathology. FTD is more heritable than Alzheimer disease, with variations in C9orf72, MAPT, or GRN (which encodes progranulin) occurring in more than 10% of FTD cases.
ESSENTIAL POINTS: The framework described here will provide clinicians with a foundation for understanding the complex and heterogeneous set of FTD syndromes. There are currently no disease-modifying or US Food and Drug Administration (FDA)-approved treatments for FTD, but clinical trials are underway, including some targeting presymptomatic genetic variation carriers. Available FTD treatments address deficits in behavior or language nonpharmacologically or through the off-label use of medications approved for other indications. Improvements in biomarkers will accelerate the discovery of new pharmacologic treatments.

DOI: https://doi.org/10.1212/CON.0000000000001506
J Cell Mol Med. 2024 Dec;28(23): e70261

Cytoplasmic Aggregates of Splicing Factor Proline-Glutamine Rich Disrupt Nucleocytoplasmic Transport and Induce Persistent Stress Granules.

Zicong Huang, Hanbin Zhang, Chuyu Huang, Runduan Yi, Xiaoyuan Zhang, Ke Ma, Wei Huang, Qingqing Wu, Yuge Zhuang, Jinsheng Liu, Wenyuan Liu, Yunhui Guo, Xiangjin Kang, Xiao Hu, Xiaochun Bai, Zhenguo Chen.

  Splicing factor proline-glutamine rich (SFPQ), a multifunctional RNA-binding protein (RBP), shows cytoplasmic colocalisation with stress granule (SG) markers; however, the causative relationship and mechanism underlying this coalescence of SFPQ aggregates and SGs remain unclear. In this study, we demonstrate that SFPQ lacking its nuclear localisation sequence spontaneously forms cytoplasmic aggregates that abnormally incorporate immature RNA and induce persistent SGs. mRNA profiling showed that SFPQ mislocalisation induced extensive changes in RNA processing, with a subset of alternatively spliced transcripts associated with nucleocytoplasmic transport. Notably, these altered transporters were sequestered into SFPQ aggregates, constituting aberrant protein-RNA complexes. Importantly, suppression of SG nucleation could not block cytoplasmic SFPQ aggregation with immature RNA and nucleocytoplasmic transporters, both of which, however, were moderately ameliorated by the inhibition of alternative splicing or nuclear export. Our results unveil the physiopathological role and mechanism for mislocalised SFPQ in the RNA metabolism, nucleocytoplasmic transport and pathological SGs.

Keywords:  RNA metabolism; SFPQ; cytoplasmic aggregate; nucleocytoplasmic transport; stress granule

DOI:  https://doi.org/10.1111/jcmm.70261
Nat Rev Mol Cell Biol. 2024 Dec 03.

Calcium connects lysosomal damage to stress granule formation.

Kim Baumann.

DOI: https://doi.org/10.1038/s41580-024-00817-w
Stem Cell Res Ther. 2024 Dec 05. 15(1): 465

Using magnetic resonance relaxometry to evaluate the safety and quality of induced pluripotent stem cell-derived spinal cord progenitor cells.

Jerome Tan, Jiahui Chen, Daniel Roxby, Wai Hon Chooi, Tan Dai Nguyen, Shi Yan Ng, Jongyoon Han, Sing Yian Chew.

   BACKGROUND: The emergence of induced pluripotent stem cells (iPSCs) offers a promising approach for replacing damaged neurons and glial cells, particularly in spinal cord injuries (SCI). Despite its merits, iPSC differentiation into spinal cord progenitor cells (SCPCs) is variable, necessitating reliable assessment of differentiation and validation of cell quality and safety. Phenotyping is often performed via label-based methods including immunofluorescent staining or flow cytometry analysis. These approaches are often expensive, laborious, time-consuming, destructive, and severely limits their use in large scale cell therapy manufacturing settings. On the other hand, cellular biophysical properties have demonstrated a strong correlation to cell state, quality and functionality and can be measured with ingenious label-free technologies in a rapid and non-destructive manner.
METHOD: In this study, we report the use of Magnetic Resonance Relaxometry (MRR), a rapid and label-free method that indicates iron levels based on its readout (T2). Briefly, we differentiated human iPSCs into SCPCs and compared key iPSC and SCPC cellular markers to their intracellular iron content (Fe3+) at different stages of the differentiation process.
RESULTS: With MRR, we found that intracellular iron of iPSCs and SCPCs were distinctively different allowing us to accurately reflect varying levels of residual undifferentiated iPSCs (i.e., OCT4+ cells) in any given population of SCPCs. MRR was also able to predict Day 10 SCPC OCT4 levels from Day 1 undifferentiated iPSC T2 values and identified poorly differentiated SCPCs with lower T2, indicative of lower neural progenitor (SOX1) and stem cell (Nestin) marker expression levels. Lastly, MRR was able to provide predictive indications for the extent of differentiation to Day 28 spinal cord motor neurons (ISL-1/SMI-32) based on the T2 values of Day 10 SCPCs.
CONCLUSION: MRR measurements of iPSCs and SCPCs has clearly indicated its capabilities to identify and quantify key phenotypes of iPSCs and SCPCs for end-point validation of safety and quality parameters. Thus, our technology provides a rapid label-free method to determine critical quality attributes in iPSC-derived progenies and is ideally suited as a quality control tool in cell therapy manufacturing.

Keywords:  Cell manufacturing; Cell therapy; Induced pluripotent stem cells; Label-free technology; Magnetic resonance relaxometry; Neural progenitors; Spinal cord injury

DOI:  https://doi.org/10.1186/s13287-024-04070-y
Mol Cell Neurosci. 2024 Dec 03. pii: S1044-7431(24)00067-8. [Epub ahead of print]132 103982

Microfluidics in neural extracellular vesicles characterization for early Alzheimer's disease diagnosis.

Hossein Zare, Michelle M Kasdorf, Amirala Bakhshian Nik.

  Dementia is a general term for conditions impairing cognitive abilities including perception, reasoning, attention, judgment, memory, and daily brain function. Early diagnosis of Alzheimer's disease (AD), the most common form of dementia, using neural extracellular vesicles (nEVs) is the focus of the current study. These nEVs carry AD biomarkers including β-amyloid proteins and phosphorylated tau proteins. The novelty of this review lies in developing a microfluidic perspective by introducing the techniques using a microfluidic platform for early diagnosis of AD. A microfluidic device can detect small sample sizes with significantly low concentrations. These devices combine nEV isolation, enrichment, and detection, which makes them ideal candidates for early AD diagnosis.

Keywords:  Alzheimer's disease; Early diagnosis; Extracellular vesicle; Microfluidic

DOI:  https://doi.org/10.1016/j.mcn.2024.103982