bims-axbals 2024-12-15 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2024–12–15
37 papers selected by
TJ Krzystek, ALS Therapy Development Institute

KIF5A regulates axonal repair and time-dependent axonal transport of SFPQ granules and mitochondria in human motor neurons.
The ALS-associated co-chaperone DNAJC7 mediates neuroprotection against proteotoxic stress by modulating HSF1 activity.
The kinase LRRK2 is required for the physiological function and expression of the glial glutamate transporter EAAT2 (SLC1A2).
Cytoskeleton dysfunction of motor neuron in spinal muscular atrophy.
Selective Inhibition of Cytosolic PARylation via PARG99: A Targeted Approach for Mitigating FUS-associated Neurodegeneration.
Heterogeneous nuclear ribonucleoprotein D - an understudied subfamily affected in sporadic TDP-43 proteinopathies.
Antibody-mediated degradation of 4R-tau restores mitochondrial membrane polarization in human induced pluripotent stem cell-derived neurons with the MAPT 10+16 mutation.
Distinct roles of ascorbic acid in extracellular vesicles and free form: Implications for metabolism and oxidative stress in presymptomatic Huntington's Disease.
Immune cells and the trajectories of depression, anxiety, and cognitive function among people with amyotrophic lateral sclerosis.
hPSCs-derived brain organoids for disease modeling, toxicity testing and drug evaluation.
Evaluation of a biomarker for amyotrophic lateral sclerosis derived from a hypomethylated DNA signature of human motor neurons.
Exploring clinical chemistry markers in amyotrophic lateral sclerosis: insights into survival and disease trajectories.
Therapeutic targeting of the oxidative stress generated by pathological molecular pathways in the neurodegenerative diseases, ALS and Huntington's.
Frequency and neuropathology of HTT repeat expansions in FTD/ALS: co-existence rather than causation.
ALS molecular subtypes are a combination of cellular, genetic, and pathological features learned by deep multiomics classifiers.
Exploring the Function of OPTN From Multiple Dimensions.
Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function.
Extracellular Vesicles from Human Induced Pluripotent Stem Cells Exhibit a Unique MicroRNA and CircRNA Signature.
Altered calcium responses and antioxidant properties in Friedreich's ataxia-like cerebellar astrocytes.
Current concepts and molecular pathology of neurodegenerative diseases.
Turning garbage into gold: Autophagy in synaptic function.
Mitochondrial dynamics govern whole-body regeneration through stem cell pluripotency and mitonuclear balance.
Inhibition of Bone Morphogenetic Protein Signaling Prevents Tau Pathology in iPSC Derived Neurons and PS19 Mice.
Genetic and functional analyses of SPTLC1 in juvenile amyotrophic lateral sclerosis.
Organoid generation from trophoblast stem cells highlights distinct roles for cytotrophoblasts and stem cells in organoid formation and expansion.
Induced pluripotent stem cell-related approaches to generate dopaminergic neurons for Parkinson's disease.
Repeat Expansion Disorders Likely Underdiagnosed.
A human-induced pluripotent stem cell (iPSC) line (SMUSHi006-A) from an ALS patient carrying a mutation c.1126C > T in the FUS gene.
Reduced Neurite Arborization in Primary Dopaminergic Neurons in Autism-Like Shank3B-Deficient Mice.
Generation of self-organized neuromusculoskeletal tri-tissue organoids from human pluripotent stem cells.
Lysosomal Trafficking and Degradation of Extracellular Proteins via Multivalent Small Molecule Ligand Display on Dextran Scaffolds.
Making blood from iPSCs: Reaching for the most sought-after prize.
Unraveling the molecular mechanisms of ALS: a network biology and structural modeling approach of investigating the impact of C9orf72 mutations.
Retinal organoid chip: engineering a physiomimetic oxygen gradient for optimizing long term culture of human retinal organoids.
Inhibition of miR-4763-3p expression activates the PI3K/mTOR/Bcl2 autophagy signaling pathway to ameliorate cognitive decline.
Reactivation of mTOR signaling slows neurodegeneration in a lysosomal sphingolipid storage disease.
Dopamine transporter endocytic trafficking: Neuronal mechanisms and potential impact on DA-dependent behaviors.

Neurobiol Dis. 2024 Dec 05. pii: S0969-9961(24)00361-9. [Epub ahead of print] 106759

KIF5A regulates axonal repair and time-dependent axonal transport of SFPQ granules and mitochondria in human motor neurons.

Irune Guerra San Juan, Jessie Brunner, Kevin Eggan, Ruud F Toonen, Matthijs Verhage.

  Mutations in the microtubule-binding motor protein kinesin 5 A (KIF5A) are impacted in several adult-onset motor neuron diseases, including Amyotrophic Lateral Sclerosis, Spastic Paraplegia Type 10 and Charcot-Marie-Tooth Disease Type 2. While KIF5 family members transport a variety of cargos along axons, the specific cargos affected by KIF5A mutations remain poorly understood. Here, we generated KIF5A null mutant human motor neurons and analyzed the impact on axonal transport and motor neuron outgrowth and regeneration in vitro. KIF5A deficiency caused reduced neurite complexity in young neurons (DIV14) and defects in axonal regeneration. KIF5A deficiency did not affect neurofilament transport but impaired mitochondrial motility and anterograde speed at DIV42. Notably, KIF5A deficiency strongly reduced anterograde transport of splicing factor proline/glutamine-rich (SFPQ)-associated RNA granules in DIV42 axons. Hence, KIF5A plays a critical role in promoting axonal regrowth after injury and in driving the anterograde transport of mitochondria and especially SFPQ-associated RNA granules in mature neurons.

Keywords:  Aging; Axonal injury and repair; Axonal transport; Human neuron development; Human neurons; KIF5A; Kinesins; Mitochondria; Motor neurons; Neurodegeneration; Neurofilaments; RNA granules

DOI:  https://doi.org/10.1016/j.nbd.2024.106759
bioRxiv. 2024 Dec 01. pii: 2024.12.01.626216. [Epub ahead of print]

The ALS-associated co-chaperone DNAJC7 mediates neuroprotection against proteotoxic stress by modulating HSF1 activity.

Andrew C Fleming, Nalini R Rao, Matthew Wright, Jeffrey N Savas, Evangelos Kiskinis.

The degeneration of neurons in patients with amyotrophic lateral sclerosis (ALS) is commonly associated with accumulation of misfolded, insoluble proteins. Heat shock proteins (HSPs) are central regulators of protein homeostasis as they fold newly synthesized proteins and refold damaged proteins. Heterozygous loss-of- function mutations in the DNAJC7 gene that encodes an HSP co-chaperone were recently identified as a cause for rare forms of ALS, yet the mechanisms underlying pathogenesis remain unclear. Using mass spectrometry, we found that the DNAJC7 interactome in human motor neurons (MNs) is enriched for RNA binding proteins (RBPs) and stress response chaperones. MNs generated from iPSCs with the ALS-associated mutation R156X in DNAJC7 exhibit increased insolubility of its client RBP HNRNPU and associated RNA metabolism alterations. Additionally, DNAJC7 haploinsufficiency renders MNs increasingly susceptible to proteotoxic stress and cell death as a result of an ablated HSF1 stress response pathway. Critically, expression of HSF1 in mutant DNAJC7 MNs is sufficient to rescue their sensitivity to proteotoxic stress, while postmortem ALS patient cortical neurons exhibit a reduction in the expression of HSF1 pathway genes. Taken together, our work identifies DNAJC7 as a crucial mediator of HNRNPU function and stress response pathways in human MNs and highlights HSF1 as a therapeutic target in ALS.

DOI: https://doi.org/10.1101/2024.12.01.626216
J Neurochem. 2025 Jan;169(1): e16265

The kinase LRRK2 is required for the physiological function and expression of the glial glutamate transporter EAAT2 (SLC1A2).

Angela Di Iacovo, Chiara D'Agostino, Manan Bhatt, Tiziana Romanazzi, Stefano Giovannardi, Raffaella Cinquetti, Cristina Roseti, Elena Bossi.

  Neurotransmitter transporters (NTTs) control synaptic responses by modulating the concentration of neurotransmitters at the synaptic cleft. Glutamate is the most abundant excitatory neurotransmitter in the brain and needs to be finely tuned in time and space to maintain a healthy brain and precise neurotransmission. The glutamate transporter EAAT2 (SLC1A2) is primarily responsible for glutamate clearance. EAAT2 impairment has been associated with Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD). Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to both monogenic and sporadic forms of PD, of which the common substitution Gly2019Ser is associated with a significant deficit in EAAT2 expression. The role of pathological mutants of the LRRK2 is intensively studied and reviewed. Here we have focused the attention on the physiological role of LRRK2 on EAAT2, comparing the activity of NTTs with or without the LRRK2 kinase. By heterologous expression in Xenopus laevis oocytes and two-electrode voltage clamp, the current amplitudes of the selected NTTs and kinetic parameters have been collected in the presence and absence of LRRK2. The results show that EAAT2 expression and function are impaired in the absence of the kinase and also under its pharmacological inhibition via MLi-2 treatment. LRRK2 stabilizes EAAT2 expression increasing the amount of transporter at the plasma membrane. Interestingly, the LRRK2 action is EAAT2-specific, as we observed no significant changes in the transport current amplitude and kinetic parameters obtained for the other excitatory and inhibitory NTTs studied. This study, for the first time, demonstrates the physiological importance of LRRK2 in EAAT2 function, highlighting the specificity of LRRK2-mediated modulation of EAAT2 and suggesting a potential role for the kinase as a checkpoint for preserving neurons from excitotoxicity. In brain conditions associated with impaired glutamate clearance, targeting LRRK2 for EAAT2 regulation may offer novel therapeutic opportunities.

Keywords:  EAAT2 (SLC1A2); LRRK2; SLC1; SLC6; dopamine transporter; excitatory/inhibitory balance

DOI:  https://doi.org/10.1111/jnc.16265
J Neurol. 2024 Dec 12. 272(1): 19

Cytoskeleton dysfunction of motor neuron in spinal muscular atrophy.

Tianyu Shi, Zijie Zhou, Taiyang Xiang, Yinxuan Suo, Xiaoyan Shi, Yaoyao Li, Peng Zhang, Jun Dai, Lei Sheng.

  Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by deletions or mutations of survival of motor neuron 1 (SMN1) gene. To date, the mechanism of selective cell death of motor neurons as a hallmark of SMA is still unclear. The severity of SMA is dependent on the amount of survival motor neuron (SMN) protein, which is an essential and ubiquitously expressed protein involved in various cellular processes including regulation of cytoskeletal dynamics. In this review, we discuss the effect of SMN ablation on cytoskeleton organization including actin dynamics, growth cone formation, axonal stability, neurite outgrowth, microtubule stability, synaptic vesicle dynamics and neurofilament protein release in SMA. We also summarized a list of critical proteins such as profilin-2 (PFN2), plastin-3 (PLS3), stathmin-1 (STMN1), microtubule-associated protein 1B (MAP1B) and neurofilament which play an important role in modulating cytoskeleton in SMA. Our aim is to highlight how cytoskeletal defects contribute to motor neuron degeneration in SMA disease progression and concentrating on cytoskeleton dynamics may be a promising approach to develop new therapy or biomarker.

Keywords:  Actin; Cytoskeleton defect; Microtubule; Microtubule-associated protein; Plastin-3; Profilin-2; Spinal muscular atrophy

DOI:  https://doi.org/10.1007/s00415-024-12724-3
bioRxiv. 2024 Nov 26. pii: 2024.11.25.625276. [Epub ahead of print]

Selective Inhibition of Cytosolic PARylation via PARG99: A Targeted Approach for Mitigating FUS-associated Neurodegeneration.

Manohar Kodavati, Vikas H Maloji Rao, Joy Mitra, Muralidhar L Hegde.

Neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) are characterized by complex etiologies, often involving disruptions in functions of RNA/DNA binding proteins (RDBPs) such as FUS and TDP-43. The cytosolic mislocalization and aggregation of these proteins are linked to accumulation of unresolved stress granules (SGs), which exacerbate the disease progression. Poly-ADP-ribose polymerase (PARP)-mediated PARylation plays a critical role in this pathological cascade, making it a potential target for intervention. However, conventional PARP inhibitors are limited by their detrimental effects on DNA repair pathways, which are already compromised in ALS. To address this limitation, we investigated a strategy focused on targeting the cytosolic compartment by expressing the cytosol-specific, natural PAR- glycohydrolase (PARG) isoform, PARG99. Using ALS patient derived FUS mutant induced pluripotent cells (iPSCs) and differentiated neurons, we observed elevated levels of FUS in insoluble fractions in mutant cells compared to mutation-corrected isogenic lines. The insoluble FUS as well as TDP-43 levels increased further in sodium arsenite-treated or oxidatively stressed cells, correlating with accumulation of unresolved SGs. Notably, both PARG99 and PARP inhibitors reduced SG formation and insoluble FUS levels, however, PARG99 treated cells exhibited significantly lower DNA damage markers and improved viability under oxidative and arsenite stress. This study highlights the potential of PARG99 as a cytosol-specific intervention to mitigate FUS-associated toxicity while preserving critical nuclear DNA repair mechanisms, offering a promising strategy for addressing the underlying pathology of ALS and potentially other SG-associated neurodegenerative diseases.

DOI: https://doi.org/10.1101/2024.11.25.625276
Brain Commun. 2024 ;6(6): fcae352

Heterogeneous nuclear ribonucleoprotein D - an understudied subfamily affected in sporadic TDP-43 proteinopathies.

Monica Pinkerton, Gabrielle L Adler, Mallory Ledger, Chen Yue Ni, Yue Yang, Rachel H Tan.

  Despite the recognition that heterogeneous nuclear ribonucleoproteins (hnRNPs) modulate TDP-43 and can limit aberrant splicing events to compensate for TDP-43 loss, their role in TDP-43 proteinopathies remains poorly understood and studies in patient tissue are lacking. This study assesses seven heterogeneous nuclear ribonucleoproteins from the A/B, C, D and H subfamilies in two cortical regions implicated in early TDP-43 dysfunction versus late TDP-43 dysfunction in sporadic amyotrophic lateral sclerosis and/or frontotemporal lobar degeneration. Our results reveal significant nuclear loss of hnRNPD, hnRNPC and hnRNPA1 in the frontal cortex of frontotemporal lobar degeneration compared to amyotrophic lateral sclerosis but not in the motor cortical neurons or Betz cells of amyotrophic lateral sclerosis cases. Cytoplasmic co-occurrence was observed between hnRNPA1 and hnRNPC but not with phosphorylated TDP-43 (pTDP-43). Interestingly, nuclear hnRNPD loss associated with increasing cytoplasmic pTDP-43, highlighting an understudied subfamily in sporadic TDP-43 proteinopathies. In summary, this study identifies the nuclear loss of hnRNPD, C and A1 in a predilection brain region of TDP-43 in frontotemporal lobar degeneration compared to amyotrophic lateral sclerosis cases without significant pTDP-43 in this region. This highlights the need for further investigation into the involvement of these heterogeneous nuclear ribonucleoproteins in disease pathogenesis and potential to serve as modulatory targets and/or proximal markers of TDP-43 dysfunction in sporadic TDP-43 proteinopathies.

Keywords:  amyotrophic lateral sclerosis; frontotemporal lobar degeneration; pTDP-43

DOI:  https://doi.org/10.1093/braincomms/fcae352
MAbs. 2024 Jan-Dec;16(1):16(1): 2436102

Antibody-mediated degradation of 4R-tau restores mitochondrial membrane polarization in human induced pluripotent stem cell-derived neurons with the MAPT 10+16 mutation.

Dale O Starkie, Charles Arber, Terry Baker, Daniel J Lightwood, Selina Wray.

  Microtubule-associated protein tau is inextricably linked to a group of clinically diverse neurodegenerative diseases termed tauopathies. The ratio balance of the major tau splicing isoform groups (3 R- and 4 R-tau) is critical in maintaining healthy neurons. An imbalance causing excess 4 R tau is associated with diseases such as progressive supranuclear palsy and frontotemporal dementia. The mechanisms by which increased 4 R results in neuronal dysfunction and neurodegeneration are not fully understood, and progress has been limited partly by a lack of suitable tools to investigate tau isoform imbalance. This work generated novel 3 R- and 4 R-specific antibody tools and 4 R-tau degrading intracellular antibody fragment "degrabodies". These were used to probe the molecular mechanisms of excess 4 R-tau in disease-mutant induced pluripotent stem cell-derived neurons. For the first time, we demonstrate a causative link between excess 4 R-tau and mitochondrial membrane hyperpolarization with wide-ranging potential for elucidating novel therapeutic approaches to treat neurodegenerative disease.

Keywords:  Antibody Discovery; degrabodies; degrading antibody fragments; iPSC-derrived neurons; intracellular antibody technologies; targeted protein degradation; tau

DOI:  https://doi.org/10.1080/19420862.2024.2436102
Free Radic Biol Med. 2024 Dec 09. pii: S0891-5849(24)01099-2. [Epub ahead of print]

Distinct roles of ascorbic acid in extracellular vesicles and free form: Implications for metabolism and oxidative stress in presymptomatic Huntington's Disease.

Felipe A Beltrán, Leandro Torres, Paulina Troncoso-Escudero, Juan Villalobos-González, Gonzalo Mayorga-Weber, Marcelo Lara, Adriana Covarrubias-Pinto, Sharin Valdivia, Isidora Vicencio, Eduardo Papic, Carolina Paredes-Martínez, Mara E Silva-Januàrio, Alejandro Rojas, Luis L P daSilva, Felipe Court, Abraham Rosas-Arellano, Federico Bátiz, Patricio Rojas, Francisco J Rivera, Maite A Castro.

  Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the first exon of the huntingtin gene. The huntingtin protein (Htt) is ubiquitously expressed and localized in several organelles, including endosomes, where it plays an essential role in intracellular trafficking. Presymptomatic HD is associated with a failure in energy metabolism and oxidative stress. Ascorbic acid is a potent antioxidant that plays a key role in modulating neuronal metabolism and is highly concentrated in the brain. During synaptic activity, neurons take up ascorbic acid released by glial cells; however, this process is disrupted in HD. In this study, we aim to elucidate the molecular and cellular mechanisms underlying this dysfunction. Using an electrophysiological approach in presymptomatic YAC128 HD slices, we observed decreased ascorbic acid flux from astrocytes to neurons, which altered neuronal metabolic substrate preferences. Ascorbic acid efflux and recycling were also decreased in cultured astrocytes from YAC128 HD mice. We confirmed our findings using GFAP-HD160Q, an HD mice model expressing mutant N-terminal Htt mainly in astrocytes. For the first time, we demonstrated that ascorbic acid is released from astrocytes via extracellular vesicles (EVs). Decreased number of particles and exosomal markers were observed in EV fractions from cultured YAC128 HD astrocytes and Htt-KD cells. We observed reduced number of multivesicular bodies (MVBs) in YAC128 HD striatum via electron microscopy, suggesting mutant Htt alters MVB biogenesis. EVs containing ascorbic acid effectively reduced reactive oxygen species, whereas "free" ascorbic acid played a role in modulating neuronal metabolic substrate preferences. These findings suggest that the early redox imbalance observed in HD arises from a reduced release of ascorbic acid-containing EVs by astrocytes. Meanwhile, a decrease in "free" ascorbic acid likely contributes to presymptomatic metabolic impairment.

Keywords:  ascorbic acid; exosomes; glucose; lactate; neurodegeneration

DOI:  https://doi.org/10.1016/j.freeradbiomed.2024.12.001
Brain Behav Immun Health. 2024 Dec;42 100907

Immune cells and the trajectories of depression, anxiety, and cognitive function among people with amyotrophic lateral sclerosis.

Yihan Hu, Elie Deeba, Ulf Kläppe, Linn Öijerstedt, John Andersson, Nicolas Ruffin, Fredrik Piehl, Caroline Ingre, Fang Fang, Christina Seitz.

   Background: Amyotrophic lateral sclerosis (ALS) represents a complex syndrome characterized by motor, psychiatric, and cognitive symptoms, where associations between cellular immune features and non-motor manifestations remain unknown.
Methods: In this cohort study, we enrolled 250 incident people with ALS (pwALS) assessed with the Hospital Anxiety and Depression Scale, and 226 pwALS with the Montreal Cognitive Assessment, including 218 overlapping pwALS. All individuals were diagnosed between January 2015 and January 2023 in Stockholm, Sweden. We applied joint latent class models to delineate distinct trajectories of anxiety, depression, and cognition, incorporating survival outcomes. A majority of the pwALS had data on leukocyte counts and flow cytometric analyses using a comprehensive T cell panel. We then used immune cell subtypes measured at diagnosis to predict trajectories of these outcomes following ALS diagnosis.
Results: We identified two distinct trajectories for anxiety, depression, and cognitive function following ALS diagnosis. PwALS with longer survival displayed more stable trajectories, while those with shorter survival showed decreasing anxiety symptom, increasing depressive symptom, and declining cognitive function. Higher count of leukocytes at the time of ALS diagnosis tended to associate with anxiety and depression trajectories related to shorter survival. Among T cell subpopulations, several CD8+ T cell subsets were associated with a stable trajectory of depressive symptom, and, in turn, better survival.
Conclusion: ALS-associated psychiatric and cognitive trajectories vary significantly between pwALS with different prognosis. Certain T cell subsets measured at diagnosis might be indicative of depression trajectories post-diagnosis.

Keywords:  Amyotrophic lateral sclerosis; Anxiety; Cognitive function; Depression; Leukocytes; T cells

DOI:  https://doi.org/10.1016/j.bbih.2024.100907
Exp Neurol. 2024 Dec 10. pii: S0014-4886(24)00436-9. [Epub ahead of print]385 115110

hPSCs-derived brain organoids for disease modeling, toxicity testing and drug evaluation.

Na Xie, Jinrong Bai, Ya Hou, Jia Liu, Yi Zhang, Xianli Meng, Xiaobo Wang.

  Due to the differences and variances in genetic background, in vitro and animal models cannot meet the modern medical exploration of real human brain structure and function. Recently, brain organoids generated by human pluripotent stem cells (hPSCs) can mimic the structure and physiological function of human brain, being widely used in medical research. Brain organoids generated from normal hPSCs or patient-derived induced pluripotent stem cells offer a more promising approach for the study of diverse human brain diseases. More importantly, the use of the established brain organoid model for drug evaluation is conducive to shorten the clinical transformation period. Herein, we summarize methods for the identification of brain organoids from cellular diversity, morphology and neuronal activity, brain disease modeling, toxicity testing, and drug evaluation. Based on this, it is hoped that this review will provide new insights into the pathogenesis of brain diseases and drug research and development, promoting the rapid development of brain science.

Keywords:  Brain organoid; Disease modeling; Drug evaluation; Human pluripotent stem cells; Toxicity testing

DOI:  https://doi.org/10.1016/j.expneurol.2024.115110
Res Sq. 2024 Nov 26. pii: rs.3.rs-5397445. [Epub ahead of print]

Evaluation of a biomarker for amyotrophic lateral sclerosis derived from a hypomethylated DNA signature of human motor neurons.

Calum Harvey, Alicja Nowak, Sai Zhang, Tobias Moll, Annika K Weimer, Aina Mogas Barcons, Cleide Dos Santos Souza, Laura Ferraiuolo, Kevin Kenna, Noah Zaitlen, Christa Caggiano, Pamela J Shaw, Michael P Snyder, Jonathan Mill, Eilis Hannon, Johnathan Cooper-Knock.

Amyotrophic lateral sclerosis (ALS) lacks a specific biomarker, but is defined by relatively selective toxicity to motor neurons (MN). As others have highlighted, this offers an opportunity to develop a sensitive and specific biomarker based on detection of DNA released from dying MN within accessible biofluids. Here we have performed whole genome bisulfite sequencing (WGBS) of iPSC-derived MN from neurologically normal individuals. By comparing MN methylation with an atlas of tissue methylation we have derived a MN-specific signature of hypomethylated genomic regions, which accords with genes important for MN function. Through simulation we have optimised the selection of regions for biomarker detection in plasma and CSF cell-free DNA (cfDNA). However, we show that MN-derived DNA is not detectable via WGBS in plasma cfDNA. In support of our experimental finding, we show theoretically that the relative sparsity of lower MN sets a limit on the proportion of plasma cfDNA derived from MN which is below the threshold for detection of WGBS. Our findings are important for the ongoing development of ALS biomarkers. The MN-specific hypomethylated genomic regions we have derived could be usefully combined with more sensitive detection methods and perhaps with study of CSF instead of plasma. Indeed we demonstrate that neuronal-derived DNA is detectable in CSF. Our work is relevant for all diseases featuring death of rare cell-types.

DOI: https://doi.org/10.21203/rs.3.rs-5397445/v1
J Neurol. 2024 Dec 12. 272(1): 7

Exploring clinical chemistry markers in amyotrophic lateral sclerosis: insights into survival and disease trajectories.

Ioannis Psychogios, Yihan Hu, Christina Seitz, Emily E Joyce, Anikó Lovik, Caroline Ingre, Fang Fang.

   OBJECTIVE: Commonly measured clinical chemistry markers might be indicative of survival and disease progression in amyotrophic lateral sclerosis (ALS).
METHODS: In a cohort study of 270 ALS patients diagnosed from April 2014 to May 2021 in Stockholm, Sweden, we examined the link between 29 clinical chemistry markers at diagnosis and mortality risk at 6 months, 1 year, and 3 years after diagnosis. Summary variables from exploratory factor analysis (EFA) assessed the markers' collective impact on survival. We integrated ALS functional rating scale-revised (ALSFRS-R) scores with survival data using a joint latent class model to identify patterns of functional decline. Multinomial logistic regression determined how the EFA-derived factors predicted the decline trajectories post-diagnosis.
RESULTS: Higher levels of total cholesterol, low-density lipoprotein cholesterol (LDL-C), apolipoprotein B, and albumin at diagnosis were linked to lower mortality in ALS patients, while increased neurofilament light chain (NfL), leukocyte count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and carbon dioxide (CO2) levels indicated higher mortality. The 'Red blood cell profile' factor, derived from EFA, emerged as a significant predictor of survival, independent of other prognostic indicators. The joint latent class model identified three distinct patient groups based on functional decline, with 'Red blood cell profile' suggesting a lower likelihood of being in the groups with slower progression.
CONCLUSION: Clinical chemistry markers, including NfL, lipids, albumin, leukocyte count, MCV, MCH, CO2, and the 'Red blood cell profile,' were associated with ALS survival. As these markers represent broader bodily functions, integrating them in ALS patient care could improve disease management.

Keywords:  ALS; Blood markers; Clinical chemistry; Prognosis; Survival

DOI:  https://doi.org/10.1007/s00415-024-12774-7
Eur J Pharmacol. 2024 Dec 05. pii: S0014-2999(24)00877-X. [Epub ahead of print] 177187

Therapeutic targeting of the oxidative stress generated by pathological molecular pathways in the neurodegenerative diseases, ALS and Huntington's.

Akarsh Bajpai, Vidhya Bharathi, Basant K Patel.

  Neurodegenerative disorders are characterized by a progressive decline of specific neuronal populations in the brain and spinal cord, typically containing aggregates of one or more proteins. They can result in behavioral alterations, memory loss and a decline in cognitive and motor abilities. Various pathways and mechanisms have been outlined for the potential treatment of these diseases, where redox regulation is considered as one of the most common druggable targets. For example, in amyotrophic lateral sclerosis (ALS) with superoxide dismutase-1 (SOD1) pathology, there is a downregulation of the antioxidant response nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. TDP-43 proteinopathy in ALS is associated with elevated levels of reactive oxygen species and mitochondrial dyshomeostasis. In ALS with mutant FUS, poly ADP ribose polymerase-dependent X ray repair cross complementing 1/DNA-ligase recruitment to the sites of oxidative DNA damage is affected, thereby causing defects in DNA damage repair. Oxidative stress in Huntington's disease (HD) with mutant huntingtin accumulation manifests as protein oxidation, metabolic energetics dysfunction, metal ion dyshomeostasis, DNA damage and mitochondrial dysfunction. The impact of oxidative stress in the progression of these diseases further warrants studies into the role of antioxidants in their treatment. While an antioxidant, edaravone, has been approved for therapeutics of ALS, numerous antioxidant molecules failed to pass the clinical trials despite promising initial studies. In this review, we summarize the oxidative stress pathways and redox modulators that are investigated in ALS and HD using various models.

Keywords:  Amyotrophic lateral sclerosis; Huntington's disease; Oxidative stress; ROS; mitochondria

DOI:  https://doi.org/10.1016/j.ejphar.2024.177187
J Neurol. 2024 Dec 12. 272(1): 58

Frequency and neuropathology of HTT repeat expansions in FTD/ALS: co-existence rather than causation.

Milan Zimmermann, David Mengel, Katrin Raupach, Tobias Haack, Manuela Neumann, Matthis Synofzik.

   INTRODUCTION: While ≥ 40 CAG repeat expansions in HTT present a well-established cause of Huntington's disease (HD), an enrichment of HTT repeat expansions was recently reported also in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), including FTD/ALS patients with additional HD neuropathology. This raises the question whether the phenotypic spectrum of HTT expansions can be extended to ALS and FTD, and whether HTT should be considered as a new causative gene of FTD/ALS. If HTT repeat expansions were indeed systematically related to FTD/ALS, one would expect an increased frequency of HTT carriers in FTD/ALS, who can clinically/neuropathologically not be explained better than by the presence of the HTT repeat expansions.
METHODS: Screening of HTT repeat expansions in 249 consecutive patients with ALS or FTD by short-read genome sequencing took place. The post-mortem neuropathological examination was performed in the identified HTT repeat expansion carrier.
RESULTS: One HTT repeat expansion [40/22 repeats (± 1)] was identified in an ALS patient, giving a frequency of 0.4% (1/249) (frequency in the general population: 0.03-0.18%). This patient showed a classic ALS phenotype, but no clinical or imaging signs of HD. Post-mortem brain examination revealed-in addition to ALS-typical degeneration of upper and lower motor neurons with TDP-43 inclusions-HD-typical polyQ-aggregates in gyrus cinguli, striatum and frontal lobe, yet without evidence of striatal degeneration.
CONCLUSIONS: Our study does not support the notion of an increased frequency of HTT repeat expansions in FTD/ALS. Moreover, the phenotype of the HTT carrier identified can be better explained by two co-existent, but independent diseases: (i) ALS and (ii) presymptomatic HD, which-given the low repeat number-is likely to become manifest only later in life. These findings corroborate the concept that HTT repeat expansions are likely co-existent/coincidental, but not causative in FTD/ALS.

Keywords:  ALS; Amyotrophic lateral sclerosis; Frontotemporal dementia; HTT; Huntington; MAPT

DOI:  https://doi.org/10.1007/s00415-024-12822-2
bioRxiv. 2024 Jul 20. pii: 2024.07.19.603731. [Epub ahead of print]

ALS molecular subtypes are a combination of cellular, genetic, and pathological features learned by deep multiomics classifiers.

NYGC ALS Consortium

Amyotrophic Lateral Sclerosis (ALS) is a complex syndrome with multiple genetic causes and wide variation in disease presentation. Despite this general heterogeneity, several common factors have been identified. For example, nearly all patients show pathological accumulations of phosphorylated TDP-43 protein in affected regions of the motor cortex and spinal cord. Moreover, large patient cohort studies have revealed that most patient samples can be grouped into a small number of ALS subtypes, as defined by their transcriptomic profiles. These ALS molecular subtypes can be grouped by whether postmortem motor cortex samples display signatures of: mitochondrial dysfunction and oxidative stress (ALS-Ox), microglial activation and neuroinflammation (ALS-Glia), or dense TDP-43 pathology and associated transposable element de-silencing (ALS-TE). In this study, we have built a deep layer ALS neural network classifier (DANcer) that has learned to accurately assign patient samples to these ALS subtypes, and which can be run on either bulk or single-cell datasets. Upon applying this classifier to an expanded ALS patient cohort from the NYGC ALS Consortium, we show that ALS Molecular Subtypes are robust across clinical centers, with no new subtypes appearing in a cohort that has quadrupled in size. Signatures from two of these molecular subtypes strongly correlate with disease duration: ALS-TE signatures in cortex and ALS-Glia signatures in spinal cord, revealing molecular correlates of clinical features. Finally, we use single nucleus RNA sequencing to reveal the cell type-specific contributions to ALS subtype, as determined by our single-cell classifier (scDANCer). Single-cell transcriptomes reveal that ALS molecular subtypes are recapitulated in neurons and glia, with both ALS-wide shared alterations in each cell type as well as ALS subtype-specific alterations. In summary, ALS molecular subtypes: (1) are robust across large cohorts of sporadic and familial ALS patient samples, (2) represent a combination of cellular, genetic, and pathological features, and (3) correlate with clinical features of ALS.
Abstract Figure:

DOI: https://doi.org/10.1101/2024.07.19.603731
Cell Biochem Funct. 2024 Dec;42(8): e70029

Exploring the Function of OPTN From Multiple Dimensions.

Yanan Guo, Yixiao Tian, Peng Xia, Xinyue Zhou, Xiaohui Hu, Zhao Guo, Pengfei Ji, Xinyi Yuan, Daosen Fu, Keyu Yin, Rong Shen, Degui Wang.

  Autophagy is an essential intracellular degradation system responsible for delivering cytoplasmic components to lysosomes. Within this intricate process, optineurin (OPTN), an autophagy receptor, has attracted extensive attention due to its multifaceted roles in the autophagy process. OPTN is regulated by various posttranslational modifications and actively participates in numerous signaling pathways and cellular processes. By exploring the regulatory mechanism of OPTN posttranslational modification, we can further understand the critical role of protein posttranslational modification in biological progress, such as autophagy. Additionally, OPTN is implicated in many human diseases, including rheumatoid arthritis, osteoporosis, and infectious diseases. And we delve into the inflammatory pathways regulated by OPTN and clarify how it regulates inflammatory diseases and cancer. We aim to enhance the understanding of OPTN's multifaceted functions in cellular processes and its implications in the pathogenesis of inflammatory diseases and cancer.

Keywords:  OPTN; autophagy; cancer; inflammatory diseases; posttranslational modification; signaling pathway

DOI:  https://doi.org/10.1002/cbf.70029
J Vis Exp. 2024 Nov 29.

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function.

Annika Kolodziejczyk, Janine Hoffmann, Paula Cubillos, Mareike Albert.

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. Studies analyzing gene function by overexpression or knockout have been instrumental in animal models to provide mechanistic insights into the regulation of neocortex development. Here, we present a detailed protocol for CRISPR/Cas9-mediated acute gene knockout by electroporation of sliced human cortical organoids. The slicing of cortical organoids aids the identification of ventricle-like structures for injection and subsequent electroporation, making this a particularly well-suited model for acute genetic manipulation during human cortical development. We describe the design of guide RNAs and the validation of targeting efficiency in vitro and in cortical organoids. Electroporation of cortical organoids is performed at mid-neurogenic stages, enabling the targeting of most major cell classes in the developing neocortex, including apical radial glia, basal progenitor cells, and neurons. Taken together, the electroporation of sliced human cortical organoids represents a powerful technique to investigate gene function, gene regulation, and cell morphology during cortical development.

DOI: https://doi.org/10.3791/67598
Int J Biol Sci. 2024 ;20(15): 6255-6278

Extracellular Vesicles from Human Induced Pluripotent Stem Cells Exhibit a Unique MicroRNA and CircRNA Signature.

Mario Barilani, Valeria Peli, Paolo Manzini, Clelia Pistoni, Francesco Rusconi, Eva Maria Pinatel, Francesca Pischiutta, Dorian Tace, Maria Chiara Iachini, Noemi Elia, Francesca Tribuzio, Federica Banfi, Alessandro Sessa, Alessandro Cherubini, Vincenza Dolo, Valentina Bollati, Luisa Fiandra, Elena Longhi, Elisa R Zanier, Lorenza Lazzari.

  Extracellular vesicles (EV) have emerged as promising cell-free therapeutics in regenerative medicine. However, translating primary cell line-derived EV to clinical applications requires large-scale manufacturing and several challenges, such as replicative senescence, donor heterogeneity, and genetic instability. To address these limitations, we used a reprogramming approach to generate human induced pluripotent stem cells (hiPSC) from the young source of cord blood mesenchymal stem/stromal cells (CBMSC). Capitalizing on their inexhaustible supply potential, hiPSC offer an attractive EV reservoir. Our approach encompassed an exhaustive characterization of hiPSC-EV, aligning with the rigorous MISEV2023 guidelines. Analyses demonstrated physical features compatible with small EV (sEV) and established their identity and purity. Moreover, the sEV-shuttled non-coding (nc) RNA landscape, focusing on the microRNA and circular RNA cargo, completed the molecular signature. The kinetics of the hiPSC-sEV release and cell internalization assays unveiled robust EV production and consistent uptake by human neurons. Furthermore, hiPSC-sEV demonstrated ex vivo cell tissue-protective properties. Finally, via bioinformatics, the potential involvement of the ncRNA cargo in the hiPSC-sEV biological effects was explored. This study significantly advances the understanding of pluripotent stem cell-derived EV. We propose cord blood MSC-derived hiPSC as a promising source for potentially therapeutic sEV.

Keywords:  circRNA; cord blood; exosomes; extracellular vesicles; human-induced pluripotent stem cells; miRNA; nanoparticles

DOI:  https://doi.org/10.7150/ijbs.100113
J Cell Sci. 2024 Dec 09. pii: jcs.263446. [Epub ahead of print]

Altered calcium responses and antioxidant properties in Friedreich's ataxia-like cerebellar astrocytes.

Chiara Marullo, Laura Croci, Iris Giupponi, Claudia Rivoletti, Sofia Zuffetti, Barbara Bettegazzi, Ottavio Cremona, Paola Giunti, Alessandro Ambrosi, Filippo Casoni, Gian Giacomo Consalez, Franca Codazzi.

  Friedreich's ataxia (FRDA) is a neurodegenerative disorder characterized by severe neurological signs, affecting the peripheral and central nervous system, caused by reduced frataxin protein (FXN) levels. While several studies highlight cellular dysfunctions in neurons, there is limited information on the effects of FXN depletion in astrocytes and on the potential non-cell autonomous mechanisms affecting neurons in FRDA. In this study, we generated a model of FRDA cerebellar astrocytes to unveil phenotypic alterations that might contribute to cerebellar atrophy. We treated primary cerebellar astrocytes with an RNA interference-based approach, to achieve a reduction of FXN comparable to that observed in patients. These FRDA-like astrocytes display some typical features of the disease, such as an increase of oxidative stress and a depletion of glutathione content. Moreover, FRDA-like astrocytes exhibit decreased calcium responses to purinergic stimuli. Our findings shed light on cellular changes caused by FXN downregulation in cerebellar astrocytes, likely impairing their complex interaction with neurons. The potentially impaired ability to provide neuronal cells with glutathione or to release neuromodulators in a calcium-dependent manner could affect neuronal function, contributing to neurodegeneration.

Keywords:  Calcium signalling; Cerebellar astrocytes; Friedreich's ataxia; Mitochondria; Oxidative stress

DOI:  https://doi.org/10.1242/jcs.263446
Pathology. 2024 Nov 14. pii: S0031-3025(24)00297-6. [Epub ahead of print]

Current concepts and molecular pathology of neurodegenerative diseases.

Shelley L Forrest, Gabor G Kovacs.

  Neurodegenerative diseases are a pathologically, clinically and genetically diverse group of diseases characterised by selective dysfunction, loss of synaptic connectivity and neurodegeneration, and are associated with the deposition of misfolded proteins in neurons and/or glia. Molecular studies have highlighted the role of conformationally altered proteins in the pathogenesis of neurodegenerative diseases and have paved the way for developing disease-specific biomarkers that capture and differentiate the main type/s of protein abnormality responsible for neurodegenerative diseases, some of which are currently used in clinical practice. These proteins follow sequential patterns of anatomical involvement and disease spread in the brain and may also be detected in peripheral organs. Recent studies suggest that glia are likely to have an important role in pathological spread throughout the brain and even follow distinct progression patterns from neurons. In addition to morphological and molecular approaches to the classification of these disorders, a further new stratification level incorporates the structure of protein filaments detected by cryogenic electron microscopy. Rather than occurring in isolation, combined deposition of tau, amyloid-β, α-synuclein and TDP-43 are frequently observed in neurodegenerative diseases and in the ageing brain. These can be overlooked, and their clinicopathological relevance is difficult to interpret. This review provides an overview of disease pathogenesis and diagnostic implications, recent molecular and ultrastructural classification of neurodegenerative diseases, how to approach ageing-related and mixed pathologies, and the importance of the protein-based classification system for practising neuropathologists and clinicians. This review also informs general pathologists about the relevance of ongoing full body autopsy studies to understand the spectrum and pathogenesis of neurodegenerative diseases.

Keywords:  disease classification; mixed pathology; neurodegenerative disease; proteinopathy

DOI:  https://doi.org/10.1016/j.pathol.2024.10.006
Curr Opin Neurobiol. 2024 Dec 11. pii: S0959-4388(24)00099-0. [Epub ahead of print]90 102937

Turning garbage into gold: Autophagy in synaptic function.

Erin Marie Smith, Maeve Louise Coughlan, Sandra Maday.

Trillions of synapses in the human brain enable thought and behavior. Synaptic connections must be established and maintained, while retaining dynamic flexibility to respond to experiences. These processes require active remodeling of the synapse to control the composition and integrity of proteins and organelles. Macroautophagy (hereafter, autophagy) provides a mechanism to edit and prune the synaptic proteome. Canonically, autophagy has been viewed as a homeostatic process, which eliminates aged and damaged proteins to maintain neuronal survival. However, accumulating evidence suggests that autophagy also degrades specific cargoes in response to neuronal activity to impact neuronal transmission, excitability, and synaptic plasticity. Here, we will discuss the diverse roles, regulation, and mechanisms of neuronal autophagy in synaptic function and contributions from glial autophagy in these processes.

DOI: https://doi.org/10.1016/j.conb.2024.102937
Nat Commun. 2024 Dec 13. 15(1): 10681

Mitochondrial dynamics govern whole-body regeneration through stem cell pluripotency and mitonuclear balance.

Xue Pan, Yun Zhao, Yucong Li, Jiajia Chen, Wenya Zhang, Ling Yang, Yuanyi Zhou Xiong, Yuqing Ying, Hao Xu, Yuhong Zhang, Chong Gao, Yuhan Sun, Nan Li, Liangyi Chen, Zhixing Chen, Kai Lei.

Tissue regeneration is a complex process involving large changes in cell proliferation, fate determination, and differentiation. Mitochondrial dynamics and metabolism play a crucial role in development and wound repair, but their function in large-scale regeneration remains poorly understood. Planarians offer an excellent model to investigate this process due to their remarkable regenerative abilities. In this study, we examine mitochondrial dynamics during planarian regeneration. We find that knockdown of the mitochondrial fusion gene, opa1, impairs both tissue regeneration and stem cell pluripotency. Interestingly, the regeneration defects caused by opa1 knockdown are rescued by simultaneous knockdown of the mitochondrial fission gene, drp1, which partially restores mitochondrial dynamics. Furthermore, we discover that Mitolow stem cells exhibit an enrichment of pluripotency due to their fate choices at earlier stages. Transcriptomic analysis reveals the delicate mitonuclear balance in metabolism and mitochondrial proteins in regeneration, controlled by mitochondrial dynamics. These findings highlight the importance of maintaining mitochondrial dynamics in large-scale tissue regeneration and suggest the potential for manipulating these dynamics to enhance stem cell functionality and regenerative processes.

DOI: https://doi.org/10.1038/s41467-024-54720-1
Ann Neurol. 2024 Dec 07.

Inhibition of Bone Morphogenetic Protein Signaling Prevents Tau Pathology in iPSC Derived Neurons and PS19 Mice.

Amira Affaneh, Anne K Linden, Elif Tunc-Ozcan, Yung-Hsu Tsai, Chian-Yu Peng, John A Kessler.

OBJECTIVE: Many neurodegenerative disorders share a common pathologic feature involving the deposition of abnormal tau protein in the brain (tauopathies). This suggests that there may be some shared pathophysiologic mechanism(s). The largest risk factor for the majority of these disorders is aging, suggesting involvement of the aging process in the shared pathophysiology. We test the hypothesis that an increase in bone morphogenetic protein (BMP) signaling that occurs during aging contributes to the onset and progression of tauopathies.
METHODS: Human induced pluripotent stem cell (iPSC)-derived neurons from patients with Alzheimer's disease (AD) were used to investigate the effects of BMP signaling on tau phosphorylation and release and the mechanisms underlying these effects. Wildtype mice were used to examine effects of BMP signaling in vivo. P301S (PS19) mice were examined for the effects of BMP signaling in a model of tauopathy.
RESULTS: Here, we show that BMP signaling, mediated by non-canonical p38 signaling, increases tau phosphorylation and release of p-tau in human iPSC-derived AD neurons. Further, there is an interaction between BMP signaling and apolipoprotein E4 (ApoE4) that significantly increases tau phosphorylation and release compared with ApoE3 neurons. Inhibiting BMP signaling reduces the changes in tau in the cultured human neurons, and it limits tau pathology and prevents cognitive decline in PS19 mice.
INTERPRETATION: Our study suggests that the age-related increase in BMP signaling may participate in the onset and progression of tau pathology. Thus, therapeutic interventions that reduce BMP signaling in the aging brain could potentially slow or prevent development of diseases involving tau hyperphosphorylation. ANN NEUROL 2024.

DOI: https://doi.org/10.1002/ana.27149
J Neurol. 2024 Dec 12. 272(1): 36

Genetic and functional analyses of SPTLC1 in juvenile amyotrophic lateral sclerosis.

So Okubo, Hiroya Naruse, Hiroyuki Ishiura, Atsushi Sudo, Kayoko Esaki, Jun Mitsui, Takashi Matsukawa, Wataru Satake, Peter Greimel, Nanoka Shingai, Yasushi Oya, Takeo Yoshikawa, Shoji Tsuji, Tatsushi Toda.

   INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder of the motor system. Pathogenic variants in SPTLC1, encoding a subunit of serine palmitoyltransferase, cause hereditary sensory and autonomic neuropathy type 1 (HSAN1), and have recently been associated with juvenile ALS. SPTLC1 variants associated with ALS cause elevated levels of sphinganines and ceramides. Reports on ALS associated with SPTLC1 remain limited. This study aimed to investigate the frequency of SPTLC1 variants in ALS and relevant clinical characteristics.
METHODS: We analyzed whole-exome and whole-genome sequence data from 40 probands with familial ALS and 413 patients with sporadic ALS without previously identified causative variants. Reverse transcription polymerase chain reaction (RT-PCR) analysis and droplet digital PCR (ddPCR) were used to assess splicing and mosaicism, respectively. Plasma sphingolipid levels were quantified to analyze biochemical consequences.
RESULTS: The heterozygous c.58G>A, p.Ala20Thr variant was identified in a 21-year-old Japanese female patient presenting with symmetric weakness which slowly progressed over 15 years. RT-PCR analysis showed no splice defects. Plasma sphingolipid levels in the patient were significantly increased compared to her asymptomatic parents. ddPCR revealed that the asymptomatic father harbored a mosaic variant with 17% relative mutant allele abundance in peripheral blood leukocytes.
CONCLUSIONS: We identified a pathogenic c.58G>A, p.Ala20Thr SPTLC1 variant in a patient with juvenile ALS, likely inherited from an asymptomatic parent with mosaicism. Lipid analysis results are consistent with previous findings on SPTLC1-associated ALS. Further studies are necessary to determine the clinical effect of mosaic variants of SPTLC1.

Keywords:   SPTLC1 ; Juvenile amyotrophic lateral sclerosis; Mosaicism; Sphingolipids

DOI:  https://doi.org/10.1007/s00415-024-12776-5
Placenta. 2024 Dec 05. pii: S0143-4004(24)00791-4. [Epub ahead of print]

Organoid generation from trophoblast stem cells highlights distinct roles for cytotrophoblasts and stem cells in organoid formation and expansion.

Cherry Sun, Lawrence W Chamley, Joanna L James.

   BACKGROUND: Organoids are stem-cell derived, self-organised, three-dimensional cultures that improve in vitro recapitulation of tissue structure. The generation of trophoblast organoids using primary placental villous digests (containing cytotrophoblasts and trophoblast stem cells (TSC)) improved high-throughput assessment of early trophoblast differentiation. However, the relative contributions of cytotrophoblasts and TSCs to trophoblast organoid growth and differentiation remain unclear, with implications for model interpretation. Here we sought to generate organoids from side-population trophoblasts (SpTSCs) to better understand the contribution of TSC to trophoblast organoid formation.
METHODS: Methods were adapted from Haider et al., 2018 to generate organoids from Okae TSCs (OkTSCs) or SpTSCs. Organoid growth was compared with primary villous trophoblast organoids and cellular composition interrogated by immunohistochemistry.
RESULTS: Organoids can be derived from first-trimester SpTSCs that exhibit similar architecture to those from primary villous trophoblast. However, organoids established from pure TSC populations (OkTSC or SpTSC) have different growth dynamics to primary placental villous digest-derived organoids - with OkTSCs developing faster and spontaneously generating migratory cells, whilst SpTSC organoids grow more slowly. Importantly, depletion of SpTSC from first-trimester villous digests ablates organoid formation. Finally, the capacity of the side-population technique to isolate late-gestation TSC enabled the generation of trophoblast organoids from term placentae, although these were significantly smaller than their first-trimester SpTSC counterparts.
DISCUSSION: Together, this work highlights the requirement of TSC for organoid formation, and the functional distinction between TSC and cytotrophoblasts. Proof-of-principle data demonstrating organoid generation from late gestation TSC isolated directly from the placenta lays the groundwork for future disease models.

Keywords:  3D culture; Organoid; Placenta; Side-population trophoblasts; Transit amplifying cell; Trophoblast stem cell

DOI:  https://doi.org/10.1016/j.placenta.2024.12.003
Neural Regen Res. 2024 Dec 07.

Induced pluripotent stem cell-related approaches to generate dopaminergic neurons for Parkinson's disease.

Ling-Xiao Yi, Hui Ren Woon, Genevieve Saw, Li Zeng, Eng King Tan, Zhi Dong Zhou.

The progressive loss of dopaminergic neurons in affected patient brains is one of the pathological features of Parkinson's disease, the second most common human neurodegenerative disease. Although the detailed pathogenesis accounting for dopaminergic neuron degeneration in Parkinson's disease is still unclear, the advancement of stem cell approaches has shown promise for Parkinson's disease research and therapy. The induced pluripotent stem cells have been commonly used to generate dopaminergic neurons, which has provided valuable insights to improve our understanding of Parkinson's disease pathogenesis and contributed to anti-Parkinson's disease therapies. The current review discusses the practical approaches and potential applications of induced pluripotent stem cell techniques for generating and differentiating dopaminergic neurons from induced pluripotent stem cells. The benefits of induced pluripotent stem cell-based research are highlighted. Various dopaminergic neuron differentiation protocols from induced pluripotent stem cells are compared. The emerging three-dimension-based brain organoid models compared with conventional two-dimensional cell culture are evaluated. Finally, limitations, challenges, and future directions of induced pluripotent stem cell- based approaches are analyzed and proposed, which will be significant to the future application of induced pluripotent stem cell-related techniques for Parkinson's disease.

DOI: https://doi.org/10.4103/NRR.NRR-D-24-00771
Am J Med Genet A. 2025 Jan;197(1): e63728

Repeat Expansion Disorders Likely Underdiagnosed.

DOI: https://doi.org/10.1002/ajmg.a.63728
Stem Cell Res. 2024 Nov 09. pii: S1873-5061(24)00302-7. [Epub ahead of print]82 103604

A human-induced pluripotent stem cell (iPSC) line (SMUSHi006-A) from an ALS patient carrying a mutation c.1126C > T in the FUS gene.

Chunyan Huang, Liying Qiu, Wenyan Zhou, Congwen Shao, Xichun Wang, Qiwen Zhang, Weilin Chen, Min Xiong, Min Huang, Mei Tang, Liangyu Zou, Xueqing Xu.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease. Four major genes associated with ALS-SOD1, TARDBP, FUS, and C9orf72-have been identified, with the fused in sarcoma (FUS) gene demonstrating considerable genetic heterogeneity. Our research group has previously established an induced pluripotent stem (iPS) cell line harboring the c.1562G > A mutation in the FUS gene. The objective of this study is to create another iPS cell line featuring the pathogenic c.1126C > T mutation in the FUS gene. This research aims not only to establish a disease model for ALS linked to FUS mutations but also to pave the way for potential therapeutic interventions.

DOI: https://doi.org/10.1016/j.scr.2024.103604
Mol Neurobiol. 2024 Dec 09.

Reduced Neurite Arborization in Primary Dopaminergic Neurons in Autism-Like Shank3B-Deficient Mice.

Zuzana Bacova, Tomas Havranek, Denisa Mihalj, Veronika Borbelyova, Kristina Kostrubanicova, Michaela Kramarova, Daniela Ostatnikova, Jan Bakos.

  Despite many studies on dopamine changes in autism, specific alterations in midbrain dopamine neurons projecting to the striatum and cortex remain unclear. Mouse models with diverse SH3 domain and ankyrin repeat containing protein 3 (Shank3) deficiencies are used for investigating autistic symptoms and underlying neurobiological mechanisms. SHANK3 belongs to postsynaptic proteins crucial for synapse formation during development, and disruptions in SHANK3 structure could lead to impaired neurite outgrowth and altered dendritic arborization and morphology. Therefore, we aimed to investigate whether Shank3 deficiency (Shank3B) leads to changes in the morphology of primary neuronal cell cultures from dopaminergic brain regions of neonatal mouse pups and whether it results in alterations in synaptic proteins in dopaminergic nerve pathway projection areas (striatum, frontal cortex). Significantly reduced neurite outgrowth was observed in primary dopaminergic neurons from the midbrain and striatum of Shank3-deficient compared to WT mice. A decrease in Synapsin I immunofluorescence signal in the cortical neurons isolated from Shank3-deficient mice was found, although neurite arborization changes were less severe. Importantly, the deficit in the length of the longest neurite was confirmed in primary cortical neurons isolated from Shank3-deficient mice. No changes in the gene expression of synaptic proteins were observed in the striatum and frontal cortex of Shank3-deficient mice, but an altered gene expression profile of dopaminergic receptors was found. These results show structural changes of dopaminergic neurons, which may explain autistic symptomatology in the used model and provide a basis for understanding the long-term development of autistic symptoms.

Keywords:   Shank3 ; Autism spectrum disorder; Dopaminergic neurons; Neurite outgrowth

DOI:  https://doi.org/10.1007/s12035-024-04652-0
Cell Stem Cell. 2024 Dec 06. pii: S1934-5909(24)00404-1. [Epub ahead of print]

Generation of self-organized neuromusculoskeletal tri-tissue organoids from human pluripotent stem cells.

Yao Yin, Wei Zhou, Jinkui Zhu, Ziling Chen, Linlin Jiang, Xuran Zhuang, Jia Chen, Jianfeng Wei, Xiaoxiang Lu, Yantong Liu, Wei Pang, Qinzhi Zhang, Yajing Cao, Zhuoya Li, Yuyan Zhu, Yangfei Xiang.

  The human body function requires crosstalk between different tissues. An essential crosstalk is in the neuromusculoskeletal (NMS) axis involving neural, muscular, and skeletal tissues, which is challenging to model using human cells. Here, we describe the generation of three-dimensional, NMS tri-tissue organoids (hNMSOs) from human pluripotent stem cells through a co-development strategy. Staining, single-nucleus RNA sequencing, and spatial transcriptome profiling revealed the co-emergence and self-organization of neural, muscular, and skeletal lineages within individual organoids, and the neural domains of hNMSOs obtained a ventral-specific identity and produced motor neurons innervating skeletal muscles. The neural, muscular, and skeletal regions of hNMSOs exhibited maturation and established functional connections during development. Notably, structural, functional, and transcriptomic analyses revealed that skeletal support in hNMSOs benefited human muscular development. Modeling with hNMSOs also unveiled the neuromuscular alterations following pathological skeletal degeneration. Together, our study provides an accessible experimental model for future studies of human NMS crosstalk and abnormality.

Keywords:  co-development; hPSCs; neural organoid; neuromusculoskeletal interaction; organoid; spinal cord

DOI:  https://doi.org/10.1016/j.stem.2024.11.005
Biomacromolecules. 2024 Dec 12.

Lysosomal Trafficking and Degradation of Extracellular Proteins via Multivalent Small Molecule Ligand Display on Dextran Scaffolds.

Benoit Louage, Demi Defreyne, Heleen Lauwers, Jamie De Baere, Annemiek Uvyn, Haixia Peng, Yong Chen, Bruno G De Geest.

Targeted protein degradation (TPD) marks a shift in drug development from conventional inhibition to the complete removal of pathological proteins. Traditional TPD technologies target intracellular proteins of interest (POIs) for degradation but are ineffective against extracellular cell surface and soluble proteins, a significant portion of the human proteome. Recent advances involve the formation of ternary complexes between a POI and a cell surface lysosomal trafficking receptor, directing POIs to lysosomes for degradation. We report on DEXtran TRAfficking Chimeras (DEXTRACs) comprising multiple copies of synthetic small molecule ligands for a model POI and the cation-independent mannose-6-phosphate receptor (CI-M6PR) lysosomal trafficking receptor. These ligands are arranged along the dextran backbones. We demonstrate that DEXTRACs leverage multivalency with their efficacy dependent on the dextran chain length and ligand density to form high-avidity ternary complexes. Our in vitro studies confirmed that DEXTRACs traffic the target POI to lysosomes and facilitate its degradation.

DOI: https://doi.org/10.1021/acs.biomac.4c01603
Cell Stem Cell. 2024 Dec 05. pii: S1934-5909(24)00407-7. [Epub ahead of print]31(12): 1730-1731

Making blood from iPSCs: Reaching for the most sought-after prize.

Anna Bigas, Gayathri M Kartha.

After many years of little progress, there is now hope for improved in vitro production of hematopoietic stem cells (HSCs). Ng et al.1 have developed defined conditions that allow the generation of HSCs from induced pluripotent stem cells (iPSCs) with unprecedented blood-repopulating capacity.

DOI: https://doi.org/10.1016/j.stem.2024.11.008
J Biomol Struct Dyn. 2024 Dec 11. 1-14

Unraveling the molecular mechanisms of ALS: a network biology and structural modeling approach of investigating the impact of C9orf72 mutations.

A Khuzaim Alzahrani, Alshrari A S, Mohd Imran.

  C9orf72 is a major genetic factor in Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disorder affecting brain and spinal cord neurons, and comprehending its mutational impact is crucial for developing ALS therapies. Therefore, the current study's protein-protein interaction (PPI) network for C9orf72 was meticulously mapped to identify key interactors that might influence the disease mechanism. Among the identified proteins, SMCR8 emerged as a prominent candidate due to its high connectivity (total network contribution = 7.896) within the C9orf72-associated network, suggesting a potential role in modulating the effects of C9orf72 mutations. Analysis of C9orf72 mutations highlighted the I525T mutation, which significantly destabilizes the protein, as indicated by a ΔΔG value of -2.02 kcal/mol. Further investigation involved comparing the structural dynamics of the wild-type C9orf72 and its mutant variants through molecular docking and dynamics simulations. The wild-type demonstrated more stable structural conformation over time, as shown by its RMSD profile than its mutant counterpart. However, after 80 nanoseconds, the mutant variant achieved a similar RMSD stability level. Intriguingly, the mutant formed a more stable complex with SMCR8, evident from its lower binding free energy (-64.18 kcal/mol compared to the wild type's -34.82 kcal/mol). Moreover, per-residue decomposition analysis further revealed critical interactions at specific residues. The wild-type protein showed a significant stabilizing interaction at Arg785, whereas the mutant favored Arg262, indicating a potential shift in binding affinity and site due to the mutation. This shift suggests an altered binding landscape in the mutant C9orf72, which might contribute to the dysregulated protein interactions and cellular processes associated with ALS pathology. The study thus underscores the pathological hyper-stability of the mutant C9orf72, highlighting its potential role in the progression of ALS.

Keywords:  Amyotrophic lateral sclerosis; C9orf72; neurodegenerative disorder; protein–protein interaction (PPI) network

DOI:  https://doi.org/10.1080/07391102.2024.2437682
Lab Chip. 2024 Dec 11.

Retinal organoid chip: engineering a physiomimetic oxygen gradient for optimizing long term culture of human retinal organoids.

Emma Drabbe, Daniel Pelaez, Ashutosh Agarwal.

An oxygen gradient across the retina plays a crucial role in its development and function. The inner retina resides in a hypoxic environment (2% O2) adjacent to the vitreous cavity. Oxygenation levels rapidly increase towards the outer retina (18% O2) at the choroid. In addition to retinal stratification, oxygen levels are critical for the health of retinal ganglion cells (RGCs), which relay visual information from the retina to the brain. Human stem cell derived retinal organoids are being engineered to mimic the structure and function of human retina for applications such as disease modeling, development of therapeutics, and cell replacement therapies. However, rapid degeneration of the retinal ganglion cell layers are a common limitation of human retinal organoid platforms. We report the design of a novel retinal organoid chip (ROC) that maintains a physiologically relevant oxygen gradient and allows the maturation of inner and outer retinal cell phenotypes for human retinal organoids. Our PDMS-free ROC holds 55 individual retinal organoids that were manually seeded, cultured for extended periods (over 150 days), imaged in situ, and retrieved. ROC was designed from first principles of liquid and gas mass transport, and fabricated from biologically- and chemically inert materials using rapid prototyping techniques such as micromachining, laser cutting, 3D printing and bonding. After computational and experimental validation of oxygen gradients, human induced pluripotent stem cell derived retinal organoids were transferred into the ROC, differentiated, cultured and imaged within the chip. ROCs that maintained active perfusion and stable oxygen gradients were successful in inducing higher viability of RGCs within retinal organoids than static controls, or ROC without oxygen gradients. Our physiologically relevant and higher-throughput retinal organoid culture system is well suited for applications in studying developmental perturbations to primate retinogenesis, including those driven by inherited traits, fetal environmental exposure to toxic agents, or acquired by genetic mutations, such as retinoblastoma.

DOI: https://doi.org/10.1039/d4lc00771a
Int J Biol Sci. 2024 ;20(15): 5999-6017

Inhibition of miR-4763-3p expression activates the PI3K/mTOR/Bcl2 autophagy signaling pathway to ameliorate cognitive decline.

Wenxin Qi, Yiwei Ying, Peiru Wu, Naijun Dong, Wenjun Fu, Qian Liu, Natalie Ward, Xin Dong, Robert Chunhua Zhao, Jiao Wang.

  Cognitive decline and memory impairment are subsequently result in neuronal apoptosis and synaptic damage. Aberrant regulation of microRNAs has been implicated in the pathogenesis of Alzheimer's disease (AD) and may play a pivotal role in the early stages of the disease. In this study, we identified the critical role of miR-4763-3p in AD pathogenesis, focusing on early-stage mild cognitive impairment (AD-MCI). Leveraging fluorescence in situ hybridization, we observed miR-4763-3p upregulation in AD hippocampal tissue, colocalizing with Aβ and Tau. Antagomir-mediated inhibition of miR-4763-3p ameliorated cognitive decline in AD-MCI mice. RNA-seq and functional assays revealed that miR-4763-3p targets ATP11A, and antagomir enhancing inward flipping of the "eat me" phosphatidylserine signal on the surface of neuronal cells, autophagy, and clearance of Aβ/lipofuscin, while reducing neuroinflammation and neuronal apoptosis. Mechanistically, miR-4763-3p modulates the PI3K/AKT/mTOR/Bcl2 pathway, thereby promoting neuronal autophagy and reducing apoptotic crosstalk. These findings underscore miR-4763-3p as a therapeutic target for AD-MCI, offering a novel strategy to enhance neuronal autophagy, alleviate inflammation, and improve cognitive function.

Keywords:  AD-MCI; apoptosis; autophagy; phosphatidylserine

DOI:  https://doi.org/10.7150/ijbs.103225
Neurobiol Dis. 2024 Dec 06. pii: S0969-9961(24)00362-0. [Epub ahead of print]204 106760

Reactivation of mTOR signaling slows neurodegeneration in a lysosomal sphingolipid storage disease.

Hongling Zhu, Y Terry Lee, Colleen Byrnes, Jabili Angina, Danielle A Springer, Galina Tuymetova, Mari Kono, Cynthia J Tifft, Richard L Proia.

  Sandhoff disease, a lysosomal storage disorder, is caused by pathogenic variants in the HEXB gene, resulting in the loss of β-hexosaminidase activity and accumulation of sphingolipids including GM2 ganglioside. This accumulation occurs primarily in neurons, and leads to progressive neurodegeneration through a largely unknown process. Lysosomal storage diseases often exhibit dysfunctional mTOR signaling, a pathway crucial for proper neuronal development and function. In this study, Sandhoff disease model mice exhibited reduced mTOR signaling in the brain. To test if restoring mTOR signaling could improve the disease phenotype, we genetically reduced expression of the mTOR inhibitor Tsc2 in these mice. Sandhoff disease mice with reactivated mTOR signaling displayed increased survival rates and motor function, especially in females, increased dendritic-spine density, and reduced neurodegeneration. Tsc2 reduction also partially rescued aberrant synaptic function-related gene expression. These findings imply that enhancing mTOR signaling could be a potential therapeutic strategy for lysosomal-based neurodegenerative diseases.

Keywords:  Ganglioside; Glycosphingolipid; Lysosomal storage disease; Lysosome; Neurodegeneration; Sandhoff disease; Sphingolipid; Synaptic function; mTOR

DOI:  https://doi.org/10.1016/j.nbd.2024.106760
J Neurochem. 2025 Jan;169(1): e16284

Dopamine transporter endocytic trafficking: Neuronal mechanisms and potential impact on DA-dependent behaviors.

Nicholas C Bolden, Rebecca G Pavchinskiy, Haley E Melikian.

  The dopamine (DA) transporter (DAT) is a major determinant of DAergic neurotransmission, and is a primary target for addictive and therapeutic psychostimulants. Evidence accumulated over decades in cell lines and in vitro preparations revealed that DAT function is acutely regulated by membrane trafficking. Many of these findings have recently been validated in vivo and in situ, and several behavioral and physiological findings raise the possibility that regulated DAT trafficking may impact DA signaling and DA-dependent behaviors. Here we review key DAT trafficking findings across multiple systems, and discuss the cellular mechanisms that mediate DAT trafficking, as well as the endogenous receptors and signaling pathways that drive regulated DAT trafficking. We additionally discuss recent findings that DAT trafficking dysfunction correlates to perturbations in DA signaling and DA-dependent behaviors.

Keywords:  dopamine; endocytosis; locomotion; reuptake; reward

DOI:  https://doi.org/10.1111/jnc.16284