During anaphase, the nonkinetochore microtubules in the spindle midzone become compacted into the central spindle, a structure which is required to both initiate and complete cytokinesis. We show that Tektin 2 (Tek2) associates with the spindle poles throughout mitosis, organizes the spindle midzone microtubules during anaphase, and assembles into the midbody matrix surrounding the compacted midzone microtubules during cytokinesis. Tek2 small interfering RNA (siRNA) disrupts central spindle organization and proper localization of MKLP1, PRC1, and Aurora B to the midzone and prevents the formation of a midbody matrix. Video microscopy revealed that loss of Tek2 results in binucleate cell formation by aberrant fusion of daughter cells after cytokinesis. Although a myosin II inhibitor, blebbistatin, prevents actin-myosin contractility, the microtubules of the central spindle are compacted. Strikingly, Tek2 siRNA abolishes this actin-myosin–independent midzone microtubule compaction. Thus, Tek2-dependent organization of the central spindle during anaphase is essential for proper midbody formation and the segregation of daughter cells after cytokinesis.

Midbody

Astral microtubules

Cleavage furrow

Aurora B kinase

Spindle pole body

10.1083/jcb.200711160

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Citations (30)

KCC2 as a novel biomarker and therapeutic target for motoneuron degenerative disease

bioRxiv (Cold Spring Harbor Laboratory) (2023)

C. Sahara Khademullah Julien Bourbonnais Mathilde Chaineau María José Castellanos‐Montiel Iason Keramidis

Abstract Hyperexcitability in cells throughout the corticospinal tract is a presymptomatic feature of amyotrophic lateral sclerosis (ALS) associated with lethal motor degeneration 1–6 . Disinhibition is a possible cause of this hyperexcitability, potentially implicating the central nervous system-specific potassium-chloride cotransporter, KCC2, a core regulator of the strength of GABAergic neurotransmission linked to several neurological disorders 7–11 . Here, we show that KCC2 is downregulated in the membrane of motor cortex neurons from post-mortem SOD1-, C9orf72- and sporadic ALS is patients. Increased protein levels of KCC2 were found in plasma and cerebral spinal fluid of ALS patients and mice harbouring the SOD1*G93A mutation. Longitudinal analysis of disease progression in both SOD1*G93A and Prp-TDP43*A315T mice revealed a decrease of KCC2 membrane levels in cortical and spinal motor neurons which were already present at the presymptomatic phase. Using KCC2-enhancing compounds, CLP290 and prochlorperazine (PCPZ) restored KCC2 membrane expression and function, delayed motor deficit onset, and extended lifespan up to two months in mutant mice. Human-derived neurons differentiated from iPSC harbouring the SOD1*G93A mutation displayed KCC2 deficits which PCPZ treatment rescued. Acute administration of KCC2 enhancers restored chloride transport in presymptomatic and symptomatic mice and reversed motor neuron hyperexcitability in awake behaving mutant mice. These findings identify KCC2 as both an early biomarker and a disease-modifying therapeutic target for ALS.

Corticospinal tract

10.1101/2023.08.24.554410

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Citations (2)

The three ‘P’s of mitophagy: PARKIN, PINK1, and post-translational modifications

Genes & Development (2015)

Thomas M. Durcan Edward A. Fon

Two Parkinson's disease (PD)-associated proteins, the mitochondrial kinase PINK1 and the E3-ubiquitin (Ub) ligase PARKIN, are central to mitochondrial quality control. In this pathway, PINK1 accumulates on defective mitochondria, eliciting the translocation of PARKIN from the cytosol to mediate the clearance of damaged mitochondria via autophagy (mitophagy). Throughout the different stages of mitophagy, post-translational modifications (PTMs) are critical for the regulation of PINK1 and PARKIN activity and function. Indeed, activation and recruitment of PARKIN onto damaged mitochondria involves PINK1-mediated phosphorylation of both PARKIN and Ub. Through a stepwise cascade, PARKIN is converted from an autoinhibited enzyme into an active phospho-Ub-dependent E3 ligase. Upon activation, PARKIN ubiquitinates itself in concert with many different mitochondrial substrates. The Ub conjugates attached to these substrates can in turn be phosphorylated by PINK1, which triggers further cycles of PARKIN recruitment and activation. This feed-forward amplification loop regulates both PARKIN activity and mitophagy. However, the precise steps and sequence of PTMs in this cascade are only now being uncovered. For instance, the Ub conjugates assembled by PARKIN consist predominantly of noncanonical K6-linked Ub chains. Moreover, these modifications are reversible and can be disassembled by deubiquitinating enzymes (DUBs), including Ub-specific protease 8 (USP8), USP15, and USP30. However, PINK1-mediated phosphorylation of Ub can impede the activity of these DUBs, adding a new layer of complexity to the regulation of PARKIN-mediated mitophagy by PTMs. It is therefore evident that further insight into how PTMs regulate the PINK1–PARKIN pathway will be critical for our understanding of mitochondrial quality control.

PINK1

Deubiquitinating enzyme

10.1101/gad.262758.115

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Citations (361)

A dual hit of α-synuclein internalization and immune challenge leads to the formation and maintenance of Lewy body-like inclusions in human dopaminergic neurons

bioRxiv (Cold Spring Harbor Laboratory) (2023)

Armin Bayati Riham Ayoubi Adriana Aguila Cornelia E. Zorca Chanshuai Han

SUMMARY Lewy bodies (LBs), rich in α-synuclein, are a hallmark of Parkinson’s disease (PD). Understanding their biogenesis is likely to provide insight into the pathophysiology of PD, yet a cellular model for LB formation remains elusive. The realization that the immune challenge is a trigger for neurodegenerative diseases has been a breakthrough in the understanding of PD. Here, iPSC-derived human dopaminergic (DA) neurons from multiple healthy donors were found to form LB-like inclusions following treatment with α- synuclein preformed fibrils, but only when coupled to an immune challenge (interferon-gamma or interleukin-1 beta) or when co-cultured with activated microglia. Human cortical neurons derived from the same iPSC lines did not form LB-like inclusions. Exposure to interferon-gamma impairs autophagy in a lysosomal-specific manner in vitro, similar to the disruption of proteostasis pathways that contribute to PD. We find that lysosomal membrane proteins LAMP1 and LAMP2 and transcription factors regulating lysosomal biogenesis and function are downregulated in DA but not cortical neurons. Finally, due to the excellent sample preservation afforded by cells compared to post-mortem PD brain tissue, we conclude that the LB-like inclusions in DA neurons are membrane-bound, suggesting they are not limited to the cytoplasmic compartment. In Brief Bayati et al. identify that iPSC-derived dopaminergic neurons undergoing a dual hit treatment of exogenous α-synuclein fibrils and proinflammatory cytokines form Lewy body-like inclusions. The dual hit treatment also led to the downregulation of lysosomal proteins. Characterization of inclusions revealed that inclusions were membrane-bound and LC3B-positive, suggesting they are dysfunctional autophagosomes. Highlights α-synuclein preformed fibril administration coupled with Interferon-gamma exposure leads dopaminergic neurons to form Lewy body-like inclusions Inclusions are filamentous, membranous, and filled with aberrant organelles Impaired autophagic flux and downregulation of TFEB, NRF2, LAMP1, and LAMP2 correlated with inclusion formation Activation of NRF2 through the treatment of neurons with the antioxidant perillaldehyde, prevents inclusion formation

Proteostasis

Proinflammatory cytokine

Internalization

Lewy body

10.1101/2023.05.29.542776

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Citations (3)

CelltypeR: A flow cytometry pipeline to annotate, characterize and isolate single cells from brain organoids

bioRxiv (Cold Spring Harbor Laboratory) (2022)

Rhalena A. Thomas Julien Sirois Shu‐Ming Li Alexandre Gestin Ghislaine Deyab

Abstract Motivated by the growing number of single cell RNA sequencing datasets (scRNAseq) revealing the cellular heterogeneity in complex tissues, particularly in brain and induced pluripotent stem cell (iPSC)-derived brain models, we developed a high-throughput, standardized approach for reproducibly characterizing cell types in complex neuronal tissues based on protein expression levels. Our approach combines a flow cytometry (FC) antibody panel targeting brain cells with a computational pipeline called CelltypeR, with functions for aligning and transforming datasets, optimizing unsupervised clustering, annotating and quantifying cell types, and statistical comparisons. We applied this workflow to human iPSC-derived midbrain organoids and identified the expected brain cell types, including neurons, astrocytes, radial glia, and oligodendrocytes. Defining gates based on the expression levels of our protein markers, we performed Fluorescence-Activated Cell Sorting of astrocytes, radial glia, and neurons, cell types were then confirmed by scRNAseq. Among the sorted neurons, we identified three subgroups of dopamine (DA) neurons; one reminiscent of substantia nigra DA neurons, the cell type most vulnerable in Parkinson’s disease. Finally, we use our workflow to track cell types across a time course of organoid differentiation. Overall, our adaptable analysis framework provides a generalizable method for reproducibly identifying cell types across FC datasets.

Cell type

Organoid

Cell Sorting

10.1101/2022.11.11.516066

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Citations (2)

Auto-qPCR: A Python-based web app for automated and reproducible analysis of qPCR data

bioRxiv (Cold Spring Harbor Laboratory) (2021)

Gilles Maussion Rhalena A. Thomas Iveta Demirova Gracia Gu Eddie Cai

Abstract Quantifying changes in DNA and RNA levels is essential in numerous molecular biology protocols. Quantitative real time PCR (qPCR) techniques have evolved to become commonplace, however, data analysis includes many time-consuming and cumbersome steps, which can lead to mistakes and misinterpretation of data. To address these bottlenecks, we have developed an open-source Python software to automate processing of result spreadsheets from qPCR machines, employing calculations usually performed manually. Auto-qPCR is a tool that saves time when computing qPCR data, helping to ensure reproducibility of qPCR experiment analyses. Our web-based app ( https://auto-q-pcr.com/ ) is easy to use and does not require programming knowledge or software installation. Using Auto-qPCR, we provide examples of data treatment, display and statistical analyses for four different data processing modes within one program: (1) DNA quantification to identify genomic deletion or duplication events; (2) assessment of gene expression levels using an absolute model, and relative quantification (3) with or (4) without a reference sample. Our open access Auto-qPCR software saves the time of manual data analysis and provides a more systematic workflow, minimizing the risk of errors. Our program constitutes a new tool that can be incorporated into bioinformatic and molecular biology pipelines in clinical and research labs.

Python

10.1101/2021.01.14.426748

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Citations (0)

Isolation of human proteasomes and putative proteasome-interacting proteins using a novel affinity chromatography method

Experimental Cell Research (2008)

Thomas C. Scanlon Bruce Gottlieb Thomas M. Durcan Edward A. Fon Lenore K. Beitel

Tandem affinity purification

Protein purification

F-box protein

Protein Degradation

Organelle

10.1016/j.yexcr.2008.10.027

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Citations (30)

Auto-qPCR; a python-based web app for automated and reproducible analysis of qPCR data

Scientific Reports (2021)

Gilles Maussion Rhalena A. Thomas Iveta Demirova Gracia Gu Eddie Cai

Python

10.1038/s41598-021-99727-6

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Citations (14)

Characterization of human-iPSCs derived spinal motor neurons by single-cell RNA sequencing

bioRxiv (Cold Spring Harbor Laboratory) (2019)

Louise Thiry Regan Hamel Stefano Pluchino Thomas M. Durcan Stefano Stifani

Abstract Human induced pluripotent stem cells (iPSCs) offer the opportunity to generate specific cell types from healthy and diseased individuals, allowing the study of mechanisms of early human development, modelling a variety of human diseases, and facilitating the development of new therapeutics. Human iPSC-based applications are often limited by the variability among iPSC lines originating from a single donor, as well as the heterogeneity among specific cell types that can be derived from iPSCs. The ability to deeply phenotype different iPSC-derived cell types is therefore of primary importance to the successful and informative application of this technology. Here we describe a combination of motor neuron (MN) derivation and single-cell RNA sequencing approaches to generate and characterize specific MN subtypes obtained from human iPSCs. Our studies provide evidence for rapid and robust generation of MN progenitor cells that can give rise to a heterogenous population of brainstem and spinal cord MNs. Approximately 58% of human iPSC-derived MNs display molecular characteristics of lateral motor column MNs, ∼19% of induced MNs resemble hypaxial motor column MNs, while ∼6% of induced MNs have features of medial motor column MNs. The present study has the potential to improve our understanding of iPSC-derived MN subtype function and dysfunction, possibly leading to improved iPSC-based applications for the study of human MN biology and diseases.

Cell type

Spinal column

10.1101/2019.12.28.889972

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Citations (1)