bims-axbals 2024-02-04 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2024–02–04
24 papers selected by
TJ Krzystek, ALS Therapy Development Institute

Cyclin F can alter the turnover of TDP-43.
Betz cells of the primary motor cortex.
Disruption of the autism-associated gene SCN2A alters synaptic development and neuronal signaling in patient iPSC-glutamatergic neurons.
A cell therapy approach based on iPSC-derived midbrain organoids for the restoration of motor function in a Parkinson's disease mouse model.
eIF5 stimulates the CUG initiation of RAN translation of poly-GA dipeptide repeat protein (DPR) in C9orf72 FTLD/ALS.
CAG repeat expansions create splicing acceptor sites and produce aberrant repeat-containing RNAs.
Host-to-graft propagation of inoculated α-synuclein into transplanted human induced pluripotent stem cell-derived midbrain dopaminergic neurons.
The LRRK2 kinase substrates RAB8a and RAB10 contribute complementary but distinct disease-relevant phenotypes in human neurons.
Cell biology of Parkinson's disease: Mechanisms of synaptic, lysosomal, and mitochondrial dysfunction.
An epigenetic barrier sets the timing of human neuronal maturation.
Identification of gene fusions associated with amyotrophic lateral sclerosis.
Increased neuronal nitric oxide synthase in Alzheimer's disease mediates spontaneous calcium signalling and divergent glutamatergic calcium responses.
Inducing Pluripotency in Somatic Cells: Historical Perspective and Recent Advances.
The synaptic drive of central pattern-generating networks to leg motor neurons of a walking insect is motor neuron pool specific.
Validation of a newly developed immunoassay for TDP-43 in human plasma.
Genetically encoded lysine photocage for spatiotemporal control of TDP-43 nuclear import.
NMNAT2 supports vesicular glycolysis via NAD homeostasis to fuel fast axonal transport.
Molecular and cellular mechanisms underlying the failure of mitochondrial metabolism drugs in cancer clinical trials.
Modeling and Correction of Protein Conformational Disease in iPSC-derived Neurons through Personalized Base Editing.
DENND6A links Arl8b to a Rab34/RILP/dynein complex, regulating lysosomal positioning and autophagy.
Axonal distribution of mitochondria maintains neuronal autophagy during aging via eIF2β.
Identification of small molecule inhibitors of G3BP-driven stress granule formation.
Estradiol enhanced neuronal plasticity and ameliorated astrogliosis in human iPSC-derived neural models.
Maintaining Toll signaling in Drosophila brain is required to sustain autophagy for dopamine neuron survival.

Neurobiol Dis. 2024 Jan 27. pii: S0969-9961(24)00020-2. [Epub ahead of print] 106421

Cyclin F can alter the turnover of TDP-43.

Stephanie L Rayner, Alison Hogan, Jennilee M Davidson, Tyler Chapman, Flora Cheng, Luan Luu, Sharlynn Wu, Selina Zhang, Shu Yang, Ian Blair, Marco Morsch, Roger Chung, Albert Lee.

  Previously, we demonstrated that the SCFcyclin F complex directly mediates the poly-ubiquitylation of TDP-43, raising the question of whether cyclin F can be used to enhance the turnover of TDP-43. A hurdle to the use of cyclin F, however, is that the overexpression of cyclin F can lead to the initiation of cell death pathways. Accordingly, the aim of this study was to identify and evaluate a less toxic variant of cyclin F. To do so, we first confirmed and validated our previous findings that cyclin F binds to TDP-43 in an atypical manner. Additionally, we demonstrated that mutating the canonical substrate region in cyclin F (to generate cyclin FMRL/AAA) led to reduced binding affinity to known canonical substrates without impacting the interaction between cyclin F and TDP-43. Notably, both wild-type and cyclin FMRL/AAA effectively reduced the abundance of TDP-43 in cultured cells whilst cyclin FMRL/AAA also demonstrated reduced cell death compared to the wild-type control. The decrease in toxicity also led to a reduction in morphological defects in zebrafish embryos. These results suggest that cyclin F can be modified to enhance its targeting of TDP-43, which in turn reduces the toxicity associated with the overexpression of cyclin F. This study provides greater insights into the interaction that occurs between cyclin F and TDP-43 in cells and in vivo.

Keywords:  Amyotrophic lateral sclerosis; Cyclin F; Frontotemporal dementia; TDP-43; Ubiquitylation

DOI:  https://doi.org/10.1016/j.nbd.2024.106421
J Comp Neurol. 2024 Jan;532(1): e25567

Betz cells of the primary motor cortex.

Matthew Nolan, Connor Scott, Patrick R Hof, Olaf Ansorge.

  Betz cells, named in honor of Volodymyr Betz (1834-1894), who described them as "giant pyramids" in the primary motor cortex of primates and other mammalian species, are layer V extratelencephalic projection (ETP) neurons that directly innervate α-motoneurons of the brainstem and spinal cord. Despite their large volume and circumferential dendritic architecture, to date, no single molecular criterion has been established that unequivocally distinguishes adult Betz cells from other layer V ETP neurons. In primates, transcriptional signatures suggest the presence of at least two ETP neuron clusters that contain mature Betz cells; these are characterized by an abundance of axon guidance and oxidative phosphorylation transcripts. How neurodevelopmental programs drive the distinct positional and morphological features of Betz cells in humans remains unknown. Betz cells display a distinct biphasic firing pattern involving early cessation of firing followed by delayed sustained acceleration in spike frequency and magnitude. Few cell type-specific transcripts and electrophysiological characteristics are conserved between rodent layer V ETP neurons of the motor cortex and primate Betz cells. This has implications for the modeling of disorders that affect the motor cortex in humans, such as amyotrophic lateral sclerosis (ALS). Perhaps vulnerability to ALS is linked to the evolution of neural networks for fine motor control reflected in the distinct morphomolecular architecture of the human motor cortex, including Betz cells. Here, we discuss histological, molecular, and functional data concerning the position of Betz cells in the emerging taxonomy of neurons across diverse species and their role in neurological disorders.

Keywords:  Betz cell; amyotrophic lateral sclerosis; motor cortex; neuroanatomy; projection neurons

DOI:  https://doi.org/10.1002/cne.25567
Front Cell Neurosci. 2023 ;17 1239069

Disruption of the autism-associated gene SCN2A alters synaptic development and neuronal signaling in patient iPSC-glutamatergic neurons.

Chad O Brown, Jarryll A Uy, Nadeem Murtaza, Elyse Rosa, Alexandria Alfonso, Biren M Dave, Savannah Kilpatrick, Annie A Cheng, Sean H White, Stephen W Scherer, Karun K Singh.

  SCN2A is an autism spectrum disorder (ASD) risk gene and encodes a voltage-gated sodium channel. However, the impact of ASD-associated SCN2A de novo variants on human neuron development is unknown. We studied SCN2A using isogenic SCN2A-/- induced pluripotent stem cells (iPSCs), and patient-derived iPSCs harboring a de novo R607* truncating variant. We used Neurogenin2 to generate excitatory (glutamatergic) neurons and found that SCN2A+/R607* and SCN2A-/- neurons displayed a reduction in synapse formation and excitatory synaptic activity. We found differential impact on actional potential dynamics and neuronal excitability that reveals a loss-of-function effect of the R607* variant. Our study reveals that a de novo truncating SCN2A variant impairs the development of human neuronal function.

Keywords:  autism; dendrite; human neuron; sodium channel; synapse

DOI:  https://doi.org/10.3389/fncel.2023.1239069
Heliyon. 2024 Jan 30. 10(2): e24234

A cell therapy approach based on iPSC-derived midbrain organoids for the restoration of motor function in a Parkinson's disease mouse model.

Chong-Lei Fu, Bo-Cheng Dong, Xi Jiang, Dan Li, Jun Yao.

  Parkinson's disease (PD) is a neurodegenerative disease characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra and loss of DA transmission in the striatum, thus making cell transplantation an effective treatment strategy. Here, we develop a cellular therapy based on induced pluripotent stem cell (iPSC)-derived midbrain organoids. By transplanting midbrain organoid cells into the striatum region of a 6-OHDA-lesioned PD mouse model, we found that the transplanted cells survived and highly efficiently differentiated into DA neurons. Further, using a dopamine sensor, we observed that the differentiated human DA neurons could efficiently release dopamine and were integrated into the neural network of the PD mice. Moreover, starting from four weeks after transplantation, the motor function of the transplanted mice could be significantly improved. Therefore, cell therapy based on iPSC-derived midbrain organoids can be a potential strategy for the clinical treatment of PD.

Keywords:  Cell therapy; Midbrain organoids; Parkinson's disease; iPSC

DOI:  https://doi.org/10.1016/j.heliyon.2024.e24234
J Biol Chem. 2024 Jan 30. pii: S0021-9258(24)00079-6. [Epub ahead of print] 105703

eIF5 stimulates the CUG initiation of RAN translation of poly-GA dipeptide repeat protein (DPR) in C9orf72 FTLD/ALS.

Shiho Gotoh, Kohji Mori, Yuzo Fujino, Yuya Kawabe, Tomoko Yamashita, Tsubasa Omi, Kenichi Nagata, Shinji Tagami, Yoshitaka Nagai, Manabu Ikeda.

Tandem GGGGCC repeat expansion in C9orf72 is a genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Transcribed repeats are translated into dipeptide repeat proteins (DPR) via repeat-associated non-AUG (RAN) translation. However, the regulatory mechanism of RAN translation remains unclear. Here, we reveal a GTPase-activating protein, eukaryotic initiation factor 5 (eIF5), which allosterically facilitates the conversion of eIF2-bound GTP into GDP upon start codon recognition, as a novel modifier of C9orf72 RAN translation. Compared to global translation, eIF5, but not its inactive mutants, preferentially stimulates poly-GA RAN translation. RAN translation is increased during integrated stress response (ISR), but the stimulatory effect of eIF5 on poly-GA RAN translation was additive to the increase of RAN translation during ISR, with no further increase in phosphorylated eIF2α. Moreover, an alteration of the CUG near cognate codon to CCG or AUG in the poly-GA reading frame abolished the stimulatory effects, indicating that eIF5 primarily acts through the CUG-dependent initiation. Lastly, in a Drosophila model of C9orf72 FTLD/ALS that expresses GGGGCC repeats in the eye, knockdown of endogenous eIF5 by two independent RNAi strains significantly reduced poly-GA expressions, confirming in vivo effect of eIF5 on poly-GA RAN translation. Together, eIF5 stimulates the CUG initiation of poly-GA RAN translation in cellular and Drosophila disease models of C9orf72 FTLD/ALS.

DOI: https://doi.org/10.1016/j.jbc.2024.105703
Mol Cell. 2024 Jan 30. pii: S1097-2765(24)00007-8. [Epub ahead of print]

CAG repeat expansions create splicing acceptor sites and produce aberrant repeat-containing RNAs.

Rachel Anderson, Michael R Das, Yeonji Chang, Kelsey Farenhem, Cameron O Schmitz, Ankur Jain.

  Expansions of CAG trinucleotide repeats cause several rare neurodegenerative diseases. The disease-causing repeats are translated in multiple reading frames and without an identifiable initiation codon. The molecular mechanism of this repeat-associated non-AUG (RAN) translation is not known. We find that expanded CAG repeats create new splice acceptor sites. Splicing of proximal donors to the repeats produces unexpected repeat-containing transcripts. Upon splicing, depending on the sequences surrounding the donor, CAG repeats may become embedded in AUG-initiated open reading frames. Canonical AUG-initiated translation of these aberrant RNAs may account for proteins that have been attributed to RAN translation. Disruption of the relevant splice donors or the in-frame AUG initiation codons is sufficient to abrogate RAN translation. Our findings provide a molecular explanation for the abnormal translation products observed in CAG trinucleotide repeat expansion disorders and add to the repertoire of mechanisms by which repeat expansion mutations disrupt cellular functions.

Keywords:  Huntington's disease; RAN translation; RNA splicing; polyglutamine diseases; repeat expansion disorders; repeat-associated non-AUG translation; spinocerebellar ataxia

DOI:  https://doi.org/10.1016/j.molcel.2024.01.006
Regen Ther. 2024 Mar;25 229-237

Host-to-graft propagation of inoculated α-synuclein into transplanted human induced pluripotent stem cell-derived midbrain dopaminergic neurons.

Serina Gima, Kazuya Oe, Kaneyasu Nishimura, Takashi Ohgita, Haruka Ito, Hiroyuki Kimura, Hiroyuki Saito, Kazuyuki Takata.

   Introduction: Cell therapeutic clinical trials using fetal mesencephalic tissue provided a proof-of-concept for regenerative therapy in patients with Parkinson's disease. Postmortem studies of patients with fetal grafts revealed that α-synuclein+ Lewy body (LB)-like inclusions emerged in long-term transplantation and might worsen clinical outcomes even if the grafts survived and innervated in the recipients. Various studies aimed at addressing whether host-derived α-synuclein could be transferred to the grafted neurons to assess α-synuclein+ inclusion appearance in the grafts. However, determining whether α-synuclein in the grafted neurons has been propagated from the host is difficult due to the intrinsic α-synuclein expression.
Methods: We induced midbrain dopaminergic (mDA) neurons from human induced pluripotent stem cells (hiPSCs) and transplanted them into the striatum of immunodeficient rats. The recombinant human α-synuclein preformed fibrils (PFFs) were inoculated into the cerebral cortex after transplantation of SNCA-/- hiPSC-derived mDA neural progenitors into the striatum of immunodeficient rats to evaluate the host-to-graft propagation of human α-synuclein PFFs. Additionally, we examined the incorporation of human α-synuclein PFFs into SNCA-/- hiPSC-derived mDA neurons using in vitro culture system.
Results: We detected human α-synuclein-immunoreactivity in SNCA-/- hiPSC-derived mDA neurons that lacked endogenous α-synuclein expression in vitro. Additionally, we observed host-to-graft α-synuclein propagation into the grafted SNCA-/- hiPSC-derived mDA neurons.
Conclusion: We have successfully proven that intracerebral inoculated α-synuclein PFFs are propagated and incorporated from the host into grafted SNCA-/- hiPSC-derived mDA neurons. Our results contribute toward the basic understanding of the molecular mechanisms related to LB-like α-synuclein deposit formation in grafted mDA neurons.

Keywords:  Cell transplantation; Dopaminergic neurons; Human induced pluripotent stem cells; α-synuclein

DOI:  https://doi.org/10.1016/j.reth.2023.12.019
Stem Cell Reports. 2024 Jan 18. pii: S2213-6711(24)00004-3. [Epub ahead of print]

The LRRK2 kinase substrates RAB8a and RAB10 contribute complementary but distinct disease-relevant phenotypes in human neurons.

Adamantios Mamais, Anwesha Sanyal, Austin Fajfer, Catherine G Zykoski, Michael Guldin, Alexis Riley-DiPaolo, Nitya Subrahmanian, Whitney Gibbs, Steven Lin, Matthew J LaVoie.

  Mutations in the LRRK2 gene cause familial Parkinson's disease presenting with pleomorphic neuropathology that can involve α-synuclein or tau accumulation. LRRK2 mutations are thought to converge upon a pathogenic increase in LRRK2 kinase activity. A subset of small RAB GTPases has been identified as LRRK2 substrates, with LRRK2-dependent phosphorylation resulting in RAB inactivation. We used CRISPR-Cas9 genome editing to generate a novel series of isogenic iPSC lines deficient in the two most well-validated LRRK2 substrates, RAB8a and RAB10, from deeply phenotyped healthy control lines. Thorough characterization of NGN2-induced neurons revealed opposing effects of RAB8a and RAB10 deficiency on lysosomal pH and Golgi organization, with isolated effects of RAB8a and RAB10 ablation on α-synuclein and tau, respectively. Our data demonstrate largely antagonistic effects of genetic RAB8a or RAB10 inactivation, which provide discrete insight into the pathologic features of their biochemical inactivation by pathogenic LRRK2 mutation in human disease.

Keywords:  LRRK2; Parkinson Disease; Rab10; Rab8a; Tau; iPSC; synuclein

DOI:  https://doi.org/10.1016/j.stemcr.2024.01.001
Curr Opin Neurobiol. 2024 Feb 01. pii: S0959-4388(24)00003-5. [Epub ahead of print]85 102841

Cell biology of Parkinson's disease: Mechanisms of synaptic, lysosomal, and mitochondrial dysfunction.

Sarah M Brooker, Grace E Naylor, Dimitri Krainc.

Parkinson's disease (PD) is a growing cause of disability worldwide and there is a critical need for the development of disease-modifying therapies to slow or stop disease progression. Recent advances in characterizing the genetics of PD have expanded our understanding of the cell biology of this disorder. Mitochondrial oxidative stress, defects in synaptic function, and impaired lysosomal activity have been shown to be linked in PD, resulting in a pathogenic feedback cycle involving the accumulation of toxic oxidized dopamine and alpha-synuclein. In this review, we will highlight recent data on a subset of PD-linked genes which have key roles in these pathways and the pathogenic cycle. We will furthermore discuss findings highlighting the importance of dynamic mitochondria-lysosome contact sites that mediate direct inter-organelle cross-talk in the pathogenesis of PD and related disorders.

DOI: https://doi.org/10.1016/j.conb.2024.102841
Nature. 2024 Jan 31.

An epigenetic barrier sets the timing of human neuronal maturation.

Gabriele Ciceri, Arianna Baggiolini, Hyein S Cho, Meghana Kshirsagar, Silvia Benito-Kwiecinski, Ryan M Walsh, Kelly A Aromolaran, Alberto J Gonzalez-Hernandez, Hermany Munguba, So Yeon Koo, Nan Xu, Kaylin J Sevilla, Peter A Goldstein, Joshua Levitz, Christina S Leslie, Richard P Koche, Lorenz Studer.

The pace of human brain development is highly protracted compared with most other species1-7. The maturation of cortical neurons is particularly slow, taking months to years to develop adult functions3-5. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cells (hPSCs) during in vitro differentiation or upon transplantation into the mouse brain4,8,9. Those findings suggest the presence of a cell-intrinsic clock setting the pace of neuronal maturation, although the molecular nature of this clock remains unknown. Here we identify an epigenetic developmental programme that sets the timing of human neuronal maturation. First, we developed a hPSC-based approach to synchronize the birth of cortical neurons in vitro which enabled us to define an atlas of morphological, functional and molecular maturation. We observed a slow unfolding of maturation programmes, limited by the retention of specific epigenetic factors. Loss of function of several of those factors in cortical neurons enables precocious maturation. Transient inhibition of EZH2, EHMT1 and EHMT2 or DOT1L, at progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Thus our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an epigenetic barrier in progenitor cells. Mechanistically, this barrier holds transcriptional maturation programmes in a poised state that is gradually released to ensure the prolonged timeline of human cortical neuron maturation.

DOI: https://doi.org/10.1038/s41586-023-06984-8
Muscle Nerve. 2024 Feb 02.

Identification of gene fusions associated with amyotrophic lateral sclerosis.

NYGC ALS Consortium

   INTRODUCTION/AIMS: Genetics is an important risk factor for amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting motor neurons. Recent findings demonstrate that in addition to specific genetic mutations, structural variants caused by genetic instability can also play a causative role in ALS. Genomic instability can lead to deletions, duplications, insertions, inversions, and translocations in the genome, and these changes can sometimes lead to fusion of distinct genes into a single transcript. Gene fusion events have been studied extensively in cancer; however, they have not been thoroughly investigated in ALS. The aim of this study was to determine whether gene fusions are present in ALS.
METHODS: Gene fusions were identified using STAR Fusion v1.10.0 software in bulk RNA-Seq data from human postmortem samples from publicly available data sets from Target ALS and the New York Genome Center ALS Consortium.
RESULTS: We report the presence of gene fusion events in several brain regions as well as in spinal cord samples in ALS. Although most gene fusions were intra-chromosomal events between neighboring genes and present in both ALS and control samples, there was a significantly greater number of unique gene fusions in ALS compared to controls. Lastly, we identified specific gene fusions with a significant burden in ALS, that were absent from both control samples and known cancer gene fusion databases.
DISCUSSION: Collectively, our findings reveal an enrichment of gene fusions in ALS and suggest that these events may be an additional genetic cause linked to ALS pathogenesis.

Keywords:  RNA-Seq; amyotrophic lateral sclerosis; gene fusion; genetics; neurodegenerative diseases

DOI:  https://doi.org/10.1002/mus.28043
Antioxid Redox Signal. 2024 Feb 01.

Increased neuronal nitric oxide synthase in Alzheimer's disease mediates spontaneous calcium signalling and divergent glutamatergic calcium responses.

Rachelle Balez, Claire H Stevens, Kerstin Lenk, Kuldip Sidhu, Greg Sutherland, Lezanne Ooi.

Nitrosative stress is a feature of Alzheimer's disease, however the underlying cause(s) driving nitrosative stress and the impact of nitric oxide (NO) on neuronal function in Alzheimer's disease is still largely unknown. We analysed neuronal nitric oxide synthase (nNOS) protein levels in post mortem tissue and induced pluripotent stem cell (iPSC) derived neurons from Alzheimer's patients and controls by immunohistochemistry and western blots. Furthermore, we assessed the impact of modulating nNOS function or NO levels on neuronal glutamatergic signalling using calcium imaging. We show that nNOS protein levels are increased in early and severely affected brain regions of Alzheimer's disease post mortem tissue, but not late and mildly affected regions, or cognitively normal individuals. The increased nNOS phenotype was also present in iPSC-derived neurons from late-onset Alzheimer's disease (LOAD) patients compared to controls, along with increased levels of nitrite, a stable marker of NO. We observed a divergent functional impact of NO that included strengthening the calcium response in control neurons, while dysregulating calcium signaling and altering the amplitude and kinetics of the calcium responses to glutamate in the Alzheimer's disease neurons. Pharmacological scavenging of NO or inhibition of nNOS prevented aberrant spontaneous calcium signalling in Alzheimer's disease neurons. Together these data identify increases in nNOS protein in Alzheimer's disease. Functional data suggest NO modulation of glutamatergic calcium signaling is neuroprotective under non-pathogenic conditions, with increased nNOS and NO contributing to dysregulated spontaneous calcium signaling in Alzheimer's disease neurons.

DOI: https://doi.org/10.1089/ars.2023.0395
Int J Stem Cells. 2024 Jan 29.

Inducing Pluripotency in Somatic Cells: Historical Perspective and Recent Advances.

Junmyeong Park, Jueun Kim, Borami Shin, Hans R Schöler, Johnny Kim, Kee-Pyo Kim.

  Inducing pluripotency in somatic cells is mediated by the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc. The resulting induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine by virtue of their ability to differentiate into different types of functional cells. Specifically, iPSCs derived directly from patients offer a powerful platform for creating in vitro disease models. This facilitates elucidation of pathological mechanisms underlying human diseases and development of new therapeutic agents mitigating disease phenotypes. Furthermore, genetically and phenotypically corrected patient-derived iPSCs by gene-editing technology or the supply of specific pharmaceutical agents can be used for preclinical and clinical trials to investigate their therapeutic potential. Despite great advances in developing reprogramming methods, the efficiency of iPSC generation remains still low and varies between donor cell types, hampering the potential application of iPSC technology. This paper reviews histological timeline showing important discoveries that have led to iPSC generation and discusses recent advances in iPSC technology by highlighting donor cell types employed for iPSC generation.

Keywords:  Cellular reprogramming; Embryonic stem cells; Induced pluripotent stem cells; Octamer-binding transcription factor 4 (OCT4); Pluripotency

DOI:  https://doi.org/10.15283/ijsc23148
Curr Biol. 2024 Jan 24. pii: S0960-9822(24)00026-5. [Epub ahead of print]

The synaptic drive of central pattern-generating networks to leg motor neurons of a walking insect is motor neuron pool specific.

Angelina Ruthe, Charalampos Mantziaris, Ansgar Büschges.

  Rhythmic locomotor activity, such as flying, swimming, or walking, results from an interplay between higher-order centers in the central nervous system, which initiate, maintain, and modify task-specific motor activity, downstream central pattern-generating neural circuits (CPGs) that can generate a default rhythmic motor output, and, finally, feedback from sense organs that modify basic motor activity toward functionality.1,2,3 In this context, CPGs provide phasic synaptic drive to motor neurons (MNs) and thereby support the generation of rhythmic activity for locomotion. We analyzed the synaptic drive that the leg MNs supplying the three main leg joints receive from CPGs in pharmacologically activated and deafferented preparations of the stick insect (Carausius morosus). We show that premotor CPGs pattern the tonic activity of five of the six leg MN pools by phasic inhibitory synaptic drive. These are the antagonistic MN pools supplying the thoraco-coxal joint and the femur-tibial joint4,5 and the levator MN pool supplying the coxa-trochanteral (CTr) joint. In contrast, rhythmic activity of the depressor MN pool supplying the CTr joint was found to be primarily based on a phasic excitatory drive. This difference is likely related to the pivotal role of the depressor muscle in generating leg stance during any walking situation. Thus, our results provide evidence for qualitatively differing mechanisms to generate rhythmic activity between MN pools in the same locomotor system.

Keywords:  CPG; locomotion; motor control; motor neurons; pattern generation; walking

DOI:  https://doi.org/10.1016/j.cub.2024.01.026
Heliyon. 2024 Jan 30. 10(2): e24672

Validation of a newly developed immunoassay for TDP-43 in human plasma.

Sayo Matsuura, Harutsugu Tatebe, Makoto Higuchi, Takahiko Tokuda.

  The level of TAR DNA-binding protein 43 (TDP-43) in human blood was reported to have potential for use as a specific fluid biomarker, which represents disease-specific pathologies, for TDP-43 proteinopathies, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), which involves the aggregation and deposition of TDP-43 in the nervous system. However, at present, no reliable immunoassay can precisely quantify TDP-43 in human plasma and detect the difference in plasma TDP-43 levels between patients with ALS and controls. We recently developed a novel ultrasensitive immunoassay to quantify TDP-43 in human plasma, and in this study, we analytically validated this assay for application as a diagnostic biomarker for TDP-43 proteinopathies. The novel TDP-43 assay was assessed for the limit of detection, lower limit of quantification, intra- and interassay variation, linearity, parallelism, and analytical spike recoveries. Additionally, 17 pilot plasma samples obtained from patients with ALS and age-matched controls were analyzed using the assay. Our novel TDP-43 assay showed sufficient analytical performance to quantify TDP-43 in human plasma, with high sensitivity (LOD and LLOQ of 0.109 and 0.759 pg/mL, respectively) and high intra- and interassay precision (%CV) below 15 %. The experimental results for spike recovery, parallelism, and dilution linearity were also acceptable. In addition, despite a small sample size, significant differences in the plasma levels of TDP-43 were found between patients with ALS and controls (ALS, 66.63 ± 20.52 pg/mL; control, 42.70 ± 23.06 pg/mL, p = 0.0330). These results support that our novel TDP-43 assay is a reliable and innovative method for the quantification of TDP-43 in human plasma and can be a potential blood-based biomarker for the diagnosis of TDP-43 proteinopathies. Further large-scale studies are warranted to validate its usefulness.

Keywords:  Amyotrophic lateral sclerosis; Immunoassay; Plasma biomarker; TDP-43; Validation

DOI:  https://doi.org/10.1016/j.heliyon.2024.e24672
Biophys Chem. 2024 Jan 23. pii: S0301-4622(24)00020-6. [Epub ahead of print]307 107191

Genetically encoded lysine photocage for spatiotemporal control of TDP-43 nuclear import.

Jared A Shadish, Jennifer C Lee.

  Intracellular aggregation of transactive response DNA binding protein of 43 kDa (TDP-43) is a hallmark of neurodegenerative diseases such as amyotrophic lateral sclerosis. While primarily a nuclear protein, TDP-43 translocates to the cytosol during cellular stress. Consequences of cytosolic accumulation of TDP-43 is difficult to evaluate in the absence of exogenous toxins. Here, we demonstrate spatiotemporal control over the nuclear import of TDP-43 by installing a photocage (ortho-nitrobenzyl ester) on a single lysine residue (K84) through amber codon suppression in HEK293T cells. Translocation of this cytosolic construct is photo-triggerable in a dose-dependent manner with 355 nm light. Interestingly, both fluid- and solid-like puncta were found based on fluorescence recovery after photobleaching experiments, similar to what is expected of stress granules and intracellular aggregates, respectively. This optogenetic method is advantageous as it is minimally perturbative and broadly applicable to other studies of protein translocation between cellular compartments.

Keywords:  Amyotrophic lateral sclerosis; FRAP; O-nitrobenzyl-oxycarbonyl-Nε-l-lysine; Optogenetics; Stress granules

DOI:  https://doi.org/10.1016/j.bpc.2024.107191
Mol Neurodegener. 2024 Jan 29. 19(1): 13

NMNAT2 supports vesicular glycolysis via NAD homeostasis to fuel fast axonal transport.

Sen Yang, Zhen-Xian Niou, Andrea Enriquez, Jacob LaMar, Jui-Yen Huang, Karen Ling, Paymaan Jafar-Nejad, Jonathan Gilley, Michael P Coleman, Jason M Tennessen, Vidhya Rangaraju, Hui-Chen Lu.

BACKGROUND: Bioenergetic maladaptations and axonopathy are often found in the early stages of neurodegeneration. Nicotinamide adenine dinucleotide (NAD), an essential cofactor for energy metabolism, is mainly synthesized by Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) in CNS neurons. NMNAT2 mRNA levels are reduced in the brains of Alzheimer's, Parkinson's, and Huntington's disease. Here we addressed whether NMNAT2 is required for axonal health of cortical glutamatergic neurons, whose long-projecting axons are often vulnerable in neurodegenerative conditions. We also tested if NMNAT2 maintains axonal health by ensuring axonal ATP levels for axonal transport, critical for axonal function.
METHODS: We generated mouse and cultured neuron models to determine the impact of NMNAT2 loss from cortical glutamatergic neurons on axonal transport, energetic metabolism, and morphological integrity. In addition, we determined if exogenous NAD supplementation or inhibiting a NAD hydrolase, sterile alpha and TIR motif-containing protein 1 (SARM1), prevented axonal deficits caused by NMNAT2 loss. This study used a combination of techniques, including genetics, molecular biology, immunohistochemistry, biochemistry, fluorescent time-lapse imaging, live imaging with optical sensors, and anti-sense oligos.
RESULTS: We provide in vivo evidence that NMNAT2 in glutamatergic neurons is required for axonal survival. Using in vivo and in vitro studies, we demonstrate that NMNAT2 maintains the NAD-redox potential to provide "on-board" ATP via glycolysis to vesicular cargos in distal axons. Exogenous NAD+ supplementation to NMNAT2 KO neurons restores glycolysis and resumes fast axonal transport. Finally, we demonstrate both in vitro and in vivo that reducing the activity of SARM1, an NAD degradation enzyme, can reduce axonal transport deficits and suppress axon degeneration in NMNAT2 KO neurons.
CONCLUSION: NMNAT2 ensures axonal health by maintaining NAD redox potential in distal axons to ensure efficient vesicular glycolysis required for fast axonal transport.

DOI: https://doi.org/10.1186/s13024-023-00690-9
J Clin Invest. 2024 Feb 01. pii: e176736. [Epub ahead of print]134(3):

Molecular and cellular mechanisms underlying the failure of mitochondrial metabolism drugs in cancer clinical trials.

Karthik Vasan, Navdeep S Chandel.

DOI: https://doi.org/10.1172/JCI176736
bioRxiv. 2024 Jan 18. pii: 2024.01.17.576134. [Epub ahead of print]

Modeling and Correction of Protein Conformational Disease in iPSC-derived Neurons through Personalized Base Editing.

Colin T Konishi, Nancy Moulayes, Tanvi Butola, Vincent Zhang, Dana Kagan, Qiaoyan Yang, Mariel Pressler, Brooke G Dirvin, Orrin Devinsky, Jayeeta Basu, Chengzu Long.

Altered protein conformation can cause incurable neurodegenerative disorders. Mutations in SERPINI1 , the gene encoding neuroserpin, alter protein conformation resulting in cytotoxic aggregation in neuronal endoplasmic reticulum. Aggregates cause oxidative stress impairing function, leading to neuronal death. Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a rare autosomal dominant progressive myoclonic epilepsy. Patients present with seizures and cognitive impairments that progress to dementia and premature death. We developed HEK293T and induced pluripotent stem cell (iPSC) models of FENIB, harboring the patient's pathogenic SERPINI1 variant or stably overexpressing mutant neuroserpin fused to GFP (MUT NS-GFP). FENIB cells form neuroserpin inclusions which increase in size and number. Here, we utilized a personalized adenine base editor (ABE)-mediated approach to efficiently correct the pathogenic variant and to restore neuronal dendritic morphology. ABE-treated MUT NS-GFP cells demonstrated reduced inclusion size and number. Using an inducible MUT NS-GFP neuron system, we identified early prevention of toxic protein expression allowed aggregate clearance, while late prevention halted neuronal impairments. To address several challenges for clinical applications of gene correction, we developed a neuron-specific engineered virus-like particle to optimize neuronal ABE delivery. Preventing mutant protein with altered conformation production improved toxic protein clearance. Our findings provide a targeted strategy and may treat FENIB and potentially other neurodegenerative diseases due to altered protein conformation such as Alzheimer's and Huntington's diseases.

DOI: https://doi.org/10.1101/2024.01.17.576134
Nat Commun. 2024 Jan 31. 15(1): 919

DENND6A links Arl8b to a Rab34/RILP/dynein complex, regulating lysosomal positioning and autophagy.

Rahul Kumar, Maleeha Khan, Vincent Francis, Adriana Aguila, Gopinath Kulasekaran, Emily Banks, Peter S McPherson.

Lysosomes help maintain cellular proteostasis, and defects in lysosomal positioning and function can cause disease, including neurodegenerative disorders. The spatiotemporal distribution of lysosomes is regulated by small GTPases including Rabs, which are activated by guanine nucleotide exchange factors (GEFs). DENN domain proteins are the largest family of Rab GEFs. Using a cell-based assay, we screened DENND6A, a member of the DENN domain protein family against all known Rabs and identified it as a potential GEF for 20 Rabs, including Rab34. Here, we demonstrate that DENND6A activates Rab34, which recruits a RILP/dynein complex to lysosomes, promoting lysosome retrograde transport. Further, we identify DENND6A as an effector of Arl8b, a major regulatory GTPase on lysosomes. We demonstrate that Arl8b recruits DENND6A to peripheral lysosomes to activate Rab34 and initiate retrograde transport, regulating nutrient-dependent lysosomal juxtanuclear repositioning. Loss of DENND6A impairs autophagic flux. Our findings support a model whereby Arl8b/DENND6A/Rab34-dependent lysosomal retrograde trafficking controls autophagy.

DOI: https://doi.org/10.1038/s41467-024-44957-1
bioRxiv. 2024 Jan 20. pii: 2024.01.20.576435. [Epub ahead of print]

Axonal distribution of mitochondria maintains neuronal autophagy during aging via eIF2β.

Kanako Shinno, Yuri Miura, Koichi M Iijima, Emiko Suzuki, Kanae Ando.

Neuronal aging and neurodegenerative diseases are accompanied by proteostasis collapse, while cellular factors that trigger it are not identified. Impaired mitochondrial transport in the axon is another feature of aging and neurodegenerative diseases. Using Drosophila , we found that genetic depletion of axonal mitochondria causes dysregulation of translation and protein degradation. Axons with mitochondrial depletion showed abnormal protein accumulation, and autophagic defects. Lowering neuronal ATP levels by blocking glycolysis did not reduce autophagy, suggesting that autophagic defects are associated with mitochondrial distribution. We found eIF2β was upregulated by depletion of axonal mitochondria via proteome analysis. Phosphorylation of eIF2α, another subunit of eIF2, was lowered, and global translation was suppressed. Neuronal overexpression of eIF2β phenocopied the autophagic defects and neuronal dysfunctions, and lowering eIF2β expression rescued those perturbations caused by depletion of axonal mitochondria. These results indicate the mitochondria-eIF2β axis maintains proteostasis in the axon, of which disruption may underly the onset and progression of age-related neurodegenerative diseases.
Highlights: Loss of axonal mitochondria impairs autophagy and accumulates proteins in the axonLoss of axonal mitochondria upregulates eIF2β and downregulates p-eIF2αNeuronal upregulation of eIF2β induces autophagic defects and locomotor dysfunctionLowering eIF2β rescues autophagic defects caused by loss of axonal mitochondria.

DOI: https://doi.org/10.1101/2024.01.20.576435
J Cell Biol. 2024 Mar 04. pii: e202308083. [Epub ahead of print]223(3):

Identification of small molecule inhibitors of G3BP-driven stress granule formation.

Brian D Freibaum, James Messing, Haruko Nakamura, Ugur Yurtsever, Jinjun Wu, Hong Joo Kim, Jeff Hixon, Rene Marc Lemieux, Jay Duffner, Walter Huynh, Kathy Wong, Michael White, Christina Lee, Rachel E Meyers, Roy Parker, J Paul Taylor.

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the RNA-binding proteins G3BP1/2. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting the co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress and dissolve pre-existing stress granules. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent powerful tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.

DOI: https://doi.org/10.1083/jcb.202308083
Regen Ther. 2024 Mar;25 250-263

Estradiol enhanced neuronal plasticity and ameliorated astrogliosis in human iPSC-derived neural models.

Sopak Supakul, Chisato Oyama, Yuki Hatakeyama, Sumihiro Maeda, Hideyuki Okano.

   Introduction: 17β-Estradiol (E2) is a sex hormone that has been previously demonstrated to have neurotherapeutic effects on animal models of Alzheimer's disease (AD). However, clinical trials on E2 replacement therapy for preventing AD onset yielded inconsistent results. Therefore, it is imperative to clarify the therapeutic effects of E2 on human cells. In this study, we utilized induced pluripotent stem cells (iPSCs) derived from multiple AD donors to explore the therapeutic effects of E2 on the in vitro model of human cells.
Methods: We conducted a systematic review and meta-analysis using a random-effects model of the previously reported AD clinical trials to summarize the effects of E2 replacement therapy on AD prevention. Subsequently, we induced iPSCs from the donors of the healthy control (1210B2 line (female) and 201B7 line (female)), the familial AD (APP V717L line (female) and APP KM670/671NL line (female)), and the sporadic AD (UCSD-SAD3.7 line (APOE ε3/ε3) (male), UCSD-SAD7D line (APOE ε3/ε4) (male), and TMGH-1 line (APOE ε3/ε3) (female)), then differentiated to neurons. In addition to the mono-culture model of the neurons, we also examined the effects of E2 on the co-culture model of neurons and astrocytes.
Results: The meta-analysis of the clinical trials concluded that E2 replacement therapy reduced the risk of AD onset (OR, 0.69; 95 % confidence interval [CI], 0.53-0.91; I2 = 82 %). Neural models from the iPSCs of AD donors showed an increase in secreted amyloid-beta (Aβ) levels in the mono-culture model and an astrogliosis-like phenotype in the co-culture model. E2 treatment to the neuronal models derived from the iPSCs enhanced neuronal activity and increased neurite complexity. Furthermore, E2 treatment of the co-culture model ameliorated the astrogliosis-like phenotype. However, in contrast to the previous reports using mouse models, E2 treatment did not change AD pathogenesis, including Aβ secretion and phosphorylated tau (pTau) accumulation.
Conclusion: E2 treatment of the human cellular model did not impact Aβ secretion and pTau accumulation, but promoted neuronal plasticity and alleviated the astrogliosis-like phenotype. The limited effects of E2 may give a clue for the mixed results of E2 clinical trials.

Keywords:  17β-estradiol; Alzheimer's disease; Astrocytes; Induced pluripotent stem cells (iPSCs); Neurons

DOI:  https://doi.org/10.1016/j.reth.2023.12.018
iScience. 2024 Feb 16. 27(2): 108795

Maintaining Toll signaling in Drosophila brain is required to sustain autophagy for dopamine neuron survival.

Jie Zhang, Ting Tang, Ruonan Zhang, Liang Wen, Xiaojuan Deng, Xiaoxia Xu, Wanying Yang, Fengliang Jin, Yang Cao, Yuzhen Lu, Xiao-Qiang Yu.

  Macroautophagy/autophagy is a conserved process in eukaryotic cells to degrade and recycle damaged intracellular components. Higher level of autophagy in the brain has been observed, and autophagy dysfunction has an impact on neuronal health, but the molecular mechanism is unclear. In this study, we showed that overexpression of Toll-1 and Toll-7 receptors, as well as active Spätzle proteins in Drosophila S2 cells enhanced autophagy, and Toll-1/Toll-7 activated autophagy was dependent on Tube-Pelle-PP2A. Interestingly, Toll-1 but not Toll-7 mediated autophagy was dMyd88 dependent. Importantly, we observed that loss of functions in Toll-1 and Toll-7 receptors and PP2A activity in flies decreased autophagy level, resulting in the loss of dopamine (DA) neurons and reduced fly motion. Our results indicated that proper activation of Toll-1 and Toll-7 pathways and PP2A activity in the brain are necessary to sustain autophagy level for DA neuron survival.

Keywords:  Cell biology; Neuroscience

DOI:  https://doi.org/10.1016/j.isci.2024.108795