bims-axbals 2024-08-04 papers

bims-axbals

Biomed News

on Axonal Biology and ALS

Issue of 2024‒08‒04
34 papers selected by
TJ Krzystek, ALS Therapy Development Institute

EVolution in ALS diagnosis: molecular markers in extracellular vesicles.
Early nuclear phenotypes and reactive transformation in human iPSC-derived astrocytes from ALS patients with SOD1 mutations.
Inflammasome-Inhibiting Nanoligomers Are Neuroprotective against Space-Induced Pathology in Healthy and Diseased Three-Dimensional Human Motor and Prefrontal Cortex Brain Organoids.
Mechanisms of lysosomal tubulation and sorting driven by LRRK2.
Why should we care about astrocytes in a motor neuron disease?
A genetic-epigenetic interplay at 1q21.1 locus underlies CHD1L-mediated vulnerability to primary progressive multiple sclerosis.
Increased copy-number variant load of associated risk genes in sporadic cases of amyotrophic lateral sclerosis.
Excitatory to inhibitory synaptic ratios are unchanged at presymptomatic stages in multiple models of ALS.
Potential contribution of spinal interneurons to the etiopathogenesis of amyotrophic lateral sclerosis.
TDP-43 proteinopathy in frontotemporal lobar degeneration and amyotrophic lateral sclerosis: From pathomechanisms to therapeutic strategies.
Extracellular Vesicles from hiPSC-derived NSCs Protect Human Neurons against Aβ-42 Oligomers Induced Neurodegeneration, Mitochondrial Dysfunction and Tau Phosphorylation.
tRNA Arg binds in vitro TDP-43 RNA recognition motifs and ligand of Ate1 protein LIAT1.
Phagocytosis of aggrecan-positive perineuronal nets surrounding motor neurons by reactive microglia expressing MMP-9 in TDP-43Q331K ALS model mice.
Isogenic patient-derived organoids reveal early neurodevelopmental defects in spinal muscular atrophy initiation.
Loss of primary cilia and dopaminergic neuroprotection in pathogenic LRRK2-driven and idiopathic Parkinson's disease.
Human iPSC-derived myelinating organoids and globoid cells to study Krabbe Disease.
Electroacupuncture alleviates motor dysfunction by regulating neuromuscular junction disruption and neuronal degeneration in SOD1G93A mice.
Motor Cortical Neuronal Hyperexcitability Associated with α-Synuclein Aggregation.
Huntingtin contains an ubiquitin-binding domain and regulates lysosomal targeting of mitochondrial and RNA-binding proteins.
Generation of a gene-corrected human isogenic iPSC line from an Alzheimer's disease iPSC line carrying the PSEN1 H163R mutation.
Visualizing traumatic brain injury: ocular clues for diagnosis and assessment.
Loss of MEF2C function by enhancer mutation leads to neuronal mitochondria dysfunction and motor deficits in mice.
Fluorescent Nanocable as a Biomedical Tool: Intracellular Self-Assembly Formed by a Natural Product Interconnects and Synchronizes Mitochondria.
RAB12-LRRK2 Complex Suppresses Primary Ciliogenesis and Regulates Centrosome Homeostasis in Astrocytes.
Drosophila larval motor patterning relies on regulated alternative splicing of Dscam2.
A functional aged human iPSC-cortical neuron model recapitulates Alzheimer's disease, senescence, and the response to therapeutics.
Identification of novel neuroprotectants against vincristine-induced neurotoxicity in iPSC-derived neurons.
Increase of HCN current in SOD1-associated amyotrophic lateral sclerosis.
Synergistic effect of PAK and Hippo pathway inhibitor combination in NF2-deficient Schwannoma.
Efficient fabrication of 3D bioprinted functional sensory neurons using an inducible Neurogenin-2 human pluripotent stem cell line.
Differential roles of lysosomal cholesterol transporters in the development of C. elegans NMJs.
HaloTag as a substrate-based macroautophagy reporter.
Novel Imaging Tools to Study Mitochondrial Dynamics in Caenorhabditis elegans.
Rapid iPSC inclusionopathy models shed light on formation, consequence, and molecular subtype of α-synuclein inclusions.

Trends Mol Med. 2024 Jul 27. pii: S1471-4914(24)00192-8. [Epub ahead of print]

EVolution in ALS diagnosis: molecular markers in extracellular vesicles.

Philippe Codron, Stéphanie Millecamps, Philippe Corcia.

  The identification of biomarkers for amyotrophic lateral sclerosis (ALS) is a central issue in disease research. In a recent article, Chatterjee et al. show that blood extracellular vesicles (EVs) with high levels of transactive response DNA-binding protein 43 (TDP-43) accurately discriminate patients with ALS from controls and correlate with disease severity, providing a promising biomarker for early diagnosis and monitoring.

Keywords:  TDP-43; amyotrophic lateral sclerosis; biomarker; cryptic exons; extracellular vesicles

DOI:  https://doi.org/10.1016/j.molmed.2024.07.006
Glia. 2024 Aug 02.

Early nuclear phenotypes and reactive transformation in human iPSC-derived astrocytes from ALS patients with SOD1 mutations.

Vincent Soubannier, Mathilde Chaineau, Lale Gursu, Sarah Lépine, David Kalaydjian, Julien Sirois, Ghazal Haghi, Guy Rouleau, Thomas M Durcan, Stefano Stifani.

  Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive death of motor neurons (MNs). Glial cells play roles in MN degeneration in ALS. More specifically, astrocytes with mutations in the ALS-associated gene Cu/Zn superoxide dismutase 1 (SOD1) promote MN death. The mechanisms by which SOD1-mutated astrocytes reduce MN survival are incompletely understood. To characterize the impact of SOD1 mutations on astrocyte physiology, we generated astrocytes from human induced pluripotent stem cell (iPSC) derived from ALS patients carrying SOD1 mutations, together with control isogenic iPSCs. We report that astrocytes harboring SOD1(A4V) and SOD1(D90A) mutations exhibit molecular and morphological changes indicative of reactive astrogliosis when compared to isogenic astrocytes. We show further that a number of nuclear phenotypes precede, or coincide with, reactive transformation. These include increased nuclear oxidative stress and DNA damage, and accumulation of the SOD1 protein in the nucleus. These findings reveal early cell-autonomous phenotypes in SOD1-mutated astrocytes that may contribute to the acquisition of a reactive phenotype involved in alterations of astrocyte-MN communication in ALS.

Keywords:  Cu/Zn superoxide dismutase 1; DNA damage; amyotrophic lateral sclerosis; astrocyte; nuclear oxidative stress; reactive astrogliosis

DOI:  https://doi.org/10.1002/glia.24598
ACS Chem Neurosci. 2024 Jul 31.

Inflammasome-Inhibiting Nanoligomers Are Neuroprotective against Space-Induced Pathology in Healthy and Diseased Three-Dimensional Human Motor and Prefrontal Cortex Brain Organoids.

Sadhana Sharma, Vincenzo S Gilberto, Jon Rask, Anushree Chatterjee, Prashant Nagpal.

  The microgravity and space environment has been linked to deficits in neuromuscular and cognitive capabilities, hypothesized to occur due to accelerated aging and neurodegeneration in space. While the specific mechanisms are still being investigated, spaceflight-associated neuropathology is an important health risk to astronauts and space tourists and is being actively investigated for the development of appropriate countermeasures. However, such space-induced neuropathology offers an opportunity for accelerated screening of therapeutic targets and lead molecules for treating neurodegenerative diseases. Here, we show a proof-of-concept high-throughput target screening (on Earth), target validation, and mitigation of microgravity-induced neuropathology using our Nanoligomer platform, onboard the 43-day SpaceX CRS-29 mission to the International Space Station. First, comparing 3D healthy and diseased prefrontal cortex (PFC, for cognition) and motor neuron (MN, for neuromuscular function) organoids, we assessed space-induced pathology using biomarkers relevant to Alzheimer's disease (AD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS). Both healthy and diseased PFC and MN organoids showed significantly enhanced neurodegeneration in space, as measured through relevant disease biomarkers, when compared to their respective Earth controls. Second, we tested the top two lead molecules, NI112 that targeted NF-κB and NI113 that targeted IL-6. We observed that these Nanoligomers significantly mitigate the AD, FTD, and ALS relevant biomarkers like amyloid beta-42 (Aβ42), phosphorylated tau (pTau), Kallikrein (KLK-6), Tar DNA-binding protein 43 (TDP-43), and others. Moreover, the 43-day Nanoligomer treatment of these brain organoids did not appear to cause any observable toxicity or safety issues in the target organoid tissue, suggesting good tolerability for these molecules in the brain at physiologically relevant doses. Together, these results show significant potential for both the development and translation of NI112 and NI113 molecules as potential neuroprotective countermeasures for safer space travel and demonstrate the usefulness of the space environment for rapid, high-throughput screening of targets and lead molecules for clinical translation. We assert that the use of microgravity in drug development and screening may ultimately benefit millions of patients suffering from debilitating neurodegenerative diseases on Earth.

Keywords:  3D human organoids; Alzheimer’s disease (AD); International Space Station; Nanoligomer; amyotrophic lateral sclerosis (ALS); brain organoids; microgravity; space biology; space-induced neurodegeneration

DOI:  https://doi.org/10.1021/acschemneuro.4c00160
Biochem Soc Trans. 2024 Jul 31. pii: BST20240087. [Epub ahead of print]

Mechanisms of lysosomal tubulation and sorting driven by LRRK2.

Luis Bonet-Ponce, Jillian H Kluss, Mark R Cookson.

  Lysosomes are dynamic cellular structures that adaptively remodel their membrane in response to stimuli, including membrane damage. Lysosomal dysfunction plays a central role in the pathobiology of Parkinson's disease (PD). Gain-of-function mutations in Leucine-rich repeat kinase 2 (LRRK2) cause familial PD and genetic variations in its locus increase the risk of developing the sporadic form of the disease. We previously uncovered a process we term LYTL (LYsosomal Tubulation/sorting driven by LRRK2), wherein membrane-damaged lysosomes generate tubules sorted into mobile vesicles. Subsequently, these vesicles interact with healthy lysosomes. LYTL is orchestrated by LRRK2 kinase activity, via the recruitment and phosphorylation of a subset of RAB GTPases. Here, we summarize the current understanding of LYTL and its regulation, as well as the unknown aspects of this process.

Keywords:  Parkinson's disease; RAB proteins; leucine-rich repeat kinase

DOI:  https://doi.org/10.1042/BST20240087
Front Mol Med. 2023 ;3 1047540

Why should we care about astrocytes in a motor neuron disease?

Katarina Stoklund Dittlau, Ludo Van Den Bosch.

  Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease in adults, causing progressive degeneration of motor neurons, which results in muscle atrophy, respiratory failure and ultimately death of the patients. The pathogenesis of ALS is complex, and extensive efforts have focused on unravelling the underlying molecular mechanisms with a large emphasis on the dying motor neurons. However, a recent shift in focus towards the supporting glial population has revealed a large contribution and influence in ALS, which stresses the need to explore this area in more detail. Especially studies into astrocytes, the residential homeostatic supporter cells of neurons, have revealed a remarkable astrocytic dysfunction in ALS, and therefore could present a target for new and promising therapeutic entry points. In this review, we provide an overview of general astrocyte function and summarize the current literature on the role of astrocytes in ALS by categorizing the potentially underlying molecular mechanisms. We discuss the current efforts in astrocyte-targeted therapy, and highlight the potential and shortcomings of available models.

Keywords:  amyotrophic lateral sclerosis; disease mechanism; disease model; gain-of-toxicity; induced pluripotent stem cell; loss-of-support

DOI:  https://doi.org/10.3389/fmmed.2023.1047540
Nat Commun. 2024 Jul 30. 15(1): 6419

A genetic-epigenetic interplay at 1q21.1 locus underlies CHD1L-mediated vulnerability to primary progressive multiple sclerosis.

Majid Pahlevan Kakhki, Antonino Giordano, Chiara Starvaggi Cucuzza, Tejaswi Venkata S Badam, Samudyata Samudyata, Marianne Victoria Lemée, Pernilla Stridh, Asimenia Gkogka, Klementy Shchetynsky, Adil Harroud, Alexandra Gyllenberg, Yun Liu, Sanjaykumar Boddul, Tojo James, Melissa Sorosina, Massimo Filippi, Federica Esposito, Fredrik Wermeling, Mika Gustafsson, Patrizia Casaccia, Jan Hillert, Tomas Olsson, Ingrid Kockum, Carl M Sellgren, Christelle Golzio, Lara Kular, Maja Jagodic.

Multiple Sclerosis (MS) is a heterogeneous inflammatory and neurodegenerative disease with an unpredictable course towards progressive disability. Treating progressive MS is challenging due to limited insights into the underlying mechanisms. We examined the molecular changes associated with primary progressive MS (PPMS) using a cross-tissue (blood and post-mortem brain) and multilayered data (genetic, epigenetic, transcriptomic) from independent cohorts. In PPMS, we found hypermethylation of the 1q21.1 locus, controlled by PPMS-specific genetic variations and influencing the expression of proximal genes (CHD1L, PRKAB2) in the brain. Evidence from reporter assay and CRISPR/dCas9 experiments supports a causal link between methylation and expression and correlation network analysis further implicates these genes in PPMS brain processes. Knock-down of CHD1L in human iPSC-derived neurons and knock-out of chd1l in zebrafish led to developmental and functional deficits of neurons. Thus, several lines of evidence suggest a distinct genetic-epigenetic-transcriptional interplay in the 1q21.1 locus potentially contributing to PPMS pathogenesis.

DOI: https://doi.org/10.1038/s41467-024-50794-z
Cell Mol Life Sci. 2024 Jul 27. 81(1): 316

Increased copy-number variant load of associated risk genes in sporadic cases of amyotrophic lateral sclerosis.

Maria Guarnaccia, Giovanna Morello, Valentina La Cognata, Vincenzo La Bella, Francesca Luisa Conforti, Sebastiano Cavallaro.

  Amyotrophic lateral sclerosis (ALS) is an age-related neurodegenerative disease characterized by selective loss of motor neurons in the brainstem and spinal cord. Several genetic factors have been associated to ALS, ranging from causal genes and potential risk factors to disease modifiers. The search for pathogenic variants in these genes has mostly focused on single nucleotide variants (SNVs) while relatively understudied and not fully elucidated is the contribution of structural variants, such as copy number variations (CNVs). Here, we applied an exon-centric aCGH method to investigate, in sporadic ALS patients, the load of CNVs in 131 genes previously associated to ALS. Our approach revealed that CNV load, defined as the total number of CNVs or their size, was significantly higher in ALS cases than controls. About 87% of patients harbored multiple CNVs in ALS-related genes, and 75% structural variants compromised genes directly implicated in ALS pathogenesis (C9orf72, CHCHD10, EPHA4, FUS, HNRNPA1, KIF5A, NEK1, OPTN, PFN1, SOD1, TARDBP, TBK1, UBQLN2, UNC13A, VAPB, VCP). CNV load was also associated to higher onset age and disease progression rate. Although the contribution of individual CNVs in ALS is still unknown, their extensive load in disease-related genes may have relevant implications for the diagnostic, prognostic and therapeutical management of this devastating disorder.

Keywords:  Amyotrophic lateral sclerosis; CNVs load; Copy number variant; Customized aCGH; Diagnostics

DOI:  https://doi.org/10.1007/s00018-024-05335-8
PLoS One. 2024 ;19(8): e0306423

Excitatory to inhibitory synaptic ratios are unchanged at presymptomatic stages in multiple models of ALS.

Calum Bonthron, Sarah Burley, Matthew J Broadhead, Vanya Metodieva, Seth G N Grant, Siddharthan Chandran, Gareth B Miles.

Hyperexcitability of motor neurons and spinal cord motor circuitry has been widely reported in the early stages of Amyotrophic Lateral Sclerosis (ALS). Changes in the relative amount of excitatory to inhibitory inputs onto a neuron (E:I synaptic ratio), possibly through a developmental shift in synapse formation in favour of excitatory transmission, could underlie pathological hyperexcitability. Given that astrocytes play a major role in early synaptogenesis and are implicated in ALS pathogenesis, their potential contribution to disease mechanisms involving synaptic imbalances and subsequent hyperexcitability is also of great interest. In order to assess E:I ratios in ALS, we utilised a novel primary spinal neuron / astrocyte co-culture system, derived from neonatal mice, in which synapses are formed in vitro. Using multiple ALS mouse models we found that no combination of astrocyte or neuron genotype produced alterations in E:I synaptic ratios assessed using pre- and post-synaptic anatomical markers. Similarly, we observed that ephrin-B1, a major contact-dependent astrocytic synaptogenic protein, was not differentially expressed by ALS primary astrocytes. Further to this, analysis of E:I ratios across the entire grey matter of the lumbar spinal cord in young (post-natal day 16-19) ALS mice revealed no differences versus controls. Finally, analysis in co-cultures of human iPSC-derived motor neurons and astrocytes harbouring the pathogenic C9orf72 hexanucleotide repeat expansion showed no evidence of a bias toward excitatory versus inhibitory synapse formation. We therefore conclude, utilising multiple ALS models, that we do not observe significant changes in the relative abundance of excitatory versus inhibitory synapses as would be expected if imbalances in synaptic inputs contribute to early hyperexcitability.

DOI: https://doi.org/10.1371/journal.pone.0306423
Front Neurosci. 2024 ;18 1434404

Potential contribution of spinal interneurons to the etiopathogenesis of amyotrophic lateral sclerosis.

Luca Goffin, Damien Lemoine, Frédéric Clotman.

  Amyotrophic lateral sclerosis (ALS) consists of a group of adult-onset fatal and incurable neurodegenerative disorders characterized by the progressive death of motor neurons (MNs) throughout the central nervous system (CNS). At first, ALS was considered to be an MN disease, caused by cell-autonomous mechanisms acting specifically in MNs. Accordingly, data from ALS patients and ALS animal models revealed alterations in excitability in multiple neuronal populations, including MNs, which were associated with a variety of cellular perturbations such as protein aggregation, ribonucleic acid (RNA) metabolism defects, calcium dyshomeostasis, modified electrophysiological properties, and autophagy malfunctions. However, experimental evidence rapidly demonstrated the involvement of other types of cells, including glial cells, in the etiopathogenesis of ALS through non-cell autonomous mechanisms. Surprisingly, the contribution of pre-motor interneurons (INs), which regulate MN activity and could therefore critically modulate their excitability at the onset or during the progression of the disease, has to date been severely underestimated. In this article, we review in detail how spinal pre-motor INs are affected in ALS and their possible involvement in the etiopathogenesis of the disease.

Keywords:  amyotrophic lateral sclerosis; motor neurons; neurodegenerative disease; non-cell autonomous mechanisms; pre-motor interneurons; spinal cord

DOI:  https://doi.org/10.3389/fnins.2024.1434404
Ageing Res Rev. 2024 Jul 27. pii: S1568-1637(24)00259-9. [Epub ahead of print]100 102441

TDP-43 proteinopathy in frontotemporal lobar degeneration and amyotrophic lateral sclerosis: From pathomechanisms to therapeutic strategies.

Pei-Chuan Ho, Tsung-Chi Hsieh, Kuen-Jer Tsai.

  Proteostasis failure is a common pathological characteristic in neurodegenerative diseases. Revitalizing clearance systems could effectively mitigate these diseases. The transactivation response (TAR) DNA-binding protein 43 (TDP-43) plays a critical role as an RNA/DNA-binding protein in RNA metabolism and synaptic function. Accumulation of TDP-43 aggregates in the central nervous system is a hallmark of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Autophagy, a major and highly conserved degradation pathway, holds the potential for degrading aggregated TDP-43 and alleviating FTLD/ALS. This review explores the causes of TDP-43 aggregation, FTLD/ALS-related genes, key autophagy factors, and autophagy-based therapeutic strategies targeting TDP-43 proteinopathy. Understanding the underlying pathological mechanisms of TDP-43 proteinopathy can facilitate therapeutic interventions.

Keywords:  Amyotrophic lateral sclerosis; Autophagy; Frontotemporal lobar degeneration; TDP-43

DOI:  https://doi.org/10.1016/j.arr.2024.102441
bioRxiv. 2024 Jul 16. pii: 2024.07.11.603159. [Epub ahead of print]

Extracellular Vesicles from hiPSC-derived NSCs Protect Human Neurons against Aβ-42 Oligomers Induced Neurodegeneration, Mitochondrial Dysfunction and Tau Phosphorylation.

Shama Rao, Leelavathi N Madhu, Roshni Sara Babu, Advaidhaa Nagarajan, Raghavendra Upadhya, Esha Narvekar, Ashok K Shetty.

Background: One of the hallmarks of Alzheimer's disease (AD) is the buildup of amyloid beta-42 (Aβ-42) in the brain, which leads to various adverse effects. Therefore, therapeutic interventions proficient in reducing Aβ-42-induced toxicity in AD are of great interest. One promising approach is to use extracellular vesicles from human induced pluripotent stem cell-derived neural stem cells (hiPSC-NSC-EVs) because they carry multiple therapeutic miRNAs and proteins capable of protecting neurons against Aβ-42-induced pathological changes. Therefore, this in vitro study investigated the proficiency of hiPSC-NSC-EVs to protect human neurons derived from two distinct hiPSC lines from Aβ-42o-induced neurodegeneration.Methods: We isolated hiPSC-NSC-EVs using chromatographic methods and characterized their size, ultrastructure, expression of EV-specific markers and proficiency in getting incorporated into mature human neurons. Next, mature human neurons differentiated from two different hiPSC lines were exposed to 1 µM Aβ-42 oligomers (Aβ-42o) alone or with varying concentrations of hiPSC-NSC-EVs. The protective effects of hiPSC-NSC-EVs against Aβ-42o-induced neurodegeneration, increased oxidative stress, mitochondrial dysfunction, impaired autophagy, and tau phosphorylation were ascertained using multiple measures and one-way ANOVA with Newman-Keuls multiple comparisons post hoc tests.
Results: Significant neurodegeneration was observed when human neurons were exposed to Aβ-42o alone. Notably, neurodegeneration was associated with elevated levels of oxidative stress markers malondialdehyde (MDA) and protein carbonyls (PCs), increased expression of proapoptotic Bax and Bad genes and proteins, reduced expression of the antiapoptotic gene and protein Bcl-2, increased expression of genes encoding mitochondrial complex proteins, decreased expression of autophagy-related proteins Beclin-1 and microtubule-associated protein 1 light chain 3B, and increased phosphorylation of tau. However, the addition of an optimal dose of hiPSC-NSC-EVs (6 x 10 9 EVs) to human neuronal cultures exposed to Aβ-42o significantly reduced the extent of neurodegeneration, along with diminished levels of MDA and PCs, normalized expressions of Bax, Bad, and Bcl-2, and genes linked to mitochondrial complex proteins, and reduced tau phosphorylation.
Conclusions: The findings demonstrate that an optimal dose of hiPSC-NSC-EVs could significantly decrease the degeneration of human neurons induced by Aβ-42o. The results also support further research into the effectiveness of hiPSC-NSC-EVs in AD, particularly their proficiency in preserving neurons and slowing disease progression.

DOI: https://doi.org/10.1101/2024.07.11.603159
MicroPubl Biol. 2024 ;2024

tRNA Arg binds in vitro TDP-43 RNA recognition motifs and ligand of Ate1 protein LIAT1.

Ljiljana Sjekloća, Emanuele Buratti.

Transactive response DNA-binding protein 43 (TDP-43) is important for RNA metabolism in all animals and its malfunctions are linked to neurodegenerative and myodegenerative diseases in humans. Arginyl transferase Ate1 transfers an arginyl group from arginylated tRNA Arg to proteolytic fragments of the C-terminal region of TDP-43, prompting their degradation by the ubiquitin proteasome system, thus contributing to TDP-43 proteostasis. To gain more insight into the molecular basis of TDP-43 arginylation, we tested if tRNA Arg could bind in vitro to a panel of recombinant multidomain constructs of human TDP-43 or to the arginylation cofactor protein LIAT1. We observed that in vitro- transcribed human tRNA Arg directly interacts with the RNA recognition motifs of TDP-43 and that their binding is stabilized by dimerization, which is promoted by the amino-terminal domain and the nuclear localization signal sequence of TDP-43. Moreover, the same human TDP-43 constructs that bind tRNA Arg bind native fungal tRNA Phe , suggesting that TDP-43 can bind different populations of tRNAs. Interestingly, human tRNA Arg is also able to bind recombinant mouse LIAT1 suggesting, for the first time, that LIAT1 is an RNA-binding protein. Our findings open a new perspective on the intricate crosstalk between protein and tRNA metabolism, which may eventually contribute to the understanding of the role of TDP-43 proteostasis in health and disease.

DOI: https://doi.org/10.17912/micropub.biology.001224
Neurobiol Dis. 2024 Jul 25. pii: S0969-9961(24)00214-6. [Epub ahead of print]200 106614

Phagocytosis of aggrecan-positive perineuronal nets surrounding motor neurons by reactive microglia expressing MMP-9 in TDP-43Q331K ALS model mice.

Sang Won Cheung, Emily F Willis, David G Simmons, Mark C Bellingham, Peter G Noakes.

  Perineuronal nets (PNNs) are extracellular matrix structures that surround excitable neurons and their proximal dendrites. PNNs play an important role in neuroprotection against oxidative stress. Oxidative stress within motor neurons can act as a trigger for neuronal death, and this has been implicated in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). We therefore characterised PNNs around alpha motor neurons and the possible contributing cellular factors in the mutant TDP-43Q331K transgenic mouse, a slow onset ALS mouse model. PNNs around alpha motor neurons showed significant loss at mid-stage disease in TDP-43Q331K mice compared to wild type strain control mice. PNN loss coincided with an increased expression of matrix metallopeptidase-9 (MMP-9), an endopeptidase known to cleave PNNs, within the ventral horn. During mid-stage disease, increased numbers of microglia and astrocytes expressing MMP-9 were present in the ventral horn of TDP-43Q331K mice. In addition, TDP-43Q331K mice showed increased levels of aggrecan, a PNN component, in the ventral horn by microglia and astrocytes during this period. Elevated aggrecan levels within glia were accompanied by an increase in fractalkine expression, a chemotaxic protein responsible for the recruitment of microglia, in alpha motor neurons of onset and mid-stage TDP-43Q331K mice. Following PNN loss, alpha motor neurons in mid-stage TDP-43Q331K mice showed increased 3-nitrotyrosine expression, an indicator of protein oxidation. Together, our observations along with previous PNN research provide suggests a possible model whereby microglia and astrocytes expressing MMP-9 degrade PNNs surrounding alpha motor neurons in the TDP-43Q331K mouse. This loss of nets may expose alpha-motor neurons to oxidative damage leading to degeneration of the alpha motor neurons in the TDP-43Q331K ALS mouse model.

Keywords:  Aggrecan; Amyotrophic lateral sclerosis (ALS); Astrocytes; Matrix metallopeptidase-9 (MMP-9); Microglia; Motor neuron; Motor neuron death; Motor neuron disease (MND); Oxidative stress; Perineuronal net (PNN); TDP-43(Q331K)

DOI:  https://doi.org/10.1016/j.nbd.2024.106614
Cell Rep Med. 2024 Jul 24. pii: S2666-3791(24)00373-2. [Epub ahead of print] 101659

Isogenic patient-derived organoids reveal early neurodevelopmental defects in spinal muscular atrophy initiation.

Tobias Grass, Zeynep Dokuzluoglu, Felix Buchner, Ines Rosignol, Joshua Thomas, Antonio Caldarelli, Anna Dalinskaya, Jutta Becker, Fabian Rost, Michele Marass, Brunhilde Wirth, Marc Beyer, Lorenzo Bonaguro, Natalia Rodriguez-Muela.

  Whether neurodevelopmental defects underlie postnatal neuronal death in neurodegeneration is an intriguing hypothesis only recently explored. Here, we focus on spinal muscular atrophy (SMA), a neuromuscular disorder caused by reduced survival of motor neuron (SMN) protein levels leading to spinal motor neuron (MN) loss and muscle wasting. Using the first isogenic patient-derived induced pluripotent stem cell (iPSC) model and a spinal cord organoid (SCO) system, we show that SMA SCOs exhibit abnormal morphological development, reduced expression of early neural progenitor markers, and accelerated expression of MN progenitor and MN markers. Longitudinal single-cell RNA sequencing reveals marked defects in neural stem cell specification and fewer MNs, favoring mesodermal progenitors and muscle cells, a bias also seen in early SMA mouse embryos. Surprisingly, SMN2-to-SMN1 conversion does not fully reverse these developmental abnormalities. These suggest that early neurodevelopmental defects may underlie later MN degeneration, indicating that postnatal SMN-increasing interventions might not completely amend SMA pathology in all patients.

Keywords:  isogenic SMA model; neurodevelopmental defects; neuromesodermal progenitors; organoids; spinal cord

DOI:  https://doi.org/10.1016/j.xcrm.2024.101659
Proc Natl Acad Sci U S A. 2024 Aug 06. 121(32): e2402206121

Loss of primary cilia and dopaminergic neuroprotection in pathogenic LRRK2-driven and idiopathic Parkinson's disease.

Shahzad S Khan, Ebsy Jaimon, Yu-En Lin, Jonas Nikoloff, Francesca Tonelli, Dario R Alessi, Suzanne R Pfeffer.

  Activating leucine-rich repeat kinase 2 (LRRK2) mutations cause Parkinson's and phosphorylation of Rab10 by pathogenic LRRK2 blocks primary ciliogenesis in cultured cells. In the mouse brain, LRRK2 blockade of primary cilia is highly cell type specific: For example, cholinergic interneurons and astrocytes but not medium spiny neurons of the dorsal striatum lose primary cilia in LRRK2-pathway mutant mice. We show here that the cell type specificity of LRRK2-mediated cilia loss is also seen in human postmortem striatum from patients with LRRK2 pathway mutations and idiopathic Parkinson's. Single nucleus RNA sequencing shows that cilia loss in mouse cholinergic interneurons is accompanied by decreased glial-derived neurotrophic factor transcription, decreasing neuroprotection for dopamine neurons. Nevertheless, LRRK2 expression differences cannot explain the unique vulnerability of cholinergic neurons to LRRK2 kinase as much higher LRRK2 expression is seen in medium spiny neurons that have normal cilia. In parallel with decreased striatal dopaminergic neurite density, LRRK2 G2019S neurons show increased autism-linked CNTN5 adhesion protein expression; glial cells show significant loss of ferritin heavy chain. These data strongly suggest that loss of cilia in specific striatal cell types decreases neuroprotection for dopamine neurons in mice and human Parkinson's.

Keywords:  Hedgehog signaling; LRRK2 kinase; Parkinson’s disease; Rab GTPase; primary cilia

DOI:  https://doi.org/10.1073/pnas.2402206121
bioRxiv. 2024 Jul 23. pii: 2024.07.19.604372. [Epub ahead of print]

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

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

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

DOI: https://doi.org/10.1101/2024.07.19.604372
Brain Res Bull. 2024 Jul 29. pii: S0361-9230(24)00169-2. [Epub ahead of print]216 111036

Electroacupuncture alleviates motor dysfunction by regulating neuromuscular junction disruption and neuronal degeneration in SOD1G93A mice.

Junyang Liu, Weijia Zhao, Jie Guo, Kaiwen Kang, Hua Li, Xiaohang Yang, Jie Li, Qiang Wang, Haifa Qiao.

  Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by the progressive destruction of the neuromuscular junction (NMJ) and the degeneration of motor neurons, eventually leading to atrophy and paralysis of voluntary muscles responsible for motion and breathing. NMJs, synaptic connections between motor neurons and skeletal muscle fibers, are extremely fragile in ALS. To determine the effects of early electroacupuncture (EA) intervention on nerve reinnervation and regeneration following injury, a model of sciatic nerve injury (SNI) was first established using SOD1G93A mice, and early electroacupuncture (EA) intervention was conducted at Baihui (DU20), and bilateral Zusanli (ST36). The results revealed that EA increased the Sciatic nerve Functional Index, the structural integrity of the gastrocnemius muscles, and the cross-sectional area of muscle fibers, as well as up-regulated the expression of acetylcholinesterase and facilitated the co-location of α7 nicotinic acetate choline receptors and α-actinin. Overall, these results suggested that EA can promote the repair and regeneration of injured nerves and delay NMJ degeneration in SOD1G93A-SNI mice. Moreover, analysis of the cerebral cortex demonstrated that EA alleviated cortical motor neuron damage in SOD1G93A mice, potentially attributed to the inhibition of the cyclic GMP-AMP synthase-stimulator of interferon genes pathway and the release of interferon-β suppressing the activation of natural killer cells and the secretion of interferon-γ, thereby further inhibiting microglial activation and the expression of inflammatory factors. In summary, EA delayed the degeneration of NMJ and mitigated the loss of cortical motor neurons, thus delaying disease onset, accompanied by alleviation of muscle atrophy and improvements in motor function in SOD1G93A mice.

Keywords:  Amyotrophic lateral sclerosis; Cyclic GMP-AMP synthase; Electroacupuncture; Motor cortex; Neuromuscular junction; Stimulator of interferon genes

DOI:  https://doi.org/10.1016/j.brainresbull.2024.111036
bioRxiv. 2024 Jul 24. pii: 2024.07.24.604995. [Epub ahead of print]

Motor Cortical Neuronal Hyperexcitability Associated with α-Synuclein Aggregation.

Liqiang Chen, Hiba Douja Chehade, Hong-Yuan Chu.

Dysfunction of the cerebral cortex is thought to underlie motor and cognitive impairments in Parkinson disease (PD). While cortical function is known to be suppressed by abnormal basal ganglia output following dopaminergic degeneration, it remains to be determined how the deposition of Lewy pathology disrupts cortical circuit integrity and function. Moreover, it is also unknown whether cortical Lewy pathology and midbrain dopaminergic degeneration interact to disrupt cortical function in late-stage. To begin to address these questions, we injected α-synuclein (αSyn) preformed fibrils (PFFs) into the dorsolateral striatum of mice to seed αSyn pathology in the cortical cortex and induce degeneration of midbrain dopaminergic neurons. Using this model system, we reported that αSyn aggregates accumulate in the motor cortex in a layer- and cell-subtype-specific pattern. Particularly, intratelencephalic neurons (ITNs) showed earlier accumulation and greater extent of αSyn aggregates relative to corticospinal neurons (CSNs). Moreover, we demonstrated that the intrinsic excitability and inputs resistance of αSyn aggregates-bearing ITNs in the secondary motor cortex (M2) are increased, along with a noticeable shrinkage of cell bodies and loss of dendritic spines. Last, neither the intrinsic excitability of CSNs nor their thalamocortical input was altered by a partial striatal dopamine depletion associated with αSyn pathology. Our results documented motor cortical neuronal hyperexcitability associated with αSyn aggregation and provided a novel mechanistic understanding of cortical circuit dysfunction in PD.

DOI: https://doi.org/10.1101/2024.07.24.604995
Proc Natl Acad Sci U S A. 2024 Aug 06. 121(32): e2319091121

Huntingtin contains an ubiquitin-binding domain and regulates lysosomal targeting of mitochondrial and RNA-binding proteins.

Gianna M Fote, Vinay V Eapen, Ryan G Lim, Clinton Yu, Lisa Salazar, Nicolette R McClure, Jharrayne McKnight, Thai B Nguyen, Marie C Heath, Alice L Lau, Mark A Villamil, Ricardo Miramontes, Ian H Kratter, Steven Finkbeiner, Jack C Reidling, Joao A Paulo, Peter Kaiser, Lan Huang, David E Housman, Leslie M Thompson, Joan S Steffan.

  Understanding the normal function of the Huntingtin (HTT) protein is of significance in the design and implementation of therapeutic strategies for Huntington's disease (HD). Expansion of the CAG repeat in the HTT gene, encoding an expanded polyglutamine (polyQ) repeat within the HTT protein, causes HD and may compromise HTT's normal activity contributing to HD pathology. Here, we investigated the previously defined role of HTT in autophagy specifically through studying HTT's association with ubiquitin. We find that HTT interacts directly with ubiquitin in vitro. Tandem affinity purification was used to identify ubiquitinated and ubiquitin-associated proteins that copurify with a HTT N-terminal fragment under basal conditions. Copurification is enhanced by HTT polyQ expansion and reduced by mimicking HTT serine 421 phosphorylation. The identified HTT-interacting proteins include RNA-binding proteins (RBPs) involved in mRNA translation, proteins enriched in stress granules, the nuclear proteome, the defective ribosomal products (DRiPs) proteome and the brain-derived autophagosomal proteome. To determine whether the proteins interacting with HTT are autophagic targets, HTT knockout (KO) cells and immunoprecipitation of lysosomes were used to investigate autophagy in the absence of HTT. HTT KO was associated with reduced abundance of mitochondrial proteins in the lysosome, indicating a potential compromise in basal mitophagy, and increased lysosomal abundance of RBPs which may result from compensatory up-regulation of starvation-induced macroautophagy. We suggest HTT is critical for appropriate basal clearance of mitochondrial proteins and RBPs, hence reduced HTT proteostatic function with mutation may contribute to the neuropathology of HD.

Keywords:  Huntingtin; RNA-binding proteins; autophagy; ubiquitin; ubiquitin-binding domain

DOI:  https://doi.org/10.1073/pnas.2319091121
Stem Cell Res. 2024 Sep;pii: S1873-5061(24)00193-4. [Epub ahead of print]79 103495

Generation of a gene-corrected human isogenic iPSC line from an Alzheimer's disease iPSC line carrying the PSEN1 H163R mutation.

Dominantly Inherited Alzheimer Network (DIAN)

We report the generation of a gene-edited human induced pluripotent stem cell (iPSC) line from an Alzheimer's disease patient-derived iPSC line harbouring the PSEN1 H163R mutation. This line demonstrates pluripotent stem cell morphology, expression of pluripotency markers, and maintains a normal karyotype.

DOI: https://doi.org/10.1016/j.scr.2024.103495
Neural Regen Res. 2025 May 01. 20(5): 1399-1400

Visualizing traumatic brain injury: ocular clues for diagnosis and assessment.

Morteza Abyadeh, Vivek Gupta, Yuyi You, Joao A Paulo, Mehdi Mirzaei.

DOI: https://doi.org/10.4103/NRR.NRR-D-23-01907
bioRxiv. 2024 Jul 17. pii: 2024.07.15.603186. [Epub ahead of print]

Loss of MEF2C function by enhancer mutation leads to neuronal mitochondria dysfunction and motor deficits in mice.

Ali Yousefian-Jazi, Suhyun Kim, Seung-Hye Choi, Jiyeon Chu, Phuong Thi-Thanh Nguyen, Uiyeol Park, Kayeong Lim, Hongik Hwang, Kyungeun Lee, Yeyun Kim, Seung Jae Hyeon, Hyewhon Rhim, Hannah L Ryu, Grewo Lim, Thor D Stein, Hoon Ryu, Junghee Lee.

Genetic changes and epigenetic modifications are associated with neuronal dysfunction in the pathogenesis of neurodegenerative disorders. However, the mechanism behind genetic mutations in the non-coding region of genes that affect epigenetic modifications remains unclear. Here, we identified an ALS-associated SNP located in the intronic region of MEF2C (rs304152), residing in a putative enhancer element, using convolutional neural network. The enhancer mutation of MEF2C reduces own gene expression and consequently impairs mitochondrial function in motor neurons. MEF2C localizes and binds to the mitochondria DNA, and directly modulates mitochondria-encoded gene expression. CRISPR/Cas-9-induced mutation of the MEF2C enhancer decreases expression of mitochondria-encoded genes. Moreover, MEF2C mutant cells show reduction of mitochondrial membrane potential, ATP level but elevation of oxidative stress. MEF2C deficiency in the upper and lower motor neurons of mice impairs mitochondria-encoded genes, and leads to mitochondrial metabolic disruption and progressive motor behavioral deficits. Together, MEF2C dysregulation by the enhancer mutation leads to mitochondrial dysfunction and oxidative stress, which are prevalent features in motor neuronal damage and ALS pathogenesis. This genetic and epigenetic crosstalk mechanism provides insights for advancing our understanding of motor neuron disease and developing effective treatments.

DOI: https://doi.org/10.1101/2024.07.15.603186
ACS Nano. 2024 Jul 30.

Fluorescent Nanocable as a Biomedical Tool: Intracellular Self-Assembly Formed by a Natural Product Interconnects and Synchronizes Mitochondria.

Xueqian Zhao, Fei Wang, Chuen Kam, Ming-Yu Wu, Jianyu Zhang, Changhuo Xu, Kai Bao, Qiyuan He, Ruquan Ye, Ben Zhong Tang, Sijie Chen.

  Self-assembly processes commonly occur in various biological contexts to form functional biological structures. However, the self-assembly of nanofibers within cells by heterologous molecules showing a biological function is rare. In this work, we reported the intracellular formation of fluorescent nanofibers by a natural small molecule, lycobetaine (LBT), which facilitated the direct physical connection between mitochondria and synchronized their membrane potential oscillations. The luminescent properties of LBT enabled the real-time observation of nanofiber formation, while the semiconductive nature of the LBT nanofiber facilitated electrical signal transduction among the connected mitochondria. This study introduces an approach to modulate mitochondrial connectivity within cells using "nano-cables" which facilitate studies on synchronized mitochondrial operations and the underlying mechanisms of drug action.

Keywords:  fluorescent probe; intracellular assembly; mitochondrial membrane potential oscillations; mitochondrial network; nanofiber

DOI:  https://doi.org/10.1021/acsnano.4c06186
bioRxiv. 2024 Jul 19. pii: 2024.07.17.603999. [Epub ahead of print]

RAB12-LRRK2 Complex Suppresses Primary Ciliogenesis and Regulates Centrosome Homeostasis in Astrocytes.

Xingjian Li, Hanwen Zhu, Bik Tzu Huang, Xianting Li, Heesoo Kim, Haiyan Tan, Yuanxi Zhang, Insup Choi, Junmin Peng, Pingyi Xu, Ji Sun, Zhenyu Yue.

Leucine-rich repeat kinase 2 (LRRK2) phosphorylates a subset of RAB GTPases, and the phosphorylation levels are elevated by Parkinson's disease (PD)-linked mutations of LRRK2. However, the precise function of the specific RAB GTPase targeted by LRRK2 signaling in the brain remains to be elucidated. Here, we identify RAB12 as a robust LRRK2 substrate in the mouse brains through phosphoproteomics profiling and solve the structure of RAB12-LRRK2 protein complex through Cryo-EM analysis. Mechanistically, RAB12 cooperates with LRRK2 to inhibit primary ciliogenesis and regulate centrosome homeostasis in astrocytes through enhancing the phosphorylation of RAB10 and recruiting Rab interacting lysosomal protein like 1 (RILPL1), while the functions of RAB12 require a direct interaction with LRRK2 and LRRK2 kinase activity. Furthermore, the ciliary deficits and centrosome alteration caused by the PD-linked LRRK2-G2019S mutation are prevented by the deletion of Rab12 in astrocytes. Thus, our study reveals a physiological function of the RAB12-LRRK2 complex in regulating ciliogenesis and centrosome homeostasis. The RAB12-LRRK2 structure offers a guidance in the therapeutic development of PD by targeting the RAB12-LRRK2 interaction.

DOI: https://doi.org/10.1101/2024.07.17.603999
Front Mol Neurosci. 2024 ;17 1415207

Drosophila larval motor patterning relies on regulated alternative splicing of Dscam2.

G Lorenzo Odierna, Sarah K Kerwin, Grace Ji-Eun Shin, S Sean Millard.

  Recent studies capitalizing on the newly complete nanometer-resolution Drosophila larval connectome have made significant advances in identifying the structural basis of motor patterning. However, the molecular mechanisms utilized by neurons to wire these circuits remain poorly understood. In this study we explore how cell-specific expression of two Dscam2 isoforms, which mediate isoform-specific homophilic binding, contributes to motor patterning and output of Drosophila larvae. Ablating Dscam2 isoform diversity resulted in impaired locomotion. Electrophysiological assessment at the neuromuscular junction during fictive locomotion indicated that this behavioral defect was largely caused by weaker bouts of motor neuron activity. Morphological analyses of single motor neurons using MultiColour FlpOut revealed severe errors in dendrite arborization and assessment of cholinergic and GABAergic projections to the motor domain revealed altered morphology of interneuron processes. Loss of Dscam2 did not affect locomotor output, motor neuron activation or dendrite targeting. Our findings thus suggest that locomotor circuit phenotypes arise specifically from inappropriate Dscam2 interactions between premotor interneurons and motor neurons when they express the same isoform. Indeed, we report here that first-order premotor interneurons express Dscam2A. Since motor neurons express Dscam2B, our results provide evidence that Dscam2 isoform expression alternates between synaptic partners in the nerve cord. Our study demonstrates the importance of cell-specific alternative splicing in establishing the circuitry that underlies neuromotor patterning without inducing unwanted intercellular interactions.

Keywords:  A02; Drosophila; Dscam2; alternative splicing; dendrite targeting; fictive locomotion; looper; motor neuron

DOI:  https://doi.org/10.3389/fnmol.2024.1415207
Alzheimers Dement. 2024 Jul 30.

A functional aged human iPSC-cortical neuron model recapitulates Alzheimer's disease, senescence, and the response to therapeutics.

Leandro H Gallo, Nesar Akanda, Kaveena Autar, Aakash Patel, Ian Cox, Haley A Powell, Marcella Grillo, Natali Barakat, Dave Morgan, Xiufang Guo, James J Hickman.

  INTRODUCTION: The degeneration of cortical layers is associated with cognitive decline in Alzheimer's disease (AD). Current therapies for AD are not disease-modifying, and, despite substantial efforts, research and development for AD has faced formidable challenges. In addition, cellular senescence has emerged as a significant contributor to therapy resistance.METHODS: Human iPSC-derived cortical neurons were cultured on microelectrode arrays to measure long-term potentiation (LTP) noninvasively. Neurons were treated with pathogenic amyloid-β (Aβ) to analyze senescence and response to therapeutic molecules.
RESULTS: Microphysiological recordings revealed Aβ dampened cortical LTP activity and accelerated neuronal senescence. Aging neurons secreted inflammatory factors previously detected in brain, plasma, and cerebral spinal fluid of AD patients, in which drugs modulated senescence-related factors.
DISCUSSION: This platform measures and records neuronal LTP activity in response to Aβ and therapeutic molecules in real-time. Efficacy data from similar platforms have been accepted by the FDA for neurodegenerative diseases, expediting regulatory submissions.
HIGHLIGHTS: This work developed a progerontic model of amyloid-β (Aβ)-driven cortical degeneration. This work measured neuronal LTP and correlated function with aging biomarkers. Aβ is a driver of neuronal senescence and cortical degeneration. Molecules rescued neuronal function but did not halt Aβ-driven senescence. Therapeutic molecules modulated secretion of inflammatory factors by aging neurons.

Keywords:  Alzheimer's disease; aging; amyloid; electrophysiology; inflammation; microelectrodes; neurodegeneration; senescence; tau; therapeutics

DOI:  https://doi.org/10.1002/alz.14044
Cell Mol Life Sci. 2024 Jul 27. 81(1): 315

Identification of novel neuroprotectants against vincristine-induced neurotoxicity in iPSC-derived neurons.

Veselina Petrova, Andrew R Snavely, Jennifer Splaine, Shannon Zhen, Bhagat Singh, Roshan Pandey, Kuchuan Chen, Anya Cheng, Crystal Hermawan, Lee B Barrett, Jennifer A Smith, Clifford J Woolf.

  Chemotherapy-induced peripheral neuropathy (CIPN) is a disabling side effect of cancer chemotherapy that can often limit treatment options for cancer patients or have life-long neurodegenerative consequences that reduce the patient's quality of life. CIPN is caused by the detrimental actions of various chemotherapeutic agents on peripheral axons. Currently, there are no approved preventative measures or treatment options for CIPN, highlighting the need for the discovery of novel therapeutics and improving our understanding of disease mechanisms. In this study, we utilized human-induced pluripotent stem cell (hiPSC)-derived motor neurons as a platform to mimic axonal damage after treatment with vincristine, a chemotherapeutic used for the treatment of breast cancers, osteosarcomas, and leukemia. We screened a total of 1902 small molecules for neuroprotective properties in rescuing vincristine-induced axon growth deficits. From our primary screen, we identified 38 hit compounds that were subjected to secondary dose response screens. Six compounds showed favorable pharmacological profiles - AZD7762, A-674563, Blebbistatin, Glesatinib, KW-2449, and Pelitinib, all novel neuroprotectants against vincristine toxicity to neurons. In addition, four of these six compounds also showed efficacy against vincristine-induced growth arrest in human iPSC-derived sensory neurons. In this study, we utilized high-throughput screening of a large library of compounds in a therapeutically relevant assay. We identified several novel compounds that are efficacious in protecting different neuronal subtypes from the toxicity induced by a common chemotherapeutic agent, vincristine which could have therapeutic potential in the clinic.

Keywords:  Chemotherapy; High-throughput screening; Human motor and sensory neurons; Neuroprotective small molecules; Vincristine-induced growth deficits

DOI:  https://doi.org/10.1007/s00018-024-05340-x
Brain. 2024 Aug 01. pii: awae248. [Epub ahead of print]

Increase of HCN current in SOD1-associated amyotrophic lateral sclerosis.

Hsing-Jung Lai, Yih-Chih Kuo, Chen-Hung Ting, Chih-Chao Yang, Chia-Hsin Kao, Yi-Chieh Tsai, Chi-Chao Chao, Hsueh-Wen Hsueh, Pei-Feng Hsieh, Hsiang-Yu Chang, I-Fan Wang, Li-Kai Tsai.

  The clinical manifestations of sporadic amyotrophic lateral sclerosis (ALS) vary widely. However, the current classification of ALS is mainly based on clinical presentations, while the roles of electrophysiological and biomedical biomarkers remain limited. Herein, we investigated a group of patients with sporadic ALS and an ALS mouse model with superoxide dismutase 1 (SOD1)/G93A transgenes using nerve excitability tests (NET) to investigate axonal membrane properties and chemical precipitation, followed by enzyme-linked immunosorbent assay analysis to measure plasma misfolded protein levels. Six of 19 patients (31.6%) with sporadic ALS had elevated plasma misfolded SOD1 protein levels. In sporadic ALS patients, only those with elevated misfolded SOD1 protein levels showed an increased inward rectification in the current-threshold (I/V) curve and an increased threshold reduction in the hyperpolarizing threshold electrotonus (TE) in the NET study. Two familial ALS patients with SOD1 mutations also exhibited similar electrophysiological patterns of NET. For patients with sporadic ALS showing significantly increased inward rectification in the I/V curve, we noted an elevation in plasma misfolded SOD1 level, but not in total SOD1, misfolded C9orf72, or misfolded phosphorylated TDP43 levels. Computer simulations demonstrated that the aforementioned axonal excitability changes are likely associated with an increase in hyperpolarization-activated cyclic nucleotide-gated (HCN) current. In SOD1/G93A mice, NET also showed an increased inward rectification in the I/V curve, which could be reversed by a single injection of the HCN channel blocker, ZD7288. Daily treatment of SOD1/G93A mice with ZD7288 partially prevented the early motor function decline and spinal motor neuron death. In summary, sporadic ALS patients with elevated plasma misfolded SOD1 exhibited similar patterns of motor axonal excitability changes as familial ALS patients and ALS mice with mutant SOD1 genes, suggesting the existence of SOD1-associated sporadic ALS. The observed NET pattern of increased inward rectification in the I/V curve was attributable to an elevation in the HCN current in SOD1-associated ALS.

Keywords:  amyotrophic lateral sclerosis; animal model; hyperpolarization-activated cyclic nucleotide-gated channel; nerve excitability test; superoxide dismutase 1

DOI:  https://doi.org/10.1093/brain/awae248
PLoS One. 2024 ;19(7): e0305121

Synergistic effect of PAK and Hippo pathway inhibitor combination in NF2-deficient Schwannoma.

Dorothy Benton, Hoi Yee Chow, Sofiia Karchugina, Jonathan Chernoff.

Neurofibromatosis type 2 is a genetic disorder that results in the formation and progressive growth of schwannomas, ependymomas, and/or meningiomas. The NF2 gene encodes the Merlin protein, which links cell cortical elements to the actin cytoskeleton and regulates a number of key enzymes including Group I p21-activated kinases (PAKs), the Hippo-pathway kinase LATS, and mTORC. While PAK1 and PAK2 directly bind Merlin and transmit proliferation and survival signals when Merlin is mutated or absent, inhibition of Group 1 PAKs alone has not proven sufficient to completely stop the growth of NF2-deficient meningiomas or schwannomas in vivo, suggesting the need for a second pathway inhibitor. As the Hippo pathway is also activated in NF2-deficient cells, several inhibitors of the Hippo pathway have recently been developed in the form of YAP-TEAD binding inhibitors. These inhibitors prevent activation of pro-proliferation and anti-apoptotic Hippo pathway effectors. In this study, we show that PAK inhibition slows cell proliferation while TEAD inhibition promotes apoptotic cell death. Finally, we demonstrate the efficacy of PAK and TEAD inhibitor combinations in several NF2-deficient Schwannoma cell lines.

DOI: https://doi.org/10.1371/journal.pone.0305121
Biofabrication. 2024 Jul 31.

Efficient fabrication of 3D bioprinted functional sensory neurons using an inducible Neurogenin-2 human pluripotent stem cell line.

Mitchell Blake St Clair-Glover, Rocio K Finol-Urdaneta, Marnie Maddock, Eileen R Wallace, Sara Miellet, Gordon G Wallace, Zhilian Yue, Mirella Dottori.

  Three-dimensional (3D) tissue models have gained recognition for their improved ability to mimic the native cell microenvironment compared to traditional two-dimensional (2D) models. This progress has been driven by advances in tissue-engineering technologies such as 3D bioprinting, a promising method for fabricating biomimetic living tissues. While bioprinting has succeeded in generating various tissues to date, creating neural tissue models remains challenging. In this context, we present an accelerated approach to fabricate 3D sensory neuron (SN) structures using a transgenic human pluripotent stem cell (hPSC)-line that contains an inducible neurogenin-2 (NGN2) expression cassette. The NGN2 hPSC line was first differentiated to neural crest cell (NCC) progenitors, then incorporated into a cytocompatible GelMA-based bioink for 3D bioprinting. Upregulated NGN2 expression in the bioprinted NCCs resulted in induced SN (iSN) populations that exhibited specific cell markers, with 3D analysis revealing widespread neurite outgrowth through the scaffold volume. Calcium imaging demonstrated functional activity of iSNs, including membrane excitability properties and voltage-gated sodium channel (NaV) activity. This efficient approach to generate 3D bioprinted iSN structures streamlines the development of neural tissue models, useful for the study of neurodevelopment and disease states and offering translational potential.

Keywords:  3D bioprinting; gelatin methacryloyl; human pluripotent stem cells; neural crest; sensory neurons

DOI:  https://doi.org/10.1088/1758-5090/ad69c4
Life Sci Alliance. 2024 Oct;pii: e202402584. [Epub ahead of print]7(10):

Differential roles of lysosomal cholesterol transporters in the development of C. elegans NMJs.

Amin Guo, Qi Wu, Xin Yan, Kanghua Chen, Yuxiang Liu, Dingfa Liang, Yuxiao Yang, Qunfeng Luo, Mingtao Xiong, Yong Yu, Erkang Fei, Fei Chen.

Cholesterol homeostasis in neurons is critical for synapse formation and maintenance. Neurons with impaired cholesterol uptake undergo progressive synapse loss and eventual degeneration. To investigate the molecular mechanisms of neuronal cholesterol homeostasis and its role during synapse development, we studied motor neurons of Caenorhabditis elegans because these neurons rely on dietary cholesterol. Combining lipidomic analysis, we discovered that NCR-1, a lysosomal cholesterol transporter, promotes cholesterol absorption and synapse development. Loss of ncr-1 causes smaller synapses, and low cholesterol exacerbates the deficits. Moreover, NCR-1 deficiency hinders the increase in synapses under high cholesterol. Unexpectedly, NCR-2, the NCR-1 homolog, increases the use of cholesterol and sphingomyelins and impedes synapse formation. NCR-2 deficiency causes an increase in synapses regardless of cholesterol concentration. Inhibiting the degradation or synthesis of sphingomyelins can induce or suppress the synaptic phenotypes in ncr-2 mutants. Our findings indicate that neuronal cholesterol homeostasis is differentially controlled by two lysosomal cholesterol transporters and highlight the importance of neuronal cholesterol homeostasis in synapse development.

DOI: https://doi.org/10.26508/lsa.202402584
Proc Natl Acad Sci U S A. 2024 Aug 06. 121(32): e2322500121

HaloTag as a substrate-based macroautophagy reporter.

Qiang Xiao, Gabrielle Cruz, Rachel Botham, Susan G Fox, Anan Yu, Seth Allen, Richard I Morimoto, Jeffery W Kelly.

  Macroautophagy is a conserved cellular degradation pathway that, upon upregulation, confers resilience toward various stress conditions, including protection against proteotoxicity associated with neurodegenerative diseases, leading to cell survival. Monitoring autophagy regulation in living cells is important to understand its role in physiology and pathology, which remains challenging. Here, we report that when HaloTag is expressed within a cell of interest and reacts with tetramethylrhodamine (TMR; its ligand attached to a fluorophore), the rate of fluorescent TMR-HaloTag conjugate accumulation in autophagosomes and lysosomes, observed by fluorescence microscopy, reflects the rate of autophagy. Notably, we found that TMR-HaloTag conjugates were mainly degraded by the proteasome (~95%) under basal conditions, while lysosomal degradation (~10% upon pharmacological autophagy activation) was slow and incomplete, forming a degraded product that remained fluorescent within a SDS-PAGE gel, in agreement with previous reports that HaloTag is resistant to lysosomal degradation when fused to proteins of interest. Autophagy activation is distinguished from autophagy inhibition by the increased production of the degraded TMR-HaloTag band relative to the full-length TMR-HaloTag band as assessed by SDS-PAGE and by a faster rate of TMR-HaloTag conjugate lysosomal puncta accumulation as observed by fluorescence microscopy. Pharmacological proteasome inhibition leads to accumulation of TMR-HaloTag in lysosomes, indicating possible cross talk between autophagy and proteasomal degradation.

Keywords:  HaloTag; autophagy; lysosome; macroautophagy; reporter

DOI:  https://doi.org/10.1073/pnas.2322500121
bioRxiv. 2024 Jul 16. pii: 2024.07.16.603730. [Epub ahead of print]

Novel Imaging Tools to Study Mitochondrial Dynamics in Caenorhabditis elegans.

Miriam Valera-Alberni, Pallas Yao, Silvia Romero-Sanz, Anne Lanjuin, William B Mair.

Mitochondria exhibit a close interplay between their structure and function. Understanding this intricate relationship requires advanced imaging techniques that can capture the dynamic nature of mitochondria and their impact on cellular processes. However, much of the work on mitochondrial dynamics has been done in single celled organisms or in vitro cell culture. Here, we introduce novel genetic tools for live imaging of mitochondrial networks in the nematode Caenorhabditis elegans , addressing a pressing need for advanced techniques in studying organelle dynamics within live intact multicellular organisms. Through a comprehensive analysis, we directly compare our tools with existing methods, demonstrating their advantages for visualizing mitochondrial morphology and contrasting their impact on organismal physiology. We reveal limitations of conventional techniques, while showcasing the utility and versatility of our approaches, including endogenous CRISPR tags and ectopic labeling. By providing a guide for selecting the most suitable tools based on experimental goals, our work advances mitochondrial research in C. elegans and enhances the strategic integration of diverse imaging modalities for a holistic understanding of organelle dynamics in living organisms.

DOI: https://doi.org/10.1101/2024.07.16.603730
Neuron. 2024 Jul 23. pii: S0896-6273(24)00409-4. [Epub ahead of print]

Rapid iPSC inclusionopathy models shed light on formation, consequence, and molecular subtype of α-synuclein inclusions.

Isabel Lam, Alain Ndayisaba, Amanda J Lewis, YuHong Fu, Giselle T Sagredo, Anastasia Kuzkina, Ludovica Zaccagnini, Meral Celikag, Jackson Sandoe, Ricardo L Sanz, Aazam Vahdatshoar, Timothy D Martin, Nader Morshed, Toru Ichihashi, Arati Tripathi, Nagendran Ramalingam, Charlotte Oettgen-Suazo, Theresa Bartels, Manel Boussouf, Max Schäbinger, Erinc Hallacli, Xin Jiang, Amrita Verma, Challana Tea, Zichen Wang, Hiroyuki Hakozaki, Xiao Yu, Kelly Hyles, Chansaem Park, Xinyuan Wang, Thorold W Theunissen, Haoyi Wang, Rudolf Jaenisch, Susan Lindquist, Beth Stevens, Nadia Stefanova, Gregor Wenning, Wilma D J van de Berg, Kelvin C Luk, Rosario Sanchez-Pernaute, Juan Carlos Gómez-Esteban, Daniel Felsky, Yasujiro Kiyota, Nidhi Sahni, S Stephen Yi, Chee Yeun Chung, Henning Stahlberg, Isidro Ferrer, Johannes Schöneberg, Stephen J Elledge, Ulf Dettmer, Glenda M Halliday, Tim Bartels, Vikram Khurana.

  The heterogeneity of protein-rich inclusions and its significance in neurodegeneration is poorly understood. Standard patient-derived iPSC models develop inclusions neither reproducibly nor in a reasonable time frame. Here, we developed screenable iPSC "inclusionopathy" models utilizing piggyBac or targeted transgenes to rapidly induce CNS cells that express aggregation-prone proteins at brain-like levels. Inclusions and their effects on cell survival were trackable at single-inclusion resolution. Exemplar cortical neuron α-synuclein inclusionopathy models were engineered through transgenic expression of α-synuclein mutant forms or exogenous seeding with fibrils. We identified multiple inclusion classes, including neuroprotective p62-positive inclusions versus dynamic and neurotoxic lipid-rich inclusions, both identified in patient brains. Fusion events between these inclusion subtypes altered neuronal survival. Proteome-scale α-synuclein genetic- and physical-interaction screens pinpointed candidate RNA-processing and actin-cytoskeleton-modulator proteins like RhoA whose sequestration into inclusions could enhance toxicity. These tractable CNS models should prove useful in functional genomic analysis and drug development for proteinopathies.

Keywords:  CRISPR screen; Lewy body; Parkinson’s disease; Rab protein; RhoA; actin cytoskeleton; aggregation; dementia with Lewy bodies; glia; iPSC; inclusion; lipid; neurodegeneration; neuron; p62; piggyBac; proximity labeling; synucleinopathy; ubiquitin; α-synuclein

DOI:  https://doi.org/10.1016/j.neuron.2024.06.002